U.S. patent application number 11/193748 was filed with the patent office on 2005-11-24 for conjugate between a modified superantigen and a target-seeking compound and the use of the conjugate.
Invention is credited to Abrahmsen, Lars, Bjork, Per, Dohlsten, Mikael, Kalland, Terje.
Application Number | 20050260215 11/193748 |
Document ID | / |
Family ID | 20394684 |
Filed Date | 2005-11-24 |
United States Patent
Application |
20050260215 |
Kind Code |
A1 |
Abrahmsen, Lars ; et
al. |
November 24, 2005 |
Conjugate between a modified superantigen and a target-seeking
compound and the use of the conjugate
Abstract
Methods of lysing cells associated with a disease condition, and
method of treating a disease condition by lysing cells associated
with the condition, by administering to a mammal a therapeutically
effective amount of a conjugate comprising a biospecific affinity
counterpart and a peptide that is derived from Staphylococcal
enterotoxin A, that has the ability to bind to a V.beta. of a T
cell receptor and has been modified at amino acid position 47, 128,
187, 225 or 227, in order to have reduced ability to bind to MHC
class II antigens.
Inventors: |
Abrahmsen, Lars; (Bromma,
SE) ; Bjork, Per; (Helsingborg, SE) ;
Dohlsten, Mikael; (Lund, SE) ; Kalland, Terje;
(Loddekopinge, SE) |
Correspondence
Address: |
Fulbright & Jaworski L.L.P.
Fulbright Tower
Suite 5100
1301 McKinney
Houston
TX
77010-3095
US
|
Family ID: |
20394684 |
Appl. No.: |
11/193748 |
Filed: |
July 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11193748 |
Jul 29, 2005 |
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08765695 |
Jul 25, 1997 |
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08765695 |
Jul 25, 1997 |
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PCT/SE95/00681 |
Jun 7, 1995 |
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Current U.S.
Class: |
424/178.1 ;
530/391.1 |
Current CPC
Class: |
A61K 47/6863 20170801;
C07K 16/30 20130101; A61K 2039/505 20130101; C07K 2319/00 20130101;
A61P 35/00 20180101; C07K 19/00 20130101; C07K 2317/55 20130101;
Y10S 514/885 20130101; C07K 16/3046 20130101; A61K 38/00 20130101;
C07K 14/31 20130101; A61P 31/12 20180101; A61P 37/02 20180101; C07K
2319/30 20130101 |
Class at
Publication: |
424/178.1 ;
530/391.1 |
International
Class: |
A61K 039/395; C07K
016/46 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 1994 |
SE |
9402430-4 |
Claims
1-13. (canceled)
14. A method for the lysis of cells associated with a disease
condition in a mammal, which condition is associated with the
presence of cells expressing a specific cell surface structure,
comprising the step of: administering to the mammal a
therapeutically effective amount of a comprising a bacterial
superantigen and a biospecific affinity counterpart, wherein (1)
the biospecific affinity counterpart is capable of binding to a
specific cell surface structure, and (2) the superantigen comprises
a peptide that: i. contains an amino acid sequence that is derived
from Staphylococcal enterotoxin A, wherein said peptide has the
ability to bind to a V.beta. chain of a T cell receptor, and ii.
has been modified at an amino acid position 47, 128, 187, 225 or
227, in order to have reduced ability to bind to MHC class II
antigens.
15. The method of claim 14, wherein said amino acid modification is
a substitution to alanine.
16. The method of claim 14, wherein said amino acid modification is
a substitution that is not a conserved substitution of the amino
acid at that position in Staphylococcal enterotoxin A.
17. The method of claim 14, wherein the specific cell surface
structure is a cancer specific epitope.
18. The method of claim 17, wherein the specific cell surface
structure is the C242 epitope.
19. The method of claim 14, wherein the biospecific affinity
counterpart is an antibody or an antigen-binding fragment of an
antibody.
20. The method of claim 19, wherein the biospecific affinity
counterpart is an antigen-binding fragment of an antibody.
21. The method of claim 20, wherein the fragment of an antibody is
selected from the group consisting of Fab, F(ab).sub.2, Fv, and
single chain antibody.
22. The method of claim 19, wherein the biospecific affinity
counterpart is monoclonal antibody C242.
23. The method of claim 19, wherein the biospecific affinity
counterpart is monoclonal antibody C215.
24. The method of claim 14, wherein the modification is at amino
acid position 47.
25. The method of claim 14, wherein the modification is at amino
acid position 128.
26. The method of claim 14, wherein the modification is at amino
acid position 187.
27. The method of claim 14, wherein the modification is at amino
acid position 225.
28. The method of claim 14, wherein the modification is at amino
acid position 227.
29. The method of claim 14, wherein the modification is a
substitution to serine at amino acid position 227.
30. The method of claim 14, wherein the modification is at amino
acid positions 47, 128, 187, 225 and 227.
31. The method of claim 15, wherein the modification is at amino
acid positions 47.
32. The method of claim 15, wherein the modification is at amino
acid position 128.
33. The method of claim 15, wherein the modification is at amino
acid position 187.
34. The method of claim 15, wherein the modification is at amino
acid position 225.
35. The method of claim 15, wherein the modification is at amino
acid position 227.
36. The method of claim 15, wherein the modification is at amino
acid positions 47, 128, 187, 225 and 227.
37. The method of claim 16, wherein the modification is at amino
acid position 47.
38. The method of claim 16, wherein the modification is at amino
acid position 128.
39. The method of claim 16, wherein the modification is at amino
acid position 187.
40. The method of claim 16, wherein the modification is at amino
acid position 225.
41. The method of claim 16, wherein the modification is at amino
acid position 227.
42. The method of claim 16, wherein the modification is at amino
acid positions 47, 128, 187, 225 and 227.
43. A method for the lysis of cells associated with a disease
condition in a mammal, which condition is associated with the
presence of cells expressing a specific cell surface structure,
comprising the step of: administering to the mammal a
therapeutically effective amount of a pharmaceutical composition
comprising: (A) a conjugate comprising a bacterial superantigen and
a biospecific affinity counterpart, wherein (1) the biospecific
affinity counterpart is capable of binding to a specific cell
surface structure, and (2) the superantigen comprises a peptide
that: i. contains an amino acid sequence that is derived from
Staphylococcal enterotoxin A, wherein said peptide has the ability
to bind to a V.beta. chain of a T cell receptor, and ii. has been
modified at an amino acid position 47, 128, 187, 225 or 227, in
order to have reduced ability to bind to MHC class II antigens; and
(B) a pharmaceutically acceptable vehicle.
44. The method of claim 43, wherein said amino acid modification is
a substitution to alanine.
45. The method of claim 43, wherein said amino acid modification is
a substitution that is not a conserved substitution of the amino
acid at that position in Staphylococcal enterotoxin A.
46. The method of claim 43, wherein the specific cell surface
structure is a cancer specific epitope.
47. The method of claim 46, wherein the specific cell surface
structure is the C242 epitope.
48. The method of claim 43, wherein the biospecific affinity
counterpart is an antibody or an antigen-binding fragment of an
antibody.
49. The method of claim 48, wherein the biospecific affinity
counterpart is an antigen-binding fragment of an antibody.
50. The method of claim 49, wherein the fragment of an antibody is
selected from the group consisting of Fab, F(ab).sub.2, Fv, and
single chain antibody.
51. The method of claim 48, wherein the biospecific affinity
counterpart is monoclonal antibody C242.
52. The method of claim 48, wherein the biospecific affinity
counterpart is monoclonal antibody C215.
53. The method of claim 43, wherein the modification is at amino
acid position 47.
54. The method of claim 43, wherein the modification is at amino
acid position 128.
55. The method of claim 43, wherein the modification is at amino
acid position 187.
56. The method of claim 43, wherein the modification is at amino
acid position 225.
57. The method of claim 43, wherein the modification is at amino
acid position 227.
58. The method of claim 43, wherein the modification is a
substitution to serine at amino acid position 227.
59. The method of claim 43, wherein the modification is at amino
acid positions 47, 128, 187, 225 and 227.
60. The method of claim 44, wherein the modification is at amino
acid position 47.
61. The method of claim 44, wherein the modification is at amino
acid position 128.
62. The method of claim 44, wherein the modification is at amino
acid position 187.
63. The method of claim 44, wherein the modification is at amino
acid position 225.
64. The method of claim 44, wherein the modification is at amino
acid position 227.
65. The method of claim 44, wherein the modification is at amino
acid positions 47, 128, 187, 225 and 227.
66. The method of claim 45, wherein the modification is at amino
acid position 47.
67. The method of claim 45, wherein the modification is at amino
acid position 128.
68. The method of claim 45, wherein the modification is at amino
acid position 187.
69. The method of claim 45, wherein the modification is at amino
acid position 225.
70. The method of claim 45, wherein the modification is at amino
acid position 227.
71. The conjugate of claim 45, wherein the modification is at amino
acid positions 47, 128, 187, 225 and 227.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation application of U.S. patent
application Ser. No. 08/765,695, filed Jul. 25, 1997, which is a
U.S. national phase Section 371 application of PCT Patent
Application Number PCT/SE95/00681, filed Jun. 7, 1995, which claims
priority to Swedish patent application number 9402430-4 filed Jul.
11, 1994, each of which are incorporated herein by reference in
their entireties, and all priorities are claimed.
TECHNICAL FIELD
BACKGROUND OF THE INVENTION
[0002] Superantigens are primarily proteins of viral or bacterial
origin and are capable of simultaneous binding to MHC class II
antigens on mammalian cells and the T cell receptor V.beta. chain.
The binding leads to activation of T-lymphocytes and lysis of the
MHC class II bearing cells. The moderate degree of polymorphism of
the binding part of the V.beta. chain causes a relatively large
portion of the T-lymphocytes to be activated when contacted with a
superantigen (in comparison with activation through normal
antigen-processing).
[0003] Initially the superantigen concept was associated with
various staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED, and
SEE). Recently a new staphylococcal enterotoxin named SEH has been
discovered (Keyong et al., J. Exp. Med. 180 (1994) 1675-1683).
After the interest had been raised, further superantigens were
discovered. Examples are Toxic Shock Syndrome Toxin 1 (TSST-1),
Exfoliating Toxins (Exft) that are associated with scalded skin
syndrome, 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) among others.
For a review of superantigens and their properties see Kotzin et
al. (Adv. Immunol. 54 (1993) 99-166).
[0004] Pseudomonas exotoxin A has been looked upon as a functional
superantigen because there are results indicating that this toxin
may be processed intracellularly by accessory cells to fragments
that are expressed on the cell surface with the ability to bind to
the V.beta. chain and a subsequent activation of T cells.
(Pseudomonas exotoxin A. Legaard et al., Cell. Immunol. 135 (1991)
372-382).
[0005] Superantigens as such have been suggested for therapy of
various diseases with curative effects being accomplished through a
general activation of the immune system (Kalland et al., WO
9104053; Terman et al., WO 9110680; Terman et al., WO 9324136;
Newell et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1074-1078).
[0006] In connection with vaccines it has been suggested to use
superantigens that have been mutated so as to lose their TCR
binding ability (Kappler & Marrack, WO 9314634).
[0007] The mutation of superantigens has previously been described
(Kappler & Marrack, WO 9314634; Kappler et al., J. Exp. Med.
175 (1992) 387-396; Grossman et al., J. Immunol. 147 (1991)
32743281; Huffnagle et al., Infect. Immun. 59 (1991)
2126-2134).
[0008] We ourselves have previously suggested to employ conjugates
between a superantigen and an antibody for therapy in order to lyse
cells that express the structure towards which the antibody is
directed (Dohlsten et al., WO 9201470; Lando et al., Cancer
Immunol. Immunother. 36 (1993) 223-228; Kalland et al., Med. Oncol.
Tumor Pharmacother. 10 (1993) 37-47; Lando et al., J. Immunol. 150
(8 part 2) (1993) 114A (Joint Meeting of the American Association
of Immunologists and the Clinical Immunology Society, Denver,
Colo., USA, May 21-25 (1993)); Lando et al., Proc. Am. Assoc.
Cancer Res. Annu. Meet. 33(0) (1992) 339 (Annual meeting of the
American Association for Cancer Research, San Diego, Calif., USA,
May 20-23 (1992)); Dohlsten et al., Proc. Natl. Acad. Sci. USA 88
(1991) 9287-9291). Diseases suggested to be treated have been
cancers, viral infections, parasitic infestations, autoimmune
diseases and other diseases associated with cells expressing
disease-specific surface structures. The experimental work carried
out so far has focused on conjugates containing recombinant SEA and
various anti-cancer antibodies. The conjugates as such have had a
somewhat reduced ability to bind MHC class II antigens compared to
the non-conjugated form of the superantigen. It has not been
determined if a decreased MHC class II antigen binding ability is
beneficial or not for achieving an optimal lyse and an optimal
therapeutic effect.
[0009] Immune therapy experiments with SEB chemically conjugated to
a tumor specific anti-idiotype antibody have previously been
described by Ochi et al., (J. Immunol. 151 (1993) 3180-3186).
[0010] During the prosecution of the priority application the
Swedish Patent Office has additionally cited Buelow et al. (J.
Immunol. 148 (1992) 1-6) that describes fusions between Protein A
and fragments of SEB without emphasis of the MHC classs II binding
or use of the fusion for cell killing; and Hartwig et al. (Int.
Immunol. 5 (1993) 869-875) that describes mutations affecting MHC
class II binding of the non-fused form of the superantigen
streptococcal erythrogenic toxin A.
BRIEF SUMMARY OF THE INVENTION
[0011] A first objective of the invention is to improve previously
known superantigen-antibody conjugates with respect to general
immune stimulation versus directed cytotoxicity. Stimulation
results in activated T-lymphocytes and is dependent on the ability
of the superantigen to bind to both the T cell receptor and an MHC
class II antigen.
[0012] A second objective of the invention is to provide conjugates
between biospecific affinity counterparts (e.g. antibodies) and
superantigens with a modified affinity for NHC class II antigens.
This has now been shown to improve the selectivity for superantigen
antibody dependent cell cytolysis (SADCC) of cells exposing the
antigen (against which the antibody/biospecific affinity
counterpart of the conjugate is directed) over other cells exposing
MHC class II antigens.
[0013] A third objective of the invention is to provide conjugates
that can be used as the active principle in the treatment of
mammals suffering from cancers, autoimmune diseases, parasitic
infestations, viral infections or other diseases associated with
cells that on their surface express structures that are specific
for respective disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] General
[0015] The mutant SEA(D227A) (=SEA(m9) or mutant m9) was at the
priority date the most promising SEA variant. We have therefore
selected to present in vitro and in vivo results with this variant
(FIGS. 3-6).
[0016] FIG. 1 is a schematic outline of the plasmids used to
express SEA and C215Fab-SEA. The coding regions and the two
transcription terminators following the product genes are indicated
by boxes. The gene encoding the kanamycin resistance protein is
labeled Km. lacI is the lac repressor gene. V.sub.H and C.sub.H1
indicates the gene encoding the F.sub.d fragment of the heavy chain
of the murine antibody C215. Likewise V.sub.K and C.sub.K indicates
the gene encoding the kappa chain. Rop is the gene encoding the
replication control protein from pBR322. The promoters directing
transcription of product genes are shown as arrows, in pKP889 the
trc promoter and in the other two vectors the promoter from
staphylococcal protein A (spa). The region containing the origin of
replication is indicated by ori. The only difference between SEA
encoded by pKP943 and pKP1055 is a glycine residue added at the
N-terminus of the latter. The SEA gene contained in the latter
vector also contains more unique restriction enzyme sites,
introduced by silent mutations.
[0017] FIG. 2 is a circular dichroism spectra for wild-type SEA and
for the mutants F47A and D227A, representing the most severely
reduced mutations in each MHC class II binding region. The solid
line is the curve for wild-type SEA. The curves for the mutants are
dotted or center, F47A respectively D227A.
[0018] FIG. 3 shows the concentration dependency of superantigen
dependent mediated cellular cytotoxicity (SDCC) for SEA(wt) and
SEA(D227A).
[0019] FIG. 4 shows the concentration dependency of superantigen
dependent cell mediated cytotoxicity (SDCC) for C215Fab-SEA(wt) and
C215Fab-SEA(D227A).
[0020] FIG. 5 shows the concentration dependency of superantigen
mAb dependent cell mediated cytotoxicity (SADCC) for
C215Fab-SEA(wt) and C215Fab-SEA(D227A) compared to free
SEA(wt).
[0021] FIG. 6A compares the therapeutic effects obtained in C57B1/6
mice carrying lung metastases of B16-C215 melanoma cells by
treatment with C215Fab-SEA(wt) and C215Fab-SEA(D227A).
[0022] FIG. 6B shows toxicity of C215-SEA(wt) and C215-SEA(D227A)
for the treatments represented in FIG. 6a.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The main aspect of the invention is a conjugate
comprising:
[0024] a. a biospecific affinity counterpart that is directed
towards a structure to which one intends to bind to the
conjugate,
[0025] b. a peptide that
[0026] i. is derived from a superantigen,
[0027] ii. has the ability to bind to the V.beta. chain of the T
cell receptor, and
[0028] iii. has a modified ability to bind to MHC class II antigens
compared to the superantigen from which the peptide is derived
(wild-type of superantigen=SA(wt)).
[0029] The peptide and the affinity counterpart are covalently
linked to each other via a bridge (B).
[0030] The preferred conjugates have the ability to activate and
direct T-lymphocytes to selective lysis of cells that on their
surface expose the structure against which the affinity counterpart
is directed. This means that the conjugates shall cause cytolysis
in an SADCC mediated method (Superantigen Antibody Dependent
Cellular Cytotoxicity). See the experimental part below and our
previous publications concerning conjugates between superantigens
and antibodies (e.g. Dohlsten et al., WO 9201470).
[0031] The inventive conjugates have a structure that is analogous
to the superantigen-antibody conjugates described in the prior art
(Dohlsten et al., WO 9201470 which hereby is incorporated by
reference), i.e. the conjugates complies with the formula:
T-B-SA(M)
[0032] where T represents the biospecific affinity counterpart,
SA(m) is the modified superantigen (the above-mentioned peptide),
and B is a covalent bridge linking T and SA(m) together.
[0033] T can in principle be any structure that binds via
biospecific affinity. In most important cases, T is capable of
binding to a cell surface structure, preferably a disease specific
structure as given above. The structure against which T is directed
is usually different from (a) the V.beta. chain epitope to which
the superantigen derived peptide (SA(m)) binds and (b) the MHC
class II antigen epitope to which the unmodified superantigen
binds. The biospecific affinity counterpart T may primarily be
selected among interleukins (e.g. interleukin-2), hormones,
antibodies and antigen binding fragments of antibodies, growth
factors etc. See for instance Woodworth, Preclinical and Clinical
Development of Cytokine Toxins presented at the conference
"Molecular Approaches to cancer Immunotherapy", Ashville, N.C.,
Nov. 7-11, 1993. Polypeptides binding to the constant domains of
immunoglobulins (e.g. Proteins A and G and L), lectins,
streptavidin, biotin etc were at the priority date considered to be
of minor importance.
[0034] At the priority date, it was preferred that T was an
antibody or an antigen binding fragment of an antibody (including
Fab, F(ab).sub.2, Fv, single chain antibody etc), with particular
emphasis of an antibody active fragment (such as Fab) of antibodies
directed against the so called C242 epitope (Lindholm et al., WO
9301303) or against other cancer specific epitopes.
[0035] In case T is an antibody it is primarily monoclonal or a
mixture of a defined number of monoclonals (e.g. 2, 3, 4, 5 or
more). T may be a polyclonal antibody, in case the use is
nontherapeutical.
[0036] It is not imperative for T to have a polypeptide
structure.
[0037] The modified superantigen SA(m) is primarily a mutated
superantigen but may potentially also be a chemically modified
superantigen, including fragments of superantigens retaining the
ability to bind to the V.beta. chain of the T cell receptor.
[0038] The expression "mutated superantigen" means that the native
ability of the superantigen to bind to MHC class II antigens has
been modified on the genomic level by replacing, inserting or
removing one or more amino acids in the native superantigen.
[0039] Superantigen fragments obtained by mutations removing parts
of the full amino acid sequence and fragments obtained by enzymatic
or chemical cleavage of superantigens may be used equivalently in
chemical conjugates of the invention.
[0040] The modified superantigen SA(m) may comprise one or more
amino acid sequences that are derived from different superantigens
and that may have been mutated, for instance combinations of the
preferred superantigens mentioned below.
[0041] The modified superantigen SA(m) as such may exhibit a
decreased immunogenicity and toxicity compared to the native
superantigen.
[0042] Other groups/substances that are capable of cross reacting
with the VP chain of the T cell receptor may potentially also be
employed equivalently with the mutated superantigen (SA(m)) as
given above. Such groups/substances may be of non-polypeptide
structure.
[0043] At the end of the priority year the most interesting product
candidates of the invention comprised mutated forms of
superantigens having multiple MHC class II binding sites and/or the
ability to coordinate Zn.sup.2+, for instance SEA, SED, SEE and
SEH.
[0044] T as well as SA(m) may be prepared by recombinant
techniques.
[0045] The bridge B may be selected as previously described
(Dohlsten et al., WO 9201470), i.e. it shall preferably be
hydrophilic and exhibit one or more structure(s) selected among
amide, thioether, ether, disulfide etc. In case the bridge have
unsubstituted unbroken hydrocarbon chains they preferably lack
aromatic rings, such as phenyl. The most important bridges are
those obtained by recombinant techniques, i.e. when the conjugation
takes places on the genomic level. In such cases oligopeptide
bridges containing hydrophilic amino acid residues, such as Gln,
Ser, Gly, Glu and Arg, are preferred. Pro and His may also be
included. During the priority year it has been decided that the
preferred bridge is a peptide comprising three amino acid residues
(GlyGlyPro).
[0046] The inventive conjugate may comprise one or more modified
superantigen(s) per biospecific affinity counterpart and vice
versa. This means that T in the formula above may contain one or
more modified super antigens in addition to the biospecific
counterpart. In analogy SA(m) may contain one or more biospecific
affinity counterpart(s) T. The affinity counterpart T and SA(m) may
also comprise other structures. The number of modified
superantigens per affinity counterpart is preferably one or
two.
[0047] The synthesis of the novel inventive conjugates may be
carried out in principle according to two main routes: 1. by
recombinant techniques and 2. chemical linking of T to SA(m). The
methods are well recognized for the ordinary skilled worker in the
field and comprise a large number of variants. It follows that the
invention primarily concerns artificial conjugates, i.e. conjugates
that are not found in nature.
[0048] Chemical linking of a modified superantigen to the
biospecific affinity counterpart T often utilizes functional groups
(e.g. primary amino groups or carboxy groups) that are present at
many positions in each compound. It follows that the final product
will contain a mixture of conjugate molecules differing with
respect to the position at which linking has taken place.
[0049] For recombinant conjugates (fusion proteins) the obtained
conjugate substance will be uniform with respect to the linking
position. Either the amino terminal of the modified superantigen is
linked to the carboxy terminal of the biospecific affinity
counterpart or vice versa. For antibodies, such as intact
antibodies and antigen binding fragments (Fab, Fv etc), either the
light or the heavy chain may be utilized for such fusions. At
present time recombinant conjugates are preferred, with preference
for Fab fragments and linking of the amino terminal of the modified
superantigen to the first constant domain of the heavy antibody
chain(CH1), without exclusion of the analogous linking to the light
chain or to the VH and VL domain that also may give quite good
results.
[0050] There are two different methods for obtaining large amounts
of superantigens (including modified and fused forms) in E. coli:
intracellular production or secretion. The latter method is
preferred for the inventive conjugates because it offers
purification of correctly folded protein from the periplasma and
from the culture medium. Intracellular production results in a
complicated purification procedure and often needs refolding in
vitro of the protein (in order for the protein to obtain the
correct tertiary structure). 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.
[0051] The production of mutated superantigens and selection of
mutants having a modified ability to bind (affinity) to MHC class
II antigens maybe carried out according to known techniques (See
e.g. Kappler et al., J. Exp. Med. 165 (1992) 387-396). See also our
experimental part.
[0052] The ability of the conjugate to bind to the T cell receptor
V.beta. chain, to the target structure and to cause lysis of the
target cell depends on i.a. the peptide (SA(m)) that is derived
from a superantigen, the biospecific affinity counterpart (T) and
the structure and length of the bridge (B). A person ordinary
skilled in the art is able to optimize the inventive conjugates
with respect to the binding ability and the ability to cause lysis
by studying the relationship between effect and structure with the
aid of those models that have been disclosed in connection with
previously known superantigen antibody conjugates (see the
above-referred publications). See also the experimental part below.
The ability of the conjugate to bind to the T cell receptor V.beta.
chain, to the target structure and to cause lysis of the target
cell depends on i.a. the peptide (SA(m)) that is derived from a
superantigen, the biospecific affinity counterpart (T) and the
structure and length of the bridge (B). A person ordinary skilled
in the art is able to optimize the inventive conjugates with
respect to the binding ability and the ability to cause lysis by
studying the relationship between effect and structure with the aid
of those models that have been disclosed in connection with
previously known superantigen antibody conjugates (see the
above-referred publications). See also the experimental part
below.
[0053] By modified ability to bind MHC class II antigens is
primarily intended that the ratio
IC.sub.50(SA(wt)):IC.sub.50(SA(m)) is <0.9 (90%), such as
<0.5 (<50%) and possibly also <0.01 (<1%). In the
alternative the modified binding ability of the inventive
conjugates can be measured as the ratio of the dissociation
constants K.sub.d(SA(wt)):K.sub.d(SA(m)) with K.sub.d measured in
nM and with the same limits as for the ratio
IC.sub.50(SA(wt)):IC.sub.50(SA(m)). For the determination of
IC.sub.50(SA(wt), IC.sub.50(SA(m)), K.sub.d(SA(m)) and
K.sub.d(SA(m)) see the experimental part below.
[0054] It is previously known that certain superantigens may have
two or more sites that bind to MHC class II antigen (Fraser et al.,
In: Superantigens: A pathogens view on the immune system. Eds.
Huber & Palmer, Current Communications in Cell Molecular
Biology 7 (1993) 7-29). For this type of superantigens the binding
ability shall be modified at least one of the binding sites, e.g.
as a reduction of the above-mentioned size. Possibly it may suffice
with a superantigen modification that create a changed difference
in affinity for two MHC class II binding sites, tentatively >10%
and preferably by reducing the affinity of at least one site.
[0055] Superantigens bind to TCR V.beta. chains of different
subgroups with varying affinities. In the inventive fusion
proteins/conjugates, the superantigen employed may have been
modified so as to show an altered subgroup specificity or an
altered affinity to one or more members of the subgroup. There are
strong reasons to believe that a parabolic relationship exists
between the affinity for TCR V.beta. and stimulation via TCR, i.e.
a moderate affinity will give the maximal stimulation. Accordingly
an appropriate affinity of a modified superantigen for TCR V.beta.
may be at hand as soon as the fusion protein/conjugate comprising
the modified superantigen is able to significantly stimulate a
resting T cell population representing essentially the distribution
of all human V.beta. subgroups to proliferate. The T cell
population may be pooled T cells from randomly selected human
individuals. By significantly is meant that the stimulation is
possible to measure. The results presented in Table II (right
column) in the experimental part indicate that the ability to cause
SADCC of the inventive conjugates/fusion proteins often is
essentially the same as for the fusion comprising the wild-type
superantigen.
[0056] Main Use of the Conjugates/Fusion Proteins of the
Invention
[0057] The conjugates according to the invention are primarily
intended for the treatment of the same diseases as the conjugates
between normal superantigens and antibodies. See the abovementioned
publications. Thus the inventive conjugates may be administered
either as the main therapy or as adjuvant therapy in connection
with surgery or other drugs.
[0058] The pharmaceutical composition of the invention comprises
formulations that as such are known within the field but now
containing our novel conjugate. Thus 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 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 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 shall not adversely affect the activity of the
conjugate. Water as such is comprised within the expression
vehicles.
[0059] Normally the conjugates will be sold and administered in
predispensed dosages, each one containing an effective amount of
the conjugate that, based on the result now presented, is believed
to be within the range of 10 .mu.g-50 mg. The exact dosage varies
from case to case and depends on the patient's weight and age,
administration route, type of disease, antibody, superantigen,
linkage (--B--) et.
[0060] The administration routes are those commonly known within
the field, i.e. a target cell lysing effective amount or a
therapeutically effective amount of a conjugate according to the
invention is contacted with the target cells. For the indications
specified above this mostly means parenteral administration, such
as injection or infusion (subcutaneously, intravenously,
intra-arterial, intramuscularly) to a mammal, such as a human
being. The conjugate may be administered locally or
systemically.
[0061] By "target cell lysing effective amount" is contemplated
that the amount is effective in activating and directing
T-lymphocytes to destroy the target cell.
[0062] At the end of the priority year it had been decided that the
preferred administration route for conjugates/fusion proteins
comprising unmodified superantigens is 3 hours' intravenous
infusion per day combined with a fever-reducing agent
(paracetamol). The administration is to be repeated during 4 days
and stopped before secondary antibodies are raised against the
fusion protein/conjugate in the patient. This dosage schedule is
likely to be applicable also to the present inventive
conjugates/fusion proteins.
[0063] Alternative Fields of Use
[0064] The inventive conjugates can also be employed to
quantitatively or qualitatively detect the structure against which
the target-seeking group (T) is directed. In general these methods
are well-known to people in the field. Thus, the modified
superantigen may function as a marker group within immunoassays
including immunohistochemistry meaning that the marker group in
turn is detected by for instance an antibody that is directed
towards the peptide (SA(m)) and labeled with an enzyme, isotope,
fluorophor or some other marker group known per se. Another
immunoassay method is to detect in a cell population cells that on
their surface express a structure capable of binding to the
target-seeking group (T). This use means that a sample from the
cell population is incubated with T-lymphocytes together with the
present inventive conjugate as in an SADCC assay. In case the
incubation leads to cell lysis this is an indication that the
population contains cells that on their surface express the
structure.
Experimental Part
Manufacture of Recombinant Proteins
[0065] Antibodies
[0066] The experimental work in connection with the invention has
primarily been done with monoclonal antibody C215 as a model
substance. This antibody is directed against an antigen in the
GA-733 family (see for instance EP 376,746) and references cited
therein and Larsson et al., Int. J. Canc. 32 (1988) 877-82). The
C215 epitope has been judged not to be sufficiently specific for
cancer treatment in humans. At the priority date mab C242 (Lindholm
et al., WO 9301303) was believed to be a better candidate, as
judged from experiments with its fusion product with wild-type
SEA.
[0067] Bacterial Strains and Plasmids
[0068] The E. coli strains UL635 (xy1-7, ara-14, T4.sup.R,
.DELTA.ompT) and HB101 (Boyer and Roulland-Dessoix, J. Mol. Biol.
41 (1969) 459-472) were used for the expression and cloning,
respectively. The vector pKP889 was used for expression of Fab-SEA
fusion proteins (derived from the murine antibody C215) and the
vectors pKP943 and pKP1055 for secretion of SEA (FIG. 1). The
Fab-SEA expression vector pKP889 is identical to pKP865 (Dohlsten
et al, Proc. Natl. Acad. Sci. USA (1994) in press) except that the
spacer between CH1 and SEA is GlyGlyAlaAlaHisTyrGly. Expression
from pKP943 yields SEA with the native amino terminus. The use of
pKP1055 results in SEA having a Gly residue added at the amino
terminus. In both vectors the signals from staphylococcal protein A
(Uhln et al., J. Biol. Chem. 259 (1984) 1695-1702) are used for
transcription and translation and a synthetic signal peptide for
secretion (L. Abrahmsn, unpublished).
[0069] In Vitro Mutagenesis
[0070] Mutations were made by polymerase chain reactions run on a
Perkin Elmer Thermocycler. The reaction mixture (100 .mu.l)
contained: 1.times.PCR buffer from Perkin Elmer Cetus (10 mM
Tris/HCl pH 8.3, 1.5 mMMgCl.sub.2, 0.001% (w/v) gelatine, an
additional 2 mM MgCl.sub.2, 0.4 mM dNTPs (Perkin Elmer Cetus), 2.5
units of Ampli Taq DNA polymerase (Perkin Elmer Cetus, USA) and 100
ng DNA template. Primers were added to a final concentration of 0.8
.mu.M. The original template was a plasmid containing
Staphylococcus aureus enterotoxin A gene identical to the one
published by Betley et al. (J. Bacteriol. 170 (1988) 34-41), except
that the first codon (encoding Ser) was changed to TCC to furnish a
Bam HI site at the 5' end of the gene. Later a derivative
containing more unique restriction enzyme sites introduced by
silent mutations was used. Mutations introduced next to a
restriction site were made with one set of primers, one of these
spanning the mutation and the restriction site. For most mutations
two set of primers had to be used and the PCR was performed in two
consecutive steps. A new restriction enzyme site was introduced
together with each mutation to enable facile identification.
Oligonucleotides used as primers were synthesized on a Gene
Assembler (Pharmacia Biotech AB, Sweden). To confirm each mutation
the relevant portion of the nucleotide sequence was determined on
an Applied Biosystems DNA-Sequenser using their Taq DyeDeoxy
Termination Cycle Sequencing Kit.
[0071] Protein Production and Analysis
[0072] E. coli cells harboring the different gene constructs were
grown overnight at room temperature (Fab-SEA vectors) and at
24-34.degree. C. (secretion vectors, the optimum depends on the
mutation). The broth was 2.times.YT (16 g/l Bacto trypton, 10 g/l
Bacto yeast extract, 5 g/l NaCl) supplemented with kanamycin (50
mg/l). Fusion proteins were induced by addition of
isopropyl-.beta.-D-thiogalactoside to a final concentration of 100
.mu.M. (The protein A promotor used in the expression of non-fused
SEA is constitutive). The cells were pelleted at 5000.times.. g and
the periplasmic contents were released by gently thawing the
previously frozen cell pellet in 10 mM Tris-HCl (pH 7.5) on ice
during agitation for 1 hour. The periplasmic extracts were
clarified by centrifugation at 9500.times.g for 15 minutes. The
Fab-SEA proteins were used without further purification. SEA and
Gly-SEA were further purified by affinity chromatography on an
anti-SEA antibody column. Polyclonal rabbit anti-SEA antibodies
were previously collected from rabbits preimmunized with SEA and
purified by affinity chromatography on protein G Sepharoses.RTM.
(Pharmacia Biotech).
[0073] Protein Analysis
[0074] The proteins were separated in precast polyacrylamide SDS
Tris-Glycine Novex gels (gradient 4-20% or homogenous 12%, Novex
novel experimental technology) and either stained with Coomassie
Blue or used in Western blot. Polyclonal rabbit anti-SEA antibodies
(above) were used to detect SEA in Western blot analysis, followed
by porcine anti-rabbit Ig antibodies, and rabbit anti-horseradish
peroxidase antibodies and peroxidase. With Fab-SEA fusion proteins
peroxidase conjugated rat antibodies recognizing the kappa chain
were also used (AAC 08P, Serotech LTD, England).
3,3'-diaminobenzidine (Sigma) was used for visualization of
peroxidase.
[0075] Circular dichroism (CD) spectra were collected in a J-720
spectropolarimeter (JASCO, Japan) at room temperature
(22-25.degree. C.) in 10 mM phosphate buffer, pH 8.2, with 0.02 mM
ZnSO.sub.4 and 0.005% (v/v) Tween.RTM. 20. The scanning speed was
10 nm/min and each spectrum was averaged from five subsequent
scans. The cell path length was 1 mm and the protein concentration
0.2 to 0.5 mg/ml. Guanidine hydrochloride (Gdn-HCl) denaturations
at equilibrium were measured at 23.degree. C. by CD at 222 nm with
a protein concentration of 0.3 mg/ml and a cell path length of 1
mm. These data were used to calculate the apparent fraction of
unfolded protein (F.sub.app). Equilibrium unfolding parameters were
derived by fitting the data to a two-site folding process (Hurle et
al., Biochemistry 29 (1990) 4410-4419).
Binding and Functional Assays In Vitro
[0076] Materials
[0077] Reagents: RPMI 1640 medium obtained from Gibco, Middlesex,
England was used. The medium had a pH of 7.4 and contained 2 mM
L-glutamine (Gibco, Middlesex, England), 0.01M HEPES (Biological
Industries, Israel), 1 mM NaHCO.sub.3 (Biochrom AG, Germany), 0.1
mg/ml Gentamycin sulphate (Biological Industries, Israel), 1 mM
Na-pyruvate (JRH Biosciences Industries, USA), 0.05 mM
mercaptoethanol (Sigma Co., USA), 100 times concentrated
nonessential amino acids (Flow Laboratories, Scotland) and was
supplemented with 10% fetal bovine serum (Gibco, Middesex,
England). Recombinant SEA(wt), SEA(m) and the fusion products
C215Fab-SEA(wt) and C215Fab-SEA(m) were obtained as described
above. Human recombinant IL-2 was from Cetus Corp., USA. Mitomycin
C was from Sigma Co., USA. Na.sub.2.sup.51CrO.sub.4 was obtained
from Merck, Germany. Phosphate buffered saline (PBS) without
magnesium and calcium was received from Imperial, England.
[0078] Cells: The human colon carcinoma cell line Colo205 and the B
cell lymphoma cell line Raji were obtained from American Type Cell
Culture Collection (Rockville, Md., USA) (expressing HLA-DR3/w10,
-DP7, -DQw1/w2). The EBV-transformed lymphoblastoid B cell line BSM
was a generous gift from Dr van De Griend, Dept of Immunology, Dr
Daniel den Hoed Cancer Center, Leiden, the Netherlands. The cells
were repeatedly tested for mycoplasma contamination with Gen-Probe
Mycoplasma T.C. test, Gen-Probe Inc., San Diego, USA.
[0079] SEA activated T cell lines were produced by activation of
mononuclear cells from peripheral blood. The blood was received as
buffy coats from blood donors at the University Hospital of Lund.
The PBMs were stimulated at a concentration of 2.times.10.sup.6
cells/ml with mitomycin C treated SEA coated BSM cells
(preincubated with 100 ng/ml SEA) in medium with 10% FCS. The T
cell lines were restimulated biweekly with 20 U/ml human
recombinant IL-2 and weekly with mitomycin C treated SEA coated BSM
cells. The cell lines were cultivated for 4-12 weeks before being
used in the assay.
[0080] The viability of the effector cells, as determined by trypan
blue exclusion, exceeded 50%.
Determination of MHC Class II Binding Characteristics of Wild-Type
and Mutant Sea
[0081] Radioiodination Procedure
[0082] Appropriate amounts of wild-type or mutant SEA were
radiolabeled with 10 to 25 mCi Na.sup.125I using enzymobeads with
the lactoperoxidase technique (NEN, Boston, Mass.). The reaction
was stopped by quenching with sodium azide and protein-bound
radioactivity was separated from free iodine by filtration through
a PD-10 column (Pharmacia Biotech AB, Sweden) with R10 medium as
elution buffer. Conditions were chosen to obtain a stoichiometric
ratio between iodine-125 and protein of .ltoreq.2:1. The
radiochemical purity was verified by size-exclusion chromatography
on a TSK SW 3000 HPLC column. The effect of the radioiodination on
the binding activity was only tested for wild-type SEA and found
not to be affected (data not shown).
[0083] Direct Binding Assay
[0084] Raji cells, 6.times.10.sup.4/100 .mu.l, previously
cultivated in R10 medium, were added to conical polypropylene tubes
and incubated (22.degree. C./45 min) in triplicate with 100
.mu.l/tube of serially diluted .sup.125I-labeled wild-type or
mutant SEA. The cells were washed with 2 ml 1% (w/v) bovine serum
albumin (BSA) in 10 mM phosphate-buffered saline (PBS), pH 7.4,
centrifugated at 300.times.g for 5 minutes and aspirated. This
procedure was repeated twice. Finally, the cells were analyzed for
cell-bound radioactivity in a gamma counter (Packard Instruments
Co, Downers Grove, Ill., USA). The apparent dissociation constant,
K.sub.d, and the number of binding sites, N, at saturation were
calculated according to Scatchard (Ann. N.Y. Acad. Sci. 51 (1949)
660-72) after subtraction of non-specific binding (i.e. binding
after incubation with R10 medium alone.
[0085] Inhibition Assay
[0086] Inhibition assay (inhibition of .sup.125I-labeled wild-type
SEA binding by mutant SEAs). These inhibition experiments were
carried out as is described for the direct binding assay with
slight modifications. Briefly, 50 .mu.l of .sup.125I-labeled
wild-type SEA was allowed to compete with an excess of unlabeled
wild-type or mutant SEA (50 .mu.l/tube) for binding to
6.times.10.sup.4/100 .mu.l Raji cells. A tracer concentration
yielding.apprxeq.40% bound radioactivity in the direct assay was
used to obtain maximal sensitivity in the inhibition assay. The
displacement capacity of the competitor was expressed as the
concentration yielding 50% inhibition (IC.sub.50) of bound
radioactivity. The binding affinity of the mutants relative to
wild-type SEA was calculated using the equation:
IC.sub.50(SEA(wt)):IC.sub.50(SEA(m))
[0087] In order to analyze whether the mutants compete for binding
to the same site on Raji cells as wild-type SEA, the binding data
obtained with SEA mutants were plotted as a log-logit function and
tested for parallelism with the corresponding data for wild-type
SEA.
[0088] Inhibition Assay
[0089] Inhibition assay (inhibition of the binding of
fluorescent-labeled wild-type SEA by unlabeled wild-type SEA and
SEA mutants). Raji cells (2.5.times.10.sup.5) were incubated with
inhibitor (wild-type or mutant SEA; 0-6000 nM) diluted in 50 .mu.l
CO.sub.2-independent medium (Gibco) supplemented with 10% FCS,
glutamine and gentamycin at 37.degree. C. for 30 minutes.
Fluorescein conjugated wild-type SEA was added to a final
concentration of 30 nM and the samples were incubated for an
additional half hour at 37.degree. C. The samples were washed three
times with ice cold PBS supplemented with 1% BSA (PBS-BSA) and
finally kept in 0.4 ml PBS-BSA on ice until they were analyzed.
From each sample 10,000 live cells were analyzed for green
fluorescence on a FACStar.RTM. (Becton Dickinson) flow cytometer
and the mean fluorescence value was calculated using the LYSIS II
program.
SDCC and SADCC Assays of SEA(wt), SEA(m) and Their Fusion Proteins
with C215FAB
[0090] SDCC-Assays
[0091] The cytotoxicity of SEA(wt), SEA(m) and their fusions with
C215Fab against MHC class II.sup.+ Raji cells was analyzed in a
standard 4 hour .sup.51Cr.sup.3+-release assay, using in vitro
stimulated SEA specific T cell lines as effector cells. Briefly,
.sup.51Cr labeled Raji cells were incubated at 2.5.times.10.sup.3
cells per 0.2 ml medium (RPMI, 10% FCS) in microtitre wells at
defined effector to target cell ratio in the presence or absence
(control) of the additives. Percent specific cytotoxicity was
calculated as 100.times.([cpm experimental release-cpm background
release]/[cpm total release-cpm background release]). The effector
to target cell ratio was 30:1 for unfused SEAs and 40:1 for fusion
proteins.
[0092] SADCC Against of Human Colon Cancer Cells
[0093] The cytotoxicity of C215Fab-SEA(wt), C215Fab-SEA(m), SEA(wt)
and SEA mutants against C.sup.215+ MHC class II.sup.- colon
carcinoma cells SW 620 was analyzed in a standard 4 hour
.sup.51Cr.sup.3+-release assay, using in vitro stimulated SEA
specific T cell lines as effector cells. Briefly,
.sup.51Cr.sup.3+-labeled SW 620 cells were incubated at
2.5.times.10.sup.3 cells per 0.2 ml medium (RPMI, 10% FCS) in
microtitre wells at effector to target cell ratio 30:1 in the
presence or absence (control) of the additives. Percent specific
cytotoxicity was calculated as for SDCC assays.
In Vivo Functional Experiments
[0094] Tumor Cells
[0095] B16-F1 melanoma cells transfected with a cDNA encoding the
human tumor associated antigen C215 (B16-C215) (Dohlsten et al.,
Monoclonal antibody-superantigen fusion proteins: Tumor specific
agents for T cell based tumor therapy; Proc. Natl. Acad. Sci. USA,
In press, 1994), were grown as adherent cells to subconfluency. The
culture medium consisted of RPMI 1640 (GIBCO, Middlesex, UK)
supplemented with 5.times.10.sup.-5 .beta.-mercaptoethanol (Sigma,
St Louis, Mo., USA), 2 mM L-glutamine (GIBCO), 0.01 M Hepes
(Biological Industries, Israel) and 10% fetal calf serum (GIBCO).
The cells were detached by a brief incubation in 0.02% EDTA and
suspended in ice cold phosphate buffered saline with 1% syngeneic
mouse serum (vehicle) to 4.times.10.sup.5 cells/ml.
[0096] Animals and Animal Treatment
[0097] The mice were 12-19 weeks old C57B1/6 mice transgeneic for a
T cell receptor V.beta.3 chain (Dohlsten et al., Immunology 79
(1993) 520-527). One hundred thousand B16-C215 tumor cells were
injected IV in the tail vein in 0.2 ml vehicle. On day 1, 2 and 3,
the mice were given IV injections of C215Fab-SEA(wt) or
C215Fab-SEA(D227A) in 0.2 ml vehicle at doses indicated in the
FIGS. 5a and 5b. Control mice were given only vehicle according to
the same schedule. On day 21 after tumor cell injection, the mice
were killed by cervical dislocation, the lungs removed, fixed in
Bouin's solution and the number of lung metastases counted.
Results
[0098] "Alanine Scanning" of Staphylococcal Enterotoxin A
[0099] Initially the structure of SEA was unknown and only
speculations could be done about what side chains were surface
accessible. Therefore, the majority of the mutants were chosen from
alignments of homologous superantigens (Marrack and Kappler,
Science 248 (1990) 705-711). Conserved (mainly polar) residues were
chosen on the rational that some of these superantigens are
expected to bind to HLA-DR in a conserved fashion (Chitagumpala et
al., J. Immunol. 147 (1991) 3876-3881). Alanine replacements were
used according to published strategies (Cunnningham and Wells,
Science 244 (1988) 1081-1085). During the course of this work the
available information increased: i) it was shown that a Zn.sup.2+
ion is important for the interaction between SEA and MHC class II
(HLA-DR) (Fraser et al., Proc. Natl. Acad. Sci. USA 89 (1991)
5507-5511), ii) a mutational analysis of staphylococcal enterotoxin
B (SEB) was presented (Kappler et al., J. Exp. Med. 175 (1992)
387-396), and iii) the structure of SEB was presented (Swaminathan
et al., Nature 359 (1992) 801-806).
[0100] Our first mutant showing a severely reduced affinity for HLA
DR, D227A, was found to coordinate the Zn.sup.2+ ion very poorly
(data not shown). Assuming a common fold for SEA and SEB, the new
data suggested two MHC class II binding regions; one involving the
Zn.sup.2+ ion and one corresponding to the site defined in SEB. A
second set of mutations were made on these assumptions. This second
set of mutants were expressed in the form of SEA carrying a glycine
added at the amino terminus. First the extension was shown to have
no effects on the binding properties of wild-type SEA (next
section).
[0101] Most of the mutants were expressed and secreted by E. coli
in a functional form as judged by analysis of the binding of
monoclonal antibodies (Table I). Very low amounts were obtained of
the mutants E154A/D156A and R160A. Consequently these were excluded
from the study. The mutants having an Ala substitution in residues
128, 187, 225 or 227 were not recognized by the monoclonal antibody
1E. The latter two mutants showed a reduced level of expression
(more pronounced at 34.degree. C. than at 24.degree. C.) and
migrated faster during SDS-PAGE, under denaturing but not reducing
conditions (all other mutants migrated as wild-type SEA, data not
shown). As judged by CD spectra analysis the structure of D227A
could differ slightly from native SEA (FIG. 2), but the stability
was very close to wild-type SEA (measured as resistance towards
guanidine hydrochloride denaturation). The calculated
.DELTA..DELTA.G between the mutant and native SEA (SEA(wt)) was
0.16 kcal/mol and is only about 4% of the .DELTA.G.degree. values
(data not shown). Overall the signals in the CD analysis were low,
as expected from a mostly .beta.-sheet structure. It was recently
reported that His 225 coordinates Zn.sup.2+ (unpublished data in
Fraser et al (Proc. Natl. Acad. Sci. USA 89 (1991) 5507-5511).
Since Asp 227 is involved in Zn.sup.2+ coordination (above) and
presumably located in the same .beta.-sheet as His 225 this
suggests that these two residues constitutes the zinc-binding
nucleus found in zinc-coordinating proteins (Vallee and Auld,
Biochemistry 29 (1990) 5647-5659).
[0102] Binding to MHC class II and T Cell Receptor
[0103] The MHC class II affinity was calculated from the amounts
needed to compete with fluorescein-labeled wild-type SEA for Raji
cell exposing large amounts of MHC class II. The displacement
capacity of a mutant was calculated from the concentration yielding
50% inhibition (IC.sub.50) of bound fluorescence compared with the
concentration needed with wild-type SEA as the competitor. For
wild-type SEA and for some mutants, the result from this analysis
was compared with the result from an analysis where .sup.125I
labeled wild-type SEA was used as the tracer. As may be seen in
Table II, the values obtained from these two inhibition analyses
correlate well.
[0104] For six selected mutants the binding to MHC class II was
measured directly using .sup.125I labeled mutant SEA (Table II).
With the mutant H50A the values obtained from the direct binding
assay and the inhibition assays correlated well but with the mutant
F47A a large discrepancy was found: the direct binding indicated
only 7 times weaker binding than wild-type SEA but both competition
analyses demonstrated around 70 times reduced binding. The data
from two of the other mutants indicated two separate binding
interactions. For the mutants H225A and D227A the affinity was
below the detection limit also in this analysis.
[0105] We previously showed that fusion proteins composed of the
Fab fragment of a carcinoma reactive antibody and SEA could be used
to direct cytotoxic T cells to specifically lyse cancer cells,
while the interaction between SEA and the T cell receptor (TCR) was
too weak to be detected by itself (Dohlsten et al., Proc. Natl.
Acad. Sci. USA, in press). Thus, in contrast to analyses involving
the isolated superantigen the Fab fusion context enables a
functional assay for the interaction between SEA and the TCR,
independent of the MHC class II binding. Consequently, the
efficiency of the different conjugates to direct T cells to lyse
cells recognized by the Fab moiety was monitored in a chromium
release assay. This analysis confirmed that the mutations shown to
affect the MHC class II binding did not affect the TCR binding
(Table II).
[0106] Biological Effects of the Mutations
[0107] The proliferative effect was measured as the ability to
stimulate peripheral lymphocytes to divide. All three mutants that
competes very poorly for MHC class II induced little or no
proliferation and the intermediate mutant H187A displayed some
proliferative capacity, whereas the other investigated mutants were
indistinguishable from the wild-type (table III). Harris et al
(Infect. Immun. 61 (1993) 3175-3183) recently reported a similar
severe reduction in T cell stimulatory activity for the SEA mutants
F47G and L48G. Clearly a strong reduction in any of the two
suggested binding regions results in a severe effect on the ability
to induce proliferation. This suggests that SEA cross-links two
molecules of MHC class II leading to dimerization of the TCR and
that this is needed to yield a signal transduction.
[0108] In contrast the efficiency of the different mutants in
directing in vitro stimulated SEA T cells to lyse MHC class II
bearing target cells shows correlation with the binding affinity,
rather than to the ability to compete (Table III). For example, the
efficiency of F47A and D227A are only reduced 2.5 times and 300
times, respectively. Thus, here no inherent requirement for
divalency too is obvious. The increase in multivalency resulting
from the significantly larger number of TCRs on the surface of
activated T cells might partially shield the effect of a lower
avidity in the SEA/MHC class II interaction. That dimerization is
not needed to direct T cell cytotoxicity has previously been
demonstrated by the use of carcinoma specific bifunctional
antibodies containing one anti-CD3 moiety and one anti-carcinoma
moiety (Renner et al., Science 264 (1994) 833-35).
[0109] In Vivo Functional Experiments
[0110] The results are represented in FIGS. 6a and 6b. Treatment of
mice with C215Fab-SEA(wt) and C215Fab-SEA(D227A) were both highly
effective in reducing the number of lung metastases of B16-C215
melanoma cells. The therapeutic effect was essentially identical
for the two variants of the targeted superantigens. Treatment with
C215Fab-SEA(wt) resulted in 70% lethality at doses of 5
.mu.g/injection. In contrast, no mice died when the same dose of
C215Fab-SEA(D227A) were used. Taken together, SEA(D227A) is an
example of a mutant with reduced toxicity and retained therapeutic
efficiency when incorporated in a Fab-SEA fusion protein.
Discussion
[0111] The structure of the complex between SEB and HLA-DR was
recently reported (Jardetzky et al., Nature 368 (1994) 711-718).
Most of the SEB residues identified to be involved in this
interaction are conserved in SEA. Our data on mutant D227A
indicates a weak affinity for the interaction between this site of
SEA (the amino proximal site) and the MHC class II, having a
K.sub.d value higher than 8 .mu.M. The K.sub.d for the interaction
between SEB and HLA-DR was recently reported to be 1.7 .mu.M (Seth
et al., Nature 369 (1994) 324-27). The different interactions
between SEB, TCR and HLA-DR were investigated and it was shown that
the complex between SEB and HLA-DR was not stably maintained in the
absence of TCR. Plasmon resonance experiments indicated that this
was because of a very fast off-rate. The avidity effects obtained
if SEA cross-links two molecules of MHC class II followed by a
subsequent dimerization of the TCR could explain how SEA may induce
proliferative effects at concentrations well below the K.sub.d.
Assuming that the mutation F47A reduces the affinity of the amino
proximal site below significance, the K.sub.d of the Zn.sup.2+ site
is around 95 nM. This hypothesis was recently strengthened by the
observation that the mutants F47R, F47R/H50A and F47R/L48A/H50D
show identical affinity for MHC class II as F47A (unpublished).
[0112] Based on the-SEB structure (Kappler et al., J. Exp. Med. 175
(1992) 387-396) and on homology alignments (Marrack and Kappler,
Science 248 (1990) 705-711), it is strongly suggested that His225
and Asp227 are located in the same .beta.-sheet and thus the side
chains could be proximal. Thus, most likely these two residues
constitute the zinc-binding nucleus found in zinc-coordinating
proteins (Vallee and Auld, Biochemistry 29 (1990) 5647-5659).
Similarly to these mutants, the mutants with a replacement at
residue 128 or 187 are also recognized by all monoclonals except
1E. Fraser et al (Proc. Natl. Acad. Sci. USA 89 (1991) 5507-5511)
showed that Zn.sup.2+ is bound to SEA and is needed for a high
affinity interaction with MHC class II. The affinity for zinc was
not affected by the addition of HLA-DR. Based on this observation
and the high affinity for Zn.sup.2+ (K.sub.d of around 1 .mu.M) a
coordination exclusively provided by SEA and involving 4 fold
coordination was suggested. Our data indicates an involvement of
the four residues N128, H187, H225 and D227. The function of the
former two residues is not yet clear; instead of providing a ligand
N128 could help in the deprotonation of D227. One argument for this
is that the effect of replacing D227 is more severe that when
replacing H225.
[0113] It was previously reported that there is a lack of
correlation between the affinity of different superantigens for the
MHC class II and the capacity to stimulate T cells to proliferate
(Chintagumpala et al., J. Immunol. 147 (1991) 3876-3881). These
results might partly be explained by different affinities of the
superantigens towards different TCR V .beta.-chains. Here we have
observed the same lack of correlation but in contrast to separate
superantigens the mutants display identical TCR affinity as shown
in the Fab-SEA context (measured as SADCC). The most likely
explanation for the lack of correlation is that two binding regions
identified in this analysis represent two separate binding sites
that yields not only a co-operative binding, but which results in
the cross-linking of two molecules of MHC class II, which in turn
yields dimerization of two molecules of the T cell receptor. This
would imply that the affinity of both sites are important to obtain
the proliferative effect. A high avidity results from the
interactions within a hexameric complex involving two molecules of
SEA, TCR and MHC class II. Thus the strong affinity/avidity of SEA
towards MHC class II enables SEA interaction with the TCR despite a
low direct affinity.
[0114] Other Biospecific Affinity Counterparts
[0115] A fusion protein of SEA(D227A) and an IgG-binding domain of
staphylococcal protein A has been produced by recombinant
technology and expressed in E. coli. This reagent has successfully
been used to target T-lymphocytes to Mot 4 and CCRF-CEM cells
(obtained from ATCC) that are CD7 and CD38 positive but HLA-DP, -DQ
and -DR negative. The Mot 4 and CCRF-CEM cells were preincubated
with anti-CD7 or anti-CD38 mouse monoclonals (Dianova, Hamburg,
Germany). In order to enhance binding between the mouse monoclonals
and the IgG-binding part of the fusion protein rabbit anti-mouse Ig
antibody was also added.
[0116] In comparison with protein A-SEA(wt), protein A-SEA(D227A)
had a decreased ability to bind to Daudi cells expressing MHC class
II antigen.
1TABLE I Confirmation of mutant structural integrity. The binding
of six monoclonal antibodies was monitored. Monoclonal antibody
Mutation 1A 2A 3A 1E 4E EC-A1 Wild-type + + + + + + D11A/K14A + + +
+ + + D45A + + + + + + F47A + + + + + + H50A (+) + (+) + + + K55A +
+ + + + + H114A + + + + + + K123A/D132G + + + + + + N128A + + + - +
+ K147A/K148A + + + + - + E154A/D156A ND ND ND + ND ND R160A ND ND
ND + ND ND H187A + + + - + + E191A/N195A + + + + + + D197A + + + +
+ + H225A + + + - + + D227A + + + - + + Footnotes: A plus sign
indicates binding, parenthesis indicate 50 to 90% binding compared
with wild-type SEA. ND means not determined.
[0117]
2TABLE II Binding of SEA mutants to the MHC class II and the T cell
receptor. The latter was monitored as the ability to direct
activated cytotoxic T-cells specifically to lyse carcinoma cells
using Fab-SEA fusions of the different mutants (SADCC).
IC.sub.50(nM) IC.sub.50(nM) K.sub.d(nM) SADCC(% of Mutation
SEA-FITC.sup.1 125.sub.I-SEA.sup.1 125.sub.I labeled.sup.1
wild-type.sup.1 wild-type 50 38 13 100.sup.2 Gly-SEA 50 ND ND
100.sup.2 D11A/K14A 50 ND ND ND D45A 53 ND ND ND F47A 3150 2943 95
100 H50A 150 132 32 100 K55A 44 ND ND ND H114A 48 ND ND ND
K123A/D132G 188 75 12/237 100 N128A 1150 ND 2.9/76 100 K147A/K148A
58 ND ND ND H187A 1030 602 97 100 E191A/N195A 51 ND ND ND D197A 78
ND ND ND H225A >9000 9600 ND ND D227A >9000 >10000
>8000 100 Footnotes: .sup.1ND means not determined. .sup.2In the
Fab-SEA context the spacer between C.sub.Hi and SEA ends with a
Gly.
[0118]
3TABLE III Biological effects of the mutations. The ability to
stimulate resting T cells to proliferate and the ability to direct
cytotoxic cells to lyse MHC class II exposing target cells were
monitored (SDCC = Superantigen Dependent mediated Cellular
Cytotoxicity). Proliferation SDCC Mutation % EC.sub.50 (relative)
wild-type 100 1 Gly-SEA ND 1 D11A/K14A ND 0.8 D45A 50 1.3 F47A
<0.2 2.5 H50A 20 1.4 K55A 100 1.3 H114A ND 1 K123A/D132G 40 2.1
N128A 40 1.2 K147A/K148A ND 0.7 E154A/D156A ND ND R160A ND ND H187A
15 4 E191A/N195A 100 1.1 D197A ND 1.3 H225A <0.2 3 .times.
10.sup.2 D227A <0.01 3 .times. 10.sup.2 Footnotes: ND means not
determined.
* * * * *