MODIFIED DIPHTHERIA TOXINS
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.
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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 INVENTIONVascular 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.
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).
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.
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.
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 DAB389IL-2 (Foss et al., Clin Lymphoma 1(4):298-302 (2001), Figgitt et al., Am J Clin Dermatol., 1(1):67-72 (2000)). DAB389IL-2, is interchangeably referred to in this application as DT387-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)].
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)].
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 INVENTIONProvided 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.
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.
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.
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.
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.
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, INFα, INFγ, 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, Fab2, F(ab′)2, scFv, scFv2, single chain binding polypeptide, VH, or VL. 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.
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.
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.
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.
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, INFα, INFγ, 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.
Provided herein are pharmaceutical compositions comprising a fusion protein and a pharmaceutically acceptable carrier or excipient.
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.
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.
Non-malignant diseases include, for example, GVHD, aGVHD and psoriasis.
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.
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.
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.
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.
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.
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.
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.
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.
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, INFα, INFγ, 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 REFERENCEAll 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.
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).
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. OverviewCell 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.
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).
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.
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.
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.
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.
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)). DAB389IL-2 (ONTAK®) 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® is variously referred to as denileukin diftitox, DAB389-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® contains 521 amino acids. It should be noted that, as a result of the methionine residue added at the N terminus of ONTAK®, numbering in the sequence of diphtheria is out of register with that of ONTAK® by one.
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):C1-6 (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 ToxinDiphtheria 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
A. Modified Diphtheria Toxins
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.”
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.
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.
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.
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.
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.
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 α4β1 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.
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.
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.
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.
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.
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.
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.
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.
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.
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 DAB398IL-2 produced in E. coli generally yields an IC50 of between about 5×10−11 M to about 1×10−12 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 IC50 of between about 5×10−11 M to about 1×10−12 M, of about 1×10−10 M to about 1×−10 M, of about 1×10−9 M to about 1×10−10 M, or of about 1×10−8 M to about 1×10−9 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.
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, INFα, INFγ, 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.
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.
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.
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.
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.
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.
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.
B. Methods of Making Modified Polypeptides
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.
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).
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.
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.
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.
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 ProteinsThe 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.
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.
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, Fab2, a F(ab′)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.
“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).
Cell-specific binding ligands can also include, but are not limited to: polypeptide hormones, e.g., those made using the binding domain of α-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, β-interferon, α-interferon (INF-α), INF-γ, angiostatin, thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, tumor necrosis factor (TNF), SVEGF, TGF-β, 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.
In IL-2, a LDL sequence (a “VLS” motif) at residues 19-21 (SEQ ID NO: 3) is located in an α-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.
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.
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.
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.
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.
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.
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 DAB389IL-2 (also known as denileukin diflitox and ONTAK®) produced in E. coli generally yields an IC50 of between about 5×10−11 M to about 1×10−12 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 IC50 of between about 5×10−11 M to about 1×10−12 M, of about 1×10−10 M to about 1×10−10 M, of about 1×10−9 M to about 1×10−10 M, or of about 1×10−8 M to about 1×10−9 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.
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.
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, INFα, INFγ, GM-CSF, G-CSF, M-CSF, TNF, VEGF, Ephrin, βFGF, 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.
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.
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).
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).
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.
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.
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 DAB486IL-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 DAB486IL-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, DAB389IL-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 DAB389 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, DAB389 (vanderSpek et al., Methods in Molecular Biology, Bacterial Toxins: Methods and Protocols., 145:89-99, Humana Press, Totowa, N.J. (2000)). Denileukin diftitox (DAB389IL-2, ONTAK®) 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.
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.
In one embodiment, provided herein are fusion proteins containing modified versions of a DAB389IL-2 where one or more VLS motifs have been modified as described herein.
IV. Nucleic Acids, Vectors and Host CellsAnother aspect of the present invention pertains to vectors containing a polynucleotide (nucleic acid, DNA) encoding a modified DT variant or a fusion protein thereof.
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.).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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™-11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as E. coli LE392.
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 β-galactosidase, ubiquitin, and the like.
Promoters that are most commonly used in recombinant DNA construction include the β-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.
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.
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.
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.
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 β-mercaptoethanol or DTT (dithiothreitol).
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.
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).
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Provided herein is a fusion protein comprising a modified diphtheria toxin made by such a method and a non-diphtheria toxin polypeptide.
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, INFα; INFγ, 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.
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 UsesEach 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.
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.
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®, PLURONICS® or polyethylene glycol (PEG).
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.
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.
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.
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.
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.
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.
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.
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™ (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.
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.
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.
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).
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.
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.
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.
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.
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)).
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.
The invention is further directed to pharmaceutical compositions comprising a modified DT or fusion protein thereof described hereinabove and a pharmaceutically acceptable carrier.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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, INFα, INFγ, 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.
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.
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).
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 μg/kg/day, 9 μg/kg/day or 18 μg/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.
Provided herein is a method of treating a subject having cutaneous T-cell lymphoma by administering a modified DT fusion protein as described herein
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 μg/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.
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.
In a Phase II clinical study, it was shown that ONTAK.® 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.
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 μg/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.
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.
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.
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.
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 μg/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).
Provided herein is a method of treating a subject having chronic lymphocytic leukemia by administering a modified DT fusion protein as described herein.
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.
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.
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.
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 μg/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 μg/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).
Provided herein is a method of treating a subject having aGVHD by administering a modified DT fusion protein as described herein.
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® (0.5, 1.5 or 5 μg/kg/day) and received three doses per week for eight weeks. Eight out of 15 patients (treated with 5 or 1.5 μg/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 μg/kg/day, benefited from a 2-grade improvement on the 5-grade PGA scale.
Provided herein is a method of treating a subject having psoriasis by administering a modified DT fusion protein as described herein.
Also contemplated herein is a method of providing maintenance therapy by administering a non-immunogenic DT fusion protein as described herein.
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 α-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.
Recombinant IL-2 diphtheria toxin conjugate DAB389IL-2 (also known as denileukin diftitox and ONTAK®) 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.
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.
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®) 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.
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.
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®), improves immunity.
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.
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.
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.
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.
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.
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 LD50 (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 LD50/ED50. 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.
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 ED50 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 IC50 (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.
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.
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.
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.
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.
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.
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 KitsIn 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.
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.
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.
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).
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.
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.).
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.
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.
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.
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.
EXAMPLESThe 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 ProteinsConstruction of DT Variant and DT-Fusion Proteins
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.
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.
SEQ ID NOS: 4-147 provides a list of non-limiting exemplary modified DTs and the corresponding amino acid sequences.
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.
Expression and Purification of DT Variants and DT-Fusion Proteins
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.
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 AssaysHuman 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.
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° 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.
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° 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 [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 [125I]albumin as well as the media in the lower well after the end of the study is demonstrated >95% of the 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 125I probe. The 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 [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 [125I]bovine serum albumin in the upper well. The [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.
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 μm 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 CaCl2, 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 μl 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 3This 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 μM [32P]-NAD+(10 mCi/ml, 1000 Ci/mmol, Amersham-Pharmacia). The purified mutant proteins are tested in a final reaction volume of 40 μl. 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 μl 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 ProteinsThe 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.
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.
Cytotoxicity Assays
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 μg/ml streptomycin. The cells are seeded at a density of 5×104/well into 96 well, V-microtiter plates. The fusion protein toxins are typically added to the wells in molarities ranging from 10−7 M down to 10−12 M. Final volume in the wells is 200 μl. The plates are incubated for 18 hours, at 37° C. in a 5% CO2 environment. The plates are subjected to centrifugation to pellet the cells, the media removed and replaced with 200 μl leucine-free, minimal essential medium containing 1.0 μCi/ml[14C] leucine (<280 mCi/mmol, Amersham-Pharmacia) and 21 mM glutamine, 50 IU/ml penicillin and 50 μg/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 μl, 0.4 M KOH followed by a 10 minute incubation at room temperature. 140 μl 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) [14C]-leucine incorporation. Toxilight™, Vialight™ and ALAMARBLUE™ 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 (10−7-10−12 M) over time using susceptible and resistant cell lines.
Pharmaceutical grade GMP purified DAB389IL-2 produced from E. coli typically yields an IC50 of between 5×10−11 M to 1×10−12 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 DAB389IL-2. These assays demonstrate comparable activity of the VLS modified DT387linker IL-2 fusion to DAB389IL-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.
The relative amounts of non-fusion toxins protein in each sample could artificially alter the IC50 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 IC50.
Purified DAB389IL-2 produced in E. coli typically yields an IC50 of between 5×10−11 M and 1×10−12 M.
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.
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 DT387 or DT387linker 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 DT387 and DT387linker IL2 will also separate the effects of modified sequences of the toxophore alone from the IL-2 targeting ligand present in DT387linker IL-2. In another example, using modified DT mutants derived from both DT389 and DT389linker 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 DT389 and DT389linker IL2 will also separate the effects of modified sequences of the toxophore alone from the IL-2 targeting ligand present in DT389linker IL-2.
Example 5This 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.
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 125I-albumin injected intravenously (Smallshaw et al. Nature Biotechnology 21:387-391 (2003)).
Example 6Multiple 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.
In Vitro Cytotoxicity Assays
The cytotoxic activities of the different modified DT fusion proteins are determined using CD22+ Daudi cells and [3H]-leucine incorporation as described previously (Ghetie et al., (1988) Cancer Res. 48:2610). The concentration of fusion protein which reduces [3H]-leucine incorporation by 50% relative to an untreated control culture is defined as the IC50.
Vascular Toxicity
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).
In Vivo Assays
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 5×106 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 μg modified DT fusion protein/g of mouse weight.
Example 7The methods described herein below are to generate variants of DT with reduced VLS using the following stages:
Stage 1—assays for DT activity;
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);
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
Stage 4—construction and expression of variant DT-IL2.
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 (ΔR)) 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 8VLS Assays
Various assays can be used to assess VLS activity of DT variants as described herein.
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.
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.
(a) Detection with Antibodies
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.
(b) Direct Conjugates
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.
The assay achieved good detection of ONTAK-A1488 and control DT-Glu52-A1488 binding to HUVEC cells as illustrated in
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
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.
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 (
For potential to induce VLS, a HUVEC cell DT-binding assay is tested using truncated DT (ΔR) comprising the C and I domains only. DT molecules are labeled for analysis of binding to HUVEC. This step utilizes expression of DT(ΔR) 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.
DT Activity/Cytotoxicity Assays
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.
(a) In Vitro Transcription-Translation (IVTT) Assay
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 IC50 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.
Using the IVTT assay described herein, it was shown that WT ΔR DT demonstrated dose-dependant inhibition of transcription/translation of T7-luc plasmid approaching 90% while the null (Glu52E) plateaued at approximately 20% inhibition (
(b) Luminescent Cytotoxicity Assay
Toxilight™, Vialight™ and ALAMARBLUE™ 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 (10−7-10−12 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 (
(c) Ribosyltransferase Assay
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.
New FACS-based methods such as those described by Kunzmann et al. (2006 Immunity & Ageing) are also available.
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 PurificationDT 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(ΔR) (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 DTVariants 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 (ΔR) in E. coli (stage 2).
Example 11 DT VLS Variants Inhibit Protein SynthesisA 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.
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.
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 μl. Reactions were incubated at 30° 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° 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.).
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 (
Percentage inhibition of protein synthesis was plotted against DNA concentration in the reaction and the resulting curves were used to calculate the IC50 for each variant. IC50s were normalized to allow for inter-assay variation by dividing the IC50 of wild type DT (included on every assay plate) with the IC50 of the DT variant.
Table 2 provides IC50 data for two VLS modified DT variants compared to wild-type and a null DT variant.
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.
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% NaN3. 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% NaN3 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.
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 ProteinsClinical Trial Design and Patient Eligibility
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 μg/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 1×107 cells suspended in 200 μl 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.
DT variant-IL2 Fusion Protein and Composition Preparation
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 μg/ml. After thawing, DT variant-IL2 fusion protein is diluted with sterile normal saline to a final concentration of 15 μg/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 4×107 cells/ml in ViaSpan (Barr Laboratories). Cells are then co-incubated for 5 minutes with 5 μg RNA per 1×106 cells and electroporated in 0.4 cm cuvettes via exponential decay delivery at 300 V and 150 μF (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-α, 10 ng/ml IL-1, 150 ng/ml IL-6 (R&D Systems), and 1 μg/ml prostaglandin E2 (PGE2; Cayman Chemical Co.). Prior to administration, cells are characterized to ensure that they met the typical phenotype of fully mature DCs: Linneg, HLA class I and IIhigh, CD86high, and CD83high.
Evaluation of Immune Status
IFN-γ 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, 1×105 T cells and 1×104 RNA-transfected DCs are added to each well of 96-well nitrocellulose plates (Multiscreen-IP; Millipore) precoated with 2 μg/ml IFN-, capture antibody (Pierce Biotechonology Inc.) or with IL4 capture antibody (BD Biosciences Pharmingen). Plates are incubated for 20 hours at 37° C., and biotinylated IFN-γ 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 μg/ml; Sigma-Aldrich) is added; plates are developed with substrate (KPL). After washing, spots are counted using an automated ELISPOT reader (Zeiss).
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 μCi of Na2[51CrO4] (PerkinElmer) in 200 μl of complete RPMI 1640 for 1 hour at 37° C. in 5% CO2, and 51 Cr-labeled target cells are incubated in complete RPMI 1640 medium with effector cells for 5 hours at 37° C. Then 50 μl of supernatant is harvested, and release of 51Cr is measured with a. scintillation counter.
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 μCi 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.
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 5×103 cells/well. After 48 hours of incubation, 20 μL MTT from a 5 mg/ml stock is added. After 4 hours, the formazan crystals are solubilized by adding 100 μl isopropanol/0.1 M hydrochloric acid. The absorbance of the formazan product is measured on an ELISA plate reader at 570 nm.
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.
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 μg/ml digitonin for 45 minutes at 4° C. Subsequently, cells are stained with anti-FoxP3 antibody (Abcam), and R-phycoerythrin anti-goat IgG in the presence of 10 μg/ml digitonin for 30 minutes 4° 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 1×106 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° C. with the antibody.
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 μg/well) (CALTAG Laboratories) CD4+/CD25− cells are plated at 2.0×104/well alone or in combination with CD4+/CD25+ cells in triplicate wells at a ratio of 1:2 (CD4+/CD25:CD4+CD25+). On day 5, 1 μCi 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.
Details of real-time PCR-based quantification of β-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.
T cell analysis before and after treatment is performed by IFN-γ 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.
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.
Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.
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, INFα, INFγ, 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, INFα; INFγ, 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.
Type: Application
Filed: Jun 20, 2008
Publication Date: Jan 8, 2009
Applicant: ANGELICA THERAPEUTICS, INC. (Emeryville, CA)
Inventors: Claude Geoffrey Davis (San Mateo, CA), Deepshikha Datta (San Francisco, CA)
Application Number: 12/143,581
International Classification: A61K 39/05 (20060101); C07K 14/195 (20060101); A61P 35/04 (20060101);
