U.S. patent application number 11/754792 was filed with the patent office on 2008-12-11 for bispecific antibodies.
Invention is credited to Patrick Baeuerle, Meera Berry, Christian Itin, Peter Kufer.
Application Number | 20080305105 11/754792 |
Document ID | / |
Family ID | 34678708 |
Filed Date | 2008-12-11 |
United States Patent
Application |
20080305105 |
Kind Code |
A1 |
Kufer; Peter ; et
al. |
December 11, 2008 |
BISPECIFIC ANTIBODIES
Abstract
The present invention discloses bispecific antibodies comprising
two antibody variable domains on a single polypeptide chain,
wherein a first portion of the bispecific antibody is capable of
recruiting the activity of a human immune effector cell by
specifically binding to an effector antigen on the human immune
effector cell, the first portion consisting of one antibody
variable domain, and a second portion of the bispecific antibody
specifically binding to a target antigen other than the effector
antigen, the target antigen on a target cell other than the human
immune effector cell, the second portion comprising one antibody
variable domain.
Inventors: |
Kufer; Peter; (Munich,
DE) ; Berry; Meera; (Munich, DE) ; Baeuerle;
Patrick; (Munich, DE) ; Itin; Christian;
(Munich, DE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE., SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
34678708 |
Appl. No.: |
11/754792 |
Filed: |
May 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09460292 |
Dec 10, 1999 |
6835866 |
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11754792 |
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Current U.S.
Class: |
424/136.1 ;
530/387.3 |
Current CPC
Class: |
A61P 31/00 20180101;
A01K 2227/105 20130101; A61P 37/06 20180101; A61P 37/00 20180101;
C12N 15/8509 20130101; A01K 2267/0306 20130101; A61P 43/00
20180101; C07K 16/2809 20130101; C12N 2830/48 20130101; A61P 33/00
20180101; A01K 2217/075 20130101; A61P 37/08 20180101; C07K 2319/21
20130101; A01K 2267/0362 20130101; A61P 31/12 20180101; C07K
2317/31 20130101; A01K 2227/10 20130101; A61P 35/00 20180101; C07K
2317/622 20130101; C07K 2317/569 20130101; A61P 29/00 20180101;
C07K 16/30 20130101; C12N 2830/008 20130101; A01K 2217/05 20130101;
A01K 67/0276 20130101; C07K 14/70567 20130101; C07K 2319/00
20130101; C07K 2319/33 20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; A61P 31/00 20060101
A61P031/00 |
Claims
1-24. (canceled)
25. A bispecific antibody comprising two antibody variable domains
on a single polypeptide chain, wherein: a first portion of the
bispecific antibody is capable of recruiting the activity of a
human immune effector cell by specifically binding to an effector
antigen located on the human immune effector cell, said first
portion comprising an antibody variable domain; and a second
portion of the bispecific antibody is capable of specifically
binding to a target antigen other than the effector antigen, said
target antigen being located on a target cell other than said human
immune effector cell, and said second portion consisting of one
antibody variable domain.
26. The bispecific antibody of claim 25, wherein the first portion
comprises two antibody variable domains.
27. The bispecific antibody of claim 25, wherein the first and
second portions are derived from the same species.
28. The bispecific antibody of claim 25, wherein the first and
second portions are derived from the same species.
29. The bispecific antibody of claim 25, wherein first and/or
second portion are/is independently derived from a species of
primate, rodent, tylopoda or cartilaginous fish.
30. The bispecific antibody of claim 29, wherein the
primate-derived first and/or second portion are/is derived from
man.
31. The bispecific antibody of claim 29, wherein the rodent-derived
first and/or second portion are/is derived from mouse or rat.
32. The bispecific antibody of claim 31, wherein the mouse- or
rat-derived first and/or second portion are/is a variable domain
from the heavy chain (VH) derived from mouse or rat.
33. The bispecific antibody of claim 29, wherein the
tylopoda-derived first and/or second portion are/is derived from
camel, llama or dromedary.
34. The bispecific antibody of claim 33, wherein the camel-, llama-
or dromedary-derived first and/or second portion is a VHH
domain.
35. The bispecific antibody of claim 25, wherein the bispecific
antibody has undergone an alteration to render it less immunogenic
when administered to humans.
36. The bispecific antibody of claim 35, wherein the alteration
comprises one or more technique selected from the group consisting
of chimerization, humanization, CDR-grafting, deimmunization, and
mutation of framework amino acids to correspond to the closest
human germline sequence (germlining).
37. The bispecific antibody of claim 25, wherein the human effector
cell is a member of the human lymphoid lineage.
38. The bispecific antibody of claim 37, wherein the effector cell
is capable of exerting a cytotoxic or an apoptotic effect on a
target cell.
39. The bispecific antibody of claim 37, wherein the effector
antigen is chosen from one or more of the human CD3 antigen, the
human CD16 antigen, the human NKG2D antigen, the human CD2 antigen,
the human CD28 antigen and the human CD25 antigen.
40. The bispecific antibody of claim 39, wherein the effector
antigen is the human CD3 antigen.
41. The bispecific antibody of any of claim 25, wherein the human
effector cell is a member of the human myeloid lineage.
42. The bispecific antibody of claim 41, wherein the effector cell
is capable of exerting a cytotoxic or an apoptotic effect on a
target cell.
43. The bispecific antibody of claim 41, wherein the effector
antigen is chosen from one or more of the human CD64 antigen or the
human CD89 antigen.
44. The bispecific antibody of claim 25, wherein the target antigen
is selected from EpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR,
PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5.sub.AC,
MUC5.sub.B, MUC7, .beta.hCG, Lewis-Y, CD20, CD33, CD30, ganglioside
GD3, 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2,
Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1,
Plasma Cell Antigen, (membrane-bound) IgE, Melanoma Chondroitin
Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP,
mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6;
desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor,
CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory
Substance (MIS) Receptor type II, sTn (sialylated Tn antigen;
TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII,
LG, SAS and CD63, and wherein all said antigens are human
antigens.
45. The bispecific antibody of any claim 25, wherein the target
antigen is a cancer-related antigen.
46. The bispecific antibody of claim 45, wherein the target antigen
is the human CD19 antigen and the effector antigen is the human CD3
antigen.
47. The bispecific antibody of claim 45, wherein the target antigen
is the human EpCAM antigen and the effector antigen is the human
CD3 antigen.
48-53. (canceled)
54. A method for the prevention, treatment or amelioration of a
proliferative disease, a tumorous disease, an inflammatory disease,
an immunological disorder, an autoimmune disease, an infectious
disease, a viral disease, an allergic reaction, a parasitic
reaction, a graft-versus-host disease or a host-versus-graft
disease in a subject in the need thereof, said method comprising
the step of administration of an effective amount of a bispecific
antibody of claim 25.
55-56. (canceled)
57. A kit comprising a bispecific antibody of claim 25.
Description
BACKGROUND OF THE INVENTION
[0001] A. Field of the Invention
[0002] The invention relates to the field of antibodies.
Specifically, the invention relates to a bispecific antibody
comprising two antibody variable domains on a single polypeptide
chain. The invention further relates to the use of such a
bispecific antibody for the preparation of a pharmaceutical
composition. The invention further relates to a method for the
prevention, treatment or amelioration of a disease comprising
administration of an effective amount of such a bispecific
antibody. Finally, the invention relates to a kit comprising such a
bispecific antibody.
[0003] B. Related Art
[0004] Unifying two antigen binding sites of different specificity
into a single construct, bispecific antibodies have the ability to
bring together two discreet antigens with exquisite specificity and
therefore have great potential as therapeutic agents. This
potential was recognized early on, leading to a number of
approaches for obtaining such bispecific antibodies. Bispecific
antibodies were originally made by fusing two hybridomas, each
capable of producing a different immunoglobulin. The resulting
hybrid-hybridoma, or quadroma, was capable of producing antibodies
bearing the antigen specificity of the first parent hybridoma as
well as that of the second parent hybridoma (Milstein et al.
(1983), Nature 305:537). However, the antibodies resulting from
quadromas often exhibited undesired properties due to the presence
of an Fc antibody portion.
[0005] Largely due to such difficulties, attempts later focused on
creating antibody constructs resulting from joining two scFv
antibody fragments while omitting the Fc portion present in full
immunoglobulins. Each scFv unit in such constructs was made up of
one variable domain from each of the heavy (VH) and light (VL)
antibody chains, joined with one another via a synthetic
polypeptide linker, the latter often being genetically engineered
so as to be minimally immunogenic while remaining maximally
resistant to proteolysis. Respective scFv units were joined by a
number of techniques including incorporation of a short (usually
less than 10 amino acids) polypeptide spacer bridging the two scFv
units, thereby creating a bispecific single chain antibody. The
resulting bispecific single chain antibody is therefore a species
containing two VH/VL pairs of different specificity on a single
polypeptide chain, wherein the VH and VL domains in a respective
scFv unit are separated by a polypeptide linker long enough to
allow intramolecular association between these two domains, and
wherein the thusly formed scFv units are contiguously tethered to
one another through a polypeptide spacer kept short enough to
prevent unwanted association between, for example, the VH domain of
one scFv unit and the VL of the other scFv unit.
[0006] Bispecific single chain antibodies of the general form
described above have the advantage that the nucleotide sequence
encoding the four V-domains, two linkers and one spacer can be
incorporated into a suitable host expression organism under the
control of a single promoter. This increases the flexibility with
which these constructs can be designed as well as the degree of
experimenter control during their production.
[0007] Remarkable experimental results have been obtained using
such bispecific single chain antibodies designed for the treatment
of malignancies (Mack, J. Immunol. (1997), 158:3965-70; Mack, PNAS
(1995), 92:7021-5; Kufer, Cancer Immunol. Immunother. (1997),
45:193-7; Loffler, Blood (2000), 95:2098-103) and non-malignant
diseases (Bruhl, J. Immunol. (2001), 166:2420-6). In such
bispecific single chain antibodies, one scFv unit is capable of
activating cytotoxic cells, for example cytotoxic T cells, within
the immune system by specifically binding to an antigen on the
cytotoxic cells, while the other scFv unit specifically binds an
antigen on a malignant cell intended for destruction. In this way,
such bispecific single chain antibodies have been shown to activate
and redirect the immune system's cytotoxic potential to the
destruction of pathological, especially malignant cells. In the
absence of such a bispecific single chain antibody construct,
malignant cells would otherwise proliferate uninhibited.
[0008] However, bispecific single chain antibodies must fulfil
additional requirements. In order to achieve the desired activity,
each scFv unit of a bispecific single chain antibody should remain
properly folded, something which often proves unrealisable in
conventional bacterial expression systems such as E. coli. The need
to use less conventional, more cumbersome and more costly
eukaryotic--even mammalian--expression systems often complicates
the production of bispecific single chain antibodies and/or reduces
the amount of product obtainable to levels lower than desired for
therapeutic application.
[0009] In the event that a bispecific antibody is intended for
therapeutic use, it is desirable to produce high amounts of this
antibody solubly and in the desired functional form. The production
of functionally active antibody becomes especially critical when
producing bispecific antibodies of which one portion is able to
activate and recruit the cytotoxic potential of human immune
effector cells. For example, a produced antibody devoid of
functional activity will not lead to the desired activation of
human immune effector cells, while a bispecific antibody which is
functionally active, albeit not in the desired manner, as for
example may be the case when the bispecific antibody is produced in
a heterogeneous form containing multiple isomers, may activate and
recruit the cytotoxic potential of human immune effector cells in
unforeseeable and/or unintended manners.
[0010] One example of the sort of unintended activation mentioned
above is the possibility of activation of human immune effector
cells to exert an effect on other human immune effector cells
instead of on a target cell intended for destruction. This type of
immune effector cell fratricide may jeopardize the effectiveness of
a regimen of therapy depending on the activity of human immune
effector cells.
[0011] However, reliable production of large amounts of functional
single chain antibody, especially large amounts of functional
bispecific single chain antibody, from prokaryotic expression
systems such as E. coli is often limited, necessitating costly
optimization (Baneyx (1999), Curr. Opinions Biotechnol.
10:411-21).
[0012] In summary, bispecific antibody constructs can be of great
therapeutic use in redirecting the powerful potential of the body's
own immune system to achieve the destruction of diseased cells. By
the same token, however, the activation of such a powerful means of
eradicating or neutralizing unwanted cells requires that this power
be controlled as precisely as possible so that the cytotoxic
potential of the immune system is recruited and applied only in the
direction intended and no other.
[0013] Clearly, when one specific binding arm of a bispecific
single chain antibody is to recruit the activity of a human immune
effector cell, for example a cytotoxic T cell, there exists an
especially heightened and, as yet, unmet need for bispecific single
chain antibodies which overcome limitations as described above.
SUMMARY OF THE INVENTION
[0014] The present inventors have found that the above limitations
can be overcome with a bispecific antibody comprising two antibody
variable domains on a single polypeptide chain, wherein: [0015] a
first portion of the bispecific antibody is capable of recruiting
the activity of a human immune effector cell by specifically
binding to an effector antigen located on the human immune effector
cell, said first portion consisting of one antibody variable
domain; and [0016] a second portion of the bispecific antibody is
capable of specifically binding to a target antigen other than the
effector antigen, said target antigen being located on a target
cell other than said human immune effector cell, and said second
portion comprising an antibody variable domain (first aspect of the
present invention).
[0017] According to one embodiment of this first aspect of the
invention, the second portion of the bispecific antibody comprises
two antibody variable domains. According to another embodiment of
the first aspect of the invention, the second portion of the
bispecific antibody comprises one antibody variable domain.
[0018] A second aspect of the invention provides a bispecific
antibody comprising two antibody variable domains on a single
polypeptide chain, wherein: [0019] a first portion of the
bispecific antibody is capable of recruiting the activity of a
human immune effector cell by specifically binding to an effector
antigen located on the human immune effector cell, said first
portion comprising an antibody variable domain, and [0020] a second
portion of the bispecific antibody is capable of specifically
binding to a target antigen other than the effector antigen, said
target antigen being located on a target cell other than said human
immune effector cell, and said second portion consisting of one
antibody variable domain.
[0021] According to one embodiment of the second aspect of the
invention, the first portion of the bispecific antibody comprises
two antibody variable domains.
[0022] In its minimal form, the total number of antibody variable
regions in the bispecific antibody according to the invention is
thus only two. Here, not two variable domains, but rather only one
variable domain, is necessary to specifically bind to each antigen
of interest. The bispecific antibody of the invention is thus
approximately half the size of conventional bispecific single chain
antibodies containing four antibody variable domains.
[0023] The greater simplicity in molecular design of the bispecific
antibody of the invention correlates to greater possible simplicity
in the host expression system used for its production in
functionally active form. As such, the small size of the inventive
bispecific antibody opens up avenues of production hitherto closed
to conventional bispecific single chain antibodies with four
antibody variable domains. For example, the bispecific antibody of
the invention may be easily produced in conventional, well
understood and cheap bacterial expression systems such as E. coli
in amounts which are desired for therapeutic applications.
[0024] Increased productivity has at least two highly advantageous
effects. First, larger amounts of the bispecific antibody of the
invention can be produced in functional form per batch than
previously possible for single chain bispecific antibodies with
four antibody variable domains, allowing greater efficiency and,
ultimately, economy in production. Second, a greater number of
constructs in the format of the bispecific antibody of the
invention may now be considered as therapeutic candidates since a
low cytotoxic activity of a bispecific construct with four antibody
variable domains may now be offset by higher amounts of available
therapeutic agent using the bispecific antibody of the invention.
The palette of possible therapeutic applications for the bispecific
antibody of the invention is thereby expanded relative to that of
single chain bispecific antibodies with four antibody variable
domains.
[0025] At the same time, less complexity in molecular design also
correlates to fewer possibilities in which undesired intermolecular
association may take place. That is to say, the bispecific antibody
of the invention can be produced more homogeneously than possible
for single chain antibody formats with four antibody variable
domains. As explained above, product heterogeneity may threaten the
therapeutic prognosis and/or product safety profile which can be
expected from a bispecific antibody capable of binding to an immune
effector cell. Decreasing the number of antibody variable domains
in the bispecific antibody of the invention decreases the number of
potential partners for intermolecular association. This eliminates
pathways by which intermolecular association can take place. A
bispecific antibody is thus obtained which retains the intended
therapeutic profile while minimizing or even abolishing formation
of intermolecular association products which might mobilize the
host immune response in unintended manners.
[0026] In one embodiment, when either the second or the first
portion of a bispecific antibody of the invention comprises two
antibody variable domains as described above, these two antibody
variable domains are a VH- and VL-domain which are associated With
one another. However, it is also contemplated that the two antibody
variable domains comprised in either the second or the first
portion may be two VH domains or two VL regions which are
associated with one another. In the event that the two antibody
variable domains of the first or second portion are covalently
associated with one another, the two antibody variable domains may
be designed as an scFv fragment, meaning that the two domains are
separated from one another by a peptide linker long enough to allow
intermolecular association between these two domains. The design of
linkers suitable for this purpose is described in the prior art,
for example in the granted patents EP 623 679 B1, U.S. Pat. No.
5,258,498, EP 573 551 B1 and U.S. Pat. No. 5,525,491.
[0027] In other words, a bispecific antibody according to this
embodiment of the invention is a construct with a total of three
antibody variable domains. Here, one antibody variable domain
specifically binds alone, i.e., without being paired with another
antibody variable domain (a) either to a human immune effector cell
by specifically binding to an effector antigen on the human immune
effector cell or to a target cell, while the remaining two antibody
variable domains together specifically bind (b) either to the
target antigen on the target cell or to a human immune effector
cell by specifically binding to an effector antigen on the human
immune effector cell, respectively.
[0028] The inventors have found that the presence of three antibody
variable domains in the bispecific antibody entails unique
advantages. Often, an scFv exhibiting the desired binding
specificity for a target antigen is already known and optimized,
and omitting one of its two antibody variable domains would abolish
or at least attenuate its binding characteristics. Such an scFv may
make up part of the bispecific antibody according to the present
embodiment of the invention. Specifically, such a three-domain
antibody may advantageously comprise an entire scFv as either its
effector antigen- or target antigen-conferring portion.
[0029] Effectively, then, the present embodiment of the invention
allows a bispecific antibody to be formed starting from a desired
scFv by simple incorporation of only one additional antibody
variable domain into the same polypeptide chain as the scFv,
wherein the one additional antibody variable domain incorporated
has an antigen binding specificity different than that of the
scFv.
[0030] In this context, it has been found that such incorporation
of a third antibody variable domain to form a three-domain
bispecific single chain antibody according to this embodiment leads
to the same, or substantially the same, production characteristics
as described above for the two-domain bispecific antibodies of the
invention. For example, problems such as low yield, restriction to
complicated expression systems, heterogeneous products, etc.,
recounted above for bispecific antibodies with four antibody
variable domains pose little to no problem when expressing
three-domain bispecific antibodies according to this
embodiment.
[0031] It would seem, then, that a bispecific antibody according to
this embodiment of the invention and including no more than three
antibody variable domains would represent the upper limit in number
of antibody variable domains for which high yielding, homogeneous
production is possible while still allowing the researcher to
employ preexisting binding molecules such as scFv constructs. As
such, the molecular architecture according to this embodiment
allows for savings in research time and resources while still
conferring the advantages associated with the bispecific antibody
of the invention in its minimal form.
[0032] According to a further embodiment of the invention, the
first and second portions of the bispecific antibody according to
the invention or according to any of the above embodiments of the
invention may be separated from one another by a synthetic
polypeptide spacer moiety, which covalently (i.e., peptidically)
links either the C-terminus of the first portion with the
N-terminus of the second portion, or the C-terminus of the second
portion with the N-terminus of the first portion. As such, the
portions of the bispecific antibody according to this embodiment
may be arranged, as either N-(first portion)-(second portion)-C or
N-(second portion)-(first portion)-C.
[0033] The term "human immune effector cell" refers to a cell
within the natural repertoire of cells in the human immune system
which, when activated, is able to bring about a change in the
viability of a target cell. The term "viability of a target cell"
may refer within the scope of the invention to the target cell's
ability to survive, proliferate and/or interact with other cells.
Such interaction may be either direct, for example when the target
cell contacts another cell, or indirect, for example when the
target cell secretes substances which have an influence on the
functioning of another distant cell. The target cell may be either
native or foreign to humans. In the event that the cell is native
to humans, the target cell is advantageously a cell which has
undergone transformation to become a malignant cell. The native
cell may additionally be a pathologically modified native cell, for
example a native cell infected with an organism such as a virus, a
plasmodium or a bacterium. In the event that the cell is foreign to
humans, the target cell is advantageously an invading pathogen, for
example an invading bacterium or plasmodium.
[0034] According to a further embodiment of the invention, the
antibody variable domains of the first and/or second portions may
be derived from identical or separate animal species. This has the
advantage that for each portion of the bispecific antibody, optimal
antibody variable domain/s can be chosen to be derived from the
animal species known to yield the best antibodies against a
particular effector and/or target antigen. In this way, this
embodiment allows the researcher to capitalize on already known,
developed and/or optimized specificities such that the efficiency
of workflow in developing bispecific antibodies as described herein
is maximized.
[0035] In one preferred embodiment, the first and/or second portion
of the bispecific antibody are/is independently derived from an
antibody produced in primate, rodent, tylopoda or cartilaginous
fish.
[0036] The first and/or second portion of a bispecific antibody
according to this embodiment may be either naturally occurring or
genetically engineered. Alternatively, it is within the scope of
the present embodiment that part of a naturally occurring antibody
is used as a substrate on which further genetic engineering is
performed, to finally yield a derivative of the naturally occurring
part of the antibody for use in the first or second portion of a
bispecific antibody according to this embodiment.
[0037] In the event that the first and/or second portion of the
bispecific antibody are/is derived from rodent, said first and/or
second portion may advantageously be derived independently from
mouse or rat antibodies. In this way, one seeking to develop and/or
optimize bispecific antibodies according to this embodiment of the
invention can benefit from the preexisting and highly diverse
palette of known murine and rat antibody sequences which bind
relevant human antigens.
[0038] In the event that a primate antibody is used as a basis for
the first and/or second portion of the bispecific antibody, said
first and/or second portion are/is advantageously derived
independently from human antibodies. Besides benefiting from the
ever-growing diversity of known human antibodies, use of human
antibody variable domains entails the further advantage that the
resulting bispecific antibodies will elicit little to no
immunogenic response when administered as part of a therapeutic
regimen in human patients. Such bispecific antibodies are thus
especially suitable as therapeutic agents for use in humans.
[0039] In the event that a tylopoda-derived antibody variable
domain is used in the first and/or second portion of a bispecific
antibody according to this embodiment of the invention, said first
and/or second portion may advantageously be derived independently
from camel, llama or/and dromedary. This use of such "camelid"
antibodies allows the researcher seeking to develop or optimize
bispecific antibodies according to this embodiment of the invention
to capitalize on the unique types of antibodies known to be
produced by these species. These species are namely known to
produce high affinity antibodies of only a single variable domain.
In the event that a tylopoda antibody is used as the source for the
antibody variable domain in the first and/or second portion of the
bispecific antibody, it is advantageous to use the VHH domain or a
modified variant thereof.
[0040] The term "VHH" denotes a variable region of a heavy chain of
a so-called "camelid" antibody. Camelid antibodies comprise a heavy
chain, but lack a light chain. As such, a VHH region from such a
camelid antibody represents the minimal structural element required
to specifically bind to an antigen of interest in these species.
Camelid VHH domains have been found to bind to antigen with high
affinity (Desmyter et al. (2001), J. Biol. Chem. 276:26285-90) and
possess high stability in solution (Ewert et al. (2002),
Biochemistry 41:3628-36).
[0041] In the event that said first and/or second portion of the
bispecific antibody is derived from a cartilaginous fish, said
cartilaginous fish is advantageously a shark.
[0042] In the event that a rodent or primate antibody is used as
the source for the antibody variable domain in the first and/or
second portion of a bispecific antibody according to this
embodiment of the invention, it is advantageous to use the VH
domain or a modified variant thereof. The VH domain of antibodies
in these species is known to contribute significantly to the
binding specificity and affinity observed for a given antibody. At
an absolute minimum, it is advantageous to use at least the third
complementarity determining region (CDR) from a VH domain of such a
parent antibody in designing the first and/or second portion of the
bispecific antibody. This is due to the fact that the VH-CDR3 is
known to play a major role in the specificity and affinity of
binding of all the CDR regions, of which there are three in each of
VH and VL.
[0043] According to a further embodiment of the invention, the
bispecific antibody may be subjected to an alteration to render it
less immunogenic when administered to a human. Such an alteration
may comprise one or more of the techniques commonly known as
chimerization, humanization, CDR-grafting, deimmunization and/or
mutation of framework region amino acids to correspond to the
closest human germline sequence (germlining). Subjecting the
bispecific antibody of the invention to such an alteration/s has
the advantage that a bispecific antibody which would otherwise
elicit a host immune response is rendered more, or completely
"invisible" to the host immune system, so that such an immune
response does not occur or is reduced. Bispecific antibodies which
have been altered as described according to this embodiment will
therefore remain administrable for a longer period of time with
reduced or no immune response-related side effects than
corresponding bispecific antibodies which have not undergone any
such alteration(s). One of ordinary skill in the art will
understand how to determine whether, and to what degree an antibody
must be altered in order to prevent it from eliciting an unwanted
host immune response.
[0044] According to another embodiment of the invention, the human
immune effector cell is a member of the human lymphoid cell
lineage. In this embodiment, the effector cell may advantageously
be a human T cell, a human B cell or a human natural killer (NK)
cell. Advantageously, such cells will have either a cytotoxic or an
apoptotic effect on the target cell. Especially advantageously, the
human lymphoid cell is a cytotoxic T cell which, when activated,
exerts a cytotoxic effect on the target cell. According to this
embodiment, then, the recruited activity of the human effector cell
is this cell's cytotoxic activity.
[0045] According to a preferred embodiment, activation of the
cytotoxic T cell may occur via binding of the CD3 antigen as
effector antigen on the surface of the cytotoxic T cell by a
bispecific antibody of this embodiment of the invention. The human
CD3 antigen is present on both helper T cells and cytotoxic T
cells. Human CD3 denotes an antigen which is expressed on T cells
as part of the multimolecular T cell complex and which comprises
three different chains: CD3-epsilon, CD3-delta and CD3-gamma.
[0046] The activation of the cytotoxic potential of T cells is a
complex phenomenon which requires the interplay of multiple
proteins. The T cell receptor ("TCR") protein is a membrane bound
disulfide-linked heterodimer consisting of two different
glycoprotein subunits. The TCR recognizes and binds foreign
peptidic antigen which itself has been bound by a member of the
highly diverse class of major histocompatibility complex ("MHC")
proteins and has been presented, bound to the MHC, on the surface
of antigen presenting cells ("APCs").
[0047] Although the variable TCR binds foreign antigen as outlined
above, signaling to the T cell that this binding has taken place
depends on the presence of other, invariant, signaling proteins
associated with the TCR. These signaling proteins in associated
form are collectively referred to as the CD3 complex, here
collectively referred to as the CD3 antigen.
[0048] The activation of T cell cytotoxicity, then, normally
depends first on the binding of the TCR with an MHC protein, itself
bound to foreign antigen, located on a separate cell. Only when
this initial TCR-MHC binding has taken place can the CD3-dependent
signaling cascade responsible for T cell clonal expansion and,
ultimately, T cell cytotoxicity ensue.
[0049] However, binding of the human CD3 antigen by the first or
second portion of a bispecific antibody of the invention activates
T cells to exert a cytotoxic effect on other cells in the absence
of independent TCR-MHC binding. This means that T cells may be
cytotoxically activated in a clonally independent fashion, i.e., in
a manner which is independent of the specific TCR clone carried by
the T cell. This allows an activation of the entire T cell
compartment rather than only specific T cells of a certain clonal
identity.
[0050] In light of the foregoing discussion, then, an especially
preferred embodiment of the invention provides a bispecific
antibody in which the effector antigen is the human CD3 antigen.
The bispecific antibody according to this embodiment of the
invention may have a total of either two or three antibody variable
domains.
[0051] According to further embodiments of the invention, other
lymphoid cell-associated effector antigens bound by a bispecific
antibody of the invention may be the human CD16 antigen, the human
NKG2D antigen, the human NKp46 antigen, the human CD2 antigen, the
human CD28 antigen or the human CD25 antigen.
[0052] According to another embodiment of the invention, the human
effector cell is a member of the human myeloid lineage.
Advantageously, the effector cell may be a human monocyte, a human
neutrophilic granulocyte or a human dendritic cell. Advantageously,
such cells will have either a cytotoxic or an apoptotic effect on
the target cell. Advantageous antigens within this embodiment which
may be bound by a bispecific antibody of the invention may be the
human CD64 antigen or the human CD89 antigen.
[0053] According to another embodiment of the invention, the target
antigen is an antigen which is uniquely expressed on a target cell
in a disease condition, but which remains either non-expressed,
expressed at a low level or non-accessible in a healthy condition.
Examples of such target antigens which might be specifically bound
by a bispecific antibody of the invention may advantageously be
selected from EpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR,
PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7,
.beta.hCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3,
9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2,
Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1,
Plasma Cell Antigen, (membrane-bound) IgE, Melanoma Chondroitin
Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP,
mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6;
desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor,
CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory
Substance (MIS) Receptor type II, sTn (sialylated Tn antigen;
TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII,
LG, SAS and CD63.
[0054] According to a specific embodiment, the target antigen
specifically bound by a bispecific antibody may be a cancer-related
antigen, that is an antigen related to a malignant condition. Such
an antigen is either expressed or accessible on a malignant cell,
whereas the antigen is either not present, not significantly
present, or is not accessible on a non-malignant cell. As such, a
bispecific antibody according to this embodiment of the invention
is a bispecific antibody which recruits the activity of a human
immune effector cell against the malignant target cell bearing the
target antigen, or rendering the target antigen accessible.
[0055] In a particular embodiment of the invention, the bispecific
antibody specifically binds to the human CD3 antigen as effector
antigen and to the human CD19 antigen as target antigen. The human
CD19 antigen is expressed in the whole human B lineage from the pro
B cell to the mature B cell, it is not shed, is uniformly expressed
on all lymphoma cells, and is absent from stem cells. Thus, a
bispecific antibody according to this embodiment, namely one which
specifically binds to the human CD3 antigen as effector antigen and
to the human CD19 antigen as target antigen, is of great potential
value as a therapeutic for the eradication of malignant B cells. A
bispecific antibody according to this embodiment consists of two or
three antibody variable domains, separated by spacer and possibly
linker polypeptides as described above.
[0056] In a further particular embodiment of the invention, the
bispecific antibody specifically binds to the human CD3 antigen as
effector antigen and to the human EpCAM antigen as target antigen.
EpCAM ("Epithelial cell adhesion molecule", also called 17-1A
antigen, KSA, EGP40, GA733-2, ks1-4 or esa) is a 40 kDa membrane
integrated glycoprotein of 314 amino acids with specific expression
in certain epithelia and on many human carcinomas. EpCAM has been
shown in various studies to be beneficial in diagnosis and therapy
of various carcinomas. Furthermore, in many cases, tumor cells were
observed to express EpCAM to a much higher degree than their
parental epithelium or less aggressive forms of said cancers. Thus,
a bispecific antibody according to this embodiment, namely one
which specifically binds to the human CD3 antigen as effector
antigen and to the human EpCAM antigen as target antigen is of
great potential value as a therapeutic for the eradication of
malignant epithelial cells. A bispecific antibody according to this
embodiment consists of two or three antibody variable domains,
separated by spacer and possibly linker polypeptides as described
above.
[0057] An anti-CD3.times.anti-EpCAM bispecific antibody according
to this latter embodiment may advantageously have the amino acid
sequence as set out in SEQ ID NO: 1. A bispecific antibody
according to this embodiment has as its first portion a
murine-derived VH specifically binding the human CD3 antigen as
effector antigen and, as its second portion, an scFv unit
specifically binding the human EpCAM antigen as target antigen. As
such, SEQ ID NO: 1 represents a bispecific antibody with three
antibody variable domains. The advantages of this type of construct
are described hereinabove.
[0058] A further aspect of the invention provides a use of a
bispecific antibody as disclosed hereinabove for the preparation of
a pharmaceutical composition for the prevention, treatment or
amelioration of a proliferative disease, a tumorous disease, an
inflammatory disease, an immunological disorder, an autoimmune
disease, an infectious disease, a viral disease, an allergic
reaction, a parasitic reaction, a graft-versus-host disease or a
host-versus-graft disease.
[0059] A further aspect of the invention provides a method for the
prevention, treatment or amelioration a proliferative disease, a
tumorous disease, an inflammatory disease, an immunological
disorder, an autoimmune disease, an infectious disease, a viral
disease, an allergic reaction, a parasitic reaction, a
graft-versus-host disease or a host-versus-graft disease in a
subject in the need thereof, said method comprising the step of
administration of an effective amount of a bispecific antibody as
disclosed hereinabove.
[0060] According to a preferred embodiment, the prevention,
treatment or amelioration occurs in a human. The tumorous disease
is preferably selected from the group of B cell disorders, for
example a lymphoma, a B cell lymphoma and a Hodgkin's lymphoma. In
a further embodiment, the B cell lymphoma is a non-Hodgkin's
lymphoma. In a further embodiment, the autoimmune disease is
selected from rheumatoid arthritis, multiple sclerosis, type 1
diabetes mellitus, inflammatory bowel disease, systemic lupus
erythematosus, psoriasis, scleroderma and autoimmune thyroid
diseases.
[0061] According to a further embodiment, any administration of a
bispecific antibody as described hereinabove may advantageously be
coupled with the administration of a proteinaceous compound capable
of providing an activation signal for immune effector cells. Such a
proteinaceous compound may advantageously be administered
simultaneously or non-simultaneously with the bispecific
antibody.
[0062] A further aspect of the invention is a kit comprising a
bispecific antibody as disclosed hereinabove.
[0063] Throughout the instant application, it is to be understood
that use of a term in the singular may imply, where appropriate,
use of the respective term in the plural. Similarly, use of a term
in the plural may imply, where appropriate, use of the respective
term in the singular.
BRIEF DESCRIPTION OF THE DRAWING
[0064] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0065] FIG. 1-Cytotoxic activity of an anti-EpCAM.times.anti-CD3
antibody comprising three variable domains
EXAMPLES
Example 1
Design, Prokaryotic Expression and Purification of a Bispecific
Antibody with Three Antibody Variable Domains
[0066] The DNA encoding an anti-EpCAM.times.anti-CD3 bispecific
antibody with a VL and VH in the anti-EpCAM portion of the molecule
and only one antibody variable domain (VH) in the anti CD3 portion
of the molecule are cloned in the multiple cloning site (MCS) of
pET-20b(+) vector (Novagen). The expression of the bispecific
antibody with a Histidine (x6) tag is induced with IPTG. The choice
of the vector facilitates the transport of the recombinant proteins
into the periplasm. Other cloning vectors such as pBAD-gIII
(Invitrogen), pET-32 series (+) vector (Novagen) may also be used.
For pBAD-gIII-based expression, arabinose is used to induce
recombinant gene expression instead of IPTG. In any case it is
important to ensure that the DNA encoding the bispecific antibody
is cloned in-frame with the sequence encoding the signal peptide
(e.g., PelB, OmpA) that mediates the transport of the recombinant
protein into the periplasm.
[0067] The pET-20b (+) containing the DNA encoding the
anti-EpCAM.times.anti-CD3 bispecific antibody described above is
cloned and propagated in the bacterial host strain DH5. The
recombinant bispecific antibody is expressed using the BL21 (DE3)
bacterial host strain (Novagen). Alternatively, the Rosetta (DE3)
bacterial host strain (Novagen) works when using a pET vector as
described above. Alternatively, the pBAD-gIII vector may be used
with the TOP 10 E. coli strain (Invitrogen).
[0068] A single colony of host cell transformed with the vector
pET-20b(+) containing DNA encoding the anti-EpCAM.times.anti-CD3
bispecific antibody described above is selected and inoculated into
50 ml LB containing the essential antibiotics. Cells are grown and
harvested according to the supplier's instruction manual. The
culture is incubated at 37.degree. C. until an OD600 of 0.4 to 1.0
is reached (0.6 is an ideal value), followed by induction of
expression by addition of appropriate amounts of IPTG. The
incubation is continued for an additional 2-3 h.
[0069] Cultures as described are harvested by centrifugation. The
cell pellet is suspended in 30 ml of 30 mM Tris-HCl pH 8, 20%
sucrose. To this suspension, 60 .mu.l of EDTA (0.5 M, pH 8) is
added to a final concentration of 1 mM. The cells are collected by
centrifugation and the cell-pellet is subjected to shock by
re-suspending the pellet thoroughly for 10 min in the cold with
chilled MgSO.sub.4 (5 mM, 30 ml) solution. The shocked cells are
subjected to centrifugation in order to separate the periplasmic
(supernatant) and cellular (pellet) fractions. The supernatant is
then further analysed by SDS-PAGE and is also checked for
activity.
[0070] Bispecific antibody produced as described above with a His
tag is purified using a Ni-NTA spin column kit (Qiagen, catalog no.
31314) following the protocol provided in the Qiagen instruction
manual. Alternatively, the Ni-NTA magnetic agarose beads (Qiagen,
catalog no. 36113) can also be used.
[0071] The polypeptide thus purified may be described as bispecific
antibody with three antibody variable domains located on the same
polypeptide chain. Progressing from the amino- to carboxy terminus,
the bispecific single chain antibody contains the following
elements: anti-human EpCAM VL; 15 amino acid linker of sequence
(Gly.sub.4Ser).sub.3; anti-human EpCAM VH; 5 amino acid spacer of
sequence Gly.sub.4Ser; anti-human CD3 VH; His.sub.6. The sequence
is as set out in SEQ ID NO: 1.
Example 2
Cytotoxicity Assay
[0072] The ability of the bispecific antibody with the sequence set
out in SEQ ID NO: 1 to recruit the cytotoxic potential of human
cytotoxic T cells to effect the killing of cells bearing the human
EpCAM antigen was measured in a cytotoxicity assay as follows.
[0073] CHO cells from the American Type Cell Culture Collection
(ATCC, USA) were transfected to express, human epithelial cell
adhesion molecule (EpCAM) as the target antigen. Cells cultured
from the resulting cell clone, referred to as CHO-EpCAM cells, were
subsequently used in the cytotoxicity experiments as the target
cells. The human cell line MC15 was used as a source of effector
cells bearing the effector antigen CD3. The cell clone was derived
from the cell clone CB15, which is a CD4-positive human T cell
clone kindly provided by Dr. Fickenscher at the University of
Erlangen/Nurnberg, Germany. Cells were cultured as recommended by
the respective suppliers.
[0074] 1.5.times.10.sup.7 target cells were washed twice with
phosphate-buffered saline (PBS) and were labeled with PKH26 dye
(Sigma-Aldrich Co.) according to the manufacturers instructions.
After staining, the cells were washed two times with 20 ml of PBS.
Labeled CHO-EpCAM cells (target cells) and MC15 cells (effector
cells) were mixed together in a ratio of 1:5, respectively. The
resulting cell suspension contained 400,000 target and
2.times.10.sup.6 effector cells per ml. BiTEs were diluted to
different concentrations in alpha MEM/10% FCS-medium.
[0075] Typically, each reaction (of volume 100 .mu.l) contained a
mixture of 20,000 target cells, 1.times.10.sup.5 effector cells and
a specific concentration of the bispecific antibody set out as in
SEQ ID NO: 1. Measurements at each concentration of bispecific
antibody were performed in triplicate. Reactions were incubated for
about 20 h at 37.degree. C./5% CO.sub.2.
[0076] Propidium iodide was added to a final concentration of 1
.mu.g/ml. Propidium iodide stains dead cells. The reaction samples
were analyzed by flow cytometry (e.g., FACS-Calibur Becton
Dickinson). The population of PKH26-labeled target cells was gated
in an FSC versus FL-2 plot and subsequent analysis of cells was
carried out only with the cell population identified within this
gate. The percent of dead cells (propidium iodide stained) was
determined in an FSC (forward scatter) versus FL-3 plot. Mean
values were plotted against concentrations of bispecific antibody
on a logarithmic scale, resulting in a typical dose response curve
(see FIG. 1). The EC.sub.50 (the concentration of bispecific
antibody required to elicit a half-maximal cytotoxic response)
values were obtained after non-linear fitting of the data obtained
with the GraphPad Prism software.
[0077] As can be seen in FIG. 1, the bispecific antibody with the
sequence as set out in SEQ ID NO: 1 showed activity as a recruiter
of cytotoxic T cells. This follows from the fact that the target
cells are efficiently killed (with an EC.sub.50 value of about 12
ng/ml) in a manner depending on the concentration of bispecific
antibody added to a respective reaction mixture in the presence of
cytotoxic T cells.
[0078] All of the methods and compositions disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the methods and compositions and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
Sequence CWU 1
1
11378PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 1Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr
Val Thr Ala Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln
Ser Leu Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala
Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala
Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr Ser Tyr Pro
Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110Lys Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Glu
Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 130 135
140Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr
Asn145 150 155 160Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His
Gly Leu Glu Trp 165 170 175Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn
Ile His Tyr Asn Glu Lys 180 185 190Phe Lys Gly Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ser Ser Thr Ala 195 200 205Tyr Met Gln Leu Ser Ser
Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 210 215 220Cys Ala Arg Leu
Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln225 230 235 240Gly
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys 245 250
255Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys
260 265 270Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr
Met His 275 280 285Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile Gly Tyr Ile 290 295 300Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn
Gln Lys Phe Lys Asp Lys305 310 315 320Ala Thr Leu Thr Thr Asp Lys
Ser Ser Ser Thr Ala Tyr Met Gln Leu 325 330 335Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr 340 345 350Tyr Asp Asp
His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu 355 360 365Thr
Val Ser Ser His His His His His His 370 375
* * * * *