WO2001012661A2 - RECEPTOR-MEDIATED UPTAKE OF AN EXTRACELLULAR BCL-xL FUSION PROTEIN INHIBITS APOPTOSIS - Google Patents

Abstract

Apoptosis-modifying fusion polypeptides, and the corresponding nucleic acid molecules, are disclosed. Pharmaceutical compositions comprising these polypeptides, and the use of these polypeptides to modify apoptosis are also provided.

Description

RECEPTOR-MEDIATED UPTAKE OF AN EXTRACELLULAR BCI_-x_L FUSION PROTEIN INHIBITS APOPTOSIS

RELATED APPLICATIONS This application claims pnonty based on U S provisional application number 60/149.220, filed August 16. 1999

FIELD

This invention relates to modification of the apoptotic response of target cells, for instance target cells a subiect More specifically, it relates to apoptosis-modifymg fusion proteins with at least two domains, one of which targets the fusion protein to a target cell, and another of which modifies an apoptotic response of the target cell

BACKGROUND Tissue and cell homeostasis m multicellular organisms is largely influenced

apoptosis. the phenomenon of programmed cell death by which an mtra- or extra-cellular trigger causes a cell to activate a biochemical "suicide^" pathway Morphological indicia of apoptosis include membrane blebbmg. chromatm condensation and fragmentation, and formation of apoptotic bodies, all of which take place relatively earlv in the process of programmed cell death Degradation of genomic DNA during apoptosis results m formation of characteristic, nucleosome sized DNA fragments, this degradation produces a diagnostic ~180bp laddenng pattern when analyzed by gel electrophoresis A later step in the apoptotic process is degradation of the plasma membrane, rendering apoptotic cells leaky to various dyes (e.g., trypan blue and propidium iodide) Apoptotic cells are usual engulfed and destroyed early in the death process; thus, apoptosis tends not to be associated v>ιth inflammation caused by cytoplasm leakage, as is found in necrosis

Various m vivo triggers can induce apoptosis. the paradigmatic trigger is a shortage of one or more necessary growth factors Apoptosis plavs a significant role m development of the neural system (reviewed in Cowan et al . Science 225 1258-1265. 1984. Davies. Development 101 -185-208. 1987; Oppenheim. Annu. Rev. Neurosci. 14 453-501. 1991) and lymphoid system (reviewed m Blackman et al.. Science 248 1335-1341. 1990. Rothenberg. ___.v. mmunol. 51 85-214. 1992) of vertebrates System development occurs through selective apoptotic extinction of certain cell populations

In spite of much study, the molecular mechanisms of apoptosis are not fully elucidated It does appear, however, that different apoptosis mducers may tngger different apoptotic pathwa} s For instance, certain pathways are transcription-dependent, in that apoptosis requires the synthesis of new proteins after stimulation by. for instance, withdrawal of growth factors Staurosponne. a non-specific kmase inhibiter. in contrast, stimulates a transcnption-independent pathway Transcription dependent and independent pathways appear to share downstream components, including the ICE family of proteases (caspases) See Rubm. British Med. Bulk , 53 617-631. 1997. for a review of apoptosis m neurons. More general reviews include Ashkenazi and Dixit Science 281 1305-1308. Thomberry and Lazebmk. Science 281 1312-1316. and Adams and Con Science 2^,1 1322-1326

Apoptosis is recognized as a gene-directed event, controlled by a complex set of interacting gene products that inhibit or enhance apoptosis (Williams and Smith. Cell 1 111-119. 1993. reviewed in White. Genes Dev. 10 1-15. 1996) Extensiλ e effort is currently underw av to identify and characterize the genes involved in this process The first protein charactenzed as influencing apoptosis was Bcl-2 (Cleary et al . Cell 47 19-28. 1986. Tsuμmoto and Croce, Proc Natl. Acad Sci. USA 83 5214-5218, 1986) Since its discovery, several Bcl-2-related proteins (the Bcl-2 family of proteins) have been identified as being involved m regulation of apoptosis (White. Genes Dev 10 1-15, 1996. Yang et al . Cell 80 285-291. 1995) One such is Bcl-x. w ich is expressed in two different forms, long (Bcl-x_L) and short (Bcl-x_s) (Boise et al . Cell 14 597-608. 1993)

BC1-X_L and certain other members of the Bcl-2 family are. like Bcl-2 itself, powerful inhibitors of cell death (the "anti-death" Bcl-2 family members) Genetic overexpression of Bcl-2 has been shown to block apoptosis in the nervous system of transgemc mice (Chen et al . Nature 385 434-439 \997. Ueakarl. Immunity 4 195-201, 1996, Lippmcott- Schwartz et al . Cell 67 601-616. 1991

Hunziker et al . Ce 1167 617-627. 1991. Krajewski et al , Cancer Research 53 4701-4714. 1993. Martmou et al , Neuron 13 1017-1030, 1994)

Other members of the Bcl-2 protein family, including Bcl-x_s. Bad and Bax. are potent enhancers of apoptosis and therefore toxic to cells ("pro-death" Bcl-2 family members) Though the mechanism of apoptosis induction by these proteins remains unknown, it has been suggested that Bad binding to Bcl-x_Lmay promote cell death (Yang et al . Cell 80 285-291. 1995. Zha et al , JBwl. Chem 272 24101-24104. 1997) and that phosphorylation of Bad may prevent its binding to Bel -x_L. thereby blocking cell death (Zha et al. JBiol Chem 272 24101 -24104. 1997. Zha et al . Cell 87 619-628. 1996) In addition to its involvement in neuronal and lymphoid system development and overall cell population homeostasis. apoptosis also plays a substantial role in cell death that occurs in conjunction with various disease and lniury conditions For instance, apoptosis is involved in the damage caused by neurodegenerative disorders, including Alzheimer's disease (Bannaga. Science 281 1303-1304), Huntmgton^'s disease, and spmal-muscular atrophy There is also a substantial apoptotic component to the neuronal damage caused dunng stroke episodes (reviewed in Rubm. British Med Bulle ,

53(3) 617-631, 1997. and Bannaga. Science 281 1302-1303). and transient lschemic neuronal m]ury. as in spinal cord injury It ould be of great benefit to prevent undesired apoptosis m various disease and miury situations

Treatment with standard apoptosis inhibitory molecules for instance peptide-tvpe caspase inhibitors (e.g , DEVD-ttpe). though useful for laboratorv experiments where micromiection can be employed, has proven unsatisfactory for clinical work due to low membrane permeability of these inhibitors Transfection of cells ith various native proteins, including members of the Bcl-2 family of regulatoπ proteins, has dual disadvantages First, transfection is usually not cell-specific, and thus ma} disrupt apoptotic processes non-specifically in all cells Second, transfection tends to provide long term alterations in the apoptotic process, in that once a transgene is integrated and functional m the genome of target cells, it may be difficult to turn off Especially in instances of stroke episodes or transient lschemic neuronal injury, it would be more ad\ antageous to be able to apply apoptosis regulation for short penods of time Therefore, there is still a strong need to develop pharmaceutical agents that overcome these disadvantages

Cancer and other hyper-proliferative cell conditions can be viewed as inappropriate escape from appropriate cell death As such, it would be advantageous to be able to enhance apoptosis m certain of these cells to stop unregulated or undesired growth Various attempts have been made to selectively eliminate cancerous cells through the use of targeted lmmunotoxms (genetic or biochemical fusions between a toxic molecule, for instance a bactenal toxm. and a targeting domain denved. tvpically from an antibody molecule)

One bactenal toxm that has been employed in attempts to kill cancerous cells is diphtheria toxm (DT) Diphtheria toxm has three structurally and functionallv distinct domains (1 ) a cell surface receptor binding domain (DTR), (2) a translocation domain (DTT) that allows passage of the active domain across the cell membrane, and (3) the A (enzymatically active) chain that, upon delivery to a cell, ADP-nbosylates elongation factor 2 and thereby inactivates translation Altenng the receptor specificity of the diphtheria toxm has been used to generate toxms that may selectively kill cancer cells in vitro (Thorpe et al , Nature 271 752-755. 1978) and in man (Laske et al , Nature Medicine 3 1362- 1368, 1997) Promising though they might have seemed, these and similar hvbπd lmmunotoxms have proven to be substantially less effective at targeted cell death than the toxms from which they were generated This is perhaps due to difficulties in translocation of the fusion protein into the target cell (Columbatti et al . J Biol Chem. 261 3030-3035. 1986) In addition, in vivo results have been particularly poor using such hybrid constructs (Fulton et al Fed Proc 461 1507. 1987)

It is to biological molecules that overcome deficiencies m the prior art that the present invention is directed

SUMMARY OF THE DISCLOSURE Disclosed herein are apoptosis-modifymg fusion proteins constructed by fusing a protein, or an apoptosis-modifymg fragment or vanant thereof from the Bcl-2 protein family with a cell-binding, targeting domain such as one derived from a bacterial toxm Using this approach, apoptosis-modifymg fusion proteins can be delivered effectively throughout the bodv and targeted to select tissues and cells In certain embodiments, fusing various cell-binding domains to Bcl-2 famil) proteins (such as Bcl-x_L or Bad) allows targeting to specific subsets of cells in vivo permitting treatment and/or prevention of the cell-death related consequences of various diseases and injuries The delivery of other Bcl-2 homologues to the cell permits regulation of cell either positively (using anti-death Bcl-2 family members) or negatively (using pro-death members of the Bcl-2 family) The apoptosis-modifi. mg fusion proteins disclosed herein have specifiable cell-targeting and apoptosis-modifymg activities Thus, they may be used clinically to treat various disease and injury conditions, through inhibition or enhancement of an apoptotic cellular response For instance, apoptosis-inhibitmg fusion proteins are beneficial to minimize or prevent apoptotic damage that can be caused by neurodegenerative disorders (e.g.. Alzheimer^'s disease. Huntington^'s disease, spmal- muscular atrophy), stroke episodes, and transient lschemic neuronal injury (e.g , spmal cord injury) The apoptosis-enhancmg fusion proteins n can be used to inhibit cell growth, for instance uncontrolled cellular proliferation

Accordingly, a first embodiment is a functional apoptosis-modifymg fusion protein capable of binding a target cell, having a first domain capable of modifying apoptosis in the target cell, and a second domain capable of specifically targeting the fusion protein to the target cell This fusion protein further integrates into or other ise crosses a cellular membrane of the target cell upon binding to that cell

Certain embodiments w ill also include a linker between these two domains This linker will usually be at least 5 ammo acids long, for example between 5 and 100 ammo acids m length, and

for instance include the ammo acid sequence shown in SEQ ID NO 6 Appropnate linkers may be 6.

7. or 8 ammo acids in length, and so forth, including linkers of about 10, 20. 30. 40 or 50 ammo acids long

The apoptosis modifying fusion proteins may also include a third domain from one of the two ongmal proteins, or from a third protein This third domain may improve the fusion protein's ability to be integrated into or otherwise cross a cellular membrane of the target cell An example of such a third domain is the translocation region (domain or sub-domain) of diphtheria toxin

Target cells for the fusion proteins disclosed herein include, but are not limited to. neurons, lymphocytes, stem cells, epithelial cells, cancer cells, neoplasm cells, and others, including other hyper-proliferative cells The target cell chosen will depend on what disease or injury condition the fusion protein is intended to treat

Receptor-bmdmg domains may be denved from \ anous cell-type specific binding proteins, including for instance bactenal toxms (e.g., diphtheria toxm or anthrax toxm). growth factors (e.g.. epidermal growth factor), monoclonal antibodies, or single-chain antibodies denved from antibody genes Further, variants or fragments of such protems ma} also be used, where these fragments or variants maintain the ability to target the fusion protein to the appropnate target cell

Further specific embodiments employ essentially the entire Bcl-x_L protein as the apoptosis- modifymg domain of the fusion protein, or variants or fragments thereof that maintain the ability to inhibit apoptosis in a target cell to which the protein is exposed Examples of such proteins are fusion proteins made of the Bcl-x_L protein, functionally linked to the diphtheria toxm receptor binding domain through a peptide linker of about six ammo acids One such protein is Bcl-x_L-DTR. which consists of Bcl-x_L and DTR, without the translocation domain of diphtheria toxm The nucleotide sequence of this fusion protein is shown in SEQ ID NO 1. and the corresponding ammo acid sequence m SEQ ID NOs 1 and 2

Another such example is LF_n-Bcl-x_L which includes the ammo terminal portion (residues 1 - 255) of mature anthrax lethal factor (LF). coupled to residues 1 -209 of Bcl-x_L The nucleotide sequence of this fusion protein is shown in SEQ ID NO 7. and the corresponding ammo acid sequence

Also encompassed are fusion proteins wherein the apoptosis-modifymg domain is an apoptosis-enhancmg domain Such domains include the vanous pro-death members of the Bcl-2 family of proteins, for instance Bad. and variants or fragments thereof that enhance apoptosis m a target cell A specific appropriate vanant of the Bad protein has an ammo acid other than senne at ammo acid position 1 12 and/or position 136. to provide constitutivelv reduced phosphorvlation

Thus, one specific embodiment is a functional apoptosis-enhancmg fusion protein capable of binding a target cell, compns g the Bad protein and the diphtheria toxm translocation and receptor binding domains, functionally linked to each other The Bad protein of this embodiment can also contain a mutatιon(s) at position 112 and/or 136 to change the senne residue to some other ammo acid, to reduce phosphorvlation of the protein One such protein is Bad-DTTR the nucleotide sequence of this protein is shown in SEQ ID NO 3, and the corresponding ammo acid sequence m SEQ ID NOs 3 and 4

Also disclosed herein are nucleic acid molecules encoding apoptosis-modifymg fusion proteins, for instance the nucleic acid sequences in SEQ ID NOs 1, 3. and 7, and nucleic acid sequences having at least 90% sequence identity to these sequences, for instance those encoding for proteins containing one or more conservative ammo acid substitutions Other nucleic acid sequences may have 95% or 98% sequence identity with SEQ ID NO 1. 3. or 7 Also encompassed are recombmant nucleic acid molecules in which such a nucleic acid sequence is operablv linked to a promoter, vectors containing such a molecule, and transgenic cells comprising such a molecule

Methods also are provided for producing functional recombmant apoptosis-modifymg fusion proteins capable of binding to a target cell, integrating into or otherwise translocating across the cell membrane, and modifying an apoptotic response of the target cell Such a protein can be produced in a prokaryotic or eukaryotic cell, for instance by transforming or transfectmg such a cell ith a recombmant nucleic acid molecule compnsmg a sequence which encodes a disclosed bispecrfic fusion protein Appropriate eukaryotic cells include veast. algae, plant or animal cells Such transformed cells can then be cultured under conditions that cause production of the fusion protein, hich is then recovered through protein purification means The protein can include a molecular tag. such as a six histidme (hexa-his) tag. to facilitate its recoven Protein analogs, derivatives, or mimetics of the disclosed proteins, which retain the ability to target to appropriate target cells and modify apoptosis in those cells, are also encompassed m embodiments Compositions containing these apoptosis modifvmg fusion proteins and analogs, derivatives or mimetics of these proteins, are further aspects of this disclosure Such compositions may further contain a pharmaceuticallv acceptable earner, various other medical or therapeutic agents, and/or additional apoptosis modifying substances Methods for modifying apoptosis in a target cell are also encompassed wherein a sufficient amount of a fusion protein of the current disclosure to modify apoptosis in the target cell is contacted with a target cell Modification of apoptosis can be by either inhibition or enhancement of an apoptotic response of the target cell The fusion protein can be administered to the target cell m the form of a pharmaceutical composition, and can further be administered with vanous medical or therapeutic agents, and/or additional apoptosis modifying substances Such agents mav include, for instance, chemotherapeutic. anti-inflammatory, anti-viral, and antibiotic agents

BCI-X -DTR. LF_n-Bcl-X - or related fusion proteins can be used to inhibit apoptosis in a target cell by contacting the target cell with an amount of this protein sufficient to inhibit apoptosis Alternatively Bad-DTTR or related fusion proteins can be used to enhance apoptosis in a target cell by contacting the target cell with an amount of this protein sufficient to enhance apoptosis

A specific aspect disclosed herem is the method of reducing apoptosis m a subject after transient ischemic neuronal injury, for instance a spinal cord injury, compnsmg admimstenng to the subject a therapeutically effective amount of an apoptosis-mhibiting protein according to this disclosure Examples of such fusion proteins include Bcl-x -DTR and LF_n-Bcl-x_L These proteins can be administered in the form of a pharmaceutical composition and can be co-administered with vanous medical or therapeutic agents, and/or additional apoptosis modifying substances

The foregoing and other features and advantages of the invention will become more apparent from the following detailed descnption of several embodiments, which proceeds with reference to the accompanying figures and tables

BRIEF DESCRIPTION OF THE FIGURES FIG 1 shows the construction, production and bioactivity of Bcl-x_L-DTR and Bcl-x_L transfected into HeLa cells FIG 1A is a schematic representation of construction of Bcl-x_L-DTR FIG IB is a Western blot of the lvsates of HeLa cells transiently transfected with Bcl-x_L (lane b) and Bcl- X_L-DTR (lane c) Lane a contains untransfected cells as a control A small amount of endogeneous Bcl-x_Lιs present in lanes a and c FIG 1C is a graph that shows transient transfection of Bcl-x (O) and BCI-X_L-DTR ( ) genes into HeLa cells inhibits apoptotic cell death induced by the addition of STS Apoptosis m control cells transfected with the vector (pcDNA3) vector is shown for companson (D)

FIG 2 is a graph that shows the results of a diphtheria toxm receptor competitrv e binding assay Cold competitor proteins [native DT (Δ). Bcl-x_L-DTR (A). Bcl-x_L (O). and DTR (•)] were used to displace I¹²³ labeled diphthena toxm (DT) tracer, and the amount of bound labeled tracer was measured Native DT and the fusion protein Bcl-x_L-DTR compete for DT receptor binding m the nanomolar concentration ranεe FIG 3 depicts the results of several expenments that demonstrate the apoptosis-mhibitmg character of the fusion construct Bcl-x_L-DTR Panel A is a graph of a time course of apoptosis induced bv staurosponne (STS) Cells were treated with 0 1 μM STS (O). 0 1 μM STS plus 4 8 μM Bcl-x_L- DTR protein medium (Δ). or 20 μl of PBS (D) Results are presented as the average number of apoptotic cells per field (magnification 160 x) For each point, at least 5 fields were counted in each of at least 3 wells FIG 3B is a SDS-PAGE gel that shows that Bcl-x_L-DTR prevents PARP cleavage Lane a contains control HeLa cells not incubated with STS (umnduced cells). Lane b HeLa cells treated with STS plus 1 μM Bcl-x_L-DTR protein. Lane c. HeLa cells freated with STS plus 1 48 μM BCI-X_L-DTR protein, and Lane d. HeLa cells treated with STS and no fusion protein FIG 4 shows that Bcl-x_L-DTR inhibits of apoptosis induced by γ-radiation. but not that induced by α-Fas antibody FIG 4A is a graph showing that the addition of Bcl-x_L-DTR prior to irradiation of Jurkat cells reduces apoptotic death in response to γ-radiation Control cells were not irradiated and not treated with Bcl-x_L-DTR FIG 4B is a graph that shows that, m Jurkat cells. Bcl-x_L- DTR had little inhibitory effect on apoptosis induced bv anti-Fas antibody Control cells were treated with PBS and no anti-Fas antibod}

FIG 5 shows that Bcl-x_L-DTR inhibits apoptosis induced by pohovirus

FIG 6 is a graph showing the time course of viability of cells treated with Bad-DTTR

FIG 7 shows the results of expenments that demonstrate that Bad-DTTR combined with STS triggers massive cell death FIG 7A is a graph quantifying cell death after treatment of U251 MG cells with various combinations of STS and Bad-DTTR Apoptosis is most enhanced when cells are freated with 0 lμM STS plus 0 65 μM Bad-DTTR. and cells begin to die about 12 hours after treatment In the experiment depicted FIG 7B. the use of 1 μM STS in combination with various concentrations of Bad-DTTR cause an earlier onset of apoptosis in U251 MG cells Key D = PBS. 0 = 0 1 μM STS O = 0 65 μM Bad-DTTR. Δ = 0 065 μM Bad-DTTR. m = 0 1 μM STS + 0 65 μM Bad-DTTR e = 0 1 μM STS + 0 065 μM Bad-DTTR

FIG 8 is a schematic diagram of the chimera LF_n-Bcl-x_L The fusion gene, LF_n-Bcl-x_L. was inserted into the vector. pETl 5b. yielding a histidme tag sequence at the N terminus of the LF_n-Bcl- x_L gene

FIG 9 is a graph showing the time course of apoptosis induced by STS in J774 cells, with or without LF_n-Bcl-x_L protein J774 cells at 3 x 10⁴ / cm² were treated with 0 1 μM staurosponne alone 0 1 μM staurosponne along with LF_n-Bcl-x_L (28 μg / ml) plus PA (33 μg / ml), or with PBS alone The apoptotic and living cells were stained with Hoechst 33342 and counted at the indicated times, and the data were calculated as reported (Liu et al . Proc Natl Acad Set USA 96 9563-9567, 1999)

FIG 10 is a bar graph showing the effect of LF_n-Bcl-x_L against J774 treated with STS J774 cells at 10⁴ / cm^" were treated with PBS. 0 1 μM staurosponne alone. 0 1 μM staurosponne along with LF_n (28 μg/ml) 0 1 μM staurosponne along with Bcl-x_L (28 μg/ml). 0 1 μM staurosponne along with LF_n-Bcl-x_L (28 μg / ml). 0 1 μM staurosponne along with LF_n-Bcl-x_L (28 μg/ml) plus PA (33 μg/ml) 0 1 μM staurosponne along with PA (33 μg/ml) and 0 1 μM staurosponne along with LF_n (28 μg/ml) plus PA (33 μg/ml) The apoptotic and living cells w ere stained with Hoechst 33342 48 hours later and counted, and the data were calculated as for FIG 9

FIG 11 is a bar graph showing the effect of LF_n-Bcl-x_L against Jurkat cells treated with STS Jurkat cells at 10⁵ / ml were treated with 0 1 μM staurosponne alone 0 1 μM staurosponne along with LF_n-Bcl-x_L (28 μg / ml) plus PA (33 μg / ml) or with PBS The apoptotic and living cells were stained with Hoechest 33342 21 hours later and counted, and the data were calculated as for FIG 9

FIG 12 is a bar graph showing that the fusion protein LF_n-Bcl-x_L prevents apoptosis bv in neonatal rat retinal ganglion cells 24 hours after optic nerve section The apoptotic and living cells in retinal ganglion lavers were counted 24 hours after optic nerve section immediatelv followed bv the injection of PBS or the indicated protem(s) The percentage of apoptotic cells versus total retinal ganglion cells per retina is represented

SEQUENCE LISTING The nucleic and ammo acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide base, and three letter code for ammo acids Onlv one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included bv any reference to the displayed strand

SEQ ID NO 1 shows the DNA coding sequence and corresponding ammo acid sequence of Bcl-x_L-DTR

SEQ ID NO 2 shows the ammo acid sequence of Bcl-x_L-DTR

SEQ ID NO 3 shows the DNA coding sequence and corresponding amino acid sequence of Bad-DTTR

SEQ ID NO 4 shows the ammo acid sequence of Bad-DTTR SEQ ID NO 5 shows the nucleotide sequence of the linker used to link Bcl-x_L to DTR in the fusion construct BCI-X_L-DTR

SEQ ID NO 6 shows the ammo acid sequence of the linker used to link Bcl-x_L to DTR to form Bcl-x_L-DTR

SEQ ID NO 7 shows the DNA coding sequence and corresponding ammo acid sequence of LF_n-Bcl-x_L

SEQ ID NO 8 shows the ammo acid sequence of LF_n-Bcl-x_L

DETAILED DESCRD7TION OF THE INVENTION I. Abbreviations and Definitions

A. Abbreviations

DT diphtheria toxm

DTR diphtheria toxm receptor binding domain DTT diphtheria toxm translocation domain

DTTR diphthena toxm translocation and receptor binding domams

E. co Escherichia coll

EF anthrax edema factor LF anthrax lethal factor

LF_n first 255 residues of anthrax lethal factor moi multiplicity of infection

PA anthrax protect-', e antigen

PCR polvmerase chain reaction RE restriction endonuclease

SDS-PAGE sodium dodecyl sulfate-polyacr lamide gel electrophoresis

STS staurosponne

TdT terminal deoxynbonucleotidyl transferase

TUNEL TdT-dependent dUTP-biot nick end labeling

B. Definitions

Unless otherwise noted, technical terms are used according to conventional usage Definitions of common terms in molecular biology may be found in Lew . Genes V published

Oxford University Press. 1994 (ISBN 0-19-854287-9). Kendrew et al., (eds ). The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd . 1994 (ISBN 0-632-02182-9). and Robert A Meyers (ed ). Molecular Biology and Biotechnology: a Comprehensive Desk Reference , published by VCH Publishers. Inc , 1995 (ISBN 1 -56081 -569-8) The nomenclature for DNA bases and the three- letter code for ammo acid residues, as set forth at 37 CFR § 1 822. are used herein

In order to facilitate review of the various embodiments of the invention, the following definitions of terms are provided These definitions are not intended to limit such terms to a scope narrower than would be known to a person of ordinary skill in the field

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds The term mammal includes both human and non-human mammals Similarly, the term "subject^" includes both human and veterinary subjects

Apoptosis-modifying ability: A protein has apoptosis-modifymg ability if it is capable of modifying apoptosis in a cell This ability is usually measurable, either in vivo or in vitro, using am one of myriad apoptosis assays The art is replete with methods for measuring apoptosis Appropriate techniques include dve exclusion (e g Hoechst dve No 33342). assaying for caspase activity, and TUNEL-sta mg The specific ability of a fusion protein to modify the apoptotic response of a cell to vanous apoptosis-mducmg stimuli can be determined by running standard apoptosis assays in the absence of or presence of vanous concentrations of the fusion proteins The results of the assay are then compared, and can be reported for instance by presenting the percentage ol apoptosis that occurs m the presence of the fusion protein

The invention also includes analogs derivatives or mimetics of the disclosed fusion proteins, and which have apoptosis-modifymg ability Such molecules can be screened for apoptosis-modifymg ability by assaying a protein similar to the disclosed fusion protein, in that it has one or more conservative ammo acid substitutions or short m-frame deletions or insertions or analogs, denvatives or mimetics thereof, and determining whether the similar protein, analog, denvative or mimetic provides modification of apoptosis in a desired target cell The apoptosis-modifymg ability and target cell binding affinity of these derivative compounds can be measured by any known means, including those discussed in this application

Apoptosis-modify ing fusion protein: Proteins that have at least tw o domains fused together, at least one domain compnsmg a cell binding region capable of targeting the fusion protein to a target cell (the targeting or cell-binding domain), and at least one domain capable of modifying apoptosis m the target cell (the apoptosis-modifymg domain) The apoptosis-modifymg fusion proteins of the current invention are further charactenzed by their ability to integrate into or otherwise cross a cellular membrane of the target cell when delivered extracellularly An apoptosis-modifymg fusion protein is considered functional if it targets to the correct target cell, and modifies an apoptotic response of that cell

In general, the two domains of the disclosed fusions are genetically ftised together, m that nucleic acid molecules that encode each protein domain are functionally linked together, for instance directly or through the use of a linker ohgonucleotide. thereby producing a single fusion-encoding nucleic acid molecule The translated product of such a fusion-encoding nucleic acid molecule is the apoptosis-modifvmg fusion protein

Apoptosis-modift mg fusion proteins can be labeled according to how they influence apoptosis in the target cell For instance, an apoptosis-modifymg fusion protein according to the current invention that inhibits apoptosis m the target cell can be referred to as an apoptosis-mhibitmg fusion protein (_ g . Bcl-x_L-DTR and LF_n-Bcl-x ) Likewise, if the fusion protein enhances apoptosis in the target cell, it can be referred to as an apoptosis-enhancmg fusion protein (e g . Bad-DTTR) Specific apoptosis-modifymg fusion proteins are usually named for the proteins from w ich domains are taken to form the fusion, or from the domains actually used For instance. "Bcl-x_L-DTR" (SEQ ID NOs 1 and 2) consists of the entire Bcl-x_L protein fused in frame to the receptor-bmdmg domain of diphtheria toxin (DTR) via a short linker

A Bcl-2 protein: A Bcl-2 protein is a protein from the Bcl-2 famih of proteins and includes those proteins related to Bcl-2 by sequence homology which affect apoptosis Bv

of example, the family includes Bcl-2. Bcl-x (both the long and short forms). Bax. and Bad Additional members of the Bcl-2 family of proteins are known (Adams and Cory. Science 281 1322-1326. 1998)

Molecules that are derived from proteins of the Bcl-2 family include fragments of such proteins (<? g . fragments of Bcl-x_L or Bad), generated either bv chemical (e g enzymatic) digestion or genetic engmeenng means Such fragments may comprise nearh all of the native protein, with one or a few ammo acids being genetically or chemically removed from the ammo or carboxy terminal end of the protein, or genetically removed from an internal region of the sequence

Derived molecules, or derived from The term ' X-denved molecules^'" or "denved from X.^" where X is a protein also encompasses analogs (non-protem organic molecules), denvatives

(chemically functiona zed protein molecules obtained starting with the disclosed protein sequences) or mimetics (three-dimensionally similar chemicals) of the native protein structure, as well as proteins sequence vanants or genetic alleles. that maintain biological functionality Where the derived molecule is used as the targeting domain of an apoptosis-modifymg fusion protein, the biological functionality maintained is the ability to target to fusion protein to the desired target cell Likewise, where the denved molecule is used as the apoptosis-modifymg domain of the fusion, the functionality maintained is the ability to affect apoptosis in the target cell Each of these functionalities can be measured in various ways, including specific protein binding and apoptosis assays, respectively

Injectable composition: A pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g . an apoptosis-modifymg fusion protein The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffenng agents and the like Such mjectable compositions that are useful for use with the fusion proteins of this invention are conventional, appropriate formulations are well known m the art

Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, t.e . other chromosomal and extra- chromosomal DNA and RNA, proteins and organelles Nucleic acids and proteins that have been "isolated^"" include nucleic acids and proteins purified bv standard punfication methods The term also embraces nucleic acids and proteins prepared by recombmant expression in a host cell as well as chemically synthesized nucleic acids

Linker: A peptide. usually between two and 150 ammo acid residues in length, which serves to join two protein domains in a multi-domain fusion protein Peptide linkers are generally encoded for by a corresponding oligonucleotide linker This can be genetically fused, in frame, between the nucleotides that encode the domains of a fusion protein

Oligonucleotide: A linear polynucleotide sequence of between six and 300 nucleotide bases m length

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence For instance, a promoter is operablv linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence Generally, operably linked DNA sequences are contiguous and. where necessary to join two protem-coding regions, in the same reading frame

Parenteral: Administered outside of the intestine, e g.. not via the alimentary tract Generalh . parenteral formulations are those that will be administered through any possible mode except ingestion This term especially refers to injections, whether administered intravenously. mtrathecally. intramuscularly, mtrapeπtoneally. or subcutaneously. and vanous surface applications including intranasal. mtradermal. and topical application, for instance

Pharmaceutically acceptable carriers: The pharmaceutically acceptable earners useful m this invention are conventional Martin. Remington '„ Pharmaceutical Sciences, published by Mack Publishing Co , Easton, PA. 15th Edition. 1975. descnbes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed

In general, the nature of the carrier will depend on the particular mode of administration being employed For instance, parenteral formulations usualh comprise mjectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle For solid compositions (e.g.. powder, pill, tablet, or capsule forms), conventional non-toxic solid earners can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate In addition to biologically- neutral earners, pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffenng agents and the like, for example sodium acetate or sorbitan monolaurate

Purified: The term punfied does not require absolute punty. rather, it is intended as a relative term Thus, for example, a purified fusion protem preparation is one in which the fusion protein is more ennched than the protem is m its generative environment, for instance withm a cell or in a biochemical reaction chamber Preferably, a preparation of fusion protem is punfied such that the fusion protem represents at least 50% of the total protein content of the preparation More punfied preparations will have fusion protem that represents at least 60%. 70%. 80% or 90% of the total protem content

Recombinant: A recombmant nucleic acid molecule is one that has a sequence that is not naturalh occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence This artificial combination can be accomplished by chemical synthesis or. more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g . by genetic engineering techniques

Similarly, a recombinant protem is one encoded for bv a recombmant nucleic acid molecule Sequence identity: The similanty between two nucleic acid sequences, or two ammo acid sequences is expressed in terms of the similanty betw een the sequences, otherwise referred to as sequence identify Sequence identity is frequently measured in terms of percentage identity (or similanty or homology). the higher the percentage, the more similar the two sequences are Homologs of the apoptosis-modifymg fusion protem will possess a relatπ ely high degree of sequence identity when ahgned using standard methods For instance, encoding sequences encompassed in the current invention include those that share about 90% sequence identity with SEQ ID NO 1 and NO 3

Methods of alignment of sequences for companson are well known in the art Various programs and alignment algonthms are described m Smith and Waterman. Adv. Appl. Math 2 482. 1981. Needleman and Wunsch. J. Mol. Biol. 48 443. 1970. Pearson and Lipman. PNAS USA 85 2444. 1988. Higgms and Sharp. Gene 73 237-244. 1988. Higgms and Sharp. CABIOS 5 151 -153. 1989. Coτpet et al., Nuc. Acids Res. 16 10881-90. 1988. Huang et al, Comp. Appls Biosci 8 155-65. 1992. and Pearson et al, Meth. Mol Biol. 24 307-31. 1994 Altschul et al, Nature Genet 6 1 19-29. 1994. presents a detailed consideration of sequence alignment methods and homology calculations The alignment tools ALIGN (Myers and Miller. CABIOS 4 1 1 -17. 1989) or LFASTA (Pearson and Lipman. PNAS USA 85 2444. 1988) may be used to perform sequence compansons (Internet Program © 1996. W R Pearson and the University of Virginia, "fasta20u63" version 2 0u63. release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity⁷ These alignment tools and their respective tutonals are available on the Internet at the NC SA web-site

For comparisons of ammo acid sequences of greater than about 30 ammo acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 1 1. and a per residue gap cost of 1 ) When aligning short peptides (fewer than around 30 ammo acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matnx set to default parameters (open gap 9. extension gap 1 penalties) Proteins with even greater similanty to the reference sequences ill show increasing percentage identities when assessed by this method, such as at least 90%. at least 92%. at least 94%. at least 95%. at least 97%. at least 98%. or at least 99% sequence identity

An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybndize to each other under stnngent conditions Stnngent conditions are sequence- dependent and are different under different environmental parameters Generally, stnngent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybndizes to a perfectly matched probe Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al, In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York. 1989. and Tijssen, Laboratory Techniques m Biochemistry and Molecular Biology Part i. Ch 2. Elseλier. New York. 1993 Nucleic acid molecules that hybndize to the disclosed apoptosis-modifi mg fusion protem sequences under stringent conditions will typicallv hybridize to a probe (based on the entire fusion protem encoding sequence, an entire domain, or other selected portions of the encoding sequence) under wash conditions of 0 2 x SSC. 0 1% SDS at 65°C

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar ammo acid sequences, due to the degeneracy of the genetic code It is understood that changes in nucleic acid sequence can be made using this degeneracλ to produce multiple nucleic acid sequences that encode substantially the same protem

Specific binding agent: An agent that binds substantially only to a defined target Thus a Bcl- X -DTR-specific binding agent binds substantially only the BCI-X_L-DTR protem m a specific preparation As used herein the term "Bcl-x_L-DTR-specιfic binding agent" includes Bcl-x_L-DTR antibodies and other agents that bind substantially only to a Bcl-x_L-DTR protem in that preparation

Antι-Bcl-x_L-DTR antibodies may be produced using standard procedures descnbed in a number of texts, including Harlow and Lane (Using Antibodies, A Laboratory Manual. CSHL. New York. 1999. ISBN 0-87969-544-7) The determination that a particular agent binds substantially only to Bcl-x_L-DTR protem may readih be made bv using or adapting routine procedures One suitable in vitro assay makes use of the Western blotting procedure (descnbed in many standard texts, including Harlow and Lane. 1999) Western blotting may be used to determine that a given protein binding agent such as an anti- Bcl-x_L-DTR monoclonal antibody, binds substantially only to the Bcl-x_L-DTR protem

Alternately, because the disclosed apoptosis-modifymg proteins are fusion proteins, thev can be detected using antibodies to one or the protein domains used in their construction For instance, fusions containing BC1-X_L can be detected using the monoclonal antibody 2H12 (Hsu and Youle. J. Biol Chem 272 13829-13834. 1997. now available from Neo Markers. Union City, CA clone #2H121 -3) or other professionally available antibody preparations, for instance, polyclonal antι-Bcl-x_L/x_s #06-851 from Upstate Biotechnology, Lake Placid. NY. polyclonal rabbit antι-Bcl-x_L #65189E from PharMmgen, San Diego. CA. and rabbit polyclonal ( B22630-050/B22630-150) or mouse monoclonal (B61220- 050/B61220- 150) antι-Bcl-x_L from Transduction Laboratories. Lexington. KY) Antibodies that recognize diphtheria toxm are. for instance, available from the Centers for Disease Control. Atlanta, GA Shorter fragments of antibodies can also serve as specific binding agents For instance FAbs. Fvs. and single-chain Fvs (SCFvs) that bind to Bcl-x_L-DTR would be Bcl-x -DTR-specific binding agents

Target cell binding affinity: The physical interaction between a target cell and an apoptosis-modifλmg fusion protem as disclosed in this invention can be examined by λ'anous methods Alternatively, the ability of fusion protem to compete for binding to its target cell with either native targeting domain or antibody that recognizes the targeting domain binding site on the target cell can be measured This allows the calculation of relative binding affinities through standard techniques

Therapeutically effective amount of an apoptosis-modifying fusion protein: A quantity of apoptosis-modifymg fusion protem sufficient to achieve a desired effect m a subject being treated For mstance. this can be the amount necessary to measurably inhibit or enhance apoptosis in a target cell An effective amount of apoptosis-modifymg fusion protem may be administered in a single dose, or in several doses, for example daily, dunng a course of treatment However, the effective amount of fusion protem will be dependent on the fusion protem applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the fusion protem For example, a therapeutically effective amount of fusion protem can vary from about 0 01 mg/kg body weight to about 1 g/kg body weight

The fusion proteins disclosed in the present invention have equal application m medical and veterinary settings Therefore, the general term "subject being treated' is understood to include all animals (e g., humans, apes. dogs. cats, horses, and cows), and particularly mammals, that are or may suffer from a chronic or acute condition or injury that causes apoptosis or a lack thereof, susceptible to modification using molecules of the current invention

Transformed: A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, hpofection. and particle gun acceleration

Transgenic cell: A transgemc cell is one that has been transformed with a recombmant nucleic acid molecule Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an ongm of replication A vector may also include one or more selectable marker genes and other genetic elements known in the art

II. Construction, Expression, and Purification of

Apoptosis-Modifying Fusion Proteins.

A. Selection of component domains.

This invention provides generally an apoptosis-modifymg fusion protem that binds to a target cell, translocates across or otherwise integrates into the membrane(s) of the target cell, and modifies an apoptotic response of the target cell As such, any target cell which it is desirous to modify (either inhibit or enhance) apoptosis is an appropriate target for a bispecific fusion protem The choice of appropriate protem binding domain for incorporation into the disclosed apoptosis-modifymg fusion protem will be dictated by the target cell or cell population chosen Examples of targeting domains include, for instance, nontoxic cell binding domains or components of bactenal toxms (such as diphtheria toxm or anthrax toxm). growth factors (such as epidermal growth factor), monoclonal antibodies, cytokmes. and so forth, as well as targeting competent variants and fragments thereol

The choice of appropnate Bcl-2 family member-derived apoptosis-modifymg domain will depend on the manner m which the target cell s response to apoptosis is to be modified Where apoptosis is to be inhibited bv the resultant fusion protein, anti-death members of the Bcl-2 protein family are appropriate sources for apoptosis-modifymg domains One such fusion protem is BC1-X - DTR. which employs the long form of Bcl-x. Bcl-x_L. as the apoptosis-modifymg domain Alternately, where enhancement of apoptosis is desired pro-death members of the Bcl-2 family of proteins will be appropnate For instance. Bad-DTTR employs the pro-death protem Bad as its apoptosis-modifymg domain

Translocation of the apoptosis-modifymg fusion protem into the target cell is important A translocation domain may be included m the fusion protem as a separate, third domain This could be supplied from a third protem. unrelated to the cell-binding and apoptosis-modifymg domains, or be a translocation domain of one of these proteins (e.g.. the diphthena toxm translocation (DTT) domain used m Bad-DTTR) The DTT domain contains several hydrophobic and amphipathic alpha helices and. after insertion into cell membranes, creates voltage dependent ion channels (Kagan et al . Proc NatlAcadSci USA 78 4950-4954, 1981. Donovan et al, Proc NatlAcadSci USA 78 172-176. 1981)

Alternately, the translocation function can be provided through the use of a cell-binding domain or apoptosis-modifymg domain that confers the additional functionality' of membrane translocation or integration This is true m Bcl-x_L-DTR. wherein Bcl-x_L provides both the apoptosis- modifymg ability and translocation into the cell

B. Assembh

The construction of fusion proteins from domains of known proteins is w ell known. In general, a nucleic acid molecule that encodes the desired prote domains are jomed using standard genetic engineering techniques to create a single, operably linked fusion oligonucleotide Appropnate molecular biological techniques may be found in Sambrook et al, In. Molecular Cloning: A Laboratory Manual, Cold Spnng Harbor. New York. 1989 Specific examples of genetically engineered multi-domain proteins, including those joined by vanous linkers, can be found m the following patent documents

U S Patent No 5.834.209 to Korsmeyer U S Patent No 5.821.082 to Chinnadurai.

U S Patent No 5.696.237 to FitzGerald et al .

U S Patent No 5.668.255 to Murphy.

U S Patent No 5.587.455 to Berger e/ α/..

WO 98/17682 to Korsmeyer, and WO 98/12328 to Home et al.

It will usually be convenient to generate vanous control molecules for comparison to an apoptosis-modifymg fusion protem. m order to measure the specificity of the apoptosis modification provided by each fusion protem Appropnate control molecules may include one or more of the native proteins used in construction of the fusion, or fragments or mutants thereof

C. Expression

One skilled m the art will understand that there are mynad ways to express a recombmant protem such that it can subsequently be purified In general, an expression vector carrying the nucleic acid sequence that encodes the desired protem will be transformed into a microorganism for expression Such microorganisms can be prokaryotic (bacteria) or eukaryotic (e g . } east) One appropnate species of bactena is Escherichia coli (__. coh). which has been used extensively as a laboratorv expenmental expression system A eukaryotic expression system will be preferred where the protein of interest requires eukarvote-specific post-translational modifications such as glycosvlation Also, protem can be expressed using a viral (e g . vaccinia) based expression SΛ stem

Protem can also be expressed in animal cell tissue culture, and such a system will be appropriate where animal-specific protem modifications are desirable or required in the recombmant protem The expression vector can include a sequence encoding a synthesis targeting peptide positioned m such a way as to be fused to the coding sequence of the apoptosis-modifvmg fusion protem This allows the apoptosis-modifymg fusion protein to be targeted to specific sub-cellular or extra-cellular locations Vanous appropnate prokarvotic and eukaryotic targeting peptides, and nucleic acid molecules encodmg such, are well known to one of ordinary skill in the art In a prokaπotic expression system a signal sequence can be used to secrete the newly synthesized protein In a eukaryotic expression

stem, the targeting peptide would specify targeting of the hybrid protem to one or more specific sub-cellular compartments, or to be secreted from the cell, depending on which peptide is chosen Through the use of a eukaryotic secretion signal sequence, the apoptosis-modifymg fusion protem can be expressed in a transgenic animal (for instance a cow. pig. or sheep) in such a manner that the protem is secreted into the milk of the animal

Vectors suitable for stable transformation of culturable cells are also well known Typically, such vectors include a multiple-clonmg site suitable for inserting a cloned nucleic acid molecule, such that it y\ ill be under the franscnptional control of 5' and 3' regulatory sequences In addition, transformation vectors include one or more selectable markers, for bacterial transformation this is often an antibiotic resistance gene Such transformation y ectors typically also contain a promoter regulatory region (e g . a regulatory region controlling mducible or constitutive expression), a transcnption initiation start site, a nbosome binding site, an RNA processing signal, and a transcnption termination site, each functionally arranged in relation to the multiple-clonmg site For production of large amounts of recombinant proteins an mducible promoter is preferred This permits selective production of the recombmant protem. and alloyvs both higher levels of production than constitutive promoters, and enables the production of recombinant proteins that mav be toxic to the expressing cell if expressed constitutively

In addition to these general guidelines, protem expression/purification kits are produced commercially See. for instance, the QIAexpress™ expression system from QIAGEN (Chatsyy orth. CA) and vanous expression systems provided by INVITROGEN (Carlsbad. CA) Depending on the details provided by the manufactures, such kits can be used for production and purification of the disclosed apoptosis-modifymg fusion proteins D. Purification

One skilled m the art will understand that there are myriad ways to punfy recombinant polypeptides, and such typical methods of protem purification may be used to purity the disclosed apoptosis-modifymg fusion proteins Such methods include, for instance, protein chromatographic methods including ion exchange, gel filtration, HPLC. monoclonal antibody affinity chromatography and isolation of insoluble protem inclusion bodies after over production In addition purification affinity-tags, for instance a six-histidme sequence, may be recombinantly fused to the protem and used to facilitate polypeptide purification A specific proteolytic site, for instance a thrombm-specific digestion site, can be engineered into the protem between the tag and the fusion itself to facilitate removal of the tag after punfication

Commercially produced protein expression/purification kits provide tailored protocols for the purification of proteins made using each system See. for instance the QIAexpress™ expression system from QIAGEN (Chatsworth CA) and various expression svstems provided bv INVITROGEN (Carlsbad. CA) Where a commercial kit is employed to produce a bispecific fusion protem the manufacturer s purification protocol is a preferred protocol for purification of that protem For instance, proteins expressed with an ammo-termmal hexa-histidine tag can be purified by binding to nickel-nitrilotnacetic acid (Ni-NTA) metal affinity chromatography matnx (The QIAexpressiomst QIAGEN, 1997)

Alternately, the binding specificities of the cell-bmdmg/targetmg domain of the disclosed apoptosis-modifymg protem may be exploited to facilitate specific punfication of the proteins A preferred method of performing such specific punfication yvould be column chromatography using column resin to which the target cell surface receptor or an appropriate epitope or fragment or domain of the target molecule, has been attached

If the apoptosis-modifymg fusion protem is produced in a secreted form, e g secreted into the milk of a transgenic animal purification will be from the secreted fluid Alternately, purification may be unnecessary if it is appropnate to apply the fusion protem directly to the subject in the secreted fluid

UI. Variation of a Bispecific Fusion Protein A. Sequence Variants

The binding and apoptosis-modifymg charactenstics of the apoptosis-modifymg fusion proteins disclosed herein lies not in the precise ammo acid sequence but rather in the three- dimensional structure inherent m the ammo acid sequences encoded by the DNA sequences It is possible to recreate the functional characteristics of any of these proteins or protem domains of this invention by recreating the three-dimensional structuie without necessanly recreating the exact ammo acid sequence This can be achieved by designing a nucleic acid sequence that encodes for the three- dimensional structure, but yvhich differs, for instance by reason of the redundancy of the genetic code Similarly. the DNA sequence mav also be varied, while still producing a functional apoptosis- modifymg fusion protein

Variant apoptosis-modifymg fusion proteins include proteins that differ in ammo acid sequence from the disclosed sequence but that share structurally significant sequence homology with any of the provided proteins Vanation can occur m any single domain of the fusion protein (e.g.. the binding or apoptosis-modifymg domain, or. yvhere appropriate, the linker) Variation can also occur in more than one of such domains in any particular variant protem Such variants may be produced by manipulating the nucleotide sequence of, for instance, a Bcl-x_L-encodmg sequence, using standard procedures, such as site-directed mutagenesis or PCR The simplest modifications involve the substitution of one or more ammo acids for ammo acids having similar biochemical properties These so-called conservative substitutions are likely to have minimal impact on the activity of the resultant prote , especially when made outside of the binding site or active site of the respective domain The regions or sub-domains of DTR that are essential to targeted cell binding are known m the art (see. Choe et al.. Nature 357 216-222, 1992. Parker and Pattus. TIBS 18 391-395 1993) Regions or sub- domains of Bcl-2 proteins responsible for apoptosis modification are under intense study, much of this work is reviewed m Adams and Cory, Science 281 1322-1326

Table 1 shows ammo acids that may be substituted for an ongmal ammo acid m a protem, and which are regarded as conservative substitutions

Table 1 Original Residue Conservative Substitutions

Ala ser

Arg lys Asn gin. his

Asp glu

Cys ser

Gin asn

Leu lie. val

Phe met. leu. tyr

Ser thr

Thr ser

More substantial changes in protem structure may be obtained bv selecting ammo acid substitutions that are less conservative than those listed in Table 1 Such changes include changing residues that differ more significantly in their effect on maintaining polypeptide backbone structure (e.g.. sheet or helical conformation) near the substitution, charge or hydrophobicity of the molecule at the target site, or bulk of a specific side chain The following substitutions are generally expected to produce the greatest changes m protem properties (a) a hydrophihc residue (e.g.. servl or threonvl) is substituted for (or by) a hydrophobic residue (e g.. leucyl. isoleucyl. phenvlala l. valyl or alanyl). (b) a cysteme or prolme is substituted for (or by) any other residue, (c) a residue having an electropositive side chain (e.g.. lysyl. argmyl. or histadyl) is substituted for (or by) an electronegative residue (e.g.. glutarml or aspartyl). or (d) a residue having a bulky side chain (e.g., phenylalanme) is substituted for (or by) one lacking a side chain (e.g . glycme)

Vanant binding domain, apoptosis-modifymg domam. or fusion protem-encodmg sequences may be produced by standard DNA mutagenesis techniques, for example. Ml 3 primer mutagenesis Details of these techniques are provided m Sambrook et a!., In Molecular Cloning A Laboratory Manual. Cold Spring Harbor. Neyv York. 1989. Ch 15 By the use of such techniques, variants may be created which differ in minor ways from the apoptosis-modifymg fusion protem-encodmg sequences disclosed DNA molecules and nucleotide sequences yvhich are derivatives of those specifically disclosed herein and that differ from those disclosed bv the deletion, addition, or substitution of nucleotides while still encoding a protem that binds to a target cell, translocates or otherwise integrates into the target cell membrane(s). and thereby modifies an apoptotic response m the target cell, are comprehended by this invention In their most simple form, such vanants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced

Alternatively, the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protem having an ammo acid sequence substantially similar to the disclosed fusion sequences For example, the 57th ammo acid residue of the Bcl-x_L-DTR protem is alamne The nucleotide codon tnplet GCC encodes this alamne residue Because of the degeneracy of the genetic code, three other nucleotide codon tnplets - (GCG. GCT and GCA) - also code for alamne Thus, the nucleotide sequence of the disclosed Bcl-x_L-DTR encodmg sequence could be changed at this position to any of these three alternative codons without affecting the ammo acid composition or charactenstics of the encoded protem Based upon the degeneracy of the genetic code, vanant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as descnbed above, or by synthesis of DNA sequences Thus, this invention also encompasses nucleic acid sequences which encode an apoptosis-modifvmg fusion protem. but which vary from the disclosed nucleic acid sequences bv virtue of the degeneracy of the genetic code Apoptosis assays, including those discussed herein, can be used to determine the ability of the resultant variant protem to modify apoptosis B. Peptide Modifications

The present invention includes biologically active molecules that mimic the action of the apoptosis-modifymg fusion proteins of the present invention, and specifically modify apoptosis in a target cell The proteins of the invention include synthetic versions of naturally -occurring proteins described herein, as well as analogues (non-peptide organic molecules), derivatives (chemically functionahzed protem molecules obtained starting y ith the disclosed peptide sequences) and y anants (homologs) of these proteins that specifically bind to a chosen target cell and modify apoptosis in that target cell Each protem of the invention is comprised of a sequence of ammo acids, which mav be either L- and/or D- ammo acids, naturally occurnng and otherwise

Proteins may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified proteins and optionally having other desirable properties For example, carboxvhc acid groups of the protem, whether carboxvl-termmal or side chain, may be provided m the form of a salt of a pharmaceutically-acceptable cation or estenfied to form a -Cι₆ ester, or converted to an amide of formula NRιR₂ wherein Rj and R₂ are each independently H or Cι-C_]6 alkyl. or combined to form a heterocychc nng. such as a 5- or 6- membered ring Amino groups of the protem. whether ammo-termmal or side chain, may be m the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic. toluene sulfomc maleic tartaric and other organic salts, or mav be modified to C]-C_{] 6} alkyl or dialkyl ammo or further converted to an amide

Hydroxyl groups of the prote side chains may be converted to C]-C]₆ alkoxy or to a Cι-C_{] 6} ester using well-recognized techniques Phenyl and phenolic rings of the protem side chains mav be substituted with one or more halogen atoms, such as fluonne. chlorine, bromine or iodine, or yyith Ci- C]₆ alk\l. Cι-C_]6 alkoxv, carboxyhc acids and esters thereof, or amides of such carboxyhc acids Methvlene groups of the protein side chains can be extended to homologous C -C alkylenes Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups Those skilled m the art will also recognize methods for introducing cyclic structures into the proteins of this invention to select and provide conformational constraints to the structure that result in enhanced stability Peptidomimetic and organomimetic embodiments are also withm the scope of the present invention, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the protem backbone and component ammo acid side chains in the apoptosis-modify mg fusion protein, resulting in such peptido- and organomimetics of the proteins of this invention hay mg measurable or enhanced neutralizing ability For computer modeling applications, a pharmacophore is an idealized, three-dimensional defimtion of the structural requirements for biological activity Peptido- and organomimetics can be designed to fit each pharmacophore with cunent computer modeling software (using computer assisted drug design or CADD) See Walters, Computer- Assisted Modeling of Drugs, in Klegerman & Groves (eds ) Pharmaceutical Biotechnology. Interpharm Press Buffalo Grove, IL. 165-174. 1993. and Munson (ed ) Principles of Pharmacology, Ch 102, 1995. for descnptions of techniques used in CADD Also included withm the scope of the invention are mimetics prepared using such techniques that produce apoptosis-modifymg fusion proteins

IV. Activity of Fusion Proteins

Because the apoptosis modifying fusion proteins provided m this invention are at least bi- functional. having one domain required for cell targeting and another for modification of apoptosis in the target cell, there are at least two activities for each fusion protem These include the affinity of the fusion protein for a specific target cell, class of target cells, tissue type, etc , (the binding ability), and the ability of the targeted fusion to effect apoptosis in the targeted cell (the apoptosis-modifymg ability) Various techniques can be used to measure each of these activities

A. Fusion protein affinity for target cells Fusion protem affinity for the target cell, or to a specific cell surface protem. can be determined using vanous techniques known in the art One common method is a competitive binding assay (Greenfield et al . Science 238 536-539. 1987) In a competitive binding assay, radiolabeled receptor binding protem. or a denvative or fragment thereof, is exposed to the target native cell in the presence of one or varying concentrations of cold fusion protem and other competitive proteins being assayed The amount of bound, labeled binding protem can be measured through standard techniques to determine the relative cell-binding affinity of the fusion

B. Apoptosis inhibition or enhancement

Several in vitro systems are used to study the process of apoptosis These include growth factor deprivation in culture, treatment of cells with staurosponne (a non-specific protem kmase inhibitor), application of γ-radiation. and infection by viruses Apoptosis as stimulated bv anv signal can be examined or measured in a variety of ways Detection of morphological indicia of apoptosis (e.g., membrane blebbing. chromatin condensation and fragmentation, and formation of apoptotic bodies) can provide qualitative information More quantitative techniques include TUNEL staining, measurement of DNA laddenng, measurement of known caspase substrate degradation (e g . PARP. Taylor et al . J Neurochem 68 1598-605. 1997) and counting dying cells, which have become susceptible to dye uptake Many companies (e g.. Trevigen. Gaithersburg MD. and R&D Systems Minneapolis MN) also supply kits useful for the measurement of apoptosis by vanous methods, many of these kits can be used to measure the effect of disclosed apoptosis-modifymg fusion proteins on apoptosis in a variety of cell types By way of example the following techniques can be used to measure the modification of apoptosis caused m a target cell after it is contacted with an apoptosis-modifymg fusion protein of the present invention TUNEL staining: Terminal end-labelmg of broken DNA fragments with labeled nucleotides. the reaction is catalyzed bv terminal nucleotide transferase (TdT) Vanous kits are available for measurement of TUNEL staining, including the TdT in situ TUNEL-based Kit (R&D Systems. Minneapolis. MN) Measurement of Caspase Activity: Another common system for measunng the amount of apoptosis occurring in an in vitro cell system is to measure the poly-ADP πbose Polymerase (PARP) cleavage after treatment of the cells with various stimulators of apoptosis PARP is a known substrate for a caspase (CPP-32) involved m the apoptotic kmase cascade This technique can be carried out using essentially the folloyvmg protocol HeLa cells are plated m growth media (e g . EMEM containing 10% FBS at 2 x 10 cells/ml) and treated with one or more concentrations of an apoptosis- modifymg fusion protem according to the current invention The appropriate concentration for each fusion protem will depend on various factors, including the fusion protem in question, target cell, and apoptosis stimulator emploved Appropriate concentrations may include for instance, about 0 5 μM to about 3 μM final It mav be beneficial to treat the target cells multiple times with the fusion protein. usually after a penod of incubation ranging from one to several hours For instance, cells can be exposed to the fusion protem a second time about fifteen hours after the ongmal treatment Usually the same concentratιon(s) of fusion protem is used in the second treatment

Apoptosis is induced immediately the last treatment of the target cells with apoptosis modifying fusion protem The method of application of the apoptosis stimulus amount applied, appropriate incubation time with the mducer. etc will be specific to the type of apoptosis induction used (e g , staurosponne γ-radiation. vmons. caspase inhibitor, etc ) Such details are in general well known to those of ordinary skill in the art After an appropnate incubation period, cell lvsates are prepared from the treated target cells, and aliquots loaded onto SDS-PAGE for analysis The resultant gels can be examined using anv of vanous well-known techniques, for instance by performing a Western analysis lmmunoblotted with anti-PARP polyclonal antibody (Boehnnger Mannheim GmbH. Germany), developed with enhanced chemilummescence

Known inhibitors of apoptotic pathways, for instance caspase inhibitors, can be used to compare the effectiveness of apoptosis-modifymg fusion proteins of this invention Appropnate inhibitors include viral caspase inhibitors like crmA and baculovirus p35. and peptide-ly pe caspase inhibitors including zVAD-frnk. YVAD- and DEVD-type inhibitors See Rubm. British Pled Bulk . 53 617-631, 1997

V. Incorporation of Apoptosis-Modifying Fusion

Proteins into Pharmaceutical Compositions

Pharmaceutical compositions that comprise at least one apoptosis modifying fusion protein as descnbed herein as an actiy e ingredient will normally be formulated yvith an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen The pharmaceutically acceptable carriers and excipients useful m this invention are conventional For instance, parenteral formulations usually compnse mjectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological salme. other balanced salt solutions, aqueous dextrose, glvcerol or the like Excipients that can be included are. for instance, other proteins, such as human serum albumin or plasma preparations If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffenng agents and the like, for example sodium acetate or sorbitan monolaurate

One or more other medicinal and pharmaceutical agents, for instance chemotherapeutic. anti- inflammatory, anti-viral or antibiotic agents, also may be included The dosage form of the pharmaceutical composition will be determined bv the mode of administration chosen For instance, in addition to mjectable fluids, topical and oral formulations can be employed Topical preparations can include eye drops, ointments, sprays and the like Oral formulations may be liquid (e g . syrups solutions or suspensions), or solid (e g.. powders, pills, tablets, or capsules) For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of manmtol. lactose, starch, or magnesium stearate Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art

The pharmaceutical compositions that compnse apoptosis modifying fusion protem will preferably be formulated m unit dosage form, suitable for individual administration of precise dosages One possible unit dosage contains approximately 100 μg of protein The amount of active compound administered will be dependent on the subject being treated, the seventy of the affliction, and the manner of administration, and is best left to the judgment of the prescnbmg clinician Withm these bounds, the formulation to be administered will contain a quantity of the active component(s) in an amount effective to achieve the desired effect in the subject being treated Ideally, a sufficient amount of the protein is administered to achieve tissue a concentration at the site of action that is at least as great as in vitro concentrations that have been shown to be effective

VI. Clinical Use of Apoptosis-Modifying Fusion

Proteins

The targeted apoptosis-regulatmg activity exhibited bv the disclosed fusion proteins makes these fusions useful for treating neurodegenerative diseases, transient lschemic injuries, and unregulated cell growth (as mav for instance be found in tumors and various cancers

The apoptosis-modifymg fusion proteins of this invention may be administered to humans, or other animals on whose cells they are effective, m various manners such as topically, orally, intravenously, intramuscularly, lntrapeπtoneally. mtranasallv. lntradermally . mtrathecally. and subcutaneously Administration of apoptosis-modifymg fusion protem composition is indicated for patients with a neurodegenerative disease, suffering from stroke episodes or transient lschemic injury, or experiencing uncontrolled or unwanted cell growth, such as malignancies or neoplasms More generally, treatment is appropriate for any condition in

it y ould be beneficial to alter (either lnhibit or enhance) an apoptotic response of a subject's target cells The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g.. the patient, the disease, and the disease-state involved) By way of example, when apoptosis is being generally inhibited over the short term, for instance after transient lschemic neuronal injury, it mav be advantageous to administer relatively large doses of fusion protein repeatedly for a few days In contrast, if apoptosis is being enhanced m specific cell types, for instance in hyper-proliferative cells, it may be of greater benefit to apply a relatively small dose of fusion prote repeatedly, e.g., daily, weekly . or monthly, over a much longer period of treatment

In addition to their individual use. apoptosis-modifymg fusion proteins as disclosed in the current invention may be combined with vanous therapeutic agents For instance, an apoptosis- enhancmg fusion protem such as Bad-DTTR may be combined with or used in association with other chemotherapeutic or chemopreventive agents for providing therapy against neoplasms or other hyper- pro ferative cellular growth conditions Vanous such anti-cancer agents are well knoyvn to those of ordinary skill in the art Apoptosis-modifymg fusion proteins according to this invention also can be supplied in the form of kits, the construction of kits appropriate for fherapeuticallv actiy e proteins known

EXAMPLE 1 Construction of functional apoptosis-modifying fusion proteins A. Bcl-x_L-DTR

The human Bcl-x_L gene from codon 1 through 233 (provided bv Dr Craig Thompson) and the diphthena toxm gene from codon 384 through 535 (receptor binding domain. DTR). containmg mutations in codons 508 and 525, were amplified by PCR so that the DT mutation at codon 525 yvas mutated to the wild-type by the PCR pnmer The two PCR products, Bcl-x_Ll -233 and DT384-535 (DTR) were digested with Ndel / Notl and Notl / Xhol restriction enzymes, respectively Bcl-x_L vas fused to the 5' end of the DTR gene with a linker (GCG TAT TCT GCG GCC GCG. SEQ ID NO 5) to encode for Ala Tyr Ser Ala Ala Ala (SEQ ID NO 6) between the two peptide domains The two digested fragments were ligated into the prokaryotic expression vector pET16b (Novagen. Inc . Madison. WI) cut with Ndel and Xhol (FIG 1 A) The codon 508 of DTR was mutated to the v ld-type form (Phe → Ser) and the first three nucleotides (CAT) of Ndel were deleted by double-stranded, site- directed mutagenesis FIG 1 A shows a schematic representation of the resultant apoptosis-modifymg fusion protem. Bcl-x_L-DTR

As controls, human Bcl-x_L (codons 1-233) and DTR (codons 384-535 of DT) genes y ere separately subcloned into pETl 6b vectors through Ndel and Xhol sites The histidme tag and Factor Xa digestion site sequences from the expression vector yy ere upstream of Bcl-x_L. DTR and Bcl-x_L- DTR coding sequences All three expression constructs \y ere venfied by sequencing

For expression m eukaryotic cells. Bcl-x_L-DTR and Bcl-x_L gene constructs yvere inserted m the eukaryotic vector pcDNA3 (Invitrogen. Carlsbad. CA) and the constructs verified by sequencing B. Bad-DTTR

The full-length mouse Bad gene with two Ser → Ala mutations at codons 112 and 136 (Schendel et al . Proc Natl Acad Set. USA 94 5113-5118 1997). and the diphthena toxm gene from codons 194 through 535 (translocation and receptor-bmdmg domains, DTTR without the catalytic domain) were amplified by PCR The two PCR products. Bad and DT194-535 (DTTR). were used as templates to directly fuse the Bad gene to the 5' end of DTTR gene by a second round of PCR The Bad- DTTR gene fragment was digested with Ndel and Xhol and hgated into the prokaryotic expression vector pETlόb (Novagen, Inc . Madison. WI) digested with Ndel and Xhol The histidme tag and Factor Xa digestion site sequences from the expression vector were upstream of the Bad-DTTR coding sequence The expression construct was verified by sequencing

EXAMPLE 2 Expression and Purification of functional apoptosis-modifying fusion proteins A. Prokaryotic Expression To produce proteins for extracellular addition to cells, the BC1-X gene, the DTR domain gene and the Bcl-x_L-DTR fusion gene were cloned into pET 16b E colt BL21 (DE3) strain yy as used to express Bcl-x_L-DTR. Bad-DTTR, Bcl-x_L and DTR. with addition of ImM IPTG when the OD260 reached 0 5-0 7 After two hours incubation and lysis bv French press the inclusion bodies were collected and dissolved in 6M guamdme-HCl B. Eukaryotic Expression

Transfection of HeLa cells with the fusion constructs was performed as reported previously (Wolter et al , J Cell Biol 139 1281 - 1292. 1997) HeLa cells were harvested and lysed in 1 ml buffer containing 100 μg / ml leupeptm 20 hours after transfection centnfuged to remove cell debris, and 15 μl aliquots of the supernatant loaded onto 10-20% SDS-PAGE The plasmid encoded proteins were visualized by lmmunoblotting with anti-Bcl-X_L monoclonal antibody (2H12. Trevigen Gaithersburg. MD) and developed using enhanced chemilummescence (Amersham Inc . Arlington Heights. IL) Results are shown in FIG IB

C. Purification

Histidme tag binding resm (Novagen. Inc . Madison. WI) was used to purify Bcl-x -DTR. Bad-DTTR. Bcl-x_L. and DTR Proteins were refolded by dialysis against, or dilution into. 100 mM Tns- Acetate (pH 8 0) / 0 5 M argimne. concentrated w ith PEG 15.000-20.000 and dialvzed against PBS This yielded protem purified to greater than 90% homogeneity The four proteins were subjected to 10-20% SDS-PAGE, visualized bv immunoblottmg with either antι-Bcl-x_L monoclonal (2H12) or horse anti-DT polyclonal antibodies (Centeis for Disease Control Atlanta. GA) and developed as above They were of the expected molecular yveight on SDS PAGE and of the expected lmmunoreactivity to antibodies against Bcl-x_L or DT on Western blots EXAMPLE 3

Assays for measuring fusion protein binding to, and translocation into, target cells

A. Competitive Binding Assay Protem binding to the diphtheria toxm receptor was performed as previously reported

(Greenfield et al . Science 238 536-539, 1987) with the following modifications DT was radiolabeled yvith I¹²⁵ using lodobeads (Pierce Chem Co . Rockford. IL) as described by the manufacturer Cos-7 cells, grown to confluency m 12 well costar plates yy ere analyzed for receptor binding and competition by incubation for three hours on ice Results are reported in FIG 2 Cold competitor proteins, native DT (Δ). Bcl-x_L-DTR (A). Bcl-x_L (O), and DTR (•). were used to displace I¹²³ labeled DT tracer

Native DT and Bcl-x_L-DTR compete for DT receptor binding in the nanomolar concentration range DT and the Bcl-x_L-DTR fusion protem competed for I¹²⁵-DT binding to its receptor to a similar extent although the affinity of the fusion was three times lower than that of native DT (FIG 2) Neither the Bcl-x_L domain alone nor the DTR domain alone w as able to compete for DT receptor binding The more complete protem (Bcl-x_L-DTR). where Bcl-x_L is substituted for the DT translocation domain, folded such that DT receptor binding activity was retained whereas the isolated binding domain (DTR) did not Addition of the DT A chain domain to the N-termmus of Bcl-x_L-DTR further increased the affinity of the chimera to the DT receptor B. Assays for effective transport of the fusion protein into the target cell

Diphthena toxm is endocvtosed by cells and reaches low pH intracellular compartments The low pH triggers a conformational change in the translocation domain which alloyvs this domain to insert into membranes and form channels The toxicity of DT is blocked by lvsosomotropic agents such as chloroqume. which mcrease the pH of intracellular compartments Chloroqume at a concentration that blocks diphthena toxm toxicity ( 10 μM) did not block the activity of Bcl-x_L-DTR to inhibit pohovirus-mduced cell death Thus the mechanism of membrane interaction of Bcl-x_L-DTR differs to some extent from that of DT However, brefeld A. an inhibitor of y esicle traffic between the ER and the Golgi apparatus (Lippmcott- Schwartz et al Cell 67 601 -616 1991 Hunziker et al . Cell 67 617-627, 1991) does block the anti-apoptosis activity of Bcl-x_L-DTR (Table 3) These results indicate that Bcl-x_L-DTR must be endocytosed and suggest that Bcl-x_L-DTR must reach the Golgi apparatus or the ER to prevent cell death The subcellular location from

native Bcl-2 family members regulate apoptosis is currently under scrutiny (Hunziker et al Cell 67 617-627 1991) Several intracellular membrane locations including the ER. appear able to mediate Bcl-2 family regulation of cell death (Krajewski et al CancerRes 53 4701-4714. 1993) Bcl-x_L-DTR mav reach the ER to translocate into the cell cvtosol or perhaps Bcl-x_L-DTR. when bound close to a membrane, can insert into that membrane and inhibit apoptosis m the membrane-intercalated form EXAMPLE 4 Measurement of Bcl-x -DTR apoptosis-inhibiting activity

A. Apoptosis inhibition after transient cell transfection To demonstrate that Bcl-x_L-DTR is effective at inhibiting apoptosis when expressed from withm the target cell, this construct and the control construct containing Bcl-x_L were transiently transfected into HeLa cells Assay of apoptosis inhibition after transient transfection was performed as reported previously (Wolter et al , J Cell Biol 139 1281 -1292 1997) The Bcl-x_L-DTR fusion gene blocked apoptosis after transient transfection into HeLa cells (FIG 1 C) to an extent similar to that of the Bcl-x_L gene after C-termmal tail truncation (Wolter et al , J Cell Biol 139 1281-1292. 1997)

B. Inhibition of STS-induced apoptosis by extracellular treatment with Bcl-x_L-DTR

Hoechst dve no 33342 staining The effectiveness of extracellular delivery of Bcl-x or the

Bcl-x_L-DTR fusion protem for inhibiting the rate of cell death by apoptosis was examined as follows Cos-7 cells at 3 x 10⁴ cells/cm² in 100 μl DMEM with 10% FBS yvere incubated with 0 1 μM STS (O), 0 1 μM STS plus 4 8 μM Bcl-x_L-DTR protem added to the medium (Δ) or 20 μl of PBS (D) Apoptotic cells were quantified by staining with Hoechst dye no 33342 Results in FIG 3A are presented as the average number of cells per field (magnification 160 x) For each point, at least 5 fields were counted in each of at least 3 wells Bcl-x_L-DTR dramatically decreased the rate of apoptosis in Cos-7 cells Six different preparations of Bcl-x_L-DTR yvere found to have activity and the apoptosis prevention activity was stable for at least 5 months when Bcl-x_L-DTR was stored at 4 ^CC Addition of Bcl-x_L-DTR minutes before the addition of STS blocked more than 70% of Cos-7 cell death after 6 hours and more than 50% of cell death after 12 hours of STS exposure (FIG 3A)

Jurkat. HeLa and U251 cells were also protected from STS-mduced apoptosis by Bcl-x_L- DTR (Table 2) BC1-X protem added to Cos-7 cells, however, did not alter the extent of cell death induced by STS A nontoxic DT mutant able to bind the DT receptor CRMl 97, also had no effect on apoptosis induced by STS To further test the role of DT receptor binding m apoptosis inhibition, cells expressing DT receptors yy ere compared with cells lacking DT receptors Mouse and rat cells are thousands of times less sensitive to DT than human or monkey cell lines due to a lack of the DT receptor (Pappenheimer The Harvey Lectures 76 45-73. 1982) Companng human, monkey, mouse and rat cell lines revealed that those cells lacking the DT receptor. WEHI-7 1 and 9L. yvere insensitive to apoptosis protection by Bcl-x_L-DTR (Table 2) The sensitivity of the six cell lines to DT toxicity', thought to reflect DT receptor levels, correlated with sensitivity to apoptosis prevention by Bcl-x_L- DTR (Table 2) The magnitude of apoptosis inhibition by extracellular Bcl-x_L-DTR (FIG 3 A. Table 2) was similar to that found by transfection of the fusion gene into cells (FIG 1 C) Although fusion to the C- termmus of Bcl-x_L inhibited bioactivity relative to native Bcl-x_L after transfection (FIG 1 C). a very substantial prevention of cell death was obtained at both the gene level and the protem level (FIG 3 A) Thus the delivery of Bcl-x_L-DTR is efficient and apoptosis can be prevented bv dehverv of BC1-X_L from the outside of cells

Measurement of caspase activity To confirm the results of cell death measurements by

Hoechst staining and trypan blue dye exclusion, y e examined caspase-mduced cleavage of poly-ADP ribose polymerase (PARP) HeLa cells were plated in EMEM containing 10% FBS at 2 x 10⁵ cells/ml and treated with two different preparations of Bcl-x_L-DTR at 1 48 μM or 1 μM Fifteen hours later. cells were treated again with Bcl-x_L-DTR at 1 48 μM or 1 μM Immediately after the second treatment. 0 8 μM STS was added Three hours later, cell lysates were made and aliquots were loaded onto SDS-PAGE. immunoblotted with anti-PARP polvclonal antibody (Boehnnger Mannheim GmbH, Germany) and developed with enhanced chemilumnescence Lane a contains control HeLa cells not incubated with STS (unmduced cells), Lane b. HeLa cells treated with STS plus 1 μM Bcl-x_L-DTR protem. Lane c. HeLa cells treated with STS plus 1 48 μM Bcl-x_L-DTR protein, and Lane d. HeLa cells treated with STS and no fusion protem HeLa cells incubated with Bcl-x_L-DTR showed significantly less cleavage of PARP after apoptosis induction with STS (FIG 3B) C. Inhibition of γ-radiation-induced apoptosis by extracellular treatment with Bcl-x_L-DTR

Radiation is a potent mducer of apoptosis in many hematopoetic cell types The ability of

BCI-X_L-DTR to prevent radiation-mduced apoptosis yy as examined in the human T cell line. Jurkat

When added to the media (serum-free RPMI-1640 medium with insulin and transfernn) of Jurkat cells plated at 10³ cells/ml a few minutes prior to induction of apoptosis by 10 gray γ-radiation. Bcl-x_L-DTR

(4 63 μM) blocked almost half of the ensuing cell death (FIG 4A) Apoptotic cells were counted using

Hoechst dve no 33342 Control cells were not irradiated and not treated with Bcl-x_L-DTR

In a clonogenic assay measuring long term survival. Jurkat cells showed more than a 3 -fold greater survival when Bcl-x_L-DTR yvas added to the media immediately prior to 5 gray γ-radiation D. Inhibition of anti-Fas-induced apoptosis by extracellular treatment with Bcl-x_L-DTR

Jurkat cells are also sensitive to apoptosis induced by antibody binding to the Fas/APO-

1/CD95 receptor The Fas pathway of apoptosis is one of the few pathways shoyvn to be less sensitive or insensitive to apoptosis protection by Bcl-2 and Bcl-x_L (Boise & Thompson Proc Natl. Acad Sci USA 94 3759-3764, 1997. Memon et al . J Immunol 155 4644-4652, 1995) and contrasts with radiation- mduced apoptosis in this regard Jurkat cells yvere plated at 10⁵ cells/ml in serum-free RPMI-1640 medium with insulin and transfernn. and treated with 100 ng/ml anti-Fas antibody (CHI 1. Upstate Biotechnology, Lake Placid. NY) minutes after addition of Bcl-x_L-DTR to a concentration 4 68 μM Control cells were treated with PBS and no anti-Fas antibody Fas antigen-mduced apoptosis (measured by counting dying cells using Hoechst dye no 33342) showed very little inhibition by Bcl- x_L-DTR. although there was a statistically significant decrease in apoptosis between 2 and 4 hours m some expenments (FIG 4B) The degree of protection of different apoptosis pathways by extracellular Bcl-x_L-DTR corresponded with that seen by transfection yvith the Bcl-x_L gene E. Inhibition of poliovirus-induced apoptosis by extracellular treatment with Bcl-x_L-DTR

Viruses mduce a powerful apoptosis response in certain cells and prevention of this apoptosis may have therapeutic utility (Hardwick, Adv Pharm 41 295-336, 1997) Pohovirus-mduced apoptosis of HeLa cells was also examined for sensitivity to exfracellular BCI-X_L-DTR, a system where inhibition of cell death by transfection with the Bcl-x_L gene has been demonstrated (Castel et al , J Exp Med 186 967-972, 1997) Addmg Bcl-x_L-DTR 30 mmutes after infection of cells with low titers (MOI of 1 pfu / cell) of poliovirus (FIG 5) or with moderatelv high titers (MOI of 20 pfu / cell) of poliovirus prevented more than half of the cell death for up to 24 hours Addition of extracellular Bcl- X_L or the DTR domain proteins alone had no affect on pohovirus-mduced apoptosis

F. Competition of apoptosis inhibition

Caspase inhibitors block many pathways of apoptosis and are bemg explored for pharmacologic potential to inhibit cell death (Chen et al , Nature 385 434-439 1997) zVAD-fmk and Boc-D-fink are powerful, broad specificity caspase inhibitors that block many apoptosis pathways

(Henkaτt, Immunity 4 195-201, 1996) Apoptosis inhibition activity of zVAD-fmk and Boc-D-fmk was compared with that of Bcl-x_L-DTR HeLa cells were plated at a density of 1 x 10⁵ cells/well in EMEM containing 10% FBS and antibiotics, infected with poliovirus at an MOI of 1 pfu/cell as reported previously (Castelh et αl , J Exp Med 186 967-972, 1997) and immediately treated with negative control peptide zFA-fmk at 20 μM, Bcl-x_L-DTR at 0 48 μM or peptides zVAD-fink or Boc- D-fmk at 20 μM Cell viability was assessed by trypan blue dve exclusion 24 hours following addition of virus zFA-fmk, zVAD-fmk and Boc-D-fink were from Enzyme Systems Products Dublm, CA

BCI-X_L-DTR at 0 48 μM blocked cell death to a greater extent than either zVAD-fmk or Boc- D-fink at 20 μM (FIG 5) Bcl-x_L-DTR showed a strong inhibition of a potent and pathologically important apoptosis pathway Interestingly, Bcl-x appears to act at an early step in the cell death pathway when intervention can permit long term viability of cells whereas caspase inhibitors appear to work relatively more downstream in the apoptosis pathway (Chmnaiyan et αl J Biol Chem 271 4573- 4576, 1996, Xiang e. α/ , Proc Nαtl Acαd Set USA 93 14559-14563, 1996 Miller et αl J Cell Biol 139 205-217, 1997)

EXAMPLE 5

Measurement of Bad-DTTR apoptosis-enhancing activity

A. Stimulation of apoptosis by extracellular treatment with Bad-DTTR To determine the effectiveness of the fusion protem Bad-DTTR at triggering apoptosis cell survival after exposure to Bad-DTTR was examined U251 MG cells at 3 x 10⁴ cells/cm" m 100 μl DMEM with 10% FBS were incubated with 0 65 μM Bad-DTTR protem added to the medium or 20 μl ofPBS Total and apoptotic cells were quantified by staining with Hoechst dye no 33342 Results are presented in FIG 6 as the average number of cells per field (magnification 160 x) Bad-DTTR decreases cell viability 12 hours after treatment

B. Enhancement of STS-triggered apoptosis by extracellular treatment with Bad-DTTR To examine the abihtv of Bad-DTTR to enhance apoptosis tnggered by STS, cell survival was determined after exposure to vanous concentrations of STS, m combination with vanous combmations of Bad-DTTR U251 MG cells at 3 x 10⁴ cells/cm² m 100 μl DMEM with 10% FBS were treated with PBS, 0 lμM STS, 0 65 μM Bad-DTTR 0 065 μM Bad-DTTR, 0 lμM STS plus 0 65 μM Bad-DTTR and 0 1 μM STS plus 0 065 μM Bad-DTTR Apoptotic death cells were quantified at different times b} staining with Hoechst dve no 33342 Results are presented as the average number of cells per field (magnification 160 x) Apoptosis is most enhanced when cells are freated with 0 lμM STS plus 0 65 μM Bad-DTTR, and cells begin to die about 12 hours after treatment

U251 MG cells at 3 x 10⁴ cells/cm² in 100 μl DMEM with 10% FBS were treated with PBS, 1 μM STS 0 65 μM Bad-DTTR 0 065 μM Bad-DTTR 1 μM STS plus 0 65 μM Bad-DTTR and 1 μM STS plus 0 065 μM Bad-DTTR Apoptotic cells were quantified and presented as above The combination of 1 μM STS and Bad-DTTR at vanous concentrations causes an earlier onset of apoptosis in U251 MG cells

EXAMPLE 6

LF--BC1-X_L Inhibits Neuron, Macrophage, and Lymphocyte Apoptosis

Anthrax toxm includes three components lethal factor (LF), edema factor (EF) and protective antigen (PA) (Leppla Anthrax toxin In Handbook of Natural Toxms, Moss et al , Eds , Dekker, New York, Vol 8, pp 543-572, 1995) PA bmds simultaneously to LF and to a cell surface receptor existing on the cells of almost all species mcludmg rodents (Leppla, 1995, Fnedlander, J Biol Chem 261 7123-7126, 1986) and transports LF mto cells where LF causes toxic effects PA alone, however, is not toxic It has been found that the first 255 residues (LF_n) of LF which constitute the PA-bmdmg domam and are not toxic to cells are sufficient for delivery of heterologous peptides to the cytosol Cytotoxms have been fused to LF_n (Leppla 1995, Arora et al ,JBιol Chem 269 26165- 26171 1994 Milne et al , Mol Microbwl 15 661-666 1995) Administration of a fusion protem containing LF_n and the gp 120 envelope glycoprotem of HIV- 1 along with PA to antigen-presenting cells sensitized them to cytolysis by cytotoxic T-lvmphocvtes (CTL) specific to gpl20 (Goletz et al , Proc NatlAcad Set USA 94 12059-12064 1997) In vivo LF_n-fused to CTL epitopes injected along with PA has been shown to stimulate a CTL response agamst the antigens in mice (Ballard et al Proc Natl Acad Sci USA 93 12531-12534, 1996 Ballard et al Infect Immun 66 615-619, 1998, Ballard et al Infect Immun 66 4696-4699, 1998 Doling er a/ Infect Immun 67 3290-3296, 1999) To inhibit neuron apoptosis, another protem delivery system was engineered bv fusing a nontoxic domam of anthrax toxm to BC1-X_L, to create the LF_Π-BC1-X_L chimenc fusion protem Macrophage and lymphocyte death in culture, and neuron death in vivo in a retinal ganglion cell model of apoptosis mduced by axotomy. can be prevented by application of this fusion protem A. Construction of LF_n-Bcl-x_L in a prokaryotic expression plasmid

The codmg sequence for lethal factor (LF) from codons 34 to 288 (LF-} (Bragg et al , Gene

81 45-54, 1989), which is the amino-terrmnal domain (residues 1-255) of mature LF (Leppla. 1995), was amplified us g PCR with the template of pET15b/LF_n (Milne et al , Mol Microbiol 15 661-666, 1995) The gene of human Bcl-x_L from Codons 1 to 209 (Bcl-x_L(l -209)) (Boise et al . Cell 74 597- 608, 1993) was amplified by PCR Then the LF_n encoding sequence was fused to the 5 end of Bcl- x_L(l -209) encoding sequence by a second round of PCR A stop codon was introduced immediately after Codon 209 of Bcl-x_L The fused DNA fragment, LF_n-Bcl-x_L, was cut with Ndel and Xho I. and inserted into prokaryotic expression vector pET 15b cut with Nde I and Xho I (FIG 8) A histidme tag and thrombin cleavage site were linked to the N-termmal of LF_n-Bcl-x_L Similarly, the Bcl-x_L gene from codons 1 to 209 was also genetically inserted into pETl 5b at the sites of Nde I and Xho I All the constructs were venfied by DNA sequencing

B. Construction of eukaryotic expression plasmids, transfection, Western blotting and biologic activity assay

The sequences encoding LF_Π-BC1-X_L, BC1-X from codons 1 to 209, and full-length BC1-X_L, were separately engmeered mto eukaryotic expression vector pcDNA3 1 + and venfied by DNA sequencmg Cos-7 cells were co-transfected with plasmid EGFP-C3 and one of the three plasmids as reported (Keith et al., J Cell Biol 139 1281-1292, 1997) The cells were treated with 0 1 μM staurosponne (STS) 12 hours later The dead and living cells were counted with Hoechst 33342 at different times after STS treatment (Liu et al . Proc NatlAcadSci USA 96 9563-9567. 1999. Keith et al , J Cell Biol 139 1281-1292. 1997) The cells were harvested and lysed 20 hours after transfection, and aliquots were loaded onto SDS/10-20% PAGE gels The plasmid-encoded protems were visualized by immunoblottmg with antι-Bcl-x Ab (Trevigen, Gaithersburg, MD) and developed by us g enhanced chemiluminescence (Amersham Pharmacia)

C. Protein expression, purification, SDS-PAGE and Western blotting

The protems LF_n, LF_n-Bcl-x_L and Bcl-x_L from codons 1 to 209 were individually expressed in

E colt BL21 (DE3) (Novagen. Inc ) and punfied with a Hιs*Tag bmdmg punfication kit (Novagen. Inc ) The transformed BL21 (DE3) was cultured at 37°C in LB medium until the OD600 reached 0 5- 0 8, and treated with lmM IPTG. and then cultured for 3 more hours The cells was pelleted, suspended in lx Hιs^«Tag bmdmg buffer with 1 mM phenvlmethylsulfonyl fluonde (PMSF), 1 mM aprotirun and 1 mM leupeptm. and disrupted with French Press The cytosol was separated from cell debns and undisrupted cells by centnfugation at 20.000 x g for 30 minutes and loaded on the His'Tag bmdmg column The eluted proteins were dialyzed against 1 x PBS and stenhzed with 0 22-um filter Protective antigen (PA) was punfied as reported (Milne et al. Mol Microbiol 15 661-666, 1995) The protems were run on SDS-PAGE gels, and stained with Coomassie Blue or visualized by immunoblottmg with antι-Bcl-x_L antibody, and developed as above

D. J744 macrophage-like cell culture, treatment and apoptosis assay

J744 macrophage-like cells at 10⁵/ ml were placed in 96-well plates ( 100 μl per well), and cultured overnight in RPMI 1640 with 10% FCS The cells were treated with PBS, 0 1 μM staurosponne alone or 0 1 μM staurosponne along with the different combinations of the protems LF_n- Bcl-x_L (28 μg/ml). PA (33 μg/ml), LF_n (28 μg/ml) and Bcl-x_L (28 μg/ml) The apoptotic and living cells were counted with Hoechst dye no 33342 as reported (Liu et al . Proc NatlAcadSci USA 96 9563-9567. 1999)

E. Optic nerve section and intra-ocular protein injection

The P0 pups of Fisher 344 rat strain were used for the present study P0 is defined as the day of birth The mtracramal lesion of unilateral optic nerve was performed as reported (Rabachi et al , J Neurosci 14 5292-301, 1994) Bnefly, a P0 pup was anesthetized by hypothermia Under a dissectmg microscope, an incision over the nght eye was cut and a piece of bone flipped up The nght optic nerve was sectioned after suctioning the overlying cerebral cortex The section site of optic nerve is about 3 mm away from the eyeball A piece of gelfoam was put m the hole, and the flipped bone replaced, and the incision repaired with SUPERGLUE™ Immediately after the operation, seven, ten and four mice were respectively treated with administration of PBS, LF_n-Bcl-x_L (0 65 μg) plus PA (0 35 μg) and PA (0 35 μg) in a volume of 350 nanoliters (nl) per eye through ora serrata mto the posterior chamber of the nght eyes by using a micro-mjector with a pulled microprpette The pups were warmed up with a light lamp until the recovery, and then sent back to the mother Four pups from the same litters, which were not operated and not treated were used for normal control

F. Histology

About 24 hours after sectioning of the optic nerve, the nght eyes were removed under deep anesthesia with sodium pentobarbital, fixed in 4% paraformaldehyde for approximately 30 hours, embedded in paraffin and cut at 6 μm The eyes taken from the normal pups in the same litters were processed in the same way to serve as confrols The sections were rehydrated. stained with 0 2% cresyl violet, dehydrated, and mounted with DPX mountant The number of pyknotic cells and the number of living cells were counted by the use of 40 x objective in the entire retinal ganglion cell layer of three sections per retma The pyknotic cells were identified as reported (Rabachi et al . J Neurosci 14 5292-301, 1994) The values were presented as the percentage of pyknotic cells versus total cells per retma (FIG 12)

G. Results

The PA protein from the Anthrax bacillus binds cell receptors and can mediate the delivery of the anthrax LF protem to the cell cytosol where LF effects toxicity to cells The N-termmal domain of LF binds to PA When exogeneous peptides are fused to the N-termmal domain of LF (LF-), they can be delivered to the cell cytosol by PA Deletion of the C-termmal region of LF prevents toxicity to cells To deliver Bcl-x_L to cells, the N-termmal 255 ammo acids of LF were fused to Bcl-x_L without mcludmg the C-terminal 24 hydrophobic ammo acids of Bcl-x . as shown schematically in FIG 8 The nucleotide and ammo acid sequences of the fusion protem, LF_n-Bcl-x_L, are shown in SEQ ID NOs 7 and 8 The fusion protem was expressed m E coh and punfied to near homogeneity

The bioactivity of the LF_n-Bcl-x_L was explored in J774 cells in tissue culture LF_n-Bcl-x_L, at 28 micrograms per ml plus PA at 33 micrograms per ml was added to the media of cells at the time of apoptosis induction with 0 1 μM staurosponne (STS) Cells treated with staurosponne alone died by apoptosis over the following 36 hours as shown in FIG 9 When the cells were treated with LF_n-Bcl-x_L plus PA, most of the cell death was inhibited

Controls were performed to explore the requirements for apoptosis inhibition FIG 10 shows data demonstrating that J774 cells treated with LF_n alone, Bcl-x alone, LF_n-Bcl-x_L without PA, and PA without LF_n-Bcl-X were not protected from apoptosis induced by staurosponne, whereas LF_n-Bcl- x_L plus PA prevented more than half of the cell death Jurkat cells yvere also protected from apoptosis by LF_n-Bcl-x_L plus PA (FIG 11 )

This new strategy to block cell death was explored in an in vivo model of neuron apoptosis Retinal ganglion cells were axotomized and immediately afterwards a mixture containing 0 35 μg of PA and 0 65 μg of LF_n-Bcl-x_L was mjected into the eye Control mice were either not axotomized, axotomized and injected with PBS, or axotomized and injected with PA alone Mice were sacnficed 24 hours later, and the eyes exammed histologically An increase m pyknotic cells, I e , apoptotic cells (Rabachi et al , JNeurosci 14 5292-301, 1994), occurs in the ganglion layer 24 hours after axotomy However, when eyes are mjected with LF_n-Bcl-x_L and PA, much of the cell death is inhibited PA alone did not prevent cell death To quantitate the extent of cell death the number of living and pyknotic cells in three entire ganglion layers in one eye from each of 4-10 mice was counted The quantified results are shown in FIG 12 LF_n-Bcl-x_L inhibited more than half of the cell death due to neuron axotomy in vivo

In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention, and should not be taken as limitations on its scope Rather the scope of the invention is defined by the following claims We therefore claim as our invention all that comes within the scope and spmt of these claims Table 2 Inhibition of Apoptosis by Bcl-x -DTR

Cell line Apoptosis Concentration of Time of STS Apoptosis DT mducer Bcl-x_L-DTR Treatment Prevention IOso CM)

(μM) (Hrs) (%*)

Cos-7 0 1 μM STS 4 8 12 58 4 10 ¹²- 10 ⁿ

(monkey kidney)

U251 0 1 μM STS 4 68 16 57 5 ιo ¹²- io-^H

(human ghoma)

HeLa 0 2 μM STS 2 17 10 32 4 ιo ^I2- io-ⁿ

(human cerucal Ca)

Jurkat 0 1 μM STS 4 68 12 21 2 10 ⁹

(human T leukemia)

9L 0 lμM STS 4 68 12 -5 4 > 10

(rat ghosarcoma)

WEH7 1 0 lμM STS 4 68 12 0 5 > 10⁷

(mouse T lymphoma)

*Apoptotιc cells were counted with Hoechst dye no 33342 and the percent prevention from apoptosis was calculated as 1 - (number of apoptotic cells with STS and BCI-XL-DTR - number of apoptotic cells without STS and BCI-X -DTR) / (number of apoptotic cells with STS - number of apoptotic cells without STS and BCI-X_L-DTR) except for the non- adherent Jurkat and WEHI7 1 cells which were counted by trypan blue dye exclusion and % apoptosis prevention calculated as (number of living cells with STS and BCI-XL-DTR - number of living cells with STS) / (number of living cells without STS and Bcl-x_L-DTR)

Table 3

Brefeldin A prevents Bcl-x_L-DTR blockade of apoptosis

Apoptotic cells were counted with Hoechst dye no 33342 14 hours after addition of STS and / or brefeldm A minutes after BCI-XL-DTR was added to Cos-7 cells The protection percentage was calculated as 1 - (number of apoptotic cells with STS and BCI-XL-DTR - number of apoptotic cells without STS and Bel- X -DTR) / (number of apoptotic cells with STS - number of apoptotic cells without STS and BCI-XL-DTR)

Claims

We claim

1 A functional apoptosis-modifymg fusion protem capable of bmdmg a target cell compnsmg (a) a first domam capable of modifying apoptosis in the target cell, and

(b) a second domam capable of specifically targeting the fusion protein to the target cell, wherein the fusion protein integrates into or otherwise crosses a cellular membrane of the target cell upon bindmg

2 The fusion protem of claim 1 wherein the first domain is capable of inducing or enhancing apoptosis

3 The fusion protem of claim 1 , wherem the first domam is capable of inhibiting or reducmg apoptosis

4 The functional punfied apoptosis-modifymg fusion protem of claim 1. compnsmg an ammo acid sequence selected from the group consisting of (a) the ammo acid sequence shown in SEQ ID NO 2

(b) the ammo acid sequence shown in SEQ ID NO 4,

(c) the ammo acid sequence shown in SEQ ID NO 8, and

(d) ammo acid sequences that differ from those specified in (a), (b), or (c) by one or more conservative ammo acid substitutions, but which retain targeting and apoptosis-modifymg abilities 5 An isolated nucleic acid molecule encodmg a protem according to claim 4

6 The isolated nucleic acid molecule of claim 5 wherem the molecule compnses a sequence selected from the group consistmg of

(a) SEQ ID NO 1 , (b) SEQ TD NO 3, (c) SEQ ID NO 7, and

(d) nucleic acid sequences having at least 90% sequence identity to the sequences specified m (a), (b). or (c)

7 A recombinant nucleic acid molecule compnsmg a promoter sequence operably linked to a nucleic acid sequence accordmg to claim 5 8 A transgenic cell compnsmg a recombmant nucleic acid molecule accordmg to claim 7

9 The transgenic cell of claim 8 wherem the cell is a bactena, a yeast, an algae, a plant, or an animal cell

10 The functional apoptosis-modifving fusion protem of claim 1 further compnsmg (c) a linker connecting the first domain to the second domain

11 The protem of claim 1. wherem the first domain is a Bcl-2 family protem, or a vanant or fragment thereof that retains an apoptosis-modifving property

12 The protem of claim 11 , wherem the first domain is pro-apoptotic 13 The protem of claim 1 1 wherem the first domain is anti-apoptotic

14 The protein of claim 11 , wherein the first domain is Bcl-x_L. or a vanant or fragment thereof that inhibits apoptosis m the target cell to which the protem is exposed

15 The protem of claim 14, wherem the first domain consists essentially of BC1-X_L 16 The protem of claim 11. wherem the first domam is Bad, or a vanant or fragment thereof that enhances apoptosis in the target cell to which the protem is exposed

17 The protem of claim 16, wherem the first domain is a vanant of Bad having an ammo acid other than senne at ammo acid position 112 and/or position 136

18 The protem of claim 16, wherem the first domain consists essentially of Bad 19 The protem of claim 14, wherein the target cell is a neuron, a lymphocyte, a macrophage, an epithelial cell, or a stem cell

20 The protein of claim 17. wherem the target cell is a tumor cell, a cancer cell, a neoplasm cell, a hyper-proliferative cell, or an adipocyte

21 The protein of claim 1 , wherem the second domain compnses a receptor-bmdmg domain denved from a bactenal toxm, a monoclonal antibody, a growth factor, or a cytoktne

22 The protem of claim 21 , wherem the second bmdmg domain compnses a receptor- bmdmg domain denved from diphthena toxm or anthrax tox

23 The protem of claim 21 wherem the second bmdmg domam compnses a receptor- binding domain denved from epidermal growth factor 24 The protem of claim 21. wherein the receptor-bmdmg domam compnses diphthena toxm receptor bmdmg domain, or a vanant or fragment thereof that targets the fusion protein to the target cell to which the protem is exposed

25 The protein of claun 21 , wherem the second domain further compnses a translocation domain of diphthena toxm 26 An isolated nucleic acid molecule encoding a fusion protem accordmg to claim 1

27 The protem of claim 10, wherem the linker is 5 - 100 ammo acid residues m length

28 The protem of claim 10, wherem the linker compnses the ammo acid sequence shown in SEQ ID NO 6

29 The protem of claim 22, wherem the linker consists essentially of the ammo acid sequence shown m SEQ ID NO 6

30 The functional apoptosis-modifymg fusion protem of claim 1 compnsmg

(a) Bcl-x_L,

(b) a bactenal toxm receptor bmdmg domain, and

(c) a peptide linker of about 6 ammo acids in length, functionally linking (a) to (b)

31 The fusion protem of claim 30, wherem (b) is a diphthena toxm or anthrax toxm receptor bmdmg domain 32 The fusion protem of claim 30, which does not include a functional diphthena toxm translocation domam

33 The fusion of claim 31. wherein (b) compnses LF_n

34 The fusion protem of claim 32. consisting essentially of (a) Bcl-x ,

(b) a diphthena toxm receptor bmdmg domain, and

(c) a peptide linker of about 6 ammo acids in length, functionally linking (a) to (b)

35 The protem of claim 30, wherem the linker has an ammo acid sequence shown in SEQ ID NO 6

36 An isolated nucleic acid molecule encodmg a protem accordmg to claim 30

37 The nucleic acid molecule of claim 36, wherem the nucleic acid sequence is represented by SEQ ID NO 1

38 The functional apoptosis-modifymg fusion protem of claim 1. compnsmg (a) Bad,

(b) a diphthena toxm translocation domain, and

(c) a bactenal toxm receptor binding domam, wherein (a), (b), and (c) are functionally linked

39 The fusion protem of claim 38. wherem (c) is a diphtheria toxm or anthrax toxm receptor bmdmg domam

40 An isolated nucleic acid molecule encodmg a protem according to claim 38

41 The nucleic acid molecule of claun 40, where the nucleic acid sequence is shown m SEQ ID NO 3

42 A method for producmg in a cell a functional apoptosis-modifymg fusion protein capable of binding a target cell, compnsmg the steps of

(a) transfect g a cell with an isolated recombinant nucleic acid molecule of claim 25 to produce a transgenic cell,

(b) cultunng the fransgemc cell under conditions that cause production of the protem. and (c) recovering the protein produced by the fransgemc cell

43 The method of claim 42, wherem the cell is a eukaryotic cell

44 The method of claim 43, wherem the eukaryotic cell is a mammalian cell

45 The method of claim 42, wherem recovering the protem compnses a) identifying the protein by the presence of a molecular tag. and b) separating the protem having the molecular tag so identified from molecules without the tag. so as to recover the protem produced by the cultured transgenic cell

46 A composition compnsmg the protem according to claim 1. or an analog or mimetic thereof 47 A pharmaceutical composition compnsmg the composition according to claim 46 and a pharmaceutically acceptable earner

48 A combined pharmaceutical composition compnsmg a fusion protem accordmg to claim 33. and a sufficient amount PA to enable measurable fransport of the fusion protein into a target cell

49 A method for modifying apoptosis in a target cell, compnsmg the step of contacting the target cell with an amount of the protem of claim 1 sufficient to modify apoptosis in the target cell

50 The method of claim 49, wherem the protem is administered in the form of a pharmaceutical composition

51 The method of claim 49, further compnsmg the step of co-admmistenng an agent selected from the group consistmg of a chemotherapeutic agent, an anti-inflammatoiy agent, an antiviral agent, and an antibiotic agent

52 The method of claim 49, wherem apoptosis in the target cell is inhibited 53 The method of claim 49. where apoptosis in the target cell is enhanced

54 A method for inhibiting apoptosis m a target cell, compnsmg the step of contacting the target cell with an amount of the protem of claim 14, sufficient to inhibit apoptosis

55 A method for enhancing apoptosis in a target cell, compnsmg the step of contacting the target cell with an amount of the protem of claim 17, sufficient to enhance apoptosis

56 A method of reducing apoptosis in a subject after transient lschemic neuronal injury, compnsmg administering to the subject a therapeutically effective amount of a protem of claim 14

57 The method of claim 56, wherem the transient lschemic neuronal injury is a sp al cord injury

58 The method of claim 56, wherem the protem is administered in the form of a pharmaceutical composition

59 The method of claim 56, further compnsmg the step of co-admmistenng an agent selected from the group consistmg of a chemotherapeutic agent, an anti-inflammatory agent, an anti- viral agent, and an antibiotic agent

60 A protem analog, denvative, or mimetic of the protem of claim 1

61 The protem of any one of claims 1-4 for use in modifying apoptosis a target cell