U.S. patent application number 17/068538 was filed with the patent office on 2021-09-02 for therapeutic antibodies.
The applicant listed for this patent is CytomX Therapecutics, Inc.. Invention is credited to Mark Raymond Frewin, Lisa Kim Gilliland, Luis Ricardo Simoes Da Silva Graca, Herman WALDMANN.
Application Number | 20210269544 17/068538 |
Document ID | / |
Family ID | 1000005586824 |
Filed Date | 2021-09-02 |
United States Patent
Application |
20210269544 |
Kind Code |
A1 |
WALDMANN; Herman ; et
al. |
September 2, 2021 |
THERAPEUTIC ANTIBODIES
Abstract
A pharmaceutical comprising a therapeutic protein that binds to
a therapeutic target, the protein being modified with a compound
that inhibits binding of the protein to the therapeutic target, the
modified protein being effective for reducing an immune response
against the protein and for producing a therapeutic effect by
binding to the therapeutic target. The therapeutic protein may be
an antibody that includes an antibody combining site that binds to
the therapeutic target.
Inventors: |
WALDMANN; Herman; (Oxford,
GB) ; Frewin; Mark Raymond; (Oxford, GB) ;
Gilliland; Lisa Kim; (Balemo, GB) ; Simoes Da Silva
Graca; Luis Ricardo; (Oxford, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CytomX Therapecutics, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000005586824 |
Appl. No.: |
17/068538 |
Filed: |
October 12, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14974042 |
Dec 18, 2015 |
10800850 |
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17068538 |
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13955785 |
Jul 31, 2013 |
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14974042 |
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12316621 |
Dec 15, 2008 |
8623357 |
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13955785 |
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09979948 |
Jul 29, 2002 |
7465790 |
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PCT/GB01/04518 |
Oct 9, 2001 |
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12316621 |
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60242143 |
Oct 23, 2000 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
C07K 16/28 20130101; C07K 2318/10 20130101; C07K 2317/24 20130101;
A61K 47/6849 20170801; C07K 2317/34 20130101; C07K 14/70592
20130101; C07K 2317/515 20130101; C07K 2317/56 20130101; C07K
2317/41 20130101; C07K 2319/00 20130101; A61K 2039/505 20130101;
C07K 16/2893 20130101; C07K 2317/51 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/68 20060101 A61K047/68; C07K 14/705 20060101
C07K014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 2000 |
GB |
0024673.6 |
Claims
1-34. (canceled)
35. A method of treating a disease selected from the group
consisting of cancer, rheumatoid arthritis, diabetes, psoriasis,
multiple sclerosis, systemic lupus, asthma, myocardial infarction,
stroke, and infectious diseases in an animal, the method
comprising: administering to said animal a modified therapeutic
antibody, wherein said modified therapeutic antibody is
administered in an amount effective to treat said disease in said
animal, wherein the modified therapeutic antibody comprises a
cell-binding antibody that includes an antibody combining site that
binds to a cell-bound target antigen, said antibody being modified
with a peptide that inhibits binding of the antibody to the target
antigen, wherein the peptide comprises the target antigen or a
domain or mimotope thereof which is reversibly bound to the
antibody combining site of the antibody, said modified antibody
being effective for reducing an immune response against the
antibody and for producing a therapeutic effect by binding to the
target antigen.
36. The method of claim 35, wherein the peptide bound to the
antibody combining site also is linked to the antibody.
37. The method of claim 35, wherein the antibody includes a light
chain and a heavy chain, and wherein only one of the chains of the
antibody has a peptide linked thereto that binds to the antibody
combining site.
38. The method of claim 35, wherein the affinity of the modified
antibody combined with the peptide for the target antigen is 5 fold
less to 100 fold less than the affinity of the unmodified antibody
for the target antigen.
39. The method of claim 38, wherein the modified antibody has an
affinity for the target antigen that is 20 fold less to 100 fold
less than the affinity of the unmodified antibody for the target
antigen.
40. The method of claim 35, wherein the antibody is an
aglycosylated antibody.
41. The method of claim 35, wherein the Fc portion of the antibody
is aglycosylated.
42. The method of claim 35, wherein the antibody does not bind to
the Fc receptor.
43. The method of claim 35, wherein the antibody is a non-human
antibody.
44. The method of claim 35, wherein the antibody is a chimeric
antibody.
45. The method of claim 35, wherein the antibody has a peptide
reversibly bound to the antibody combining site whereby said target
antigen competes for and displaces the peptide from the antibody
combining site, said peptide inhibiting binding of the antibody to
the target antigen, said modified antibody initially binding to the
target antigen in an amount that is lower than the unmodified
antibody, with said binding to the target antigen increasing as a
result of peptide being displaced from the antibody combining site
as the antibody becomes bound to the target antigen.
46. The method of claim 35, wherein the antibody is a modified
alemtuzumab (CAMPATH-1H) antibody.
47. The method of claim 35, wherein the antibody comprises the CD52
mimotope having the amino acid sequence QTSSPSAD tethered to
alemtuzumab (CAMPATH-1H) light chain V-region by a flexible
Glycine4 Serine x2 Linker and (CAMPATH-1H) heavy chain with wild
type human IgG1 constant region.
48. The method of claim 35, wherein the antibody comprises the CD52
mimotope having the amino acid sequence QTSSPSAD tethered to
alemtuzumab (CAMPATH-1H) light chain V-region by a flexible
Glycine4 Serine x2 Linker and (CAMPATH-1H) heavy chain with an
aglycosyl human IgG1 constant region.
49. The method of claim 35, wherein the antibody comprises the CD52
mimotope having the amino acid sequence QTSSPSAD tethered to
alemtuzumab (CAMPATH-1H) light chain V-region by a flexible
Glycine4 Serine x2 Linker and (CAMPATH-1H) heavy chain with an Fc
mutated human IgG1 constant region.
50. The method of claim 47, wherein the light chain of the antibody
comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid
sequence encoded by the nucleotide sequence of SEQ ID NO: 2.
51. The method of claim 48, wherein the light chain of the antibody
comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid
sequence encoded by the nucleotide sequence of SEQ ID NO: 2.
52. The method of claim 49, wherein the light chain of the antibody
comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid
sequence encoded by the nucleotide sequence of SEQ ID NO: 2.
53. The method of claim 35, wherein the peptide that is reversibly
bound to the antibody combining site of the antibody is
displaceable from the antibody combining site in the presence of
the target antigen, whereby said target antigen when present
displaces the peptide from the antibody combining site as a result
of competitive binding.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
14/974,042, filed Dec. 18, 2015, which is a continuation of
application Ser. No. 13/955,785, filed Jul. 31, 2013, now
abandoned, which is a continuation of application Ser. No.
12/316,621, filed Dec. 15, 2008, now U.S. Pat. No. 8,623,357,
issued on Jan. 7, 2014, which is a continuation of application Ser.
No. 09/979,948, filed Jul. 29, 2002, now U.S. Pat. No. 7,465,790,
issued Dec. 16, 2008, which is the national phase application PCT
Application No. PCT/GB2001/004518, filed Oct. 9, 2001, which claims
priority based on U.S. Provisional Application Ser. No. 60/242,143,
filed Oct. 23, 2000, and United Kingdom Application No. 0024673.6,
filed Oct. 9, 2000, the contents of which are incorporated by
reference in their entireties.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file named
"CYTM_048_C04US_SeqList_ST25.txt", which was created on Apr. 27,
2021 and is 13.9 KB in size, are hereby incorporated by reference
in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to therapeutic antibodies and
to a method for reducing or eliminating their immunogenicity
[0004] Tolerance to foreign antigen or tissue is a state whereby an
otherwise normal, mature immune system is specifically unable to
respond aggressively to that antigen/tissue which it therefore
treats like a normal (non-diseased) body tissue/component. At the
same time the immune system is competent to respond aggressively to
foreign or diseased antigens/tissues to which it has not
specifically tolerant either by the natural process of
self-tolerance or by therapeutic tolerance induction procedures. A
test for tolerance usually requires a demonstration that the
tolerant individual fails to become immune to the specific
antigen/tissue when one or preferably more attempts to immunize are
made at a later time when the same individual can be shown to
respond to an irrelevant antigen/tissue. As used herein, reference
to induction of tolerance is also intended to encompass both
complete and partial/incomplete tolerance induction. Complete
tolerance induction involves the removal of the immune response to
the antigen/tissue to which tolerance is to be induced whereas
partial or incomplete tolerance induction involves a significant
reduction in this immune response.
PRIOR ART
[0005] One of the major problems with the use of antibodies in
therapy is the immune response mounted against them. As humans are
naturally tolerant of their immunoglobulins, a number of strategies
have been used to create human forms of therapeutic antibodies,
strategies such as humanisation, phage display from human
libraries, or the use of mice carrying human immunoglobulin gene
repertoires. Although useful, these procedures cannot guarantee
that patients do not still react against unique features of the
therapeutic antibody, features such as the allotypic determinants
in the constant regions, and idiotypic determinants encoded by the
complementary-determining regions (CDRs).
[0006] Chiller and Weigle (1970) PNAS 65:551 showed in rodents that
tolerance to foreign immunoglobulins can be induced by deaggregated
monomers of those immunoglobulins whilst aggregates of such
immunoglobulins were potentially immunogenic. Benjamin and Waldmann
et al (1986) J. Exp. Med. 163:1539 showed that cell-binding
antibodies could also be immunogenic compared to non-cell binding
antibodies. Isaacs and Waldman (1994) Therapeutic Immunology
1:363-312 showed that the humoral response against therapeutic
antibodies is CD4+ T-cell-dependent. To ensure that therapeutic
antibodies are not immunogenic it would be desirable to induce
tolerance in the CD4 T-cell population to all potentially
immunogenic determinants of those therapeutic antibodies that
host-cells might recognise.
[0007] Gilliland et al (1999) The Journal of Immunology
162:3663-3671, described a alternative route to prevent immune
response against therapeutic antibodies by pre-tolerising the host
with a monomeric preparation of non-cell-binding antibody mutants.
Specifically, this study showed that mutants of the anti-CD52
antibody CAMPATH-1H which are non-cell-binding lose immunogenicity
and can consequently induce tolerance to wild-type binding
antibodies. CAMPATH-1 is the generic name given to the CD52
glycoprotein antigen and to the family of antibodies that recognize
this. CAMPATH is a registered trade mark. The unique ability of
CAMPATH-1H antibodies to kill lymphocytes by both
complement-mediated lysis and cell-mediated lysis has led to the
extensive use of these antibodies for the serotherapy of lymphoma,
marrow and organ transplantation and in the treatment of autoimmune
diseases. The observation that some patients mount antiglobulin
responses to the therapeutic antibody led to research aimed at
abolishing immunogenicity. Gilliland et al. showed in murine models
that the antiglobulin response to a cell-binding form of the
CAMPATH-1H antibody could be abolished by first tolerizing with a
non-cell binding mutant. However, to use this method
therapeutically would require the application of two products, the
non-binding tolerogen and the actual therapeutic antibody. This is
a costly process and has the disadvantage that as the mutant and
therapeutic antibodies differ in a few amino-acid residues and in
some cases tolerance may not extend to the difference, so that an
antiglobulin response could still arise to the wild-type
(unmutated) antibody. There is therefore a need to ensure tolerance
to the whole therapeutic antibody.
[0008] It has thus been a long-term goal in immunology to find a
means to abolish the potential to mount an immune response to
certain therapeutic proteins which may have amino-acid sequences
different to the host. This would have major implications in a
broad range of therapeutic areas ranging from cancer, to autoimmune
disease to transplantation.
STATEMENT OF THE INVENTION
[0009] In accordance with one aspect of the present, there is
provided a modified therapeutic antibody wherein the modified
therapeutic antibody as compared to the unmodified antibody has a
reduced binding to its target antigen. The reduced binding is such
that over time the binding of the antibody to the target is
increased.
[0010] According to one aspect, the present invention is directed
to a therapeutic tolerising antibody which comprises a therapeutic
antibody having a specific therapeutic effect wherein the antibody
has been subject to a temporary obstruction of its
antibody-combining site which reduces the binding of the antibody
for its natural target and wherein following administration to a
host the antibody is capable of regenerating sufficient of a
functionally-competent form of the therapeutic antibody to achieve
the said therapeutic effect, whereby the reduction of the binding
of the antibody for its natural target renders the modified
antibody tolerogenic to itself and to its functionally-competent
form. In this respect, tolerogenic means that an immunogenic immune
response (an antibody response) against the antibody is inhibited,
reduced in severity and/or essentially eliminated.
[0011] Using this antibody the immunogenicity of cell-binding
antibodies may be reduced or circumvented so that antibody therapy
can be used to its full potential. Only one product is used which
is one able to tolerise itself and produce the desired therapeutic
effect. This eliminates the need for two products as used
previously. The temporary blockade of the antibody combining site
(ACS) of the antibody must be for a sufficient time to induce
tolerance within the host immune system, i.e., inhibit the
immunogenic immune response against the antibody, but once this has
been achieved the antibody should revert to or regenerate a form
which can interact with the therapeutic target by increasing the
amount of antibody bound thereto. Thus, immunologically foreign
antibodies may be given to produce the desired therapeutic effect
with a reduction of and/or essentially eliminating a host
immunogenic immune response to them. Thus, the generation of
antibodies against the therapeutic antibody is reduced and/or
essentially eliminated.
[0012] Thus, in accordance with an aspect of the invention, there
is provided a pharmaceutical in the form of a therapeutic antibody
wherein the therapeutic antibody includes an antibody combining
site (ACS) for a therapeutic target and the antibody is modified
with a compound that inhibits the binding of the therapeutic
antibody to the therapeutic target.
[0013] In one such embodiment there is provided a therapeutic
antibody that is modified to include a compound that is reversibly
bound to the antibody combining site of the antibody, with the
target antigen competing with the compound for binding to the ACS
upon administration of the antibody, whereby binding of the
antibody to the target is inhibited. In this manner, the amount of
the modified antibody that becomes bound to the target antigen in
the initial period after administration is less than would have
become bound if the antibody was administered in its non-modified
form. As the compound is displaced from the ACS as a result of
competitive binding, the amount of antibody that becomes bound to
the target antigen increases. By inhibiting the binding of the
antibody, with the amount of antibody that is bound to the target
increasing over time, the modified antibody is capable of reducing
and/or essentially eliminating an antibody response thereto and is
also capable of accomplishing the desired therapeutic effect.
[0014] In one embodiment, the modified antibody has an avidity for
the target that is less than the avidity for the target of the
unmodified antibody. The avidity is reduced in an amount that is
effective for reducing and/or eliminating an antibody response
against the therapeutic antibody while producing the desired
therapeutic effect by binding to the therapeutic target.
[0015] The term "therapeutic" as used herein encompasses both
treating an existing disease condition or disorder and preventing
and/or reducing the severity of a disease, condition or
disorder.
[0016] A therapeutic target is the antigen to which the antibody
binds, which antigen may or may not be present on a tissue or
cells. The compound that is combined with the therapeutic antibody
for inhibiting binding to the target may inhibit such binding by
binding to the ACS and/or by binding or blocking access to the ACS;
e.g., by steric hindrance.
[0017] The compound may be combined with the antibody by linking
the compound to the antibody and/or by binding of the compound to
the ACS. In one embodiment, the compound is linked or tethered to
the antibody and also binds to the ACS. In another embodiment, the
compound is linked to the antibody without binding to the ACS and
inhibits binding of the antibody to the target by inhibiting access
to the ACS; e.g., by steric hindrance. In one non-limiting
embodiment, the compound is linked to only one of the chains of the
antibody.
[0018] The therapeutic antibody may be used as a therapeutic in
humans and may be a non-human antibody e.g. one raised in a
rodent.
[0019] Chimeric and humanised, e.g. CDR-grafted, antibodies may be
used in accordance with the present invention. These antibodies are
less immunogenic than the corresponding rodent antibodies and thus
temporary ACS blockade of such antibodies in accordance with the
present invention may further reduce immunogenicity and enhance
tolerogenicity.
[0020] The compound functions to inhibit binding of the antibody to
the target whereby immediately after administration there is a
reduction of the amount of antibody that binds to the target as
compared to the amount of antibody that would bind without the
presence of the compound. The amount of antibody that becomes bound
to the target increases over time whereby in effect there is a
temporary blocking of the ACS that inhibits the amount of antibody
that binds to the target.
[0021] The temporary blockade of the ACS (a blockade that initially
reduces the amount of antibody that binds to the target, with such
amount increasing with time) may be effected by the following,
including; [0022] (i) Temporary occupancy with molecules such as
the defined antigen or a domain thereof, low affinity antigenic
peptides or mimotopes by pre-incubation in-vitro, that might
gradually dissociate in-vivo, such that the antibody would
gradually accumulate on cell-bound or other "target" antigen if the
association and dissociation constants were favourable by
comparison with the "obstructive" element; or [0023] (ii) Temporary
occupancy with molecules such as the defined antigen or a domain
thereof, low affinity antigenic peptides or mimotopes which may be
attached by flexible linkers. Once administered in-vivo the
antibody would gradually accumulate on cell-bound or other "target"
antigen if the association and dissociation constants were
favourable by comparison with the "obstructive" element; or [0024]
(iii) Chemical drugs which may bind non-covalently in the ACS and
be able to dissociate in-vivo; or [0025] (iv) Other changes that
might temporarily obstruct the ACS.
[0026] Such a modification would interfere with antibody
accumulation on the target antigen for a limited period, which
would be enough to ensure that the administered therapeutic
antibody has a tolerizing effect (which is at least a partial
tolerizing effect) while allowing for the antibody to revert to or
regenerate sufficient of its functionally-competent form to achieve
the desired therapeutic effect, i.e., accumulate on the target
antigen in an amount to produce such effect. Removal of the
modification may also occur by the host's own physiological and
biochemical processes such as pH changes, enzymatic cleavage within
the host, natural competition with host antigens bound to cells.
For example a peptide mimotope could be linked to the antibody H or
L chain by a linker which carries an enzyme degradable motif,
susceptible to cleavage by host enzymes, such as for example,
leukocyte elastase, in-vivo.
[0027] According to one particularly advantageous embodiment of the
invention the linker is cleaved by an enzyme which occurs only or
preferentially at the desired site of action of the therapeutic
antibody thereby providing selective delivery of the therapeutic
antibody to the desired site of action. For example a linker
cleaved by leukocyte elastase would be appropriate for an antibody
whose intended site of action was the joints. Alternatively,
soluble antigen or mimotope might dissociate more easily at low pH
within the site of a tumour which may also provide selective
delivery of the antibody to the desired site of action.
Alternatively, a low affinity mimotope attached by an inert linker
may naturally dissociate in-vivo, and reassociation may be
prevented by the ACS interacting with the natural antigen on host
cells
[0028] Preferably, the native antigen, domains thereof, and peptide
fragments or mimotopes are used to modify the ACS. The latter may
be generated from peptide libraries either synthetically or
biologically-derived. Non-covalently binding chemicals can be
screened from natural or synthetic libraries or from other
available products, for their ability to inhibit antibody binding
to its antigen or a surrogate equivalent. The linkers which may be
used are preferably flexible, but could be enzymatically cleavable
and/or degradable by the body over a set time period.
[0029] The present invention is also directed to antibodies as
described above further comprising an Fc region designed to reduce
interaction with the complement system and with specialised cell
receptors for the Fc region of immunoglobulins (FcR receptors).
Part of the immunogenicity of cell-binding antibodies may come from
their capacity to biologically activate the complement system and
other cells which bind through FcR receptors. The removal of the
biological effector functions in the Fc region of the antibody may
reduce immunogenicity as compared to the unmodified antibody and
thus enhance tolerogenicity. This will be useful for many
antibodies where cell lysis is not essential, such as blocking or
agonist antibodies. Thus, the addition of mutations in the ACS
together with those in the Fc region may be the most effective at
tolerisation towards Fc mutated antibodies designed to block or
enhance cell-function.
[0030] According to a further aspect, the invention provides an
antibody as defined above for use in therapy.
[0031] According to a still further aspect, the invention provides
the use of an antibody as defined above in the manufacture of a
medicament for use in the treatment of a mammal to achieve the said
therapeutic effect. The treatment comprises the administration of
the medicament in a dose sufficient to achieve the desired
therapeutic effect. The treatment may comprise the repeated
administration of the antibody.
[0032] According to a still further aspect, the invention provides
a method of treatment of a human comprising the administration of
an antibody as defined above in a dose sufficient to achieve the
desired therapeutic effect and reduce and/or eliminate an antibody
response to the therapeutic antibody. The therapeutic effect may be
the alleviation or prevention of diseases which may include cancer,
chronic inflammatory diseases such rheumatoid arthritis, autoimmune
diseases such as diabetes, psoriasis, multiple sclerosis, systemic
lupus and others, allergic diseases such as asthma, cardiovascular
diseases such as myocardial infarction, stroke and infectious
diseases. Indeed any disease where continuous or repeated doses of
a therapeutic antibody are contemplated.
[0033] Temporary modification of the type described above may also
be applicable to therapeutic proteins other than antibodies whose
activity depends on a biologically active site which can be
transiently blocked and where the activity of this site determines
immunogenicity. Examples of such therapeutic proteins include
hormones, enzymes, clotting factors, cytokines, chemokines,
immunoglobulin-based fusion proteins.
[0034] When covalently linking the compound to the antibody, in one
embodiment, the compound is preferably linked to only one of the
two arms of the antibody.
[0035] The term "antibody" as used herein includes all forms of
antibodies such as recombinant antibodies, humanized antibodies,
chimeric antibodies, single chain antibodies, monoclonal antibodies
etc. The invention is also applicable to antibody fragments that
are capable of binding to a therapeutic target.
[0036] In one embodiment, a compound (which may be a peptide or
other molecule that is capable of binding to the ACS of the
antibody) is reversibly bound to the antibody binding or combining
site of the antibody that is to be administered to a person. The
compound occupies the binding site of the antibody for the antigen
and thereby inhibits binding of the antibody to the antigen. Since
the compound is reversibly bound to the antibody binding site, the
antibody is capable of binding to the antigen against which the
antibody is directed.
[0037] In one embodiment, the compound that is selected for binding
to the antibody combining site of the antibody is one whereby the
antibody avidity for the compound is less than the antibody avidity
for the antigen. In this manner, when the antibody is initially
administered, there will be reduced binding of the antibody to the
antigen, as compared to the binding that would occur in the absence
of the compound, with such binding increasing over time.
[0038] Applicant has found that reduction of an antibody response
to a therapeutic antibody can be accomplished by administering an
antibody that is capable of effectively binding to the antigen for
producing the desired therapeutic effect, provided that during the
period that immediately follows administration of the antibody, the
amount of the antibody that binds to the antigen is reduced, with
such amount being increased from the reduced amount over time.
[0039] Thus, unlike the prior art, in accordance with the
invention, an antibody that is capable of performing its
therapeutic function also reduces the immunogenic immune response
against the antibody by initially reducing the amount of the
therapeutic antibody binding to the antigen followed by an increase
in the amount of the therapeutic antibody binding.
[0040] The compound that is used for binding to the antibody
combining site in a manner that initially reduces the amount of
antibody binding to the antigen may be a peptide. The peptide may
be identical to or different from a corresponding peptide portion
of the antigen to which the therapeutic antibody binds. The
appropriate peptide for an antibody may be selected by testing a
panel of peptides in an inhibition of binding assay with respect to
the antibody and its antigen. These and other procedures should be
known to those skilled in the art based on the teachings
herein.
[0041] In one embodiment, the antibody combined with the compound
has an avidity for the target antigen that is less than the avidity
of the non-modified antibody for the target antigen. The relative
avidity of the modified antibody and the unmodified antibody may be
determined by an inhibition of binding assay using fifty percent
binding inhibition as an end point. A modified antibody has a
reduced avidity if there is an increase in the amount of modified
antibody as compared to the amount of unmodified antibody required
to produce a fifty percent inhibition of the binding of a labeled
unmodified antibody to the target antigen.
[0042] The avidity of the modified antibody is reduced in an amount
that is effective for reducing and/or essentially eliminating an
antibody response against the antibody and the modified antibody
has an avidity for the target that is effective for producing the
desired therapeutic effect.
[0043] By way of non-limiting examples, the modified antibody as
compared to the unmodified antibody has an avidity for the target
antigen that is at least 4-fold less, and in many cases at least
50-fold less or at least 100-fold less than the avidity of the
unmodified antibody for the target antigen.
[0044] In one non-limiting embodiment, the compound may inhibit
binding of the modified antibody by providing a modified antibody
with a reduced affinity for the target antigen as compared to the
unmodified antibody. In one non-limiting embodiment, the modified
antibody may have an affinity for the antigen to which it is to be
bound that is at least two or at least five-fold less than the
affinity of the unmodified antibody.
[0045] In many cases, the modified antibody may have an affinity
that is at least ten-fold less or at least 20-fold less or at least
100 fold less than the unmodified antibody.
[0046] In one embodiment of the invention, the amount of the
modified antibody that is administered is coordinated with the
inhibition of binding of the modified antibody to the therapeutic
target such that during the first 24 hours after administration the
amount of modified antibody that is bound to the target antigen is
less than the amount of modified antibody that is not bound to the
target antigen, with such relative amounts being effective for
reducing or eliminating the antibody response against the
therapeutic antibody.
[0047] In many cases, without limiting the present invention, the
modified antibody during the first twenty four hours or in some
cases in the first 48 or 72 hours after administration thereof
binds to the target antigen in an amount such that the ratio of the
antibody that is not bound to the target to the antibody that is
bound to the therapeutic target is at least 10:1 and in many cases
is at least 50:1 or at least 100:1.
[0048] The modified antibody is employed in an amount that is
effective for both producing the desired therapeutic effect and for
inducing a reduced immune response against the antibody. In
general, without limiting the present invention, the modified
antibody is administered in an amount such that the quantity of the
antibody administered during the 24-hour period that begins when
the antibody is first administered is at least 50 mg and in general
at least 100 mg and more generally at least 200 mg. The modified
therapeutic antibody in many cases is used in an amount that is
greater than the amount of the unmodified form required to achieve
the desired therapeutic effect with such increased amount being
used to provide an amount of modified therapeutic antibody that is
not bound to the target antigen and is effective for reducing
and/or essentially eliminating an immune response against the
antibody in the recipient.
[0049] Thus, in accordance with an aspect of the present invention,
there is a reduced immune response against a therapeutic antibody
by modifying the antibody in a manner such that the antibody binds
to its antigen, in vivo, in a reduced amount with such amount
increasing over time. Applicant has found that a modified
therapeutic antibody can perform its therapeutic function in vivo
while also inducing a reduced immunogenic immune response against
the antibody in vivo, provided that binding of the antibody to its
antigen is inhibited or reduced immediately after administration
thereof, with the binding increasing over time.
[0050] The therapeutic antibody may be employed in combination with
a pharmaceutically acceptable carrier. The use of a suitable
carrier is deemed to be within the skill of the art from the
teachings herein.
[0051] The present invention is also directed to a therapeutic
tolerising protein which comprises a protein having a specific
therapeutic effect wherein the protein has a biologically active
site which has been subject to a temporary obstruction which
reduces the binding of the protein for its natural target and
wherein following administration to a host the protein is capable
of regenerating sufficient of a functionally-competent form of the
therapeutic protein to achieve the said therapeutic effect, whereby
the reduction of the binding of the protein for its natural target
renders the modified protein tolerogenic to itself and to its
functionally competent form.
[0052] The present invention is also directed to a method of
modifying the pharmacokinetics of a therapeutic antibody or other
protein such that its half-life is extended through longer-term
presence as a free monomer. This is advantageous as a form of "slow
release depot" in terms of cumulative doses and frequency of
administration of the therapeutic protein needed to achieve desired
therapeutic effects. In addition it also allows better tumour
penetration and minimizes some of the side-effects that follow
antibody administration, effects resulting from immediate and
massive accumulation of antibody on target cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] The invention now will be described with respect to the
drawings wherein:
[0054] FIG. 1 shows the results of binding studies which show that
the form of CAMPATH-1H, with the mimotope bound by a flexible
linker, is not able to bind to human T-cell line HUT78 which
carries CD52 by comparison with forms of CAMPATH-1H carrying the
linker alone (linker), an irrelevant peptide linked in the same way
(p61-IgGI), the linker with mimotope attached (MIM-IgGI), as well
as aglycosylated (removal of asparagine at position 297 of the
H-chain) forms of the various antibodies (AG etc). It should be
noted that AG.MIM-IgG1 form is also non-cell binding, and that the
mere insertion of the linker itself reduces binding of CAMPATH-1H
by about 4 fold.
[0055] FIGS. 2A-2B show a Fluorescent Activated cell Sorter (FACS)
dot-plot examining the binding of CAMPATH-1H antibody on the
lymphocytes of CP-1-transgenic mice given various antibody
constructs (0.5 mg) intraperitoneally (IP) 3 hours earlier.
Peripheral blood and splenic lymphocytes were stained with an
anti-human IgG1 to show up any accumulated antibody on their
surface. In FIG. 2A we examined peripheral blood lymphocytes. Mice
treated with the CAMPATH-1H and the AG-CAMPATH-1H form were very
brightly stained, in fact saturated with antibody. Indeed some
depletion of T-cells from the blood is seen at this stage with both
constructs (4% and 32% of the lymphocytes being CD3+). The p61-IgG1
and AG-p61-IgG1 constructs also stain strongly, and achieve some
depletion at this time (13.5% and 23% of the blood lymphocytes
being CD3+). Mim-IgG1 stains the T-cells in the blood, albeit less
effectively than the above constructs, and very little depletion is
seen at this stage (65.7%) of the lymphocytes are CD3+). Finally,
the AG-MIM-IgGI binds very weakly to blood lymphocytes and that
weak binding is not associated with any T-cell depletion at this
stage. In FIG. 2B comparable data are presented on splenic
lymphocytes. Here we can see that both MIM-IgG1 and AG-MIM-IgG1 are
extremely inefficient at binding and depletion unlike the other
constructs that have achieved around 50% depletion by this
stage.
[0056] FIG. 3 shows that even though the MIM-IgG1 and AG.MIM-IgG1
antibodies bind poorly to antigen in-vitro, they do bind to CD52+
cells (in CP-1 transgenic mice) in-vivo. 7 days after the
administration of 500 .mu.g of each antibody spleen and blood
lymphocytes were analysed by flow cytometry. This figure shows that
AG.MIM-IgG1 has bound to the CD3+ cells of the animal, and that the
intensity of staining is higher than in FIG. 2. MIM-IgGI has done
the same but clearly some depletion has taken place as the
percentage of CD3+ cells in the animals is less (1.7% in spleen vs
36.6% for AG.MEVI-IgGl; and 16.1% in blood vs 78.9% for
AG.MIM-IgG1).
[0057] FIGS. 4A-4B show the effects, on peripheral blood lymphocyte
counts, of treating mice transgenic for the CAMPATH-1 antigen (CP-1
mice) with different doses of CAMPATH-1H with (MIM-IgG1) or without
the bound mimotope (CAMPATH-1H)). Peripheral blood lymphocytes
(PBL) were analysed by flow cytometry. The left column shows the
results of mice treated with 1 .mu.g to 50 .mu.g of antibody and
the right column shows the results of the second experiment where
animals were treated with 0.1 mg to 0.5 mg of antibody. The
therapeutic antibody can kill host lymphocytes within 24 hours at
doses down to 51.1 g/ml whereas the antibody with mimotope bound is
not able to do so with doses up to 250 .mu.g/ml. In contrast at 21
days there are clear effects of depletion seen at the 250 .mu.g and
500 .mu.g doses of "with mimotope" while with the therapeutic
antibody CAMPATH-1H lymphocytes are beginning to replenish the
blood.
[0058] FIGS. 5A-5B. FIG. 5A shows the immunogenicity of the various
antibody constructs in CP-1 transgenic mice. Sera were taken from
CP-1 mice treated with different doses of test antibodies. Sera
were collected 21 days (expt. A) or 28 days (expt. B) after
administration and assessed for the presence of anti-CAMPATH-1H Abs
by ELISA. Serum samples were diluted 1:20 in PBS 1% BSA and
subsequently in two-fold dilutions. All doses of the therapeutic
antibody CAMPATH-1H were immunogenic, while responses to all other
modified forms were much lower (including p61-IgGI). Remarkably,
500 .mu.g of the aglycosylated form with the mimotope (AG.MIM-IgG1)
bound generated absolutely no response whatsoever. In Fig. B it can
be seen that the failure of AG.MIM.IgG1 to immunise is not just the
result of the mutation to remove the glycosylation of the FC
region, as AG-CAMPATH-1H proved very immunogenic. The specificity
of the effect for the mimotope was also clearly established as
AG-p61-IgG1 was also quite immunogenic.
[0059] FIGS. 6A-6B. FIG. 6A examines the tolerogenicity of the
various antibody constructs in CP-1 transgenic mice and shows the
results of sera from CP1 mice treated with different doses of Ab at
day 0 which were collected 30 days after challenge with 5 daily
intraperitoneal injections of 50 .mu.g of CAMPATH-1H and assessed
for the presence of anti-CAMPATH-1H Abs by ELISA. Serum samples
were diluted 1:20 in phosphate buffered saline (PBS) containing 1%
BSA and subsequently titrated out in twofold dilutions. In the left
hand figure mice were left 60 days before receiving the challenge
CAMPATH-1H antibody, while in the right-hand figure they were left
21 days. The left panel of FIG. 6A shows that animals pretreated
with any of 100, 250 or 500 m doses of the mimotope were very
impaired in their humoral response to CAMPATH-1H. This indicates
some level of tolerisation. However, the right panel of FIG. 6A
shows that mice were completely tolerised with the aglycosylated
form of the MIM-binding antibody, but only partially impaired with
the antibody binding the irrelevant peptide. FIG. 6B examines the
tolerogenic potential of the constructs are repeat boosting with
the challenge antibody CAMPATH-1H. These are the results for the
same animals seen in FIG. 5A, which had received a further
challenge with 5 doses of 50 .mu.g CAMPATH-1H antibody at the time
of the previous sera collection. Sera from these animals were then
collected 30 days after the rechallenge and analysed as described
in FIGS. 5A-5B. The conclusions are similar to those in FIG.
6A.
[0060] FIGS. 7, 8A and 8B show the nucleotide and amino acid
sequence for the construct MIM-IgG1 used in the following
examples.
[0061] FIGS. 9A, 9B and 10 show the nucleotide and amino acid
sequence for the linker used in the following examples.
[0062] FIGS. 11A, 11B and 12 show the nucleotide and amino acid
sequence for P61-IgG1 used in the following examples.
[0063] The following examples illustrate the invention.
EXAMPLES
Materials and Methods
[0064] The humanised anti-CD52 antibody CAMPATH-1H was used in the
following experiments. Various constructs were made using the
CAMPATH-1H antibody and the following methodology.
Generation of Non-Binding Variants of CAMPATH-1H:
[0065] The cloning of the V-regions of the humanised antibody
CAMPATH-1H specific for the human CD52 antigen is performed as
described in Gilliland et al (1999) The Journal of Immunology
162:33663-3671. The methodology is based on that of Orlandi et al.,
1989, PNAS 86: 3833, using the polymerase chain reaction (PCR). The
wild-type humanised CAMPATH-1 light chain was cloned into the
vector pGEM 9 (Promega) and used as a PCR template for
site-directed mutagenesis.
[0066] A flexible linker (Gly4Ser x 2) was added to the
amino-terminal end of the light chain between the CAMPATH-1H leader
sequence and CAMPATH-1H VL sequence using the oligonucleotide
primers PUCSE2 and Link L-3'+Link-L-5' and PUC SE REV. The
resulting fragments were PCR assembled using primers PUCSE2+PUCSE
REV to give full length Linker-CP-1H light chain which could be
cloned into expression vector as Hind111/Hind 111 fragment.
[0067] The Linker-CP-1H light chain construct was then used as a
PCR template to generate the CD52 Mimotope QTSSPSAD (amino acid
residues 33-40 of SEQ ID NO: 1) and P61 SLLPAIVEL (amino acid
residues 27-35 of SEQ ID NO: 6) peptide constructs. Primers PUCSE2
and MIM-3'+CD52Mim-5' and PUC SE REV were used to give
Mimotope-CP-1H light chain construct. Primers PUCSE2 and
P61-3'+HuP61-5' and PUCSE REV were used to give P61-CP-1H light
chain construct.
[0068] Linker-CP-1H, Mimotope-CP-1H, P61-CP-1H mutants were
transferred to pBAN-2, a derivative of the pNH316 mammalian
expression vector containing neomycin selection (Page et al. 1991
Biotechnology 9:64-68). and PEE 12 a mammalian expression vector
containing the Glutamine Synthetase gene for selection Bebbington
et al. 1992 Biotechnology 10:169-175.
[0069] Subconfluent dhfr.sup.- Chinese Hamster Ovary cells (Page et
al. 1991 Biotechnology 9:64-68) or NSO mouse myeloma cells (ECACC
cat no 8511503, Meth Enzymol 1981, 73B,3) were co-transfected with
the light chain mutants and the CAMPATH-1H heavy chain construct
with wild type human IgG1 constant region, aglycosyl human IgG1
constant region and Non FcR binding human IgG1 constant region.
[0070] CAMPATH 1H heavy chain constructs were expressed in pRDN-1 a
variant of the pLD9 mammalian expression vector with a dhfr
selectable marker (Page et al. 1991 Biotechnology 9:64-68) and PEE
12.
[0071] Transfection was carried out using LipofectAMINE PLUS
reagent (Life Technologies) following the manufacturers
recommendations.
[0072] Human IgG1 constant was derived from the wild type Glm
(1,17) gene described by Takahashi et al., 1982 Cell 29, 671-679.
Aglycosyl mutation at position 297 from asparagine to an alanine
residue. Oligosaccharide at Asn-297 is a characteristic feature of
all normal human IgG antibodies (Kabat et al, Sequence of proteins
of immunological interest US Department of Health human services
publication). Substitution of asparagine with alanine prevents the
glycosylation of the antibody (Routledge and Waldman,
Transplantation, 1995, 60). Non FcR binding mutation at position
235 from leucine to alanine and position 237 from glycine to
alanine Xu et al. 1993 J Immunology 150: 152A. Substitution of
leucine and glycine at positions 235 and 237 prevents complement
fixation and activation.
[0073] Heavy and Light chain transfectants are selected for in
hypoxanthine free IMDM containing 1 mg G418+5% (v/v) dialysed
foetal calf serum. Resulting selected cells are screen for antibody
production by ELISA and for antigen binding to human T cell clone
HUT 78 Gootenberg J E et al. 1981 J. Exp. Med. 154: 1403-1418 and
CD52 transgenic mice.
[0074] Cells producing antibody were cloned by limiting dilution,
and then expanded into roller bottles cultures. The immunoglobulin
from approximately 15 litres of tissue culture supernatant from
each cell line is purified on protein A, dialysed against PBS and
quantified.
List of Primers Used
TABLE-US-00001 [0075] PUCSE-2 5'-CAC AGA TGC GTA AGG AGA AAA TAC-3'
PUCSE REV 5'-GCA GTG AGC GCA ACG CAA T-3' LINK-L3' 5'-GCT TCC GCC
TCC ACC GGA TCC GCC ACC TCC TTG GGA GTG GAC ACC TGT AGC TGT TGC
TAC-3' LINK-L5' 5-GGA GGT GGC GGA TCC GGT GGA GGC GGA AGC GAC ATC
CAG ATG ACC CAG AGC CCA AG-3' MIM-3' 5'-GTC TGC TGA TGG GCT GCT GGT
TTG GGA GTG GAC ACC TGT AGC TGT TGC-3' CD52Mim-5' 5'-CAA ACC AGC
AGC CCA TCA GCA GAC GGA GGT GGC GGA TCC GGT GGA GGA-3' P61-3'
5'-CTC CAC GAT TGC TGG CAG CAG GCT TTG GGA GTG GAC ACC TGT AGC TGT
TG-3' HuP61- 5'AGC CTG CTG CCA GCA ATC GTG GAG CTG GGA GGT GGC GGA
TCC GGT GGA G-3'
[0076] A blocking ligand was based on a published sequence of
antibody peptide mimotope (Hale G 1995 Immunotechnology 1,175-187)
and was engineered into the wild-type CAMPATH-1H antibody as a cDNA
sequence with a generic linker to attach the peptide product to the
antibody light chain so as to enable the antibody to be secreted
with its ligand bound in the antibody combining site. A similar
antibody also had its Fc-region mutated so as to remove the
glycosylation site at position 297.
Constructs/Cell Lines Produced
TF CHO/CP-1H IgGl/MIM and TF NSO/CP-1H IgGl/MIM (MIM IgG1)
[0077] CD52 Mimotope QTSSPSAD (amino acid residues 33-40 of SEQ ID
NO: 1) tethered to CAMPATH-1H light chain V-region by flexible
Glycine4 Serine x2 Linker+Campath-1H heavy chain with wild type
human IgG1 constant region. Cloned into Celltech expression vector
PEE12 for NSO produced antibody and Wellcome pRDN-1 and pBAN-2
expression vectors for CHO produced antibody.
TF NSO/CP-1H AG IgGl/MIM (AG MIM IgG1)
[0078] CD52 Mimotope QTSSPSAD (amino acid residues 33-40 of SEQ ID
NO: 1) tethered to CAMPATH-1H light chain V-region by flexible
Glycine4 Serine x2 Linker+CAMPATH-1H heavy chain Aglycosyl human
IgG1 constant region. Cloned into Celltech expression vector PEE12
for NSO produced antibody.
TF NSO/CP-1H FCR IgGl/MIM (FcRmutMIM IgG1)
[0079] CD52 Mimotope QTSSPSAD (amino acid residues 33-40 of SEQ ID
NO: 1) tethered to CAMPATH-1H light chain V-region by flexible
Glycine4 Serine x2 Linker+CAMPATH-1H heavy chain FcR-MUTATED human
IgG1 constant region. Cloned into Celltech expression vector PEE12
for NSO produced antibody.
TF CHO/CP-1H IgGl/Link (Linker)
[0080] Flexible Glycine4 Serine x2 Linker only on CAMPATH-1H light
chain V-region+CAMPATH-1H heavy chain with wild type human IgG1
constant region. Cloned into Wellcome expression vectors pRDN-1 and
pBAN-2 for CHO produced antibody.
TF CHO/CP-1H IgGl/P61 (P61-IgG1)
[0081] HLA P61 binding peptide SLLPAIVEL (amino acid residues 27-35
of SEQ ID NO: 6) (Hunt et al Science 1992 255 1261-1263) tethered
to CAMPATH-1H light chain V-region by flexible Glycine4 Serine x2
Linker+CAMPATH-1H heavy chain with wild type human IgG1 constant
region. Cloned into Wellcome expression vectors pRDN-1 and pBAN for
CHO produced antibody.
TF NSO/CP-1H AG IgGl/P61 (AGP61 IgG1)
[0082] HLA P61 binding peptide SLLPAIVEL (amino acid residues 27-35
of SEQ ID NO: 6) tethered to CAMPATH-1H light chain V-region by
flexible Glycine4 Serine x2 Linker+CAMPATH-1H heavy chain with
aglycosyl human IgG1 constant region. Cloned into Celltech
expression vector PEE12 for NSO produced antibody.
TF NSO/CP-1H FCR IgGl/P61 (FcRmut P61 IgG1)
[0083] HLA P61 binding peptide SLLPAIVEL (amino acid residues 27-35
of SEQ ID NO: 6) tethered to CAMPATH-1H light chain V-region by
flexible Glycine4 Serine x2 Linker+CAMPATH-1H heavy chain with no
FCR human IgG1 constant region. Cloned into Celltech expression
vector PEE12 for NSO produced antibody.
TF CHO/CO-1H IgG1 (CAMPATH-1H)
[0084] Wild type CAMPATH-1H light chain V-region+CAMPATH-1H heavy
chain with wild type human IgG1 constant region. Cloned into
Wellcome expression vectors pRDN-1 and pBAN-2 for CHO produced
antibody.
TF NSO/CP-1H AG IgG1 (AG-IgG1)
[0085] Wild type CampathOlH light chain V-region+CAMPATH-1H heavy
chain with aglycosyl human IgG1 constant region. Cloned into
Celltech expression vector PEE12 for NSO produced antibody.
Results
[0086] A high dose of the purified, secreted products (CAMPATH-1H,
MIM-IgG1, AG.MIM-IgG1) was injected into mice made transgenic for
human CD52 (Gilliland et al). After one week the antibody could be
found binding to cells in all 3 groups, whereas lymphocyte
depletion could only be seen in the CAMPATH-1H and MIM-IgG1
groups.
[0087] Mice were then challenged with the wild-type antibody on
multiple occasions and could mount only poor xenogenic humoral
responses, unlike mice which had not received the tolerogen or mice
that had, instead been treated with the wild-type CAMPATH-1H
antibody from the outset. Mice tolerised with the aglycosylated
form of MIM-IgG1 (AG.MIM-IgG1) were completely unable to mount a
xenogenic response even after 10 challenge doses of the therapeutic
CAMPATH-1H antibody.
[0088] FIG. 1 shows the binding abilities of the various antibody
constructs to CD52-bearing HUT cells. CAMPATH-1H binds with an
efficiency approximately 2000 times superior to MIM-IgGl, 5 times
than CAMPATH-1H-p61 (both P61-IgG1 and AG.P61-IgG1), and >10,000
times better than AG.MIM-IgGl.
[0089] FIGS. 2A-2B shows a Fluorescent Activated cell Sorter (FACS)
dot-plot examining the binding of CAMPATH-1H antibody on the
lymphocytes of CP-1-transgenic mice given various antibody
constructs (0.5 mg) intraperitoneally (IP) 3 hours earlier.
Peripheral blood and splenic lymphocytes were stained with an
anti-human IgG1 to show up any accumulated antibody on their
surface. In FIG. 2A we examined peripheral blood lymphocytes. Mice
treated with the CAMPATH-1H and the AG-MIM-IgGI form were very
brightly stained, in fact saturated with antibody. Indeed some
depletion of T-cells from the blood is seen at this stage with both
constructs (4% and 32% of the lymphocytes being CD3+). The p61-IgG1
and AG-p61-IgG1 constructs also stain strongly, and achieve some
depletion at this time (13.5% and 23% of the blood lymphocytes
being CD3+). Mim-IgG1 stains the T-cells in the blood, albeit less
effectively than the above constructs, and very little depletion is
seen at this stage (65.7%) of the lymphocytes are CD3+). Finally,
the AG-MIM-IgG1 binds very weakly to blood lymphocytes and that
weak binding is not associated with any T-cell depletion at this
stage. In FIG. 2B comparable data are presented on splenic
lymphocytes. Here we can see that both MIM-IgG1 and AG-MIM-IgG1 are
extremely inefficient at binding and depletion unlike the other
constructs that have achieved around 50% depletion by this
stage.
[0090] FIG. 3 shows that even though the MIM-IgG1 and AG.MIM-IgG1
antibodies bind poorly to antigen in-vitro, they do bind well to
CD52+ cells (in CP-1 transgenic mice) in-vivo. 7 days after the
administration of 500 ug of each antibody spleen and blood
lymphocytes were analysed by flow cytometry. The figure shows that
AG.MIM-IgG1 has bound to the CD3+ cells of the animal. MIM-IgGI has
done the same but clearly some depletion has taken place as the
percentage of CD3+ cells in the animals is less (1.7% in spleen vs
36.6% for AG.MIM-IgGl; and 16.1% in blood vs 78.9% for
AG.MIM-IgG1).
[0091] FIGS. 4A-4B shows that mimotope-binding form of CAMPATH-1H
(MIM-IgG1) is lytic for blood lymphocytes. After the first 24 hrs
there is only limited cell-depletion in the blood. However after 7
days it can see that the high doses of MIM-IgGI antibody do
eliminate a significant number of blood lymphocytes. By 1 month the
lymphocyte counts in treated hosts are comparable between the two
forms of antibody at the high doses (250 .mu.g and 500 .mu.g). The
left column (FIG. 4A) shows the level of blood lymphocyte depletion
achieved in mice treated with 1 .mu.g to 50 .mu.g of antibody. At
these doses, the mimotope-binding form did not deplete while
CAMPATH-1H treated animals showed a dose-dependent depletion of
T-cells. In the right column (FIG. 4B) CAMPATH-1H shows a fast and
efficient depletion of T-cells, whilst the form with bound mimotope
achieved a slower depletion that at 7 days was not as complete as
with CAMPATH-1H treatment, but was maintained for a longer period.
The decrease of hCD52+ cells was not due to coating of the antigen
with the injected antibody as the results were confirmed by an
equivalent decrease of CD4+ and CD8+ cells.
[0092] FIG. 5A shows that the mimotope-binding antibody (MIM-IgG1)
is poorly immunogenic, and that the aglycosylated form of
CAMPATH-1H mimotope is not immunogenic at all. Animals treated with
CAMPATH-1H had high titres of anti-CAMPATH-1H Abs, while the titres
of mice treated with MIMOTOPE-bound form are far lower. Animals
that received the aglycosylated form of the mimotope antibody that
is not depleting, had no detectable antiglobulin response. In FIG.
5B it can be seen that the failure of AG.MIM.IgG1 to immunise is
not just the result of the mutation to remove the glycosylation of
the FC region, as AG-CAMPATH-1H proved very immunogenic. The
specificity of the effect for the mimotope was also clearly
established as AG-p61-IgG1 was also quite immunogenic.
[0093] FIG. 6A shows that agylcosylated form of the
mimotope-binding CAMPATH-1H antibody (AG.MIM-IgG1) is profoundly
tolerogenic. The animals treated at day 0 with CAMPATH-1H linked to
the control peptide, or the ones that received no treatment also
had high titres of antiglobulin. The mice treated with the
mimotope-binding antibody (MIM-IgGI) had much lower titres of
antiglobulin, while animals that received the aglycoslylated form
of the mimotope-binding antibody (AG.MIM-IgGI) that is not
depleting, had no detectable antiglobulin in the sera.
[0094] FIG. 6B confirms further that the aglycosylated form of
mimotope-binding CAMPATH-1H (AG.MIM-IgG1) is profoundly
tolerogenic. The results from FIG. 6B are similar to FIG. 6A with a
larger difference in the antiglobulin titres between the groups
treated at day 0 with CAMPATH-1H, CAMPATH-1H-p61 or untreated and
those groups treated with the mimotope-binding antibodies. Again
there were no detectable anti-globulins in mice treated with
aglycosyl-form (AG.MIM-IgG1)
[0095] Numerous modifications and variations of the embodiments
described herein are possible based on the teachings herein;
therefore, the scope of the invention is not limited to such
embodiments.
Sequence CWU 1
1
211263PRTArtificial SequenceMIM-IgG1 synthetic construct 1Ser Leu
Ala Leu Gln Leu Leu Ser Thr Gln Asp Leu Thr Met Gly Trp1 5 10 15Ser
Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His Ser 20 25
30Gln Thr Ser Ser Pro Ser Ala Asp Gly Gly Gly Gly Ser Gly Gly Gly
35 40 45Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser 50 55 60Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn
Ile Asp65 70 75 80Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu 85 90 95Leu Ile Tyr Asn Thr Asn Asn Leu Gln Thr Gly
Val Pro Ser Arg Phe 100 105 110Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Phe Thr Ile Ser Ser Leu 115 120 125Gln Pro Glu Asp Ile Ala Thr
Tyr Tyr Cys Leu Gln His Ile Ser Arg 130 135 140Pro Arg Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Thr Val Ala145 150 155 160Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 165 170
175Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
180 185 190Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser 195 200 205Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 210 215 220Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val225 230 235 240Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys 245 250 255Ser Phe Asn Arg Gly
Glu Cys 2602850DNAArtificial SequenceDNA encoding MIM-IgG1 protein
and cloning vector sequence 2gaattcgagc tcggtacccg gggatcctct
agagtcgacc tgcaggcatg caagcttggc 60tctacagtta ctgagcacac aggacctcac
catgggatgg agctgtatca tcctcttctt 120ggtagcaaca gctacaggtg
tccactccca aaccagcagc ccctcagcag acggaggtgg 180cggatccggt
ggaggcggaa gcgacatcca gatgacccag agcccaagca gcctgagcgc
240cagcgtgggt gacagagtga ccatcacctg taaagcaagt cagaatattg
acaaatactt 300aaactggtac cagcagaagc caggtaaggc tccaaagctg
ctgatctaca atacaaacaa 360tttgcaaacg ggtgtgccaa gcagattcag
cggtagcggt agcggtaccg acttcacctt 420caccatcagc agcctccagc
cagaggacat cgccacctac tactgcttgc agcatataag 480taggccgcgc
acgttcggcc aagggaccaa ggtggaaatc aaaactgtgg ctgcaccatc
540tgtcttcatc ttcccgccat ctgatgagca gttgaaatct ggaactgcct
ctgttgtgtg 600cctgctgaat aacttctatc ccagagaggc caaagtacag
tggaaggtgg ataacgccct 660ccaatcgggt aactcccagg agagtgtcac
agagcaggac agcaaggaca gcacctacag 720cctcagcagc accctgacgc
tgagcaaagc agactacgag aaacacaaag tctacgcctg 780cgaagtcacc
catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg
840ttagaagctt 8503780DNAArtificial SequenceDNA encoding linker
peptide and cloning vector sequence 3aagcttggct ctacagttac
tgagcacaca ggacctcacc atgggatgga gctgtatcat 60cctcttcttg gtagcaacag
ctacaggtgt ccactcccaa ggaggtggcg gatccggtgg 120aggcggaagc
gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggtga
180cagagtgacc atcacctgta aagcaagtca gaatattgac aaatacttaa
actggtacca 240gcagaagcca ggtaaggctc caaagctgct gatctacaat
acaaacaatt tgcaaacggg 300tgtgccaagc agattcagcg gtagcggtag
cggtaccgac ttcaccttca ccatcagcag 360cctccagcca gaggacatcg
ccacctacta ctgcttgcag catataagta ggccgcgcac 420gttcggccaa
gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat
480cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa 540taacttctat cccagagagg ccaaagtaca gtggaaggtg
gataacgccc tccaatcggg 600taactcccag gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag 660caccctgacg ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac 720ccatcagggc
ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttagaagct
7804258PRTArtificial Sequencelinker peptide 4Val Ser Leu Ala Leu
Gln Leu Leu Ser Thr Gln Asp Leu Thr Met Gly1 5 10 15Trp Ser Cys Ile
Ile Leu Phe Leu Val Ala Thr Ala Thr Gly Val His 20 25 30Ser Gln Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 35 40 45Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60Ile
Thr Cys Lys Ala Ser Gln Asn Ile Asp Lys Tyr Leu Asn Trp Tyr65 70 75
80Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asn Thr Asn
85 90 95Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly 100 105 110Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu
Asp Ile Ala 115 120 125Thr Tyr Tyr Cys Leu Gln His Ile Ser Arg Pro
Arg Thr Phe Gly Gln 130 135 140Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser Val Phe145 150 155 160Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200
205Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
210 215 220Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
Glu Val225 230 235 240Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly 245 250 255Glu Cys5821DNAArtificial SequenceDNA
encoding P61-IgG1 protein and cloning vector sequence 5gcatcactag
taagcttggc tctacagtta ctgagcacac aggacctcac catgggatgg 60agctgtatca
tcctcttctt ggtagcaaca gctacaggtg tccactccca aagcctgctg
120ccagcaatcg tggagctggg aggtggcgga tccggtggag gcggaagcga
catccagatg 180acccagagcc caagcagcct gagcgccagc gtgggtgaca
gagtgaccat cacctgtaaa 240gcaagtcaga atattgacaa atacttaaac
tggtaccagc agaagccagg taaggctcca 300aagctgctga tctacaatac
aaacaatttg caaacgggtg tgccaagcag attcagcggt 360agcggtagcg
gtaccgactt caccttcacc atcagcagcc tccagccaga ggacatcgcc
420acctactact gcttgcagca tataagtagg ccgcgcacgt tcggccaagg
gaccaaggtg 480gaaatcaaac gaactgtggc tgcaccatct gtcttcatct
tcccgccatc tgatgagcag 540ttgaaatctg gaactgcctc tgttgtgtgc
ctgctgaata acttctatcc cagagaggcc 600aaagtacagt ggaaggtgga
taacgccctc caatcgggta actcccagga gagtgtcaca 660gagtaggaca
gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca
720gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct
gagctcgccc 780gtcacaaaga gcttcaacag gggagagtgt tagaagcttt g
8216259PRTArtificial SequenceP61-IgG1 - synthetic construct 6Ser
Thr Gln Asp Leu Thr Met Gly Trp Ser Cys Ile Ile Leu Phe Leu1 5 10
15Val Ala Thr Ala Thr Gly Val His Ser Gln Ser Leu Leu Pro Ala Ile
20 25 30Val Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
Gln 35 40 45Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp
Arg Val 50 55 60Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asp Lys Tyr
Leu Asn Trp65 70 75 80Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr Asn Thr 85 90 95Asn Asn Leu Gln Thr Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser 100 105 110Gly Thr Asp Phe Thr Phe Thr Ile
Ser Ser Leu Gln Pro Glu Asp Ile 115 120 125Ala Thr Tyr Tyr Cys Leu
Gln His Ile Ser Arg Pro Arg Thr Phe Gly 130 135 140Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val145 150 155 160Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 165 170
175Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
180 185 190Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
Ser Val 195 200 205Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser Thr Leu 210 215 220Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr Ala Cys Glu225 230 235 240Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg 245 250 255Gly Glu
Cys724DNAArtificial SequencePCR primer 7cacagatgcg taaggagaaa atac
24819DNAArtificial SequencePCR primer 8gcagtgagcg caacgcaat
19960DNAArtificial SequencePCR primer 9gcttccgcct ccaccggatc
cgccacctcc ttgggagtgg acacctgtag ctgttgctac 601056DNAArtificial
SequencePCR primer 10ggaggtggcg gatccggtgg aggcggaagc gacatccaga
tgacccagag cccaag 561148DNAArtificial SequencePCR primer
11gtctgctgat gggctgctgg tttgggagtg gacacctgta gctgttgc
481248DNAArtificial sequencePCR primer 12caaaccagca gcccatcagc
agacggaggt ggcggatccg gtggagga 481350DNAArtificial SequencePCR
primer 13ctccacgatt gctggcagca ggctttggga gtggacacct gtagctgttg
501449DNAArtificial SequencePCR primer 14agcctgctgc cagcaatcgt
ggagctggga ggtggcggat ccggtggag 491516PRTArtificial Sequencecloning
vector sequence 15Lys Leu Cys Ser Arg Leu Glu Phe Val Asp Glu Leu
Pro Ile Val Ser1 5 10 151618PRTArtificial SequenceCloning vector
sequence 16Lys Leu Cys Ser Arg Leu Glu Phe Val Asp Glu Leu Pro Ile
Val Ser1 5 10 15Arg Ile178PRTArtificial SequenceMIM-IgG1 synthetic
construct 17Gln Thr Ser Ser Pro Ser Ala Asp1 5189PRTArtificial
SequenceP61-IgG1 - synthetic construct 18Ser Leu Leu Pro Ala Ile
Val Glu Leu1 51917PRTArtificial SequenceCloning vector sequence
19Asn Ser Ser Ser Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His1
5 10 15Ala204PRTArtificial SequenceCloning vector sequence 20Ala
Ser Leu Val12110PRTArtificial Sequencelinker 21Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser1 5 10
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