U.S. patent application number 11/023086 was filed with the patent office on 2005-10-13 for cytolysis of target cells by superantigen conjugates inducing t-cell activation.
This patent application is currently assigned to ACTIVE BIOTECH AB. Invention is credited to Abrahmsen, Lars, Dohlsten, Mikael, Forsberg, Goran, Kalland, Terje, Lando, Peter, Soegaard, Morten.
Application Number | 20050226885 11/023086 |
Document ID | / |
Family ID | 26663128 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050226885 |
Kind Code |
A1 |
Soegaard, Morten ; et
al. |
October 13, 2005 |
Cytolysis of target cells by superantigen conjugates inducing
T-cell activation
Abstract
A method for inactivating target cells in the presence of T
cells by bringing the two types of cells in contact with a
superantigen (SAG) in the presence of an immune modulator,
characterized in that at least one of the superantigen and the
immune modulator is in the form of a conjugate between a "free"
superantigen (Sag) and a moiety targeting the conjugate to the
target cells. A superantigen conjugate complying with the formula
(1): (T).sub.x(Sag).sub.y(IM).sub.z; a) T is a targeting moiety,
Sag corresponds to a free superantigen, IM is an immune modulator
that is not a superantigen and T, Sag and IM are linked together
via organic linkers B; b) x, y and z are integers that typically
are selected among 0-10 and represent the number of moieties T, Sag
and IM, respetively, in a given conjugate molecule, with the
provision that y>0 and also one or both of x and z>0. The
superantigen conjugate is preferably a triple fusion protein. A
targeted immune modulator, characterized in that it is a conjugate
between a targeting moiety (T'") and a modified immune modulator
(IM'"). The conjugate complies with a formula analogous to formula
(I) except for the imperative presence of the modified immune
modulator. A superantigen moiety may be present. A DNA molecule
encoding a superantigen and an immune modulator.
Inventors: |
Soegaard, Morten;
(Kopenhamn, DK) ; Abrahmsen, Lars; (Bromma,
SE) ; Lando, Peter; (Malmo, SE) ; Forsberg,
Goran; (Eslov, SE) ; Kalland, Terje;
(Loddekopinge, SE) ; Dohlsten, Mikael; (Lund,
SE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Assignee: |
ACTIVE BIOTECH AB
Lund
SE
|
Family ID: |
26663128 |
Appl. No.: |
11/023086 |
Filed: |
December 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11023086 |
Dec 27, 2004 |
|
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09463470 |
Jan 21, 2000 |
|
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09463470 |
Jan 21, 2000 |
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PCT/EP98/04219 |
Jul 2, 1998 |
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60053211 |
Jul 21, 1997 |
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Current U.S.
Class: |
424/184.1 ;
424/277.1 |
Current CPC
Class: |
A61K 47/642 20170801;
A61K 47/6829 20170801; A61K 47/6863 20170801; A61P 35/00 20180101;
C07K 14/47 20130101; C07K 2319/035 20130101; A61K 47/6801 20170801;
A61P 37/02 20180101; A61K 47/6813 20170801; A61P 37/00
20180101 |
Class at
Publication: |
424/184.1 ;
424/277.1 |
International
Class: |
A61K 039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 14, 1997 |
SE |
SE-9704170-1 |
Claims
1-34. (canceled)
35. A pharmaceutical composition comprising a superantigen, an
immune modulator and a targeting moiety, wherein at least one of
the superantigen and immune modulator is conjugated to a targeting
moiety.
36. The pharmaceutical composition of claim 35, wherein the
superantigen is conjugated to the targeting moiety.
37. The pharmaceutical composition of claim 35, wherein the immune
modulator is conjugated to the targeting moiety.
38. The pharmaceutical composition of claim 35, wherein the
superantigen and immune modulator are both conjugated to the same
targeting moiety.
39. The pharmaceutical composition of claim 36, wherein the immune
modulator is not conjugated to the targeting moiety.
40. The pharmaceutical composition of claim 37, wherein the
superantigen is not conjugated to the targeting moiety.
41. The pharmaceutical composition of claim 35, wherein the
targeting moiety binds to a cell surface antigen.
42. The pharmaceutical composition of claim 36, wherein the
targeting moiety binds to a cell surface antigen.
43. The pharmaceutical composition of claim 41, wherein the cell
surface antigen is associated with a disease.
44. The pharmaceutical composition of claim 43, wherein the disease
is a cancer.
45. The pharmaceutical composition of claim 42, wherein the cell
surface antigen is associated with a disease.
46. The pharmaceutical composition of claim 45, wherein the disease
is a cancer.
47. The pharmaceutical composition of claim 35, wherein the
targeting moiety is selected from the group consisting of an
antibody, an antigen-binding fragment of an antibody, an Fab
fragment of an antibody, an Fab.sub.2 fragment of an antibody, or a
single chain antibody.
48. The pharmaceutical composition of claim 36, wherein the
targeting moiety is selected from the group consisting of an
antibody, an antigen-binding fragment of an antibody, an Fab
fragment of an antibody, an Fab.sub.2 fragment of an antibody, or a
single chain antibody.
49. The pharmaceutical composition of claim 46, wherein the
targeting moiety is selected from the group consisting of an
antibody, an antigen-binding fragment of an antibody, an Fab
fragment of an antibody, an Fab.sub.2 fragment of an antibody, or a
single chain antibody.
50. The pharmaceutical composition of claim 35, wherein the
superantigen is modified to have a decreased ability to bind to MHC
class II antigen compared to a corresponding wild-type
superantigen.
51. The pharmaceutical composition of claim 35, wherein the
superantigen is modified to have decreased seroreactivity in human
sera compared to a corresponding wild-type superantigen.
52. The pharmaceutical composition of claim 35, wherein the
superantigen is chimeric comprising sequences derived from two or
more wild type superantigens.
53. The pharmaceutical composition of claim 49, wherein the
superantigen is modified to have a decreased ability to bind to MHC
class II antigen compared to a corresponding wild-type
superantigen.
54. The pharmaceutical composition of claim 49, wherein the
superantigen is modified to have decreased seroreactivity in human
sera compared to a corresponding wild-type superantigen.
55. The pharmaceutical composition of claim 49, wherein the
superantigen is chimeric comprising sequences derived from two or
more wild type superantigens.
56. The pharmaceutical composition of claim 35, wherein the
superantigen is obtained from Staphylococcal enterotoxin.
57. The pharmaceutical composition of claim 56, wherein the
superantigen is obtained from Staphylococcal enterotoxin A.
58. The pharmaceutical composition of claim 57, wherein the
superantigen is Staphylococcal enterotoxin A.
59. The pharmaceutical composition of claim 49, wherein the
superantigen is obtained from Staphylococcal enterotoxin.
60. The pharmaceutical composition of claim 59, wherein the
superantigen is obtained from Staphylococcal enterotoxin A.
61. The pharmaceutical composition of claim 60, wherein the
superantigen is Staphylococcal enterotoxin A.
62. The pharmaceutical composition of claim 35, wherein the immune
modulator is selected from the group consisting of cytokines,
chemokines, and extracellular parts of lymphocyte bound receptors
and ligands.
63. The pharmaceutical composition of claim 35, in the immune
modulator is IL-2.
64. The pharmaceutical composition of claim 49, wherein the immune
modulator is selected from the group consisting of cytokines,
chemokines, and extracellular parts of lymphocyte bound receptors
and ligands.
65. The pharmaceutical composition of claim 49, in the immune
modulator is IL-2.
66. A conjugate composition comprising a superantigen, an immune
modulator and a targeting moiety, wherein at least one of the
superantigen and immune modulator is conjugated to a targeting
moiety.
67. The conjugate composition of claim 66, wherein the superantigen
is conjugated to the targeting moiety.
68. The conjugate composition of claim 66, wherein the immune
modulator is conjugated to the targeting moiety.
69. The conjugate composition of claim 66, wherein the superantigen
and immune modulator are both conjugated to the same targeting
moiety.
70. The conjugate composition of claim 66, wherein the targeting
moiety binds to a cell surface antigen.
71. The conjugate composition of claim 70, wherein the cell surface
antigen is associated with a disease.
72. The conjugate composition of claim 71, wherein the disease is a
cancer.
73. The conjugate composition of claim 66, wherein the targeting
moiety is selected from the group consisting of an antibody, an
antigen-binding fragment of an antibody, an Fab fragment of an
antibody, an Fab.sub.2 fragment of an antibody, or a single chain
antibody.
74. The conjugate composition of claim 66, wherein the superantigen
is modified to have a decreased ability to bind to MHC class II
antigen compared to a corresponding wild-type superantigen.
75. The conjugate composition of claim 66, wherein the superantigen
is modified to have decreased seroreactivity in human sera compared
to a corresponding wild-type superantigen.
76. The conjugate composition of claim 66, wherein the superantigen
is chimeric comprising sequences derived from two or more wild type
superantigens.
77. The conjugate composition of claim 66, wherein the superantigen
is obtained from Staphylococcal enterotoxin.
78. The conjugate composition of claim 77, wherein the superantigen
is obtained from Staphylococcal enterotoxin A.
79. The conjugate composition of claim 78, wherein the superantigen
is Staphylococcal enterotoxin A.
80. The conjugate composition of claim 66, wherein the immune
modulator is selected from the group consisting of cytokines,
chemokines, and extracellular parts of lymphocyte bound receptors
and ligands.
81. The conjugate composition of claim 66, in the immune modulator
is IL-2.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to inactivation/cytolysis of
target cells caused by T cells activated by functional
superantigens. Cytolysis can be applied to therapy and to in vitro
assays.
DEFINITIONS
[0002] Superantigens. According to the very first definition
(around 1988-1993), superantigens are bacterial or viral proteins
capable of binding to MHC class II antigens without prior
intracellular processing and activate T cells by binding to the
V.beta.-chain variable region (V.beta.) of the T cell receptor
(TCR). The binding leads to a V.beta. family restricted activation
of a relatively large oroportion/subset of T cells and lysis of MHC
Class II expressing cells (superantigen dependent cell-mediated
cytolysis SDCC). Normally the superantigen activated subset of T
cells constitutes about 1-30% of the total amount of T cells of an
individual.
[0003] Well known wild-type superantigens according to the
definition above are the staphylococcal enterotoxins (SEA, SEB,
SEC1, SEC2, SED, SEE and SEH). Further examples are Toxic Shock
Syndrome Toxin 1 (TSST-1, also of staphylococcal origin),
Exfoliating Toxins (EXft), Streptococcal Pyrogenic Exotoxin A, B
and C(SPE A, B and C), Mouse Mammary Tumor Virus proteins (MMTV),
Streptococcal M proteins, Clostridial Perfringens Enterotoxin
(CPET), mycoplasma arthritis superantigens etc. For a review of
superantigens and their properties see Kotzin et al 1993.
[0004] Wild-type and chimeric superantigens have also been mutated
to have a reduced or no MHC class II binding and/or TCRV.beta.
binding (Kappler et al WO 9314264; Kappler et al 1993; Blanco et
al; Abrahmsn et al., WO9601650; Antonsson et al WO 9736932;
Antonsson et al 1997). This type of superantigens becomes less
toxic. In case they completely lack ability to bind to MHC class II
or to TCRV.beta. they no longer are functional superantigens
because they then lose their T cell activating ability.
[0005] By mutating structurally similar wild-type superantigens it
has been possible to construct chimeric functionally active
superantigens (hybrid superantigens) (Lamphaer et al., 1996 and
Antonsson et al WO 9736932).
[0006] It has been discovered that activation and subsequent cell
lysis can occur in a MHC class II independent manner in case the
wild-type superantigen was conjugated with a target-seeking moiety
capable of binding to a cell surface strcture (Dohlsten et al
WO9201470). This novel effector mechanism has been termed
superantigen antibody dependent cell-mediated cytolysis (=SADCC).
It includes the analogous mechanisms for targeting moieties other
than antibodies (Abrahmsn et al., WO9601650; Antonsson et al WO
9736932).
[0007] Accordingly the superantigen concept of today encompasses
any compound (preferably of polypeptide structure) that without
intracellular processing is capable of binding to a cell surface
structure (target structure) and to one or more olymorphic TCR
chains, in particular the V.beta. chain, thereby activating a
subset of T cells expressing the specific TCR chain involved in the
binding. The T cells then become cytotoxic and direct their
cytotoxicity against cells carrying the surface structure (target
structure, target cells). The definition of superantigen (SAG) as
used in the context of he invention and if not otherwise specified
thus will encompass conjugates between a targeting moiety and a
free superantigen as discussed above for SADCC.
[0008] By the term superantigen is contemplated, if not otherwise
specified, only functional superantigens.
[0009] A free superantigen (Sag) is a wild-type, possibly mutated
or otherwise modified, superantigen that is not conjugated to a
targeting moiety or to an immune modulator. The MHC class II
binding ability of free superantigens is an inherent targeting
property. Since free superantigens lacI conjugated targeting
moieties they will only exert SDCC.
[0010] A conjugated superantigen is a conjugate between a free
superantigen and a targeting moiety or an immune modulator. A
conjugated superantigen exerts either or both of SDCC and
SADCC.
[0011] An immune modulator (IM) is a compound capable of regulating
the immune system. In the context of the invention superantigens
are treated separately and are not included when the term immune
modulator is used. An immune modulator often has an inherent
targeting ability, such as for a corresponding lymphocyte receptor.
Unless otherwise specified, an immune modulator is in un-conjugated
form.
[0012] A targeting moiety (T) is a moiety that is capable of
binding to a cell surface structure and/or a tissue structure.
[0013] A conjugate is composed of two or more moieties Sag, IM, T
etc that are linked to each other covalently.
[0014] By soluble forms of active ingredients means forms that are
soluble in body derived fluids such as serum and plasma.
BACKGROUND ART--THERAPEUTIC USE OF SUPERANTIGENS
[0015] Non-conjugated wild-type and mutated superantigens have been
suggested for therapy with curative effect presumably being
accomplished through an activation of the immune system, either
locally at Class II expressing cell associated with the disease to
be treated or as a systemic activation (Kalland et al WO9104053;
Terman et al WO9110680 and WO9324136; Antonsson et al WO 9736932;
and Newell et al 1991). Due to the extreme toxicity of wild-type
superantigens this approach, with respect to cancer treatment,
should only be applicable to a very minor fraction of all
cancers.
[0016] It has also been suggested to use superantigens conjugated
to target-seeking moieties (Dohlsten et al WO9201470; Abrahmsn et
al WO9601650, Antonsson et al WO 9736932 and Ochi et al 1993), all
three publications being incorporated by reference)
[0017] In connection with studies on prevention of superantigen
induced down-regulation of T cell mediated cytotoxic activity by
IL-2 in vivo it has been speculated that it should be beneficial to
coadminister IL-2 with unconjugated wild-type superantigens and
wild-type superantigens conjugated to antibodies (Belfrage Thesis
Augusti/September 1996; Belfrage et al 1994; Belfrage at al 1995;
Belfrage et al 1997a; Belfrage et al 1997b (wild-type
superantigens))
[0018] It has also been suggested that cell membrane anchored CD80
has a role in superantigen activation of T cells in the absence of
MHC class II antigens (Lando et al 1993 and 1996).
[0019] FIG. 4 in Lando et al 1996 shows an experiment in which the
ability of superantigen conjugated to an antibody alone or in
combination with IL-2 to induce proliferation of resting human T
cells was analyzed. In this 4-day experiment the conjugated
superantigen was presented on parent CHO-cell and CHO-cells
transfected to express Class II or C215 or Class II plus C215. The
effect of IL-2 was insignificant.
[0020] Kappler et al (WO9314634) have suggested non-conjugated
wild-type SEB mutated to have lost its V.beta. or MHC Class II
binding ability (in the context of vaccines and as an agent to
neutralize toxic effects of superantigens). Abrahmsn et al
(WO9601650) have suggested cancer therapy with conjugated
superantigens having a modified, preferably decreased; ability to
bind to Class II antigens. Antonsson et al (We) 9736932) has
suggested therapy with chimeric superantigens and superantigens
with reduced seroreactivity (see also Abrahmsn et al). Mutations as
described by Abrahmsn et al (WO9601650) and Antonsson et al (WO
9736932) will implicate superantigens with a lowered systemic
toxicity, lowered immunogenicity and/or lowered seroreactivity in
the mammal to be treated
[0021] Therapy with administration of nucleic acids encoding
wild-type superantigens have been suggested (Terman et al
WO9110680; WO9324136) and Dow et al WO9636366). Dow et al go
further on and suggest coadministration of a nucleic acid encoding
a cytokine or a chemokine with a nucleic acid encoding a
superantigen. Without enabling experimental support, WO9636366 also
suggests constructs in form of a biscistronic gene construct in
which one cistron contains the gene coding for the superantigen and
the other cistron contains the gene coding for a cytokine or a
chemokine.
[0022] Without enabling experimental support Pouletty P (Sangstat,
EP 510949) has speculated that conjugates between targeting
moieties, such as IL-2, and wild-type superantigens might be useful
for inactivating cells expressing the IL-2 receptor.
BACKGROUND ART--THERAPEUTIC USE OF IMUNE MODULATORS IN COMBINATION
WITH ANTIBODIES SPECIFIC TO CELLS THAT ARE TO BE INACTIVATED
[0023] It previously has been suggested to conjugate antibodies
with biological response modifiers, for instance a chemokine or a
cvtokine, such as interleukin-2 (Fell et al EP 439095; Rosenblum et
al EP 396387, Pancook et al 1996; and Becker et al 1996).
[0024] The Problem the Present Invention Sets Out to Solve.
[0025] The present invention sets out to provide improvements in
relation to superantigen therapy involving activation of the immune
system in order to inactivate undesired target cells in a mammal to
be treated. In particular the improvements relate to: 1. extending
the activation period locally, for instance in a tumour, during a
first treatment cycle; 2) counteracting the appearance of
hyporesponsiveness due to the tendency of activated T cells to
escape into anergy; 3) facilitating MHC class II independent T cell
activation in the tumor area; and 4) broadening the therapeutic
window for cytolysis via superantigen activation. It has now been
discovered that these improvements wholly or partly may be
accomplished provided that the administration of the superantigen
(SAG) is combined with the administration of an immune modulator in
soluble form, at least one of the superantigen and the immune
modulator being in form of a conjugate with a moiety having
targeting properties for the cell Lo be inactivated.
[0026] The First Major Aspect of the Invention: a Method of
Inactivating Target Cells.
[0027] The first aspect of the invention covers both therapy and
assays in vitro and is a method for inactivating undesired target
cells in the presence of T cells by bringing the two types of cells
in contact with a superantigen (SAG), in particular a superantigen
that activates T cells through binding to TCRV.beta., in the
presence of an immune modulator (IM) that is not a superantigen
(Sag). In its broadest aspect the method is characterized in that
at least one of the superantigen and the immune modulator is in
form of a conjugate with a moiety (T) having targeting properties
for the cell to be inactivated. In a subaspect the method is
characterized in that
[0028] a. the superantigen (SAG) and the immune modulator is used
in form of a triple conjugate comprising a superantigen (Sag), a
targeting moiety (T) for the target cells and an immune modulator
(IM) (T,IM,Sag-conjugate);
[0029] b. the superantigen (SAG) is used in form of a dual
conjugate between a superantigen (Sag) and a targeting moiety (T)
for the target cells in combination with a dual conjugate between
an immune modulator (IM) and a targeting moiety (T') for the target
cells (T,Sag-conjugate+T',IM-conjugate);
[0030] c. the superantigen (SAG) is used in form of a dual
conjugate between a superantigen (Sag) and a tarqeting moiety (T)
for the target cells and the immune modulator (IM) is used in free
form, i.e. not conjugated to a targeting moiety for the target
cells (T,Sag-conjugate+IM);
[0031] d. the superantigen (SAG) is used in free form (Sag) and the
immune modulator is used in conjugate form, i.e. a dual conjugate
between the immune modulator (IM) and the superantigen (Sag)
(Sag+T,IM-conjugate); and
[0032] e. the superantigen (SAG) and the immune modulator is used
in form of a dual conjugate between a superantigen (Sag) and an
immune modulator (IM) (Sag,IM-conjugate);
[0033] The superantigen and the immune modulator may be targeted to
the same type of cells, for instance to identical or crossreacting
structures/epitopes or to different type of cells within the same
tissue. Targeting may be for normal cells or diseased cells
associated with one and the same tissue. Either or both of the
superantigen and the immune modulator may be targeted with one or
several antibodies.
[0034] The Diseases to Be Treated by the Method of the
Invention.
[0035] The diseases to be treated are in principle the same as
those previously suggested for superantigens. See for instance
under headings "Background . . . " above. Illustrative examples are
cancers, autoimmune diseases, parasitic infestations, viral
infections and other diseases associated with cells that on their
surface express MHC class II antigens and/or other structures that
are specific for respective disease and bind to the target-seeking
moiety incorporated in the superantigen in accordance with the
inventive concept (formula I). Also bacterial infections may be
combated by the use of the invention.
[0036] Important cancer forms in the context of the invention are:
melanomas, carcinomas, hematopoetic neoplasias and fibrosarcomas
and includes specific forms such as squamous cell carcinoma, breast
cancers, head and neck carcinomas, thyroid carcinomas, soft tissue
carcinomas, bone sarcomas, testicular cancer, prostatic cancer,
ovarian cancers, bladder cancers, skin cancers, brain cancers,
angiosarcomas, hemanqiosarcomas, mast cell tumors, primary hepatic
cancers, lung cancers, cervix cancers, renal cell carcinomas,
leukemias and lymphomas. Included are any type of malignant or
benign tumors as well as multi-drug resistant cancers, metastatic
cancers, various forms of chemically or virally (herpes, SV40, HIV
etc) induced cancers.
[0037] The Second Major Aspect of the Invention: Inventive
Superantigen Conjugates.
[0038] This aspect of the invention comprises conjugates complying
with the formula
(T).sub.x(Sag).sub.y(IM).sub.z Formula I
[0039] T is a targeting moiety, Sag corresponds to a free
superantigen and IM is an immune modulator that is not a
superantigen. T, Sag and IM are linked together via organic linkers
B that may be different or equal within one and the same conjugate
molecule or substance. Conjugates according to formula I encompass
chemical conjugates as well as recombinantly produced conjugates
(fusion proteins). x, Y and z are integers that typically are
selected among 0-10, such as 0-5, and represent the number of
moieties T, Sag and IM, respectively, in a given conjugate
molecule, with the provision that y>0 and also one or both of x
and >0. Chemical conjugates are normally conjugate substances
containing a mixture of different conjugate molecules. Accordingly
in chemical conjugate substances x, v and z may also be
non-integers within the range 0-10, such as within the range
0-5.
[0040] In a first subaspect of formula I, Sag and IM and T are
present in the conjugate (x and y and z>0;T,Sag,IM-conjugates).
x, y, and z are typically integers 1-3, with preference for 1-2.
Typical relations between x, y and z are: x=y=z; x=y=0.5z;
x=0.5y=0.5z; and x=0.5v=z.
[0041] In a second subaspect of conjugates according to formula I,
the targeting moiety is absent (IM,Sag-conjugates, x=0). y and z
typically are integers 1-3. Preferred relations between x and y
are: x=y; x=0.5y, 0.5x=y; x=1/3y and 1/3x=
[0042] In both subaspects integers or relations primarily refer to
fusion proteins in which the targeting moiety may be a protein
containing 1, 2, 3 or 4 polypeptide chains and in which there are
one T in each conjugate molecule.
[0043] Formula I for conjugates according to the second subaspect
reduces to:
(Sag).sub.y(IM).sub.z Formula II
[0044] This type of conjugates are primarily adapted to the
treatment of diseases associated with cells expressing MHC class II
antigens, in particular class II expressing cancers, such as
cancers of the hematopoetic system, and certain autoimmune
diseases, viral infections and parasitic infestations, but also
with diseases associated with cell membrane anchored receptors for
the immune modulator, for instance T cell lymphoma expressing for
instance the IL-2 receptor.
[0045] A. The Immune Modulator IM in Formula I
[0046] IM stands for an immune modulator that is not a free or
conjugated superantigen.
[0047] The immune modulator may be a cytokine or a chemokine.
Illustrative cytokines are granulocyte macrophage colony
stimulating factor (GM-CSF), tumor necrosis factor .alpha. or
.beta. (TNF.alpha. or TNF.beta.), macrophage colony stimulating
factor (M-CSF), granulocyte stimulating factor (G-CSF), IL-1, IL-2,
IL-4, IL-6, IL-7, IL-12, IL-15, IL-18 and IGF. Illustrative
chemokines are C5a, IL-8, monocyte chemotactic protein 1alpha
(MIP1alpha) or monocyte chemotactic protein 1.beta. (MIP1.beta.),
monocyte chemoattractant protein 1 (MCP-1), monocytic
chemoattractant protein 2 (MCP-2), monocytic chemoattractant
protein 3 (MCP-3), platelet activating factor (PAFR),
N-formyl-methionyl-leucyl-phenylalanine (FMLPR), leukotriene
B.sub.4 (LTB.sub.4R), gastrin releasing peptide (GRP), RANTES,
eotaxin, lymphotactin, IP10, I-309, ENA78, GCP-2, NAP-2, MGSA/gro,
DC-CK1, Flt3L (ectopic domain), fractalkin, PF-4 etc.
[0048] Another type of immune modulators are those derived from
cell membrane anchored receptor/ligand pairs involved in modulation
of a triggered immune response, such as costimulation (for instance
lymphocyte surface bound receptors and corresponding cell bound
ligands). IllustratLive examples are members selected from the
pairs CD40L/CD40, 4-BB1/4-BB1L, CD28/B7, CTLA-4/B7 etc. B7 includes
variants such as CD80 and CD86 with preference for the former.
Preferred forms are soluble, contain the extracellular part
(ectopic domain) and are devoid of the intracellular and membrane
anchored parts.
[0049] Particularly preferred immune modulators are capable of
potentiating the effects of superantigens in vivo, for instance by
counteracting escape of superantigen activated T-cells into anergy.
Typical appropriate cell bound receptors/ligands are CD28/B7
including analogues and fragments as defined above. Typical
cytokines of this group are IL-2, as being the main downstream
effector of CD28/B7 signaling, and the IL-2 like cytokines IL-7 and
IL-15. Among T cell surface associated receptor/ligand pairs the
member not bound to the T cell to be activated is preferred to be
incorporated in a conjugate according to the invention. For
CD40L/CD40, 4-BB1/4-BB1L and CD28/B7 this means soluble forms CD40,
4-BB1L, and B7 with preferences as defined above. The experimental
part of this text illustrates the immune modulator variants that at
the priority date were found optimal in the invention.
[0050] Immune modulators should preferably be of the same species
origin as the individual who is intended to be treated. Native
immune modulators, such as cytokines and chemokines, often show a
high systemic toxicity and a relatively short half live in mammals.
The literature is extensive on how to modify immune modulators to
an increased stability relative to oxidation, a longer in vivo half
life, a lower toxicity, an improved refolding when produced by
recombinant techniques etc. For instance U.S. Pat. No. 5,229,109
(Grimm et al) describes low toxicity analogues of IL-2 having a
reduced affinity for the high affinity IL-2 receptor (IL-2R) by
being deficient in binding to the p55 .alpha. subunit of the
receptor. The analogues are primarily prepared by mutating the
codon for an amino acid in position 33-46 in IL-2 (for instance
Arg38Ala and Phe42Lys or Phe42Ala). The Asp20Lys mutant has 100-500
fold reduced affinity for the p75/.beta.-chain of the IL-2 receptor
without affecting binding to p55 (Collins et al 1988). Other
mutations, e.g. Asp20Ser are less severe (Berndt et al 1994).
Studies on murine IL-2 indicates that Asp84 and Asn88 of human IL-2
are also implicated in p75 binding (Zurawski et al 1993). This is
supported by modelling of the binding between human IL-2 and its
receptor (Bamborough et al 1994). The expected reduction in
affinity of these mutations is Asp20>Asn88>Asp84.
[0051] Combining the mutations in Arg38 and Phe42 with mutations in
position 88 and 20 would result in a still lower affinity for
IL-2R. Another potent and at the priority date preferred IL-2
mutation is Thr51Pro that gives an IL-2 analogue with a lowered
rate of cell internalization and a prolonged duration of its immune
modulating effect (Chang et al 1996).
[0052] The numbering of amino acid positions is according to
Taniguchi et al 1983.
[0053] The publications by Grimm et al; Collins et al 1988; Berndt
et al 1994; Zurawski et al 1993; Bamborough et al 1994; Chang et al
1996; and Taniguchi et al 1983 are incorporated by reference.
[0054] The use of cytokine and chemokine analogues with a reduced
affinity for their normal receptors in conjugates as described
above will strengthen their targeting to the preselected target
cell. It is conceivable that cytokines and chemokines mutated to
show a reduced rate of cell internalization upon binding to their
respective cell receptor and incorporated into a conjugate
according to formula I will result in a prolonged superantigen
activity compared to corresponding conjugates with the native form
of the immune modulator.
[0055] The term immune modulator (IM) thus encompasses any modified
form, for instance any mutated form, that is capable of agonizing
or antagonizing the effects of the corresponding native form of the
immune modulator.
[0056] B. The Superantigen Part SAG in Formula I
[0057] Sag in formula I represents a superantigen as defined for
free superantigens and under heading "Background . . . " above,
i.e. wild-type superantigens possibly modified, for instance by
mutation,
[0058] a. to have a decreased ability to bind to MHC class II
antigen compared to the corresponding wild type superantigen (see
for instance in Abrahmsn et al (WO9601650));
[0059] b. to have a decreased seroreactivity in human sera compared
to the corresponding wild-type superantigen (see for instance
Abrahmsn et al (WO9601650) and Antonsson et al WO 9736932 and
Antonsson et al 1997);
[0060] c. to have a decreased immunogenicity in humans compared to
the corresponding wild-type superantigen (see for instance
Antonsson et al WO 9736932 and Antonsson et al 1997);
[0061] d. to be a chimera between two or more analogous wild-type
superantigens in which one region in one first wild-type
superantigen has been replaced with the corresponding region in a
second analogous superantigen. The region in question may be a
region determining binding to TCRV.beta., e.g. as defined for
SEE/SEA and SEA/SEE chimeras (see for instance Antonsson et al WO
9736932; Antonsson et al 1997; and Lamphaer et al 1996).
[0062] Also other modifications/mutations that may be found
appropriate are included, for instance to avoid undesired
glycosylation when produced in eucaryotic cells.
[0063] Typical mutations for SEA/SEE-like superantigens at the
priority were (numbering as used by Antonsson et al, 1997 and
Antonsson et al WO 9736932):
[0064] a. decreased MHC class II binding: Asp227Ala (=SEAm9),
Phe47Ala and/or Asp70Arg. The mutant Phe47Ala/Asp227Ala SEAm23.
[0065] b. and d. chimeras between SEA and SEE, aimed at reducing
seroreactivity in humans, while retaining SADCC capability of the
corresponding conjugated superantigen: SEE with the following
substitutions Gly20Arg, Thr21Asn, Glv24Ser, Lys24Arg.
[0066] The mutants used in the experimental part are
SEA(Asp227Ala)=SEAm9, SEA(Phe47Ala/Asp227Ala) SEAm23 and
SEA(Phe47Ala/Asn102Q/Asn149Asp/Thr218V- al/Asp227Ala)=SEAm57.
[0067] At the priority filing preferred superantigens were selected
among
[0068] 1. superantigens (Sag) that exhibit two MHC class II binding
sites (for instance staphylococcal enterotoxins A and E),
[0069] 2. superantigens (Sag) that in their unmutated form required
Zn-ion for optimal binding to MHC class II (for instance SEA, SEE
and SEH),
[0070] 3. Staphylococcal enterotoxins.
[0071] A Sag molecule that is to be incorporated into a conjugate
according to the invention does not need to be a functional
superantigen, the main issue being that the final conjugate is so
by exerting either or both of SADCC and SDCC as outlined above.
[0072] C. Targeting Moiety T in formula I.
[0073] T can in principle be any structure that is able to bind to
a cell surface structure, preferably a disease specific structure.
The structure against which T is directed is usually different (a)
from the polymorphic TCR chain epitope to which Sag binds, and (b)
from the MHC class II epitopes to which Sag binds. The
target-seeking moiety may be selected among interleukins (e.g.
interleukin-2), hormones, antibodies including antigen binding
fragments of antibodies, growth factors etc. See for instance
Woodworth 1993 (hereby incorporated by reference).
[0074] The targeting moiety may thus be a protein containing 1, 2,
3 or 4 polypeptide chains.
[0075] At the priority date, it was preferred that T was an
antibody (full length antibody, Fab, F(ab).sub.2, Fv, ScFv (single
chain antibody), multiple single chain antibodies (ScFv).sub.n and
any other antigen binding antibody fragment), including any
functionally active truncated form of the antibody forms mentioned
above. Other variants are monospecilic and bispecific. The antibody
may in principle be directed towards any disease
associated/specific cell surface structure, for instance structures
linked to any of the cancer forms given above, with particular
emphasis for antibody active fragments (such as Fab). Typically the
antibody may be directed towards a colon and/or pancreatic specific
epitope, for instance the so called C242 epitope. (Lindholm et al
WO9301303), a lung cancer specific epitope for instance the epitope
for the 5T4 antibody (Stern et al WO8907947), a lymphoma specific
epitope for instance on CD19, a melanoma specific epitope for
example HMW-MAA, etc.
[0076] The term "antibody" comprises monocional as well as
polyclonal variants, with preference for monoclonal
preparations.
[0077] In case the target moiety is a Fab fragment the cysteine
residues normally linking the heavy and light Fab chains together
preferably have been replaced by an amino acid not permitting
disulfide formation, for instance serine. See also Antonsson et al
WO 9736932.
[0078] What has been said above also includes that T may be
directed towards unique structures on more or less healthy cells
that regulate or control the development of a disease.
[0079] D. The Linker B.
[0080] The linker B may be selected as previously described
(Dohlsten et al WO9201470; Abrahmsn et al WO9601650; and Antonsson
et al WO 9736932), i.e. B shall preferably be hydrophilic and
exhibit one or more structure(s) selected among amide, thioether,
disulphide etc. The most prominent linkers are those obtained by
recombinant techniques, i.e. conjugation takes place at the genomic
level resulting in oligopeptide linkers. Typically oligopeptide
linkers contain 1-30, such as 1-20, amino acid residues that
preferably are selected so that the linker in total is hydrophilic.
The linker residues thus preferably are selected among hydrophilic
amino acid residues, such as Gln, Ser, Gly, Glu, Pro, His and Arg.
Typical oligopeptide linkers comprise the tripeptide GlyGlyPro or
the so called Q linker (Wootton et al 1989 hereby incorporated by
reference) possibly modified with gly-pro at the amino terminal
end.
[0081] E. Attachment Points for T, SAG and IM
[0082] Chemical conjugates will typically contain a mixture of
conjugate molecules differing in linking positions. The conjugate
substance will contain hetero- as well as homo-conjugates.
[0083] For recombinant conjugates (fusion proteins) the obtained
conjugate substance will be uniform with respect to the linking
position. For each individual subunit (T, Sag, IM) the amino
terminal is fused to the carboxy termina of another subunit or vice
versa, preferably via an inserted oligopeptide bridge. The
combinations in case the conjugate contains one each of T, IM, Sag
will be T-IM-Sag, IM-T-Sag, Sag-IM-T, Sag-T-IM, T-Sag-IM, IM-Sag-T
(the occurrence of linker structure of B is not shown). In case one
or more of the subunits contains two or more polypeptide chains the
number of possibilities increase. For T being an antibody Fab
fragment the possibilities will be (the oligopeptide linkers B are
not shown):
1 1. Sag-Fab(light chain)-IM 2. Fab(light chain) Fab(heavy chain)
Sag-Fab(heavy chain)-IM 3. Sag-Fab(light chain 4. Fab(light
chain)-IM Fab (heavy chain)-IM Sag-Fab(heavy chain) 5.
Sag-Fab(light chain) 6. IM-Fab(light chain) IM-Fab(heavy chain)
Sag-Fab(heavy chain) 7. Fab(light chain)-Sag 8. Fab(light chain)-IM
Fab(heavy chain)-IM Fab(heavy chain)-Sag
[0084] In further variants the immune modulator and the
superantigen may be fused in sequence at any end of any of the
chains in the antibody.
[0085] At the priority date recombinant conjugates were preferred,
with utmost preference for Fab fragments as targeting moiety and
linking of the amino terminal of the free superantiger to the first
constant domain of the heavy (C.sub.Hl) or light antibody chain and
the immune modulator to the remaining carboxy terminal (valid for
formulas I-IV).
[0086] For optimal production and function the fusion protein is
expressed recombinantly as a two chain product in which the
superantigen is fused C-terminally to the C.sub.Hl-domain of the
antibody Fab fragment via a flexible hydrophilic amino acid linker
of 3-11 residues. This linker may have the sequence Gly-Gly-Pro or
Pro-Ala-Ser-Gly-Gly-Gly-Gly-Ala-Gly-Gly-- Pro (SEQ ID NO: 19) or
4-9 residues based on SEQ ID NO: 19, with SEQ ID NO: 19 being
preferred The immune modulator moiety is fused C-terminally to the
light chain via a hydrophilic and neutral or positively charged
linker of 10-20 residues (linker Q). Preferably, linker Q may have
the following sequences
Gly-Pro-Arg-Gln-Ala-Asn-Glu-Leu-Pro-Gly-Ala-Pro-Ser-G-
ln-Glu-Glu-Arg (SEQ ID NO: 23),
Gly-Pro-Arg-Gln-Ser-Asn-Glu-Thr-Pro-Gly-Se-
r-Pro-Ser-Gln-Glu-Glu-Arg (SEQ ID NO: 20),
Gly-Pro-Arg-Gln-Ala-Lys-Thr-Leu-
-Pro-Gly-Ala-Pro-Ser-Gln-Thr-Thr-Arg (SEQ ID NO: 21) or
Gly-Pro-Thr-Glu-Ala-Asp-Glu-Leu-Pro-Gly-Ala-Pro-Ser-Glu-Glu-Glu-Thr
(SEQ ID NO: 22), with SEQ ID NO: 20 and 21 being most preferred
(see Example 2 for more details).
[0087] The analogous combinations at the amino terminal or
combination of attachments at the amino and carboxy terminals of
the VH and VL domain were at this stage believed to result in
active but less efficient conjugates.
[0088] F. Active Entities Not Complying with Formula I But Used
According to the Combinations a-e Above in the Inventive
Method.
[0089] Free superantigens (Sag): See under heading "Definition".
Typical Sags are given under the headings "Background" and "B. The
superantigen part of Sag in formula I".
[0090] Unconjugated immune modulators: See under heading 1'
"Definition". In principle the same immune modulators as given
under the heading "A. The immune modulator IM in formula I" can be
used. Cytokines and chemokines are preferred with emphasis for IL-2
and IL-2-like cytokines.
[0091] Targeted immune modulators (T,IM-conjugates): These
conjugates comply with the general formula
(T').sub.x(IM').sub.z Formula III
[0092] in which T' and IM' are linked together by an organic linker
B'. T', IM' and B' are selected among the same groups of
compounds/structures as T, IM and B. x and z are defined in the
same way as in formula I (y=0), with preference for one or two IM'
per T' and conjugate molecule. The attachment points between T' and
IM' are as defined for formula I. See also under heading "E.
Attachment points . . . " in which the formulas 1-8 and comments
thereto are applicable also to T,IM conjugates except that Sag is
omitted. Conjugates of formula III can be manufactured according to
known techniques, i.e. conventional chemical linking or recombinant
techniques (fusion proteins), with preference for the latter (Fell
et al EP 439095; Rosenblum et al EP 396387). Particularly important
T,IM-conjugates comprise an IM'-moiety that has been modified, for
instance mutated, to a reduced affinity and/or reduced rate of
internalization as defined above.
[0093] Targeted superantigens (T,Sag-conjugates). These conjugates
comply with the general formula
(T").sub.x(Sag").sub.y Formula IV
[0094] in which T" and Sag' are linked together by organic linkers
B". T", Sag" and B" are selected among the same groups of
compounds/structures as T, IM and B. x and y are defined in the
same way as in formula I (z=0), with preference for one or two Sag"
per T" and conjugate molecule. The attachment points between T" and
Sag" are as defined for formula I. See also under heading "E.
Attachment points . . . " in which the formulas 1-8 and comments
thereto are applicable also to T,Sag conjugates except that IM is
omitted from the formula. Conjugates of formula II can be
manufactured according to known techniques, i.e. conventional
chemical linking or recombinant techniques (fusion proteins), with
preference for the former. See for instance Dohlsten et al
WO9201470; Abrahmsn et al WO 9601650, Antonsson et al WO WO
9736932.
[0095] Third Major Aspect of the Invention: Conjugates Containing a
Modified Immune Modulator.
[0096] These novel conjugates complies with the formula:
(T'").sub.x(Sag'").sub.y(IM'").sub.z Formula V
[0097] where T'" and Sag'" are selected among the same compounds as
T and Sag for formula I. IM'" is an immune modulator that has been
modified, for instance by mutation, to exhibit a lowered affinity
to its cell membrane anchored receptor and/or a lowered rate of
internalization via binding to its receptor (compared to
corresponding native forms). IM" is preferably a cytokine or a
chemokine. See further under heading "A. The immune modulator IM in
formula I". One important immune modulator for this aspect of the
invention is modified IL-2. x, y and z are defined in the same way
as in formula I.
[0098] In a first subaspect of conjugates according to formula V,
T'", Sag'" and IM'" are always present (all x, y and z 0), i.e.
T,Sag,IM-conjugates. x, y and z are typically integers 1-3, with
preference for 1-2. Typical relations between x, y and z are:
x=y=z; x=v=0.5z; x=0.5y=0.5z; and x=0.5y=z.
[0099] In a second subaspect of conjugates according to formula V,
the superantigen moiety is absent (y=0), i.e. T,IM-conjugates. x
and z are typically integers 1-3. Preferred relations between x and
z are: x=z; x=0.5z, 0.5x=z; x=1/3z and 1/3x=z.
[0100] In both subaspects of conjugates according to formula V, the
ranges given for integers and their interrelations primarily refer
to fusion proteins in which the targeting moiety may be a protein
containing 1, 2, 3 or 4 polypeptide chains and in which there are
one T in each conjugate molecule. The attachment points in fusion
proteins of formula V are the same as for corresponding fusion
proteins complying with formula I. See also under heading "E.
Attachment points . . . " in which the formulas 1-8 and comments
thereto are applicable also to T,IM conjugates except that, for the
second subaspect, Sag is omitted.
[0101] Manufacture of Conjugates Defined Above.
[0102] The manufacture of conjugates may be carried out in
principle according to two main routes:
[0103] 1. Chemical linking of the individual subunits T, Sag and IM
together. Each individual subunit or combination of them may have
been produced by recombinant techniques.
[0104] 2. Recombinant techniques directly providing a conjugate as
defined in any of formulas given above.
[0105] The factual methods are well recognized for the average
skilled worker and comprise a large number of variants.
[0106] Chemical linking typically utilizes functional groups (e.g.
primary amino groups, carboxy groups, mercapto, carbohydrate
groups) that typically are natively present in several positions of
superantigens, proteinic immune modulators and proteinic targeting
moieties. The techniques are well-known in the art.
[0107] The main host cell for large scale recombinant production of
the inventive conjugates between a superantigen and an immune
modulator (fused forms as well as non-conjugated forms) is E. coli.
This host provides for in principle two routes: intracellular
production and secretion. The latter variant is preferred because
it offers purification of correctly folded proteins from the
periplasma and from the culture medium. The above does not exclude
that it is possible to produce active conjugates also in other host
cells, e.g. eukaryotic cells, such as yeast or mammalian cells.
Preliminary results have so far indicated that eukaryotic cells,
such as mammalian cells, may be preferred for incorporating immune
modulators interacting with CD28, for instance B7 and its
analogues.
[0108] The Fourth Aspect of the Invenion: Gene Constructs Encoding
the Novel Inventive Conjugates.
[0109] A fourth aspect of the invention is a recombinant nucleic
acid molecule, preferably DNA, such as cDNA, or RNA, encoding a
superantigen and an immune modulator as defined above, with
preference for IL-2, IL-7, IL-12, IL-15, IL-18, RANTES, ectopic
domain of CD80, ectopic domain of CD86, 4-BB1L and Flt3L and their
analogues as defined above. In this aspect of the invention, the
nucleic acid typically should contain regulatory control sequences
such as translation regulatory sequences, origin of replication,
secretory signal sequences either for one or both of the immune
modulator and the superantigen, etc that are compatible with the
host cell in which the immune modulator and the superantigen are to
be expressed. The region between the part sequences encoding the
superantigen and the immune modulator may comprise a sequence
encoding a ribosome entry sites such as in biscistronic gene
constructs. The latter will allow separate expression of each
polypeptide chain comprised within the conjugate. In case of
multichain conjugate combinations containing IM, Sag, targeting
moiety conjugate and the proper signal sequences, biscistronic
constructs will facilitate folding and assembly to fully active
conjugates with IM bound to one of the chains. This will be
important for cases in which the targeting moiety is a two chain
antibody molecule and at least one of the superantigen (Sag) and
the immune modulator is fused to a terminal end of the antibody
chain. See above.
[0110] Pharmaceutical Compositions, Dosage and Routes of
Administration.
[0111] A fourth aspect of the instant invention is a pharmaceutical
composition containing the inventive combination of superantigen
(Sag), targeting moiety (T) and immune modulator (IM). The
characteristic feature of this aspect of the invention is that at
least one of the superantigen and the immune modulator is in the
form of a conjugate permitting targeting to cells that are to be
inactivated as described above.
[0112] The compositions contemplated are known in the field, except
that now they contain the inventive conjugate combination. In a
particular composition the combination may comprise:
[0113] a. a triple conjugate comprising a superantigen (Sag), a
targeting moiety (T) for the target cells and an immune modulator
(IM) (T,IM,Sag-conjugate);
[0114] b. two dual conjugates--one between a targeting antibody (T)
and a superantigen and one between a targeting antibody (T') an
immune modulator (i.e. T,Sag-conjuqate+T',IM-conjugate). T and T'
may be different or equal as regard to epitope specificity;
[0115] c. on dual conjugate between a superantigen (Sag) and a
targeting moiety (T), and an immune modulator (IM) in free form
(T,Sag-conjugate+IM);
[0116] d. one dual conjugate between an immune modulator (IM) and a
targeting moiety (T), and a superantigen in free form (Sag)
(T,IM-conjugate+Sag); or
[0117] e. a superantigen (Sag) and an immune modulator (IM) in form
of a dual conjugate (Sag,IM-conjugate);
[0118] See further above in the context of the inventive methods.
In case the compositions contain two active components (b-d above)
the composition may allow for keeping the components apart up to
the occasion for administration.
[0119] The compositions may be in the form of a lyophilized
particulate material, a sterile or aseptically produced solution, a
tablet, an ampoule etc. Vehicles such as water (preferably buffered
to a physiologically acceptable pH value by for instance PBS) or
other inert solid or liquid material may be present. In general
terms the compositions are prepared by the conjugate, possibly in
combination with an unconjugated active component, being mixed
with, dissolved in, bound to, or otherwise combined with one or
more water-soluble or water-insoluble aqueous or non-aqueous
vehicles, if necessary together with suitable additives and
adjuvants. It is imperative that the vehicles and conditions must
not adversely affect the activity of active components as defined
in a-e above.
[0120] Normally the superantigens (SAG) to be used in the invention
will be sold and administered in predispensed dosages, each one
containing an effective amount of SAG that, based on the result now
presented, is believed to be within the range of 1 pg-50 mg. The
exact dosage will vary from case to case depending on the patient's
weight and age and pretiter of antibodies specific for the SAG
used, route of administration, type of disease, target-seeking
moiety, superantigen, linkage (--B--), immune modulator etc.
[0121] An important factor to account for in determining the dose
for a a combination to be used in the inventive method is that
superantigens and immune modulators exert optimal dose ranges. A
too low dose will result in no or a suboptimal effect and a too
high dose will give unacceptable side effects such as toxicity that
may be lethal. Thus it has to be emphasized the broad range given
above is an attempt to encompass all ranges possible for all
variants of the inventive method. Thus, each specific combination
according to the inventive method has a dose subrange within the
range of 0.1 pg to 50 mg. This does not exclude that future
developments and results may lead to dose levels outside this
range.
[0122] The administration routes will be those commonly
contemplated within the field, i.e. a target killing effective
amount or therapeutically active amount of a superantigen-immune
modulator combination according to the invention is brought into
contact with the target cells. For the indications specified above
this mostly means parenteral administration, such as injection or
infusion (subcutaneously, intravenously, intraarterial,
intramuscularly, intraperitoneal) to a mammal, such as a human
being. The conjugate combination of the invention may be
administered locally or systemically.
[0123] By the term "target killing effective amount" is
contemplated that the amount is effective in activating and
directing T cells to destroy target cells.
[0124] The preferred administration routes at the priority date are
the same as contemplated for the superantigen conjugates according
to Dohlsten et al WO9201470; Abrahmsn et al WO9601650 and Antonsson
et al PCT/97/00537. This means 1-5 hours' intravenous infusion
(preferably 4 hours) per day combined with a fever-reducing agent
(paracetamol). The administration is to be repeated during some
days, for instance 5-8 days, with care consideration taken for the
risk of boostering antibodies directed towards the conjugate.
Optimally, several cycles of therapy with each cycle containing
treatment during one or more days followed by a rest period during
one or more days, e.g. cycles with treatment and resting during 5
and 2 days, respectively.
[0125] The inventive compositions may be administered either as the
main therapy or in preferred modes as adjuvant therapy in
connection with surgery or with other drugs.
EXPERIMENTAL PART
LEGENDS TO THE FIGURES
[0126] FIG. 1. FACS analysis of CHO-CD28 cells stained with
CD80-C215Fab, CD80-C215Fab-SEAm57 or C215Fab-SEA followed by
incubation with anti-mouse kappa chain mAb labeled with PE. The
staining were done at 4.degree. C. without any washes. Ordinate:
mean channel.
[0127] FIG. 2. FACS analysis of Colo205 cells stained with
CD80-C215Fab, CD80-C215Fab-SEAm5.sup.7 or C215Fab-SEA followed by
incubation with anti-mouse kappa chain mAb labeled with PE. The
staining were done at 4.degree. C. with three washes between each
staining step. Ordinate: mean channel. Abscissa: Effector (E) to
Target cell (T) ratio.
[0128] FIG. 3. Proliferation. T cells were incubated with Colo205
cells and 4 uM C242Fab-SEA and varying amounts of CD80-C215Fab for
4 days after which the incorporated .sup.3H-thymidine was counted.
The activity obtained without any CD80-C215Fab was 20039
cpm+/-1750.
[0129] FIG. 4 IL-2 production. T cells were incubated with Colo205
cells and 4 uM C242Fab-SEA and varying amounts of CD80-C215Fab for
4 days after which the supernatant was harvested and the amount
IL-2 was determined. The amount IL-2 obtained without any
CD80-C215Fab was 2849 pg/ml
[0130] FIG. 5. Proliferation. T cells were incubated with Colo205
cells and varying amounts of CD80-C215Fab-SEAm57 or C215Fab-SEAm23
for 4 days after which the incorporated .sup.3H-thymidine was
counted.
[0131] FIG. 6. IL-2 production. T cells were incubated with Colo205
cells and varying amounts of CD80-C215Fab-SEAm57 or C215Fab-SEAm23
for 4 days after which the supernatants were harvested and the IL-2
contend determined.
[0132] FIG. 7. Proliferative capacity of human blood T cells
incubated for 7 days with the indicated proteins and irradiated,
transfected CHO cells (for presentation of SEA). Ordinate:
.sup.3H-Thymidine incorporation (cpm)
[0133] FIG. 8. Simultaneous administration of targeted SEA and IL2
leads to enhanced T cell activation in vivo. Cytotoxicity
(expressed as percentage) against SEA-coated MHC class II.sup.+
Raji cells of spleenocytes from mice treated with 1 or 3 injections
of C215FabSEA (FS), C215Fab-Q-hIL2 (FI), combination of the two
(FS+FI) or C215FabSEA-Q-hIL2 (FSI). Effector:target cell ratio was
30:1, and cytotoxicity was measured in a standard 4 hr .sup.51Cr
release assay
[0134] FIG. 9. Therapy of day 5 B16-C215 tumors in C57Bl/6 mice
with three injections (days 5, 6 and 7 after tumor inoculation) of
C215FabSEA (FS), C215Fab-Q-hIL2 (FI), C215FabSEA+C215Fab-Q-hIL2
(FS+FI) or C215Fab-Q-hIL2 (FSI). Equimolar amounts of FabSEA and
Fab-Q-hIL2 were used. Abscissa: amount of injected protein.
Ordinate: tumor reduction expressed in percentage.
[0135] FIG. 10. Increased tumor infiltration of CD25 T cells
following C215Fab-SEA (FS), C215Fab-Q-IL2 (FI) or C215FabSEA-Q-IL2
treatment. CD25-positive cells infiltrating the lung of mice
carrying established B16-GA733 lung tumors were estimated
byimmunohistochemistry. Ordinate: percentage of stained area.
Abscissa: 1=PBS; 2=first injection; 3=second injection; 4=third
injection; 5=fourth injection.
[0136] FIG. 11. Sustained levels of Interferon y following up to
four injections, once daily (37 mg/injection), of C215FabSEA-Q-hIL2
(FSI) (37 mg/). Blood was collected four hours after the last
injection and the The interferon .gamma. content (in the ordinate)
was determined by ELISA measurement using recombinant murine
Interferon .gamma. (Pharmingen) as standard. A similar experiment
was performed with eguimolar amounts (30 mg/injection) of
C215FabSEA (FS).
[0137] FIG. 12. Cytotoxicity (expressed as percentage in the
ordinate) against SEA-coated MHC class II Raji cells of splenocytes
from mice treated with PBS or 1, 4 or 6 injections of
C215FabSEA.sub.D227A-Q-hIL2 (FSm9-IL2). Cytotoxicity was measured
in a standard 4 hr Cr release assay. PBS is used as negative
control.
[0138] FIG. 13. Therapy of day 3 B16-C215 tumors in C57 Bl/6 mice
following treatment with 8 injections C215FabSEA.sub.D227A(=FSm9)
or C215FabSEA.sub.D227A-Q-hIL2 (.dbd.FSm9-IL2). Treatment given
daily for 8 consecutive days. On day 21 animals were sacrificed,
and lung metastases counted. Ordinate: tumor reduction expressed in
percentage.
[0139] FIG. 14. Therapy of day 3 B16-C215 tumors in Vb3 TCR
transgenic mice following treatment with 8 injections of
C215FabSEA.sub.D227A-Q-hIL2- .sub.F42A(=FSm9-IL2(F42A)),
C215FabSEA.sub.D227A-Q-hIL2(.dbd.FSm9-IL2), or
C.sup.215FabSEA.sub.D227A(=FSm9-IL2). Treatment was given daily for
8 consecutive days. On day 21 animals were sacrificed, and lung
metastases counted. Ordinate: tumor reduction expressed in
percentage.
[0140] FIG. 15. Therapy of day 3 B16-C215 tumors in Vb3 TCR
transgenic mice following treatment with 8 injections of
C215FabSEA.sub.D227A-Q-hIL2- .sub.F42A (F42A),
C215FabSEA.sub.D227A-Q-hIL2.sub.F42F F42K or
C215FabSEA.sub.D227A-Q-hIL2.sub.F42A/D20S (F42A/D20S). Treatment
was given daily for 8 consecutive days. On day 21 animals were
sacrificed, and lung metastases counted. Ordinate: tumor reduction
expressed in percentage.
EXAMPLE 1
Biological Activity of CD80-C215FAB Fusion Proteins for Use in
Costimulation of Tumor Therapy with Sag Targeted FAB
[0141] Summary: CD80-C215Fab and CD80-C215Fab-SEAm57 fusion
proteins have been constructed to contain the extracellular domain
of human CD80. The fusion proteins were produced in mammalian
cells, and tested for binding to CD28 using CHO cell transfectants
and C215 antigen using Colo205 cells. Both fusion proteins bound to
CD28 and C215 antigen, the later binding being 100-fold reduced
compared to C215Fab-StA. Since CD80 is linked to the N-terminal
part of the L-chain it is possible that the CD80 moiety interferes
with the C215Fab binding. Both fusion proteins costimulate SAG
activated human T cells showing that the soluble CD80 retains its
biological function.
[0142] Material and Methods
[0143] Recombinant DNA techniques and enzymes: Plasmid DNA
preparations and other operations were performed essentially
according to Sambrook et al. (Sambrook et al 1989). E. coli HB101
(Boyer et al 1969) was used as the host strain. Restriction
endonucleases and the Klenow fragment of DNA polymerase I were
obtained from Boehringer Mannheim or New England Biolabs, and used
according to the suppliers recommendations. Taq-polymerase was
obtained from Perkin Elmer. cDNA was made from the total RNA using
the GeneAmp RNA PCR kit (Perkin-Elmer). Oligonucleotides were
synthesized on a Gene Assembler (Pharmacia Biotech AB) or an ABI
392 DNA/RNA synthesizer (Applied Biosystems), and purified by
reversed phase chromatography on the FPLC system (Pharmacia
Biotech). Sequencing was done according to the dideoxy
chain-termination principle (Sanger et al 1977) using Applied
Biosystems Taq DyeDeoxy Termination Cycle Sequencing Kit and the
products separated and detected on a DNA sequencer ABI 373A
(Applied Biosystems). Bacteria harboring different plasmids were
selected on plates containing 2.times.YT and 15 g agar base per
liter, supplemented with 70 mg/L kanamycin, or 100 mg/L ampicillin.
The liquid broth was 2.times.YT (per liter: 10 g yeast extract
(Difco), 16 g tryptone (Difco) and 5 g NaCl).
2TABLE I (SEQ ID NO 1) LAKQ5 ATA TAA GCT TCC ACC ATG GGC CAC ACA
CGG AGG (SEQ ID NO 2) LAKQ7 ACG CAG ATC TTT AGT TAT CAG GAA AAT GCT
CTT GC (SEQ ID NO 3) LAKQ3O TCA AAG CTT CTC GAG CGC GCT GTT ATC AGG
AAA ATG CTC (SEQ ID NO 4) LAKQ37 CGC GCG TCA GGC TAA CGA ACT GCC
AGG CGC CCC GTC ACA GAG ACG A (SEQ ID NO 5) LAKQ38 AGC TTC GTC TCA
CGC GCG TTC TTC CTG TGA CGG GGC GCC TGG CAG TTC GTT AGC CTG ACG
(SEQ ID NO 6) LAKQ88 TGG TAC ACC ACA GAA GAC AGC TTG TAT GTA TG
(SEQ ID NO 7) LAKQ89 CAT ACA TAC AAG CTG TCT TCT GTG GTG TAC CA
(SEQ ID NO 8) LAKQ90 CGA ATA AGA AAG ACG TCA CTG TTC AGG AGT TGG
(SEQ ID NO 9) LAKQ91 CCA ACT CCT GAA CAG TGA CGT CTT TCT TAT TCG
(SEQ ID NO 10) LAKQ92 GAG ATA ATA AAG TTA TTA ACT CAG AAA ACA TG
(SEQ ID NO 11) LAKQ93 CAT GTT TTC TGA GTT AAT AAC TTT ATT ATC TC
(SEQ ID NO 12) LAKQ108 CGC GGA TCC GCG CGG CAC CAG GCC GCT GTT ATC
CGG AAA ATG CTC TTG C (SEQ ID NO 13) LAKQ117 CCG GAT AAC AGC GCG
CGT CAG CTA ACG AAC TCC AGG CGC CCC GTC ACA GGA AGAA CGC CCG CAG
GTC CAA CTG CA (SEQ ID NO 14) LAKQ118 GTT GGA CCT GCG GGC GTT GTT
CCT GTG ACG GGG CGC CTG GCA GTT CGT TAG CCT GAC GCG CGC TGT TAT
[0144] Plasmid Constructions
[0145] The genes encoding a Fab fragment containing the variable
domains of the murine antibody C215 were assembled as described
previously (Dohlsten et al 1994). Cloning and mutagenesis of the
staphylococcal enterotoxin (SEA) gene yielding the replacements
F47A and D227A has been described previously (Abrahmsn et al 1995).
Three other mutations were introduced in the SEA gene using the
primers LAKQ88-93 (all oligonucleotides used are compiled in Table
I) to obtain SEA mutant 57. LAKQ5 and LAKQ7 were used in RT-PCR to
introduce a Kozak box upstream, and to clone the gene encoding
signal peptide and the extracellular part of human CD80 from human
spleen total RNA. The nucleotide sequence was found to correspond
to the gene bank sequence of CD80 (Accession number M27533). Two
variants of the CD80 gene were made, having different cloning sites
at the 3'-end: in separate PCRs the primers LAKQ30 and LAKQ108 were
used to obtain a BssHII or a MroI site, respectively. A gene fusion
encoding a CD80 fused before the kappa chain by a Q-linker (Wooton
et al 1989), was constructed by inserting the DNA linker LAKQ37/38
BssHII-- Esp3I, between the relevant CD80 gene and a DsaI site
directly preceding the kappa gene. The plasmid pKGE987 was obtained
by inserting the gene fusion encoding CD80-(Q-linker)-kappa,
preceded by the CD80 signal peptide into a vector, which in
addition to a CMV promoter and a poly A tail contains a neomycin
gene to be used for selection of transformants. The last version of
the CD80 gene was used to construct a gene fusion where it precedes
the Fd-SEA mutant 57 gene fusion: a DNA fragment (LAKQ117/118)
encoding a Q-linker was inserted between a MroI site in the CD80
gene and a PstI site at codon 4 and 5 in the C215 VH gene. This
gene fusion was inserted in a second CMV promoter vector to yield
the plasmid pMB189, thus encoding the CD80 signal peptide and
extracellular portion followed by Fd and SEA mutant 57, connected
by the three residue spacer GGP. In the plasmid pKGE961 the Fd gene
has been inserted following a signal sequence (derived from another
murine VH gene). The plasmid pMB156 encodes the native kappa chain,
preceded by its native signal peptide, and contains the neomycin
gene.
[0146] Production
[0147] Hamster embryonic kidney 293 cells were transfected with
either pKGE961 and pKGE987, or with pMB156 and pMB189, to obtain
cell lines producing CD80-C215Fab, and CD80-C215Fab-SEAm57,
respectively. To obtain stable cell lines the selection medium was
DMEM without phenol red (Pharmacia no MS 0127) supplemented with
L-glutamine (GIBCO BRL no 25030-24), 10% bovine calf serum and
Geneticin 1 mg/ml. The production medium was DMEM without phenol
red supplemented with L-glutamin and 0.1% HSA (Pharmacia &
Upjohn AB, Sweden). Fusion proteins were purified from the
filtrated (Sartobran 0.65-0.4 um) culture media by affinity
chromatography on protein G Sepharose FF (Pharmacia Biotech AB),
followed by anti-CD80 affinity purification (immobilized anti-human
CD80 antibody; Camfolio L307.4) or ion-exchange chromatography on
SP Sepharose FF (Pharmacia Biotech).
[0148] Reagents. RPMII 1640 medium (Gibco, Middlesex, UK)
supplemented with 2 mM L-glutamin (Gibco, Middlesex, UK), 0.01 M
HEPES (Biological Industries, Israel), 1 mM NaHCO.sub.3 (Biochrom
KG, Berlin, Germany), 0.1 mg/ml gentamicin sulfate (Biological
Industries, Kibbutz Beit Haemek, Israel), 1 mM sodium pyruvate (JRH
Biosciences Industries, USA) and 10.degree. heat inactivated fetal
bovine serum (Gibco Middlesex, UK) was used as complete medium for
all cell cultures.
[0149] Antibodies. mAbs directed to human CD57 (HNK1) and CD56
(HB55) were obtained from the mAb producing hybridoma cells
(American Type Culture Collection, Rockville, Md.). Anti-mouse
kappa chain mAb labelled with PE was obtained from Becton Dickinson
(San Jose, Calif.)
[0150] Cells. Chinese hamster ovary (CHO) K1 cells were transfected
with human cDNA encoding the CD28 gene at Pharmacia and Upjohn,
Stockholm, Sweden. The transfectants were routinely analyzed for
CD28 expression and maintained by FACS sorting at similar antigen
expression levels. The human colon carcinoma cell line Colo205 was
obtained from ATCC. All cell lines were free of mycoplasma.
[0151] T lymphocyte proliferation assay. T cells were obtained from
human peripheral blood mononuclear cells (PBM) as previously
described (Lando et al 1996) by negative selection panning with
CD57, HLA-DR4, CD14 and CD56 mAbs. All tests on T cells were
performed with 0.1.times.10.sup.6 cells/well in 200 .mu.l volumes,
using flat-bottomed 96-well plates (Nunc, Roskilde, Denmark)
DNA-synthesis was studied after exposure of cultures to
[.sup.3H]-thymidine [.sup.3H]TdR (0.5mCi/well) as described earlier
(10).
[0152] Analysis by flow cytometry. Flow cytometric analysis and
sorting were performed according to standard setting on a
FACStar.sup.Plus flow cytometer (Becton Dickinson, Mountain View,
Calif.). Due to the low affinity of CD80 to CD28 staining of
CHO-CD28 cells with CD80-C215Fab fusion proteins were done omitting
washes of the cells.
[0153] Cytokine assay. The production of IL-2 was analyzed using a
IL-2 ELISA kit (huIL-2 Duoset, Genzyme).
[0154] Results.
[0155] Description of the CD80-Fab fusion proteins. The Fab moiety
is of murine IgG1/k isotype although it contains the variable
domains of the IgG2A/k monoclonal antibody C215 (Dohisten et al
1995). The tripeptide sequence GGP follows the inter-chain
disulphide forming cysteine in the CH1 domain. In the
CD80-C215Fab-SEAm57 triple fusion protein, this functions as a
spacer between Fd and a mutant of staphylococcal enterotoxin A
(SEA; Betley et al 1988), having five substitutions. The
replacements F47A and D227A were introduced to diminish affinity
for MHC class II (Abrahmsn et al 1995), and the replacements N102Q,
N149D and T218V were introduced to avoid fortuitous glycosylation
when produced in eukaryotic cells. These latter replacements were
selected with the aid of the X-ray structure (Sundstrom et al
1996). The final penta mutant was designated SEA mutant 57. Both
fusion proteins contain the extracellular domain of human CD80
(defined to end FPDN). The native CD80 signal peptide was used and
the mature protein found to start VIHV, as determined by amino acid
sequencing of purified CD80-C215Fab. A spacer of 18 amino acids
connects CD80 with the kappa chain in CD80-C215Fab, or the Fd
portion of the Fab fragment in the CD80-C215Fab-SEAm57 triple
fusion protein. The spacers resemble a Q-linker (Wooton et al 1996)
and have the sequences SARQANELPGAPSQEERA (SEQ ID NO 15) and
SARQANELPGAPSQEERP (SEQ ID NO 16), respectively.
[0156] Facs analysis: Binding of CD80-C21SFab fusion proteins to
CD28 positive cells and to C215 positive cells.
[0157] CD28 positive cells: CHO-CD28 cells were stained with
CD80-C215Fab, CD80-C215Fab-SEAm57 or C215Fab-SEA followed by
incubation with anti-mouse kappa chain mAb labeled with PE. The
staining were done at 4.degree. C. without any washes.
[0158] Both the CD80-C215Fab and the triple fusion protein bound to
CD28 expressed on the CHO cells in a dose dependent manner. No
staining was, as expected, seen with the control fusion protein
C215Fab-SEA.
[0159] C215 positive cells. The binding of the fusion proteins
against the C215 antigen was evaluated against Colo205 cells.
Colo205 cells were stained with CD80-C215Fab, CD80-C215Fab-SEAm57
or C215Fab-SEA followed by incubation with anti-mouse kappa chain
mAb labeled with PE. The staining were done at 4.degree. C. with
three washes between each staining step.
[0160] Results (FIGS. 1-2): Both CDB0-C215Fab and
CD80-C215Fab-SEAm57 bound to the C215 antigen positive Colo205
cells in a dose dependent manner. The binding was 50-100 fold lower
than that of C215Fab-SEA. This indicates that the introduction of
CD80 in the N-terminal part of the fusion protein might interfere
with the binding of the C215Fab part to the C215 antigen.
[0161] Dual fusions. Costimulation of superantigen activated T
cells.
[0162] To determine the biological activity of the fusion proteins
they were tested for costimulation of superantigen activated T
cells.
[0163] Proliferation: T cells were incubated with Colo205 cells and
4 uM C242Fab-SEA and varying amounts of CD80-C215Fab for 4 days
after which proliferation measured as incorporated
.sup.3H-thymidine was counted. The activity obtained without any
CD80-C215Fab was 20039 cpm+/-1750.
[0164] IL-2 production: T cells were incubated with Colo205 cells
and 4 uM C242Fab-SEA and varying amounts of CD80-C215Fab for 4 days
after which the supernatant was harvested and the amount IL-2 was
determined. The amount of IL-2 obtained without any CD80Fab-C215Fab
was 2849 pg/ml.
[0165] Results (FIGS. 3-4): CD80-C215Fab costimulated the
C242Fab-SEA induced activation of the T cells in a dose dependent
manner both seen as proliferation and IL-2 production.
[0166] Triple Fusions. Costimulation of Superantigen Activated T
Cells.
[0167] To test the biological activity of the triple fusion
protein, purified T cells were incubated with C215Fab-SEAm23 (m23
being the same SEA mutant affecting MHC class II binding that was
used in the triple fusion protein, i.e. Phe47Ala/Asp227Ala) or
CD80-C215Fab-SEAm57 presented on Colo205 cells. Proliferation: The
incorporated .sup.3H-thymidine was counted after 4 days. IL-2
production: The supernatants were harvested and the IL-2 content
determined after 4 days.
[0168] Results (FIGS. 5-6): The triple fusion protein induced T
cell activation and IL-2 production when presented on Colo205
cells. No such activity was seen with the C215Fab-SEAm23 indicating
the importance of costimulation by CD80 for activation. Due to the
lower binding affinity of the triple fusion protein (50-100.times.
lower than that of C21sFab-SEA, see FACS data), the actual amount
of C215Fab-SEAm23 bound to cells is likely to be substantially
higher than that of the triple fusion protein.
EXAMPLE 2
Targeted IL-2 Potentiates and Prolongs the Effects of FabSEA on T
Cell Activation and in Tumor Therapy
[0169] Methods and Materials:
[0170] Construction of IL2 expression plasmids. The IL2 cDNA was
cloned by RT-PCR using mRNA isolated from human peripheral blood
mononuclear cells (PBM) which had been stimulated with the
superantigen SEA for 24 hours. mRNA was isolated from
5.times.10.sup.6 cells using a mRNA Direct kit from Dynal, Oslo
according to the manufacturer's instructions. mRNA annealed to
oligo(dT).sub.26-coated magnetic beads was eluted by heating to
95.degree. C. Subsequently a PCR product was obtained by RT-PCR
taking advantage of the RT and DNA polymerase activities of Taq
polymerase. {fraction (1/10)} of the eluted mRNA was mixed with PCR
primers IL2-1 and IL2-2 and a standard PCR reaction performed (30
rounds). The PCR product was subjected to agarose gel
electrophoresis, the band excised from the gel and purified using
the Prep-a-gene kit (Bio-Rad) kit. Following digestion with EcoRI
and BamHI the fragment was cloned into EcoRI/BamHI-digested
pBluescript KS II. The insert was sequenced and confirmed to be
identical to the previously reported IL2 cDNA Taniguchi et al
1983). However, as a result of the PCR reaction a DNA segment
encoding the
Gly-Pro-Arg-Gln-Ala-Asn-Glu-Leu-Pro-Gly-Ala-Pro-Ser-Gln-Glu--
Glu-Arg (SEQ ID NO: 23) "Gly-Pro-Q-linker" had been added 5' to the
segment encoding mature human IL-2. Q-hIL2 was cloned into a
plasmid from a house collection (cut with RsrII-XbaI) as a
RsrII-NheI fragment. The resulting plasmid, pMS306, directs the
secretion of C21SFabSEA-Q-hIL2 to the periplasma of E. coli. The
linkers between the moieties were further optimized as follows. A
linker-encoding region, coding for
Pro-Ala-Ser-Gly-Gly-Gly-Gly-Ala-Gly-Gly-Pro (SEQ ID NO: 19)
(replacing the original Gly-Gly-Pro) was introduced between the
superantigen and the Fab moiety-encoding regions using
site-directed mutagenesis. Similarly, the linkers
Gly-Pro-Arg-Gln-Ser-Asn-Glu-Thr-Pro-Gly-Ser-Pro-Ser-Gln-Glu-G-
lu-Arg (SEQ ID NO: 20),
Gly-Pro-Arg-Gln-Ala-Lys-Thr-Leu-Pro-Gly-Ala-Pro-Se-
r-Gln-Thr-Thr-Arg (SEQ ID NO: 21) or
Gly-Pro-Thr-Glu-Ala-Asp-Glu-Leu-Pro-G-
ly-Ala-Pro-Ser-Glu-Glu-Glu-Thr (SEQ ID NO: 22) replaced the
original Q-linker between the IL-2 and the Fab moiety. For
combinations of dual fusion proteins, also constructs with IL-2
fused to the heavy or light chain, respectively, were compared.
[0171] Primers:
3 IL2-1: (SEQ ID NO 17) 5'-GCG GAT CCC GGT CCG CGT CAG GCT AAC GAA
CTG CCA GGA GCT CCG TCT CAG GAA GAG CGT GCA CCT AC TTC AAG TTC TAC
AAA G-3' IL2-2: (SEQ ID NO 18) 5'-CCG AAT TCG CTA GCT TAT CAA GTT
AGT GTT GAG ATG AT-3'
[0172] Expression of the fusion proteins in fermenter. Fusion
proteins were expressed in the E. coli K-12 strain UL 635 (xyl-7,
ara-14, T4.sup.R, deltaompT) using a plasmid with a kanamycin
resistance gene and lacUV5-promoter. Bacteria from frozen stock
were incubated at 25.degree. C. for approximately 21 h in shaker
flasks containing (g/l) (NH.sub.4).sub.2SO.sub.4, 2.5;
KH.sub.2PO.sub.4, 4.45; K.sub.2HPO.sub.4, 11.85; sodium citrate,
0.5; MgSO.sub.4.7H.sub.2O, 1; glucose monohydrate, 11, 0.11 mM
kanamycin and 1 ml/l trace element solution. (Forsberg et al.,
1989), however without Na.sub.2MoO.sub.4.2H.sub.2O. The cells were
grown to an OD.sub.600 of 1-2 and 450 ml culture medium was used to
inoculate a fermenter (Chemap, Switzerland) to a final volume of 5
l. The fermenter medium contained (g/l) (NH.sub.4).sub.2SO.sub.4,
2.5; KH.sub.2PO.sub.4, 9; K.sub.2HPO.sub.4, 6; sodium citrate, 0.5;
glucose monohydrate, 22; MgSO.sub.4.7H.sub.2O, 1; 0.11 mM
kanamycin; 1 ml adecanol (Asahi Denka Kogyo K.K, Japan) and 1 ml/l
trace element solution. The pH was kept at 7.0 by titration with
25% ammonia, the temperature was 25.degree. C. and aeration with
atmospheric air 5 l/min. The partial pressure of dissolved O.sub.2
was controlled to 30% by increasing agitation from 300 to 1000 rpm
during batch phase and regulating the feed of 60% (w/v) glucose
during fed batch phase. Product formation was induced at an
OD.sub.600 of 50 by adding 0.1 mM isopropyl
.beta.-D-thiogalactopyranoside, IPTG. After fermentation, the cells
were removed by centrifugation at 8000.times.g for 40 min at 40C.
The clarified medium was either analysed and purified directly or
stored at -20.degree. C.
[0173] Purification of fusion proteins. DNA present in the
clarified medium was removed using precipitation with 0.19%
polyethylenimine and 0.2 M NaCl during 30 min (Atkinson and Jack,
1973). After centrifugation as above, the supernatant was collected
and the NaCl concentration adjusted to 0.5 M. This medium was
applied to a Protein G Sepharose column (Pharmacia Biotech AB,
Uppsala, Sweden) equilibrated with 10 mM sodium phosphate, 150 mM
NaCl, pH 7.4 containing 0.05% Tween 80, PBST. The column was then
washed with 5 column volumes PBST and bound protein was eluted with
0.1 M acetic acid, 0.02% Tween 80 pH 3.2. The pH of the sample was
adjusted to 5.0 using 1 M Tris-HCl, pH 8.0, and applied to an SP
Sepharose HP column (Pharmacia Biotech) equilibrated with 50 mM
ammonium acetate, 0.02% Tween 80. The column was then washed with 2
column volumes equilibration buffer and the fusion protein eluted
using a linear gradient from 50 to 500 mM ammonium acetate over 10
column volumes. For the C215Fab-IL2 fusion proteins, a pH of 6.0
was utilised while for the C215Fab-SEA-IL2 triple fusion proteins,
the pH was 5.7 during the separation. The fusion proteins were
filtered through a 0.22 um filter and stored at -70.degree. C. If
more dilute, the eluate is concentrated to a final concentration of
0.5-1 mg/ml using Centricon 30 (Amicon) according to the
manufacturer's instructions
[0174] Cytotoxicity assays. MHC class II dependent and independent
cytotoxicity assays were performed as previously described
(Dohlsten et al., 1990). Briefly for MHC class II dependent assays
.sup.51Cr-labelled Raji cells (2500 cells per well in a final
volume of 200 ul) were mixed with an SEA-dependent effector cell
line generated by incubation of human PBM in the presence of low
levels of recombinant hIL-2. Effector:Target cell ratio was 30:1.
For MHC classII-independent assays .sup.51Cr-labelled C215+colo205
cells (2500) were incubated with SEA-dependent effector cells at an
E:T ratio of 45:1. .sup.51Cr released into the medium was
determined after 4 hours of incubation by scintillation
counting.
[0175] In vitro co-stimulation assay on purified human T cells.
Naive human T cells were purified essentially as described by Lando
et al. (1993). Briefly, naive human T cells were purified from
human blood PBM by Ficoll gradient centrifugation, followed by
separation over gelatine columns, and lastly negative selection by
panning in petri dishes containing HNK1 and HLA-DR mAbs.
Proliferation experiments using naive human T cells was performed
essentially as described by Lando et al. (1996). Briefly, naive T
cells (100.000 cells per well) were combined with irradiated CHO
transtectants (-10.000 cells per well) in a total volume of 200-ul
RPMI-1640 with supplements (Lando et al 1993). For experiments with
IL-2 containing fusion proteins, cells were incubated for 7 days in
the presence of 1 nM of the indicated substances. On the final day
of the experiment the cells were pulsed with .sup.3H-Thymidine to
measure incorporation into DNA of dividing cells.
[0176] Therapy of B16-C215 tumors. Therapy was performed
essentially as described previously (Dohlsten et al 1994; Hansson
et al 1997). On day 0 C57Bl/6 mice were injected i.v. into the tail
vein with 75000-150000 syngeneic B16-F10 melanoma cells. These B16
cells were expressing the human GA-733 antigen recognized by the
C215 mAb. On day 1, 3 or 5 therapy with C215Fab proteins were
initiated. On day 21 the experiment was terminated, at which time
the lungs were removed and disseminated lung tumors counted.
[0177] Immunohistochemistry was performed on lungs of animals
carrying Day 18 B16-C215 tumors essentially as described previously
(Dohlsten et al 1995). Samples were taken out 4 hours after the
final injection. Stained area was determined manually.
[0178] Immunopharmacology was performed essentially as described
(Rosendahl et al 1996). Spleens from C57 Bl/6 mice having received
1 or 3 injections, once daily, were removed 48 hours after the
final injection and SEA-dependent cytotoxicity determined, using
Raji cells as targets in a standard .sup.51Cr release assay as
described above. E:T ratio was 100:1.
[0179] Results and Discussion:
[0180] Production and purification of IL2-containing fusion
proteins. An E. coli expression vector encoding a C215FabSEA-Q-hIL2
triple fusion protein was constructed. This vector encodes the two
subunits of the triple fusion protein on a bi-cistronic mRNA
transcribed from the LacUV5 promotor. Each f the two subunits are
preceded by a signal peptide directing export to the periplasmic
space of E. coli. The first subunit is VH of the C215Fab followed
by a Gly-Gly-Pro linker (VH-C.sub.Hl-Gly-Gly-Pro-SEA). The other
subunit is VK of C21SFab followed by Ck of the C242Fab, which is
linked to human IL2 by a Gly-Pro-Q-linker (Vk-Ck-Gly-Pro-Q-hIL2).
The Gly-Pro-Q-linker sequence (SEQ ID NO: 23) is a slightly
modified version of a natural linker found in the OmpR E. coli
protein (Wootton et al 1989). See materials and methods for more
complete information. Also a C215Fab-Q-hIL2 fusion protein was
produced. It is identical to C215FabSEA-Q-hIL2 except that the
Gly-Gly-Pro-SEA moiety of the protein has been removed. The
corresponding DNA sequence in the expression vector is deleted
accordingly. Mutated derivatives of C215FabSEA-Q-hIL2 were
generated by PCR-mediated site-directed mutagenesis by standard
methods to produce a number of proteins such as
C215FabSEAD227A-Q-hIL2 and C215FabSEAD227A-Q-hIL2F42A. In later
variants of the triple fusion protein the intersubunit cystine is
replaced by two serine residues. This alteration does not affect
the biological activity.
[0181] Plasmids encoding IL2-containing proteins were transformed
into the E. coli production strain UL635, and fermentation
subsequently performed. Fusion proteins were purified from the
culture medium using protein G affinity chromatography. Degraded
variants of the fusion proteins were removed using ion exchange
chromatography. The products obtained were at least 90% full-length
fusion protein, as determined by SDS-PAGE (material and methods).
Using the optimal design of the fusion protein, up to 130 mg/l
fusion protein is obtained in the growth medium and typically 70 mg
triple fusion protein was obtained from 1 litre medium.
[0182] Functional characterization of IL2-containing Fab fusion
proteins. The ability of IL2-containing fusion proteins such as
C215FabSEA-Q-hIL2 and C215Fab-Q-hIL2 fusion proteins to induce
proliferation of the IL-2 dependent murine cell line CTLL-2 was
essentially similar to that of recombinant human IL2 on a molar
basis (data not shown) Moreover, antigen-binding and SEA activity
of C215FabSEA-Q-hIL2 and C215FabSEA were found to be
indistinguishable in a number of assays, suggesting that there were
no adverse effects of introducing IL2 into the molecule. These
assays included ability to induce MHC classII-independent killing
of the C215.sup.+ human colon cancer cell line colo205 by an
SEA-reactive T cell effector cell line in a 4 hour
.sup.51Cr-release assay. Also MHC class II-dependent killing of MHC
classII.sup.+ Raji (rat lymphoma) cells by SEA-reactive effector T
cells proceed with similar efficiencies (data not shown). More
direct evidence for uncompromised C215 antigen and MHC class II
binding was obtained by FACS analysis. The dose dependence of
C215FabSEA and C215FabSEA-Q-hIL2 binding to Raji cells was found to
be similar on a molar basis (data not shown). Also dose-dependent
binding of C215FabSEA-Q-hIL2 and C215Fab-Q-hIL2 to colo205 cells
was found to be similar to that observed for C215FabSEA (data not
shown), indicating that antigen-binding was uncompromised in the
IL2-containing fusion proteins.
[0183] IL2-dependent proliferation induced by FabSEA. Resting human
T cells require both a signal 1 and a signal 2 to trigger optimal T
cell proliferation and activation (Schwartz, 1990). Superantigens
can deliver signal 1, if presented on a cell. IL-2 being the major
downstream effector of B7/CD28 signalling is expected to give
signal 2.
[0184] Here we show using resting T cells purified from human blood
that a C215FabSEA-Q-hIL2 triple fusion protein or C215FabSEA in
combination with C215FAb-Q-hIL2 or recombinant human IL-2 does
indeed induce T cell proliferation in vitro (FIG. 7)
[0185] Resting human T cells were incubated with targeted SEA
(C215FabSEA or C215FabSEA-Q-hIL2) in the presence of IL2 (in the
form of C215Fab-Q-hIL2, C215FabSEA-Q-hIL2 or recombinant human
IL-2). SEA was presented on irradiated CHO cells transfected with
C215 antigen (via the Fab part), MHC classII/Dr which binds SEA.
Untransfected CHO cells served as control. After 7 days of
incubating T cells with CHO transfectants and the substances in
question proliferation was measured by incorporation of H-Thymidine
into DNA.
[0186] C215FabSEA-Q-hIL2 induced the proliferation of human T cells
when presented on CHO cells transfected with the human C215 antigen
(CH0--C215), which the Fab is directed against (FIG. 7). Likewise,
proliferation was induced when the protein was presented on CHO-Dr
(human MHC class II), whereas no proliferation was observed in the
presence of untransfected CHO cells (CHO) or in the absence of CHO
cells (R10). C215FabSEA or C215Fab-Q-hIL2 did not induce any
significant proliferation by themselves when presented on CHO
cells, indicating that both SEA and IL-2 are indeed necessary for
induction of proliferation. This was confirmed, as the combination
of C215FabSEA and C215Fab-Q-hIL2 or C215FabSEA and recombinant
human IL2 induced a qualitatively similar effect to the one induced
by C215FabSEA-Q-hIL2. This also shows that in this assay IL-2 is
necessary but, unlike SEA, it does not need not be cell bound.
[0187] C215FabSEA-Q-hIL2 as well as the combination of C215FabSEA
and C215Fab-Q-hIL2 causes enhanced and sustained. T cell activation
and improved tumor infiltration in vivo. Both
C215Fab-Q-hIL2+C215FabSEA, and C215FabSEA-Q-hIL2 were much more
potent inducers of T cell activation than C215FabSEA as measured by
the ability to induce SEA-dependent killing of target cells (FIG.
8). Briefly, mice received 1 or 3 injections administered daily of
the indicated proteins. Two days after the last injection spleens
from treated mice were taken out and cytotoxic activity against
SEA-coated Raji cells determined in a standard 4 hr .sup.51Cr
release assay.
[0188] Both C215FabSEA-Q-hIL2 and C215FabSEA/C215Fab-Q-hIL2 induced
not only enhanced but also sustained T cell activation. Levels of
serum cytokines such as IFNgamma., which dips drastically after the
fourth injection of C215FabSEA stays at a very high level even
after the fourth injection of C215FabSEA-Q-hIL2 (data not shown).
In general, IFNgamma and TNFalpha levels were much higher (up to
10.times.) higher than in the case of C215FabSEA.
[0189] Improved therapy of established B16-C215 tumors with
C215FabSEA and C215Fab-Q-hIL2 combination treatment. The effects of
IL2-potentiation of FabSag tumor therapy was investigated in the
murine Bl/6 melanoma model (FIG. 9). In the shown example 8-12 week
old C57 Bl/6 female mice were inoculated on day 0 with 150.000 B16
cells transfected with the human tumor antigen GA-733, which the
C215 mAb is directed against (Bl6-C215 in the following). The
indicated substances were injected on days 5, 6 and 7. Each
datapoint corresponds to 7 animals. On day 21 the animals were
sacrificed, and the number of melanin-pigmented B16 tumors
colonizing the lung counted. Combination of C215FabSEA and
C215Fab-Q-hIL2 in several experiments were shown to induce better
therapy than C215FabSEA as compared to a non-treated control (FIG.
9). In the indicated experiment, C215FabSEA/C215Fab-Q-hIL2
combination treatment gave better therapeutic effect than the
C215FabSEA-Q-hIL2 triple fusion protein.
[0190] Subsequent immunohistochemistry studies revealed that
C215FabSEA alone or combination of C215FabSEA and C215Fab-Q-h-IL2
gave rise to similar numbers of B16-C215 tumor-infiltrating CD4 and
CD8 T cells. Interestingly, however, the number of CD25 (IL2Ra)
positive cells--a good marker for T cell activation--dramatically
increased between the 3.sup.rd and 4.sup.th injection of
C215FabSEA/C215Fab-Q-hIL2 (FIG. 10). In contrast, it decreased in
the case of C215FabSEA indicating beginning anergy. The quality of
infiltrating T-cells thus seems to be higher in the case of
combination treatment than C215FabSEA alone, which may help to
explain the improved therapeutic effect. Likewise, serum cytokines
such as Interferon .gamma. produced secondary to T cell activation
de crease markedly after the fourth injection of FabSEA (FIG. 11).
With a FabSEA-Q-hIL2 triple fusion protein, however, the Interferon
levels stay at a high level even after the fourth injection. This
observation provides an additional indication that including IL-2
in the construct can counteract Fab-SEA induced T cell anergy.
[0191] Improved therapy of B16-C215 tumors with
C.sup.21.sup.5FabSEAD227A-- Q-hIL2. Superantigens are much more
toxic in humans than in mice, in part because the affinity for MHC
class II in humans is considerably higher (Hansson et al 1997).
Systemic toxicity of FabSEA proteins is thus expected to be the
major limitation for therapy in humans. One way to increase local
activation in the tumor (Fab dependent) versus systemic
immune-activation (SEA-MHC classII dependent) would be to decrease
the affinity of SEA for MHC classII. Based on the crystal structure
of SEA we made a mutant of C215FabSEA, C215FabSEA.sub.D227A, with
100-fold reduced affinity for MHC class II. Unlike wt C215Fab-SEA
it does not have the capacity to cross-link MHC classII molecules,
which is believed to be a major reason for SEA-mediated systemic
toxicity (Hansson et al 1997).
[0192] The window between efficient therapy and toxicity in
treatment of day 1 B16-C215 tumors in Vb3 TCR transgenic mice was
at least 50-fold wider for the C215FabSEA.sub.D227A mutant as
compared to C215FabSEA (Hansson et al 1997). In accordance with
this observation, immunohistochemistry revealed that at doses of
the two proteins resulting in similar therapy, comparable immune
activation in the tumor was observed, whereas much less systemic
immune activation was observed in the spleen with C215FabSEAD227A
(Hansson et al 1997). Moreover, pharmacokinetic studies in rabbits
showed a dramatic reduction in the targeting of
C215FabSEA.sub.D227A to the spleen and other lymphoid organs, where
most of the MHC classII.sup.+ cells are located, as compared to
C215FabSEA (data not shown).
[0193] For clinical use a Fab-SEA-IL2 triple fusion protein is
expected to contain a mutated superantigen. We therefore made a
C215FabSEA.sub.D227A-Q-hIL2 triple fusion protein. The produced
protein was able to induce the proliferation of resting human T
cells (data not shown)--and induced sustained SEA-dependent CTL
activity in mice for up to 6 injections (FIG. 12). In contrast,
background CTL activity was observed with C215FabSEAD227A
(<10%--data not shown) and C215Fab-Q-hIL2 (<15%--data not
shown). Therapy of established (day 3) B16-GA733 tumors in normal
C57 Bl/6 mice with 8 injections of this protein administered daily
gave as good therapy as an optimal dose of C215FabSEA (FIG. 13) In
this system C215FabSEA.sub.D227A, which is designed for optimal
activity in humans not mice, does only have minor effects (FIG.
13). At the highest dose, some toxicity of C215FabSEAD227A-Q-hIL2
was encountered, however. In contrast, several therapy experiments
indicate that combination of C215FabSEA.sub.D227A and
C215Fab-Q-hIL2 (8 injections) does also lead to substantially
improved therapy of day 3 B16-GA733 tumors in Vb3 TCR transgenic
mice when compared to C215FabSEA.sub.D227A alone (data not shown)
In Vb3 TCR transgenic mice >90% of T cells react with SEA, as
opposed to 10-20% in normal mice. Interestingly, in rabbits
repeated injections (up to 8) of C215FabSEA.sub.D227A-Q-hIL2 (0/4
rabbits dead after repaeated injections at 20 .mu.g/kg) does not
appear to be more toxic than C215FabSEA.sub.D227A without IL-2 (0/4
rabbits dead after repaeated injections at 20 .mu.g/kg; 4/4 dead at
20 .mu.g/kg) and much less toxic than C215FabSEA (1/4 rabbits dead
after repaeated injections at 1 .mu.g/kg). At the same time,
sustained immune activation (lymphocyte rebound effect) is observed
only after treatment with C215FabSEA.sub.D227A-Q-hIL2 but not with
C215FabSEA.sub.D227A or C215FabSEA--indicating that indeed
inclusion of IL-2 helps to counteract FabSEA-induced T cell anergy
(data not shown).
[0194] Therapy of B16-C215 tumors with IL-2 mutated
C215FabSEA.sub.D227A-Q-hIL2 proteins. The greater toxicity, and in
the case of C215FabSEA-Q-hIL2 lower therapeutic efficacy, of
IL2-containing triple fusion proteins may in part be due to
"retargeting" of the SEA activity to lymphoid tissues. This
retargeting effect is ascribed to the high affinity of IL2 (Kd=10
pM) for its receptor, which is mainly found on T cells in the
spleen and other lymphoid tissues. To address this question we have
made derivatives of C215FabSEA.sub.D227A-Q-hIL2 in which the IL-2
moiety is mutated in order to reduce the affinity for IL2R+cells
and thus systemic activation. In principle the same approach that
was used to improve the therapeutic window for the
C215FabSEA.sub.D227A protein.
[0195] Interaction between IL-2 and its high-affinity receptor is
very well studied. The receptor consists of three subunits a, b and
g. Cross-linking of b and g is required for biological activity,
whereas the a subunit is required for optimal binding. Our strategy
is to reduce affinity of IL-2 for its high-affinity receptor (abg)
by eliminating a-subunit binding, and subsequently reduce, but not
eliminate, b and g-subunit binding as necessary.
[0196] Three such mutants of C215FabSEA.sub.D227A-Q-hIL2 were
designed to eliminate IL2Ra-binding (F42A, F42K) and in addition
impair IL2Rb binding (F42A/D20S). These proteins have 100-(F42A),
1000-(F42K) and 3000-fold (F42A/D20S) reduced activity,
respectively, in inducing proliferation of the IL-2 dependent
murine cell-line CTLL-2. Experiments are in progress to determine
in more detail the properties of these proteins, in particular with
respect to affinity and on-rates for binding to activated human T
cells and the IL2 receptor. Initial therapy experiments indicate
that such a strategy is promising (FIG. 14; FIG. 15). In the shown
examples, 8 injections were given to Vb3 TCR transgenic mice (where
>90% of T cells are activated by SEA) carrying day 3 B16-GA733
tumors. Although some toxicity was still observed at the highest
dose, it came later when C215FabSEA.sub.D227A-Q-hIL2.sub.F42A or
C215FabSEA.sub.D227A-Q-hIL2.sub.F42K (8.sup.th injection) rather
than C215FabSEA.sub.D227A-Q-hIL2 was used (6.sup.th injection) and
treatment at the highest dose consistently leads to more than 90%
tumor reduction when compared to a PBS-treated control (FIG. 14).
We are currently exploring this highly promising approach by
introducing additional mutations into the SEA and IL2 parts to
further improve the therapy to toxicity window.
[0197] Moreover, work is in progress to make C215FabSEA-Q-hIL2
triple fusion proteins comprising a Thr51Pro mutation in the IL-2
part (Chang et al 1996). This mutation may serve to block
IL2R-mediated internalization of the fusion protein without
reducing IL-2 bioactivity. This is likely to be important because
it will reduce the removal of rusion protein by this pathway, and
thus increase local concentration and efficacy of the drug.
Thr51Pro mutated proteins may contain further mutations in the SEA
and IL-2 parts to reduce affinity for MHC classII and the IL2
receptor, respectively.
EXAMPLE 3
Experiments for Verifying Effects of (Sag,IM)-Conjugates. Target
Cells IM-Receptor Positive or MHC Class II Positive Cells
[0198] A Sag-IM molecule may bind to cells expressing MHC class II,
thus facilitating SDCC. Alternatively, cells expressing the
receptor for IM may be targeted. It is therefore possible that a
Sag-IM molecule could be useful for inducing the killing of
undesired cells expressing the receptor for IM.
[0199] A C215FabSEA-hIL2 triple fusion protein can be considered a
Sag-IM molecule in the event that neither effector nor target cell
express the antigen recognized by the C215Fab. Effector cells would
be T cells of the right V.beta. subtype. Target cells could e.g. be
abberrant hemapoietic cells expressing the IL2 receptor such as
those present in certain leukemias or lymphomas.
[0200] Exp. A: In vitro proof-of-concept: Effector T cells (human T
cells repeatedly stimulated with SEA) and target cells (f.ex. human
"Raji" B cell lymphoma cells) will be incubated with C215FabSEA-IL2
triple fusion protein mutated to reduce binding to MHC class I.
This is the standard set up for SDCC assays (effector T cells, Raji
cells, experimental substance). We have actually found that a
C215FabSEA.sub.D227A-hIL2 protein with severely reduced affinity
for MHC classII has approximately 10-fold higher potency than
C215FabSEA.sub.D227A in killing of Raji cells. An obvious
explanation for the increased potency is that the triple fusion
protein is presented on IL2 receptors expressed on Raji cells.
[0201] Exp. B: In vivo proof-of-concept: Murine lymphoma models
(such as the RBL-5 lymphoma in C57 Bl/6 mice) are well established
(Hoglund et al J Exp Med 168, 1469-1474). Targeting of the IL-2
receptor or another IM-R with a Sag-IM protein in such models could
provide proof-of-concept for targeting IM-receptor positive cells
in vivo.
[0202] Sag-IM targeting of IM-R positve cells is complicated by the
fact that both effector and target cells frequently express the
receptor for IM. Nevertheless, under certain circumstances this
mode of therapy may be efficacious. Factors such as density of IM-R
expression on target cells, number and location of target cells,
etc. is likely to play a role. It should also be emphasized that
e.g. the IL2R alpha is only up-regulated upon T cell
activation--there might thus be a temporal window during which IM-R
is expressed at high abundancy on target cells, but not on the
effector cells.
[0203] Sag-IM fusion proteins in which Sag stands for a wild-type
superantigen mutated to a reduced affinity for MHC Class II may
similarly be used.
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Sequence CWU 1
1
23 1 33 DNA ARTIFICIAL SEQUENCE DNA primer for use in RT-PCR. 1
atataagctt ccaccatggg ccacacacgg agg 33 2 35 DNA ARTIFICIAL
SEQUENCE DNA primer for use in RT-PCR. 2 acgcagatct ttagttatca
ggaaaatgct cttgc 35 3 39 DNA ARTIFICIAL SEQUENCE DNA primer for use
in RT-PCR. 3 tcaaagcttc tcgagcgcgc tgttatcagg aaaatgctc 39 4 46 DNA
ARTIFICIAL SEQUENCE DNA primer for use in RT-PCR. 4 cgcgcgtcag
gctaacgaac tgccaggcgc cccgtcacag agacga 46 5 60 DNA ARTIFICIAL
SEQUENCE DNA primer for use in RT-PCR. 5 agcttcgtct cacgcgcgtt
cttcctgtga cggggcgcct ggcagttcgt tagcctgacg 60 6 32 DNA ARTIFICIAL
SEQUENCE DNA primer for use in RT-PCR. 6 tggtacacca cagaagacag
cttgtatgta tg 32 7 32 DNA ARTIFICIAL SEQUENCE DNA primer for use in
RT-PCR. 7 catacataca agctgtcttc tgtggtgtac ca 32 8 33 DNA
ARTIFICIAL SEQUENCE DNA primer for use in RT-PCR. 8 cgaataagaa
agacgtcact gttcaggagt tgg 33 9 33 DNA ARTIFICIAL SEQUENCE DNA
primer for use in RT-PCR. 9 ccaactcctg aacagtgacg tctttcttat tcg 33
10 32 DNA ARTIFICIAL SEQUENCE DNA primer for use in RT-PCR. 10
gagataataa agttattaac tcagaaaaca tg 32 11 32 DNA ARTIFICIAL
SEQUENCE DNA primer for use in RT-PCR. 11 catgttttct gagttaataa
ctttattatc tc 32 12 49 DNA ARTIFICIAL SEQUENCE DNA primer for use
in RT-PCR. 12 cgcggatccg cgcggcacca ggccgctgtt atccggaaaa tgctcttgc
49 13 77 DNA ARTIFICIAL SEQUENCE DNA primer for use in RT-PCR. 13
ccggataaca gcgcgcgtca ggctaacgaa ctcccaggcg ccccgtcaca ggaagaacgc
60 ccgcaggtcc aactgca 77 14 69 DNA ARTIFICIAL SEQUENCE DNA primer
for use in RT-PCR. 14 gttggacctg cgggcgttct tcctgtgacg gggcgcctgg
cagttcgtta gcctgacgcg 60 cgctgttat 69 15 18 PRT ARTIFICIAL SEQUENCE
Designated Peptide to act as a spacer between the kappa chain or
the Fd portion of the Fab Fragment in a fusion protein. The spacer
resembles a Q-linker. 15 Ser Ala Arg Gln Ala Asn Glu Leu Pro Gly
Ala Pro Ser Gln Glu Glu 1 5 10 15 Arg Pro 16 18 PRT ARTIFICIAL
SEQUENCE Designated Peptide to act as a spacer between the kappa
chain or the Fd portion of the Fab Fragment in a fusion protein.
The spacer resembles a Q-linker. 16 Ser Ala Arg Gln Ala Asn Glu Leu
Pro Gly Ala Pro Ser Gln Glu Glu 1 5 10 15 Arg Pro 17 84 DNA
ARTIFICIAL SEQUENCE DNA Primer for use in RT-PCR 17 gcggatcccg
gtccgcgtca ggctaacgaa ctgccaggag ctccgtctca ggaagagcgt 60
gcacctactt caagttctac aaag 84 18 38 DNA ARTIFICIAL SEQUENCE DNA
Primer for use in RT-PCR 18 ccgaattcgc tagcttatca agttagtgtt
gagatgat 38 19 11 PRT ARTIFICIAL SEQUENCE Designated Peptide to act
as a Q-linker. 19 Pro Ala Ser Gly Gly Gly Gly Ala Gly Gly Pro 1 5
10 20 17 PRT ARTIFICIAL SEQUENCE Designated Peptide to act as a
Q-linker. 20 Gly Pro Arg Gln Ser Asn Glu Thr Pro Gly Ser Pro Ser
Gln Glu Glu 1 5 10 15 Arg 21 17 PRT ARTIFICIAL SEQUENCE Designated
Peptide to act as a Q-linker. 21 Gly Pro Arg Gln Ala Lys Thr Leu
Pro Gly Ala Pro Ser Gln Thr Thr 1 5 10 15 Arg 22 17 PRT ARTIFICIAL
SEQUENCE Designated Peptide to act as a Q-linker. 22 Gly Pro Thr
Gly Ala Asp Glu Leu Pro Gly Ala Pro Ser Glu Glu Glu 1 5 10 15 Thr
23 17 PRT ARTIFICIAL SEQUENCE Designated Peptide to act as a
Q-linker. 23 Gly Pro Arg Gln Ala Asn Glu Leu Pro Gly Ala Pro Ser
Gln Glu Glu 1 5 10 15 Arg
* * * * *