U.S. patent application number 11/179844 was filed with the patent office on 2005-11-10 for immunotoxins directed against malignant cells.
This patent application is currently assigned to The Govt. of the U.S.A as represented by the Sect. of the Dept. of Health & Human Services, Inc. Invention is credited to Goldenberg, David M., Newton, Dianne L., Rybak, Susanna M..
Application Number | 20050249738 11/179844 |
Document ID | / |
Family ID | 21945973 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249738 |
Kind Code |
A1 |
Goldenberg, David M. ; et
al. |
November 10, 2005 |
Immunotoxins directed against malignant cells
Abstract
The present invention relates to immunotoxins, that effectively
kill malignant cells having a given surface marker and nucleic acid
constructs encoding them. These reagents comprise a toxic moiety
that is derived from a Rana pipiens protein having ribonucleolytic
activity linked to an antibody capable of specific binding with a
chosen tumor cell.
Inventors: |
Goldenberg, David M.;
(Mendham, NJ) ; Rybak, Susanna M.; (Frederick,
MD) ; Newton, Dianne L.; (Rockville, MD) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
The Govt. of the U.S.A as
represented by the Sect. of the Dept. of Health & Human
Services, Inc
Rockville
MD
Immunomedics, Inc.
Morris Plains
NJ
|
Family ID: |
21945973 |
Appl. No.: |
11/179844 |
Filed: |
July 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11179844 |
Jul 11, 2005 |
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09918887 |
Jul 30, 2001 |
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09918887 |
Jul 30, 2001 |
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09071672 |
May 1, 1998 |
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6395276 |
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60046895 |
May 2, 1997 |
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Current U.S.
Class: |
424/155.1 ;
424/178.1; 530/388.8; 530/391.1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; C07K 16/2833 20130101; A61K 38/00 20130101;
C07K 16/3061 20130101; A61K 47/6817 20170801; C07K 16/2803
20130101; C07K 2319/00 20130101; A61K 47/6849 20170801; C07K 14/463
20130101; A61P 35/02 20180101 |
Class at
Publication: |
424/155.1 ;
424/178.1; 530/391.1; 530/388.8 |
International
Class: |
A61K 039/395; C07K
016/30; C07K 016/46 |
Claims
1-34. (canceled)
35. A selective cytotoxic reagent comprising an onc protein having
measurable ribonucleolytic activity covalently linked to an
antibody directed against a surface marker specific to a B
cell.
36. The reagent of claim 35, wherein the onc protein has the amino
acid sequence of SEQ ID NO:1.
37. The reagent of claim 35, wherein the onc protein is produced by
recombinant means.
38. The reagent of claim 37, wherein the onc protein has the amino
acid sequence of SEQ ID NO:3
39. The reagent of claim 35, wherein the antibody is a monoclonal
antibody.
40. The reagent of claim 39, wherein the monoclonal antibody is
humanized.
41. The reagent of claim 40, wherein the monoclonal antibody is a
single chain antibody.
42. The reagent of claim 35, wherein the antibody is specific for B
cell lymphomas.
43. The reagent of claim 42, wherein the antibody is selected from
the group consisting of RFB4 and LL2.
44. The reagent of claim 35, wherein the surface marker is
CD22.
45. The reagent of claim 35, wherein the onc protein is conjugated
to the antibody through recombinant fusion.
46. A nucleic acid sequence encoding the reagent of claim 35.
47. A pharmaceutical composition comprising a selective cytotoxic
reagent comprising an onc protein having measurable ribonucleolytic
activity covalently linked to an antibody directed against a cell
surface marker specific to a B cell together with a
pharmaceutically acceptable carrier.
48. The pharmaceutical composition of claim 47, wherein the onc
protein has the amino acid sequence of SEQ ID NO:1.
49. The pharmaceutical composition of claim 47, wherein the onc
protein is produced by recombinant means.
50. The pharmaceutical composition of claim 49, wherein the onc
protein has the amino acid sequence of SEQ ID NO:3.
51. The pharmaceutical composition of claim 47, wherein the onc
protein is conjugated to the antibody through recombinant
means.
52. The pharmaceutical composition of claim 47, wherein the
antibody is a monoclonal antibody.
53. The pharmaceutical composition of claim 52, wherein the
monoclonal antibody is humanized.
54. The pharmaceutical composition of claim 53, wherein the
monoclonal antibody is a single chain antibody.
55. The pharmaceutical composition of claim 47, wherein the
antibody is directed against a surface marker present on B cell
lymphomas.
56. The pharmaceutical composition of claim 47, wherein the
antibody is selected from the group consisting of RFB4 and LL2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
U.S. Provisional Application 60/046,895, filed May 2, 1997. The
disclosure of the following U.S. Provisional Patent Application is
incorporated herein by reference in its entirety: S. M. Rybak and
D. L. Newton, "Recombinant Anti-Tumor RNAse," filed Mar. 27, 1998
(Attorney Docket No. 15280-343000).
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] Toxic enzymes from plants and bacteria such as ricin,
diphtheria toxin and Pseudomonas toxin have been coupled to
antibodies or receptor binding ligands to generate
cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l
Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad.
Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci.
USA 77:5419 (1980)). Regardless of the fact that the
cell-recognition moiety is not always an antibody, these directed
toxins are generally known as immunotoxins. These hybrid proteins
kill cells which express the receptor or cell surface marker that
the antibody or ligand portion of the molecule recognizes.
[0004] Under appropriate conditions, depending on the particular
receptor or cell surface marker, the toxin enters the cytosol,
inactivates the protein synthesis machinery and causes death of the
target cell. Immunotoxins, which have been shown to be highly
cytotoxic to cancer cells growing in cell culture and in animal
models, demonstrate the potential of these reagents to treat blood
and lymph borne malignancies which, because of their dissemination
are not treatable by traditional surgical techniques, as well as
solid tumors in restricted compartments such as the intraperitoneal
cavity (reviewed in Griffin, et al., IMMUNOTOXINS, p 433,
Boston/Dordrecht/Lancaster, Kluwer Academic Publishers, (1988);
Vitetta, et al., Science 238:1098 (1987); Fitzgerald, et al., J.
Nat'l Cancer Inst. 81:1455 (1989)). Traditional chemotherapies,
while being effective in the treatment of some cancerous
conditions, exhibit undesired side effects due to the systemic
toxicity of the chemotherapeutic compounds.
[0005] An ideal candidate for cancer therapy, therefore, would be
an immunotoxin that would selectively be cytotoxic to cancer cells
yet remain harmless to non-cancerous cells of the patient.
Utilization of this type of anti-tumor therapy, however, has been
stymied by the development of immune responses in patients to
foreign proteins which comprise the immunotoxins. Immune responses
against murine monoclonal antibodies (Sawler, et al., J. Immunol.
135:1530 (1985); Schroff, et al., Cancer Res. 45:879 (1985)) and
anti-toxin antibodies have been detected in both animals and humans
treated with immunotoxins (Rybak, et al., Immunol. and Allergy
Clinics of North America 11(2):359 (1991); Harkonen, et al., Cancer
Res. 47:1377 (1987); Hertler, A. in IMMUNOTOXINS p. 475, Kluwer
Academic Publishers, Boston/Dordrecht/Lancaster (1988)). Advances
in humanization techniques have alleviated some of the
immunogenicity associated with the antibody portion of immunotoxins
(Bird, et al., Science 242:423 (1988); Huston, et al., Proc. Nat'l
Acad. Sci. USA 85:5879 (1988); Ward, et al., Nature 341:544 (1989);
and Jones, et al., Nature 314:522 (1986)). However, no solution has
been found to counter the immunogenicity of the toxic moiety other
than immunosuppression of the patients (Khazaeli, et al.,
Proceedings of AACR 29:418 (1988)). Thus, there has been a
continuing need for methods and compositions that would reduce the
immunogenicity of the toxic moiety of immunotoxins yet retain the
ability to selectively kill cells having a given surface
marker.
[0006] B-cell lymphomas fall under the generic rubric of
non-Hodgkin's lymphomas and can either be a disseminated or a solid
tumor within the lymph system. Radiolabeled humanized murine
antibodies which have been raised against CD22 (LymphoCide.TM.), a
surface marker on malignant B cells, are currently in clinical
trials as a treatment for B-cell lymphomas (Immunomedics, Inc.,
Press Release, http://www.immunomedic.com/- theral.html). See also,
Amlot, et al., Blood 82:2624-2633 (1993); Sausville, et al., Blood
85:3457-3465 (1995); Grossbard, et al., Blood 81:2263-2271 (1993);
Grossbard, et al., Clin. Oncol. 11:726-737 (1993). To date, some
antitumor responses have been noted but immunotoxin-mediated
toxicity to normal tissue often prevented dosing at therapeutic
levels. In addition to CD22, several B-cell-specific antigens such
as CD19 and CD40 have been targeted by immunotoxins made with plant
toxins such as ricin A-chain and bacterial toxins, such as
Pseudomonas exotoxin A (PE). Uckun, et al., Blood 79:2201-2214
(1992); Ghetie, et al., Cancer Res. 51:5876-5880 (1991); Francisco,
et al., Cancer Res. 55:3099-3104 (1995).
[0007] The cytotoxicity of RNase A toward tumor cells is well
documented from studies performed in the 1960s and 1970s. Early
work is reviewed in Roth, Cancer Res. 23:657 (1963). The relevance
of these early studies has been sustained by the discovery that an
anti-tumor protein from oocytes of Rana pipiens is homologous to
bovine pancreatic RNase A (Ardelt, et al., J. Biol. Chem. 256:245
(1991)). P-30 protein (and referred to herein as the onc protein)
was isolated from extracts of Rana pipiens early embryos based upon
anti-proliferative/cytotoxic effects toward cancer cells in vitro
(Darzynkiewicz, et al., Cell Tissue Kinet. 21:169 (1988); Mikulski,
et al., Cell Tissue Kinet. 23:237 (1990)) and in animal models
(Mikulski, et al, J. Nat l. Cancer Inst. 82:151 (1990)). Phase III
human clinical trials of the onc protein in patients with a variety
of solid tumors are currently in progress.
SUMMARY OF THE INVENTION
[0008] The present invention relates to immunotoxins, that are
useful for killing malignant B cells and other malignant cells and
are directed to a surface marker on B cells and the nucleic acid
constructs encoding the immunotoxins. These reagents comprise a
toxic moiety that is derived from a Rana pipiens protein having
ribonucleolytic activity linked to an antibody capable of specific
binding with a chosen tumor cell.
[0009] We have found that these particular immunotoxins had highly
surprising properties as they were up to 2000 fold more active
against malignant B cells than their human RNase counterparts or
than the toxin itself Further, as will be described in more detail
below, their use when administered in vivo against disseminated
tumors, resulted in dramatically lowered side effects. These highly
effective, but apparently non-toxic, immunotoxins directed against
such ubiquitous diseases as B cell lymphomas present a new and
exciting therapeutic option for patients suffering from such
diseases.
[0010] It is an object of the present invention to provide
cytotoxic RNase (onc protein) immunotoxins that selectively kill
cells having a given surface marker. These immunotoxins are
minimally immunogenic and generate less systemic toxicity than
presently known immunotoxins. In particular, it is an object of the
present invention to provide direct immunotoxins comprising protein
fragments with ribonucleolytic activity linked to humanized
antibodies that recognize specific markers on tumor cells.
[0011] In another embodiment, the present invention relates to a
pharmaceutical composition comprising an immunotoxin of the present
invention and a pharmaceutically acceptable carrier.
[0012] In a further embodiment, the present invention relates to a
method selectively killing cells. The method comprises contacting
the tumor cells to be killed with a selective immunotoxin of the
present invention under conditions such that the monoclonal
antibody binds to a surface marker on the tumor cell thereby
causing the toxic onc protein to kill the cell.
[0013] Various other objects and advantages of the present
invention will be apparent from the following description of the
invention.
DESCRIPTION OF THE FIGURES
[0014] FIG. 1 indicates that ONCONASE.RTM. is more cytotoxic to HUT
102 T cell lymphoma cells (which do not bear the CD22 marker
recognized by LL2) than the immunotoxin, LL2-ONCONASE.RTM..
[0015] FIG. 2 demonstrates the superior cytotoxicity of
LL2-ONCONASE.RTM. to Burkitt Lymphoma cell lines when compared to
ONCONASE.RTM. alone.
[0016] FIG. 3 indicates that ONCONASE.RTM. conjugated to antibodies
directed against CD22 is more inhibitory of protein synthesis than
EDN conjugated to anti-CD22 antibodies. EDN is a human non-toxic
RNase as described in the text.
[0017] FIG. 4 indicates that ONCONASE.RTM. is more inhibitory of
protein synthesis when conjugated to antibodies compared to human
pancreatic RNase.
[0018] FIGS. 5A and 5B demonstrate that .sup.125-I labeled
LL2-ONCONASE.RTM. is not degraded by the lysosomes of Daudi cells
as rapidly as the LL2 antibody or the LL2-EDN immunotoxin. FIG. 5A
shows the percentage of RNase material retained in the cells and
FIG. 5B shows the percentage of RNase material degraded and
released into the supernatant.
[0019] FIG. 6 demonstrates that LL2 antibody diminishes the
cytotoxic effect of LL2-ONCONASE.RTM.. It is believed that LL2
competes for binding to CD22 with LL2-ONCONASE.RTM. and prevents
the internalization of the ONCONASE.RTM., thus reducing
cytotoxicity.
[0020] FIG. 7 is a survival graph showing LL2-ONCONASE.RTM.
protected SCID mice from B cell lymphoma. 5.times.10.sup.6 Daudi
cells were implanted intraperitoneally in mice. 24 hours later, the
mice were treated intravenously with 500 .mu.g of the indicated
compound.
[0021] FIG. 8 is a survival graph showing that LL2-ONCONASE.RTM.
completely protected SCID mice from an intraperitoneal implantation
of 2.times.10.sup.6 of Daudi cells. The mice were treated 24 hours
after implantation with 500 .mu.g of indicated compounds; 100 .mu.g
every day for 5 days.
[0022] FIG. 9 represents the decreased toxicity of the
LL2-ONCONASE.RTM. immunotoxin when compared to ONCONASE.RTM. alone
and IT-dgRTA (RFB4-deglycosylated Ricin A chain). The drugs were
administered every 2 hours. 4.times./day for 5 days. The arrows
indicate the days the mice with each respective treatment were
found dead, i.e., the mouse treated with 30 mg/kg ONCONASE.RTM. was
found dead on day 4 and the mouse treated with 50 mg/kg IT-dgRTA
was found dead on day 7.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention relates to the use of an RNase
protein, particularly an RNase derived from Rana pipiens as a toxic
moiety in a directed immunotoxin to B cells. Immunotoxic reagents
of the present invention comprise a protein and an antibody that
specifically binds to a chosen tumor cell surface marker. In
studies detailed below, the onc protein is shown to be far superior
to other immunotoxins that comprise antibodies directed against
CD22 or CD74 and a human non-toxic RNase. The onc protein-based
immunotoxins are powerful agents against malignant B cells, such as
B cell lymphomas and leukemias and other malignancies, such as
neuroblastoma.
Definitions
[0024] The term "antibody" or "antibody peptide(s)" refers to
polyclonal and monoclonal antibodies, an entire immunoglobulin or
antibody or any functional fragment of an immunoglobulin molecule
which binds to the target antigen and is defined further below.
Examples of such functional entities include complete antibody
molecules, antibody fragments, such as Fv, single chain Fv,
complementarity determining regions (CDRs), V.sub.L (light chain
variable region), V.sub.H (heavy chain variable region), Fab,
F(ab).sub.2' and any combination of those or any other functional
portion of an immunoglobulin peptide capable of binding to target
antigen.
[0025] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially an Fab with part of
the hinge region (see, FUNDAMENTAL IMMUNOLOGY, 3RD ED., W. E. Paul,
ed., Raven Press, N.Y. (1993)). While various antibody fragments
are defined in terms of the digestion of an intact antibody, one of
skill will appreciate that such fragments may be synthesized de
novo either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein, also includes antibody
fragments either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA
methodologies.
[0026] For this invention, an antibody, is "reactive with" or
"binds to" an antigen if it interacts with the antigen. This
interaction is analogous to a chemical reaction in which two
reactants come together to form a product. In the case of the
antibody-antigen interaction, the product of the interaction is an
antibody-antigen complex. The preferred antigens which bind to
immunoglobulins of the invention are the CD22 and the CD74 cell
surface marker.
[0027] The term "binding specificity," "specifically binds to an
antibody" or "specifically immunoreactive with," when referring to
a protein or carbohydrate, refers to a binding reaction which is
determinative of the presence of the protein or carbohydrate in the
presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein or carbohydrate
and do not bind in a significant amount to other proteins or
carbohydrates present in the sample. Specific binding to an
antibody under such conditions may require an antibody selected for
its specificity towards a particular protein or carbohydrate. For
example, antibodies raised to the CD22 antigen may be selected to
provide antibodies that are specifically immunoreactive with CD22
protein and not with other proteins. A variety of immunoassay
formats may be used to select antibodies specifically
immunoreactive with a particular protein or carbohydrate. For
example, solid-phase ELISA immunoassays are routinely used to
select antibodies specifically immunoreactive with a protein or
carbohydrate. See Harlow & Lane, ANTIBODIES, A LABORATORY
MANUAL, Cold Spring Harbor Publication, New York (1988) for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
[0028] The term "humanized" refers to an antibody wherein the
constant regions have at least about 80% or greater homology to
human immunoglobulin. Additionally, some of the nonhuman, such as
murine, variable region amino acid residues can be modified to
contain amino acid residues of human origin.
[0029] Humanized antibodies have been referred to as "reshaped"
antibodies. Manipulation of the complementarity-determining regions
(CDR) is a way of achieving humanized antibodies. See, for example,
Jones, et al., Nature 321:522 (1988) and Riechmann, et al., Nature
332:323 (1988), both of which are incorporated by reference herein.
For a review article concerning humanized antibodies, see Winter
& Milstein, Nature 349:293 (1991), incorporated by reference
herein.
[0030] The terms "isolated" or "substantially purified," when
applied to a nucleic acid or protein, denotes that the nucleic acid
or protein is essentially free of other cellular components with
which it is associated in the natural state. It is preferably in a
homogeneous state, although it can be in either a dry or aqueous
solution. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein which is the predominant species present in a preparation
is substantially purified.
[0031] In particular, an isolated onc protein gene is separated
from open reading frames which flank the gene and encode proteins
other than onc protein. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the nucleic acid
or protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure. The term "nucleic acid"
refers to a deoxyribonucleotide or ribonucleotide polymer in either
single- or double-stranded form, and unless otherwise limited,
would encompass known analogs of natural nucleotides that can
function in a similar manner as naturally occurring
nucleotides.
[0032] The term "joined" in the context of the immunotoxins of this
invention encompasses the linking of moieties (typically an
antibody and a toxin) through covalent bonding, including disulfide
bonding; hydrogen bonding; electrostatic bonding; recombinant
fusion; and conformational bonding, e.g., antibody-antigen, and
biotin-avidin associations.
[0033] The terms "measurable ribonucleolytic activity" or
"significant ribonucleolytic activity" refer to a molecule which
has an IC.sub.50 of less than 40 ng/mL when added to a rabbit
reticulocyte lysate assay wherein protein synthesis is inhibited as
measured by the incorporation of [.sup.35S]methionine into acid
precipitable protein. IC.sub.50 is the concentration of protein
necessary to inhibit protein synthesis by 50% in the assay. The
lysate assay may be done as described in the Promega lysate assay
kit which is commercially available from Promega Corporation,
Madison, Wis. Ribonucleolytic activity using high molecular weight
RNA and tRNA is determined at 37.degree. C. through the formation
of perchloric acid soluble nucleotides following published
protocols (Newton, D. L., et al. Biochemistry 35:545-553 (1996)).
With poly(A,C) UpG and poly U, ribonucleolytic activity is assayed
according to DePrisco, et al., and Libonati & Floridi
(DePrisco, R., et al. Biochimica et Biophysica Acta 788:356-363
(1984); Libonati, M. et al. European J. Biochem. 8:81-87 (1969)).
Activity is assayed by measuring the increase with time in
absorbance at 260 nm. Incubation mixtures (1 mL of 10 mM imidazole,
0.1 M NaCl, pH 6.5 or pH 7) contain substrate and appropriate
amounts of enzyme solution at 25.degree. C. The in vitro
translation assay (St. Clair, D. K., et al. Proc. Nat'l Acad. Sci.
USA 84:8330-8334 (1987)) and the cell viability assays using the
(3-[4,5-dimethylthiazol-2- -yl]-2,5-diphenyltetrazolium bromide;
Thiazolyl blue; MTT) (Mossman, T. J. Immunol. Methods 65:55-63
(1983)) are performed as previously described (Pearson, J. W., et
al. J. Nat'l Cancer Inst. 83:1386-1391 (1991)).
[0034] The term "nucleic acid encoding" or "nucleic acid sequence
encoding" refers to a nucleic acid which directs the expression of
a specific protein or peptide. The nucleic acid sequences include
both the DNA strand sequence that is transcribed into RNA and the
RNA sequence that is translated into protein. The nucleic acid
sequences include both full length nucleic acid sequences as well
as shorter sequences derived from the full length sequences. It is
understood that a particular nucleic acid sequence includes the
degenerate codons of the native sequence or sequences which may be
introduced to provide codon preference in a specific host cell. The
nucleic acid includes both the sense and antisense strands as
either individual single strands or in the duplex form.
[0035] The term "onc protein" refers to an RNase A derived from
Rana pipiens that was originally designated P-30 protein and first
described in Darzynkiewicz, et al., Cell Tissue Kinet. 21:169
(1988), such as the protein having the sequence set out in SEQ ID
NO:1. A description of this protein can be found in U.S. Pat. No.
5,559,212. The term "native onc protein" refers to the protein in
its native form, purified from Rana pipiens oocytes. The term
"recombinant onc protein" refers to the protein produced by
recombinant means. Preferred embodiments of these recombinant
proteins and their nucleic sequences are described in PCT
Application No: PCT/US97/02588. It is understood that onc proteins
also encompass modifications in both the nucleic acid and the amino
acid sequences but have measurable ribonucleolytic activity.
[0036] An "one-derived" amino acid sequence includes one that
contains at least one string of six contiguous amino acids
identical to a contiguous sequence of six amino acids selected from
the group of sequences beginning at amino acid positions 1 (with
Glu replacing pyroGlu), 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15,
16, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 41, 42, 43, 44, 45, 46, 47, 50, 52, 54, 56, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 80, 81, 82,
84, 85, 86, 87, 91, 92, 93, 95, or 96 of the one amino acid
sequence (SEQ ID NO:1).
[0037] The term "pharmaceutical composition" refers to formulations
of various preparations. Parenteral formulations are known and are
preferred for use in the invention. The formulations containing
therapeutically effective amounts of the immunotoxins are either
sterile liquid solutions, liquid suspensions or lyophilized
versions and optionally contain stabilizers or excipients.
Lyophilized compositions are reconstituted with suitable diluents,
e.g., water for injection, saline, 0.3% glycine and the like, at a
level of about from 0.01 mg/kg of host body weight to 10 mg/kg or
more.
[0038] Typically, the pharmaceutical compositions containing the
immunotoxins are administered in a therapeutically effective dose
over either a single day or several days by daily intravenous
infusion.
[0039] The immunotoxins of this invention may be administered
systemically by injection, most preferably intravenously, but also
intramuscularly, subcutaneously, intrathecally, intraperitoneally,
into vascular spaces, or into joints, e.g., intraarticular
injection. The dose will be dependent upon the properties of the
immunotoxin employed, e.g., its activity and biological half-life,
the concentration of the immunotoxin in the formulation, the site
and rate of dosage, the clinical tolerance of the patient involved,
the extent of cancer afflicting the patient and the like as is well
within the skill of the physician.
[0040] The immunotoxin of the present invention may be administered
in solution. The pH of the solution should be in the range of pH 5
to 9.5, preferably pH 6.5 to 7.5. The immunotoxins or derivatives
thereof should be in a solution having a suitable pharmaceutically
acceptable buffer such as phosphate, tris (hydroxymethyl)
aminomethane-HCl or citrate and the like. Buffer concentrations
should be in the range of 1 to 100 mM. The solution of the
immunoglobulin may also contain a salt, such as sodium chloride or
potassium chloride in a concentration of 50 to 150 mM. An effective
amount of a stabilizing agent such as albumin, a globulin, a
detergent, a gelatin, a protamine or a salt of protamine may also
be included and may be added to a solution containing the
immunotoxin or to the composition from which the solution is
prepared. Systemic administration of the immunotoxin is typically
made every two to three days or once a week if a humanized form of
the antibody is used. Alternatively, daily administration is
useful. Usually administration is by either intramuscular injection
or intravascular infusion.
[0041] Administration may also be intranasal or by other
nonparenteral routes. The immunotoxin may also be administered via
microspheres, liposomes or other microparticulate delivery systems
placed in certain tissues including blood.
[0042] The immunotoxin may also be administered by aerosol to
achieve localized delivery to the lungs. This is accomplished by
preparing an aqueous aerosol, liposomal preparation or solid
particles containing or derivatives thereof. A nonaqueous (e.g.,
fluorocarbon propellent) suspension could be used. Sonic nebulizers
preferably are used in preparing aerosols. Sonic nebulizers
minimize exposing the antibody or derivatives thereof to shear,
which can result in degradation of the immunotoxin.
[0043] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the immunotoxin together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers will vary depending upon the
requirements for the particular immunotoxin, but typically include
nonionic surfactants (TWEEN-20 OR-80.RTM., PLURONIC-F128
OR-67.RTM., or polyethylene glycol), innocuous proteins like serum
albumin, or sorbitan esters, oleic acid, lecithin, amino acids such
as glycine, buffers, salts, sugars or sugar alcohols. The
formulations will be sterile. Aerosols generally will be prepared
from isotonic solutions.
[0044] The terms "recombinant DNA," "recombinant nucleic acid" or
"recombinantly produced DNA" refer to DNA which has been isolated
from its native or endogenous source and modified either chemically
or enzymatically by adding, deleting or altering
naturally-occurring flanking or internal nucleotides. Flanking
nucleotides are those nucleotides which are either upstream or
downstream from the described sequence or sub-sequence of
nucleotides, while internal nucleotides are those nucleotides which
occur within the described sequence or subsequence.
[0045] The term "recombinant means" refers to techniques where
proteins are isolated, the cDNA sequence coding the protein
identified and inserted into an expression vector. The vector is
then introduced into a cell and the cell expresses the protein.
Recombinant means also encompasses the ligation of coding or
promoter DNA from different sources into one vector for expression
of a fusion protein, constitutive expression of a protein, or
inducible expression of a protein.
[0046] The terms "recombinant protein," "recombinantly produced
protein" or "recombinantly produced immunotoxin" refer to a peptide
or protein produced using non-native cells that do not have an
endogenous copy of DNA able to express the protein. The cells
produce the protein because they have been genetically altered by
the introduction of the appropriate nucleic acid sequence. The
recombinant protein will not be found in association with proteins
and other subcellular components normally associated with the cells
producing the protein.
[0047] The term "selective cytotoxic reagent" refers to a compound
that when added to a population of different cells, e.g., within an
organism, kills one type of cell in the population based on some
physical characteristic of the cell, i.e., a surface ligand or
marker to which the cytotoxic reagent binds and then becomes
internalized.
[0048] The term "single chain antibody" refers to an antibody
wherein the genetic information encoding the functional fragments
of the antibody are located in a single contiguous length of DNA.
For a thorough description of single chain antibodies, see Bire, et
al., Science 242:423 (1988) and Huston, et al., Proc. Nat'l Acad.
Sci. USA 85:5879 (1988).
[0049] The term "surface marker" refers to a protein, carbohydrate,
or glycoprotein present on the surface of a cell. Different types
of cells express different cell surface markers and therefore cells
can be identified by the presence of a cell surface marker. For
example, malignant B cells overexpress CD22. Thus, the binding of
an antibody that recognizes CD22 identifies that cell as a B cell.
CD74, described below, is an example of a cell surface marker found
on B cells and a select group of other malignant cells.
[0050] Among various uses of the immunotoxins of the present
invention are included a variety of disease conditions caused by
specific human cells that may be eliminated by the toxic action of
the protein. One preferred application for the immunoconjugates of
the invention is the treatment of malignant B cells expressing
CD22. Exemplary malignant diseases of B cells include acute
lymphocytic leukemia (ALL), chronic B-lymphocytic leukemia (B-CLL),
chronic myelogenous leukemia, Burkitt's, AIDS-associated and
Follicular lymphomas, and hairy cell leukemias. Immunotoxins
described herein which are directed to CD74 are useful for
inhibition and treatment of melanoma, neuroblastoma and myeloma
cells.
[0051] The preferred cytotoxic reagents of this invention are at
least 100 times, preferably at least 500 times and most preferably
at least 1000 times more cytotoxic to target cells bearing a B cell
marker than a comparison reagent comprised of the same antibody
joined to EDN, a human non-toxic RNAse.
[0052] A. Antibodies to Cell Surface Markers
[0053] Antibodies refer to polypeptides substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof,
which specifically bind and recognize an analyte (antigen). The
recognized immunoglobulin genes include the kappa, lambda, alpha,
gamma, delta, epsilon and mu constant region genes, as well as the
myriad immunoglobulin variable region genes. Light chains are
classified as either kappa or lambda. Heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0054] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0055] A variety of methods for producing monoclonal antibodies are
known in the art. See, e.g., Goding, MONOCLONAL ANTIBODIES;
PRINCIPLES AND PRACTICE, Academic Press, 2nd Edition (1986); and
Harlow & Lane. A monoclonal antibody directed against or
reactive with human B cells is obtained by using combinations of
immunogens to immunize mice and screening hybridoma supernatant
against cells which express the desired antigen or by a screening
assay designed to be specific for monoclonal antibodies directed
against the antigen of interest. Useful cell lines for screening
for the antibodies of this invention are readily available or
obtained. Such cells include the Burkitt's lymphoma cell lines
Daudi, CA-46 and Raji.
[0056] CD22, a lineage-restricted B-cell antigen belonging to the
Ig superfamily, is expressed on the surface of many types of
malignant B cells, including but not limited to, acute lymphocytic
leukemia (B-ALL), chronic B-lymphocytic cells (B-CLL), B lymphoma
cells such as Burkitt's, AIDS-associated and Follicular lymphomas,
and hairy cell leukemias, as well as on normal mature B
lymphocytes. CD22 is not expressed in early stages of B-cell
development, nor is it found on the surface of stem cells or
terminal stage plasma cells. Vaickus, et al., Crit. Rev.
Oncol/Hematol. 11:267-297 (1991). Additionally, no shed antigen is
detected in normal human serum or serum from patients with CLL. Li,
et al., Cell. Immunol. 118:85-99 (1989).
[0057] CD74, also known as the MHC Class II associated invariant
chain (Ii), is found on B cells, macrophages, monocytes and other
MHC Class II positive cells. In addition to the malignant B cells
listed above, CD74 is also found on neuroblastoma, melanoma and
myeloma cells.
[0058] Production of monoclonal antibodies directed against, e.g.,
B cells, is accomplished by: 1) immunization with human B cells
followed by screening of the resultant hybridomas for reactivity
against a non-human cell line transfected with human B cell
antigens constructed in a manner similar to that described in
Nishimura, et al., Eur. J. Immunol. 18:747 (1988) which is
incorporated by reference herein; 2) immunization with a non-human
cell line (preferably autologous to the animal to be immunized)
transfected with human B cell antigens followed by screening of the
resultant hybridomas for reactivity against a human B cell line; 3)
immunization with human or non-human cell lines expressing human B
cell antigens followed by screening of the resultant hybridomas for
ability to block reactivity of existing anti-B cell monoclonal
antibodies with a human B cell line; 4) immunization with human or
non-human cell lines expressing human B cell antigens followed by
screening of the resultant hybridomas for reactivity with purified
native or recombinant B cell antigens; and 5) immunization with a
recombinant derivative of human B cell antigens followed by
screening of the resultant hybridomas for reactivity against a
human B cell line. Upon review of this disclosure, those of skill
will realize other methods of raising antibodies which can be used
in this invention.
[0059] Recombinant DNA methodologies are used to synthesize the
preferred antibodies of this invention. For example, a preferred
antibody portion of an immunotoxin for use in humans is a
"humanized" antibody against a B cell antigen which contains only
murine complementarity-determining regions (CDRs) combined with
human variable region frameworks and human constant regions.
[0060] Humanization techniques are well known in the art. See, for
example, PCT Application Publication No. WO 87/02671; U.S. Pat. No.
4,816,567; EP Patent Application 0173494; Jones, et al., Nature
321:522 (1986); and Verhoeyen, et al., Science 239:1534 (1988).
Manipulation of the CDR is a way of achieving humanized antibodies.
See, for example, Jones, et al., Nature 321:522 (1988) and
Riechmann, et al., Nature 332:323 (1988). For a review article
concerning humanized antibodies see Winter & Milstein, Nature
349:293 (1991).
[0061] In addition to humanized, the antibody moieties of this
invention are single chain antibodies. In one aspect of this
invention, single chain antibodies are cloned from the parent
hybridoma cell lines.
[0062] The Fv regions of monoclonal antibodies are cloned using the
same general strategy. Typically, for example, poly(A)+ RNA
extracted from hybridoma cells is reverse transcribed using random
hexamers as primers. The V.sub.H and V.sub.L domains are amplified
separately by two polymerase chain reactions (PCR). Heavy chain
sequences are amplified using 5' end primers which are designed
according to the amino-terminal protein sequences of the heavy
chains, and the 3' end primers according to consensus
immunoglobulin constant region sequences (Kabat, et al., SEQUENCES
OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5TH ED., U.S. Department of
Health and Human Services, Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Light chain Fv regions
are amplified using 5' end primers designed according to the
amino-termainal protein sequences of the light chains and in
combination with the primer C-kappa. One of skill in the art will
recognize other suitable primers may be used.
[0063] The crude PCR products are subcloned into suitable cloning
vectors which are well known to those of skill in the art and
commercially available. Clones containing the correct size DNA
insert are identified, for example, agarose gel electrophoresis.
The nucleotide sequence of the heavy or light chain coding regions
is then determined from double stranded plasmid DNA using the
sequencing primers adjacent to the cloning site. Commercially
available kits (e.g., the Sequenase.RTM. kit, United States
Biochemical Corp., Cleveland, Ohio) are used to facilitate
sequencing the DNA.
[0064] One of skill will appreciate that, utilizing the sequence
information provided for the Fv regions, nucleic acids encoding
these sequences are obtained using a number of methods well known
to those of skill in the art. Thus, DNA encoding the Fv regions is
prepared by any suitable method, including, for example,
amplification techniques such as ligase chain reaction (LCR) (see
Wu & Wallace, Genomics 4:560 (1989), Landegren, et al., Science
241:1077 (1988) and Barringer, et al., Gene 89:117 (1990)),
transcription amplification (see Kwoh, et al., Proc. Nat'l Acad.
Sci. USA 86:1173 (1989)), and self-sustained sequence replication
(see Guatelli, et al., Proc. Nat'l Acad. Sci. USA 87:1874 (1990)),
cloning and restriction of appropriate sequences or direct chemical
synthesis by methods such as the phosphotriester method of Narang,
et al., Meth. Enzymol. 68:90 (1979); the phosphodiester method of
Brown, et al., Meth. Enzymol. 68:109 (1979); the
diethylphosphoramidite method of Beaucage, et al., Tetra. Lett.
22:1859 (1981); and the solid support method of U.S. Pat. No.
4,458,066.
[0065] The nucleic acid sequences which encode the single chain
antibodies are identified by techniques well known in the art (see,
Sambrook, et al.). Briefly, the DNA products described above are
separated on an electrophoretic gel. The contents of the gel are
transferred to a suitable membrane (e.g., Hybond-N.RTM., Amersham)
and hybridized to a suitable probe under stringent conditions. The
probe should comprise a nucleic acid sequence of a fragment
embedded within the desired sequence.
[0066] If the DNA sequence is synthesized chemically, a single
stranded oligonucleotide will result. This may be converted into
double stranded DNA by hybridization with a complementary sequence,
or by polymerization with a DNA polymerase using the single strand
as a template. While it is possible to chemically synthesize an
entire single chain Fv region, it is preferable to synthesize a
number of shorter sequences (about 100 to 150 bases) that are later
ligated together.
[0067] Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired
DNA sequence.
[0068] Once the Fv variable light and heavy chain DNA is obtained,
the sequences may be ligated together, either directly or through a
DNA sequence encoding a peptide linker, using techniques well known
to those of skill in the art. Thus, the entire sequence encodes the
Fv domain in the form of a single chain antibody.
[0069] Alternatively, antibodies directed against B cells, for
example, are commercially available from suppliers of immunological
reagents (for example, Ancell Corp., Bayport, Minn. (RFB4); Becton
Dickinson, San Jose, Calif.; The Binding Site, Inc., San Diego,
Calif.; CalTag Laboratories, South San Francisco, Calif.;
Boehringer Mannheim Biochemicals, Indianapolis, Ind.; Pharmacia
Biotech, Piscataway, N.J.: and Zymed, Foster City, Calif.). RFB4 is
a preferred antibody of this invention which has surprising
efficacy when compared to other antibodies. It has been
characterized and is described in a PCT Patent Application, filed
Mar. 19, 1998, entitled FitzGerald, et al., "Recombinant RFB4
Immunotoxins Exhibit Potent Cytotoxic Activity for CD-22 Bearing
Cells and Tumors" as well as in Mansfield, et al., Bioconj. Chem.
7:557 (1996); Mansfield, et al., Biochem. Soc. Trans. 25:709
(1997); and Mansfield, et al., Blood 90:2020 (1997); all of which
are incorporated in this disclosure in their entirety.
[0070] B. Cytotoxic Onc Protein
[0071] This application discloses a new use for the onc protein
from Rana pipiens. The Rana pipiens onc protein is a substantially
pure protein derived from the eggs and/or embryos of Rana pipiens
having a molecular weight of about 12,000 Daltons by mass
spectrometry, and an isoelectric point of between 9.5 and 10.5. It
is also exemplified by a product sometimes referred to herein by
the trade name ONCONASE.RTM., available from Alfacell Corporation,
Bloomfield, N.J.
[0072] Preferably for this invention, the onc proteins are proteins
having the amino acid sequence set out in SEQ ID NO:1.
[0073] The onc protein used in this invention is unique compared to
other RNases used in immunotoxin construction because it is a
monomeric member of the pancreatic RNase family and is toxic to
certain cancer cells without an internalizing ligand (see U.S. Pat.
No. 5,559,212). However, it is a discovery of this invention that,
when conjugated to an antibody directed to a B cell, the
cytotoxicity of the onc protein dramatically increases up to as
much as 2,000 fold. In spite of the cytotoxicity to cancer cells,
patient toxicity and immunogenicity are expected to be low because
of the efficiency of this particular immunotoxin and the small size
of the toxin.
[0074] It will be understood by those of skill in the art that SEQ
ID NO:1 may be altered in a manner that does not substantially
affect the functional advantages of the sequence provided here. For
example, glycine and alanine are typically considered to be
interchangeable as are aspartic acid and glutamic acid and
asparagine and glutamine. Any such modification in which the
functional advantages of the sequence are maintained are intended
to be covered by the sequence described in SEQ ID NO:1.
[0075] An exemplary recombinant onc protein described and claimed
herein is defined as comprising SEQ ID NO:2. The recombinant onc
proteins of this invention have similar measurable ribonucleolytic
activity compared to native onc protein. However, one of skill in
the art will recognize that many different variations of one
sequences will encode onc proteins with roughly the same measurable
ribonucleolytic activity as native onc protein.
[0076] For a description of preferred recombinant onc proteins,
variants of recombinant onc proteins, and techniques for
synthesizing recombinant onc proteins, see PCT Application No:
PCT/US97/02588 which is incorporated by reference herein.
[0077] C. Immunotoxins
[0078] The toxic moiety and the antibody may be conjugated by
chemical or by recombinant means (see, Rybak, et al., Tumor
Targeting 1:141 (1995)). Chemical modifications include, for
example, derivitization for the purpose of linking the moieties to
each other, either directly or through a linking compound, by
methods that are well known in the art of protein chemistry. In the
presently preferred chemical conjugation embodiment, the means of
linking the toxic moiety and the recognition moiety comprises a
heterobifunctional coupling reagent which ultimately contributes to
formation of an intermolecular disulfide bond between the two
moieties. Other types of coupling reagents that are useful in this
capacity for the present invention are described, for example, in
U.S. Pat. No. 4,545,985. Alternatively, an intermolecular disulfide
may conveniently be formed between cysteines in each moiety which
occur naturally or are inserted by genetic engineering. The means
of linking moieties may also use thioether linkages between
heterobifunctional crosslinking reagents or specific low pH
cleavable crosslinkers or specific protease cleavable linkers or
other cleavable or noncleavable chemical linkages. The means of
linking moieties of the immunotoxins may also comprise a peptidyl
bond formed between moieties which are separately synthesized by
standard peptide synthesis chemistry or recombinant means.
[0079] Possible chemical modifications of the protein moieties of
the present invention also include derivitization with polyethylene
glycol (PEG) to extend time of residence in the circulatory system
and reduce immunogenicity, according to well known methods (See for
example, Lisi, et al., Applied Biochem. 4:19 (1982); Beauchamp, et
al., Anal. Biochem. 131:25 (1982); and Goodson, et al.,
Bio/Technology 8:343 (1990)).
[0080] Possible genetic engineering modifications of the proteins
of the immunotoxins include combination of the relevant functional
domains of each into a single chain multi-functional biosynthetic
protein expressed from a single gene derived by recombinant DNA
techniques. (See, for example, PCT published application
WO/88/09344). Furthermore, recombinant DNA techniques can be used
to link the recombinant onc protein and the antibody. Accordingly,
the immunotoxin can comprise a fused protein beginning at one end
with the onc protein and ending with the antibody.
[0081] Methods of producing recombinant fusion proteins are well
known to those of skill in the art. Thus, for example, Chaudhary,
et al, Nature 339:394 (1989); Batra, et al., J. Biol. Chem.
265:15198 (1990); Batra, et al., Proc. Nat'l Acad. Sci. USA 86:8545
(1989); Chaudhary, et al., Proc. Nat'l Acad. Sci. USA 87:1066
(1990), all incorporated by reference, describe the preparation of
various single chain antibody-toxin fusion proteins.
[0082] In general, producing immunotoxin fusion proteins involves
separately preparing the Fv light and heavy chains and DNA encoding
the onc protein to be used. The two sequences are combined in a
plasmid or other vector to form a construct encoding the particular
desired fusion protein. A simpler approach involves inserting the
DNA encoding the particular Fv region into a construct already
encoding the desired onc protein.
[0083] Thus, for example, DNA encoding anti-B cell single chain
antibody/onc protein immunotoxins is most easily prepared by
inserting the DNA encoding the antibody V.sub.H and V.sub.L chains
(Fv region) into constructs already containing DNA encoding the
desired onc protein or vice versa. The DNA sequence encoding the Fv
region is inserted into the construct using techniques well known
to those of skill in the art.
[0084] Mammalian cells have been used to express and secrete hybrid
molecules such as antibody-cytokines (Hoogenboom, et al., Biochem.
Biophys. Acta 1096:345 (1991); Hoogenboom, et al., Mol. Immunol.
28:1027 (1991)) and antibody-enzyme (Casadei, et al., Proc. Nat'l
Acad. Sci. USA 87:2047 (1990); Williams, et al., Gene 43:319
(1986)). In part, immunogenicity of foreign proteins is due to
incorrect glycosylation patterns present on recombinant proteins.
Therefore, eukaryotic cell lines are preferred over prokaryotic
cells as the expressed proteins are glycosylated. Human derived
cell lines are particularly preferred in that these cells
incorporate a sialic acid as the terminal glycoside. Cell lines
such as the hamster CHO and BHK, as well as the HEK-293 human
fibroblast line have been used to express recombinant human
proteins.
[0085] Other genetic engineering modifications of the protein
moieties of the immunotoxins of this invention include deletions of
functionally unnecessary domains to reduce the size of the protein
or to modify other parameters which facilitate production or
utility, such as sequence changes to affect the solubility (e.g.,
cysteine to serine) or glycosylation sites. One skilled in the art
would appreciate that many additional well known chemical and
genetic modifications of proteins may be advantageously applied to
any protein which, like the present cytotoxic reagent, is intended
for parenteral administration.
[0086] Preferred immunotoxins of the present invention are fusion
proteins containing as the toxic moiety a protein having the amino
acid sequence of SEQ ID NO:1 and a humanized antibody that binds a
specific cell surface marker on the cell of interest (more
preferably against B cells). The construction of this unique
genetic linkage of the fusion protein between the onc protein and
the antibody eliminates the heterogeneity of chemically linked
antibody/onc protein conjugates. This, it is believed, may
contribute to the increased potency and decreased immunogenicity of
the immunotoxin.
[0087] The invention includes nucleic acid constructs that encode
the novel proteins described here. A nucleic acid construct is one
which, when incorporated into an appropriate vector, is capable of
replicating in a host. The constructs may be linked to other
sequences capable of affecting the expression of the construct,
such as promoters and enhancers.
[0088] The immunotoxin of the present invention may be utilized for
the selective killing of tumor cells. This method is based on the
appropriate selection of an antibody that binds to cell surface
markers found specifically or predominantly on the type of cell
that is to be selectively killed. For example, the immunotoxin of
this invention includes those comprising an antibody that binds to
a tumor cell-specific surface marker, of which many are known in
the art. In the preferred embodiment for a human application, the
antibody is a humanized single chain protein, or a modified form
thereof, which preferentially binds B-cells, indicating
malignancy.
[0089] D. Pharmaceutical Compositions
[0090] The present invention also relates to a pharmaceutical
composition comprising immunotoxins of the present invention in a
pharmaceutically acceptable carrier. In therapeutic applications,
compositions are administered to a patient suffering from a
disease, in an amount sufficient to cure or at least partially
arrest the disease and its complications. An amount adequate to
accomplish this is defined as a therapeutically effective dose.
Amounts effective for this use will depend on the severity of the
disease and the general state of the patient's health.
[0091] Advantageously, the pharmaceutical composition is suitable
for parenteral administration. The immunotoxins of the present
invention may be administered by various means appropriate for
different purposes, for example, for treating tumors in various
parts of the body, according to methods known in the art for other
immunotoxins. (See, for example, Rybak, et al., Human Cancer
Immunology, in IMMUNOLOGY AND ALLERGY CLINICS OF AMERICA, W. B.
Saunders, 1990, and references cited therein). Accordingly, the
present invention also relates to pharmaceutical compositions
comprising an immunotoxin of this invention and a pharmaceutically
acceptable carrier, particularly such compositions which are
suitable for the above means of administration.
[0092] Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient.
[0093] Preferably, the compositions for administration will
commonly comprise a solution of the fusion protein comprising the
single chain antibody and the onc protein dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like. These solutions are sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of fusion protein in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0094] Thus, a typical pharmaceutical composition for intravenous
administration would be about 0.01 to 100 mg per patient per day.
Dosages from 0.1 up to about 1000 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a tumor or an organ
within which a tumor resides. Actual methods for preparing
parenterally administrable compositions will be known or apparent
to those skilled in the art and are described in more detail in
such publications as REMINGTON'S PHARMACEUTLCAL SCIENCE, 15TH ED.,
Mack Publishing Co., Easton, Pa., (1980).
[0095] Further, the present invention relates to a method of
selectively killing cells using a selective immunotoxin of the
present invention having an antibody specific for a target on the
surface of the cells to be killed under conditions allowing binding
of the antibody. Binding of the antibody to the surface marker on a
cell causes the onc protein of the reagent to selectively kill the
cell. This method of the present invention may be used for cell
separation in vitro by selectively killing unwanted types of cells,
for example, in bone marrow prior to transplantation into a patient
undergoing marrow ablation by radiation.
EXAMPLES
[0096] In the following non-limiting examples, the present
invention is exemplified by a immunotoxin in which the toxic moiety
is ONCONASE.RTM. and the antibodies recognize tumor cells, in
particular, B cells.
Example 1
Production of Native and Recombinant Onc Protein from Rana
pipiens
[0097] A. Isolation and Purification of Native Onc Protein
[0098] Techniques describing the isolation of oocytes from Rana
pipiens, in vitro fertilization of the eggs, and the isolation and
purification of native onc protein from frog embryos are
exquisitely detailed in U.S. Pat. Nos. 5,559,212 and 5,728,805,
which are both incorporated by reference herein.
[0099] B. Production and Assaying of Recombinant Onc Protein
[0100] The production of recombinant onc protein was done as
described in PCT application PCT/US97/02588. Ribonucleolytic
activity using high molecular weight RNA and tRNA was determined
following published protocols, Newton, et al., J. Neurosci. 14:538
(1994) at 37.degree. C. through the formation of perchloric acid
soluble nucleotides (see, Newton, et al., Biochem. 35:545 (1996)).
With poly (A,C), UpG, and poly U, ribonuclease activity was assayed
spectrophotometrically according to Libonati, et al., Biochim. et
Biophys. Acta 788:356 (1984), and Libonati & Floridi, Eur. J.
Biochem. 8:81 (1969). Briefly, activity was assayed by measuring
the increase in absorbance at 260 nm. Incubation mixtures (1 mL of
10 mM imidazole, 0.1 M NaCl, pH 6.5 or pH 7) contained substrate
and appropriate amounts of enzyme solution at 25.degree. C. The in
vitro translation assay (St. Clair, et al., Proc. Nat'l Acad. Sci.
USA 84:8330 (1987)), and the cell viability assays (Pearson, et
al., J. Nat'l Cancer Inst. 83:1386 (1991)), using the MTT method of
Mossman were performed as previously described.
Example 2
Chemical Analysis and Composition of Onc Proteins
[0101] The native onc protein described above has been well
characterized chemically. To be as fully functional as the native
onc protein, it is believed the recombinant onc protein should have
the chemistry and structure as described below.
[0102] The native onc protein was purified to homogeneity (as
established by standard tests used to assay the homogeneity of
proteins). By electrophoresis, the molecular weight of the native
onc protein was determined to be approximately 14,500 Daltons.
Calculation of the molecular weight based upon the listed amino
acid sequence (see, infra), indicated the molecular weight of
native onc protein should be 11,860 Daltons. However, because metal
ions may have bonded to the protein despite all efforts to remove
them, and because different isotopes may be involved, the molecular
weight of the native onc protein was 12,430 Daltons as determined
by mass spectroscopy. In view of this discrepancy, the molecular
weight of the pharmaceutical as determined by mass spectrometry was
considered to be approximately 12,000 Daltons. The isoelectric
point (pI) of native onc protein was found to be between about 9.5
and 10.5, as determined by isoelectric focussing. The amino
terminal group of native onc protein was blocked and was found to
be essentially free of carbohydrates (as determined by anthrone and
orcinol methods).
[0103] Table 1 indicates the amino acid composition of the native
onc protein.
1TABLE 1 Amino Acid Analysis of Native Onc Protein AMINO ACID
RESIDUE % MOL (24 HOUR ACID HYDROLYSIS) Aspartic acid/Asparagine
13.99 Threonine 9.30 (Note 1) Serine 7.78 Glutamic acid/Glutamine
6.10 Proline 4.36 Glycine 3.09 Alanine 3.09 Cysteine/2 6.92 (Note
1) Valine 8.20 Methionine 0.85 (Note 1) Isoleucine 4.86 (Note 2)
Leucine 5.22 Tyrosine 2.96 Phenylalanine 6.05 Histidine 2.88 Lysine
11.62 Arginine 2.70 Tryptophan Not Determined (Note 3) Approximate
Total 99.97% (Note 1) Threonine, cysteine/2 and methionine are
partially destroyed during hydrolysis and this value is uncorrected
for such partial destruction. (Note 2) This value is uncorrected
for incomplete hydrolysis. (Note 3) Tryptophan cannot be detected
in acid hydrolysis of proteins because it is destroyed and is
consequently shown as Not Determined. However, analysis of the
ultraviolet spectrum revealed the presence of one tryptophan
residue per molecule.
[0104]
2TABLE 2 Amino Acid Composition (as calculated from amino acid
sequence) APPROX. # OF RESIDUES (PER MOLECULE AMINO ACID OF NATIVE
ONC PROTEIN) Aspartic acid 6 Asparagine 8 Threonine 10 Serine 8
Glutamic acid 3 Pyroglutamic acid 1 Glutamine 2 Proline 4 Glycine 3
Alanine 3 Cysteine/2 8 Valine 8 Methionine 1 Isoleucine 6 Leucine 5
Tyrosine 3 Phenylalanine 6 Histidine 3 Lysine 12 Arginine 3
Tryptophan 1 Approximate Total 104
[0105] The native onc protein has been sequenced. The N-terminus of
the native protein is pyroglutamic acid (<Glu). This is a
cyclized derivative of glutamic acid which is devoid of the free
amino group necessary for direct sequencing and which therefore
"blocks" the N-terminus of the protein. The amino terminus of the
molecule has been altered to facilitate recombinant production of
the molecule as set out in previously cited PCT/US97/02588. The
preferred amino acid sequence of the cytotoxic RNase is shown as
SEQ ID NO:1.
Example 3
ANTI-CD22-ONCONASE.RTM. Immunotoxin
[0106] A. Materials and Methods
[0107] ONCONASE.RTM. (previously named P-30) was provided by
Alfacell Corp. as a lyophilized protein and was dissolved in
phosphate buffered saline (PBS). Stock solutions of at least 1
mg/mL were kept frozen at -20.degree. C. until dilutions were
prepared for assays. All other reagents were purchased from sources
previously described (Rybak, et al., J. Biol. Chem. 266:21202
(1991); Newton, et al., J. Biol. Chem. 267:19572 (1992); Mikulski,
et al., Cell Tissue Kinet. 23:237 (1990)), herein incorporated by
reference.
[0108] LL2 is a murine monoclonal antibody that recognizes and
specifically binds to CD22 on human B cells. The LL2 antibody was
provided by Immunomedics, Inc. (Morris Plains, N.J.). RFB4 is also
a murine monoclonal antibody that binds to CD22. This antibody is
available from many sources, including Ancell Corp.
[0109] Three Burkitt lymphoma cell lines (Daudi (ATCC CCL 213), CA
46 (ATCC CRL 1648), and Raji (ATCC CCL86)) were grown in RPMI 1640
media containing 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM
sodium pyruvate, and 10 .mu.g/mL gentamicin. HUT 102, a human
cutaneous T cell lymphoma cell line (ATCC TIB 162) was also grown
in supplemented RPMI medium. All cells were incubated at 37.degree.
C. in a 5% CO.sub.2 humidified atmosphere.
[0110] B. Preparation of LL2-ONCONASE.RTM. Immunotoxins
[0111] Disulfide linked conjugates were prepared as described in
Newton, et al., J. Biol. Chem., 267:19572 (1992), with the
following modifications. Antibody (12.5 nmol) was incubated with
250 nmol 2-iminothiolane and 2.5 mM 5,5'dithiobis(2-nitrobenzoic
acid) (DTNB) in 100 mM sodium borate, pH 8.5, at room temperature
for 1 hour in a final volume.ltoreq.0.5 mL. The reaction mixture
was applied to a PD-10.RTM. column (Pharmacia Biotech, Piscataway,
N.J.) equilibrated with Buffer A (0.1 M NaPO.sub.4, pH 7.5,
containing 0.1 M NaCl).
[0112] SPDP-modified ONCONASE.RTM. (0.9-1.2 mol N-succinimidyl 3
(2-pyridyldithio)propionate (SPDP)/mol ONCONASE.RTM. was prepared
as described (Newton, et al., (1992) supra). The SPDP-modified
ONCONASE.RTM. (340 nM) was reduced for 1 hour at room temperature
with dithiothreitol (DTT) at a final DTT concentration of 2 mM and
gel filtered on a PD-10.RTM. column equilibrated with Buffer A to
remove excess DTT. The modified ONCONASE.RTM. was added to the
modified antibody and the reaction incubated overnight at room
temperature. The ONCONASE.RTM. was at least a 10-fold molar excess
over the antibody.
[0113] Thioether-linked conjugates were prepared according to
Rybak, et al., Drug Delivery 1:3 (1993) and Newton, et al., Int'l
J. Oncology 8:1095 (1996) using
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Briefly, LL2
antibody (2 mg) was incubated with a 5-fold molar excess of MBS
(stock solution, 30 mM in DMF) for 10 min at room temperature. The
reaction contents were applied to a PD-10 column equilibrated with
Buffer A. Peak fractions (1.5 mL) were pooled. The SPDP-modified
ONCONASE.RTM. was dialyzed against 0.1 M sodium acetate, pH 4.5,
containing 0.1 M NaCl, followed by incubation with 25 mM DTT (final
concentration) for 30 min at room temperature. The reaction
contents were applied to a PD-10.RTM. column equilibrated with
Buffer A and the peak fractions pooled and added to the MBS
antibody. The reaction was incubated at room temperature overnight.
The ONCONASE.RTM. was present in a .gtoreq.10 fold molar excess
over antibody. The conjugates were separated from unreacted
ONCONASE.RTM. by gel filtration on a TSK-3000.RTM. HPLC column
(Toso-Haas).
[0114] The amount of protein present in the preparations was
determined by UV spectroscopy following Beer's Law:
[A=.epsilon.(conc.)] with the following extinction coefficients at
277 .mu.m: ONCONASE.RTM., .epsilon.(1%)=7.3; and immunotoxins,
.epsilon.(1%)=10.
[0115] The moles of ONCONASE.RTM. conjugated to antibody was
determined by gel electrophoresis of the reduced immunotoxins along
with standards of ONCONASE.RTM. and antibody. The gel was analyzed
using Image (NIH public domain software).
[0116] Analysis of ONCONASE.RTM. immunotoxins by SDS polyacrylamide
gel electrophoresis under reducing conditions demonstrated the
component proteins were regenerated after reduction. Under
non-reducing conditions, the antibody conjugates consisted of
multiple high molecular weight forms. The reactivity of the
cross-linker groups in the thiol-disulfide interchange reaction may
explain the heterogeneity of the conjugate. The immunotoxins
contained 1-2 moles of ONCONASE.RTM./mol of antibody. The purified
immunotoxins did not, by gel electrophoresis, appear to contain
significant amounts of free antibody, presumably because the
.gtoreq.10 fold molar excess of ONCONASE.RTM. yielded essentially
all immunotoxin and no free antibody.
[0117] C. Preparation of RFB4-ONCONASE.RTM. Immunotoxins
[0118] RFB4-ONCONASE.RTM. immunotoxins are prepared as described
above. Because RFB4 recognizes CD22, immunotoxins which contain
RFB4 are also cytotoxic to malignant B cells. Thus, the experiments
described below can be performed with RFB4-ONCONASE.RTM. as
well.
Example 4
In Vitro Cell Viability Studies
[0119] Protein synthesis was measured as described in Rybak, et
al., J. Biol. Chem. 266:21202 (1991). The same protocol was used to
measure RNA synthesis, except the cells were pulsed with 3 .mu.Ci
of [.sup.3H]uridine. Cell number was determined by a direct count
with a hemocytometer. An aliquot of cells was incubated for 5 min
with an equal volume of 0.5% Trypan Blue exclusion dye and viable
cells were scored. The MTT colorimetric assay (Mossman, T., J.
Immunol. Methods 65:55 (1983)) was performed as described.
(Mikulski, et al., Cell Tissue Kinet. 23:237 (1990)).
[0120] The IC.sub.50 for protein synthesis inhibition in Burkitt
lymphoma cells by ONCONASE.RTM.-immunotoxins is presented in Table
4.
3TABLE 4 Protein Synthesis Inhibition by ONCONASE
.RTM.-Immunotoxins IC.sub.50 Cell Line ONCONASE .RTM. LL2-ONCONASE
.RTM. Daudi >200 nM 100 pM CA 46 >200 nM 800 pM Raji >200
nM 800 pM HUT 102 30 nM >100 nM
[0121] The concentrations of immunotoxin required to inhibit
protein synthesis 50% in B cells after 24 hours are in the
picomolar range compared to the nanomolar range for unconjugated
ONCONASE.RTM.. HUT 102 cells, which do not express CD22, were not
sensitive to the LL2-ONCONASE.RTM. immunotoxin but were more
sensitive to the unconjugated ONCONASE.RTM. than the B-cell lines.
See FIG. 1.
[0122] As can be seen in FIG. 2, ONCONASE.RTM. alone was not
cytotoxic to B-lymphoma cells after 24 h compared to ONCONASE.RTM.
conjugated to the LL2 antibody. Thus, ONCONASE.RTM. (conjugated to
antibodies capable of internalization was more potent than the
unconjugated ONCONASE.RTM..
[0123] In addition to being more effective than ONCONASE.RTM.
alone, FIGS. 3 and 4 demonstrate the ONCONASE.RTM. immunotoxins
were much more effective than immunotoxins in which the toxic
moiety was either a human non-toxic RNase, eosinophil-derived
neurotoxin (EDN) (FIG. 5) or a human pancreatic RNase (FIG. 6).
[0124] In FIG. 5, LL2 or LL1 antibodies were conjugated to EDN as
described above and assayed on Daudi or CA 46 Burkitt s lymphoma
cells. [ ]It is believed that LL1 and LL2 immunotoxins are
delivered to the lysosomes where the immunotoxin is degraded to the
antibody and RNase moieties. The RNase leaves the lysosome and
enters the cytosol where it interferes with ribosomal activity.
From the data shown in FIG. 5, it is postulated that ONCONASE.RTM.
is about 2,000 fold more active than EDN because ONCONASE.RTM. is
not inactivated by degradation by the lysosome. Therefore, the
protein that enters the cytosol is an intact cytotoxin.
[0125] In FIG. 6, LL2-ONCONASE.RTM. was compared to LL2-pancreatic
RNase. Again, at concentrations of about 1 nM, LL2-ONCONASE.RTM.
completely blocked protein synthesis. At the same concentration,
only about 75% of protein synthesis had been blocked by the
addition of LL2-pancreatic RNase.
[0126] To test the hypothesis that ONCONASE.RTM. was not degraded
by the lysosomes leading to increased inhibition of protein
synthesis and cytotoxicity, .sup.125-I labeled LL2 and LL2
immunotoxins were added to Daudi cells. As can be seen in FIG. 7,
after the indicated time span, cells treated with LL2-ONCONASE.RTM.
contained more .sup.125-I labeled protein in their lysates,
indicating the immunotoxin was degraded at a slower rate than
LL2-EDN and LL2 alone. Thus, it would appear that ONCONASE.RTM. is
not degraded in the lysosomes.
[0127] To test the hypothesis that CD22 mediates the toxicity of
ONCONASE.RTM. immunotoxins via binding of the antibody portion of
the hybrid protein, the immunotoxins were assayed in the presence
of excess LL2 antibody (FIG. 6). The cytotoxicity observed in Daudi
cells after 24 h in the presence of the ONCONASE.RTM. immunotoxins
was reversed by an equimolar amount of LL2. These data show that
CD22 can mediate ONCONASE.RTM. cytotoxicity to Burkitt lymphoma
cells.
Example 5
In Vivo Efficacy of LL2-ONCONASE.RTM. Immunotoxins
[0128] To test the effect of LL2-ONCONASE.RTM. in vivo, Daudi cells
were implanted into SCID mice. One day later, the mice were treated
with ONCONASE.RTM. and LL2-ONCONASE.RTM., LL2-Pseudomonas exotoxin
and LL2-doxorubicin immunotoxins.
[0129] As can be seen in Table 5, LL2-ONCONASE.RTM. did not cause
cytotoxic side effects (death) in mice. As a comparison, the mice
were treated as well with LL2 conjugated to a mutant of domain II
of Pseudomonas exotoxin. As can be seen, this immunotoxin was
lethal. Thus, it appears that ONCONASE.RTM. as the toxic moiety of
an immunotoxin is not toxic to the treated animal and therefore
would be tolerated better as a therapeutic.
4TABLE 5 In vivo Cytotoxicity of LL2-ONCONASE Immunotoxins Toxicity
in Mice Dose Schedule Total Dose (.mu.g) Death/Total LL2-PE38KDEL*
80 .mu.g i.p. .times. 1 80 2/2 35 .mu.g i.p. QD .times. 4 140 2/2
LL2-ONCONASE 100 .mu.g i.p .times. 1 100 0/3 100 .mu.g i.p. QOD
.times. 5 500 0/3 100 .mu.g i.p. QD .times. 5 500 0/3 500 .mu.g
i.p. .times. 1 500 0/3 *Kreitman, et al., Cancer Res. 53: 819
(1993) QD = daily QOD = every other day
[0130] Table 6 shows the effects of LL2-ONCONASE.RTM. and
LL2-doxorubicin on Daudi-implanted SCID mice. The mice were
implanted with 5.times.10.sup.6 Daudi cells intravenously. 24 hours
later, treatment began with 5 equal doses daily. The doxorubicin
immunotoxin was injected intravenously and the ONCONASE.RTM.
immunotoxin was injected intraperitoneally. As can be seen, by
weight, almost one half the amount of LL2-ONCONASE.RTM.
significantly enhanced the survival of the mice compared to the
doxorubicin, a systemic chemotherapeutic reagent.
5TABLE 6 Treatment in SCID Mice with Disseminated Daudi Lymphoma %
of Mice with Enhanced Survival Relative to Immunotoxin Total Dose
Antibody Alone LL2-doxorubicin 9000 .mu.g 0 LL2-ONCONASE .RTM. 500
.mu.g 40%
[0131] In SCID mice implanted intravenously with 5.times.10.sup.6
Daudi B lymphoma cells, LL2-ONCONASE.RTM. injected
intraperitoneally proved to prolong the lives of the mice compared
to mice treated with phosphate buffered saline (PBS) or with
monoclonal antibody LL2 alone. FIG. 7 shows that all animals
treated with PBS developed severe B-cell lymphoma and were
sacrificed by day 35. All of the animals treated with LL2 were
sacrificed by day 37 due to lymphoma. On the other hand, all of the
animals treated with the immunotoxin survived through day 37. The
last animal treated with immunotoxin was sacrificed on day 46.
[0132] FIG. 8 shows that SCID mice implanted intraperitoneally with
2.times.10.sup.6 Daudi cells and then treated with 500 .mu.g
LL2-ONCONASE.RTM. intraperitoneally, 100 .mu.g per dose per day,
survived for over 100 days. The cohort of animals treated with PBS,
and unconjugated LL2 and ONCONASE.RTM. showed some indication of
disease within that time frame. The mean time of survival for the
PBS control group was 71 days, for the LL2+ONCONASE.RTM., the mean
time for survival was 80 days and the LL2-ONCONASE.RTM. treated
mice survived longer than 112 days.
[0133] Finally, FIG. 9 indicates that LL2-ONCONASE.RTM. is less
toxic than ONCONASE.RTM. alone or RFB4-deglycosylated Ricin A
chain. Compared to a lethal dose of 30 mg/kg ONCONASE.RTM., the
mouse treated with 300 mg/kg LL2-ONCONASE.RTM. not only survived
but gained weight during the course of the experiment. RFB4, when
conjugated to a Pseudomonas exotoxin fragment, had an LD.sub.50 of
1 mg/kg in a murine model wherein the immunotoxin was given only
once per day (Mansfield, et al., Bioconj. Chem. 7:557 (1996)).
[0134] These in vivo results indicate that LL2-ONCONASE.RTM. is a
superior B cell toxin compared to ONCONASE.RTM. alone, LL2 alone
and immunotoxins of LL2-Pseudomonas exotoxin and LL2-doxorubicin.
The toxicity studies show that LL2-ONCONASE.RTM. is tolerated well
with little, if any, side effects.
[0135] All publications, including patents and patent applications,
mentioned herein above are hereby incorporated by reference.
[0136] The foregoing invention has been described in some detail
for purposes of clarity and understanding. It will also be obvious
that various combinations in form and detail can be made without
departing from the scope of the invention.
6 SEQ ID NO:1 1 2 3 4 5 6 7 8 9 10
<Glu-Asp-Trp-Leu-Thr-Phe-Gln-Lys-Lys-His- 11 20
Ile-Thr-Asn-Thr-Arg-Asp-Val-As- p-Cys-Asp- 21 30
Asn-Ile-Met-Ser-Thr-Asn-Leu-Phe-His-Cys- 31 40
Lys-Asp-Lys-Asn-Thr-Phe-Ile-Tyr-Ser-- Arg- 41 50
Pro-Glu-Pro-Val-Lys-Ala-Ile-Cys-Lys-Gly- 51 60
Ile-Ile-Ala-Ser-Lys-Asn-Val-Leu-Thr-Thr- - 61 70
Ser-Glu-Phe-Tyr-Leu-Ser-Asp-Cys-Asn-Val- 71 80
Thr-Ser-Arg-Pro-Cys-Lys-Tyr-Lys-Leu-Lys- - 81 90
Lys-Ser-Thr-Asn-Lys-Phe-Cys-Val-Thr-Cys- 91 100
Glu-Asn-Gln-Ala-Pro-Val-His-Phe-Val-Gly- - 101 104 Val-Gly-Ser-Cys
SEQ ID NO:2 GATGTTGATT GTGATAATAT CATGTCAACA AACTTGTTCC ACTGCAAGGA
50 CAAGAACACT TTTATCTATT CACGTCCTGA GCCAGTGAAG GCCATCTGTA 100
AAGGAATTAT AGCCTCCAAA AATGTGTTAA CTACCTCTGA GTTTTATCTC 150
TCTGATTGCA ATGTAACAAG CAGGCCTTGC AAGTATAAAT TAAAGAAATC 200
AACTAATAAA TTTTGTGTAA CTTGTGAAAA TCAGGCACCA GTTCATTTT 249 SEQ ID
NO:3 1 10 Asp-Val-Asp-Cys-Asp-Asn-Ile-Met-Ser-Thr- 11 20
Asn-Leu-Phe-His-Cys-Lys-Asp-Lys-Asn-Thr- 21 30
Phe-Ile-Tyr-Ser-Arg-Pro-Glu-Pro-Val-Lys- 31 40
Ala-Ile-Cys-Lys-Gly-Ile-Ile-Ala-Ser-Lys- 41 50
Asn-Val-Leu-Thr-Thr-Ser-Glu-Phe-Tyr-Leu- 51 60
Ser-Asp-Cys-Asn-Val-Thr-Ser-Arg-Pro-Cys- 61 70
Lys-Tyr-Lys-Leu-Lys-Lys-Ser-Thr-Asn-Lys- 71 80
Phe-Cys-Val-Thr-Cys-Glu-Asn-Gln-Ala-Pro- 81 83 Val-His-Phe
[0137]
Sequence CWU 1
1
4 1 104 PRT Artificial Sequence Description of Artificial
SequenceRNase A derived from Rana pipiens, "onc protein" 1 Xaa Asp
Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn Thr Arg Asp 1 5 10 15
Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His Cys Lys Asp 20
25 30 Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys Ala Ile
Cys 35 40 45 Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser
Glu Phe Tyr 50 55 60 Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys
Lys Tyr Lys Leu Lys 65 70 75 80 Lys Ser Thr Asn Lys Phe Cys Val Thr
Cys Glu Asn Gln Ala Pro Val 85 90 95 His Phe Val Gly Val Gly Ser
Cys 100 2 249 DNA Artificial Sequence Description of Artificial
Sequencenucleic acid encoding "onc protein" 2 gatgttgatt gtgataatat
catgtcaaca aacttgttcc actgcaagga caagaacact 60 tttatctatt
cacgtcctga gccagtgaag gccatctgta aaggaattat agcctccaaa 120
aatgtgttaa ctacctctga gttttatctc tctgattgca atgtaacaag caggccttgc
180 aagtataaat taaagaaatc aactaataaa ttttgtgtaa cttgtgaaaa
tcaggcacca 240 gttcatttt 249 3 83 PRT Artificial Sequence
Description of Artificial Sequence"onc protein", positions 16-98 of
SEQ ID NO1 3 Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe
His Cys Lys 1 5 10 15 Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu
Pro Val Lys Ala Ile 20 25 30 Cys Lys Gly Ile Ile Ala Ser Lys Asn
Val Leu Thr Thr Ser Glu Phe 35 40 45 Tyr Leu Ser Asp Cys Asn Val
Thr Ser Arg Pro Cys Lys Tyr Lys Leu 50 55 60 Lys Lys Ser Thr Asn
Lys Phe Cys Val Thr Cys Glu Asn Gln Ala Pro 65 70 75 80 Val His Phe
4 4 PRT Artificial Sequence Description of Artificial
Sequencecarboxy terminal sequence of RFB4-PE35KDEL and LL2-PE38KDEL
chimeric immunotoxins with Pseudomonas exotoxin A (PE) modified by
KDEL endoplasmic reticulum retention sequence 4 Lys Asp Glu Leu
1
* * * * *
References