U.S. patent application number 11/501894 was filed with the patent office on 2006-11-30 for humanized anti-cd3 specific antibodies.
This patent application is currently assigned to BTG International Limited. Invention is credited to Sarah L. Bolt, Michael R. Clark, Scott D. Gorman, Edward G. Routledge, Herman Waldman.
Application Number | 20060269547 11/501894 |
Document ID | / |
Family ID | 10712757 |
Filed Date | 2006-11-30 |
United States Patent
Application |
20060269547 |
Kind Code |
A1 |
Bolt; Sarah L. ; et
al. |
November 30, 2006 |
Humanized anti-CD3 specific antibodies
Abstract
Novel aglycosylated antibodies having a binding affinity for the
CD3 antigen complex are of value for use in therapy, particularly
in immunosuppression.
Inventors: |
Bolt; Sarah L.; (Cambridge,
GB) ; Clark; Michael R.; (Cambridge, GB) ;
Gorman; Scott D.; (Oxfordshire, GB) ; Routledge;
Edward G.; (Newcastle, GB) ; Waldman; Herman;
(Oxfordshire, GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
BTG International Limited
London
GB
|
Family ID: |
10712757 |
Appl. No.: |
11/501894 |
Filed: |
August 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10743423 |
Dec 23, 2003 |
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11501894 |
Aug 10, 2006 |
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08478684 |
Jun 7, 1995 |
6706265 |
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10743423 |
Dec 23, 2003 |
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07988925 |
Nov 8, 1993 |
5585097 |
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PCT/GB92/01933 |
Oct 21, 1992 |
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08478684 |
Jun 7, 1995 |
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Current U.S.
Class: |
424/143.1 ;
530/388.22 |
Current CPC
Class: |
C07K 16/00 20130101;
A61P 37/06 20180101; C07K 2317/56 20130101; C07K 2317/565 20130101;
A61P 35/00 20180101; A61K 2039/505 20130101; C07K 2317/73 20130101;
C07K 2317/74 20130101; C07K 2317/24 20130101; C07K 2317/41
20130101; C07K 16/2809 20130101 |
Class at
Publication: |
424/143.1 ;
530/388.22 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 24, 1992 |
GB |
9206422.9 |
Claims
1. An aglycosylated IgG antibody having a binding affinity for the
CD3 antigen complex.
2. An aglycosylated antibody according to claim 1, which has a
binding affinity for the human CD3 antigen complex.
3. An aglycosylated antibody according to claim 2, in which at
least one CDR is selected from the amino acid sequence:
TABLE-US-00010 (SEQ ID NO: 1) (a) Ser-Phe-Pro-Met-Ala, (SEQ ID NO:
2) (b) Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-
Arg-Asp-Ser-VaI-Lys-Gly, (SEQ ID NO: 3) (c)
Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr, (SEQ ID NO: 4) (d)
Thr-Leu-Ser-Ser-Gly-Asn-IIe-Glu-Asn-Asn-Tyr- Val-His, (SEQ ID NO:
5) (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp, (SEQ ID NO: 6) (f)
His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
and conservatively modified variants thereof.
4. An aglycosylated antibody according to claim 2, which has a
heavy chain with at least one CDR selected from the amino acid
sequences: TABLE-US-00011 (SEQ ID NO: 1) (a) Ser-Phe-Pro-Met-Ala,
(SEQ ID NO: 2) (b) Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-
Arg-Asp-Ser-Val-Lys-Gly, (SEQ ID NO: 3) (c)
Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr,
and conservatively modified variants thereof, and/or a light chain
with at least one CDR selected from the amino acid sequences:
TABLE-US-00012 (SEQ ID NO: 4) (d) Thr-Leu-Ser-Ser-Gly-Asn-Ile
Glu-Asn-Asn-Tyr- Val-His, (SEQ ID NO: 5) (e)
Asp-Asp-Asp-Lys-Arg-Pro-Asp, (SEQ ID NO: 6) (f)
His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
and conservatively modified variants thereof.
5. An aglycosylated antibody according to claim 2, which has a
heavy chain with three CDRs comprising the amino acid sequences:
TABLE-US-00013 (SEQ ID NO: 1) (a)Ser-Phe-Pro-met-Ala, (SEQ ID NO:
2) (b)Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-
Arg-Asp-Ser-Val-Lys-Gly, (SEQ ID NO: 3)
(c)Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr,
or conservatively modified variants thereof, and a light chain with
three CDRs comprising the amino acid sequences: TABLE-US-00014 (SEQ
ID NO: 4) (d) Thr-Leu-Ser-Ser-Gly-Asn-Ile Glu-Asn-Asn-Tyr- Val-His,
(SEQ ID NO: 5) (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp, (SEQ ID NO: 6) (f)
His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
or conservatively modified variants thereof, the heavy chain CDRs
being arranged in the order (a), (b), (c) in the
leader.fwdarw.constant domain direction and the light chain CDRs
being arranged in the order (d), (e), (f) in the
leader.fwdarw.constant domain direction.
6. An aglycosylated antibody according to claim 1, in which the
variable domain framework regions are of or are derived from those
of rat or mouse origin.
7. An aglycosylated antibody according to claim 1, in which the
CDRs are of different origin to the variable framework region.
8. An aglycosylated antibody according to claim 7, in which the
variable domain framework regions are of or are derived from those
of human origin.
9. An aglycosylated antibody according to claim 8, in which the
heavy chain variable domain framework region reading from in the
leader.fwdarw.constant domain direction comprises TABLE-US-00015
Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-
Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-
Ser-Gly-Phe-Thr-Phe-Ser-/CDR/-Trp-Val-Arg-Gln-Ala-
Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-/CDR/-Arg-Phe-
Thr-Ile-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-
Leu-Gln-Met-Asn-Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-
Val-Tyr-Tyr-Cys-Ala-Lys/CDR/-Trp-Gly-Gln-Gly-Thr-
Leu-Val-Thr-Val-Ser-Ser, (SEQ ID NO: 7/CDR/ SEQ ID NO: 8/CDR/SEQ ID
NO: 9/CDR/SEQ ID NO: 10),
ID NO: 8/CDR/SEQ ID NO: 9/CDR/SEQ ID NO: 10), CDR indicating the
presence of a CDR of which at least one is (a), (b) or (c) or a
conservatively modified variant thereof.
10. An aglycosylated antibody according to claim 8, in which the
light chain variable domain framework region reading in the
leader.fwdarw.constant domain direction comprises TABLE-US-00016
Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-
Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-/CDR/-Trp-
Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-
Ile-Phe-/CDR/-Gly-Val-Pro-Asp-Arg-Phe-Ser-Gly-Ser-
Ile-Asp-Arg-Ser-Ser-Asn-Ser-Ala-Ser-Leu-Thr-Ile-
Ser-Gly-Leu-Gln-Thr-Glu-Asp-Glu-Ala-Asp-Tyr-Tyr-
Cys-/CDR/-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Thr-Val-Leu-
Gly-Gln-Pro-Lys-Ala-Ala-Pro-Ser-Val-Thr-Leu-Phe-
Pro-Pro-Ser-Ser-Glu-Glu-Leu-Gln (SEQ ID NO: 12/ CDR/SEQ ID NO:
13/CDR/SEQ ID NO: 14/CDR/ SEQ ID NO: 26),
ID NO: 14/CDR/SEQ ID NO: 26), CDR indicating the presence of a CDR
of which at least one is (d), (e) or (f) or a conservatively
modified variant thereof.
11. An aglycosylated antibody according to claim 9 having a heavy
chain variable domain which comprises TABLE-US-00017 (SEQ ID NO:
11) Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-
Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-
Ser-Gly-Phe-Thr-Phe-Ser-Ser-Phe-Pro-Met-Ala-Trp-
Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-
Ser-Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-
Arg-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Ile-Ser-Arg-
Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-
Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-
Ala-Lys-Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr-
Trp-Gly-Gln-Gly-Thr-Leu-Vat-Thr-Val-Ser-Ser.
12. An aglycosylated antibody according to claim 8 having a light
chain variable domain which comprises TABLE-US-00018 (SEQ ID NO:
25) Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-
Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-Thr-Leu-
Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His-Trp-
Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-
Ile-Phe-Asp-Asp-Asp-Lys-Arg-Pro-Asp-Gly-Val-Pro-
Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-Asn-
Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu-
Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-His-Ser-Tyr-Val-Ser-
Ser-Phe-Asn-Val-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Thr-
Val-Leu-Gly-Gln-Pro-Lys-Ala-Ala-Pro-Ser-Val-Thr-
Leu-Phe-Pro-Pro-Ser-Ser-Glu-Glu-Leu-Gln.
13. An aglycosylated antibody according to claim 1, in which the
constant domains are of or are derived from those of rat or mouse
origin.
14. An aglycosylated antibody according to claim 1, in which the
CDRs are of different origin to the constant region.
15. An aglycosylated antibody according to claim 1, in which the
constant domains are of or are derived from those of human
origin.
16. An aglycosylated antibody according to claim 1, in which the
constant region is of an IgG isotype.
17. An aglycosylated antibody according to claim 15, in which the
constant region is of an IgG1 isotype.
18. An aglycosylated antibody according to claim 15, in which
asparagine residue at position 297 of each constant region heavy
chain is replaced by an alternative amino acid residue.
19. An aglycosylated antibody according to claim 18, in which the
asparagine residue is replaced by an alanine residue.
20. An aglycosylated antibody according to claim 1, in which only
one of the arms thereof has an affinity for the CD3 antigen.
21. An aglycosylated antibody according to claim 20 which is
monovalent.
22. An aglycosylated antibody according the claim 21, in which one
half of the antibody consists of a complete heavy chain and light
chain and the other half consists of a similar but truncated heavy
chain lacking the binding site for the light chain.
23. An aglycosylated antibody according to claim 1 in the form of a
pharmaceutical composition comprising a physiologically acceptable
diluent or carrier.
24. An aglycosylated antibody according to claim 1, for use in
therapy.
25. The use of an aglycosylated antibody according to claim 1 for
the manufacture of a medicament for use in immunosuppression.
26. The use according to claim 25, in which the medicament is for
use in the treatment of recipients of a transplant.
27. A method of treating a patient having cancer or requiring
immunosuppression which comprises administering to said patient a
therapeutically effective amount of a ligand or an antibody or
fragment thereof according to claim 1.
Description
[0001] This invention relates to novel antibodies, in particular to
antibodies directed against the CD3 antigen complex.
[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 in a "Y" shaped configuration. The two "arms" of the antibody
are responsible for antigen binding, and include regions where the
polypeptide structure varies, these "arms" being termed Fab'
fragments (fragment-antigen-binding) or F(ab').sub.2 which
represents two Fab' arms linked together by disulphide bonds. The
"tail" or central axis of the antibody contains a fixed or constant
sequence of peptides and is termed the Fc fragment
(fragment-crystalline). The production of monoclonal antibodies was
first disclosed by Kohler and Milstein (Kohler & Milstein,
Nature, 256, 495-497 (1975)). Such monoclonal antibodies have found
widespread use as diagnostic agents and also in therapy.
[0003] 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 (CHl). The constant
domains in the light and heavy chains are not involved directly in
binding the antibody to antigen. The light chain constant domain
and the CHl domain of the heavy chain account for 50% of each Fab'
fragment.
[0004] The variable domains of each pair of light and heavy chains
form the antigen binding site. The domains on the light and heavy
chains have the same general structure and each domain comprises
four framework regions, whose sequences are relatively conserved,
connected by three complementarity determining regions (CDRs)
(Kabat et al, Sequences of Proteins of Immunological Interest, U.S.
Department of Health and Human Services (1987)). The four framework
regions largely adopt a beta-sheet conformation and the CDRs form
loops connecting, and in some cases forming part of, the beta-sheet
structure. The CDRs are held in close proximity by the framework
regions and, with the CDRs from the other domain, contribute to the
formation of the antigen binding site.
[0005] The human CD3 antigen consists of a minimum of four
invariant polypeptide chains, which are non-covalently associated
with the T-cell receptors on the surface of T-cells, and is
generally now referred to as the CD3 antigen complex. It is
intimately involved in the process of T-cell activation in response
to antigen recognition by the T-cell receptors.
[0006] All CD3 monoclonal antibodies can be used to sensitise
T-cells to secondary proliferative stimuli such as IL1 (interleukin
1) and IL2 (interleukin 2). In addition, certain CD3 monoclonal
antibodies are themselves mitogenic for T-cells. This property is
isotype dependent and results from the interaction of the CD3
antibody Fc domain with Fc receptors on the surface of accessory
cells.
[0007] Rodent CD3 antibodies have been used to influence
immunological status by suppressing, enhancing or re-directing
T-cell responses to antigens. They therefore have considerable
therapeutic potential in the human for use as an immunosuppressive
agent, for example for the treatment of rejection episodes
following the transplantation of renal, hepatic and cardiac
allografts. However their value is compromised by two main factors.
The first is the antiglobulin response evoked due to the xenogeneic
nature of the antibody. The second is the "first dose" syndrome
experienced by patients following the initial administration of the
antibody. The symptoms, which range in severity from fever and
chills to pulmonary edema, and which in rare cases can cause death,
are caused by the elevated levels of circulating cytokines
associated with CD3-antibody induced T-cell activation. This
phenomenon requires the cross-linking of the CD3 antigen on the
surface of T-cells to accessory cells through Fc receptors; such
proliferation does not occur with F(ab').sub.2 fragments of CD3
antibodies.
[0008] The first problem can be addressed by re-shaping or
"humanising" the variable region genes of antibodies and expressing
them in association with relevant human constant domain genes. This
reduces the non-human content of the monoclonal antibody to such a
low level that an antiglobulin response is unlikely. Such a
reshaped antibody with a binding affinity for the CD3 antigen
complex is described in UK Patent Application No. 9121126.8
(published as GB 2249310A) and its equivalents (European Patent
Application No. 91917169.4, Japanese Patent Application No.
516117/91 and U.S. patent application Ser. No. 07/862,543).
[0009] There remains however the problem of the first dose response
when these antibodies are used in therapy. Aglycosylation of
antibodies has been described to reduce their ability to bind to Fc
receptors in vitro in some cases. However, it is not predictable
that this will be true of all antibodies, particularly in vivo, and
aglycosylation may result in the introduction into the antibody of
novel and unpredictable properties including novel Fc binding
characteristics causing other undesirable effects. It is also
possible that other undesirable properties not associated with Fc
binding may be introduced to the antibody.
[0010] Moreover, it is of course of vital importance that
aglycosylation is not accompanied by the loss of certain desirable
features of Fc binding in addition to the loss of the undesirable
features such as those attributable to the first dose response.
[0011] It has now been found, however, that it is possible to
produce aglycosylated CD3 antibodies of the IgG subclass which
surprisingly retain their antigen binding specificity and
immunosuppressive properties and yet do not induce T cell
mitogenesis in vitro and induce a reduced level of cytokine release
in vivo, whilst still maintaining some Fc binding ability.
[0012] Accordingly, the invention provides an aglycosylated IgG
antibody having a binding affinity for the CD3 antigen complex.
[0013] The term aglycosylated is employed in its normal usage to
indicate that the antibodies according to the invention are not
glycosylated. Although the present invention can be applied to
antibodies having a binding affinity for a non-human CD3 antigen
complex, for example various other mammalian CD3 antigens for
veterinary use, the primary value of the invention lies in
aglycosylated antibodies having an affinity for the human CD3
antigen complex for use in the human and the following discussion
is particularly directed to that context.
[0014] Further discussion of CD3 antigens is to be found in the
report of the First International Workshop and Conference on Human
Leukocyte Differentiation Antigens and description of various
glycosylated antibodies directed against the CD3 antigen is also to
be found in the reports of this series of Workshops and
Conferences, particularly the Third and Fourth, published by Oxford
University Press. Specific examples of such antibodies include
those described by Van Ller et al., Euro. J. Immunol., 1987, 17,
1599-1604, Alegre et al., J. Immunol., 1991, 140, 1184, and by
Smith et al., ibid, 1986, 16, 478, the last publication relating to
the IgG1 antibody UCHT1 and variants thereof. However, of
particular interest as the basis for aglycosylated antibodies
according to the present invention are the CDRs contained in the
antibodies OKT3 and YTH 12.5.14.2. The antibody OKT3 is discussed
in publications such as Chatenaud et al., Transplantation, 1991,
51, 334 and the New England Journal of Medicine paper, 1985, 313,
339, and also in European Patent No. 0 018 795 and U.S. Pat. No.
4,361,539. The antibody YTH 12.5.14.2 (hereinafter referred to as
YTH 12.5) is discussed in publications such as Clark et al.,
European J. Immunol., 1989, 19, 381-388 and reshaped YTH 12.5
antibodies are the subject of UK Patent Application No. 9121126.8
and its equivalents, this application describing in detail the CDRs
present in this antibody.
[0015] Aglycosylated antibodies containing one or more of the CDRs
described in the above application are of particular interest. Thus
the antibodies of the invention preferably have at least one CDR
selected from the amino acid sequences: TABLE-US-00001 (SEQUENCE ID
NO. 1) (a) Ser-Phe-Pro-Met-Ala, (SEQUENCE ID NO. 2) (b)
Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-
Tyr-Arg-Asp-Ser-Val-Lys-Gly, (SEQUENCE ID NO. 3) (c)
Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr, (SEQUENCE ID NO. 4) (d)
Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn- Tyr-Val-His (SEQUENCE ID
NO. 5) (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp, (SEQUENCE ID NO. 6) (f)
His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
and conservatively modified variants thereof.
[0016] The term "conservatively modified variants" is one well
known in the art and indicates variants containing changes which
are substantially without effect on antibody-antigen affinity.
[0017] The CDRs are situated within framework regions of the heavy
chain (for (a), (b) and (c)) and light chain (for (d), (e) and (f))
variable domains. The antibody also comprises a constant
domain.
[0018] In a preferred embodiment the aglycosylated antibody has
three CDRs corresponding to the amino acid sequences (a), (b) and
(c) above or conservatively modified variants thereof and/or three
CDRs corresponding to amino acid sequences (d), (e) and (f) or
conservatively modified variants thereof, the heavy chain CDRs (a),
(b) and (c) being of most importance.
[0019] A preferred aglycosylated antibody with a binding affinity
for the CD3 antigen thus has a heavy chain with at least one CDR
and particularly three CDRs selected from the amino acid sequences:
TABLE-US-00002 (SEQUENCE ID NO. 1) (a) Ser-Phe-Pro-Met-Ala,
(SEQUENCE ID NO. 2) (b)
Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-
Arg-Asp-Ser-Val-Lys-Gly, (SEQUENCE ID NO. 3) (c)
Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr,
[0020] and conservatively modified variants thereof, and/or a light
chain with at least one CDR and particularly three CDRs selected
from the amino acid sequences: TABLE-US-00003 (SEQUENCE ID NO. 4)
(d) Thr-Leu-Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr- Val-His, (SEQUENCE
ID NO. 5) (e) Asp-Asp-Asp-Lys-Arg-Pro-Asp, (SEQUENCE ID NO. 6) (f)
His-Ser-Tyr-Val-Ser-Ser-Phe-Asn-Val,
and conservatively modified variants thereof.
[0021] Where an aglycosylated antibody according to the invention
contains preferred CDRs as described hereinbefore it conveniently
contains both one or more of the specified heavy chain CDRs and one
or more of the specified light chain CDRs. The CDRs (a), (b) and
(c) are arranged in the heavy chain in the sequence: framework
region 1/(a)/framework region 2/(b)/framework region
3/(c)/framework region 4 in a leader.fwdarw.constant domain
(n-terminal to C-terminal) direction and the CDRs (d), (e) and (f)
are arranged in the light chain in the sequence: framework region
1/(d)/framework region 2/(e)/framework region 3/(f)/framework
region 4 in a leader.fwdarw.constant domain direction. It is
preferred, therefore, that where all three are present the heavy
chain CDRs are arranged in the sequence (a), (b), (c) in a
leader.fwdarw.constant domain direction and the light chain CDRs
are arranged in the sequence (d), (e), (f) in a
leader.fwdarw.constant domain direction.
[0022] It should be appreciated however, that aglycosylated
antibodies according to the invention may contain quite different
CDRs from those described hereinbefore and that, even when this is
not the case, it may be possible to have heavy chains and
particularly light chains containing only one or two of the CDRs
(a), (b) and (c) and (d), (e) and (f), respectively. However,
although the presence of all six CDRs defined above is therefore
not necessarily required in an aglycosylated antibody according to
the present invention, all six CDRs will most usually be present in
the most preferred antibodies. A particularly preferred
aglycosylated antibody therefore has a heavy chain with three CDRs
comprising the amino acid sequences (a), (b) and (c) or
conservatively modified variants thereof and a light chain with
three CDRs comprising the amino acid sequences (d), (e) and (f) or
conservatively modified variants thereof in which the heavy chain
CDRs are arranged in the order (a), (b), (c) in the leader constant
region direction and the light chain CDRs are arranged in the order
(d), (e), (f) in the leader constant region direction.
[0023] The CDRs may be of different origin to the variable
framework region and/or to the constant region and, since the CDRs
will usually be of rat or mouse origin, this is advantageous to
avoid an antiglobulin response in the human, although the invention
does extend to antibodies with such regions of rat or mouse
origin.
[0024] More usually the CDRs are either of the same origin as the
variable framework region but of a different origin from the
constant region, for example in a part human chimaeric antibody,
or, more commonly, the CDRs are of different origin from the
variable framework region.
[0025] The preferred CDRs discussed hereinbefore are obtained from
a rat CD3 antibody. Accordingly, although the variable domain
framework region can take various forms, it is conveniently of or
derived from those of a rodent, for example a rat or mouse, and
more preferably of or derived from those of human origin. One
possibility is for the antibody to have a variable domain framework
region corresponding to that in the YTH12.5 hybridoma although the
constant region will still preferably be of or derived from those
of human origin. However the antibody of the invention is
preferably in the humanised form as regards both the variable
domain framework region and as discussed further hereinafter, the
constant region.
[0026] Accordingly, the invention further comprises an
aglycosylated antibody which has a binding affinity for the human
CD3 antigen and in which the variable domain framework regions
and/or the constant region are of or are derived from those of
human origin.
[0027] Certain human variable domain framework sequences will be
preferable for the grafting of the preferred CDR sequences, since
the 3-dimensional conformation of the CDRs will be better
maintained in such sequences and the antibody will retain a high
level of binding affinity for the antigen. Desirable
characteristics in such variable domain frameworks are the presence
of key amino acids which maintain the structure of the CDR loops in
order to ensure the affinity and specificity of the antibody for
the CD3 antigen, the lambda type being preferred for the light
chain.
[0028] Human variable region frameworks which are particularly
suitable for use in conjunction with the above CDRs have been
previously identified in UK Patent Application No. 9121126.8. The
heavy chain variable (V) region frameworks are those coded for by
the human VH type III-gene VH26.D.J. which is from the B cell
hybridoma cell line 18/2 (Genbank Code: Huminghat, Dersimonian et
al., Journal of Immunology, 139, 2496-2501). The light chain
variable region frameworks are those of the human V.sub.L.lamda.
type VI gene SUT (Swissprot code; LV6CSHum, Solomon et al. In
Glenner et al (Eds), Amyloidosis, Plenum Press N.Y., 1986, p.
449.
[0029] The one or more preferred CDRs of the heavy chain of the rat
anti-CD3 antibody are therefore preferably present in a human
variable domain framework which has the following amino acid
sequence reading in the leader.fwdarw.constant region direction,
CDR indicating a CDR (a), (b) or (c) as defined hereinbefore, a
conservatively modified variant thereof or an alternative CDR:--
TABLE-US-00004 Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-
Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-
Ser-Gly-Phe-Thr-Phe-Ser-/CDR/-Trp-Val-Arg-Gln-Ala-
Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-Ser-/CDR/-Arg-Phe-
Thr-Ile-Ser-Arg-Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-
Leu-Gln-Met-Asn-Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-
Val-Tyr-Tyr-Cys-Ala-Lys-/CDR/-Trp-Gly-Gln-Gly-Thr-
Leu-Val-Thr-Val-Ser-Ser (SEQUENCE ID NO. 7/CDR/ SEQUENCE ID NO.
8/CDR/SEQUENCE ID NO. 9/CDR/ SEQUENCE ID NO. 10).
[0030] In an aglycosylated antibody containing all three preferred
CDRs, the heavy chain variable region comprises the following
sequence:-- TABLE-US-00005
Glu-Val-Gln-Leu-Leu-Glu-Ser-Gly-Gly-Gly-Leu-Val-
Gln-Pro-Gly-Gly-Ser-Leu-Arg-Leu-Ser-Cys-Ala-Ala-
Ser-Gly-Phe-Thr-Phe-Ser-Ser-Phe-Pro-Met-Ala-Trp-
Val-Arg-Gln-Ala-Pro-Gly-Lys-Gly-Leu-Glu-Trp-Val-
Ser-Thr-Ile-Ser-Thr-Ser-Gly-Gly-Arg-Thr-Tyr-Tyr-
Arg-Asp-Ser-Val-Lys-Gly-Arg-Phe-Thr-Ile-Ser-Arg-
Asp-Asn-Ser-Lys-Asn-Thr-Leu-Tyr-Leu-Gln-Met-Asn-
Ser-Leu-Arg-Ala-Glu-Asp-Thr-Ala-Val-Tyr-Tyr-Cys-
Ala-Lys-Phe-Arg-Gln-Tyr-Ser-Gly-Gly-Phe-Asp-Tyr-
Trp-Gly-Gln-Gly-Thr-Leu-Val-Thr-Val-Ser-Ser (SEQUENCE ID NO.
11).
[0031] Similarly, the one or more preferred CDRs of the light chain
of the rat CD3 antibody are therefore preferably present in a human
variable domain framework which has the following amino acid
sequence reading in the leader.fwdarw.constant region direction,
CDR indicating a CDR (d), (e) and (f) as defined hereinbefore, a
conservatively modified variant thereof or an alternative
CDR:--Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-Ser-Pro-Gly-Lys-Thr-
-Val-Ile-Ile-Ser-Cys-/COR/-Trp-Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-
-Val-Ile-Phe-/COR/-Gly-Val-Pro-Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-
-Asn-Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu-Asp-Glu-Ala-Asp-Tyr-T-
yr-Cys-/CDR/-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Thr-Val-Leu (SEQUENCE ID
NO. 12/CDR/SEQUENCE ID NO. 13/CDR/SEQUENCE ID NO. 14/CDR/SEQUENCE
ID NO. 15).
[0032] In an aglycosylated antibody containing all three preferred
CDRs the light chain-variable region comprises the following
sequence:-- TABLE-US-00006
Asp-Phe-Met-Leu-Thr-Gln-Pro-His-Ser-Val-Ser-Glu-
Ser-Pro-Gly-Lys-Thr-Val-Ile-Ile-Ser-Cys-Thr-Leu-
Ser-Ser-Gly-Asn-Ile-Glu-Asn-Asn-Tyr-Val-His-Trp-
Tyr-Gln-Gln-Arg-Pro-Gly-Arg-Ala-Pro-Thr-Thr-Val-
Ile-Phe-Asp-Asp-Asp-Lys-Arg-Pro-Asp-Gly-Val-Pro-
Asp-Arg-Phe-Ser-Gly-Ser-Ile-Asp-Arg-Ser-Ser-Asn-
Ser-Ala-Ser-Leu-Thr-Ile-Ser-Gly-Leu-Gln-Thr-Glu-
Asp-Glu-Ala-Asp-Tyr-Tyr-Cys-His-Ser-Tyr-Val-Ser-
Ser-Phe-Asn-Val-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Thr- Val-Leu (SEQUENCE
ID NO. 16).
[0033] The variable domains, for example comprising one or more
preferred CDRs as described above, preferably in the humanised form
having human antibody-derived variable framework regions, are
attached to appropriate constant domains.
[0034] The heavy and light chain constant regions can be based on
antibodies of different types as desired subject to the antibody
being an IgG antibody, but although they may be of or derived from
those of rat or mouse origin they are preferably of or are derived
from those of human origin. For the light chain the constant region
is preferably of the lambda type and for the heavy chain it is
preferably of an IgG isotype, especially IgG1, modified to effect
aglycosylation as appropriate. All human constant regions of the
IgG isotype are known to be glycosylated at the asparagine residue
at position 297, which makes up part of the N-glycosylation motif
Asparagine.sup.297-X.sup.298-Serine.sup.299 or Threonine.sup.299,
where X is the residue of any amino acid except proline. The
antibody of the invention may thus be aglycosylated by the
replacement of Asparagine.sup.297 in such a constant region with
another amino acid which cannot be glycosylated. Any other amino
acid residue can potentially be used, but alanine is the most
preferred. Alternatively, glycosylation at Asparagine.sup.297 can
be prevented by altering one of the other residues of the motif,
e.g. by replacing residue 298 by proline, or residue 299 by any
amino acid other than serine or threonine. Techniques for
performing this site directed mutagenesis are well known to those
skilled in the art and may for example be performed using a site
directed mutagenesis kit such, for example, as that commercially
available from Amersham. The procedure is further exemplified
hereinafter.
[0035] It is well recognised in the art that the replacement of one
amino acid in a CDR with another amino acid having similar
properties, for example the replacement of a glutamic acid residue
with an aspartic acid residue, may not substantially alter the
properties or structure of the peptide or protein in which the
substitution or substitutions were made. Thus, the aglycosylated
antibodies of the present invention include those antibodies
containing the preferred CDRs but with a specified amino acid
sequence in which such a substitution or substitutions have
occurred without substantially altering the binding affinity and
specificity of the CDRs. Alternatively, deletions may be made in
the amino acid residue sequence of the CDRs or the sequences may be
extended at one or both of the N- and C-termini whilst still
retaining activity.
[0036] Preferred aglycosylated antibodies according to the present
invention are such that the affinity constant for the antigen is
10.sup.5 mole.sup.-1 or more, for example up to 10.sup.12
mole.sup.-1. Ligands of different affinities may be suitable for
different uses so that, for example, an affinity of 10.sup.6,
10.sup.7 or 10.sup.8 mole.sup.-1 or more may be appropriate in some
cases. However antibodies with an affinity in the range of 10.sup.6
to 10.sup.8 mole.sup.-1 will often be suitable. Conveniently the
antibodies also do not exhibit any substantial binding affinity for
other antigens. Binding affinities of the antibody and antibody
specificity may be tested by assay procedures such as those
described in the Examples section hereinafter, (Effector Cell
Retargetting Assay), or by techniques such as ELISA and other
immunoassays.
[0037] Antibodies according to the invention are aglycosylated IgG
CD3 antibodies having a "Y" shaped configuration which may have two
identical light and two identical heavy chains and are thus
bivalent with each antigen binding site having an affinity for the
CD3 antigen. Alternatively, the invention is also applicable to
antibodies in which only one of the arms of the antibody has a
binding affinity for the CD3 antigen. Such antibodies may take
various forms. Thus the other arm of the antibody may have a
binding affinity for an antigen other than CD3 so that the antibody
is a bispecific antibody, for example as described in U.S. Pat. No.
4,474,893 and European Patent Applications Nos. 87907123.1 and
87907124.9. Alternatively, the antibody may have only one arm which
exhibits a binding affinity, such an antibody being termed
"monovalent".
[0038] Monovalent antibodies (or antibody fragments) may be
prepared in a number of ways. Glennie and Stevenson (Nature, 295,
712-713, (1982)) describe a method of preparing monovalent
antibodies by enzymic digestion. Stevenson et al. describe a second
approach to monovalent antibody preparation in which enzymatically
produced Fab' and Fc fragments are chemically cross-linked
(Anticancer Drug Design, 3, 219-230 (1989)). In these methods the
resulting monovalent antibodies have lost one of their Fab' arms. A
third method of preparing monovalent antibodies is described in
European Patent No. 131424. In this approach the "Y" shape of the
antibody is maintained, but only one of the two Fab' domains will
bind to the antigen. This is achieved by introducing into the
hybridoma a gene coding for an irrelevant light chain which will
combine with the heavy chain of the antibody to produce a mixture
of products in which the monovalent antibody is the one of
interest.
[0039] More preferably, however, the monovalent aglycosylated CD3
antibodies of the invention are prepared by the following method.
This involves the introduction into a suitable expression system,
for example a cell system as described hereinafter, together with
genes coding for the heavy and light chains, of a gene coding for a
truncated heavy chain in which the variable region domain and first
constant region domain of the heavy chain are absent, the gene
lacking the exon for each of these domains. This results in the
production by the cell system of a mixture of (a) antibodies which
are complete bivalent antibodies, (b) antibody fragments consisting
only of two truncated heavy chains (i.e. an Fc fragment) and (c)
fragments of antibody which are monovalent for the CD3 antigen,
consisting of a truncated heavy chain and a light chain in
association with the normal heavy chain. Such an antibody fragment
(c) is monovalent since it has any only one Fab' arm. Production of
a monovalent antibody in the form of such a fragment by this method
is preferred for a number of reasons. Thus, the resulting antibody
fragment is easy to purify from a mixture of antibodies produced by
the cell system since, for example, it may be separable simply on
the basis of its molecular weight. This is not possible in the
method of European Patent No. 131424 where the monovalent antibody
produced has similar characteristics to a bivalent antibody in its
size and outward appearance. Additionally, the production of a
monovalent antibody fragment by the new method uses conditions
which can more easily be controlled and is thus not as haphazard as
an enzyme digestion/chemical coupling procedure which requires the
separation of a complex reaction product, with the additional
advantage that the cell line used will continue to produce
monovalent antibody fragments, without the need for continuous
synthesis procedures as required in the enzyme digestion/chemical
coupling procedure.
[0040] It is believed that aglycosylated antibodies according to
the invention do not occur in nature and these aglycosylated
antibodies may in general be produced synthetically in a number of
ways. Most conveniently, however, appropriate gene constructs for
the constant and variable regions of the heavy and light chains
which are present in the antibody are separately obtained and then
inserted in a suitable expression system.
[0041] Genes encoding the variable domains of a ligand of the
desired structure may be produced and conveniently attached to
genes encoding the constant domains of an antibody which have
undergone site directed mutagenesis. These constant genes may be
obtained from hybridoma cDNA or from the chromosomal DNA and have
undergone mutagenesis (site directed) to produce the aglycosylated
constant regions. Genes encoding the variable regions may also be
derived by gene synthesis techniques used in the identification of
the CDRs contained herein. Suitable cloning vehicles for the DNA
may be of various types.
[0042] Expression of these genes through culture of a cell system
to produce a functional CD3 ligand is most conveniently effected by
transforming a suitable prokaryotic or particularly eukaryotic cell
system, particularly an immortalised mammalian cell line such as a
myeloma cell line, for example the YB2/3.01/Ag2O (hereinafter
referred to as Y0) rat myeloma cell, or Chinese hamster ovary cells
(although the use of plant cells is also of interest), with
expression vectors which include DNA coding for the various
antibody regions, and then culturing the transformed cell system to
produce the desired antibody. Such general techniques of use for
the manufacture of ligands according to the present invention are
well known in the very considerable art of genetic engineering and
are described in publications such as "Molecular Cloning" by
Sambrook, Fritsch and Maniatis, Cold Spring Harbour Laboratory
Press, 1989 (2nd edition). The techniques are further illustrated
by the Examples contained herein.
[0043] The present invention thus includes a process for the
preparation of an aglycosylated IgG antibody having a binding
affinity for the CD3 antigen which comprises culturing cells
capable of expressing the antibody in order to effect expression
thereof. The invention also includes a cell line which expresses an
aglycosylated antibody according to the invention.
[0044] Preferred among such cell lines are those which comprise DNA
sequences encoding the preferred CDRs described hereinbefore. A
group of nucleotide sequences coding for the CDRs (a) to (f)
described hereinbefore is as indicated under (a) to (f) below,
respectively, but it will be appreciated that the degeneracy of the
genetic code permits variations to be made in these sequences
whilst still encoding for the CDRs' amino acid sequences.
TABLE-US-00007 (SEQUENCE ID NO. 17) (a) AGCTTTCCAA TGGCC (SEQUENCE
ID NO. 18) (b) ACCATTAGTA CTAGTGGTGG TAGAACTTAC TATCGAGACT
CCGTGAAGGG C (SEQUENCE ID NO. 19) (c) TTTCGGCAGT ACAGTGGTGG
CTTTGATTAC (SEQUENCE ID NO. 20) (d) ACACTCAGCT CTGGTAACAT
AGAAAACAAC TATGTGCAC (SEQUENCE ID NO. 21) (e) GATGATGATA AGAGACCGGA
T (SEQUENCE ID NO. 22) (f) CATTCTTATG TTAGTAGTTT TAATGTT
[0045] Such cell lines will particularly contain larger DNA
sequences which comprise (1) DNA expressing human heavy chain
variable framework regions and one or more of (a), (b) and (c), and
(2) DNA expressing human light chain variable framework regions and
one or more of (d), (e) and (f). A specific example of such DNA is
that sequence (1) indicated below which codes for the CDRs (a), (b)
and (c) arranged in the heavy chain framework coded for by the
human VH type III gene VH26.D.J. as discussed hereinbefore and that
sequence (2) indicated below which codes for the CDRs (d), (e) and
(f) arranged in the light chain framework coded for by the human
V.sub.L.lamda. type VI gene SUT. The CDR sequences (a), (b), (c),
(d), (e) and (f) have been underlined. TABLE-US-00008 (SEQUENCE ID
NO. 23) (1) GAGGTCCAAC TGCTGGAGTC TGGGGGCGGT TTAGTGCAGC CTGGAGGGTC
CCTGAGACTC TCCTGTGCAG CCTCAGGATT CACTTTCAGT AGCTTTCCAA TGGCCTGGGT
CCGCCAGGCT CCAGGGAAGG GTCTGGAGTG GGTCTCAACC ATTAGTACTA GTGGTGGTAG
AACTTACTAT CGAGACTCCG TGAAGGGCCG ATTCACTATC TCCAGAGATA ATAGCAAAAA
TACCCTATAC CTGCAAATGA ATAGTCTGAG GGCTGAGGAC ACGGCCGTCT ATTACTGTGC
AAAATTTCGG CAGTACAGTG GTGGCTTTGA TTACTGGGGC CAAGGGACCC TGGTCACCGT
CTCCTCA (SEQUENCE ID NO. 24) (2) GACTTCATGC TGACTCAGCC CCACTCTGTG
TCTGAGTCTC CCGGAAAGAC AGTCATTATT TCTTGCACAC TCAGCTCTGG TAACATAGAA
AACAACTATG TGCACTGGTA CCAGCAAAGG CCGGGAAGAG CTCCCACCAC TGTGATTTTC
GATGATGATA AGAGACCGGA TGGTGTCCCT GACAGGTTCT CTGGCTCCAT TGACAGGTCT
TCCAACTCAG CCTCCCTGAC AATCAGTGGT CTGCAAACTG AAGATGAAGC TGACTACTAC
TGTCATTCTT ATGTTAGTAG TTTTAATGTT TTCGGCGGTG GAACAAAGCT
CACTGTCCTT
[0046] The cell lines will of course also particularly contain DNA
sequences expressing the heavy and light chain constant
regions.
[0047] The humanised aglycosylated antibodies in accordance with
the invention have therapeutic value. In particular, such
aglycosylated antibodies, especially a humanised aglycosylated
antibody with a specificity for the human CD3 antigen, has valuable
applications in immunosuppression, particularly in the control of
graft rejection, where it is especially desirable that
immunosuppression is temporary rather than total, and thus that
T-cells are not completely destroyed, but instead rendered
non-functional by antibody blockade of the CD3 antigen-TCR complex.
In addition, the aglycosylated CD3 antibodies may have potential in
other areas such as in the treatment of cancer, specifically in the
construction of bispecific antibodies (for effector cell
retargetting) or antibody-toxin conjugates, where the efficacy of
the therapeutic agent would be compromised by Fc-mediated killing
of the effector cells or non-specific killing of Fc receptor
bearing cells respectively.
[0048] In a further aspect, the invention thus includes a method of
treating patients with cancer, particularly a lymphoma, or for
immunosuppression purposes, for instance in a case where graft
rejection may occur, comprising administering a therapeutically
effective amount of an aglycosylated antibody in accordance with
the invention.
[0049] Aglycosylated antibodies in accordance with the invention
may be formulated for administration to patients by administering
the said antibody together with a physiologically acceptable
diluent or carrier. The antibodies are preferably administered in
an injectable form together with such a diluent or carrier which is
sterile and pyrogen free. By way of guidance it may be stated that
a suitable dose of antibody is about 1-10 mg injected daily over a
time period of, for example 10 days, although due to the
elimination of the first dose response it will be possible if
desired to adminster higher amounts of the antibody, for example
even up to 100 mg daily, depending on the individual patient's
needs. Veterinary use is on a similar g/kg dosage basis.
[0050] The invention is illustrated by the following Examples which
are illustrated by the drawings listed below:--
[0051] FIGS. 1-8: These figures show the results of proliferation
assays of peripheral blood lymphocytes to CD3 antibodies. Four
different healthy volunteers were used. The humanised
anti-lymphocyte antibody CDw52 was included as a negative
control.
[0052] FIGS. 9-12: These figures show the comparison of
aglycosylated CD3 antibody and glycosylated CD3 antibody in a mixed
lymphocyte reaction. Aglycosylated antibody specific for the mouse
CD8 antigen was included as a negative control.
[0053] FIGS. 13 & 14: These figures show the results of an
Effector Cell Retargetting Assay comparing glycosylated and
aglycosylated IgG-type CD3 antibodies. The CDw52 antibody was used
as a negative control.
EXAMPLES
Example 1
Preparation of an Aglycosylated Antibody Specific for the Human CD3
Antigen Containing CDRs from the YTH 12.5 Rat Antibody in Human
Variable Framework Regions
[0054] The cloning and re-shaping of the V-region gene of the rat
antibody YTH 12.5 specific for the human CD3 antigen was performed
as described in Routledge et al., 1991, Eur. J. Immunol., 21, 2717
and in UK Patent Application No. 9121126.8 and its equivalents. YTH
12.5 is a rat hybridoma cell line secreting an IgG2b monoclonal
antibody specific for the CD3 antigen complex.
[0055] Briefly, the methodology was based on that of Orlandi et
al., 1989, PNAS USA, 86, 3833, using the polymerase chain reaction
(PCR). The V.sub.H gene (heavy chain variable region gene) was
cloned using oligonucleotide primers VH1FOR and VH1BACK. The PCR
products were ligated into the vector M13-VHPCR1 in which site
directed mutagenesis was performed using 6 oligonucleotide primers.
The V.sub.L gene (light chain variable region gene) was cloned
using primers designed based on the published V.sub.L.lamda.
sequences. The gene was cloned into the vector M13-VKPCR, together
with the human lambda light chain constant region. In this vector
mutagenesis of the V.sub.L framework was performed using 5
oligonucleotides. The humanised V.sub.L gene was then inserted into
the expression vector pH.beta.Apr-1.
[0056] A vector was generated (p316) in which the reshaped CD3 VH
gene could be expressed in conjunction with different
immunoglobulin H chain constant region genes, this vector being
based on the pH.beta.Apr-gpt vector (Gunning et al., 1987, P.N.A.S.
USA, 85, 7719-7723). A 1.65 Kb fragment of DNA carrying the
dihydrofolate reductase (dhft) gene and SV 40 expression signals
(Page & Sydenham, 1991, Biotechnology, 9, 64) was inserted into
the unique EcoRI site of pH.beta.Apr-gpt. A 700 bp HindIII-BamHI
DNA fragment encoding the reshaped CD3-VH gene was then cloned into
the vector's multiple cloning site, downstream and under the
control of the .beta. actin promoter. The desired H chain constant
region gene (in genomic configuration) could then be inserted into
the unique BamHl restriction enzyme site downstream of the CD3-VH
gene.
[0057] The aglycosyl human IgG1 constant region was derived from
the wild type Glm (1,17) gene described by Takahashi et al., (1982,
Cell, 29, 671-679) as follows. The gene was cloned into the vector
M13 tg131 where site-directed mutagenesis was performed (Amersham
International PLC) to mutate the amino acid residue at position 297
from an asparagine to an alanine residue.
[0058] Oligosaccharide at Asn-297 is a characteristic feature of
all normal human IgG antibodies (Kabat et al., 1987, Sequence of
Proteins of Immunological Interest, US Department of Health Human
Services Publication), each of the two heavy chains in the IgG
molecules having a single branched chain carbohydrate group which
is linked to the amide group of the asparagine residue (Rademacher
and Dwek, 1984, Prog. Immunol., 5, 95-112). Substitution of
asparagine with alanine prevents the glycosylation of the
antibody.
[0059] The 2.3 Kb aglycosyl IgG1 constant region was excised from
M13 by double digestion using BamHI and BgIII and ligated into the
BamHI site of vector p316 to produce clone p323.
[0060] Subconfluent monolayers of dhfr.sup.- Chinese Hamster Ovary
cells were co-transfected with the vector p323 containing the heavy
chain gene and a second vector p274 containing the re-shaped human
.lamda. light chain (Routledge et al., 1991, Eur. J. Immunol., 21,
2717-2725). Prior to tranfection both plasmid DNAs were linearised
using the restriction endonuclease PvuI. Transfection was carried
out using the DOTMA reagent (Boehringer, Germany) following the
manufacturer's recommendations.
[0061] Heavy and light chain transfectants were selected for in
xanthine/hypoxanthine free IMDM containing 5% (v/v) dialysed foetal
calf serum.
[0062] The production of the analogous wild type human IgG1-CD3
heavy chain vector p278 has been described elsewhere (Routledge et
al., 1991, Eur. J. Immunol., 21, 2717-2725). H-chain expression
vectors carrying the non-mutant human IgG2 (Flanagan &
Rabbitts, 1982, Nature 300, 709-713), IgG3 (Huck et al., 1986, Nuc.
Acid. Res., 14, 1779-1789), IgG4 (Flanagan & Rabbitts, 1982,
Nature 300, 709-713), Epsilon (Flanagan & Rabbitts, 1982, EMBO.
Journal 1, 655-660) and Alpha-2 (Flanagan & Rabbitts, 1982,
Nature 300, 709-713) constant region genes (vectors p317, p318,
p320, p321 and p325, respectively) were derived from the vector
p316. Introduction of these vectors, in conjunction with the light
chain vector p274, into dhfr.sup.- CHO cells as described earlier,
produced cell lines secreting CD3 antibody of the .gamma.1,
.gamma.2, .gamma.3, .gamma.4, .epsilon. and .alpha.-2 isotype
respectively. Cells expressing CD3 antibodies were subjected to two
rounds of cloning in soft agar, and then expanded into roller
bottle cultures. The immunoglobulin from approximately 4 litres of
tissue culture supernatant from each cell line was concentrated by
ammonium sulphate precipitation, dialysed extensively against PBS
and then quantified as follows:
[0063] As the antibody was not pure, a competition assay was
designed to specifically quantitate the concentration of antibody
with CD3 antigen binding capacity. Human T-cell blasts were
incubated with FITC labelled UCHT-1, an antibody which binds to the
same epitope of the CD3 antigen as the chimaeric panel. The
concentration of FITC reagent used had previously been determined
to be half saturating. Unlabelled YTH 12.5 (HPLC purified) was
titrated from a known starting concentration and added to wells
containing T-cells and UCHT-1 FITC. The unlabelled antibody serves
as a competitor for the antigen binding site. This is detected as
decrease in the mean fluorescence seen when the cells are studied
using FACS analysis. Thus, titration of the chimaeric antibodies
from unknown starting concentrations yields a series of sigmoidal
curves when mean fluorescence is plotted against antibody dilution.
These can be directly compared with the standard YTH 12.5
curve.
Example 2
Proliferation Assays
[0064] The capacity of a CD3 antibody to support T-cell
proliferation in solution is related to the interaction of the Fc
region of the antibody with Fc receptors on accessory cells.
[0065] The aglycosylated chimaeric CD3 antibody prepared as
described in Example 1 was compared with a panel of other chimaeric
antibodies which shared the same variable region architecture but
different H chain constant regions (see Example 1) for the ability
to induce proliferation of human peripheral blood lymphocytes.
Lymphocytes isolated from healthy donors' blood were separated on a
lymphopaque gradient, washed and resuspended in IMDM containing 5%
(v/v) heat-inactivated human AB serum and plated at
5.times.10.sup.4 to 1.times.10.sup.5 cells per well in plates
containing CD3 antibodies in solution. After 3 days in culture the
cells were pulse labelled with tritiated thymidine and harvested 6
hours later and the level of cellular .sup.3H incorporation was
determined by scintillation counting. The proliferation response to
titrated antibody was studied in four blood donors. For a fifth
donor the proliferation response was studied only at 1 .mu.g. The
results are shown in FIGS. 1 to 8.
[0066] For the donors studied, all `wild type` antibodies led to
T-cell proliferation. However the mutant, aglycosylated IgG1
isotype was never mitogenic implicating an important role for the
carbohydrate side chains.
[0067] In a separate experiment, the ability of a panel of CD3
monoclonal antibodies in solution to stimulate T-cell mitogenesis
was studied using lymphocytes isolated from the blood of 10 donors
from a variety of ethnic backgrounds. All of the naturally
occurring isotypes caused proliferation in the presence of 5% human
AB serum, although there were T-cell donor dependent variations in
the extent of the responses caused by the antibodies. In general,
the .gamma.1 and .epsilon. monoclonal antibodies were the most
mitogenic, activating cells at the lowest concentration, and the
.gamma.3 and .gamma.2 were the least active. The aglycosyl
derivative of the .gamma.1 monoclonal antibody was the only CD3
antibody which consistently failed to induce T-cell proliferation
in any of the donors tested, giving responses equivalent to those
of the non-activating control monoclonal antibody Campath-1H. In
order to exclude endotoxin contamination as the cause of the
proliferation seen with the .alpha.2 and .epsilon. preparations, it
was confirmed that proliferation could be blocked by the addition
of an excess of the aglycosyl CD3 mAb thus implicating the CD3
antigen in the activation process.
[0068] The total lack of proliferative response seen with the
aglycosyl .gamma.1 CD3 monoclonal antibody was surprising, given
its position in the ECR activity hierarchy (see Example 5 below).
The reason for this inactivity is probably due to its reduced
affinity for FcRs rather than because aglycosylation has abolished
the ability to trigger some post-binding event required for
proliferation, e.g. by destroying a secondary recognition site on
the monoclonal antibody. This is supported by the observation that
the aglycosyl .gamma.1 mAb could stimulate proliferation to a
considerable degree if the assay was performed in IgG-free medium.
There is probably a minimum thereshold level for the number of
contacts or strength of interaction between T-cell and accessory
cell which must be exceeded before proliferation can be initiated,
and this cannot be achieved by the aglycosyl .gamma.1 mAb in the
presence of significant levels of competing immunoglobulin.
Example 3
The Effect of Chimaeric CD3 Antibodies in Mixed Lymphocyte
Reactions
[0069] A series of experiments was conducted to test whether the
aglycosylated antibody IgG1Ag of Example 1 had the capacity to
block T-cell proliferation in a mixed lymphocyte reaction (MLR) and
the results of 2 experiments are shown in FIGS. 9 to 12.
[0070] Peripheral blood lymphocytes were isolated from two blood
donors. The stimulator cell population was caesium irradiated. The
responder population was incubated with titrated antibody for 30
minutes before the irradiated stimulator cells were added (FIGS. 9,
10 and 11). Control wells of responder cells were also incubated
with irradiated responder cells at each antibody condition, to
determine the specific effect of the antibody on the responder
cells (FIGS. 9, 10 and 12).
[0071] After 5 days incubation the wells were pulse labelled with
tritiated thymidine and harvested 6 hours later.
[0072] The aglycosylated antibody does block the mixed lymphocyte
reaction; as the antibody is titrated out the blockade effect is
less and the proliferation increases. The `wild type` IgG1 actually
has a mitogenic effect on the T-cells and so any blockade of the
MLR is not seen through this response.
[0073] An `irrelevant` aglycosylated antibody specific for the
murine CD8 antigen was included as a negative control. This
antibody has as expected no effect on the MLR.
Example 4
In Vivo Effect of IgG1 Antibodies
[0074] In vivo experiments were performed with chimaeric anti-human
CD3 antibodies in mice which were transgenic for the human CD3
epsilon subunit including the aglycosylated antibody of Example 1
(IgG1Ag). The ability of some of the chimaeric CD3 antibodies to
cause the release of TNF factor following a single injection was
compared.
[0075] In this set of experiments serum was collected from the mice
before injection and then at 90 minutes and 4 hours following
intravenous injection with 10 .mu.g of relevant CD3 antibody.
Groups consisted of 5 mice and the serum collected from each group
was pooled. Analysis of the level of TNF in the serum was performed
in the laboratory of Professor Jean-Francois Bach, using a bioassay
which measured the cytotoxic effect of sera on the L929 mouse
fibroblast cells as a result of the presence of TNF.
[0076] The results are shown in Table 1 below: TABLE-US-00009 TABLE
1 TNF detected in serum of hCD3 mice following injection with
chimaeric CD3 antibodies Prebleed 90 minute 4 hour Sample TNF level
TNF level TNF level Saline 0 units/ml 0 units/ml 0 units/ml IgG1 0
units/ml >400 units/ml 0 units/ml IgG2 0 units/ml >400
units/ml 0 units/ml IgG1Ag 0 units/ml 50 units/ml 0 units/ml IgE 0
units/ml 50 units/ml 25 units/ml YTH 12.5 0 units/ml 50 units/ml 0
units/ml
[0077] A significant difference is seen between the level of TNF
associated with injection of the two forms of human IgG1. The
aglycosylated form is associated with at least an eight-fold less
release of TNF than the wild type IgG1, or with the IgG2
antibody.
[0078] The results of Examples 2-4 show that the aglycosylated CD3
antibody was not mitogenic to T-cells in solution indicating that
the antibody had a reduced capacity to interact with Fc receptors
on accessory cells. The antibody retained the immunosuppressive
properties that are characteristic of CD3 antibodies. In vivo the
aglycosylated antibody led to a significantly lower release of
tumour necrosis factor in human CD3 transgenic mice than the
parental IgG1 antibody. Thus this agent may be an `improved` CD3
antibody for the purposes of immunosuppression if the decreased TNF
release seen in mice is mirrored in humans.
Example 5
Effector Cell Retargetting Assays for the Detection of CD3
Antibodies with the Ability to Direct T-Cell Killing
[0079] This was performed as described elsewhere (Gilliland et al.,
1988, PNAS USA, 85, 4419) and measures the ability of a CD3
monoclonal antibody to cross-link activated T-cells to Fc.gamma.R
bearing target cells and thus to mediate target cell lysis. Briefly
U937 human monocytic human cells which express the Fc.gamma.
receptors I, II and III were labelled with .sup.5Cr sodium chromate
and resuspended to 2.times.10.sup.5 cells per ml. These cells were
used as targets. Human T cell blasts, generated from human
peripheral blood lymphocytes by activation with mitogenic CD3
antibody followed by culture in medium containing IL-2, were used
as the effector cells. They were washed and resuspended at a
concentration of 2.times.10.sup.5 cells ml.sup.-1 prior to use in
the assay. 100 .mu.l volumes of the purified chimaeric antibody
preparations were diluted in 3-fold steps in the wells of a
microtitre plate. 50 .mu.l each of the effector and target cells
were then added to each well and the mixture was incubated at
37.degree. C. for at least 4 hours. After this time 100 .mu.l of
supernatant was removed and assayed for released .sup.51Cr. Each
antibody dilution was tested in duplicate.
[0080] The U937 monocytic cell line expresses human Fc receptors
and can be lysed by activated human T-cell blasts in the presence
of CD3 monoclonal antibodies capable of cross-linking the two cell
types. The results (FIGS. 13 and 14) show that when aglycosylated,
the human IgG1 antibody of Example 1 is still able to cross-link
1-cells to the U937 cells, albeit at a reduced level, and thus
redirect T-cell cytotoxicity. This was a surprising finding since,
given the published data, the effective killing mediated by
aglycosyl .gamma.1 monoclonal antibody was unexpected.
[0081] There existed the possibility that removal of the
carbohydrate had made this monoclonal antibody especially sticky,
and so able to bind to U937 cells without interacting with
Fc.gamma.Rs. However, it was subsequently demonstrated (results not
shown) that the ability of the aglycosyl .gamma.1 monoclonal
antibody to mediate the destruction of mouse L cell targets (a
mouse cell line which expresses the human Fc.gamma.RI) was
dependent on the expression of a transfected human Fc.gamma.RI
gene, thus confirming the Fc.gamma.R binding activity of this
monoclonal antibody. We conclude that the ECR assay is a
particularly sensitive method of detecting Fc-FcR interactions.
[0082] The ECR results indicate that the hierarchy of binding of
the IgG chimaeric antibodies is
.gamma.2<.gamma.3<Ag.gamma.1<.gamma.4<.gamma.1. If the
assumption is made that the mitogenic activity of an antibody is
predicted by its Fc receptor binding ability, then one would expect
the above hierarchy to be displayed in the T cell proliferation
assays. However, this was not the case; the order of activities in
T cell proliferation experiments (1 to 3) was
Ag.gamma.1<.gamma.2<.gamma.4<.gamma.3<.gamma.1. This
demonstrates that the mitogenicity of an antibody cannot be
predicted in a straightforward fashion from the results of assays
which measure Fc-Fc receptor interactions. This view is supported
by the behaviour of the epsilon chimaeric antibody which performed
poorly in the ECR assay and yet consistently had the highest
mitogenic activity. This suggests that antibodies can activate T
cells by binding to something other than Fc.gamma. receptors (as
displayed on U937 cells) on accessory cells, i.e. an inability to
bind to Fc.gamma. receptors is no guarantee that an antibody will
not be mitogenic.
* * * * *