U.S. patent application number 12/143581 was filed with the patent office on 2009-01-08 for modified diphtheria toxins.
This patent application is currently assigned to ANGELICA THERAPEUTICS, INC.. Invention is credited to Deepshikha Datta, Claude Geoffrey Davis.
Application Number | 20090010966 12/143581 |
Document ID | / |
Family ID | 40221619 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090010966 |
Kind Code |
A1 |
Davis; Claude Geoffrey ; et
al. |
January 8, 2009 |
MODIFIED DIPHTHERIA TOXINS
Abstract
The present application relates to compositions of modified
diphtheria toxin and fusion proteins containing modified diphtheria
toxin that reduce binding to vascular endothelium or vascular
endothelial cells, and therefore, reduce the incidence of Vascular
Leak Syndrome, as well as methods of making the compositions. The
present application also relates to a polypeptide toxophore from a
modified diphtheria toxin, where the modification is at least one
amino acid residue at the amino acid residues 6-8, 28-30 or 289-291
of an unmodified native diphtheria toxin. Also described are fusion
proteins which contain a modified diphtheria toxin and a
non-diphtheria toxin fragment which contains a cell binding
portion. The modified diphtheria toxins described can be used for
the treatment of a malignant disease or a non-malignant
disease.
Inventors: |
Davis; Claude Geoffrey; (San
Mateo, CA) ; Datta; Deepshikha; (San Francisco,
CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
ANGELICA THERAPEUTICS, INC.
Emeryville
CA
|
Family ID: |
40221619 |
Appl. No.: |
12/143581 |
Filed: |
June 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60945556 |
Jun 21, 2007 |
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60954278 |
Aug 6, 2007 |
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61032888 |
Feb 29, 2008 |
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61042178 |
Apr 3, 2008 |
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60945568 |
Jun 21, 2007 |
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60954284 |
Aug 6, 2007 |
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61032910 |
Feb 29, 2008 |
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61042187 |
Apr 3, 2008 |
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Current U.S.
Class: |
424/238.1 ;
530/350 |
Current CPC
Class: |
A61K 2039/55533
20130101; A61P 35/04 20180101; A61P 37/00 20180101; A61P 35/00
20180101; A61P 35/02 20180101; A61K 39/39 20130101; A61K 39/0011
20130101; A61K 38/16 20130101; A61K 2039/55544 20130101; A61K
2039/5156 20130101; C07K 14/34 20130101; A61K 47/6415 20170801;
A61P 43/00 20180101; A61K 47/642 20170801 |
Class at
Publication: |
424/238.1 ;
530/350 |
International
Class: |
A61K 39/05 20060101
A61K039/05; C07K 14/195 20060101 C07K014/195; A61P 35/04 20060101
A61P035/04 |
Claims
1. A composition comprising a modified diphtheria toxin, said
modified diphtheria toxin having one or more amino acid
modifications therein, wherein: at least one amino acid
modification is made within an (x)D(y) motif; said modified
diphtheria toxin exhibits cytotoxicity comparable to an unmodified
diphtheria toxin; and a modification at position (x) is a
substitution of V or I by an amino acid residue selected from among
F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, and a modified or unusual
amino acid from Table 1; a modification at position D is a
substitution of D by an amino acid residue selected from among I,
V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R and a modified or
unusual amino acid from Table 1; a modification at position (y) is
a substitution of S by an amino acid residue selected from among I,
F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K, R and a modified or
unusual amino acid from Table 1; or a combination thereof.
2. The composition of claim 1, wherein said modified diphtheria
toxin contains one or more modifications selected from among V7T,
V7N, V7D, D8N, S9A, S9T, S9G, V28N, V28D, V28T, D29N, S30G, S30N,
I290T, S292A, S292G and S292T.
3. The composition of claim 1, wherein said modified diphtheria
toxin comprises two modifications selected from among V7N V28N, V7N
V28T, V7N V28D, V7T V28N, V7T V28T and V7T V28D.
4. The composition of claim 1, wherein said modified diphtheria
toxin comprises three modifications selected from among V7N V29N
I290N, V7N V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T
I290N, V7N V29T I290T, V7N V29T S292A, V7N V29T S292T, and V7T V29T
I290T.
5. The composition of claim 1, wherein said unmodified diphtheria
toxin has an amino acid sequence of SEQ ID NO: 1, 2 or 149.
6. The composition of claim 1, wherein said unmodified diphtheria
toxin has an amino acid sequence of any one of SEQ ID NOS:
4-147.
7. The composition of claim 1, wherein said composition exhibits
reduced binding activity to human vascular endothelial cells
(HUVECs) compared to an unmodified diphtheria toxin.
8. The composition of claim 1, wherein said modified diphtheria
toxin further comprises a non-diphtheria toxin polypeptide.
9. The composition of claim 8, wherein said non-diphtheria toxin
polypeptide is among an antibody or antigen-binding fragment
thereof, EGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, INF.alpha., INF.gamma.,
GM-CSF, G-CSF, M-CSF, TNF, VEGF, Ephrin, BFGF, TGF, or a
cell-specific binding portion thereof.
10. The composition of claim 9, wherein said non-diphtheria toxin
polypeptide is IL-2 or a cell-specific binding portion thereof.
11. The composition of claim 9, wherein said non-diphtheria toxin
polypeptide is IL-3 or a cell-specific binding portion thereof.
12. A fusion protein comprising a polypeptide toxophore of a
modified diphtheria toxin, and a non-diphtheria toxin polypeptide,
said polypeptide toxophore comprising a diphtheria toxin having an
amino acid sequence as recited in SEQ ID NO. 2 or 149 having one or
more amino acid modifications therein, wherein: at least one amino
acid modification is made within an (x)D(y) motif; said modified
diphtheria toxin has cytotoxicity comparable to an unmodified
diphtheria toxin; and a modification at position (x) is a
substitution of V or I by an amino acid residue selected from among
F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, and a modified or unusual
amino acid from Table 1; a modification at position D is a
substitution of D by an amino acid residue selected from among I,
V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R and a modified or
unusual amino acid from Table 1; a modification at position (y) is
a substitution of S by an amino acid residue selected from among I,
F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K, R and a modified or
unusual amino acid from Table 1; or a combination thereof.
13. The fusion protein of claim 12, wherein said polypeptide
toxophore has reduced binding to human vascular endothelial cells
compared to a diphtheria toxin molecule having a sequence of SEQ ID
NO: 1, 2 or 149.
14. The fusion protein of claim 12, wherein said non-diphtheria
toxin polypeptide is an antibody or antigen-binding fragment
thereof, EGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, INF.alpha.; INF.gamma.,
GM-CSF, G-CSF, M-CSF, TNF, VEGF, Ephrin, BFGF, TGF, or a
cell-binding portion thereof.
15. The fusion protein of claim 14, wherein said non-diphtheria
toxin polypeptide is IL-2 or a cell binding portion thereof.
16. The fusion protein of claim 14, wherein said non-diphtheria
toxin polypeptide is IL-3 or a cell binding portion thereof.
17. The fusion protein of claim 12, further comprising a
pharmaceutically acceptable carrier or excipient.
18. A method for treating a malignant disease in a subject
comprising administering a therapeutically effective amount of a
composition of claim 17 to said subject, wherein said malignant
disease is a blood cancer, a solid tumor or a metastasis.
19. A method for treating a non-malignant disease in a subject
comprising administering a therapeutically effective amount of a
composition of claim 17 to said subject, wherein said non-malignant
disease is graft versus host disease or psoriasis.
20. A method for enhancing the activity of an anti-cancer agent
comprising administering an anti-cancer agent and a composition of
claim 17 to a subject.
21. A method for treating a malignant disease in a subject
comprising administering a therapeutically effective amount of a
composition of claim 17 and an anti-cancer agent to said subject,
wherein said malignant disease is a blood cancer, a solid tumor or
a metastasis.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/945,556, filed Jun. 21, 2007 (attorney docket
number 33094-702.101), U.S. Provisional Application No. 60/954,278,
filed Aug. 6, 2007 (attorney docket number 33094-702.102), U.S.
Provisional Application No. 61/032,888, filed Feb. 29, 2008
(attorney docket number 33094-702.103), U.S. Provisional
Application No. 61/042,178, filed Apr. 3, 2008 (attorney docket
number 33094-702.104), U.S. Provisional Application No. 60/945,568,
filed Jun. 21, 2007 (attorney docket number 33094-703.101), U.S.
Provisional Application No. 60/954,284, filed Aug. 6, 2007
(attorney docket number 33094-703.102), U.S. Provisional
Application No. 61/032,910, filed Feb. 29, 2008 (attorney docket
number 33094-703.103), and U.S. Provisional Application No.
61/042,187, filed Apr. 3, 2008 (attorney docket number
33094-703.104), each of which applications is incorporated herein
in its entirety by reference.
BACKGROUND OF THE INVENTION
[0002] Vascular Leak Syndrome (VLS) arises from protein-mediated
damage to the vascular endothelium. In the case of recombinant
proteins, immunotoxins and fusion toxins, the damage is initiated
by the interaction between therapeutic proteins and vascular
endothelial cells.
[0003] The mechanisms underlying VLS are unclear and likely involve
a cascade of events which are initiated in endothelial cells (ECs)
and involve inflammatory cascades and cytokines (Engert et al., In:
Clinical Applications of Immunotoxins, Frankel (ed.), 2:13-33,
1997. Freifelder, Physical Biochemistry, Second Edition, pages
238-246). VLS has a complex etiology involving damage to vascular
endothelial cells (ECs) and extravasation of fluids and proteins
resulting in interstitial edema, weight gain and, in its most
severe form, kidney damage, aphasia, and pulmonary edema (Sausville
and Vitetta, In: Monoclonal Antibody-Based Therapy of Cancer,
Grossbard (ed.), 4:81-89, 1997; Baluna and Vitetta, "Vascular leak
syndrome: A side effect of immunotherapy," Immunopharmacology,
37:117-132, 1996; Engert et al., 1997 supra).
[0004] It was reported that one of the VLS motifs found in ricin
toxin, the "LDV" motif, essentially mimics the activity of a
subdomain of fibronectin which is required for binding to the
integrin receptor. Integrins mediate cell-to-cell and
cell-to-extracellular matrix interactions (ECM). Integrins function
as receptors for a variety of cell surface and extracellular matrix
proteins including fibronectin, laminin, vitronectin, collagen,
osteospondin, thrombospondin and von Willebrand factor. Integrins
play a significant role in the development and maintenance of
vasculature and influence endothelial cell adhesiveness during
angiogenesis. Further, it was reported that the ricin "LDV" motif
can be found in a rotavirus coat protein, and this motif is
important for cell binding and entry by the virus. (Coulson, et
al., Proc. Natl. Acad. Sci. USA, 94(10): 5389-5494 (1997)). Thus,
it appears to be a direct link between endothelial cell adhesion,
vascular stability and the VLS motifs which mediate ricin binding
to human vascular endothelial cells (HUVECs) and vascular leak.
[0005] Mutant deglycosylated ricin toxin A chains (dgRTAs) were
constructed in which this motif was removed by conservative amino
acid substitution, and these mutants illustrated fewer VLS effects
in a mouse model (Smallshaw et al. Nat. Biotechnol., 21(4):387-91
(2003)). However, the majority of these constructs yielded dgRTA
mutants that were not as cytotoxic as wild type ricin toxin,
suggesting that significant and functionally critical structural
changes in the ricin toxophore resulted from the mutations. It
should also be noted that no evidence was provided to suggest that
the motifs in dgRTA mediated HUVEC interactions and VLS in any
other protein. Studies revealed that the majority of the mutant
dgRTAs were much less effective toxophores and no evidence was
provided to suggest that fusion toxins could be assembled using
these variant toxophores.
[0006] VLS is often observed during bacterial sepsis and may
involve IL-2 and a variety of other cytokines (Baluna and Vitetta,
J. Immunother., (1999) 22(1):41-47). VLS is also observed in
patients receiving protein fusion toxin or recombinant cytokine
therapy. VLS can manifest as hypoalbuminemia, weight gain,
pulmonary edema and hypotension. In some patients receiving
immunotoxins and fusion toxins, myalgia and rhabdomyolysis result
from VLS as a function of fluid accumulation in the muscle tissue
or the cerebral microvasculature (Smallshaw et al., Nat.
Biotechnol. 21(4):387-91 (2003)). VLS has occurred in patients
treated with immunotoxins containing ricin A chain, saporin,
pseudomonas exotoxin A and diphtheria toxin (DT). All of the
clinical testing on the utility of targeted toxins, immunotoxins
and recombinant cytokines reported that VLS and VLS-like effects
were observed in the treatment population. VLS occurred in
approximately 30% of patients treated with DAB.sub.389IL-2 (Foss et
al., Clin Lymphoma 1(4):298-302 (2001), Figgitt et al., Am J Clin
Dermatol., 1(1):67-72 (2000)). DAB.sub.389IL-2, is interchangeably
referred to in this application as DT.sub.387-IL2, is a protein
fusion toxin comprised of the catalytic (C) and transmembrane (T)
domains of DT (the DT toxophore), genetically fused to interleukin
2 (IL-2) as a targeting ligand. [Williams et al., Protein Eng.,
1:493-498 (1987); Williams et al., J. Biol. Chem., 265:11885-11889
(1990); Williams et al., J. Biol. Chem., 265 (33):20673-20677,
Waters et al., Ann. New York Acad. Sci., 30(636):403-405, (1991);
Kiyokawa. et al., Protein Engineering, 4(4):463-468 (1991); Murphy
et al., In Handbook of Experimental Pharmacology, 145:91-104
(2000)].
[0007] VLS has also been observed following the administration of
IL-2, growth factors, monoclonal antibodies and traditional
chemotherapy. Severe VLS can cause fluid and protein extravasation,
edema, decreased tissue perfusion, cessation of therapy and organ
failure. [Vitetta et al., Immunology Today, 14:252-259 (1993);
Siegall et al., Proc. Natl. Acad. Sci., 91(20):9514-9518 (1994);
Baluna et al., Int. J. Immunopharmacology, 18(6-7):355-361 (1996);
Baluna et al., Immunopharmacology, 37(2-3):117-132 (1997); Bascon,
Immunopharmacology, 39(3):255 (1998)].
[0008] Thus, there is a need to design modified diphtheria toxins
that cause reduced vascular leak syndrome compared to wild-type
diphtheria toxin.
SUMMARY OF THE INVENTION
[0009] Provided herein are modified diphtheria toxins, fusion
proteins containing the modified diphtheria toxins, compositions
thereof, methods of making modified diphtheria toxins and methods
of treating diseases such as malignant diseases or non-malignant
diseases with modified diphtheria toxins.
[0010] Provided herein are compositions of modified diphtheria
toxins, said modified diphtheria toxin comprising an amino acid
sequence as recited in SEQ ID NO. 2 or 149 with one or more amino
acid modifications therein, wherein at least one amino acid
modification is made within an (x)D(y) motif in a region selected
from among residues 7-9, 29-31 and 290-292 of SEQ ID NO 2 or 149,
and said modified diphtheria toxin has cytotoxicity comparable to
an unmodified diphtheria toxin. In one embodiment, a modification
at position (x) is a substitution of V or I by an amino acid
residue selected from among F, C, M, T, W, Y, P, H, E, Q, D, N, K,
R, and a modified or unusual amino acid from Table 1. In another
embodiment, a modification at position D is a substitution of D by
an amino acid residue selected from among I, V, L, F, C, M, A, G,
T, W, Y, P, H, Q, N, K, R and a modified or unusual amino acid from
Table 1. In another embodiment, a modification at position (y) is a
substitution of S by an amino acid residue selected from among I,
F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K, R and a modified or
unusual amino acid from Table 1. In one embodiment, a modified
diphtheria toxin comprises two, three or more modifications in one
or more (x)D(y) motifs.
[0011] Unmodified diphtheria toxins can have, for example, an amino
acid sequence of SEQ ID NO: 2, 149 or an amino acid sequence of any
one of SEQ ID NOS: 4-147.
[0012] In one embodiment, a modified diphtheria toxin contains one
or more modifications selected from among V7T, V7N, V7D, D8N, S9A,
S9T, S9G, V29N, V29D, V29T, D30N, S31 G, S31N, I290T, S292A, S292G
and S292T.
[0013] In one embodiment, a modified diphtheria toxin contains two
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N, V7N V29T,
V7N V29D, V7T V29N, V7T V29T or V7T V29D.
[0014] In one embodiment, a modified diphtheria toxin contains
three modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0015] Compositions comprising modified diphtheria toxins exhibit
(have) reduced binding activity to human vascular endothelial cells
(HUVECs) compared to an unmodified diphtheria toxin. Such
compositions can further comprise a non-diphtheria toxin
polypeptide including, but not limited to, an antibody or an
antigen-binding fragment thereof, EGF, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, INF.alpha., INF.gamma., GM-CSF, G-CSF, M-CSF, TNF, VEGF,
Ephrin, BFGF and TGF. The non-diphtheria toxin polypeptide can also
be a fragment of such polypeptides, such as a cell-binding portion
thereof. In one embodiment, the modified toxin is fusion toxin
wherein the cell binding domain is an antibody or antigen-binding
fragment thereof. An antibody can be, for example, monoclonal,
polyclonal, humanized, genetically engineered, or grafted. An
antigen-binding fragment can be, for example, a Fab, Fab.sub.2,
F(ab').sub.2, scFv, scFv2, single chain binding polypeptide,
V.sub.H, or V.sub.L. In a further embodiment, the antibody or
antigen binding fragment thereof binds to a B-cell surface molecule
such as, for example, the B-cell surface molecule CD19 or CD22.
Alternatively, the antibody or antigen binding fragment thereof,
binds to the ovarian receptor MISIIR (Mullerian Inhibitory
Substance type II receptor). In one embodiment, the non-diphtheria
toxin polypeptide is IL-2 or a cell-binding portion thereof, or
IL-3 or a cell-binding portion thereof.
[0016] Provided herein are fusion proteins of a polypeptide
toxophore from a modified diphtheria toxin, and a non-diphtheria
toxin polypeptide, said polypeptide toxophore comprising a
diphtheria toxin having an amino acid sequence as recited in SEQ ID
NO. 2 with one or more amino acid modifications therein, wherein at
least one amino acid modification is made within an (x)D(y) motif
in a region selected from among residues 7-9, 29-31 and 290-292 of
SEQ ID NO 2 or 149, and said modified diphtheria toxin has
cytotoxicity comparable to an unmodified diphtheria toxin. In one
embodiment, a modification at position (x) is a substitution of V
or I by an amino acid residue selected from among F, C, M, T, W, Y,
P, H, E, Q, D, N, K, R, and a modified or unusual amino acid from
Table 1. In one embodiment, a modification at position D is a
substitution of D by an amino acid residue selected from among I,
V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R and a modified or
unusual amino acid from Table 1. In one embodiment, a modification
at position (y) is a substitution of S by an amino acid residue
selected from among I, F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K,
R and a modified or unusual amino acid from Table 1. In one
embodiment, a modified diphtheria toxin comprises two, three or
more modifications in one or more (x)D(y) motifs.
[0017] The fusion proteins have reduced binding to human vascular
endothelial cells compared to, for example, a diphtheria toxin
molecule having a sequence of SEQ ID NO: 1, 2 or 200.
[0018] In one embodiment, a modified diphtheria toxin contains one
or more modifications selected from among V7T, V7N, V7D, D8N, S9A,
S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31N, I290T, S292A, S292G
and S292T.
[0019] In one embodiment, a modified diphtheria toxin contains two
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N, V7N V29T,
V7N V29D, V7T V29N, V7T V29T or V7T V29D.
[0020] In one embodiment, a modified diphtheria toxin contains
three modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0021] The non-diphtheria toxin polypeptide can be, for example, an
antibody or an antigen-binding fragment thereof, EGF, IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-14, IL-15, INF.alpha., INF.gamma., GM-CSF, G-CSF, M-CSF,
TNF, VEGF, Ephrin, BFGF, TGF, and a cell-binding portion thereof.
In one embodiment, the non-diphtheria toxin polypeptide is IL-2 or
a cell-binding portion thereof.
[0022] Provided herein are pharmaceutical compositions comprising a
fusion protein and a pharmaceutically acceptable carrier or
excipient.
[0023] Provided herein is a method for treating malignant diseases
and non-malignant diseases such as GVHD in a mammal comprising
administering a therapeutically effective amount of a
pharmaceutical composition described herein to said mammal.
[0024] Malignant diseases can be a blood cancer. Malignant diseases
can be a solid tumor. Malignant diseases also can be a metastasis.
Exemplary blood cancers include, but are not limited to, acute
myelogenous leukemia, cutaneous T-cell lymphoma,
relapsed/refractory T-cell non-Hodgkin lymphoma,
relapsed/refractory B-cell non-Hodgkin lymphoma, panniculitic
T-cell lymphoma, extranodal natural killer/T cell lymphoma, nasal
type, chronic lymphocytic leukemia, solid tumor and human T-cell
lymphotrophic virus 1-associated acute T cell leukemia/lymphoma.
Exemplary solid tumors include, but are not limited to, those of a
tissue or organ selected from among skin, melanoma, lung, pancreas,
breast, ovary, colon, rectum, stomach, thyroid, laryngeal,
prostate, colorectal, head, neck, eye, mouth, throat, esophagus,
chest, bone, testicular, lymph, marrow, bone, sarcoma, renal, sweat
gland, liver, kidney, brain, gastrointestinal tract, nasopharynx,
genito-urinary tract, muscle, and the like tissues. Metastasis
include, but are not limited to, metastatic tumors of any of the
solid tumors described.
[0025] Non-malignant diseases include, for example, GVHD, aGVHD and
psoriasis.
[0026] Provided herein is a method of enhancing activity of an
anti-cancer agent (e.g., RNA transfected DCs, anti-CLTA4
antibodies, MISIIR scFvs, etc.), by administering a DT variant-IL2
fusion protein described herein. In one embodiment, a DT
variant-IL2 fusion protein is administered followed by
administration of the anti-cancer agent. In one non-limiting
example, the DT variant-IL2 fusion protein is administered at least
four (4) days prior to the anti-cancer agent.
[0027] Also provided herein is a method of treating a metastatic
cancer via reduction or elimination of Tregs by administering an
anti-cancer agent (e.g., RNA transfected DCs, anti-CLTA4
antibodies, MISIIR scFvs, etc.) and a DT variant-IL2 fusion protein
described herein. Metastatic tumors include, for example,
metastatic renal cell carcinoma, metastatic prostate cancer,
metastatic ovarian cancer and metastatic lung cancer. In one
embodiment, a DT variant-IL2 fusion protein is administered
followed by administration of the anti-cancer agent. In one
non-limiting example, the DT variant-IL2 fusion protein is
administered at least four (4) days prior to the anti-cancer
agent.
[0028] In another aspect, provided herein is a method of treating a
prostate tumor, an ovarian tumor, a lung tumor or a melanoma via
reduction or elimination of Tregs by administering an anti-cancer
agent (e.g., RNA transfected DCs, anti-CLTA4 antibodies, MISIIR
scFvs, etc.) and a DT variant-IL2 fusion protein described herein.
In one embodiment, a DT variant-IL2 fusion protein is administered
followed by administration of the anti-cancer agent. In one
non-limiting example, the DT variant-IL2 fusion protein is
administered at least four (4) days prior to the anti-cancer
agent.
[0029] Provided herein is a method for making a composition
comprising the steps of: (a) constructing a vector comprising a
nucleic acid sequence which encodes a polypeptide having an amino
acid sequence of any of SEQ ID NOS: 4-147 or a polypeptide having
two or more modifications of SEQ ID NOS: 4-147, and (b) causing
said polypeptide to be expressed in a host cell comprising said
vector. In one embodiment, a composition produced by such a method,
wherein said composition has a reduced binding activity to human
vascular endothelial cells (HUVEC) compared to a DT molecule having
a sequence of SEQ ID NO: 2 or 149.
[0030] Provided herein is a method for making a modified diphtheria
toxin having a reduced binding activity to human vascular
endothelial cells (HUVEC) compared to an unmodified diphtheria
toxin, said method comprising the step of: (a) constructing a
vector comprising a nucleic acid sequence encoding a modified
diphtheria toxin, said modified diphtheria toxin comprising a
diphtheria toxin having an amino acid sequence as recited in SEQ ID
NO: 2 or 149 with one or more amino acid modifications therein,
wherein at least one amino acid modification is made within an
(x)D(y) motif in a region selected from the group consisting of
residues 7-9, 29-31 and 290-292 of SEQ ID NO: 2 or 149, and said
modified diphtheria toxin has cytotoxicity comparable to an
unmodified diphtheria toxin, wherein a modification at position (x)
is a substitution of V or I by an amino acid residue selected from
among F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, and a modified or
unusual amino acid from Table 1; or wherein a modification at
position D is a substitution of D by an amino acid residue selected
from among I, V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R and a
modified or unusual amino acid from Table 1; or wherein a
modification at position (y) is a substitution of S by an amino
acid residue selected from among I, F, C, M, A, G, T, W, Y, P, H,
E, Q, D, N, K, R and a modified or unusual amino acid from Table 1;
or a combination of two, three or more modifications in one or more
(x)D(y) motifs, and wherein said modified diphtheria toxin has
cytotoxicity comparable to that of a diphtheria toxin having a
sequence of SEQ ID NO: 2 or 149; and (b) causing said polypeptide
to be expressed in a host cell comprising said vector.
[0031] Unmodified diphtheria toxins can have, for example, an amino
acid sequence of SEQ ID NO: 2, 149 or an amino acid sequence of any
one of SEQ ID NOS: 4-147.
[0032] In one embodiment, a modified diphtheria toxin contains one
or more modifications selected from among V7T, V7N, V7D, D8N, S9A,
S9T, S9G, V29N, V29D, V29T, D30N, S31 G, S31N, I290T, S292A, S292G
and S292T.
[0033] In one embodiment, a modified diphtheria toxin contains two
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N, V7N V29T,
V7N V29D, V7T V29N, V7T V29T or V7T V29D.
[0034] In one embodiment, a modified diphtheria toxin contains
three modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0035] Provided herein is a fusion protein comprising a modified
diphtheria toxin made by such a method and a non-diphtheria toxin
polypeptide, wherein said non-diphtheria toxin polypeptide is
selected from among an antibody or antigen-binding fragment
thereof, EGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, INF.alpha., INF.gamma.,
GM-CSF, G-CSF, M-CSF, TNF, VEGF, Ephrin, BFGF, TGF, and a
cell-specific binding portion thereof. In one embodiment, the
non-diphtheria toxin polypeptide is, for example, IL-2 or a
cell-specific binding portion thereof, or IL-3 or a cell-specific
binding portion thereof.
INCORPORATION BY REFERENCE
[0036] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1. Wild-type .DELTA.R DT (diamonds) inhibited
transcription/translation of T7-luc plasmid to a greater extent
than the null construct (asterisk) in an IVTT assay.
[0038] FIG. 2. DT specific antibodies detected DT bound to the
surface of endothelial cells. Binding of a DT variant (DT-Glu52;
CRM mutant) to HUVEC cells and detection by antibodies using FACS
analysis. Black bars are results using no binding agents. Diagonal
hatching indicates assay controls using various conditions as
described. Cross-hatching indicates DT variant in the presence of
detection antibodies (DT+Goat pAb anti-DT
(Serotec)+anti-gt-PE).
[0039] FIG. 3. Binding of ONTAK-A1488 and DT-Glu52-A1488 to HUVEC
cells--detection by antibody by FACS. ONTAK.RTM.-A1488 6:1 D:P
(yellow top line; diamonds); DT-Glu52-A1488 3:1 D:P (red middle
line; circles); and DT-Glu52-A1488 2:1 D:P (green bottom line;
triangles).
[0040] FIG. 4. HUVEC cells were tested for IL-2R expression by
FACS: it was confirmed that ONTAK.RTM.-A1488 is not binding via
IL-2 receptors.
[0041] FIG. 5. Illustrates cell membrane integrity assays using
propidium iodide and FACS to measure loss of integrity of cell
membrane after short incubation with toxins. ONTAK.RTM. (diamonds),
DT-Glu52 (squares) and rhIL-2 (triangles). ONTAK (containing 4 VLS
motifs) appears to cause more membrane damage than either DT-Glu52
(3 VLS motifs) or rhIL-2 (1 VLS motif).
[0042] FIG. 6. Cytotoxicity assays are utilized to confirm activity
of DT-IL2 T cell epitope and VLS variant leads selected in IVTT
assays. ONTAK.RTM. (diamonds), recombinant human IL-2 (rhIL-2;
squares) and control (triangles) illustrate that ONTAK.RTM. is
cytotoxic.
[0043] FIG. 7. Illustrates ADP Ribosylation activity of variants
relative to wild-type (WT). Threshold for the assay was 0.5. Bars
marked with "!" or "*" indicate statistically significant
results.
[0044] FIG. 8. Inhibition of in vitro transcription/translation of
target T7-luciferase plasmid, by wild-type and epitope (EP)
variants of DT, was measured using the T7-coupled reticulocyte
lysate kit and SteadyGlo chemiluminescence reagents (Promega).
IC.sub.50s were determined and value for wild-type DT was divided
by the value for each epitope variant to calculate the activity
ratio plotted (Y axis). Mean values from replicate experiments
(n=3) are shown. 1=WT activity. 0.5=threshold of minimum acceptable
activity.
[0045] FIG. 9. Shows the relative activities of VLS DT variants
compared to wild type DT in the inhibition of protein
synthesis.
[0046] FIG. 10. Shows binding of labeled VLS variants to HUVEC
cells. DT389-IL2 is shown as closed diamonds (.diamond-solid.),
DT382 is shown as closed squares (.box-solid.), DT382(V7N V29T
S292T) is shown as closed triangles (.tangle-solidup.), DT382(V7N
V29T I290N) is shown as an "x," and BSA is shown as a closed circle
( ).
[0047] FIG. 11. Binding of ONTAK-488, control DT(.DELTA.R)488 and
BSA-488 to HUVEC cells--detection by antibody by FACS.
ONTAK.RTM.488 2.8:1 D:P (top line; plus sign "+"); control
DT(.DELTA.R)-488 3:1 D:P (second top line; circles " "); BSA-488
7:1 D:P (middle line; asterisks "*"), BSA-488 5:1 D:P (second
bottom line; "x") and BSA-488 2.6:1 D:P (bottom line; triangles
".tangle-solidup.").
[0048] FIG. 12. Provides amino acid sequences of wild type DT382,
DT382 variants, and null construct DT382(G53E). Underlined
sequences are vector/tag sequences; enterokinase cleavage site
highlighted in italicized text; and mutations from WT sequences are
shown in bold text.
DETAILED DESCRIPTION OF THE INVENTION
[0049] It is to be understood that this application is not limited
to particular formulations or process parameters, as these may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting. Further, it is understood
that a number of methods and materials similar or equivalent to
those described herein can be used in the practice of the present
invention(s).
[0050] In accordance with the present application, there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989);
"Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R.
M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes
I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology"
Volumes I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide
Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.
D. Hames & S. J. Higgins eds. (1985)]; "Transcription And
Translation" [B. D. Hames & S. J. Higgins, eds. (1984)];
"Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized
Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical
Guide To Molecular Cloning" (1984), each of which is specifically
incorporated herein by reference in its entirety.
I. Overview
[0051] Cell damage, particularly endothelial cell damage, whether
produced by toxins, such as from snake bites or molecules causing
septic shock, or therapeutic agents, such as immunotoxins or
interleukins, remains a problem for patients.
[0052] VLS is often observed during bacterial sepsis and may
involve IL-2 and a variety of other cytokines (Baluna and Vitetta,
Immunopharmacology, 37:117-132, 1996). The mechanisms underlying
VLS are unclear and are likely to involve a cascade of events which
are initiated in endothelial cells (ECs) and involve inflammatory
cascades and cytokines (Engert et al., In: Clinical Applications of
Immunotoxins, Frankel (ed.), 2:13-33, 1997). VLS has a complex
etiology involving damage to vascular endothelial cells (ECs) and
extravasation of fluids and proteins resulting in interstitial
edema, weight gain and, in its most severe form, kidney damage,
aphasia, and pulmonary edema (Sausville and Vitetta, In: Monoclonal
Antibody-Based Therapy of Cancer, Grossbard (ed.), 4:81-89, 1997;
Baluna and Vitetta, Immunopharmacology, 37:117-132, 1996; Engert et
al., In: Clinical Applications of Immunotoxins, Frankel (ed.),
2:13-33, 1997). Vascular leak syndrome (VLS) has been a major
problem with all immunotoxins thus far tested in humans, as well as
cytokines such as interleukin 2 (IL-2), TNF and adenovirus vectors
(Rosenberg et al., N. Engl. J. Med., 316:889-897, 1987; Rosenstein
et al., J. Immunol., 137:1735-1742, 1986).
[0053] Antibody-conjugated peptides from ricin toxin A chain
containing a modified sequence at residues L74, D75, V76, exhibited
reduced (Vitetta et al. U.S. Pat. No. 6,566,500). Thus, it is
contemplated that one or more amino acid deletion(s) or mutation(s)
of the (x)D(y) sequence(s), and/or one or more flanking residues of
diphtheria toxin, may reduce or prevent the ability of DT molecules
comprising these sequences to induce EC damage. It is expected that
one or more polypeptides comprising at least one mutated motif
and/or one or more flanking residues can be created that reduce or
eliminate the EC damaging activity of such agents.
[0054] Described herein below are compositions with reduced VLS
promoting abilities based upon mutations in the (x)D(y) or (x)D(y)T
sequences within polypeptides, which remove or alter such
sequences, respectively, and their methods of use. Thus, it will be
understood that all methods described herein for producing
polypeptides with reduced VLS promoting ability will be applied to
produce polypeptides with reduced EC damaging activity. All such
methods, and compositions identified or produced by such methods as
well as equivalents thereof, are encompassed by the present
invention.
[0055] In certain aspects, the application provides the use of a
modified diphtheria toxin composition that has at least one amino
acid of a sequence comprising (x)D(y) and/or (x)D(y)T removed or
altered, relative to the sequence of an unmodified diphtheria toxin
composition, for the manufacture of a medicament for the treatment
of a disease, including but not limited to malignant diseases such
as, for example acute myelogenous leukemia, cutaneous T-cell
lymphoma, relapsed/refractory T-cell non-Hodgkin lymphoma,
relapsed/refractory B-cell non-Hodgkin lymphoma, panniculitic
T-cell lymphoma, extranodal natural killer/T cell lymphoma, nasal
type, chronic lymphocytic leukemia, and human T-cell lymphotrophic
virus 1-associated acute T cell leukemia/lymphoma; non-malignant
diseases such as, for example, graft versus host disease and damage
to endothelial cells (i.e., VLS) during the progression of such
diseases.
[0056] Clearly, further development of diphtheria toxin as well, as
well as cytokines and fusion proteins thereof as clinical agents
would be greatly facilitated by the elimination or reduction of
VLS. If VLS could be avoided or reduced it would permit the use of
much higher doses of a variety of therapeutic agents without the
dose limiting side effects currently encountered.
[0057] Reduction or elimination of VLS as a side effect would
represent a significant advancement as it would improve the "risk
benefit ratio" of protein therapeutics, and in particular, the
immunotoxin and fusion toxin subclasses of protein therapeutics.
(Baluna et al., Int. J. Immunopharmacology, 18(6-7):355-361 (1996);
Baluna et al., Immunopharmacology, 37(No. 2-3):117-132 (1997);
Bascon, Immunopharmacology, 39(3): 255 (1998). The ability to
develop fusion proteins, single chain molecules comprised of a
cytotoxin and unique targeting domain (cell binding domains in the
case of immunotoxins) could facilitate the development of the
therapeutic agents for autoimmune diseases, such as rheumatoid
arthritis and psoriasis transplant rejection and other
non-malignant medical indications. (Chaudhary et al., Proc. Natl.
Acad. Sci. USA, 87(23):9491-9494 (1990); Frankel et al., In
Clinical Applications of Immunotoxins Scientific Publishing
Services, Charleston S.C., (1997), Knechtle et al.,
Transplantation, 15(63):1-6 (1997); Knechtle et al., Surgery,
124(2): 438-446 (1998); LeMaistre, Clin. Lymphoma, 1:S3740 (2000);
Martin et al., J. Am. Acad. Dermatol., 45(6):871-881, 2001)).
DAB.sub.389IL-2 (ONTAK.RTM.) is currently the only FDA approved
protein fusion toxin and employs a DT toxophore and the cytokine
IL-2 to target IL-2 receptor bearing cells and is approved for the
treatment of cutaneous T-cell lymphoma (CTCL) (Figgitt et al., Am.
J. Clin. Dermatol., 1(1):67-72 (2000); Foss, Clin. Lymphoma,
1(4):298-302 (2001); Murphy et al., In Bacterial Toxins: Methods
and Protocols, Holst O, ed, Humana Press, Totowa, N.J., pp. 89-100
(2000)). ONTAK.RTM. is variously referred to as denileukin
diftitox, DAB.sub.389-IL-2, or Onzar. Its structure is comprised
of, in order, a methionine residue, residues 1-386 of native DT,
residues 484-485 of native DT, and residues 2-133 of IL-2 (SEQ ID
NO: 148). Hence, full length ONTAK.RTM. contains 521 amino acids.
It should be noted that, as a result of the methionine residue
added at the N terminus of ONTAK.RTM., numbering in the sequence of
diphtheria is out of register with that of ONTAK.RTM. by one.
[0058] A number of other toxophores, most notably ricin toxin and
pseudomonas exotoxin A, have been employed in developing both
immuntoxins, fusion toxins and chemical conjugates; however, these
molecules have not successfully completed clinical trials and all
exhibit VLS as a pronounced side effect (Kreitman, Adv. Pharmacol.,
28:193-219 (1994); Puri et al., Cancer Research, 61:5660-5662
(1996); Pastan, Biochim Biophys Acta., 24:1333(2):C.sub.1-6 (1997);
Frankel et al., Supra (1997); Kreitman et al., Current Opin.
Invest. Drugs, 2(9): 1282-1293 (2001)). The modifications described
herein for diphtheria toxin can be extrapolated to other toxins
such as, for example, ricin and pseudomonas exotoxin A.
II. Diphtheria Toxin
[0059] Diphtheria toxin (DT) is composed of three domains: a
catalytic domain; a transmembrane domain; and a receptor binding
domain (Choe et al. Nature, 357:216-222 (1992)). The nucleic acid
and amino acid sequences of native DT were described by Greenfield
et al. PNAS (1983) 80: 6853-6857 in FIG. 2. Native DT is targeted
to cells that express heparin binding epidermal growth factor-like
receptors (Naglish et al., Cell, 69:1051-1061 (1992)). The first
generation targeted toxins were initially developed by chemically
cross-linking novel targeting ligands to toxins such as DT or
mutants of DT deficient in cell binding (e.g. CRM45). (Cawley, Cell
22:563-570 (1980); Bacha et al., Proc. Soc. Exp. Biol. Med.,
181(1):131-138 (1986); Bacha et al., Endocrinology,
113(3):1072-1076 (1983); Bacha et al., J. Biol. Chem,
258(3):1565-1570 (1983)). The native cell binding domain or a
cross-linked ligand that directs the DT toxophore to receptors on a
specific class of receptor-bearing cells must possess intact
catalytic and translocation domains. (Cawley et al., Cell,
22:563-570 (1980); vanderSpek et al., J. Biol. Chem.,
5:268(16):12077-12082 (1993); vanderSpek et al., J. Biol. Chem.,
7(8):985-989 (1994); vanderSpek et al., J. Biol. Chem., 7(8)985-989
(1994); Rosconi, J. Biol. Chem., 10; 277(19):16517-161278 (2002)).
These domains are critical for delivery and intoxification of the
targeted cell following receptor internalization (Greenfield et
al., Science, 238(4826)536-539 (1987)). Once the toxin, toxin
conjugate or fusion toxin has bound to the cell surface receptor,
the cell internalizes the toxin bound receptor via endocytic
vesicles. As the vesicles are processed, they become acidified, and
the translocation domain of the DT toxophore undergoes a structural
reorganization which inserts the 9 transmembrane segments of the
toxin into the membrane of the endocytic vesicle. This event
triggers the formation of a productive pore through which the
catalytic domain of the toxin is threaded. Once translocated, the
catalytic domain which possess the ADP-ribosyltransferase activity,
is released into the cytosol of the targeted cell, it is free to
poison translation thus effecting the death of the cell (reviewed
in vanderSpek et al., Methods in Molecular Biology, Bacterial
Toxins: methods and Protocols, 145:89-99, Humana press, Totowa,
N.J., (2000)).
[0060] A. Modified Diphtheria Toxins
[0061] Fewer than ten molecules of DT will kill a cell if they
enter the cytosol (although many times that number must bind to the
cell surface because the entry process is inefficient). This
extraordinary potency initially led to the concern that such
poisons were too powerful to control. However, toxins such as DT
can be rendered innocuous (except when directed to the target
cells) simply by removing or modifying their cell-binding domain or
subunit. The remaining portion of the toxin (lacking a cell-binding
domain) can then be coupled to a ligand (e.g., a polypeptide or
portion thereof containing a cell-binding domain) that targets the
toxic portion to a target cell. By selecting a polypeptide or
portion thereof containing a cell-binding domain lacking unwanted
cross-reactivity, fusion proteins are safer and have fewer
non-specific cytotoxic effects than most conventional anti-cancer
drugs. The other main attraction of toxins such as DT is that
because they are inhibitors of protein synthesis, they kill resting
cells as efficiently as dividing cells. Hence, tumor or infected
cells that are not in cycle at the time of treatment do not escape
the cytotoxic effect of a fusion protein.
[0062] Toxins such as DT often contain two disulfide-bonded chains,
the A and B chains. The B chain carries both a cell-binding region
and a translocation region, which facilitates the insertion of the
A chain through the membrane of an acid intracellular compartment
into the cytosol. The A chain then kills the cell after
incorporation. For their use in vivo, the ligand and toxin are
coupled in such a way as to remain stable while passing through the
bloodstream and the tissues and yet be labile within the target
cell so that the toxic portion can be released into the
cytosol.
[0063] However, it may be desirable from a pharmacologic standpoint
to employ the smallest molecule possible that nevertheless provides
an appropriate biological response. One may thus desire to employ
smaller A chain peptides or other toxins which will provide an
adequate anti-cellular response.
[0064] In certain embodiments, diphtheria toxins or compounds
modified based on one or more of the (x)D(y) and/or (x)D(y)T motifs
or its flanking sequences can be used to inhibit VLS in vivo. Thus,
it is contemplated that such mutations that affects the (x)D(y)
sequence or flanking sequence can alter the ability of a
polypeptide to induce VLS or other abilities associated with these
sequences. In one non-limiting example, diphtheria toxin is
modified to inhibit VLS in vivo.
[0065] In order to produce diphtheria toxins or compounds that have
a reduced ability to induce VLS, it is contemplated that one or
more (up to, and including all) remaining (x)D(y) and/or (x)D(y)T
sequences have a reduced exposure to the surface of the
polypeptide. For example, it is contemplated that (x)D(y) and/or
(x)D(y)T sequences that are at least partly located in the
non-exposed portions of a polypeptide, or otherwise masked from
full or partial exposure to the surface of the molecule, would
interact less with cells, receptors or other molecules to promote
or induce VLS. Thus, the complete elimination of (x)D(y) and/or
(x)D(y)T sequences from the primary structure of the polypeptide
may not be necessary to produce toxins or molecules with a reduced
ability to induce or promote VLS. However, removal of all (x)D(y)
and/or (x)D(y)T sequences is contemplated to produce a composition
that has the least ability to induce or promote VLS.
[0066] To determine whether a mutation would likely produce a
polypeptide with a less exposed (x)D(y) and/or (x)D(y)T motif, the
putative location of the moved or added (x)D(y) and/or (x)D(y)T
sequence can be determined by comparison of the mutated sequence to
that of the unmutated polypeptide's secondary and tertiary
structure, as determined by such methods known to those of ordinary
skill in the art including, but not limited to, X-ray
crystallography, NMR or computer modeling. Computer models of
various polypeptide structures are also available in the literature
or computer databases. In a non-limiting example, the Entrez
database website (ncbi.nln.nih.gov/Entrez/) can be used to identify
target sequences and regions for mutagenesis. The Entrez database
is cross-linked to a database of 3-D structures for the identified
amino acid sequence, if known. Such molecular models can be used to
identify (x)D(y), (x)D(y)T and/or flanking sequences in
polypeptides that are more exposed to contact with external
molecules, than similar sequences embedded in the interior of the
polypeptide. (x)D(y), (x)D(y)T and/or flanking sequences that are
more exposed to contact with external molecules are more likely to
contribute to promoting or reducing VLS and other toxic effects
associated with these sequences and, thus, should be primary
targets for mutagenesis. The mutated or wild-type polypeptide's
structure could be determined by X-ray crystallography or NMR
directly before use in in vitro or in vivo assays, as would be
known to one of ordinary skill in the art.
[0067] Once an amino acid sequence comprising a (x)D(y) and/or
(x)D(y)T sequence is altered in a polypeptide, changes in its
ability to induce or promote at least one toxic effect can be
assayed using any of the techniques described herein or as known to
one of ordinary skill in the art.
[0068] As used herein, "alter," "altered," "altering," and
"alteration" of an amino acid sequence comprising a (x)D(y)
sequence or a (x)D(y)T sequence can include chemical modification
of an amino acid sequence comprising a (x)D(y) and/or a (x)D(y)T
sequence in a polypeptide as known to those of ordinary skill in
the art, as well as any mutation of such an amino acid sequence
including, but not limited to, insertions, deletions, truncations
or substitutions. Such changes can alter or modify (reduce) at
least one toxic effect (i.e., the ability to promote VLS, EC
damage, etc.) of one or more amino acid sequence(s) comprising a
(x)D(y) and/or (x)D(y)T sequences. As used herein an amino acid
sequence comprising a (x)D(y) sequence or a (x)D(y)T sequence can
contain at least one flanking sequence C- and/or N-terminal to a
(x)D(y) and/or a (x)D(y)T tri- or quatra-peptide sequence. Such an
"alteration" can be made in synthesized polypeptides or in nucleic
acid sequences that are expressed to produce mutated
polypeptides.
[0069] In one aspect, the alteration of an amino acid sequence
containing a (x)D(y) and/or a (x)D(y)T sequence is by removal of
the amino acid sequence. As used herein, "remove", "removed",
"removing" or "removal" of an amino acid sequence containing a
(x)D(y) and/or a (x)D(y)T sequence refers to a mutation in the
primary amino acid sequence that eliminates the presence of the
(x)D(y) and/or a (x)D(y)T tri- or quatra-peptide sequence, and/or
at least one native flanking sequence. The terms "removed" or
"lacks" are used interchangeably.
[0070] One aspect of the present application relates to genetically
modified polypeptides of diphtheria toxin (DT) having reduced
binding to human vascular endothelial cells (HUVECs). These
modified polypeptides are hereinafter referred to as modified DTs,
modified DT polypeptides or DT variants. The present application
provides for modified DT having one or more changes within the
(x)D(y) motifs of the DT polypeptide, i.e., at residues 6-8 (VDS),
residues 28-30 (VDS), and residues 289-291 (IDS) of the native DT
sequence (SEQ ID NO: 1), or at residues 7-9 (VDS), residues 29-31
(VDS), and residues 290-292 (IDS) of SEQ ID NO: 2 or 149. Since the
(x)D(y) motifs are referred to as "VLS motifs," the modified DT
polypeptides with one or more modified (x)D(y) motifs can be
referred to as "VLS-modified DT polypeptides."
[0071] With the identification of the (x)D(y) and the (x)D(y)T
motifs as inducing VLS, inducing apoptosis, and other effects, it
is possible that the creation of a new family of molecules of VLS
inhibitors will allow these molecules to exert maximal beneficial
effects. For example, a reduced toxicity of DT therapeutic agents
using the compositions and methods disclosed herein may allow
larger patient population to be treated or more advanced disease to
be treated (e.g., a cancer or graft versus host disease). In
certain embodiments, modified proteins or fusion proteins based on
the (x)D(y) and/or (x)D(y)T motif or its flanking sequences may be
used to inhibit VLS or other activities in vivo.
[0072] To produce peptides, polypeptides or proteins that lack the
(x)D(y) and/or (x)D(y)T sequence, one could delete or mutate the
conserved aspartic acid (D), substitute another amino acid for the
aspartic acid, or insert one or more amino acids at or adjacent to
its position. Modifications contemplated herein include a
substitution of the (D) residue in the sequence by an amino acid
residue selected from among isoleucine (I), valine (V), leucine
(L), phenylalanine (F), cysteine (C), methionine (M), alanine (A),
glycine (G), threonine (T), tryptophan (W), tyrosine (Y), proline
(P), histidine (H), glutamine (Q), asparagine (N), lysine (K),
arginine (R) and a modified or unusual amino acid from Table 1, as
a consequence of a deletion or mutation event.
TABLE-US-00001 TABLE 1 Abbreviation Amino acid Abbreviation Amino
acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad
3-Aminoadipic acid Hyl Hydroxylysine BAla .beta.-alanine,
.beta.-Amino- Ahyl Allo-Hydroxylysine propionic acid Abu
2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4--Aminobutyric
acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp 6-Aminocaproic
acid Ide Isodesmosine Ahe 2-Aminoheptanoic Aile Allo-Isoleucine
acid Aib 2-Aminoisobutyric MeGly N-Methylglycine, acid sarcosine
BAib 3-Aminoisobutyric MeIle N-Methylisoleucine acid Apm
2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric
MeVal N-Methylvaline acid Des Desmosine Nva Norvaline Dpm
2,2'-Diaminopropionic Nle Norleucine acid Dpr 2,3-Diaminopropionic
Orn Ornithine acid EtGly N-Ethylglycine
[0073] Alternatively the (x) residue could be deleted, substituted,
or moved by the insertion of one or more amino acids, to remove the
(x)D(y) and/or (x)D(y)T sequence. Modifications contemplated herein
include a substitution of the (x) residue in the sequence by an
amino acid residue selected from among phenylalanine (F), cysteine
(C), methionine (M), threonine (T), tryptophan (W), tyrosine (Y),
proline (P), histidine (H), glutamic acid (E), glutamine (Q),
aspartic acid (D), asparagine (N), lysine (K), arginine (R) and a
modified or unusual amino acid from Table 1 as a consequence of the
deletion or mutation event. For example, a V or I amino acid
residue as described is replaced with any of such amino acid
residues.
[0074] Or the (y) residue could be deleted, substituted, or moved
by the insertion of one or more amino acids, to remove the (x)D(y)
and/or (x)D(y)T sequence. An amino acid that may replace the (y)
residue in the sequence as a consequence of the deletion or
mutation event is, for example, isoleucine (I); phenylalanine (F);
cysteine/cystine (C); methionine (M); alanine (A); glycine (G);
threonine (T); tryptophan (W); tyrosine (Y); proline (P); histidine
(H); glutamic acid (E); glutamine (Q); aspartic acid (D);
asparagine (N); lysine (K); and arginine (R), and including, but
not limited to, those shown at Table 1.
[0075] In one embodiment, a modified diphtheria toxin contains one
or more modifications selected from among V7T, V7N, V7D, D8N, S9A,
S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31N, I290T, S292A, S292G
and S292T.
[0076] In one embodiment, a modified diphtheria toxin contains two
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N, V7N V29T,
V7N V29D, V7T V29N, V7T V29T or V7T V29D.
[0077] In one embodiment, a modified diphtheria toxin contains
three modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0078] Other residues that are positioned in the physical region,
three-dimensional space, or vicinity of the HUVEC binding site
and/or the (x)D(y) motif may be mutated or altered to abrogate,
reduce, or eliminate VLS. The amino acids targeted for mutation in
the flanking regions include amino acids on or near the surface of
a native DT protein. The alteration may remove or substitute a
charged residue in the region of a (x)D(y) motif, which may negate
or reverse the charge in a particular area on the surface of the
protein. The alteration may also change size and/or hydrophilic
nature of an amino acid in the physical region, space or vicinity
of the (x)D(y) sequence or active site of a protein. For example,
LDV constitutes the minimal active site in the CS1 domain of
fibronectin responsible for its binding to the .alpha.4.beta.1
integrin receptor (Makarem and Humphries, 1991; Wayner and Kovach,
1992; Nowlin et al., 1993). However, fibronectin (FN) does not
damage HUVECs. Instead, FN protects HUVECs from RTA-mediated damage
(Baluna et al., 1996). Unlike RTA, FN has a C-terminal LDV-flanking
proline instead of a threonine. In disintegrins, residues flanking
RGD play a role in ligand binding (Lu et al., 1996). The difference
between the ability of an LDV or homologue-containing molecule to
promote vascular integrity (e.g., FN) or disrupt it (e.g., DT) may
depend on the orientation, or availability for interaction (i.e.,
binding), of the LDV motif and hence, on flanking sequences.
Therefore, changes in one or more flanking residues of the (x)D(y)
sequence may enhance or reduce the ability of a molecule to promote
VLS. Further, changes that expose the (x)D(y) sequence to the
external surface of the protein so as to interact with other
proteins, such as receptors, would enhance VLS promoting activity,
while conformations that are less exposed may reduce VLS promoting
activity.
[0079] At least one mutation, chemical modification, movement or
other alteration in the N- or C-terminal flanking sequences of the
(x)D(y) and/or (x)D(y)T sequence may also produce polypeptides that
have a reduced ability to promote VLS. Such mutations or
alterations can occur in one or more residues which will not affect
the active site. In other embodiments, the mutations or alterations
can occur in one or more residues of from about 1, about 2, about
3, about 4, about 5, about 6 or more N-terminal and/or C-terminal
to the (x)D(y) tripeptide sequence. In other aspects, one or more
residues that are not adjacent to the (x)D(y) tripeptide may
contribute to the function of the (x)D(y) motif. Such residues may
be identified by their proximity to the tripeptide sequence in a
3-dimensional model, as described herein and as would be known to
one of ordinary skill in the art, and are contemplated for
alteration as part of a flanking sequence. Such alterations may
include any of those described above for altering the (x)D(y) and
(x)D(y)T sequences, as long as one or more "wild type" flanking
residues are altered, removed, moved, chemically modified, etc.
[0080] Such amino acid modifications can be assayed for the ability
to effectively deliver the catalytic domain of DT to a targeted
cell within the context of a fusion protein, and not reconstitute
an intact VLS motif. Provided herein are modified diphtheria toxins
that have a reduced ability to induce VLS; any remaining (x)D(y)
and/or (x)D(y)T sequences, if possible, are to have a reduced
exposure to the surface of the polypeptide.
[0081] For example, it is contemplated that (x)D(y) and/or (x)D(y)T
sequences that are at least partly located in the non-exposed
portions of a diphtheria toxin, or otherwise masked from full or
partial exposure to the surface of the molecule, would interact
less with cells, receptors or other molecules to promote or induce
VLS. Thus, it is contemplated that the complete elimination of
(x)D(y) and/or (x)D(y)T sequences from the primary structure of the
diphtheria toxin is not necessary to produce toxins or molecules
with a reduced ability to induce or promote VLS. However, in one
embodiment, all (x)D(y) and/or (x)D(y)T sequences are removed to
generate a composition that has the least ability to induce or
promote VLS.
[0082] To determine whether a mutation would likely produce a
modified diphtheria toxin with a less exposed (x)D(y) and/or
(x)D(y)T motif, the putative location of the moved or added (x)D(y)
and/or (x)D(y)T sequence could be determined by comparison of the
mutated sequence to that of the unmutated diphtheria toxin's
secondary and tertiary structure, as determined by such methods
known to those of ordinary skill in the art including, but not
limited to, X-ray crystallography, NMR or computer modeling.
Computer models of various polypeptide and peptide structures are
also available in the literature or computer databases. In a
non-limiting example, the Entrez database
(www.ncbi.nlm.nih.gov/Entrez/) can be used to identify target
sequences and regions for mutagenesis. The Entrez database is
cross-linked to a database of 3-D structures for the identified
amino acid sequence, if known. Such molecular models can be used to
identify (x)D(y), (x)D(y)T and/or flanking sequences in diphtheria
toxin that are more exposed to contact with external molecules,
(e.g. receptors) than similar sequences embedded in the interior of
the polypeptide or polypeptide. It is contemplated that (x)D(y),
(x)D(y)T and/or flanking sequences that are more exposed to contact
with external molecules are more likely to contribute to promoting
or reducing VLS and other toxic effects associated with these
sequences, and, thus, should be primary targets for mutagenesis. In
certain embodiments, when adding at least one (x)D(y), (x)D(y)T
and/or flanking sequence is desirable, regions of the protein that
are more exposed to contact with external molecules are preferred
as sites to add such a sequence. The mutated or wild-type
diphtheria toxin's structure could be determined by X-ray
crystallography or NMR directly before use in in vitro or in vivo
assays, as would be known to one of ordinary skill in the art.
[0083] Once an amino acid sequence comprising a (x)D(y) and/or
(x)D(y)T sequence is altered in a diphtheria toxin, changes in its
ability to promote at least one toxic effect can be assayed by any
of the techniques described herein or as would be known to one of
ordinary skill in the art. Methods of altering (changing) amino
acid sequences are described in more detail below and are known in
the art.
[0084] Modifications (changes) are those amino acid substitutions,
insertions or deletions which permit the alteration of a native
sequence or a previously modified sequence within these regions but
do not impair the cytotoxicity of a DT toxophore. These
modifications would not include those that regenerate the VDS/IDS
sequences responsible for mediating the interaction with
endothelial cells. Such non-native recombinant sequences therefore
comprise a novel series of mutants that maintain the native
function of the unique domains of diphtheria toxin while
significantly decreasing their ability to interact with vascular
endothelial cells.
[0085] Provided herein are modified diphtheria toxins, said
modified diphtheria toxin comprising an amino acid sequence as set
forth in SEQ ID NO: 2 or 149 with one or more amino acid
modifications therein, wherein at least one amino acid modification
is made within an (x)D(y) motif in a region selected from among
residues 7-9, 29-31 and 290-292 of SEQ ID NO: 2 or 149, and said
modified diphtheria toxin has cytotoxicity comparable to an
unmodified diphtheria toxin. In one embodiment, a modification at
position (x) is a substitution of V or I by an amino acid residue
selected from among F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, and a
modified or unusual amino acid from Table 1. In one embodiment, a
modification at position D is a substitution of D by an amino acid
residue selected from among I, V, L, F, C, M, A, G, T, W, Y, P, H,
Q, N, K, R and a modified or unusual amino acid from Table 1. In
one embodiment, a modification at position (y) is a substitution of
S by an amino acid residue selected from among I, F, C, M, A, G, T,
W, Y, P, H, E, Q, D, N, K, R and a modified or unusual amino acid
from Table 1. In another embodiment, a modified diphtheria toxin
has a combination of two, three or more modifications in one or
more (x)D(y) motifs.
[0086] In one embodiment, a modified diphtheria toxin contains one
or more modifications selected from among V7T, V7N, V7D, D8N, S9A,
S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31N, I290T, S292A, S292G
and S292T.
[0087] In one embodiment, a modified diphtheria toxin contains two
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N, V7N V29T,
V7N V29D, V7T V29N, V7T V29T or V7T V29D.
[0088] In one embodiment, a modified diphtheria toxin contains
three modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0089] Unmodified diphtheria toxins can have, for example, an amino
acid sequence of SEQ ID NO: 2, 149 or an amino acid sequence of any
one of SEQ ID NOS: 4-147. DT387 (SEQ ID NO: 2) is a truncated DT
protein comprising an N-terminal a methionine residue, and amino
acid residues 1-386 of the native DT protein which include the
catalytic domain and the translocation domain. DT389 (SEQ ID NO:
149) is a truncated DT protein including in order, a methionine
residue, residues 1-386 of native DT and residues 484-485 of native
DT. In one embodiment, DT variants contain at least one
modifications within one of the (x)D/E(y) motifs of the DT
molecule, i.e., within residues 7-9 (VDS), residues 29-31 (VDS),
and residues 290-292 (IDS) of SEQ ID NO:2 or 149 to eliminate
motifs that are associated with VLS and thereby reduce the clinical
adverse effects commonly associated with this syndrome. In one
embodiment, a modified diphtheria toxin having cytotoxicity
comparable to an unmodified diphtheria toxin refers to a modified
diphtheria toxin having cytotoxicity substantially similar to, or
with at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, or at least about 95% or more
cytotoxicity compared to an unmodified diphtheria toxin. Purified
DAB.sub.398IL-2 produced in E. coli generally yields an IC.sub.50
of between about 5.times.10.sup.-11 M to about 1.times.10.sup.-12
M. Thus, in another embodiment, a modified diphtheria toxin having
cytotoxicity comparable to an unmodified diphtheria toxin refers to
a modified diphtheria toxin having an IC.sub.50 of between about
5.times.10.sup.-11 M to about 1.times.10.sup.-12 M, of about
1.times.10.sup.-10 M to about 1.times..sup.-10 M, of about
1.times.10.sup.-9 M to about 1.times.10.sup.-10 M, or of about
1.times.10.sup.-8 M to about 1.times.10.sup.-9 M. Cytotoxicity of a
modified diphtheria toxin compared to an unmodified diphtheria
toxin can be tested in a cytotoxicity assay such as those described
below in the Examples.
[0090] Modified diphtheria toxins provided herein have reduced
binding activity to human vascular endothelial cells (HUVECs)
compared to an unmodified diphtheria toxin. Such compositions can
further comprise a non-diphtheria toxin polypeptide including, but
not limited to, an antibody or antigen-binding fragment thereof,
EGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, INF.alpha., INF.gamma., GM-CSF,
G-CSF, M-CSF, TNF, VEGF, Ephrin, BFGF, TGF or a cell-specific
binding ligand. The non-diphtheria toxin polypeptide can also be a
fragment of such polypeptides, such as a cell-binding portion
thereof. In one embodiment, the non-diphtheria toxin polypeptide is
IL-2 or a cell-binding portion thereof.
[0091] DT variants of the present application contain at least one
modifications within one of the (x)D(y) motifs of the DT molecule,
i.e., within residues 6-8 (VDS), residues 28-30 (VDS), and residues
289-291 (IDS) of SEQ ID NO: 1, or within residues 7-9 (VDS),
residues 29-31 (VDS), and residues 290-292 (IDS) of SEQ ID NO: 2 or
149 to eliminate motifs that are associated with VLS and thereby
reduce the clinical adverse effects commonly associated with this
syndrome. The modified DTs of the present application, however, are
as effective and efficient as DT387 in their ability to facilitate
the delivery of its catalytic domain to the cytosol of targeted
eukaryotic cells when incorporated into protein fusion toxins.
[0092] In addition to the modification in the (x)D(y) motifs, the
modified DTs can further comprise a deletion or substitution of
about 1 to about 30 amino acids, about 1 to about 10 amino acids,
or about 1 to about 3 amino acids of SEQ ID NO: 2 or 149, so long
as the truncated molecule retains the ability to translocate into
cells and kill target cells when the truncated molecule is fused
with a cell binding domain.
[0093] Provided herein is a modified diphtheria toxin having one or
more amino acid modifications therein, wherein at least one amino
acid modification is made within an (x)D(y) motif in a region
selected from the group consisting of residues 7-9, 29-31 and
290-292 of SEQ ID NO 2 or 149, and wherein said modified diphtheria
toxin has cytotoxicity comparable to an unmodified diphtheria
toxin. In one embodiment, a modification at position (x) is a
substitution of V or I by an amino acid residue selected from among
F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, and a modified or unusual
amino acid from Table 1. In another embodiment, a modification at
position D is a substitution of D by an amino acid residue selected
from among I, V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R and a
modified or unusual amino acid from Table 1. In another embodiment,
a modification at position (y) is a substitution of S by an amino
acid residue selected from among I, F, C, M, A, G, T, W, Y, P, H,
E, Q, D, N, K, R and a modified or unusual amino acid from Table 1.
In another embodiment, a modified diphtheria toxin has a
combination of two, three or more modifications in one or more
(x)D(y) motifs.
[0094] In one embodiment, a modified diphtheria toxin contains one
or more modifications selected from among V7T, V7N, V7D, D8N, S9A,
S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31N, I290T, S292A, S292G
and S292T.
[0095] In one embodiment, a modified diphtheria toxin contains two
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N, V7N V29T,
V7N V29D, V7T V29N, V7T V29T or V7T V29D.
[0096] In one embodiment, a modified diphtheria toxin contains
three modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0097] Modified diphtheria toxins can contain, about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about
20 modifications within one or more (x)D/E(y) motifs. Functional
activity of such modified diphtheria toxins can be tested in a
cytotoxicity assay or another assay described herein or known in
the art. Modified diphtheria toxins containing more than one
modification can be made, using the methods described herein, by
sequentially modifying amino acid residues and comparing activity
after each modification to the previously unmodified or previously
modified diphtheria toxin. Alternatively, modified diphtheria
toxins containing more than one modification can be made, using the
methods described herein, by modifying two or more amino acid
residues at the same time and comparing activity to the previously
unmodified diphtheria toxin.
[0098] Modified diphtheria toxins can be tested for activity using
assays known in the art and described herein including, but not
limiting to, cytotoxicity assays and ADP ribosylation assays.
[0099] B. Methods of Making Modified Polypeptides
[0100] In certain aspects, mutagenesis of nucleic acids encoding
polypeptides can be used to produce the desired modifications in
the (x)D(y) and flanking sequences of the modified DTs. Mutagenesis
can be conducted by any means disclosed herein or known to one of
ordinary skill in the art.
[0101] One particularly useful mutagenesis technique is alanine
scanning mutagenesis in which a number of residues are substituted
individually with the amino acid alanine so that the effects of
losing side-chain interactions can be determined, while minimizing
the risk of large-scale perturbations in protein conformation
(Cunningham et al., Science, 2:244(4908):1081-5 (1989).
[0102] As specific amino acids can be targeted, site-specific
mutagenesis is a technique useful in the preparation of individual
peptides, or biologically functional equivalent proteins or
peptides, through specific mutagenesis of the underlying
polynucleotide. As used herein a "codon" refers to the three
nucleotides which, when transcribed and translated, encode a single
amino acid residue; or in the case of UUA, UGA or UAG encode a
termination signal. Codons encoding amino acids are well known in
the art. The technique further provides a ready ability to prepare
and test sequence variants, incorporating one or more of the
foregoing considerations, by introducing one or more nucleotide
sequence changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent nucleotides, to provide a
primer sequence of sufficient size and sequence complexity to form
a stable duplex on both sides of the mutation site being traversed.
Typically, a primer of about 17 to 25 nucleotides in length is
used, with about 5 to 10 residues on both sides of the junction of
the sequence being altered.
[0103] In general, the technique of site-specific mutagenesis is
well known in the art. Briefly, a bacteriophage vector that will
produce a single stranded template for oligonucleotide directed PCR
mutagenesis can be employed. Phage vectors (e.g., M13), are
commercially available and their use is generally well known to
those in the art. Similarly, double stranded plasmids are also
routinely employed in site directed mutagenesis, which eliminates
the step of transferring a polynucleotide of interest from a phage
to a plasmid. Synthetic oligonucleotide primers bearing the desired
mutated sequence can be used to direct the in vitro synthesis of
modified (desired mutant) DNA from this template and the
heteroduplex DNA is used to transform competent E. coli for the
growth selection and identification of desired clones.
Alternatively, a pair of primers can be annealed to two separate
strands of a double stranded vector to simultaneously synthesize
both corresponding complementary strands with the desired
mutation(s) in a PCR reaction.
[0104] In one embodiment, the Quick Change site-directed
mutagenesis method using plasmid DNA templates as described by
Sugimoto et al. can be employed (Sugimoto et al., Annal. Biochem.,
179(2):309-311 (1989)). PCR amplification of the plasmid template
containing the insert target polynucleotide of insert is achieved
using two synthetic oligonucleotide primers containing the desired
mutation. The oligonucleotide primers, each complementary to
opposite strands of the vector, are extended during temperature
cycling by mutagenesis-grade PfuTurbo DNA polymerase. On
incorporation of the oligonucleotide primers, a mutated plasmid
containing staggered nicks is generated. Amplified un-methylated
products are treated with Dpn I to digest methylated parental DNA
template and select for the newly synthesized DNA containing
mutations. Since DNA isolated from most E. coli strains is dam
methylated, it is susceptible to Dpn I digestion, which is specific
for methylated and hemimethylated DNA. The reaction products are
transformed into high efficiency strains of E. coli to obtain
plasmids containing the desired modifications. Additional methods
for introducing amino acid modifications into a polypeptide are
known in the art and can also be used herein.
[0105] The preparation of sequence variants of the selected gene
using site-directed mutagenesis is provided as a means of producing
potentially useful species and is not meant to be limiting, as
there are other ways in which sequence modifications of
polynucleotides can be obtained. For example, recombinant vectors
encoding the desired gene can be treated with mutagenic agents,
such as hydroxylamine, to obtain sequence variants. These basic
techniques, the protocols for sequence determination, protein
expression and analysis are incorporated by reference to citations
in this specification and are generally accessible to those
reasonably skilled in the art within Current Protocols in Molecular
Biology (Fred M. Ausubel, Roger Brent, Robert E. Kingston, David D.
Moore, J. G. Seidman, John A. Smith, Kevin Struhl, Editors John
Wiley and Sons Publishers (1989)). In accordance with the present
application, there may be employed conventional molecular biology,
microbiology, and recombinant DNA techniques within the skill of
the art. Such techniques are explained fully in the literature.
See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual"
(1989); "Current Protocols in Molecular Biology" Volumes I-III
[Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook"
Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in
Immunology" Volumes I-III [Coligan, J. E., ed. (1994)];
"Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid
Hybridization" [B. D. Hames & S. J. Higgins eds. (1985)];
"Transcription And Translation" [B. D. Hames & S. J. Higgins,
eds. (1984)]; "Animal Cell Culture" [R. I. Freshney, ed. (1986)];
"Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A
Practical Guide To Molecular Cloning" (1984), each of which is
specifically incorporated herein by reference in its entirety.
III. Fusion Proteins
[0106] The present invention also provides DT fusion proteins. A DT
fusion protein contains a DT-related polypeptide (e.g., a modified
DT described herein) and a non-DT polypeptide fused in-frame to
each other. The DT-related polypeptide corresponds to all or a
portion of modified DT exhibiting (having) reduced binding to human
vascular endothelial cells. In one embodiment, a DT fusion protein
comprises at least one portion of a modified DT sequence described
above such as, for example, a polypeptide having an amino acid
sequence set forth in one of SEQ ID NOS: 4-147 or a polypeptide
having two or more modifications of SEQ ID NOS: 4-147.
[0107] Modified DT polypeptides can be fused to, for example, a
non-DT polypeptide. In one embodiment, the non-DT polypeptide is a
cell-specific binding ligand. The specific-binding ligands used in
the invention can contain an entire ligand, or a portion of a
ligand which includes the entire binding domain of the ligand, or
an effective portion of the binding domain. It is most desirable to
include all or most of the binding domain of the ligand
molecule.
[0108] In another embodiment, the modified toxin is a fusion toxin
wherein the cell binding domain is an antibody or antigen-binding
fragment thereof. An antibody can be, for example, monoclonal,
polyclonal, humanized, genetically engineered, or grafted. An
antigen-binding fragment can be, for example, a Fab, Fab.sub.2, a
F(ab').sub.2, a scFv, a scFv2 (a tandem linkage of two scFv
molecules head to tail in a chain), a single chain binding
polypeptide, a VH or a VL. Methods of making antigen-binding
fragments are known in the art and are incorporated herein. Useful
antibodies include those that specifically bind to a receptor or
other moiety expressed on the surface of the target cell membrane
or tumor associated antigens.
[0109] "Specifically binds" means that the binding agent binds to
the antigen on the target cell with greater affinity than it binds
unrelated antigens. Preferably such affinity is at least about
10-fold greater, at least about 100-fold greater, or at least about
1000-fold greater than the affinity of the binding agent for
unrelated antigens. The terms "immunoreactive" and "specifically
binds" are used interchangeably herein. In certain embodiments, the
anti-tumor antibodies or antigen-binding fragments thereof (e.g.,
scFv) are those which recognize a surface determinant on the tumor
cells and are internalized in those cells via receptor-mediated
endocytosis. In a further embodiment, the antibody or antigen
binding fragment thereof binds to a B-cell surface molecule such
as, for example, the B-cell surface molecule is CD19 or CD22.
Alternatively, the antibody or antigen binding fragment thereof,
binds to the ovarian receptor MISIIR (Mullerian Inhibitory
Substance type II receptor).
[0110] Cell-specific binding ligands can also include, but are not
limited to: polypeptide hormones, e.g., those made using the
binding domain of .alpha.-MSH, can selectively bind to melanocytes,
allowing the construction of improved DT-MSH chimeric toxins useful
in the treatment of melanoma. (Murphy, J. R. et al., Proc. Natl.
Acad. Sci. U.S.A., 83(21):8258-8262 (1986)). Other cell-specific
binding ligands which can be used include insulin, somatostatin,
interleukins I and III, and granulocyte colony stimulating factor.
Other useful polypeptide ligands having cell-specific binding
domains are follicle stimulating hormone (specific for ovarian
cells), luteinizing hormone (specific for ovarian cells), thyroid
stimulating hormone (specific for thyroid cells), vasopressin
(specific for uterine cells, as well as bladder and intestinal
cells), prolactin (specific for breast cells), and growth hormone
(specific for certain bone cells). Specific-binding ligands which
can be used include cytokines including, but not limited to, IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, .beta.-interferon, .alpha.-interferon
(INF-.alpha.), INF-.gamma., angiostatin, thrombospondin,
endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, tumor necrosis
factor (TNF), SVEGF, TGF-.beta., Flt3 and B-cell growth factor that
bind to receptors on cells. IL-2 is of particular importance
because of its role in allergic reactions and autoimmune diseases
such as systemic lupus erythmatosis (SLE), involving activated T
cells. DT fusion proteins made using IL-2 B-cell growth factor can
be used as immunosuppressant reagents which kill proliferating
B-cells (cancer cells), which bear high affinity IL-2 receptors or
B-cell growth factor receptors, and which are involved in
hypersensitivity reactions, organ rejection and graft versus host
disease. Other cytokines include Substance P (Benoliel et al.,
Pain, 79(2-3):243-53 (1999)), VEGF (Hotz et al., J Gastrointest
Surg., 6(2): 159-66 (2002)), IL-3 (Jo et al., Protein Exp Purif.
33(1):123-33 (2004)) and GM-CSF (Frankel et al., Clin Cancer Res,
8(5):1004-13 (2002)). VLS modified DT fusion toxins using these
ligands are useful in treating cancers or other diseases of the
cell type to which there is specific binding.
[0111] In IL-2, a LDL sequence (a "VLS" motif) at residues 19-21
(SEQ ID NO: 3) is located in an .alpha.-helix and is also partially
exposed. A mutation in Asp-20 to Lys, in the LDL motif eliminates
binding of IL-2 to the 0 chain of the IL-2 receptor and subsequent
cell proliferation (Collins et al., 1988). It has been reported
that IL-2 directly increases the permeability of the vascular
endothelium to albumin in vitro and that this effect can be
inhibited by anti-IL-2 receptor monoclonal antibodies (Downie et
al., 1992). The LDL sequence in IL-2 damages HUVECs. The Asp-20 in
the LDL of IL-2 is involved in receptor binding and functional
activity (Collins et al., 1988). Thus, it is contemplated that in
certain embodiments, mutations in IL-2's (x)D(y) sequence and/or
flanking sequence(s) to eliminate or reduce VLS should preserve the
Asp-20 or the biological activity of IL-2 may be reduced.
[0112] For a number of cell-specific binding ligands, the region
within each such ligand in which the binding domain is located is
now known. Furthermore, advances in solid phase polypeptide
synthesis enable those skilled in this technology to determine the
binding domain of practically any such ligand, by synthesizing
various fragments of the ligand and testing them for the ability to
bind to the class of cells to be labeled using conventional methods
known in the art such as an ELISA assay. Thus, the chimeric genetic
fusion toxins described herein need not include an entire ligand,
but rather can include only a fragment of a ligand which exhibits
the desired cell-binding capacity. Likewise, analogs of the ligand
or its cell-binding region having minor sequence variations can be
synthesized, tested for their ability to bind to cells, and
incorporated into the hybrid molecules of the invention. If needed,
the amino acid sequences of cell-binding polypeptides can be
analyzed for one or more VLS motifs and modified according to the
concepts described herein. Other potential ligands include
antibodies (e.g., monoclonal) or antigen-binding, single-chain
analogs of monoclonal antibodies, where the antigen is a receptor
or other moiety expressed on the surface of the target cell
membrane. The antibodies most useful are those against tumors; such
antibodies are already well-known targeting agents used in
conjunction with covalently bound cytotoxins. In the present
invention, anti-tumor antibodies (preferably not the whole
antibody, but just an scFv derived therefrom) are those which
recognize a surface determinant on the tumor cells and are
internalized in those cells via receptor-mediated endocytosis.
[0113] In one aspect, a DT-fusion protein is produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different polypeptide sequences are ligated together in-frame
in accordance with conventional techniques, for example, by
employing blunt-ended or stagger-ended termini for ligation,
restriction enzyme digestion to provide for appropriate termini,
filling-in of cohesive ends as appropriate, alkaline phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. In
another embodiment, the fusion gene can be synthesized by
conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried
out using anchor primers which give rise to complementary overhangs
between two consecutive gene fragments which can subsequently be
annealed and reamplified to generate a chimeric gene sequence.
[0114] If needed for proper conformational folding of the fusion
protein, a peptide linker sequence can be employed to separate the
DT-related polypeptide from non-DT polypeptide components by a
distance sufficient to ensure that each polypeptide folds into its
secondary and tertiary structures. Such a peptide linker sequence
can be incorporated into the fusion protein using standard
techniques well known in the art and can be chosen based on the
following factors: (1) their ability to adopt a flexible extended
conformation; (2) its inability to adopt a secondary structure that
could interact with functional epitopes on the DT-related
polypeptide and non-DT polypeptide; and (3) the lack of hydrophobic
or charged residues that might react with the polypeptide
functional epitopes. Certain peptide linker sequences contain Gly,
Asn and Ser residues or Gly, Asp and Ser residues. Other near
neutral amino acids, such as Thr and Ala, can also be used in the
linker sequence. Another peptide linker sequence contains His and
Ala (residues 484-485 of native diphtheria toxin). Amino acid
sequences which can be usefully employed as linkers include, but
are not limited to, those disclosed in Maratea et al., Gene,
40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA,
83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No.
4,751,180. The linker sequence can generally be from 1 to about 50
amino acids in length. Linker sequences may not be required when
the DT-related polypeptide and non-DT polypeptide have
non-essential N-terminal amino acid regions that can be used to
separate the functional domains and prevent steric
interference.
[0115] Provided herein are fusion proteins comprising a polypeptide
toxophore from a modified diphtheria toxin, and a non-diphtheria
toxin polypeptide, said polypeptide toxophore comprising a
diphtheria toxin having an amino acid sequence as recited in SEQ ID
NO: 2 or 149 with one or more amino acid modifications therein,
wherein at least one amino acid modification is made within an
(x)D(y) motif in a region selected from among residues 7-9, 29-31
and 290-292 of SEQ ID NO: 2 of 149, and said modified diphtheria
toxin has cytotoxicity comparable to an unmodified diphtheria
toxin. In one embodiment, a modification at position (x) is a
substitution of V or I by an amino acid residue selected from among
F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, and a modified or unusual
amino acid from Table 1. In one embodiment, a modification at
position D is a substitution of D by an amino acid residue selected
from among I, V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R and a
modified or unusual amino acid from Table 1. In another embodiment,
a modification at position (y) is a substitution of S by an amino
acid residue selected from among I, F, C, M, A, G, T, W, Y, P, H,
E, Q, D, N, K, R and a modified or unusual amino acid from Table 1.
In another embodiment, a modified diphtheria toxin has a
combination of two, three or more modifications in one or more
(x)D(y) motifs.
[0116] In one embodiment, a modified diphtheria toxin contains one
or more modifications selected from among V7T, V7N, V7D, D8N, S9A,
S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31N, I290T, S292A, S292G
and S292T.
[0117] In one embodiment, a modified diphtheria toxin contains two
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N, V7N V29T,
V7N V29D, V7T V29N, V7T V29T or V7T V29D.
[0118] In one embodiment, a modified diphtheria toxin contains
three modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0119] In one embodiment, a modified diphtheria toxin having
cytotoxicity comparable to an unmodified diphtheria toxin refers to
a modified diphtheria toxin having cytotoxicity substantially
similar to, or with at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, or at
least about 95% or more cytotoxicity compared to an unmodified
diphtheria toxin. Purified DAB.sub.389IL-2 (also known as
denileukin diflitox and ONTAK.RTM.) produced in E. coli generally
yields an IC.sub.50 of between about 5.times.10.sup.-11 M to about
1.times.10.sup.-12 M. Thus, in another embodiment, a modified
diphtheria toxin having cytotoxicity comparable to an unmodified
diphtheria toxin refers to a modified diphtheria toxin having an
IC.sub.50 of between about 5.times.10.sup.-11 M to about
1.times.10.sup.-12 M, of about 1.times.10.sup.-10 M to about
1.times.10.sup.-10 M, of about 1.times.10.sup.-9 M to about
1.times.10.sup.-10 M, or of about 1.times.10.sup.-8 M to about
1.times.10.sup.-9 M. Cytotoxicity of a modified diphtheria toxin
compared to an unmodified diphtheria toxin can be tested in a
cytotoxicity assay such as that described below.
[0120] The fusion proteins have reduced binding to human vascular
endothelial cells compared to, for example, a diphtheria toxin
molecule having a sequence of SEQ ID NOS: 1, 2 or 149.
[0121] The non-diphtheria toxin polypeptide can be, for example, an
antibody or an antigen-binding fragment thereof, EGF, IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-14, IL-15, INF.alpha., INF.gamma., GM-CSF, G-CSF, M-CSF,
TNF, VEGF, Ephrin, .beta.FGF, TGF, or a cell-specific binding
portion thereof. In one embodiment, the non-diphtheria toxin
polypeptide is IL-2 or a cell-specific binding portion thereof.
[0122] Chemical crossing or conjugation results in a variety of
molecular species representing the reaction products, and typically
only a small fraction of these products are catalytically and
biologically active. The cross-linking of DT with the cell binding
region is one aspect of the invention. In one embodiment, a
cross-linker which presents a disulfide function can be used such
that the toxin moiety is releasable from the binding agent once the
agent has delivered the toxin inside the targeted cells. Each type
of cross-linker, as well as how the cross-linking is performed,
will tend to vary the pharmacodynamics of the resultant conjugate.
Ultimately, one desires to have a conjugate that will remain intact
under conditions found everywhere in the body except the intended
site of action, at which point it is desirable that the conjugate
have good release characteristics. Therefore, a particular
cross-linking scheme, including in particular the particular
cross-linking reagent used and the structures that are
cross-linked, are considered herein.
[0123] Cross-linking reagents are used to form molecular bridges
that tie together functional groups of two different proteins
(e.g., DT and a cell binding ligand). To link two different
proteins in a step-wise manner, heterobifunctional cross-linkers
can be used which eliminate the unwanted homopolymer formation. An
exemplary heterobifunctional cross-linker contains two reactive
groups: one reacting with primary amine group (e.g., N-hydroxy
succinimide) and the other reacting with a thiol group (e.g.,
pyridyl disulfide, maleimides, halogens, etc.). Through the primary
amine reactive group, the cross-linker can react with the lysine
residue(s) of one protein (e.g., the selected cell binding ligand)
and through the thiol reactive group, the cross-linker, already
tied up to the first protein, reacts with the cysteine residue
(free sulfhydryl group) of the other protein (e.g., DT).
[0124] The spacer arm between these two reactive groups of any
cross-linkers can have various length and chemical composition. A
longer spacer arm allows a better flexibility of the conjugate
components while some particular components in the bridge (e.g.,
benzene group) can lend extra stability to the reactive group or an
increased resistance of the chemical link to the action of various
aspects (e.g., disulfide bond resistant to reducing agents).
[0125] One routinely used cross-linking reagent is SMPT, which is a
bifunctional cross-linker containing a disulfide bond that is
sterically hindered by an adjacent benzene ring and methyl groups.
It is believed that steric hindrance of the disulfide bond serves a
function of protecting the bond from attack by thiolate anions such
as glutathione which can be present in tissues and blood, and
thereby help in preventing decoupling of the conjugate prior to its
delivery to the site of action by the binding agent. The SMPT
cross-linking reagent, as with many other known cross-linking
reagents, lends the ability to crosslink functional groups such as
the SH of cysteine or primary amines (e.g., the epsilon amino group
of lysine). Another possible type of cross-linker includes the
heterobifunctional photoreactive phenylazides containing a
cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido
salicylamido) ethyl-1,3'-dithiopropionate. The
N-hydroxy-succinimidyl group reacts with primary amino groups and
the phenylazide (upon photolysis) reacts non-selectively with any
amino acid residue.
[0126] Although hindered cross-linkers can be used, non-hindered
linkers can also be employed and advantages in accordance herewith
nevertheless realized. Other useful cross-linkers, not considered
to contain or generate a protected disulfide include, but are not
limited to, SATA, SPDP and 2-iminothiolane. The use of such
cross-linkers is well understood in the art.
[0127] In order to be biologically active, the reaction products
should be conjugated in manner that does not interfere with the
innate structure and activity of the catalytic and translocation
domains in the toxophore. Resolution of the active or highly active
species from the inactive species is not always feasible as the
reaction products often possess similar biophysical
characteristics, including for example size, charge density and
relative hydrophobicity. It is noteworthy that isolation of large
amounts of pure clinical grade active product from chemically
cross-linked toxins is not typically economically feasible for the
production of pharmaceutical grade product for clinical trials and
subsequent introduction to the clinical marketplace. To circumvent
this issue, a genetic DT-based protein fusion toxin in which the
native DT receptor-binding domain was genetically replaced with
melanocyte-stimulating hormone as a surrogate receptor-targeting
domain was created (Murphy et al., PNAS, 83:8258-8262 (1986)). This
approach was used with human IL-2 as a surrogate targeting ligand
to create DAB.sub.486IL-2 that was specifically cytotoxic only to
those cells that expressed the high-affinity form of the IL-2
receptor (Williams et al., Protein Eng., 1:493-498 (1987)).
Subsequent studies of DAB.sub.486IL-2 indicated that truncation of
97 amino acids from the DT portion of the molecule resulted in a
more stable, more cytotoxic version of the IL-2 receptor targeted
toxin, DAB.sub.389IL-2 (Williams et al., J. Biol Chem.,
265:11885-889 (1990)). The original constructs (the 486 forms)
still possessed a portion of the native DT cell binding domain. The
DAB.sub.389 amino acid residue version contains the catalytic (C)
and translocation (T) domains of DT with the DT portion of the
fusion protein ending in a random coil between the T domain and the
relative receptor binding domain. A number of other targeting
ligands have since been genetically fused to this DT toxophore,
DAB.sub.389 (vanderSpek et al., Methods in Molecular Biology,
Bacterial Toxins: Methods and Protocols., 145:89-99, Humana Press,
Totowa, N.J. (2000)). Denileukin diftitox (DAB.sub.389IL-2,
ONTAK.RTM.) is a fusion protein containing the enzymatic and
translocation domain of diphtheria toxin and the ligand binding
domain of recombinant IL-2. Denileukin diftitox binds to
intermediate or high affinity IL-2 receptors; however, only binding
of denileukin diftitox to the high affinity chain results in
receptor endocytosis. Upon acidification of the formed vesicle, the
cytotoxic A fragment of diphtheria toxin inhibits protein synthesis
by ADP ribosylation of elongation factor 2, resulting in cell
death. Similar approaches have now been employed with other
bacterial proteins and genetic fusion toxins are often easier to
produce and purify.
[0128] The present application provides fusion proteins against
target epitopes, such as epitopes expressed on a diseased tissue or
a disease causing cell (e.g., IL-2 receptors on cancer cells). In
certain embodiments the fusion protein comprises a modified DT
described herein. In other embodiments the fusion protein further
comprises a second agent. Such an agent can be a molecule or moiety
such as, for example, a reporter molecule or a detectable label.
Reporter molecules are any moiety which can be detected using an
assay. Non-limiting examples of reporter molecules which have been
conjugated to polypeptides include enzymes, radiolabels, haptens,
fluorescent labels, phosphorescent molecules, chemiluminescent
molecules, chromophores, luminescent molecules, photoaffinity
molecules, colored particles or ligands, such as biotin. Detectable
labels include compounds and/or elements that can be detected due
to their specific functional properties, and/or chemical
characteristics, the use of which allows the polypeptide to which
they are attached to be detected, and/or further quantified if
desired. Many appropriate detectable (imaging) agents are known in
the art, as are methods for their attachment to polypeptides (see,
for e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each
incorporated herein by reference). The imaging moieties used can be
paramagnetic ions; radioactive isotopes; fluorochromes;
NMR-detectable substances; X-ray imaging. Molecules containing
azido groups can also be used to form covalent bonds to proteins
through reactive nitrene intermediates that are generated by low
intensity ultraviolet light (Potter & Haley, 1983). In
particular, 2- and 8-azido analogues of purine nucleotides have
been used as site-directed photoprobes to identify nucleotide
binding proteins in crude cell extracts (Owens & Haley, 1987;
Atherton et al., 1985). The 2- and 8-azido nucleotides have also
been used to map nucleotide binding domains of purified proteins
(Khatoon et al., 1989; King et al., 1989; and Dholakia et al.,
1989) and can be used as polypeptide binding agents.
[0129] In one embodiment, provided herein are fusion proteins
containing modified versions of a DAB.sub.389IL-2 where one or more
VLS motifs have been modified as described herein.
IV. Nucleic Acids, Vectors and Host Cells
[0130] Another aspect of the present invention pertains to vectors
containing a polynucleotide (nucleic acid, DNA) encoding a modified
DT variant or a fusion protein thereof.
[0131] The nucleotide and polypeptide sequences for various genes
have been previously disclosed, and can be found at computerized
databases known to those of ordinary skill in the art. One such
database is the National Center for Biotechnology Information's
Genbank and GenPept databases. The coding regions for these known
genes can be amplified and/or expressed using the techniques
disclosed herein or by any technique that would be know to those of
ordinary skill in the art. Additionally, polypeptide sequences can
be synthesized by methods known to those of ordinary skill in the
art, such as polypeptide synthesis using automated polypeptide
synthesis machines, such as those available from Applied Biosystems
(Foster City, Calif.).
[0132] As used herein, the term "expression vector or construct"
means any type of genetic construct containing a nucleic acid
coding for a gene product in which part or all of the nucleic acid
encoding sequence is capable of being transcribed. The transcript
can be translated into a protein, but it need not be. Thus, in
certain embodiments, expression includes both transcription of a
gene and translation of a RNA into a gene product. In other
embodiments, expression only includes transcription of the nucleic
acid, for example, to generate antisense constructs.
[0133] Particularly useful vectors are contemplated to be those
vectors in which the coding portion of the DNA segment, whether
encoding a full length protein, polypeptide or smaller peptide, is
positioned under the transcriptional control of a promoter. A
"promoter" refers to a DNA sequence recognized by the synthetic
machinery of the cell, or introduced synthetic machinery, required
to initiate the specific transcription of a gene. The phrases
"operatively positioned," "under control" or "under transcriptional
control" means that the promoter is in the correct location and
orientation in relation to the nucleic acid coding for the gene
product to control RNA polymerase initiation and expression of the
gene.
[0134] The promoter can be in the form of the promoter that is
naturally associated with a gene, as can be obtained by isolating
the 5' non-coding sequences located upstream of the coding segment
or exon, for example, using recombinant cloning and/or PCR
technology, in connection with the compositions disclosed
herein.
[0135] In other embodiments, it is contemplated that certain
advantages will be gained by positioning the coding DNA segment
under the control of a recombinant, or heterologous, promoter. As
used herein, a recombinant or heterologous promoter is intended to
refer to a promoter that is not normally associated with a gene in
its natural environment. Such promoters can include promoters
normally associated with other genes, and/or promoters isolated
from any other bacterial, viral, eukaryotic, or mammalian cell,
and/or promoters made by the hand of man that are not "naturally
occurring," that is, containing difference elements from different
promoters, or mutations that increase, decrease or alter
expression.
[0136] Promoters that effectively direct the expression of the DNA
segment in the cell type, organism, or even animal, are chosen for
expression. The use of promoter and cell type combinations for
protein expression is generally known to those of skill in the art
of molecular biology, for example, see Sambrook et al., (1989),
incorporated herein by reference. The promoters employed can be
constitutive, or inducible, and can be used under the appropriate
conditions to direct high level expression of the introduced DNA
segment, such as is advantageous in the large-scale production of
recombinant proteins or peptides.
[0137] At least one module in a promoter generally functions to
position the start site for RNA synthesis. The best known example
of this is the TATA box, but in some promoters lacking a TATA box,
such as the promoter for the mammalian terminal deoxynucleotidyl
transferase gene and the promoter for the SV40 late genes, a
discrete element overlying the start site itself helps to fix the
place of initiation.
[0138] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 base pairs (bp) upstream of the start site, although
a number of promoters have been shown to contain functional
elements downstream of the start site as well. The spacing between
promoter elements frequently is flexible, so that promoter function
is preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 bp apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either co-operatively or independently to activate
transcription.
[0139] The particular promoter that is employed to control the
expression of a nucleic acid is not believed to be critical, so
long as it is capable of expressing the nucleic acid in the
targeted cell. Thus, where a human cell is targeted, it is
preferable to position the nucleic acid coding region adjacent to
and under the control of a promoter that is capable of being
expressed in a human cell. Generally speaking, such a promoter
might include either a human or viral promoter.
[0140] In expression, one will typically include a polyadenylation
signal to effect proper polyadenylation of the transcript. The
nature of the polyadenylation signal is not believed to be crucial
to the successful practice of the invention, and any such sequence
may be employed. Polyadenylation signals include, but are not
limited to the SV40 polyadenylation signal and the bovine growth
hormone polyadenylation signal, convenient and known to function
well in various target cells. Also contemplated as an element of
the expression cassette is a terminator sequence. These elements
can serve to enhance message levels and to minimize read through
from the cassette into other sequences.
[0141] A specific initiation signal also can be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon and adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals. It is well known that the initiation codon must be
"in-frame" with the reading frame of the desired coding sequence to
ensure translation of the entire insert. The exogenous
translational control signals and initiation codons can be either
natural or synthetic. The efficiency of expression may be enhanced
by the inclusion of appropriate transcription enhancer
elements.
[0142] It is contemplated that polypeptides can be co-expressed
with other selected proteins, wherein the proteins can be
co-expressed in the same cell or a gene(s) can be provided to a
cell that already has another selected protein. Co-expression can
be achieved by co-transfecting the cell with two distinct
recombinant vectors, each bearing a copy of either of the
respective DNA. Alternatively, a single recombinant vector can be
constructed to include the coding regions for both of the proteins,
which could then be expressed in cells transfected with the single
vector. In either event, the term "co-expression" herein refers to
the expression of both the gene(s) and the other selected protein
in the same recombinant cell.
[0143] As used herein, the terms "engineered" and "recombinant"
cells or host cells are intended to refer to a cell into which an
exogenous DNA segment or gene, such as a cDNA or gene encoding a
protein has been introduced. Therefore, engineered cells are
distinguishable from naturally occurring cells which do not contain
a recombinantly introduced exogenous DNA segment or gene.
Engineered cells are thus cells having a gene or genes introduced
through the hand of man. Recombinant cells include those having an
introduced cDNA or genomic gene, and also include genes positioned
adjacent to a promoter not naturally associated with the particular
introduced gene.
[0144] To express a recombinant polypeptide, whether modified or
wild-type, in accordance with the present invention one would
prepare an expression vector that comprises a wild-type, or
modified protein-encoding nucleic acid under the control of one or
more promoters. To bring a coding sequence "under the control of" a
promoter, one positions the 5' end of the transcription initiation
site of the transcriptional reading frame generally between about 1
and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen
promoter. The "upstream" promoter stimulates transcription of the
DNA and promotes expression of the encoded recombinant protein.
This is the meaning of "recombinant expression" in this
context.
[0145] Many standard techniques are available to construct
expression vectors containing the appropriate nucleic acids and
transcriptional/translational control sequences in order to achieve
protein, polypeptide or peptide expression in a variety of
host-expression systems. Cell types available for expression
include, but are not limited to, bacteria, such as E. coli and B.
subtilis transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors.
[0146] Certain examples of prokaryotic hosts are E. coli strain
RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as
well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325);
bacilli such as Bacillus subtilis; and other enterobacteriaceae
such as Salmonella typhimurium, Serratia marcescens, and various
Pseudomonas species.
[0147] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is often transformed using derivatives of pBR322,
a plasmid derived from an E. coli species. pBR322 contains genes
for ampicillin and tetracycline resistance and thus provides easy
means for identifying transformed cells. The pBR plasmid, or other
microbial plasmid or phage must also contain, or be modified to
contain, promoters which can be used by the microbial organism for
expression of its own proteins.
[0148] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, the phage lambda GEM.TM.-11 may be utilized in making a
recombinant phage vector which can be used to transform host cells,
such as E. coli LE392.
[0149] Further useful vectors include pIN vectors (Inouye et al.,
1985); and pGEX vectors, for use in generating glutathione
S-transferase (GST) soluble fusion proteins for later purification
and separation or cleavage. Other suitable fusion proteins are
those with .beta.-galactosidase, ubiquitin, and the like.
[0150] Promoters that are most commonly used in recombinant DNA
construction include the .beta.-lactamase (penicillinase), lactose
and tryptophan (trp) promoter systems. While these are the most
commonly used, other microbial promoters have been discovered and
utilized, and details concerning their nucleotide sequences have
been published, enabling those of skill in the art to ligate them
functionally with plasmid vectors.
[0151] The following details concerning recombinant protein
production in bacterial cells, such as E. coli, are provided by way
of exemplary information on recombinant protein production in
general, the adaptation of which to a particular recombinant
expression system will be known to those of skill in the art.
[0152] Bacterial cells, for example, E. coli, containing the
expression vector are grown in any of a number of suitable media,
for example, LB. The expression of the recombinant protein may be
induced, e.g., by adding IPTG to the media or by switching
incubation to a higher temperature. After culturing the bacteria
for a further period, generally of between 2 and 24 hours (h), the
cells are collected by centrifugation and washed to remove residual
media.
[0153] The bacterial cells are then lysed, for example, by
disruption in a cell homogenizer and centrifuged to separate the
dense inclusion bodies and cell membranes from the soluble cell
components. This centrifugation can be performed under conditions
whereby the dense inclusion bodies are selectively enriched by
incorporation of sugars, such as sucrose, into the buffer and
centrifugation at a selective speed.
[0154] If the recombinant protein is expressed in the inclusion
bodies, as is the case in many instances, these can be washed in
any of several solutions to remove some of the contaminating host
proteins, then solubilized in solutions containing high
concentrations of urea (e.g., 8 M) or chaotropic agents such as
guanidine hydrochloride in the presence of reducing agents, such as
.beta.-mercaptoethanol or DTT (dithiothreitol).
[0155] It is contemplated that the polypeptides produced by the
methods described herein can be overexpressed, i.e., expressed in
increased levels relative to its natural expression in cells. Such
overexpression can be assessed by a variety of methods, including
radio-labeling and/or protein purification. However, simple and
direct methods are preferred, for example, those involving SDS/PAGE
and protein staining or western blotting, followed by quantitative
analyses, such as densitometric scanning of the resultant gel or
blot. A specific increase in the level of the recombinant protein,
polypeptide or peptide in comparison to the level in natural cells
is indicative of overexpression, as is a relative abundance of the
specific polypeptides in relation to the other proteins produced by
the host cell and, e.g., visible on a gel.
[0156] Expression vectors provided herein comprise a polynucleotide
encoding modified DT or a fusion protein thereof in a form suitable
for expression of the polynucleotide in a host cell. The expression
vectors generally have one or more regulatory sequences, selected
on the basis of the host cells to be used for expression, which is
operatively linked to the polynucleotide sequence to be expressed.
It wilt be appreciated by those skilled in the art that the design
of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of expression of
protein desired, and the like. The expression vectors described
herein can be introduced into host cells to produce proteins,
including fusion proteins, encoded by polynucleotides as described
herein (e.g., a modified DT or a DT fusion protein, and the
like).
[0157] Expression vectors can be designed for expression of a
modified DT or a DT fusion protein in prokaryotic or eukaryotic
cells. The presence of a single DT molecule inside a eukaryotic
cell would kill the cell. Specifically, the toxin binds to EF-tu
which is required for translation and ribosylation. Accordingly, DT
can only be expressed in cells with modified EF-tu that is no
longer recognized by DT (see, e.g., Liu et al., Protein Expr Purif,
30:262-274 (2003); Phan et al., J. Biol. Chem., 268(12):8665-8
(1993); Chen et al., Mol. Cell. Biol., 5(12):3357-60 (1985); Kohne
et al., Somat Cell Mol. Genet., 11(5):421-31 (1985); Moehring et
al., Mol. Cell. Biol., 4(4):642-50 (1984)). In addition, a modified
DT or a fusion protein thereof can be expressed in bacterial cells
such as E. coli (Bishai et al., J Bacteriol 169(11):5140-51
(1987)). Consideration is given to the expression and activity of
the types and levels of host protease expression, and this is
dependent upon the cleavage site present in the engineered DT
toxophore. The innate expression host protease expression profile
could negatively impact the yields of DT fusion toxin produced
(Bishai et al., Supra (1987)). To the degree that this requisite
cleavage site can be altered to modulate the cell selectivity of
resultant fusion proteins, it is envisioned that such cleavage site
mutants could be in VLS-modified toxophores (Gordon et al., Infect
Immun, 63(1):82-7 (1995); Gordon et al., Infect Immun, 62(2):333-40
(1994); Vallera et al., J Natl. Cancer Inst., 94:597-606 (2002);
Abi-Habib et al., Blood., 104(7):2143-8 (2004)). Alternatively, the
expression vector can be transcribed and translated in vitro.
[0158] The present application further provides gene delivery
vehicles for the delivery of polynucleotides to cells, tissue, or a
mammal for expression. For example, a polynucleotide sequence of
the present invention can be administered either locally or
systemically in a gene delivery vehicle. These constructs can
utilize viral or non-viral vector approaches in in vivo or ex vivo
modality. Expression of such coding sequences can be induced using
endogenous mammalian or heterologous promoters. Expression of the
coding sequence in vivo can be either constitutive or regulated.
The invention includes gene delivery vehicles capable of expressing
the contemplated polynucleotides including viral vectors. For
example, Qiao et al. developed a system employing PG13 packaging
cells produce recombinant retroviruses carrying a DT fragment which
kills cancer cell and provides a method for using DT as component a
suicide vector. Qiao et al., J. Virol. 76(14):7343-8 (2002).
[0159] Expressed DT-mutants and DT-fusion proteins can be tested
for their functional activity. Methods for testing DT activity are
well-known in the art. For example, the VLS effect of DT-mutants
and DT-fusion proteins can be tested in HUVECs as described in
Example 2. The ribosyltransferase activity of DT variants or
DT-fusion proteins can be tested by the ribosyltransferase assay
described in Example 3. The cytotoxicity of DT variants or
DT-fusion proteins can be tested as described in Example 4.
[0160] The present application also provides purified, and in
preferred embodiments, substantially purified, polypeptides
expressed using one or more of the methods described herein. The
term "purified" as used herein, is intended to refer to a
proteinaceous composition, isolatable from mammalian cells or
recombinant host cells, wherein the at least one polypeptide is
purified to any degree relative to its naturally-obtainable state,
i.e., relative to its purity within a cellular extract. A purified
polypeptide therefore also refers to a wild-type or modified
polypeptide free from the environment in which it naturally
occurs.
[0161] Where the term "substantially purified" is used, this will
refer to a composition in which the specific polypeptide forms the
major component of the composition, such as constituting about 50%
of the proteins in the composition or more. In one embodiment, a
substantially purified polypeptide will constitute more than about
60%, about 70%, about 80%, about 90%, about 95%, about 99% or even
more of the polypeptides in the composition.
[0162] A polypeptide that is "purified to homogeneity," as applied
to the present invention, means that the polypeptide has a level of
purity where the polypeptide is substantially free from other
proteins and biological components. For example, a purified
polypeptide will often be sufficiently free of other protein
components so that degradative sequencing can be performed.
[0163] Various methods for quantifying the degree of purification
of polypeptides will be known to those of skill in the art in light
of the present disclosure. These include, for example, determining
the specific protein activity of a fraction, or assessing the
number of polypeptides within a fraction by gel
electrophoresis.
[0164] To purify a desired polypeptide, a natural or recombinant
composition comprising at least some specific polypeptides will be
subjected to fractionation to remove various other components from
the composition. In addition to those techniques described in
detail herein below, various other techniques suitable for use in
protein purification will be well known to those of skill in the
art. These include, for example, precipitation with ammonium
sulfate, PEG, antibodies and the like or by heat denaturation,
followed by centrifugation; chromatography steps such as ion
exchange, gel filtration, reverse phase, hydroxylapatite, lectin
affinity and other affinity chromatography steps; isoelectric
focusing; gel electrophoresis; and combinations of such and other
techniques.
[0165] Another example is the purification of a specific fusion
protein using a specific binding partner. Such purification methods
are routine in the art. As the present invention provides DNA
sequences for the specific proteins, any fusion protein
purification method can now be practiced. This is exemplified by
the generation of a specific protein-glutathione S-transferase
fusion protein, expression in E. coli, and isolation to homogeneity
using affinity chromatography on glutathione-agarose or the
generation of a polyhistidine tag on the N- or C-terminus of the
protein, and subsequent purification using Ni-affinity
chromatography. However, given many DNA and proteins are known, or
may be identified and amplified using the methods described herein,
any purification method can now be employed.
[0166] There is no general requirement that the polypeptides always
be provided in their most purified state. Indeed, it is
contemplated that less substantially purified polypeptides which
are nonetheless enriched in the desired protein compositions,
relative to the natural state, will have utility in certain
embodiments. Polypeptides exhibiting a lower degree of relative
purification may have advantages in total recovery of protein
product, or in maintaining the activity of an expressed
protein.
[0167] Provided herein is a method for making a composition
comprising: (a) constructing a vector comprising a polynucleotide
which encodes a polypeptide having an amino acid sequence of SEQ ID
NOS: 4-147 or a polypeptide having two or more of such
modifications, and (b) causing said polypeptide to be expressed in
a host cell comprising said vector. In one embodiment, a
composition produced by such a method, wherein said composition has
a reduced binding activity to human vascular endothelial cells
(HUVEC) compared to a DT molecule having a sequence of SEQ ID NO: 2
or 149.
[0168] Provided herein is a method for making a modified diphtheria
toxin having a reduced binding activity to human vascular
endothelial cells (HUVEC) compared to an unmodified diphtheria
toxin, said method comprising the step of: (a) constructing a
vector comprising a nucleic acid sequence encoding a modified
diphtheria toxin, said modified diphtheria toxin comprising a
diphtheria toxin having an amino acid sequence as recited in SEQ ID
NO: 2 or 149 with one or more amino acid modifications therein,
wherein at least one amino acid modification is made within an
(x)D(y) motif in a region selected from the group consisting of
residues 7-9, 29-31 and 290-292 of SEQ ID NO: 2 or 149, and said
modified diphtheria toxin has cytotoxicity comparable to an
unmodified diphtheria toxin. In one embodiment, a modification at
position (x) is a substitution of V or I by an amino acid residue
selected from among F, C, M, T, W, Y, P, H, E, Q, D, N, K, R, and a
modified or unusual amino acid from Table 1. In one embodiment, a
modification at position D is a substitution of D by an amino acid
residue selected from among I, V, L, F, C, M, A, G, T, W, Y, P, H,
Q, N, K, R and a modified or unusual amino acid from Table 1. In
another embodiment, a modification at position (y) is a
substitution of S by an amino acid residue selected from among I,
F, C, M, A, G, T, W, Y, P, H, E, Q, D, N, K, R and a modified or
unusual amino acid from Table 1. In another embodiment, a modified
diphtheria toxin has a combination of two, three or more
modifications in one or more (x)D(y) motifs. The modified
diphtheria toxin has cytotoxicity comparable to that of a
diphtheria toxin having a sequence of SEQ ID NO: 2 or 149; and (b)
causing said polypeptide to be expressed in a host cell comprising
said vector. In one embodiment, a modified diphtheria toxin
contains one or more modifications selected from among V7T, V7N,
V7D, D8N, S9A, S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31N, I290T,
S292A, S292G and S292T. In one embodiment, a modified diphtheria
toxin contains two modifications. Such modified diphtheria toxins
can contain a combination of mutations such as, for example, V7N
V29N, V7N V29T, V7N V29D, V7T V29N, V7T V29T or V7T V29D. In one
embodiment, a modified diphtheria toxin contains three
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0169] Unmodified diphtheria toxins can have, for example, an amino
acid sequence of SEQ ID NO: 2, 149 or an amino acid sequence of any
one of SEQ ID NOS: 4-147.
[0170] Bacterial and plant holotoxins often contain two
disulfide-bonded chains, the A and B chains. The B chain carries
both a cell-binding region (whose receptor is often
uncharacterized) and a translocation region, which facilitates the
insertion of the A chain through the membrane of an acid
intracellular compartment into the cytosol. The A chain then kills
the cell after translocation. For their use in vivo, the ligand and
toxin are coupled in such a way as to remain stable while passing
through the bloodstream and the tissues and yet be labile within
the target cell so that the toxic portion can be released into the
cytosol.
[0171] Diphtheria toxin as described herein comprises the amino
acid sequence as set forth in SEQ ID NO: 2 or 149. Additionally,
variants of diphtheria are known to contain nucleic acid residue
insertions, deletions, and/or substitutions in their nucleic acid
sequence while still retaining their biological activity. Variants
of diphtheria toxin have been characterized demonstrating nucleic
acid variation among diphtheria toxins. (Holmes, R. K., J. Infect.
Dis., 181 (Supp. 1): S156-S167 (2000)), thus diphtheria toxins can
comprise different nucleic acid and/or amino acid sequences.
Nucleic acid residue insertions, deletions, and/or substitutions
can also affect the amino acid sequence. However, not all nucleic
acid residue changes will result in a change at the amino acid
residue level of a protein due to the redundancy of the genetic
code. Nucleic acid and/or amino acid variations (i.e., insertions,
deletions, and/or substitutions) of diphtheria toxin are also
included within the definition of diphtheria toxin and contemplated
herein. As used herein, diphtheria toxin comprises the amino acid
sequence as set forth in SEQ ID NO: 2 or 149 and further includes
diphtheria toxins comprising amino acid sequences about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO:
2 or 149. C-terminal truncations of DT can also made and include
for example, deletion of about 1, about 2, about 3, about 4, about
5, about 6, about 7, about 8, about 9, about 10, about 11, about
12, about 13, about 14, about 15, about 20, about 25, about 30,
about 35, about 40, or about 50 amino acid residues of DT389 or
DT387. For example, as used herein, diphtheria toxin comprises the
amino acid sequence as set forth in amino acid residues 1-382 of
SEQ ID NO: 2 or 149 and further includes diphtheria toxins
comprising amino acid sequences about 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% homologous to amino acid residues 1-382 of
SEQ ID NO: 2 or 149. One would understand that variants of
diphtheria toxin could be modified and tested for function using
any of the methods described herein.
[0172] However, it may be desirable from a pharmacologic standpoint
to employ the smallest molecule possible that nevertheless provides
an appropriate biological response. One can, thus, desire to employ
smaller A chain peptides which will provide an adequate
anti-cellular response. To this end, DT can be "truncated" and
still retain an adequate toxin activity. It is proposed that where
desired, this truncated A chain can be employed in fusion proteins
in accordance with the embodiments described herein.
[0173] Alternatively, one may find that the application of
recombinant DNA technology to the toxin moiety may provide
additional benefits. In that biologically active DT has now been
cloned and recombinantly expressed, it is now possible to identify
and prepare smaller or otherwise variant peptides which
nevertheless exhibit an appropriate toxin activity. Moreover, the
fact that DT has now been cloned allows the application of
site-directed mutagenesis through which one can readily prepare and
screen for DT A chain, toxin-derived peptides and obtain additional
useful moieties for use in connection with the presently described
compounds. Once identified, these moieties can be mutated to
produce toxins exhibiting a reduced ability to promote VLS, EC
damaging activity and/or other effects of such sequences described
herein or known to one of skill in the art.
[0174] In one aspect, toxin as used herein contemplates fusion
proteins between toxins (e.g., diphtheria toxin) and non-toxin
polypeptides containing at least one cell binding domain. In one
non-limiting example, a diphtheria toxin or a fragment thereof is
fused to a cell-binding domain of interleukin-2 (IL-2), thus
creating a fusion toxin. As described in further detail herein,
fusion protein toxins can also comprise linker polypeptides and
conjugates. Such toxins are also contemplated as toxins to be
modified by the methods disclosed herein.
[0175] In one embodiment, a toxin is a fusion protein comprising a
modified toxin wherein the toxin binding domain has been replaced
with the binding domain of a non-toxin polypeptide. In another
embodiment, the toxin is a fusion protein comprising a modified
diphtheria toxin and a non-toxin polypeptide. In another
embodiment, the toxin is a fusion protein comprising diphtheria
toxin and IL-2.
[0176] Provided herein is a fusion protein comprising a modified
diphtheria toxin made by such a method and a non-diphtheria toxin
polypeptide.
[0177] In certain embodiments, the non-diphtheria toxin polypeptide
is, for example, an antibody or antigen-binding fragment thereof,
EGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, INF.alpha.; INF.gamma., GM-CSF,
G-CSF, M-CSF, TNF, VEGF, Ephrin, BFGF, TGF or a cell-binding
portion thereof. In one embodiment, the non-diphtheria toxin
polypeptide is IL-2 or a cell-specific binding portion thereof, or
IL-3 or a cell-specific binding portion thereof.
[0178] In certain embodiments, the fusion protein or toxin further
comprises at least another agent. Such an agent can be a molecule
or moiety including, but not limited to, at least one effector
(therapeutic moiety) or reporter molecule (a detectable label) as
described elsewhere herein.
V. Compositions and Therapeutic Uses
[0179] Each of the compounds described herein can be used as a
composition when combined with an acceptable carrier or excipient.
Such compositions are useful for in vitro analysis or for
administration to a subject in vivo or ex vivo for treating a
subject with the disclosed compounds.
[0180] Thus pharmaceutical compositions can comprise, in addition
to active ingredient, a pharmaceutically acceptable excipient,
carrier, buffer, stabilizer or other materials well known to those
skilled in the art. Such materials should be non-toxic and should
not interfere with the efficacy of the active ingredient. The
precise nature of the carrier or other material will depend on the
route of administration.
[0181] Pharmaceutical formulations comprising a protein of
interest, e.g., an antibody, identified by the methods described
herein can be prepared for storage by mixing the protein having the
desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.RTM., PLURONICS.RTM. or polyethylene glycol (PEG).
[0182] The formulation described herein can also contain more than
one active compound as necessary for the particular indication
being treated. Such molecules are suitably present in combination
in amounts that are effective for the purpose intended.
[0183] Acceptable carriers are physiologically acceptable to the
administered patient and retain the therapeutic properties of the
compounds with/in which it is administered. Acceptable carriers and
their formulations are and generally described in, for example,
Remington' pharmaceutical Sciences (18th Edition, ed. A. Gennaro,
Mack Publishing Co., Easton, Pa. 1990). One exemplary carrier is
physiological saline. The phrase "pharmaceutically acceptable
carrier" as used herein means a pharmaceutically acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting the subject compounds from the
administration site of one organ, or portion of the body, to
another organ, or portion of the body, or in an in vitro assay
system. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to a subject to whom it is administered. Nor should an
acceptable carrier alter the specific activity of the subject
compounds. Exemplary carriers and excipients have been provided
elsewhere herein.
[0184] In one aspect, provided herein are pharmaceutically
acceptable or physiologically acceptable compositions including
solvents (aqueous or non-aqueous), solutions, emulsions, dispersion
media, coatings, isotonic and absorption promoting or delaying
agents, compatible with pharmaceutical administration.
Pharmaceutical compositions or pharmaceutical formulations
therefore refer to a composition suitable for pharmaceutical use in
a subject. The pharmaceutical compositions and formulations include
an amount of a compound described herein, for example, an effective
amount of modified DT fusion protein described herein, and a
pharmaceutically or physiologically acceptable carrier.
[0185] Compositions can be formulated to be compatible with a
particular route of administration, systemic or local. Thus,
compositions include carriers, diluents, or excipients suitable for
administration by various routes. Pharmaceutical compositions for
oral administration may be in tablet, capsule, powder or liquid
form. A tablet may comprise a solid carrier such as gelatin or an
adjuvant. Liquid pharmaceutical compositions generally comprise a
liquid carrier such as water, petroleum, animal or vegetable oils,
mineral oil or synthetic oil. Physiological saline solution,
dextrose or other saccharide solution or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol may be
included.
[0186] In a further embodiment, the compositions can further
comprise, if needed, an acceptable additive in order to improve the
stability of the compounds in composition and/or to control the
release rate of the composition. Acceptable additives do not alter
the specific activity of the subject compounds. Exemplary
acceptable additives include, but are not limited to, a sugar such
as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose,
sucrose, galactose, dextran, dextrose, fructose, lactose and
mixtures thereof. Acceptable additives can be combined with
acceptable carriers and/or excipients such as dextrose.
Alternatively, exemplary acceptable additives include, but are not
limited to, a surfactant such as polysorbate 20 or polysorbate 80
to increase stability of the peptide and decrease gelling of the
solution. The surfactant can be added to the composition in an
amount of 0.01% to 5% of the solution. Addition of such acceptable
additives increases the stability and half-life of the composition
in storage.
[0187] The pharmaceutical composition can be delivered
subcutaneously, intramuscularly, intraperitoneally, orally or
intravenously. Aerosol delivery of the compositions is also
contemplated herein using conventional methods. For example,
intravenous delivery is now possible by cannula or direct injection
or via ultrasound guided fine needle. Mishra (Mishra et al., Expert
Opin. Biol., 3(7): 1173-1180 (2003)) provides for intratumoral
injection.
[0188] Formulations or enteral (oral) administration can be
contained in a tablet (coated or uncoated), capsule (hard or soft),
microsphere, emulsion, powder, granule, crystal, suspension, syrup
or elixir. Conventional non-toxic solid carriers which include, for
example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharin, talcum, cellulose, glucose,
sucrose, magnesium carbonate, can be used to prepare solid
formulations. Supplementary active compounds (e.g., preservatives,
antibacterial, antiviral and antifungal agents) can also be
incorporated into the formulations. A liquid formulation can also
be used for enteral administration. The carrier can be selected
from various oils including petroleum, animal, vegetable or
synthetic, for example, peanut oil, soybean oil, mineral oil,
sesame oil. Suitable pharmaceutical excipients include e.g.,
starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt,
rice, flour, chalk, silica gel, magnesium stearate, sodium
stearate, glycerol monostearate, sodium chloride, dried skim milk,
glycerol, propylene glycol, water, ethanol.
[0189] Compositions for injection include aqueous solutions (where
water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions. For intravenous administration, suitable carriers
include physiological saline, bacteriostatic water, Cremophor
EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. Fluidity can be maintained, for example,
by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Antibacterial and antifungal agents include, for
example, parabens, chlorobutanol, phenol, ascorbic acid and
thimerosal. Isotonic agents, for example, sugars, polyalcohols such
as manitol, sorbitol, and sodium chloride may be included in the
composition. The resulting solutions can be packaged for use as is,
or lyophilized; the lyophilized preparation can later be combined
with a sterile solution prior to administration.
[0190] Compositions can be conventionally administered
intravenously, such as by injection of a unit dose, for example.
For injection, an active ingredient can be in the form of a
parenterally acceptable aqueous solution which is substantially
pyrogen-free and has suitable pH, isotonicity and stability. One
can prepare suitable solutions using, for example, isotonic
vehicles such as Sodium Chloride Injection, Ringer's Injection,
Lactated Ringer's Injection. Preservatives, stabilizers, buffers,
antioxidants and/or other additives may be included, as
required.
[0191] In one embodiment, the composition is lyophilized and
reconstituted prior to administration to increase shelf-life of the
compound. When the compositions are considered for medicaments, or
use in any of the methods provided herein, it is contemplated that
the composition can be substantially free of pyrogens such that the
composition will not cause an inflammatory reaction or an unsafe
allergic reaction.
[0192] Acceptable carriers can contain a compound that stabilizes,
increases or delays absorption or clearance. Such compounds
include, for example, carbohydrates, such as glucose, sucrose, or
dextrans; low molecular weight proteins; compositions that reduce
the clearance or hydrolysis of peptides; or excipients or other
stabilizers and/or buffers. Agents that delay absorption include,
for example, aluminum monostearate and gelatin. Detergents can also
be used to stabilize or to increase or decrease the absorption of
the pharmaceutical composition, including liposomal carriers. To
protect from digestion the compound can be complexed with a
composition to render it resistant to acidic and enzymatic
hydrolysis, or the compound can be complexed in an appropriately
resistant carrier such as a liposome. Means of protecting compounds
from digestion are known in the art (see, e.g., Fix (1996) Pharm
Res. 13:1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 135;
and U.S. Pat. No. 5,391,377, describing lipid compositions for oral
delivery of therapeutic agents).
[0193] For intravenous, injection, or injection at the site of
affliction, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and
has suitable pH, isotonicity and stability. Those of relevant skill
in the art are well able to prepare suitable solutions using, for
example, isotonic vehicles such as Sodium Chloride Injection,
Ringer's Injection, Lactated Ringer's Injection. Preservatives,
stabilizers, buffers, antioxidants and/or other additives may be
included, as needed.
[0194] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar untoward reaction,
such as gastric upset, dizziness and the like, when administered to
a human.
[0195] The term "unit dose" when used in reference to a therapeutic
composition refers to physically discrete units suitable as unitary
dosage for humans, each unit containing a predetermined quantity of
active material calculated to produce the desired therapeutic
effect in association with the required diluent; i.e., carrier, or
vehicle.
[0196] The compositions can be administered in a manner compatible
with the dosage formulation, and in a therapeutically effective
amount. The quantity to be administered depends on the subject to
be treated, capacity of the subject's immune system to utilize the
active ingredient, and degree of binding capacity desired. Precise
amounts of active ingredient required to be administered depend on
the judgment of the practitioner and are peculiar to each
individual. Suitable regimes for initial administration and booster
shots are also variable, but are typified by an initial
administration followed by repeated doses at one or more hour
intervals by a subsequent injection or other administration.
Alternatively, continuous intravenous infusion sufficient to
maintain concentrations of ten nanomolar to ten micromolar in the
blood are contemplated.
[0197] A "therapeutically effective amount" as used herein, is an
amount that achieves at least partially a desired therapeutic or
prophylactic effect in an organ or tissue. The amount of a modified
DT necessary to bring about prevention and/or therapeutic treatment
of the disease is not fixed per se. The amount of VLS modified DT
fusion toxin administered will vary with the type of disease,
extent of the disease, and size of species of the mammal suffering
from the disease. Generally, amounts will be in the range of those
used for other cytotoxic agents used in the treatment of cancer,
although in certain instances lower amounts will be needed because
of the specificity and increased toxicity of the VLS-modified DT
fusion toxins. In certain circumstances, and as can be achieved by,
currently available techniques (for example, cannulae or convection
enhanced delivery, selective release), attempts to deliver enhanced
locally elevated fusion toxin amounts to specific sites may also be
desired. (Laske et al., J. Neurosurg., 87:586-5941(997); Laske et
al., Nature Medicine, 3:1362-1368 (1997), Rand et al., Clin. Cancer
Res., 6:2157-2165 (2000); Engebraaten et al., J. Cancer, 97:846-852
(2002), Prados et al., Proc. ASCO, 21:69b (2002), Pickering et al.,
J Clin Invest, 91(2):724-9 (1993)).
[0198] One embodiment contemplates the use of the compositions
described herein to make a medicament for treating a condition,
disease or disorder described herein. Medicaments can be formulated
based on the physical characteristics of the patient/subject
needing treatment, and can be formulated in single or multiple
formulations based on the stage of the condition, disease or
disorder. Medicaments can be packaged in a suitable package with
appropriate labels for the distribution to hospitals and clinics
wherein the label is for the indication of treating a subject
having a disease described herein. Medicaments can be packaged as a
single or multiple units. Instructions for the dosage and
administration of the compositions can be included with the
packages as described below.
[0199] The invention is further directed to pharmaceutical
compositions comprising a modified DT or fusion protein thereof
described hereinabove and a pharmaceutically acceptable
carrier.
[0200] Sterile injectable solutions can be prepared by
incorporating an active ingredient in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
ingredient into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the methods of preparation are those
such as vacuum drying and freeze drying which yields a powder of
the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0201] In certain embodiments is the further purification of this
mixture to obtain preparations essentially comprising fusion
proteins. This purification is accomplished by further
chromatographic separation which can be accomplished by affinity
chromatography for example, using a salt gradient to elute the
various species of immunotoxins and gel filtration to separate the
immunotoxins from larger molecules.
[0202] A gel to be used in purification of compounds described
herein is a three dimensional network which has a random structure.
Molecular sieve gels are those cross-linked polymers that do not
bind or react with the material being analyzed or separated. For
gel filtration purposes, a gel material is generally uncharged. The
space within the gel is filled with liquid and the liquid phase
constitutes the majority of the gel volume. Materials commonly used
in gel filtration columns include dextran, agarose and
polyacrylamide.
[0203] Dextran is a polysaccharide composed of glucose residues and
is commercially available under the name SEPHADEX (Pharmacia Fine
Chemicals, Inc.). The beads are prepared with various degrees of
cross-linking in order to separate different sized molecules by
providing various pore sizes. Alkyl dextran is cross-linked with
N,N'-methylenebisacrylamide to form SEPHACRYL-S100 to S1000 which
allows strong beads to be made that fractionate in larger ranges
than SEPHADEX can achieve.
[0204] Polyacrylamide can also be used as a gel filtration medium.
Polyacrylamide is a polymer of cross-linked acrylamide prepared
with N,N'-methylenebisacrylamide as the cross-linking agent.
Polyacrylamide is available in a variety of pore sizes from Bio-Rad
Laboratories (USA) to be used for separation of different size
particles.
[0205] The gel material swells in water and in a few organic
solvents. Swelling is the process by which the pores become filled
with liquid to be used as eluant. As the smaller molecules enter
the pores, their progress through the gel is retarded relative to
the larger molecules which do not enter the pores, forming the
basis of the separation. The beads are available in various degrees
of fineness to be used in different applications. The coarser the
bead, the faster the flow and the poorer the resolution. Superfine
can be used for maximum resolution, but the flow is very slow. Fine
is used for preparative work in large columns which require a
faster flow rate. The coarser grades are for large preparations in
which resolution is less important than time, or for separation of
molecules with a large difference in molecular weights.
[0206] Affinity chromatography is generally based on the
recognition of a protein by a substance such as a ligand or an
antibody. The column material can be synthesized by covalently
coupling a binding molecule, such as an activated dye, for example
to an insoluble matrix. The column material is then allowed to
adsorb the desired substance from solution. Next, the conditions
are changed to those under which binding does not occur and the
substrate is eluted. The requirements for successful affinity
chromatography are that the matrix must adsorb molecules, the
ligand must be coupled without altering its binding activity, a
ligand must be chosen whose binding is sufficiently tight, and it
must be possible to elute the substance without destroying it.
[0207] One embodiment of the compounds described herein is an
affinity chromatography method where the matrix is a reactive
dye-agarose matrix. Blue-SEPHAROSE, a column matrix composed of
Cibacron Blue 3GA and agarose or SEPHAROSE can be used as the
affinity chromatography matrix. Alternatively, SEPHAROSE CL-6B is
available as Reactive Blue 2 from Sigma Chemical Company. This
matrix binds fusion proteins directly and allows their separation
by elution with a salt gradient.
[0208] Provided herein are compositions containing modified
diphtheria toxins, said modified diphtheria toxin comprising an
amino acid sequence as set forth in, for example, SEQ ID NO: 2 or
149 with one or more amino acid modifications therein, wherein at
least one amino acid modification is made within an (x)D(y) motif
in a region selected from among residues 7-9, 29-31 and 290-292 of
SEQ ID NO: 2 or 149, and said modified diphtheria toxin has
cytotoxicity comparable to an unmodified diphtheria toxin. In one
embodiment, a modification at position (x) is a substitution of V
or I by an amino acid residue selected from among F, C, M, T, W, Y,
P, H, E, Q, D, N, K, R, and a modified or unusual amino acid from
Table 1. In one embodiment, a modification at position D is a
substitution of D by an amino acid residue selected from among I,
V, L, F, C, M, A, G, T, W, Y, P, H, Q, N, K, R and a modified or
unusual amino acid from Table 1. In another embodiment, a
modification at position (y) is a substitution of S by an amino
acid residue selected from among I, F, C, M, A, G, T, W, Y, P, H,
E, Q, D, N, K, R and a modified or unusual amino acid from Table 1.
In another embodiment, a modified diphtheria toxin has a
combination of two, three or more modifications in one or more
(x)D(y) motifs. In one embodiment, a modified diphtheria toxin
contains one or more modifications selected from among V7T, V7N,
V7D, D8N, S9A, S9T, S9G, V29N, V29D, V29T, D30N, S31G, S31N, I290T,
S292A, S292G and S292T. In one embodiment, a modified diphtheria
toxin contains two modifications. Such modified diphtheria toxins
can contain a combination of mutations such as, for example, V7N
V29N, V7N V29T, V7N V29D, V7T V29N, V7T V29T or V7T V29D. In one
embodiment, a modified diphtheria toxin contains three
modifications. Such modified diphtheria toxins can contain a
combination of mutations such as, for example, V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, or V7T V29T I290T.
[0209] Unmodified diphtheria toxins can have, for example, an amino
acid sequence of SEQ ID NO: 2 or 149 or an amino acid sequence of
any one of SEQ ID NOS: 4-147.
[0210] Compositions comprising modified diphtheria toxins, said
have reduced binding activity to human vascular endothelial cells
(HUVECs) compared to an unmodified diphtheria toxin. Such
compositions can further comprise a non-diphtheria toxin
polypeptide including, but not limited to, an antibody or an
antigen-binding fragment thereof, EGF, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, INF.alpha., INF.gamma., GM-CSF, G-CSF, M-CSF, TNF, VEGF,
Ephrin, BFGF or, TGF. The non-diphtheria toxin polypeptide can also
be a fragment of such polypeptides, such as a cell-specific binding
portion thereof. In one embodiment, the non-diphtheria toxin
polypeptide is IL-2 or a cell-specific binding portion thereof, or
IL-3 or a cell-specific binding portion thereof.
[0211] Modified DT and fusion proteins thereof having reduced
binding to HUVECs while maintaining the cytotoxicity can be used
for the treatment of various lymphoid-derived malignancies (e.g.,
cancers), solid tumors and non-malignant diseases such as GVHD or
psoriasis.
[0212] In an exemplary embodiment, the VLS modified DT fusion
toxins of the invention are administered to a subject such as a
mammal (e.g., a human), suffering from a medical disorder, e.g., a
cancer such as a T cell lymphoma, or non-malignant conditions
characterized by the presence of a class of unwanted cells to which
a targeting ligand can selectively bind (e.g., GVHD).
[0213] Denileukin diftitox has been shown to be effective in
treating a subject having previously been treated for cutaneous
T-cell lymphoma (Chin and Foss (2006) Clinical Lymphoma and
Myeloma, 7(3): 199-204; Talpur et al. (2006) J. Investigative
Dermatology 126: 575-583). Briefly, denileukin diftitox was
administered intravenously for 3 or 5 consecutive days at a dose of
4 .mu.g/kg/day, 9 .mu.g/kg/day or 18 .mu.g/kg/day for 3-21 cycles.
An overall response rate of 51% was observed. Denileukin diftitox
has been approved by the FDA for treatment of cutaneous T cell
lymphoma in the United States.
[0214] Provided herein is a method of treating a subject having
cutaneous T-cell lymphoma by administering a modified DT fusion
protein as described herein
[0215] Denileukin diftitox has been shown to be effective in
treating subjects having relapsed/refractory T-cell and B-cell
non-Hodgkin lymphoma (Dang et al. (2006) Br. J. Haematology 136:
439-447; Dang et al. (2004) J. Clin. Oncol. 22: 4095-4102).
Briefly, eligible patients received denileukin diftitox 18
.mu.g/kg/day for 5 days every three weeks for up to eight cycles.
Such a regimen was well tolerated in patients and was effective in
treating relapsed/refractory T-cell and B-cell non-Hodgkin
lymphoma.
[0216] Provided herein is a method of treating a subject having
relapsed/refractory T-cell or B-cell non-Hodgkin lymphoma by
administering a modified DT fusion protein as described herein.
[0217] In a Phase II clinical study, it was shown that ONTAK..RTM.
in combination with rituximab was significantly effective in
treating patients having relapsed/refractory B-cell non-Hodgkin
lymphoma. Therefore, provided herein is a method of treating a
subject having relapsed/refractory B-cell non-Hodgkin lymphoma by
administering a modified DT fusion protein as described herein.
[0218] Denileukin diftitox has been shown to be effective in
treating a subject having panniculitic T-cell lymphoma (McGinnis et
al. (2002) Arch. Dermatol. 138: 740-742). Briefly, a female patient
previously treated with bexarotene and interferon alpha relapsed
within 2 months of therapy. The patient was then treated with 1
cycle of intravenous denileukin diftitox (9 .mu.g/kg/day for 5
days). Clinical remission was observed with resolution of all
cutaneous disease, and constitutional symptoms was achieved 2 weeks
after the completion of the third cycle of denileukin diftitox.
[0219] Provided herein is a method of treating a subject having
panniculitic T-cell lymphoma by administering a modified DT fusion
protein as described herein. In one non-limiting embodiment, if
needed, the subject can be treated in combination with one or more
other therapies, such as, for example, bexarotene and/or interferon
alpha.
[0220] Denileukin diftitox has been shown to be effective in
treating a subject having extranodal natural killer/T cell
lymphoma, nasal type (Kerl et al. (2006) Br. J. Dermatology, 154:
988-991). Briefly, a 58-year old male with rapidly progressive
Epstein-Barr virus-positive nasal type extranodal natural killer/T
cell lymphoma was treated with a combination of bexarotene and
denileukin diftitox. A significant regression of the cutaneous
tumors was observed after a first cycle of denileukin diftitox and
was maintained for a period of 5 months with monthly cycles of
denileukin diftitox.
[0221] Provided herein is a method of treating a subject having
extranodal natural killer/T cell lymphoma, nasal type by
administering a modified DT fusion protein as described herein in
combination with bexarotene.
[0222] Denileukin diftitox has been shown to be effective in
treating a subject having previously treated chronic lymphocytic
leukemia (Frankel et al. (2006) Cancer, 106(10): 2158-2164).
Briefly, denileukin diftitox was administered as a 60-minute
intravenous infusion for 5 days every 21 days at a dose of 18
.mu.g/kg/day for up to 8 cycles. Overall, patients exhibited
reduction of peripheral chronic lymphocytic leukemia (CLL) cells,
reductions in lymph node size, and in some cases, remission as
identified over time from bone marrow biopsies. In one instance, a
patient treated was chemorefractory against fludarabine (Morgan et
al. (2004) Clin. Cancer Res. 9(10 Pt 1): 3555-3561).
[0223] Provided herein is a method of treating a subject having
chronic lymphocytic leukemia by administering a modified DT fusion
protein as described herein.
[0224] Denileukin diftitox has been shown to be effective in
treating a subject having human T-cell lymphotrophic virus
1-associated acute T cell leukemia/lymphoma (Venuti et al. (2003)
Clin. Lymphoma 4(3): 176-180). Briefly, 4 cycles of denileukin
diftitox was administered which resulted in restoration of normal
hematopiesis and a reduction in bone marrow myelofibrosis.
Following disease progression, 4 cycles of hyper-CVAD
(hyperfractionated
cyclophosphamide/doxorubicin/vincristine/decadron) were
administered and complete clinical remission was achieved. The
patient received maintenance therapy with denileukin diftitox for 1
year.
[0225] Provided herein is a method of treating a subject having
human T-cell lymphotrophic virus 1-associated acute T cell
leukemia/lymphoma by administering a modified DT fusion protein as
described herein in combination with hyper-CVAD therapy.
[0226] Denileukin diftitox has been shown to be effective in
treating a subject having a solid tumor (Eklund and Kuzel. Expert
Rev. Anticancer Ther., 2005 February; 5(1):33-8). Therefore,
provided herein is a method of treating a subject having one or
more solid tumors by administering a modified DT fusion protein as
described herein. Exemplary solid tumors include, but are not
limited to, those of a tissue or organ selected from among skin,
melanoma, lung, pancreas, breast, ovary, colon, rectum, stomach,
thyroid, laryngeal, ovary, prostate, colorectal, head, neck, eye,
mouth, throat, esophagus, chest, bone, testicular, lymph, marrow,
bone, sarcoma, renal, sweat gland, liver, kidney, brain,
gastrointestinal tract, nasopharynx, genito-urinary tract, muscle,
and the like tissues.
[0227] Acute Graft-versus Host Disease (aGVHD) is mediated partly
through activated T cells which express the high affinity receptor
for IL-2, which is recognized by denileukin diftitox. In a phase II
study of patients suffering from steroid-resistant aGVHD, one group
of patients were treated with a dose regimen of 4.5 .mu.g/kg daily
on days 1-5 and then weekly on study days 8, 15, 22 and 29. Another
group of patients were treated at with a dose regimen of 9 .mu.g/kg
on the same schedule. Responses were assessed at days 36 and
100.41% of the patients responded, all with a complete response at
day 36 and 27% patients responding at day 100 (4 complete responses
and 2 partial responses).
[0228] Provided herein is a method of treating a subject having
aGVHD by administering a modified DT fusion protein as described
herein.
[0229] Psoriasis is an immune-mediated skin disease in which
T-cells are chronically stimulated by antigen-presenting cells in
the skin. Psoriasis is a chronic relapsing disease that requires
intermittent treatment. Denileukin diftitox was shown to
effectively target activated T cells and improved psoriasis;
however, a side effect of the treatment was vascular leak syndrome
(Walsh and Shear. (2004) CMAJ, 170(13): 1933-1941). In a phase II
study of patients suffering from severe psoriasis, 35 patients were
treated with one of three doses of ONTAK.RTM. (0.5, 1.5 or 5
.mu.g/kg/day) and received three doses per week for eight weeks.
Eight out of 15 patients (treated with 5 or 1.5 .mu.g/kg/day)
showed more than 50% decrease in symptoms as measured by the
Psoriasis Area and Severity Index (PASI) and Physician's Global
Assessment (PGA). Four patients, all treated with a dose of 5
.mu.g/kg/day, benefited from a 2-grade improvement on the 5-grade
PGA scale.
[0230] Provided herein is a method of treating a subject having
psoriasis by administering a modified DT fusion protein as
described herein.
[0231] Also contemplated herein is a method of providing
maintenance therapy by administering a non-immunogenic DT fusion
protein as described herein.
[0232] As described by Dannull et al. (J. Clin. Invest. 115(12):
3623-3633 (2005)), immunization with RNA-transfected dendritic
cells (DCs) is an effective strategy to stimulate potent T cell
responses in patients with metastatic cancers (Su et al. 2003.
Cancer Res. 63: 3127-2133; Heiser et al. 2002. J. Clin. Invest.
109: 409-417). CD4+ T cells constitutively expressing the IL-2
receptor .alpha.-chain (CD25) act in a regulatory capacity by
suppressing the activation and function, of other T cells (Shevach,
E. M. 2001. J. Exp. Med. 193: F41-F46). Their physiological role is
to protect the host against the development of autoimmunity by
regulating immune responses against antigens expressed by normal
tissues (Jonuleit et al. 2000. J. Exp. Med. 192: 1213-1222; Read
and Powrie. 2001. Curr. Opin. Immunol. 13: 644-649). Since tumor
antigens are largely self antigens, T regulatory cells (Tregs) may
also prevent the tumor-bearing host from mounting an effective
anti-tumor immune response. Previous studies have shown that
elevated numbers of CD4+CD25+ Tregs can be found in advanced cancer
patients (Woo et al. 2002. J. Immunol. 168: 4272-4276) and that
high Treg frequencies are associated with reduced survival (Curiel
et al. 2004. Nat. Med. 10: 942-949). The important role of
CD4+CD25+ Tregs in controlling tumor growth was further highlighted
by the demonstration that depletion of Tregs using anti-CD25
antibodies can evoke effective anti-tumor immunity in mice (Shimizu
et al. 1999. J. Immunol. 163: 5211-5218; Onizuka et al. 1999.
Cancer Res. 59: 3128-3133). Moreover, anti-CD25 therapy enhanced
the therapeutic efficacy of GM-CSF-secreting B16 tumor cells and
prolonged survival of tumor-bearing animals (Sutmuller et al. 2001.
J. Exp. Med. 194: 823-832). Cumulatively, these experimental data
suggest that the efficacy of cancer treatment could be enhanced by
administration of agents that lead to the preferential depletion of
CD4+CD25+ Tregs, such as compounds that target cells expressing the
IL-2 receptor CD25 subunit.
[0233] Recombinant IL-2 diphtheria toxin conjugate DAB389IL-2 (also
known as denileukin diftitox and ONTAK.RTM.) to eliminate
CD25-expressing Tregs in metastatic renal cell carcinoma (RCC)
patients. The cytotoxic action of DAB389IL-2 occurs as a result of
binding to the high-affinity IL-2 receptor, subsequent
internalization, and enzymatic inhibition of protein synthesis,
ultimately leading to cell death.
[0234] DAB389IL-2 was shown to selectively eliminate Tregs from
human PBMCs in a dose-dependent manner without apparent bystander
toxicity to other PBMCs or CD4+ T cells with intermediate- or
low-level expression of CD25. Treg depletion resulted in enhanced
stimulation of proliferative and cytotoxic T cell responses in
vitro but only when DAB389IL-2 was used prior to and omitted during
the T cell priming phase. Depletion of Tregs in RCC patients with
DAB389IL-2 followed by immunization with tumor RNA-transfected DCs
led to improved stimulation of tumor-specific T cells when compared
with administration of tumor RNA-transfected DCs alone.
CD4+CD25high Tregs can be eliminated using a single dose of
DAB389IL-2 without apparent bystander toxicity and without having
an impact on the function of other cells expressing CD25.
DAB389IL-2 profoundly reduced the number of Tregs present in the
peripheral blood of RCC patients, reduced levels of peripheral
blood-derived FoxP3 transcripts, and abrogated Treg-mediated
immunosuppressive activity in vivo. Moreover, significantly higher
frequencies of tumor-specific CD8+ T cells could be measured in
patients treated with combined DAB389IL-2 and DC immunization when
compared with subjects receiving the DCs alone. Also, there was a
trend toward an improved CD4+ T cell response after combined
therapy.
[0235] Cognate immunity against neoplastic cells depends on a
balance between effector T cells and regulatory T (Treg) cells.
Treg cells prevent immune attack against normal and neoplastic
cells by directly suppressing the activation of effector CD4+ and
CD8+ T cells. The use of a recombinant interleukin 2/diphtheria
toxin conjugate (DAB/IL2; Denileukin Diftitox; ONTAK.RTM.) was
studied as a strategy to deplete Treg cells and break tolerance
against neoplastic tumors in humans. DAB/IL2 (12 microg/kg; four
daily doses; 21 day cycles) was administered to 16 patients with
metastatic melanoma and the effects on the peripheral blood
concentration of several T cell subsets and on tumor burden are
measured.
[0236] Rasku et al. (J. Translational Medicine; 6: 12 (2008)) found
that DAB/IL2 caused a transient depletion of Treg cells as well as
total CD4+ and CD8+ T cells (<21 days). T cell repopulation
coincided with the de novo appearance of melanoma antigen-specific
CD8+ T cells in several patients as determined by flow cytometry
using tetrameric MART-1, tyrosinase and gp100 peptide/MHC
conjugates. Sixteen patients received at least one cycle of DAB/IL2
and five of these patients experienced regressions of melanoma
metastases as measured by CT and/or PET imaging. One patient
experienced a near complete response with the regression of several
hepatic and pulmonary metastases coupled to the de novo appearance
of MART-1-specific CD8+ T cells. A single metastatic tumor remained
in this patient and, after surgical resection, immunohistochemical
analysis revealed MART1+ melanoma cells surrounded by CD8+ T cells.
The transient depletion of T cells in cancer patients may disrupt
the homeostatic control of cognate immunity and allow for the
expansion of effector T cells with specificity against neoplastic
cells.
[0237] Recent work demonstrates that lack of naturally induced
tumor associated antigen (TAA)-specific immunity is not simply a
passive process. Barnett et al. (Am J Reprod Immunol. 54(6):321
(2005)) demonstrated that tumors actively prevent induction of
TAA-specific immunity through induction of TAA-specific tolerance.
This tolerance was mediated in part by regulatory T cells (Tregs).
Barnett et al. presented evidence that depleting Tregs in human
cancer, including ovarian cancer, using denileukin diftitox
(ONTAK.RTM.), improves immunity.
[0238] CD4+CD25+Foxp3+ regulatory T (Treg) cells have been
implicated in the lack of effective antitumor immunity (Litzinger
et al. (2007) Blood; 110(9): 3192-201). Denileukin diftitox
(DAB(389)IL-2), provides a means of targeting Treg cells. Treg
cells in spleen, peripheral blood, and bone marrow of normal
C57BL/6 mice were variously reduced after a single intraperitoneal
injection of denileukin diftitox; the reduction was evident within
24 hours and lasted approximately 10 days. Injection of denileukin
diftitox 1 day before immunization with another agent enhanced
antigen-specific T-cell responses above levels induced by
immunization alone. Litzinger et al. demonstrated in a murine model
the differential effects of denileukin diftitox on Treg cells in
different cellular compartments, the advantage of combining
denileukin diftitox with another agent to enhance antigen-specific
T-cell immune responses, the lack of inhibition by denileukin
diftitox of host immune responses directed against a live viral
vector, and the importance of dose scheduling of denileukin
diftitox when used in combination with an immunogen.
[0239] Tregs have been shown to be an integral part of regulating
and even suppressing an immune response to growing tumor cells.
Matsushita et al. (J. Immunol. Methods; 333(1-2):167-79 (2008))
compared three methods of Treg depletion and/or elimination,
utilizing low dose cyclophosphamide (CY), a specific antibody
directed against the IL-2 receptor found on Tregs (PC61) and the
use of denileukin diftitox (DD). Matsushita demonstrated that
utilization of DD resulted in a >50% Treg cell reduction without
parallel cytocidal effects upon other T cell subsets but did not
enhance anti-tumor immunity against B16 melanoma. Lastly, the PC61
showed a moderate reduction of Tregs that lasted longer than the
other reagents, without a reduction in the total number of CD8(+) T
cells.
[0240] Provided herein is a method of enhancing activity of an
anti-cancer agent (e.g., RNA transfected DCs, anti-CLTA4
antibodies, MISIIR scFvs, etc.), by administering a DT variant-IL2
fusion protein described herein. In one embodiment, a DT
variant-IL2 fusion protein is administered followed by
administration of the anti-cancer agent. In one non-limiting
example, the DT variant-IL2 fusion protein is administered at least
four (4) days prior to the anti-cancer agent.
[0241] Also provided herein is a method of treating a metastatic
cancer via reduction or elimination of Tregs by administering an
anti-cancer agent (e.g., RNA transfected DCs, anti-CLTA4
antibodies, MISIIR scFvs, etc.) and a DT variant-IL2 fusion protein
described herein. Metastatic tumors include, for example,
metastatic renal cell carcinoma, metastatic prostate cancer,
metastatic ovarian cancer and metastatic lung cancer. In one
embodiment, a DT variant-IL2 fusion protein is administered
followed by administration of the anti-cancer agent. In one
non-limiting example, the DT variant-IL2 fusion protein is
administered at least four (4) days prior to the anti-cancer
agent.
[0242] In another aspect, provided herein is a method of treating a
prostate tumor, an ovarian tumor, a lung tumor or a melanoma via
reduction or elimination of Tregs by administering an anti-cancer
agent (e.g., RNA transfected DCs, anti-CLTA4 antibodies, MISIIR
scFvs, etc.) and a DT variant-IL2 fusion protein described herein.
In one embodiment, a DT variant-IL2 fusion protein is administered
followed by administration of the anti-cancer agent. In one
non-limiting example, the DT variant-IL2 fusion protein is
administered at least four (4) days prior to the anti-cancer
agent.
[0243] Toxicity and therapeutic efficacy of such ingredient can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to non-cancerous and otherwise healthy cells and, thereby, reduce
side effects.
[0244] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture and as presented below in Example 4.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0245] The embodiments of the compounds and methods of the present
application are intended to be illustrative and not limiting.
Modifications and variations can be made by persons skilled in the
art in light of the above teachings specifically those that may
pertain to alterations in the DT toxophore surrounding the
described VLS sequences that could result in reduced HUVEC binding
while maintaining near native functionally with respect to the
ability to use as a DT toxophore in protein fusion toxin
constructions.
[0246] It is also conceivable to one skilled in the art that the
compounds and methods described herein can be used for other
purposes, including, for example, the delivery of other novel
molecules to a selected cell population.
[0247] The present application contemplates compositions for use in
immunization embodiments. It is contemplated that proteinaceous
compositions that are less effective in promoting VLS or other
toxic effects by alterations in one or more (x)D(y), (x)D(y)T
and/or flanking sequences are useful as antigens to stimulate an
immune response to the toxin. In particular embodiments, DT
comprising one or more modified (x)D(y), (x)D(y)T and/or flanking
sequences are contemplated as useful antigens. Preferably the
composition is extensively dialyzed to remove undesired small
molecular weight molecules and/or lyophilized for more ready
formulation into a desired vehicle. In other embodiments, it is
also possible to use toxins lacking one or more active site
residues (i.e., a toxoid) for immunization.
[0248] The compounds and methods described herein can be employed
under those circumstances in which amounts of DT toxophore would be
used to deliver such agents in a clinical setting or in settings
where it would be desirable to reduce as much as possible the
potential for VLS. In this setting the catalytic domain or some
portion thereof would be replaced, rendered inactive and fused with
the desired agent or molecule. Acid sensitive or protease sensitive
cleavage sites could be inserted between the remnant of the
catalytic domain and the desired agent or molecule.
[0249] Agents or molecules that might be coupled to VLS modified DT
toxophore such as disclosed herein include but are not limited to;
peptides or protein fragments, nucleic acids, oligonucleotides,
acid insensitive proteins, glycoproteins, proteins or novel
chemical entities that required selective delivery.
[0250] Therefore, it should be understood that changes may be made
in the particular embodiments disclosed which are within the scope
of what is described.
VI. Packages and Kits
[0251] In still further embodiments, the present application
concerns kits for use with the compounds described above. Toxins
exhibiting reduced VLS promoting or toxic effects can be provided
in a kit. Such kits may be used to combine the toxin with a
specific cell binding ligand to produce a fusion protein that
targets a particular receptor on a cell (e.g., IL-2 receptors on
cancer cells) in a ready to use and storable container. The kits
will thus comprise, in suitable container means, a composition with
reduced VLS promoting activity. The kit may comprise a modified DT
or a fusion protein thereof in suitable container means.
[0252] The container means of the kits will generally include at
least one vial, test tube, flask, bottle, syringe and/or other
container means, into which the at least one polypeptide can be
placed, and/or preferably, suitably aliquoted. The kits can include
a means for containing at least one fusion protein, detectable
moiety, reporter molecule, and/or any other reagent containers in
close confinement for commercial sale. Such containers may include
injection and/or blow-molded plastic containers into which the
desired vials are stored. Kits can also include printed material
for use of the materials in the kit.
[0253] Packages and kits can additionally include a buffering
agent, a preservative and/or a stabilizing agent in a
pharmaceutical formulation. Each component of the kit can be
enclosed within an individual container and all of the various
containers can be within a single package. Invention kits can be
designed for cold storage or room temperature storage.
[0254] Additionally, the preparations can contain stabilizers (such
as bovine serum albumin (BSA)) to increase the shelf-life of the
kits. Where the compositions are lyophilized, the kit can contain
further preparations of solutions to reconstitute the lyophilized
preparations. Acceptable reconstitution solutions are well known in
the art and include, for example, pharmaceutically acceptable
phosphate buffered saline (PBS).
[0255] Additionally, the packages or kits provided herein can
further include any of the other moieties provided herein such as,
for example, one or more reporter molecules and/or one or more
detectable moieties/agents.
[0256] Packages and kits can further include one or more components
for an assay, such as, for example, an ELISA assay, cytotoxicity
assay, ADP-Ribosyltransferase activity assay, etc. Samples to be
tested in this application include, for example, blood, plasma, and
tissue sections and secretions, urine, lymph, and products thereof.
Packages and kits can further include one or more components for
collection of a sample (e.g., a syringe, a cup, a swab, etc.).
[0257] Packages and kits can further include a label specifying,
for example, a product description, mode of administration and/or
indication of treatment. Packages provided herein can include any
of the compositions as described herein. The package can further
include a label for treating a cancer.
[0258] The term "packaging material" refers to a physical structure
housing the components of the kit. The packaging material can
maintain the components sterilely, and can be made of material
commonly used for such purposes (e.g., paper, corrugated fiber,
glass, plastic, foil, ampules, etc.). The label or packaging insert
can include appropriate written instructions. Kits, therefore, can
additionally include labels or instructions for using the kit
components in any method of the invention. A kit can include a
compound in a pack, or dispenser together with instructions for
administering the compound in a method described herein.
[0259] Instructions can include instructions for practicing any of
the methods described herein including treatment methods.
Instructions can additionally include indications of a satisfactory
clinical endpoint or any adverse symptoms that may occur, or
additional information required by regulatory agencies such as the
Food and Drug Administration for use on a human subject.
[0260] The instructions may be on "printed matter," e.g., on paper
or cardboard within or affixed to the kit, or on a label affixed to
the kit or packaging material, or attached to a vial or tube
containing a component of the kit. Instructions may additionally be
included on a computer readable medium, such as a disk (floppy
diskette or hard disk), optical CD such as CD- or DVD-ROM RAM,
magnetic tape, electrical storage media such as RAM and ROM, IC tip
and hybrids of these such as magnetic/optical storage media.
EXAMPLES
[0261] The application may be better understood by reference to the
following non-limiting examples, which are provided as exemplary
embodiments of the application. The following examples are
presented in order to more fully illustrate embodiments of the
invention and should in no-way be construed, however, as limiting
the broad scope of the application. While certain embodiments of
the present application have been shown and described herein, it
will be obvious that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments described herein may be employed in practicing the
methods described herein.
Example 1
Construction, Expression and Purification of DT Variant and
DT-Fusion Proteins
[0262] Construction of DT Variant and DT-Fusion Proteins
[0263] A truncated DT-based toxophore comprising a methionine
residue at the N-terminus and amino acid residues 1 through 386
(SEQ ID NO: 2) of the native DT (now residues 2-387 in the
truncated toxophore) is constructed as DT387 or residues 1-382 of
DT387. The DT-based toxophore can also comprise a methionine
residue at the N-terminus, amino acid residues 1 through 386 (SEQ
ID NO: 2) of the native DT (now residues 2-387 in the truncated
toxophore), and residues 484-485 of native DT, constructed as
DT389. DT387 and DT389 contains three (x)D(y) motifs at residues
7-9 (VDS), residues 29-31 (VDS), and residues 290-292 (IDS). DT382
contains residues 1-382 of DT387 or DT389. Other C-terminal
truncated DT constructs as described herein can be used in the
assays provided herein for testing functionality of DT variants.
One would understand that modifications made to DT389 also could be
made in a truncated construct (e.g., DT382) and tested for
functionality. FIG. 12 provides amino acid sequences of wild type
DT382, DT382 variants, and null construct DT382(G53E): underlined
sequences are vector/tag sequences; enterokinase cleavage site
highlighted in italicized text; and mutations from WT sequences are
shown in bold text.
[0264] Site directed mutagenesis is employed to alter the (x)D(y)
motif in DT. A Stratagene Quickchange mutagenesis kit is used to
construct the mutations. Oligonucleotide primers are designed to
alter encoding residues within the (x)D(y) motif implicated in
VLS.
[0265] SEQ ID NOS: 4-147 provides a list of non-limiting exemplary
modified DTs and the corresponding amino acid sequences.
[0266] The mutants are tested in the context of protein fusion
toxin genetically fused to sequences encoding human interleukin 2
(SEQ ID NO: 3) or a cell binding portion thereof. DT-fusion
proteins are expressed and purified.
[0267] Expression and Purification of DT Variants and DT-Fusion
Proteins
[0268] Plasmid constructs encoding truncated DT protein, DT
mutants, and DT-fusion protein are transformed into E. coli HMS 174
(DE3) cells. E. coli HMS 174 is a protease-deficient strain in
which over-expression of recombinant proteins can be achieved.
Induction of the recombinant protein expression is obtained by
addition of isopropylthiogalactosidase (IPTG) to E. coli HMS 174.
Following incubation, the bacterial cells are harvested by
centrifugation and lysed, and the recombinant protein is further
purified from inclusion body preparations as described by Murphy
and vanderSpek, Methods in Molecular Biology, Bacterial Toxins:
Methods and Protocols, 145:89-99 Humana press, Totowa, N.J. (2000).
It may be necessary to remove endotoxin from the protein
preparations to assure that effects on HUVECs are from VLS and not
due to the presence of the endotoxin. Endotoxin is removed to
<250 EU/ml by passage over an ion-exchange resin. Separation of
breakdown products from full-length material also occurs during
ion-exchange chromatography. After another final purification over
ion exchange resin, endotoxin is reduced to <25 EU/ml and the
toxophore is tested for VLS as a function of HUVEC cell binding in
vitro. Analysis of DT387 or DT389 toxophore can be conducted using
Coomassie Blue staining and Western Blot when samples from the
process described above are resolved by SDS Polyacrylamide Gel
Electrophoresis (PAGE) using conventional techniques described
herein and known in the art.
[0269] Mutations that result in stable constructs with adequate
expression that do not affect ribosyltransferase activity of the
DT387 toxophore can be subsequently tested for targeted
cytotoxicity in the corresponding VLS modified DT-EGF and VLS
modified DT-IL-2 protein fusion toxins (Example 5
respectively).
Example 2
Cell Permeabilization Assays
[0270] Human vascular endothelial cells are maintained in EGM media
(obtained from Cambrex, Walkersville, Md.). Sub-confluent early
passage cells are seeded at equivalent cell counts onto plastic
cover slips. Purified, endotoxin free wild type DT toxophore and
mutants are labeled with the fluorescent tag F-150 (Molecular
Probes, Eugene, Oreg.) through chemical conjugation. HUVECs are
incubated with equivalent amounts of the labeled toxophores. The
media is then aspirated, and the cells are then washed, fixed and
prepared for analysis. Examination of the cells on cover slips from
different treatment groups permits the analysis of the number of
cells labeled by the fluorescent toxophore. No targeting ligand is
present on the toxophore and, consequently, the level of HUVEC
interaction is proportional only to the toxophores affinity for
HUVECs. Comparisons are carried out using a fluorescent microscope
and comparing the number of cells labeled from at least ten
independent fields, different cover slips or different slides. DAPI
stain is used to localize cells, particularly in the case of the
mutant constructs as cell labeling is not readily apparent.
4'-6-Dianidino-2-phenylindole (DAPI) is known to form fluorescent
complexes with natural double-stranded DNA; as such DAPI is a
useful tool in various cytochemical investigations. When DAPI binds
to DNA, its fluorescence is strongly enhanced. Thus, DAPI serves as
a method of labeling cell nuclei. In contrast, cells treated with
F-150DT toxophore are easily observed. To facilitate that
quantification of the mutant DT toxophore constructs, the signal
intensity and changes in background signal are also increased.
[0271] Effect of the DT variant IL-2 fusion proteins on the
morphology of HUVEC monolayers can also be assessed according to
methods described, by example, Baluna et al. (Int. J. Immunopharm.,
18:355-361, 1996) and Soler-Rodriguez et al. (Exp. Cell Res.,
206:227-234, 1993). To determine whether the VLS sequences in DT
and IL-2 damage HUVECs, monolayers are incubated with different
concentrations of DT variants, DT variant-IL-2-fusion proteins, or
controls. HUVECs are isolated, cultured and studied
microscopically. Briefly, HUVEC monolayers are incubated at
37.degree. C. for 18 hours with 10-6 M of each variant, fusion
protein, control, or medium-only and then examined by
phase-contrast microscopy (magnification at 20 times). Normal
monolayers consist of highly packed cells with elogated shapes,
whereas damaged cells round up and detach from the plate. Untreated
HUVECs consist of tightly packed elongated cells. Monolayers can be
assessed after 2 hours for cell rounding after 2 hr of incubation
and after 18 hours for formation of gaps in the monolayer. Toxic
effects on HUVECs are assessed.
[0272] Another method for measuring permeability of endothelial
monolayers in vitro has been described in detail previously
(Friedman et al. J. Cell. Physiol., 129: 237-249 (1986); Downie et
al. Am. J. Resp. Cell. Mol. Biol., 7(1): 58-65 (1992)). After
incubation with the various media described, the filters containing
confluent endothelial cells are washed 2 times with PBS. The
filters with attached endothelial cells are then mounted in
modified flux chambers, and the chambers placed in a culture dish.
The upper well of the chamber is filled with serum-free medium
containing 50 mM Hepes. The dish is filled with the same medium. A
stirring bar is added to the lower well, and the entire chamber
placed on an electrical stirring device and incubated at 37.degree.
C. The chamber is incubated until the level of media between the
upper well and the surrounding fluid in the beaker is equal. Thus,
no hydrostatic pressure difference is present between the upper and
lower wells. After this equilibration period, a small aliquot of
medium in the upper well is removed and replaced with medium
containing [.sup.125I]bovine serum albumin (30,000 cpm/ml). The
radiolabeled albumin is extensively dialyzed against 1 M PBS
immediately before use. Chromatographic monitoring of the dialyzed
[.sup.125I]albumin as well as the media in the lower well after the
end of the study is demonstrated >95% of the .sup.125I to
co-chromatograph with albumin (Friedman et al. J. Cell. Physiol.,
129: 237-249 (1986)). Small aliquots of media (in triplicate) are
removed serially from both the upper and lower wells 10, 30, 60,
120, 180, and 240 min after the addition of the .sup.125I probe.
The .sup.125I activity in each aliquot is measured in a gamma
counter, and the average cpm/ml for the samples from the upper and
lower wells is determined. Appropriate corrections are made for
background using the experimental media. The [.sup.125I]albumin
transfer rate of the BPAEC monolayers is expressed as the rate of
appearance of counts in the lower well relative to the number of
counts in the upper well/hour over the 90 to 240-min period of
steady-state clearance (Friedman et al. J. Cell. Physiol., 129:
237-249 (1986)). Each albumin transfer rate point ("n") represents
the average rate of duplicate filters within a group. Each group of
filters included duplicate control filters (i.e., monolayers on
filters incubated with diluent alone). In additional filters,
non-radiolabeled bovine serum albumin (final concentration of 1%)
is added along with [.sup.125I]bovine serum albumin in the upper
well. The [.sup.125I]albumin transfer rate across the monolayer is
determined using the previously described method. The endothelial
monolayers is expected to be more intact after exposure to
DTvariant-IL-2 fusion proteins compared to unmodified DT-IL-2
fusion proteins.
[0273] In yet another assay, channel-forming activities of the
mutants of DT-IL-2 are determined using a planar lipid bilayer
membrane system (vanderSpek et al., J. Biol. Chem. 268: 12077-12082
(1993); Silverman et al., J. Membr. Biol. 137: 17-28 (1994); Hu et
al. Prot. Eng. 11(9): 811-817 (1998)) and compared to unmodified
DT-IL-2. The membranes are formed across 50-100 .mu.m apertures are
made in polystyrene cups. A 1% hexane solution of lecithin type IIS
(Sigma) with the neutral lipids removed (Kagawa and Racker, Biol.
Chem. 246: 5477-5487 (1971)) is used to coat both sides of the
aperture and allowed to dry. The outside of the aperture is then
coated with a 1.5% squalene solution prepared in light petroleum.
The cup is placed in the back chamber of a block prepared by Warner
Instruments (Hamden, Conn.). A buffer solution (1 M KCl, 2 mM
CaCl.sub.2, 1 mM EDTA, 50 mM HEPES, pH 7.2) is added to the cup to
above the level of the aperture (0.5 ml). The front chamber of the
block is filled with 1.0 ml of the same buffer solution, except
with 30 mM MES, pH 5.3, instead of the HEPES. A 50 .mu.l aliquot of
the lecithin hexane solution is layered on top of the buffer in the
front chamber and the hexane is allowed to evaporate. The buffer in
the front chamber is then lowered and raised above the level of the
aperture and the planar lipid bilayer is formed. Unmodified DT-IL-2
fusion proteins and DT variant-IL-2 fusion proteins thereof are
added to the front chamber at concentrations ranging from 20 to 730
ng/ml. A voltage of +60 mV is applied across the membrane using
voltage clamp conditions. The back chamber of the block, containing
the cup, is held at virtual ground and the voltages refer to the
front chamber to which the proteins are added. Current is monitored
using standard methods (Jakes et al., J. Biol. Chem. 265: 6984-6991
(1989)). Channel conductances are determined using the equation
g=I/V, where g is the conductance, I is the current flowing through
the membrane and V is the voltage applied across the membrane. The
lipid bilayer is expected to be more intact after exposure to
DTvariant-IL-2 fusion proteins compared to unmodified DT-IL-2
fusion proteins.
Example 3
[0274] This example describes a method for testing
ADP-Ribosyltransferase Activity. Ribosome inactivating protein
toxins, such as diphtheria toxin, catalyze the covalent
modification of translation elongation factor 2 (EF-2).
Ribosylation of a modified histidine residue in EF-2 halts protein
synthesis at the ribosome and results in cell death.
Ribosyltransferase assays to determine catalytic activity of the
DT387 mutants are performed in 50 mM Tris-Cl, pH8.0, 25 mM EDTA, 20
mM Dithiothreitol, 0.4 mg/ml purified EF-2, and 1.0 .mu.M
[.sup.32P]-NAD+(10 mCi/ml, 1000 Ci/mmol, Amersham-Pharmacia). The
purified mutant proteins are tested in a final reaction volume of
40 .mu.l. The reactions are performed in 96 well, V-bottom
microtiter plates (Linbro) and incubated at room temperature for an
hour. Proteins are precipitated by addition of 200 .mu.l 10% TCA
and collected on glass fiber filters, and radioactivity is
determined by standard protocols. Traditional methods for measuring
ADP-ribosylation use permeabilized cells treated with double
stranded (ds) activator DNA oligonucleotide; subsequent measurement
of radiolabeled NAD+ is incorporated into acid insoluble material.
FACS-based methods such as those described by Kunzmann et al.
(Immunity & Ageing 3:8 (2006)) are also available.
Example 4
Cytotoxicity Assays on Crude Extracts of Modified DT-IL-2 Fusion
Proteins
[0275] The DT387 or DT389 construct is initially used to
demonstrate that VLS-modified toxophores can be chemically coupled
to a number of targeting ligands and yield functional targeted
toxins. Single chain fusions toxins, as exemplified by DT387linker
IL-2 or DT389linker IL-2, circumvent the scale-up purification
problems typically encountered in the development of conjugate
toxins. To confirm the effects of the engineered changes, a number
of VLS modified DT387IL-2 or DT389IL-2 fusion toxins are produced
and tested in cytotoxicity assays.
[0276] Amino acid substitutions made, as described above; to
determine that the changes do not yield inactive toxophores
incapable of producing fusion toxins, cytotoxicity assays are
performed.
[0277] Cytotoxicity Assays
[0278] Cytotoxicity assays are performed using HUT102/6TG cells, a
human HTLV1 transformed T-cell line that expresses high affinity
IL-2 receptors. HUT102/6TG cells are maintained in RPMI 1640
(Gibco) media supplemented with 10% fetal bovine serum, 2 mM
glutamine, 50 IU/ml penicillin and 50 .mu.g/ml streptomycin. The
cells are seeded at a density of 5.times.10.sup.4/well into 96
well, V-microtiter plates. The fusion protein toxins are typically
added to the wells in molarities ranging from 10.sup.-7 M down to
10.sup.-12 M. Final volume in the wells is 200 .mu.l. The plates
are incubated for 18 hours, at 37.degree. C. in a 5% CO.sub.2
environment. The plates are subjected to centrifugation to pellet
the cells, the media removed and replaced with 200 .mu.l
leucine-free, minimal essential medium containing 1.0
.mu.Ci/ml[.sup.14C] leucine (<280 mCi/mmol, Amersham-Pharmacia)
and 21 mM glutamine, 50 IU/ml penicillin and 50 .mu.g/ml
streptomycin. The cells are pulsed for 90 minutes and then the
plates subjected to centrifugation to pellet the cells. The
supernatant is removed and the cells are lysed in 60 .mu.l, 0.4 M
KOH followed by a 10 minute incubation at room temperature. 140
.mu.l of 10% TCA is then added to each well and another 10 minute,
room temperature incubation is performed. The precipitated proteins
are collected on glass fiber filters using a PHD cell harvester and
the incorporated radioactivity is determined using standard
methods. The results are reported as a percentage of control (no
fusion protein added to inhibit protein synthesis)
[.sup.14C]-leucine incorporation. Toxilight.TM., Vialight.TM. and
ALAMARBLUE.TM. kits are non-radioactive, commercial assays which
can be used to assess the variants. The assays are conducted in a
96-well plate format, titrating toxin (10.sup.-7-10.sup.-12 M) over
time using susceptible and resistant cell lines.
[0279] Pharmaceutical grade GMP purified DAB.sub.389IL-2 produced
from E. coli typically yields an IC.sub.50 of between
5.times.10.sup.-11 M to 1.times.10.sup.-12 M. Partially purified
toxins exhibit activity between 10-100 fold lower in partially
purified non-homogenous extracts. Pharmaceutical grade toxins are
purified to homogeneity and the active fractions of refolded fusion
toxins are used as biologically active drug. In the example above
we utilize a moderate through put analysis to determine the
receptor specific cytotoxicity of partially purified VLS modified
DT-IL-2 fusion toxins and compared them to the activity of
similarly purified DAB.sub.389IL-2. These assays demonstrate
comparable activity of the VLS modified DT.sub.387linker IL-2
fusion to DAB.sub.389IL-2. It should be noted that the calculation
of specific cytotoxicity was based upon the total amount of protein
in the samples of partially fusion toxin. For assays equimolar
concentrations of fusion toxins were tested.
[0280] The relative amounts of non-fusion toxins protein in each
sample could artificially alter the IC.sub.50 of any given
construct. That is, the presence of non full length or non fusion
toxin protein in the samples used in this analysis could
potentially account for small differences in IC.sub.50.
[0281] Purified DAB.sub.389IL-2 produced in E. coli typically
yields an IC.sub.50 of between 5.times.10.sup.-11 M and
1.times.10.sup.-12 M.
[0282] A moderate throughput cytotoxicity assay is used to analyze
crude purifications of VLS modified DT-IL-2 fusion toxins and
compare them to the activity of similarly purified DT387linker
IL-2.
[0283] It should be noted that there is one (x)D(y) motif in IL-2
located at residues 19-21 (LDL). The contribution of IL-2 to VLS
can be determined by modifying the (x)D(y) motif in the IL-2 and
test the modified protein using the cytotoxicity assay described
above. For example, using modified DT mutants derived from
DT.sub.387 or DT.sub.387linker IL-2, it is possible to distinguish
between effects of the mutations on catalytic activity, VLS
activity and effective delivery of the targeted toxin to the
cytosol of target cells. The comparison between modified DT mutants
of DT.sub.387 and DT.sub.387linker IL2 will also separate the
effects of modified sequences of the toxophore alone from the IL-2
targeting ligand present in DT.sub.387linker IL-2. In another
example, using modified DT mutants derived from both DT.sub.389 and
DT.sub.389linker IL-2, it is possible to distinguish between
effects of the mutations on catalytic activity, VLS activity and
effective delivery of the targeted toxin to the cytosol of target
cells. The comparison between modified DT mutants of DT.sub.389 and
DT.sub.389linker IL2 will also separate the effects of modified
sequences of the toxophore alone from the IL-2 targeting ligand
present in DT.sub.389linker IL-2.
Example 5
[0284] This example describes an in vivo method to test the effect
of fusion proteins described herein. A model has been developed to
study the effect of toxin-containing fusion proteins on human
endothelium in vivo by grafting vascularized human skin onto SCID
mice, injecting the mice with toxin-containing fusion proteins and
measuring fluid accumulation in the graft as the wet/dry weight
ratio (Baluna and Vitetta, J. Immunother., 22(1):4147 (1999)).
Fluid accumulation in the human skin is measured by weighing punch
biopsies of the skin grafts before and after freeze drying. This
model can be used to evaluate the effect of the modified DT fusion
proteins described herein in vivo.
[0285] The fluid accumulation in the lungs of normal SCID mice is
also used as a surrogate model for VLS. IL-2 has been shown to
induce fluid accumulation in the lungs of mice (Orucevic and Lala,
J Immunother Emphasis Tumor Immunol., 18(4):210-220 (1995)). The
water content of the lungs or skin grafts is calculated as the
wet/dry weight ratio. In this model, pulmonary vascular leak can
also be assessed by measuring the accumulation in the lungs of
.sup.125I-albumin injected intravenously (Smallshaw et al. Nature
Biotechnology 21:387-391 (2003)).
Example 6
[0286] Multiple assays are available to test the function of DT
variants described herein such as, for example, in vitro
cytotoxicity assays, in vitro vascular toxicity, in vivo mouse
models as well as any other assay described herein or known in the
art.
[0287] In Vitro Cytotoxicity Assays
[0288] The cytotoxic activities of the different modified DT fusion
proteins are determined using CD22.sup.+ Daudi cells and
[.sup.3H]-leucine incorporation as described previously (Ghetie et
al., (1988) Cancer Res. 48:2610). The concentration of fusion
protein which reduces [.sup.3H]-leucine incorporation by 50%
relative to an untreated control culture is defined as the
IC.sub.50.
[0289] Vascular Toxicity
[0290] As a first step in evaluating the ability of fusion proteins
prepared with modified DTs to induce vascular damage, a series of
in vitro studies using HUVECs can be conducted. For in vitro
assays, the effect of modified DT fusion proteins on the morphology
of HUVEC monolayers is tested as described previously (Baluna et
al., (1996) Immunopharmacology, 37:117-132).
[0291] In Vivo Assays
[0292] The effects of modified DT fusion proteins can be determined
in the SCID/Daudi tumor model (Ghetie et al., (1992) Blood. 80(9):
2315-2320). Briefly, female SCID mice are injected intraveneously
(i.v.; lateral tail vein) with 5.times.10.sup.6 Daudi cells on day
zero. Fusion proteins are injected i.v. on days 1, 2, 3 and 4.
Groups of 5 mice are used for each treatment and studies are
repeated. Treatment groups receive (1) no treatment (control); (2)
unmodified DT fusion proteins; or (3) modified DT fusion proteins.
Mice are followed and sacrificed when the paralysis of their hind
legs occurs. Pulmonary vascular leak in SCID mice is evaluated as
described (Baluna et al., J. Immunother., (1999) 22(1):41-47). The
water content of the lungs is calculated as the wet/dry weight
ratios of lungs removed from mice injected with 10 .mu.g modified
DT fusion protein/g of mouse weight.
Example 7
[0293] The methods described herein below are to generate variants
of DT with reduced VLS using the following stages:
[0294] Stage 1--assays for DT activity;
[0295] Stage 2--gene synthesis, expression and purification of
whole DT in E. coli in a format suitable for screening multiple
variants (approximately 100-250 variants);
[0296] Stage 3--generation and testing of DT variants for reduced
VLS (HUVEC binding assay)--two rounds of variants (single
locus/multiple loci) are planned; and
[0297] Stage 4--construction and expression of variant DT-IL2.
[0298] Due to testing multiple variants of the full DT-IL2
molecule, stage 2 uses truncated DT variants without the wild-type
receptor binding (R) domain (DT (.DELTA.R)) while stage 3 uses full
length DT. Modifications are made in the catalytic (C) and
membrane-inserting (I) domains (translocation (T) domains) and the
wild-type R domain of DT is added for cell entry. Stage 4 involves
generating a fusion of the lead DT variant by fusion with human
IL-2 (2-133).
Example 8
[0299] VLS Assays
[0300] Various assays can be used to assess VLS activity of DT
variants as described herein.
[0301] In one assay, DT-IL2 or variants are conjugated to
fluorescein. Fluorescence microscopy/manual counting of labeled
cells can be used to assess direct binding to HUVEC cells as
surrogate for VLS activity.
[0302] In vivo studies; morphological changes; cell monolayer
permeability or loss of cell membrane integrity; and correlation
with CD31 expression represent various means by which the variants
described herein can be detected can also be used to assess VLS
activity.
[0303] (a) Detection with Antibodies
[0304] DT-specific antibodies can be used to detect DT bound to
endothelial cell surface via VLS motif. Common epitopes were
directly labeled to detect differences in binding between variants.
An ELISA was used to confirm specificity/affinity of available
anti-DT antibodies. In addition to the method described here,
His-tag Abs provide another means by which DT variants can be
selected.
[0305] FIG. 2 illustrates binding of a DT variant (DT-Glu52; CRM
mutant) to HUVEC cells and detection by antibodies using FACS
analysis.
[0306] (b) Direct Conjugates
[0307] Direct conjugates of DT and toxin variants to fluorochrome
(Alexa 488) can be used to assess DT variants described herein.
Alexa dyes are highly stable and bright. The Microscale Protein
Labelling Kit (Molecular Probes/Invitrogen: A30006) allows
microscale labelling of protein (20-100 ug). The degree of
labelling (DOL) can be optimised and the dye: protein (D:P) ratio
is measured by spectrophotometer and reproducible. FACS is used to
measure fluorescence.
[0308] The assay achieved good detection of ONTAK-A1488 and control
DT-Glu52-A1488 binding to HUVEC cells as illustrated in FIG. 3. The
assay achieved good detection of ONTAK-A1488 and control
DT(.DELTA.R)-A1488 binding to HUVEC cells compared to binding of
BSA-A1488 as illustrated in FIG. 11. The assay achieved good
detection of ONTAK-A1488 and control DT(.DELTA.R)-A1488 binding to
HUVEC cells compared to binding of BSA-A1488 as illustrated in FIG.
11.
[0309] HUVEC cells were tested for IL-2R expression by FACS: it was
confirmed that ONTAK-A1488 binding to HUVEC cells is independent of
IL-R as shown in FIG. 4.
(c) Cell Membrane Integrity
[0310] Cell membrane integrity can be assessed to measure loss of
integrity of cell membranes after exposure to toxins. Peptides
encompassing VLS motifs were directly conjugated to a fluorochrome
using methods as described by Baluna et al (PNAS USA, 96: 3957
(1999)). Alternatively, other assays described herein or known in
the art can be utilized.
[0311] Cell membrane integrity assays were conducted using
propidium iodide (PI) and the effect on the toxins was assessed by
FACS to measure PI uptake as a surrogate for loss of integrity of
cell membrane after short incubation with toxins (FIG. 5).
[0312] For potential to induce VLS, a HUVEC cell DT-binding assay
is tested using truncated DT (.DELTA.R) comprising the C and I
domains only. DT molecules are labeled for analysis of binding to
HUVEC. This step utilizes expression of DT(.DELTA.R) from stage 3
(described above) and, thus, generation of HUVEC assays is
initiated after DT expression. Alternatively, other assays
described herein or known in the art can be utilized.
[0313] DT Activity/Cytotoxicity Assays
[0314] Assays suitable for measurement of DT activity have been
established. Cytotoxicity assays are utilized to confirm toxin
activity of DT-IL2 T cell epitope and VLS variant leads selected in
IVTT assays.
[0315] (a) In Vitro Transcription-Translation (IVTT) Assay
[0316] Using an in vitro transcription-translation (IVTT) assay to
measure DT activity, direct transcription/translation of DT genes
was tested using a rabbit reticulocyte lysate system. Typically
this involves coupled transcription/translation of a luciferase
gene with a chemiluminescent assay which measures IVTT of target
plasmid (T7-luciferase)--active toxin inhibits and reduces
luciferase signal. PCR products are utilized allowing medium
throughput screening (MTS) with a titration curve and all variants
are compared to WT DT in the same 96-well assay plate. A surrogate
IC.sub.50 can be determined from an inhibition curve. Since DT
binds and causes the covalent modification of elongation factor 2
(EF2), this should cause an inhibition of luciferase production for
active DT variants. Coupled transcription/translation assays can be
used for analysis of ribosome-inhibitory proteins to provide
information on activity of the C domain of DT. Various methods are
known to those of skill in the art that are useful in carrying out
such coupled transcription/translation assays reactions such as,
for example, described in U.S. Pat. Nos. 5,976,806 and 5,695,983,
each of which is hereby incorporated by reference in its
entirety.
[0317] Using the IVTT assay described herein, it was shown that WT
.DELTA.R DT demonstrated dose-dependant inhibition of
transcription/translation of T7-luc plasmid approaching 90% while
the null (Glu52E) plateaued at approximately 20% inhibition (FIG.
1).
[0318] (b) Luminescent Cytotoxicity Assay
[0319] Toxilight.TM., Vialight.TM. and ALAMARBLUE.TM. kits are
non-radioactive, commercial assays which can be used to measure
cytotoxicity. The assays are conducted in a 96-well plate format,
titrating toxin (10.sup.-7-10.sup.-12 M) over time using
susceptible and resistant cell lines. Cytotoxicity of ONTAK vs.
IL-2 to human T cell lines was assessed using the Toxilight kit at
48 hours; luminescence counts per second (LCPS) reflect the degree
of adenylate kinase release (FIG. 6).
[0320] (c) Ribosyltransferase Assay
[0321] In addition to the coupled transcription/translation assay,
a ribosyltransferase assay (such as described in Example 3) was
established in a 96-well format. This assay uses samples of DT from
expression of DT genes in E. coli (stage 3) and is tested in
conjunction with the coupled transcription/translation assay above.
Traditional methods for measuring ADP-ribosylation use
permeabilized cells treated with double stranded (ds) activator DNA
oligonucleotide; subsequent measurement of radiolabeled NAD+ is
incorporated into acid insoluble material.
[0322] New FACS-based methods such as those described by Kunzmann
et al. (2006 Immunity & Ageing) are also available.
[0323] For measurement of cytotoxicity of DT variants, a cellular
cytotoxicity assay (such as described in Example 4) is developed,
using HUT102-6TG cells in a 96-well format for analysis of full
length DT variants (HUT102-6TG is the cell line used for final
analysis of the lead DT-IL-2 protein). As the cellular cytotoxicity
assay requires expression of full length DT, this assay is used
after DT expression in stage 3.
Example 9
Gene Synthesis, Expression and Purification
[0324] DT and human IL-2 genes are synthesized at the start of
stage 2 using codons optimized for expression in E. coli using
conventional techniques known in the art. For generation of full
length DT and DT(.DELTA.R) (comprising the C and I domains only) to
be used in analysis in cellular cytotoxicity and HUVEC binding
assays (see stage 1), vector systems are used which include
secretory leader sequences for export of DT into the periplasmic
space of E. coli. Methods for purification of DT include, for
example, purification via affinity tags fused to DT (e.g., a His6
tag). The method developed in stage 2 provides for reliable
production of multiple DT variants with similar quality such that
the activities of these variants can be accurately compared in
order to identify lead candidates in stages 3 and 4.
Example 10
Design and Construction of VLS Variants of DT
[0325] Variants of DT for reduction in potential to reduce VLS are
generated by two rounds of mutation: first, with separate mutations
at each of the three (x)D(y) motifs in DT and, second, with
combinations of lead mutations with optional additional mutations.
Each DT variant is tested in the HUVEC binding assay (stage 1 or
Example 6). Generation of DT variants for these assays are by
expression of truncated DT (.DELTA.R) in E. coli (stage 2).
Example 11
DT VLS Variants Inhibit Protein Synthesis
[0326] A DT382 construct was used and contained amino acid residues
1-382 of SEQ ID NO: 2 or 149 of DT as well as IL2. A restriction
enzyme site was engineered at amino acid residue 382 for cloning in
either the R domain or the IL2 portion. Modifications are
incorporated as described below.
[0327] Variants of DT382 gene were produced where VLS motifs were
mutated such that the recognized x(D)y motif was disrupted. The
activity of variants at single and multiple loci were assessed for
activity in an in vitro transcription/translation assay using PCR
products.
[0328] Briefly, the activity of single and combined variants was
measured in an in vitro transcription/translation assay. Purified
PCR product of each variant was titrated into a TnT coupled
transcription/translation reaction mix (#L4610 Promega, Madison
Wis., according to the manufacturer's instructions) containing
rabbit reticulocyte lysate, TnT buffer, T7 RNA polymerase, amino
acid mix--Met, amino acid mix--Leu and RNasin (#N2511 Promega,
Madison Wis.) using a DNA range from 1 ng to 64 ng per reaction in
a total volume of 10.5 .mu.l. Reactions were incubated at
30.degree. C. for 30 minutes to allow for possible differences in
the rate of DT gene translation between the different variants.
T7-luciferase control plasmid (250 ng) was then added and reactions
were incubated for a further 45 minutes at 30.degree. C. Expression
of luciferase was measured by luminescence after incubating the
reaction with SteadyGlo luciferase assay reagent, according to
manufacturer's instructions (#E2510 Promega, Madison Wis.).
Luminescent readout was measured using BMG FLUOstar OPTIMA
fluorescent plate reader (BMG Labtech, Durham, N.C.).
[0329] Twenty-eight (28) VLS mutants have been designed and
constructed. Eighteen out of twenty-eight (18/28) VLS mutants have
been tested in an IVTT assay. Known VLS variants were show to have
equivalent activity to wild type (WT) and a number of alternative
VLS variants have also been identified that demonstrate activity
equivalent to or better than WT (FIG. 7). FIG. 8 shows
representative results for mutants of the epitopes, demonstrating
that mutants have been obtained for each epitope that retain wild
type activity.
[0330] Percentage inhibition of protein synthesis was plotted
against DNA concentration in the reaction and the resulting curves
were used to calculate the IC.sub.50 for each variant. IC.sub.50s
were normalized to allow for inter-assay variation by dividing the
IC.sub.50 of wild type DT (included on every assay plate) with the
IC.sub.50 of the DT variant.
[0331] Table 2 provides IC.sub.50 data for two VLS modified DT
variants compared to wild-type and a null DT variant.
TABLE-US-00002 Molecule IC.sub.50 (ng/12.5 .mu.l) Relative Activity
WT 20.27 1.00 D30N 37.93 0.53 S31G 40.57 0.5 Null 46.65 0.43
[0332] Table 3 provides a relative IVTT score for VLS mutations
compared to wild type. The relative IVTT score is determined by
dividing the IC50 of wild type DT by the IC50 of the mutant DT.
TABLE-US-00003 Mutation Relative IVTT score Activity Compared to WT
V7S 1.00 Equivalent V7T 0.93 Equivalent V7N 0.63 Reduced V7D 0.57
Reduced D8E 0.90 Equivalent D8N 0.36 Inactive S9A 2.25 Improved S9G
0.48 Inactive S9T 1.5 Equivalent V28S 3.61 Improved V29T 1.57
Equivalent V29N 2.47 Improved V29D 1.51 Equivalent D30E 2.18
Improved D30N 0.56 Reduced S31T 0.14 Inactive S31G 0.26 Inactive
S31N 1.85 Equivalent I290S 0.07 Inactive I290T 4.41 Improved I290D
0.43 Inactive D291E 1.84 Equivalent S292A 1.00 Equivalent S292T
2.11 Improved S292G 0.65 Reduced V7N V29N 1.18 Equivalent V7N V29T
1.56 Equivalent V7N V29D 1.18 Equivalent V7T V29N 0.78 Equivalent
V7T V29T 0.97 Equivalent V7T V29D 1.15 Equivalent
[0333] FIG. 9 shows the relative activities of VLS DT variants
compared to wild type DT in the inhibition of protein synthesis.
The data shows that the following VLS variants: V7N V29N I290N, V7N
V29N I290T, V7N V29N S292A, V7N V29N S292T, V7N V29T I290N, V7N
V29T I290T, V7N V29T S292A, V7N V29T S292T, and V7T V29T I290T all
show equivalent activity to DT382 in the inhibition of protein
synthesis. In contrast, a G53E substitution of DT382 results in a
decrease in activity. As described herein, a reference to a G52
modification refers to amino acid residue numbering of a DT
molecule of SEQ ID NO: 1 that does not contain the N-terminal
methionine.
Example 12
Binding of VLS Variants to HUVECs
[0334] Human vascular endothelial cells (HUVEC) were maintained in
EBM (CC-3124 Lonza, Basel, Switzerland). Before use, cells were
detached from plastic substratum using an enzyme free dissociation
buffer (C5914 Sigma, Poole, UK) and resuspended in phosphate
buffered saline containing 1% BSA and 0.05% NaN.sub.3. Cells were
then incubated in the same buffer containing 5% normal human serum
for 20 minutes before adding a titration of purified DT382 protein
(prepared as described above in Example 12) or DT382 VLS variants
that had been conjugated to Alexa488 fluorochrome (A30006
Invitrogen, Carlsbad Calif.), according to the manufacturer's
instructions. The cells were incubated with the labelled protein
for 30 minutes before being washed and resuspended in PBS+1%
BSA+0.05% NaN.sub.3 buffer. Labelled DT-389-IL2 fusion was used as
a positive control and labelled BSA was used as a negative control.
Cells were then analyzed on a FACSCalibur flow cytometer (Becton
Dickinson, Franklin Lakes, N.J.) and fluorescent staining of the
cell population was measured. The percentage of cells that showed
above background staining was then plotted against the
concentration of labelled protein used.
[0335] FIG. 10 shows binding of labelled DT382 VLS variants to
HUVEC cells. Labelled DT-389-IL2 fusion (ONTAK.RTM.) was used as a
positive control and labelled BSA was used as a negative control.
The data shows that purified Alexa488 labelled DT382 and DT389-IL2
(positive control) bind to HUVEC at similar levels. In contrast,
binding of the VLS variants V7N V29T S292T (shown), V7N V29T I290N
(shown), and V7N V29N I290N (not shown) exhibit a reduced level of
binding to HUVECs compared to either DT382 or DT389-IL2.
Example 13
Construction and Expression of Variant DT-IL2
[0336] In stage 4, one or more lead DT-IL2 variants is generated by
fusion of the lead DT variant from stage 3 with the human-IL2
(2-133) gene from stage 2. Expression of the wild-type and lead
DT-IL2 variant in E. coli follows conventional methods for, for
example, DT-IL2 involving accumulation of protein aggregates in
inclusion bodies and refolding. Wild-type and one or more lead
DT-IL2 variants are then tested in the cytotoxicity and/or
VLS-related assays as described in stage 1 and/or Example 6.
Example 14
Adjuvant Effect of DT Variant-IL2 Fusion Proteins
[0337] Clinical Trial Design and Patient Eligibility
[0338] Treatment of patients is performed following written
informed consent as part of a protocol approved by an Institutional
Review Board and the FDA. Patients with histologically confirmed
metastatic RCC are eligible for study. All patients are required to
have adequate hepatic, renal, and neurological function, a life
expectancy of more than 6 months, and a Karnofsky performance
status of greater than or equal to 70%. Patients are to have
recovered from all toxicities related to any prior therapy and not
received any chemotherapy, radiation therapy, or immunotherapy for
at least 6 weeks prior to study entry. Excluded from this study are
patients with CNS metastases, with a history of autoimmune disease,
and with serious intercurrent chronic or acute illnesses. Patients
on immunosuppressive agents are also excluded. Eligible subjects
are randomized with equal probability to receive either a single
dose of DT variant-IL2 fusion protein (18 .mu.g/kg) followed by
immunization with tumor RNA-transfected DCs or DT variant-IL2
fusion protein alone. All subjects receive 3 intradermal injections
of tumor RNA-transfected DCs. The injections are administered
intradermally at biweekly intervals and consist of 1.times.10.sup.7
cells suspended in 200 .mu.l 0.9% sodium chloride at each
injection. Following treatment, subjects are evaluated for clinical
toxicity and immunological and clinical responses. Due to
regulatory restrictions and, in some subjects, limited access to
rumor tissue, no tumor biopsies are performed.
[0339] DT variant-IL2 Fusion Protein and Composition
Preparation
[0340] DT variant-IL2 fusion protein is provided as a frozen,
sterile solution formulated in citrate buffer in 2 ml single-use
vials at a concentration of 150 .mu.g/ml. After thawing, DT
variant-IL2 fusion protein is diluted with sterile normal saline to
a final concentration of 15 .mu.g/ml and delivered by intravenous
infusion over a 30-minute period. Patients are permitted to receive
acetaminophen (600 mg) and antihistamines 30 to 60 minutes prior to
infusion. For DC culture, a concentrated leukocyte fraction is
harvested by leukapheresis. PBMCs are isolated from the
leukapheresis product by density gradient centrifugation
(Histopaque; Sigma-Aldrich). The semiadherent cell fraction is used
for DC culture in serum-free X-VIVO 15 medium (Cambrex Corp.)
supplemented with recombinant human IL-4 (500 U/ml; R&D
Systems) and recombinant human GM-CSF (rhGM-CSF; 800 U/ml; Immunex
Corp.). After 7 days, immature DCs are harvested and transfected
with total RNA extracted from tumor tissues histologically
classified as clear cell carcinoma. Control RNA used for
immunological monitoring studies is isolated from autologous benign
renal tissues (RE) or from PBMCs. Transfection of immature DCs is
carried out by electroporation. DCs are washed in PBS and
resuspended at a concentration of 4.times.10.sup.7 cells/ml in
ViaSpan (Barr Laboratories). Cells are then co-incubated for 5
minutes with 5 .mu.g RNA per 1.times.10.sup.6 cells and
electroporated in 0.4 cm cuvettes via exponential decay delivery at
300 V and 150 .mu.F (Gene Pulser II; Bio-Rad). After
electroporation, cells are re-suspended in X-VIVO 15 medium and
matured for 20 hours in the presence of 10 ng/ml TNF-.alpha., 10
ng/ml IL-1, 150 ng/ml IL-6 (R&D Systems), and 1 .mu.g/ml
prostaglandin E.sub.2 (PGE.sub.2; Cayman Chemical Co.). Prior to
administration, cells are characterized to ensure that they met the
typical phenotype of fully mature DCs: Lin.sup.neg, HLA class I and
II.sup.high, CD86.sup.high, and CD83.sup.high.
[0341] Evaluation of Immune Status
[0342] IFN-.gamma. and IL-4 ELISPOT analyses are performed using
PBMCs obtained prior to, during, and after immunization. PBMCs are
cultured overnight in complete RPMI 1640 medium. CD4+ and CD8+ T
cells are isolated from PBMCs by negative depletion (Miltenyi
Biotec). After blocking, 1.times.10.sup.5 T cells and
1.times.10.sup.4 RNA-transfected DCs are added to each well of
96-well nitrocellulose plates (Multiscreen-IP; Millipore) precoated
with 2 .mu.g/ml IFN-, capture antibody (Pierce Biotechonology Inc.)
or with IL4 capture antibody (BD Biosciences Pharmingen). Plates
are incubated for 20 hours at 37.degree. C., and biotinylated
IFN-.gamma. detection antibody (Pierce Biotechonology Inc.) or
biotinylated IL-4 antibody (BD Biosciences Pharmingen) is added to
each well. Cells are then incubated for an additional 2 hours at
room temperature, then with streptavidin-alkaline phosphatase (1
.mu.g/ml; Sigma-Aldrich) is added; plates are developed with
substrate (KPL). After washing, spots are counted using an
automated ELISPOT reader (Zeiss).
[0343] CTL assays are performed by co-culturing RNA-transfected DCs
with autologous PBMCs. Cells are re-stimulated once, and IL-2 (20
units/ml) is added after 5 days and every other day thereafter.
After 12 days of culture, effector cells are harvested. Target
cells are labeled with 100 .mu.Ci of Na.sub.2[51CrO.sub.4]
(PerkinElmer) in 200 .mu.l of complete RPMI 1640 for 1 hour at
37.degree. C. in 5% CO.sub.2, and 51 Cr-labeled target cells are
incubated in complete RPMI 1640 medium with effector cells for 5
hours at 37.degree. C. Then 50 .mu.l of supernatant is harvested,
and release of .sup.51Cr is measured with a. scintillation
counter.
[0344] For proliferation assays, purified CD3+ T cells are seeded
into round-bottomed microplates in the presence of mRNA-transfected
DCs. T cells alone are used as the background control. After 4
days, 1 .mu.Ci of [methyl-3H] thyridine (PerkinElmer) is added to
each well for an additional 16 hours. Incorporation of thymidine is
determined using a liquid scintillation counter.
[0345] Cytotoxicity of DT variant-IL2 fusion protein is determined
in MTT assays. After 6 hours incubation with varying concentrations
of DT variant-IL2 fusion protein, cells are seeded in 96-well
plates at a density of 5.times.10.sup.3 cells/well. After 48 hours
of incubation, 20 .mu.L MTT from a 5 mg/ml stock is added. After 4
hours, the formazan crystals are solubilized by adding 100 .mu.l
isopropanol/0.1 M hydrochloric acid. The absorbance of the formazan
product is measured on an ELISA plate reader at 570 nm.
[0346] Cytokine secretion by vaccine-induced CD4+ T cells is
measured using the human Th-1/Th-2 cytokine kit (Cytokine Bead
Array; BD Biosciences Pharmingen) according to the manufacturer's
instructions. Isolated CD4+ T cells are re-stimulated overnight
with RNA-transfected DCs at a ratio of 10:1.
[0347] Four-color FACS analyses are performed using the following
antibodies: anti-CD4 FITC, anti-CD45RO, anti-CD45RA (CALTAG
Laboratories), anti-CD25 PE (BD Biosciences Pharmingen), and
anti-GITR (R&D Systems) as well as isotypic controls (CALTAG
Laboratories). Sorting of CD4+/CD25neg, CD4+/CD25+int and
CD4+/CD25high T cells is performed using a BD FACSAria cell sorter
after antibody labeling. For intracellular detection of FoxP3,
cells are permeabilized with 30 .mu.g/ml digitonin for 45 minutes
at 4.degree. C. Subsequently, cells are stained with anti-FoxP3
antibody (Abcam), and R-phycoerythrin anti-goat IgG in the presence
of 10 .mu.g/ml digitonin for 30 minutes 4.degree. C. Following
staining, cells are fixed analyzed by FACS. For intracellular
CTLA-4 detection, T cells are permeabilized, fixed, and stained
with biotinylated anti-CD152 (BD Biosciences Pharmingen) followed
by APC-strepavidin (BD Biosciences Pharmingen). A total of
1.times.106 cells are suspended in staining buffer (PBS with 1%
PCS, 2 mM EDTA, and 0.1% sodium aside) and incubated for 20 minutes
at 4.degree. C. with the antibody.
[0348] The suppressive activity of Tregs isolated from PBMCs of
study subjects prior to and 4 days after DT variant-IL2fusion
protein administration is analyzed as described previously
(Tsaknaridis et al. 2003. J. Neurosci. Res. 74: 296-308).
CD4+/CD25+ T cells are isolated from the PBMCs of study subjects
using magnetic bead separation techniques. Cells are washed with
PBS, re-suspended in complete RPMI 1640 medium, and placed into
96-well round bottom plates pre-coated with anti-CD3/CD28
antibodies (0.4 .mu.g/well) (CALTAG Laboratories) CD4+/CD25- cells
are plated at 2.0.times.10.sup.4/well alone or in combination with
CD4+/CD25+ cells in triplicate wells at a ratio of 1:2
(CD4+/CD25:CD4+CD25.sup.+). On day 5, 1 .mu.Ci of 3H thymidine is
added for the final 16 hours of the cultures. Cells are then
harvested on glass fiber filters and assessed for uptake of
radiolabeled thymidine.
[0349] Details of real-time PCR-based quantification of
.beta.-actin transcripts are previously described in the
literature. FoxP3 mRNA transcripts are quantified using the
Hs00203958 ml Taq-Man gene expression assay (Applied Biosystems)
according to the protocol provided by the manufacturer. A plasmid
containing the full-length FoxP3 insert is used to generate
standard curves.
[0350] T cell analysis before and after treatment is performed by
IFN-.gamma. ELISPOT on all patients who completed immunotherapy.
Increases of antigen-specific CD4+ and CD8+ T cells after
immunization are compared using the Wilcoxon matched-pairs signed
rank test, analyzing the null hypothesis that the rates of change
in T cell response are equivalent prior to and after therapy. A
2-sided P value of less than 0.05 is considered statistically
significant.
[0351] This invention may be embodied in other forms or carried out
in other ways without departing from the spirit or essential
characteristics thereof. The present disclosure is therefore to be
considered as in all aspects illustrated and not restrictive, and
all changes which come within the meaning and range of equivalency
are intended to be embraced therein.
[0352] Various references are cited throughout this specification,
each of which is incorporated herein by reference in its entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090010966A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090010966A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References