New approaches to targeting bioactive compounds

Journal of ControlledRelease, 28 ( 1994) 15-35 0 1994 Elsevier Science B.V. All rights reserved

I5 01683659/94/$07.00 SSDr0168-3659(93)E0124-X

COREL 00939

New approaches to targeting bioactive compounds* Alexander

V. Kabanov,

Valery Yu. Alakhov

MoscowInstitute ofBiotechnology Inc., Department ofBiopolymer Chemistry and Department of Drug Delivery Systems, Russian Research Center of Molecular Diagnostics and Therapy, Moscow, Russia (Accepted 24 August 1993)

The paper reviews some of the new approaches to the targeting of bioactive compounds. In particular, the results of artificially rendering macromolecules hydrophobic by nonpolar substituents are considered. This principle enabled us to develop a method for suppressing virus reproduction in cells with fatty acylated antiviral antibodies and also to enhance antisense activity of short complementary oligonucleotides. Some ideas for creation of drug-targeting systems based on self-assembling supramolecular complexes have been tested. In this respect, a possibility of using interpolyelectrolyte complexes of nucleic acids with polycations for targeting genetic material into cells is discussed. Within the limits of this concept, a new class of highly selective immunotoxins (‘respecrins’ ) representing supramolecular protein complexes in which toxin molecules are reversibly masked by antibodies to specific antigens of target cells has been developed. Key words: Antisence Immunotoxins

oligonucleotide;

Antiviral;

Introduction The general problem of drug targeting consists of at least three basic subproblems. They are the following: lhow to ensure the most effective interaction of drugs with target cells, including their proper binding on cell membranes and intracellular transport; lhow to effectively deliver drugs towards certain target cells avoiding unfavourable drug Correspondence to: A.V. Kabanov or V.Yu. Alakhov, MIB Inc., North American Branch, 550, Rue Sherbrooke Ouest, Bureau 1205, Montreal, Quebec, H3A lB9 Canada. *Paper presented at the 6th International Symposium on Recent Advances in Drug Delivery Systems.

Drug delivery

system;

Genetic

transformation;

distribution in the organism and their disintegration on the way to the targets; lhow to avoid nonspecific nontarget cells.

action of drugs on

As a rule, researchers come across not one but a combination of these problems while developing certain drug delivery systems. In particular, fascinating tasks of antisense and gene therapy are restricted first of all by the problem of genetic material penetration into cells. Some relatively successful efforts to solve this problem have been made during the past few years. However, the solutions found remain only of academic interest unless they take into account the other problems listed above, in particular, targeted delivery of genetic material avoiding its disintegration in body fluids. Therefore, despite some progress

16

achieved in this field, no reliable tools for oligonucleotide and gene targeting have been yet developed. It often happens that solution of one of the above mentioned problems entails drastic complication of the others. Immunotoxin studies provide many good examples to this assertion. One of the main disadvantages of immunotoxins (as of many other anticancer agents) is their relatively high nonspecific toxicity. To decrease the latter, toxin fragments are used as toxic components of immunotoxins. Such constructions do not affect nontarget cells. However, they are also much less effective with respect to their targets because the toxin fragments are devoid of receptor-recognizing sites and are practically incapable of cell uptake. Meanwhile, it is perfectly well known that natural systems (such as viruses, for example) easily cope with these difficulties during their life cycles. In the course of evolution, viruses have ‘learned’ how to safely reach their target cell, bind to it and penetrate inside. There also exist many other natural molecular complexes that successfully manage to fullil the functions of drug delivery systems. The approaches to bioactive compound targeting described below exploit some features characteristic of natural objects (for reviews see also [ 1,2] ). In particular, the natural mechanism of protein anchoring on membranes, namely, its fatty acid acylation, has been used to enhance biopolymer binding and uptake into cells. This approach permitted significant increase of antiviral activity of antibodies and was then successfully applied to target antisense oligonucleotides into cells. Another approach reviewed in this paper is based on the use of self-assembling supramolecular complexes for drug targeting. It is illustrated by an example of DNA interpolyelectrolyte complexes (IPECs) with linear polycations that serve as a tool for cell transformation and gene targeted delivery. Such complexes mimic some basic features and functions of natural viruses. Possibilities of designing highly selective im-

munotoxins of the new generation (‘respecrins’ ) are also analysed. Respecrins represent supramolecular protein complexes in which the toxic component is reversibly masked by antibodies to the target cell. They are selectively activated only upon interaction with the markers of target cells. A similar structural and functional organization is characteristic of supramolecular biopolymer complexes functioning in living cells.

Fatty acylated antibodies Effect of artificial fatty acylation binding and uptake

on protein

Posttranslational modification of proteins with lipids, in particular fatty acid acylation, has been discovered over the past decade in yeast, plant and animal cells and viruses [ 3-7 1. Numerous studies have demonstrated that such modilication facilitates protein insertion in a membrane and plays a most important role in realization of their intracellular activity. Recently, this natural way of protein hydrophobization was artificially applied to enhance protein interaction with lipid and cell membranes (for review see [ 2,8 ] ). To introduce a lipid moiety in a protein molecule it is necessary to treat the latter with a waterinsoluble reagent. However, the attempts to carry out such reactions in aqueous media often meet with serious obstacles [ 2 1. Under these conditions, the reaction is uncontrollable and it appears to be very difficult to produce a protein with a low degree of modification [ 9 1. To overcome these difficulties, the system of reversed micelles (RM) of surfactant in organic solvent (Aerosol OT in octane) was used as a reaction medium for modification [ 10,111. In this system, the protein molecule is entrapped in the RM inner cavity, thus acquiring a cover of hydrated surfactant molecules (Fig. 1). The reagent becomes dissolved in the bulk phase, however it can also incorporate in the micelle and thus come into contact with the solubilized protein. After modification, the protein can be precipitated from the reaction system by addition of acetone or extraction into aqueous phase.

17 in water

A

in organic solvent

B

Fig. I. Chemical modification of a protein (P) with water insoluble reagent in water (A) and in organic solvent (B). In water, the reaction is uncontrollable: the reagent forms emulsion, the reaction proceeds on the reagent drop surface and cannot be stopped when the protein is modified to a low degree. These diffkulties can be bypassed using a microheterogeneous reversed micelle system as a reaction medium [ 11.

Proteins were effectively modified in such systems with fatty acids [ 111, lipids [ 121, hormones [ 131, fluorescent dyes (our unpublished data), organometallics [ 141 and radioactive labels [ 15 1. These reactions are characterized by high yields resulting from good solubility of modifying reagents in the RM system, their low hydrolysis rate (observed, for example, in the cases of chloranhydrides [ 91 or activated esters [ 111) and their concentration in the micelle interface in the vicinity of the modified protein group. Contrary to modification in regular aqueous solution, the proposed procedure produces products homogeneous by modification degree. The modification degree is strictly controlled by changing the [reagent ] / [protein] ratio in the system. The simplicity of preparation of RM reaction systems and protein recovery from them makes this method very convenient. A number of fatty acylated proteins (enzymes, toxins, antibodies) which retained their specific biological activity after modification were artificially produced by this method. The modified preparations contained from one to live fatty residues per one protein molecule and were soluble in aqueous solution in the absence of detergents. The effect of fatty acylation on protein interaction with various mammalian cells was studied using horse radish peroxidase (HRP) and

monoclonal antibodies (Mabs) to HRP as examples [ 16 1, These in vitro experiments were focused on HRP and antibody binding and endocytosis. These studies proved that fatty acylation of proteins significantly enhanced their binding with mammalian cells (Jurkat T-lymphoma, Chinese hamster ovarian (CHO ) , Mardine Darby canine kidney (MDCK), X63 myeloma and HepG2 hepatoma cells). This result is in good agreement with the data reported by Peacock et al. [ 17,181 who studied interaction of fatty acylated antibodies with mammalian cells. Alongside enhancement of protein adsorption on the plasma membrane, fatty acylation enhances protein internalization (endocytosis) into a cell (Table 1). While studying fatty acylated HRP, we found that the internalized protein was mainly located in endocytic vesicles and did not penetrate into the cytoplasm (at least in the amounts, detectable under experimental conditionsused) [ 161. The efficiency of fatty acylated protein binding is cell line dependent [ 161. This phenomenon is very difficult to interpret. In particular, it may be related to differences in the plasma membrane state (lipid composition, microviscosity, etc.) or to the presence of proteins (receptors), capable of specific binding of fatty residues. For instance, the most efficient binding of fatty acylated HRP was observed in the case of HepG2 cells, which can be probably explained by the presence in hepatoma of a membraneous fatty acid binding protein [ 19 1. This protein is known to play an important role in hepatocellular fatty acid uptake [ 19 1, and it cannot be excluded that it provides for efficient intemalization of fatty acylated HRP, also observed in the case of HepG2 cells [ 16 1. Protein binding depends on chemical composition of fatty acid residues. In particular, the study of Mabs revealed that palmitoylation led to more efficient antibody binding than stearoylation [ 161. Further analysis of processes underlying this phenomenon may be of considerable importance for the understanding of differences in the behaviour (e.g., cellular localization) of natural degree fatty acylated proteins [ 5 1.

18 TABLE 1 Effect of HRP stearoylation

on its binding and endocytosis

in CHO cells” (from Ref. [ 16 ] )

Cell-bound HRP ( 1O4molecules per cell) Nonmodified

Total binding Adsorbed on the cell surface Internalized

Effect of fatty acylationb

Stearoylated

4°C

37°C

4°C

37°C

4°C

37°C

0.15

0.9

4.8

5.7

32

6.3

0.15 0

0.15 0.75

4.8 0

2.1 3.6

32

14.5 4.8

“The HRP (0.5 pM) was incubated with cells ( IO6 cells/ml) at 4 or 37°C for 3.0 h in the absence of serum. Cells were then washed, lysed and the HRP activity was determined in the cell lysate. The amount of internalized HRP was determined after removal of the enzyme adsorbed on cell surface by proteinase K. The amount of adsorbed HRP equals to the difference of the amounts corresponding to the enzyme total binding and internalization. The modified HRP contains 1.Ok 0.1 stearic acid residues per one protein molecule. bRatio of the amounts of stearoylated and nonmodified HRP.

Suppression of virus reproduction with fatty acylated antiviral antibodies It is well known that antiviral antibodies may influence cell infection with a virus. In some cases, antibodies stimulate infection [ 20-23 1, for instance, by promoting virus uptake by cells via Fc-receptor mediated pathway [ 20,2 1 ] or through complement elements [ 231. In other cases they, on the contrary, neutralize viruses, inhibiting either their binding with and penetration into cells or post-penetrational steps of the infection [ 24-271. Meanwhile much less is known about the effect of antibodies on the development of infection in already infected cells. Moreover the above cited data on virus neutralization [ 24-271 were obtained using antibodies to virus surface proteins. However, the neutralizing effect of antibodies to internal virus antigens has not been described. The last two phenomena (virus inhibition in infected cells and antiviral effect of antibodies to internal antigens ) were recently observed during the study of fatty acylated antibodies to influenza antigens [ 28-3 11. The effect of antibodies to HA and M1 proteins on virus reproduction in MDCK cells was investigated. The antibodies were introduced at distinct stages of the virus

replication cycle: either 60-O min prior the infection [ 311 or several hours after it [ 28-3 1 ] (Fig. 2). Nonmodified antibodies did not practically affect the virus infection in both types of experiments. (The exception was the case of antibodies to HA which neutralized the virus when added before the infection). Meanwhile, stearoylated antibodies displayed a reliable antiviral activity which, however, depended on antibody specificity (Fig. 2 ) . In particular, polyclonal antibodies to HA interfered with the infection both at the early [ 3 1 ] and late [ 291 stages of the replication cycle. At that stage, the neutralizing activity of fatty acylated antibodies to HA was significantly higher than that of nonmodified ones. A similar effect was observed when Mabs to HA (clone C102) were used. Mabs to M, protein (clone 2E5Cl) were found to inhibit the infection only when they were added to the cells simultaneously with the virus [ 3 11. The effect of antibodies to HA at the early infection stages is consistent with the general concept of antibody neutralizing action. According to this concept [ 25 1, antibodies to envelope proteins bind on the virus surface thus sterically preventing virus adsorption and uptake (Fig. 3A). It is probable that the fatty acid anchor enhances the antibody binding with the influenza

19 The antibodies and Mlinhibit

to HA the infection

II (6-8 hr)

Replication

is inhibited

only by antibodies

to HA

Fig. 2. Influenza virus replication cycle. (I ) Adsorption and intracellular penetration. At this stage, the virus particle binds with the cell receptor which then provides for receptor-mediated uptake of the virus in endosomes. In acidic endosomal compartments, the conformation transition in the HA molecule takes place which induces the virus envelope fusion with the endosomal membrane and further release of the virus genetic content in the cytoplasm. (II) Synthesis of virus polymer components. At this stage, the transcription and replication of virus RNA and translation of virus-specific proteins take place. (III) Assembly and budding of the virions. At this stage, the virus envelope and nucleocapsid are assembled. The nucleocapside is assembled inside the cell from newly synthesized virus RNA, NP and P proteins. The envelope proteins (HA, NA and M) are transported through the Golgi complex to the plasma membrane where the final stage of virion assembly and budding takes place. The effect of stearoylated antibodies is marked by arrows.

virus membrane which enhances the neutralizing effect. At the same time, it is probable that the fatty acid anchor plays a distinctive role in the antibody antiviral action. As described in the previous section, fatty acylation stimulates protein uptake (in MDCK cells in particular). This can lead to a simultaneous appearance of antibodies and virus particles in the same endocytic compartments, where antibodies disturb the fusion, which is known to be very sensitive to HA conformation and orientation [ 33,341 (Fig. 3B). This mechanism probably plays the most important role in the case of Mabs to M, protein. Since this protein is located on the internal side

of the influenza envelope, these antibodies do not bind with the virus surface and cannot neutralize the infection according to the regular mechanism [ 251. At the same time, the interaction of Mabs with M1 protein may become possible in endocytic vesicles during fusion, which probably explains the antiviral effect observed [ 3 11. At the late stages of virus replication, when the infection process is already completed, virusspecific proteins are synthesised in the endoplasmatic reticulum (ER) and are then transported through the Golgi complex towards the plasma membrane (Fig. 3). We demonstrated that fatty acylated Mabs (clone C 102 ) or polyclonal antibodies to HA inhibited the virus infection at this replication stage [ 28-301. This effect of hydrophobized antibodies is highly specific: polyclonal antibodies to type A virus suppress reproduction of the latter, but do not produce any effect on type B virus [ 291. The study of kinetics of HA accumulation in the infected cells revealed that modified antibodies did not interfere with the synthesis of virus-specific proteins [ 301. Meanwhile, the addition of antibodies to the infected cells prevented the appearance of virus antigens in the culture medium which complies with the data of virus infectious titre determination. At first, the observed effects of fatty acylated antibodies were related to their ability to penetrate through cell membranes and interact with intracellular antigens [ 28,291. However, this hypothesis was not confirmed by further studies. We recently demonstrated, in the cooperative work with Prof. G. Buttin and Dr. B. Goud (Pasteur Institute), that fatty acylated Mabs to the cytoplasmic tail of vesicular stomatitis virus (VSV) G-protein interfered neither with the synchronized transport of this protein from the endoplasmic reticulum to the plasma membrane nor with the infection of CHO cells [ 161. At the same time, microinjection of these antibodies in the cytoplasm suppressed the transport of newly synthesized G-protein to the cell surface [ 38 1. Neither did we observe suppression of influenza virus reproduction with fatty acylated Mabs to M, protein when these antibodies were intro-

20 INFLUENZA

VIRUS

4!$q~;l~~ng; nonmcdified

I

0‘ fatty

acylated antibodies

ADSORPTION AND UPTAKE

ASSEMBLAGE AND BUDDING

PLASMA

MEMBRANE

ENDOSO

4

J

TRANSCRIPTION AND REPLICATION Fig. 3. Hypothetical mechanisms of antiviral action of fatty acylated antibodies at different stages of virus replication cycle. (A) The antibodies neutralize the virus preventing its adsorption and uptake. At this stage, only antibodies to surface virus antigens are active. (B) The antibodies are internalized simultaneously with the virus and interfere with the fusion process in acidic compartments. At this stage, both antibodies to surface (HA) and to internal (M, ) virus proteins affect the infection. (C) The antibodies interact with newly synthesized virus proteins on the external side of the plasma membrane and interfere with virion budding and assembly. This interaction is possible only if antibodies to external antigens are used.

duced at the late stages of infection [ 3 I]. It is known that M1 protein has a peripheral association with the cytoplasmic side of the plasma membrane [ 391. These findings together with the data of VSV experiments indicate that fatty acylated antibodies cannot penetrate into the cyto-

plasm and bind with intracellular antigens. A more realistic explanation of the observed phenomenon [ 28-301 is based on the recent observation that Mabs to influenza surface antigen ( M2 protein) inhibit virus reproduction [ 391. The study of the mechanism of this effect re-

21

vealed that the surface binding of Mabs to M2 protein interfered with the interaction of cytoplasmic domains of M2 and Mi proteins, and inhibited the virion assemblage and budding [ 391. It is reasonable to assume that the above discussed inhibition of virus replication with fatty acylated antibodies to HA can be also explained by surface interaction of antibodies with HA which impairs the proper assembly and/or budding of the virus (Fig. 3C). In this case, the role of fatty acylation of antibodies is probably connected with enhancement of their binding with the cell surface [ 311. In particular, we demonstrated that stearoylation of antibodies to HA drastically increased their binding with both infected and noninfected MDCK cells. Meanwhile, in the case of infected cells this increase was the most pronounced (Table 2 ) , which can be presumably explained by the fact that antibodies have two points of binding on the cell surface: one via fatty anchor; another via an antigen recognizing site. These data demonstrate that artificial fatty acylation intensities the specific interaction of antibodies with the surface of infected cells. Inhibition of virus replication with fatty acylated antibodies seems to be a general phenomenon; it was, at least, also observed in experiments on replication of respiratory-syncytial (RS) virus in HeLa cells [ 291 and Herpes simplex virus (HSV-1) in chicken embryo libroTABLE 2 Binding of nonmodified and stearoylated polyclonal antibodies to HA with noninfected and influenza virus infected MDCK cells (from Ref. [ 3 1 ] ) Antibodies studied

Infection

Cell-bound antibodies ( 1O5molecules per 1 cell)

Nonmodified

+ +

0.37 1.14 2.30 7.53

Stearoylateda

“The antibodies were radiolabelled with Bolton-Hunter reagent and their binding with infected or noninfected MDCK cells was studied. To this end the antibodies (6.7 nM) were incubated with the cells at 4°C for 5 h. Cells were then washed, lysed and the radioactivity in the lysate was determined.

blasts [40]. These studies demonstrated that, after fatty acylation, polyclonal antibodies to RS virus and Mabs to glycoprotein D of HSV-1 acquired an ability to suppress reproduction of the corresponding virus in the infected cells. Therapeutic efficiency of fatty acylated antiviral antibodies The effect of fatty acylation on antiviral activity and therapeutic efficiency of Mabs to glycoprotein D of HSV- 1 was studied [ 40 1. The in vitro experiments revealed that, in contrast to nonmodified antibodies, the hydrophobized ones were capable of suppressing reproduction of HSV-1 in chicken embryo fibroblasts. The therapeutic efficiency of these antibodies during lethal forms of herpes - meningocephalitis and generalized infection was studied by Kolomiets et al. [ 401. Native antibodies produced a reliable dose-dependent therapeutic action during the experimental disease in mice. Meanwhile, the therapeutic effect of hydrophobized antibodies was much stronger (Fig. 4 ) . Alongside with a decrease in lethality, hydrophobized antibodies caused from 3- to 8-day prolongation of the disease incubation period. Immunohistochemical determination of HSV1 antigens revealed a limited number of local clusters of virus-specific antigens in the cortex and brainstem of animals treated with hydrophobized antibodies. At the same time, in mice treated with native antibodies and especially in untreated animals, the lesion of the central nervous system was diffusive. In these cases, the titre of HSV- 1 in the central nervous system tissue of dead animals was considerably higher than in the case of mice treated with hydrophobized antibodies. Processes underlying the enhancement of therapeutic efficiency of hydrophobized antibodies may be more complicated than those that explain the in vitro effects described in the previous section. It is probable that antibody-dependent cell cytotoxicity (ADCC) and activation of the complement system may be promoted by fatty acylated Mabs. In particular, Colsky et

22

Fig. 4. Therapeutic efficiency of nonmodilied and stearoylated antibodies to glycoprotein D of HSV-1 during experimental herpetic meningocephalitis and herpetic generalized infection in mouse. In order to produce the experimental infections, HSV-1 suspension was intracerebrally (meningocephalitis) or intraperitoneally (generalized infection) inoculated in mice. The antibodies were introduced intraperitoneally during 5 days after infection in various doses (either 25 or 50 mg/day) [ 401.

al. reported that incorporation of fatty acylated antibodies to a target cell antigen in the membrane of effector cells (nylon wool-nonadherent spleen cells) induced natural killer cell-mediated cytotoxicity specific with respect to the target [ 411. In other works, these authors demonstrated that the arming of macrophages with palmitoylated antibodies specilic for chicken erythrocytes (CE) promoted both Fc-receptor dependent and independent ADCC against CE [ 42,43 1. Antisense oligonucleotides combined with a lipid moiety One of the most promising tools for the regulation of gene expression are ‘antisense’ oligonucleotides, i.e., oligonucleotides that can, in a complementary fasion, interact with intracellular nucleic acids [44,45]. At present, in more than dozens of laboratories, the possibilities of using antisense oligonucleotides as agents for in-

hibition of virus reproduction are being intensively studied [ 46-501. One of the serious obstacles impeding practical application of oligonucleotides for these purposes is connected with low efficiency of their penetration into intact cells. To overcome this difficulty, it was recently suggested to modify oligonucleotides with hydrophobic substitutes [ 5 1-54 1. Of late, a whole series of oligonucleotide derivatives attached by their 3’ or 5’-ends to steroids [ 5 1,54,55], aliphatic alcohols, amines [ 52,56-581, phospholipids [ 5 3 ] and other hydrophobes [ 59 ] (Fig. 5 ) was synthesized. Introduction of lipid substitutes into oligonucleotide molecules enhances their uptake by cells. Thus, in particular, Boutorin et al. [ 5 1,601 established that the ability of an oligonucleotide alkylating derivative to penetrate into nuclei of intact cells (carcinoma and libroblasts) and interact with intracellular DNA considerably increased as a result of derivative modification with cholesterol. Letsinger et al. [ 541 reported that an oligonucleotide attached to cholesterol produced an antiviral action, which did not depend on the oligomer sequence. The effect of enhancement of oligonucleotide antisense activity as a result of their hydrophobization with lipid substituents was demonstrated by Kabanov et al. [ 521. Eleven-chain oligonucleotides complementary to polyadenylation signal or to RNA site, which encodes polymerase III (PA) of influenza virus A, were modified by their 5’-ends with undecanol derivatives or dodecylamine. Contrary to nonmodilied antisense and modified nonsense oligonucleotides, 100 ,uM concentrations of these compounds displayed a pronounced capacity to inhibit reproduction of influenza virus A and synthesis of virus-specific proteins in MDCK cells. Soon after that, Shea et al. [ 53 ] demonstrated that the antisense activity of oligonucleotides targeted at genes of various VSV proteins substantially increased as a result of modification of oligonucleotide 5’-ends with synthetic phospholipids. Later, Abramova et al. [ 561 demonstrated the possibility of considerably enhancing antisense inhibition of human immunodefi-

23

3’-end CtlS:“a

5’-end

0)

Boutorln

et.&.

Letslnger

(1989)

er.al.

(lQf39)

(VI

(III)

Abramova

st.al.

(1990)

0

(IV)

0

nC,0H2,Ntt~TO-

(VII

o-tr-ob-

n-CmHasC

Kabanov

et.el.

~--CIBHMO

(1990)

F

Shea

(VII)

Acr-NHC6H1

er.af. (1990)

2”--‘;--”

Saison-Behmoaras (1991) 0 (VIII)

II “Cl

1 H23t+-p-O-

ollgod.oxyn”c,*o,,d,

rN-C6Hl

6NH;NH-Acr

l-

Vlnogradov (1991)

Acr =

Fig. 5. Oligonucleotides covalently modified by lipids. (I) The derivative was found to penetrate effectively into cells and modify intracellular DNA [ 511. (II) The antiviral action of this oligonucleotide derivative was observed, which, however, did not depend on the base sequence [ 541. (III-IV) The sequence-specific anti-influenza virus activity of these oligonucleotides was registered [ 521. (V) The derivative was found to be active with respect to HIV-l virus [ 561. (VI) The phospholipid derivative effectively suppressed the synthesis of VSV proteins [ 531. (VII) This type of derivative was used for inhibition of carcinoma cell proliferation [ 57 1. (VIII) The antisense oligonucleotide to protein kinase (type 1) regulatory subunit was shown to inhibit leukaemic cell proliferation [ 58 1.

24

ciency virus (HIV) reproduction by attaching oligonucleotides to octadecylamine or cholesterol. Recently, Helene et al. reported data on selective cleavage of mRNA and inhibition of carcinoma T24 cell proliferation with a hydrophobized antisense oligonucleotide directed against activated Ha-rus human oncogene [ 5 7 1. The oligonucleotide was substituted with acridine (5’end) and/or dodecanol chain (3’-end), and the effect of such substitution on its antisense activity was studied. Conjugation of oligonucleotide with either the intercalating agent or hydrocarbon chain caused an increase in its specific inhibitory effect on Ha-ras mRNA translation in rabbit reticulocyte lysate. However, the strongest inhibition of cell-free translation was observed in the case of an oligonucleotide derivative containing both acridine and dodecanol chain. This substituted oligonucleotide was also characterized by a more effective cellular uptake and higher stability than other derivatives, and its millimolar concentrations caused 60% inhibition of growth of carcinoma T24 cells, which carry an activated Ha-rus oncogene. At the same time, no antiproliferative effect was observed when a similarly substituted nonspecific oligonucleotide was used. Proliferation of nontumorigenie human mammary HBL 100 cells (which contain only normal rus) and NIH 3T3 cells remained unaffected by addition of mutated Haras-specific and nonspecific derivatives. It is likely that the most rational way to design antisense oligonucleotides is to combine them with both intercalating agents and lipid substituents. In experiments with influenza-infected MDCK cells, described in our previous work [ 52 1, we also observed an additional increase in antiviral activity of an oligonucleotide as a result of its simultaneous end modification with acridine (3 ‘-end) and undecanol hydrocarbon (5 ‘end) [ 1 lo]. A similar oligonucleotide derivative was then used for selective suppression of protein kinase (type 1) regulatory subunit and inhibition of Molt-4 leukaemic cell proliferation [581. On the one hand, the above described

phe-

nomena of an increase in antisense activity as a result of oligonucleotide conjugation with lipid substituents can be explained by enhancement of its binding with complementary regions of nucleic acids by analogy to those observed in the cases of derivatives linked to intercalating agents. However, Shea et al. [ 53 ] demonstrated that introduction of lipid anchors into oligonucleotides even decreased the stability (melting temperature) of duplexes formed by them. A more systematic study revealed that lipophilic moditication did not essentially affect the melting temperature of antisense oligonucleotide to DNA template [ 59 1. On the other hand, an increased activity of hydrophobized oligonucleotides in vitro can be explained by their higher resistance to the action of cell nucleases [56,57]. However, the data obtained by Abramova et al. [56] indicated that the antiviral activity of a derivative modified by its 5’-end with nonpolar 1,2_diaminopentane did not practically differ from that of the initial nonmodified oligonucleotide. At the same time, like the derivative linked to a long-chain hydrocarbon which displayed a pronounced antiviral effect, this compound was less resistant to nuclease cleavage, than the nonmodilied oligonucleotide. In this connection, the most probable reason of high antisense activity of hydrophobized oligonucleotides, in our opinion, is their ability to effectively penetrate into cells. At present, there is growing interest to the sequence-independent effect of hydrophobized oligonucleotides which have been shown to be active as antiviral agents [ 541. The mechanism of this effect is as yet unknown. It may result from interaction of oligonucleotides either with viral polymerases or with surfaces of cells and/or virus particles which interfere with the infection. At the moment, due to their higher stability, more efficient uptake into cells and antisense activity, hydrophobized oligonucleotides are regarded as promising tools for in vivo therapeutic application [ 6 11. DNA interpolyelectrolyte complexes as a tool for gene delivery into a cell The targeting of foreign nucleic acids into in-

25

tact cells underlies many key genetic engineering methods [62]. At present, there are several methods for introduction of DNA into cells, the most common of them being precipitation with calcium phosphate [ 631 or with DEAE-dextran [ 64 1, electroporation [ 65 1, microinjection [ 66 ] and incorporation of DNA into reconstructed virus coats [ 671 or liposomes [ 68 1. Despite the great variety of these methods, the search of new ways for transforming animal, plant and prokaryotic cells continues. On the one hand, there is the need to enhance the efficiency of transformation in comparison with that achieved by traditional approaches which can be applied only to a limited number of cell lines [ 691. On the other hand, traditional approaches seem to be ineffective for introducing RNA molecules into cells [ 701. And, finally, most of these approaches cannot be used for genetic transformation in vivo [ 7 11. Recently, a new approach has been suggested which is based on incorporation of nucleic acids into soluble interpolyelectrolyte complexes (IPECs) with polycations

[721. When an aqueous solution of nucleic acid is mixed with that of linear polycation, there follows cooperative binding of oppositely charged polyions resulting in formation of an IPEC (Fig. 6A) [ 73,741. The general regularities of formation of such complexes are described elsewhere [ 75 1. If the polycation chain has a hydrophobic backbone, its ‘sticking’ to the nucleic acid chain, accompanied by compensation of phosphate group charges, results in formation of a hydrophobic site. The length and number of such sites is determined by the polycation length (degree of polymerization) and IPEC composition (p), i.e., the ratio of polycation and DNA oppositely charged units r.u. ] ) . (q= [ Polycation r.u. ] / [DNA Therefore, the physico-chemical properties of IPECs depend strongly on their composition [ 74,761. In particular, using the example of DNA complexes with N-alkylated poly (4-vinyl-pyridine) (PVP+ ) polycations we have demonstrated that, in solutions with rather high ionic strengths

(I= 1.O- 1.5 ) , soluble IPECs were formed when ~~0.5. Under these conditions, the polycation chains were uniformly distributed among DNA molecules (the contour length of PVP+ is at least one order of magnitude smaller than that of DNA). Further addition of PVP+ caused a typical disproportionate phenomenon: parallel with the soluble IPEC an insoluble complex was formed. A similar behaviour is typical for IPECs of synthetic polyions [ 761. When q= 1.O, the polyion charges are fully compensated and IPECs are insoluble. However, when the polycation base molar concentration exceeds that of polyanion, the complex dissolution (q> 1.O) may be observed, which results from formation of positively charged polycation loops bound on the polyanion chain [ 77 1. The properties of IPEC-incorporated DNA differ significantly from those of free nucleic acid. The ultracentrifugation [ 761 and electron microscopy [ 78 ] data reveal significant condensing of DNA structure as a result of its interaction with a polycation. The complex formation is also accompanied by an increase in DNA stability with respect to nuclease treatment [ 761. In soluble IPECs with ~~0.5, nucleases cleave only those DNA sites that are free of polycation; meanwhile, the digestion of polycation-covered sites is drastically decelerated. In IPECs with 92 1.O, DNA cleavage is completely abolished. Our experiments with Bacillus subtilis [76] and various mammalian [ 791 cells demonstrated that the nucleic acid ability to bind on the cell membrane was significantly enhanced after incorporation of the former into IPEC with PVP+. On the one hand, this may be due to appearance of hydrophobic sites on DNA chain which provides for insertion of IPEC in the nonpolar part of the lipid layer. In order to enhance hydrophobic interactions with the cell membrane, PVP+s can be also additionally hydrophobized with long-chain hydrocarbons (Fig. 6B) [ 76,791. On the other hand, it is probable that especially in complexes with q> 1.O, positively charged polycation loops bind negatively charged lipids and therefore anchor IPECs on the membrane. In the case of mammalian cells, plas-

26

POLYCATION IPC

03

+

HYDROPHOBIZED POLYCATION IPC

DNA

DNA

POLYCATION CONJUGATE WITH RECEPTOR-RECOGNIZING MOLECULE

IPC

Fig. 6. Schematic representation of IPEC formation resulting from DNA interaction with (A) polycation, (B) polycation hydrophobized with long chain alkyl residues and (C) polycation conjugate with receptor-recognizing molecule [ 21.

mid incorporation into such IPECs significantly enhanced the nucleic acid uptake which was observed simultaneously with an increase in its adsorption on the plasma membrane [ 791. This uptake is strongly dependent on temperature: being observe at 37 “C it is significantly inhibited at 4°C. Experiments on transformation of competent B. subtilis cells revealed that incorporation of a plasmid into a soluble complex (q< 0.5) with PVP+ caused a drastic ( lOO-fold) increase in the plasmid transforming activity [ 72,761. Effective

gene transformation of mammalian cells with DNA-PVP+ IPECs was also observed, the effciency of which was a few times higher than that of calcium-phosphate precipitation (Table 3 ) [ 791. In this case however, transformation was observed only when IPECs with q> 1.O were used, which probably reflects a difference in physico-chemical properties of bacilla and mammalian cell membranes. Therefore, IPECs formed by DNA and carbochain polycations represent a promising tool for gene delivery into cells. Unlike calcium-phos-

21 TABLE 3 Gene transformation in 3T3 NIT cells using pPGa1 plasmid (containing B-galactosidase gene) and its IPECs with PVP+ (fromRef. [79] Transformation conditions”

Calcium-phosphate precipitation IPEC

0 (for IPECs)

1.0 3.0 5.0 10.0 15.0

BGalactosidase activity per mg cell protein (arb. units) Suspension

Monolayer

17 1.5 17 62 104 100

15 1.0 13 56 110 117

‘The IPECs are obtained by mixing the aqueous solutions of the pp-Gal plasmid and PVP+ (poly(N-ethylpyridinium bromide), weight-average polymerization degree equals to 500). The cell suspension ( lo4 cells/ml) or monolayer are incubated with the DNA IPECs of variable composition for 2 h at 37°C (total DNA concentration equals to 0.75 mg/ml), washed and then lysed. The /&galactosidase activity in the lysate is determined using 4-methyl-BD-galactopiranoside as the substrate. For comparison the cells were also transformed either in suspension or monolayer by calcium-phosphate precipitation using the same amounts of the DNA.

phate precipitation, the method based on using such IPECs is not accompanied with cytotoxic effects [ 791. Cell transformation with DNA IPECs seems to be a general phenomenon. In particular, Behr et al. [ 801 independently used a similar approach for efficient transformation of various mammalian cells by DNA complexes with lipospermines that actually represent a variety of IPECs. The mechanistic explanation of the phenomena observed has not been fully established yet. It is evident that transformation mechanisms differ for bacilli and mammalian cells. In the latter case it is probable that the first stage of cell transformation is conditioned by cooperative interaction of IPEC-incorporated polycation loops with negatively charged phospholipids of the plasma membrane. Such interaction provides for enhancement of DNA adsorption on the cell surface observed in our experiments. At the same time, the polycation interaction

with the plasma membrane can significantly change the properties of the latter (its structure, ion permeability, etc. [ 8 l-861 ) which in its turn may serve as a ‘signal’ inducing endocytosis. The phenomenon of polycation-induced endocytosis is well described in literature [ 87-901. We believe that it may be the reason for effective uptake of IPEC-incorporated DNA into cells. An additional factor which can probably facilitate such uptake is significant condensation of DNA upon its incorporation into IPECs which provides for the DNA (IPEC) incorporation into endocytic vesicles ( < 100 nm) . Therefore, we believe that the polycation role in cell transformation consists in directing IPECincorporated DNA along the endocytic pathway. An increase in DNA stability may also serve as a positive factor providing for transformation due to inhibition of nucleic acid cleavage with cell nucleases [ 76 1. The next stage of cell transfection must evidently involve DNA elaboration from endocytic vesicles into the cytoplasm. If the efficiency of this process is low, this evidently creates a serious obstacle for transformation. Therefore, further efforts aiming at development of cell transformation systems must be focused on the search of some tools facilitating passing the endocytic barrier. The polycation chemical structure seems to influence significantly the DNA uptake and cell transfection efficiencies. In particular, according to the previously reported data [ 76,781, no cell transformation was observed when free poly (lysine ) was used for complexing with DNA. The difference between our data [ 791 and the results described in [ 781 may be well due to some differences in the structures and properties of DNA IPECs formed by quaternized PVPs and polypeptides. At the same time, it may also result from a difference in interactions of these polycations with the plasma membrane. In any case, it is evident that further analysis of physicochemical regularities of IPEC formation and membrane interaction is required to understand the mechanism of cell transformation with IPECs.

28

In parallel with promoting nonspecific uptake, IPECs can also be used to direct DNA along the receptor-mediated pathway. Wu and Wu [ 7 I,9 11 suggested to conjugate a polycation with a molecule (‘vector’) capable of receptor-mediated endocytosis. The mixing of this conjugate with the DNA polyanion resulted in formation of an IPEC, carrying the ligand (Fig. 6C), which provided for IPEC receptor-mediated uptake and cell transformation. A possibility of using an asialoglycoproteinpoly (L-lysine) conjugate for effective transformation of receptor-positive HepG2 hepatoma cells was demonstrated [ 801. In further studies, transferrin [ 69,78,92,93] and insulin [ 941 were also used as vectors instead of asialoglycoprotein for transformation of various eukaryotic cells. Recently, the IPEC technique was used for receptor-mediated delivery of antisence oligonucleotide into cells [ 95 1. In this work, the complex of a transferrin-poly (L-lysine) conjugate with antisense oligonucleotide to c-myb-encoding DNA was shown to specifically inhibit human leukaemia HL-60 cell proliferation. The inhibitory effect of this IPEC was significantly stronger than that of free oligonucleotide. The most impressive achievement obtained using DNA IPECs was their application for targeted gene delivery in vivo. Wu and Wu [ 8 1 ] demonstrated that poly (L-lysine) conjugate with asialoglycoprotein could serve as vehicle for gene delivery and expression in liver. The essence of this technique is that asialoglycoprotein selectively binds with unique hepatocyte receptors that recognize and internalize galactose-terminal glycoproteins. Recently, the same authors applied this approach to partially correct genetic analbuminemia in rats [ 96 1. On the basis of these works Trubetskoy et al. [ 971 developed a system for gene targeting in mouse lung endothelial cells, using poly (L-lysine ) conjugate with Mabs to lung surface antigens as DNA carriers.

Respecrins: a new class of immunotoxins A significant progress in the field of drug delivery has been achieved with immunotoxins

representing hybrid molecules comprising conjugates of bacterial or plant toxins (or their fragments) with antibodies specific for certain antigen markers on the surface of target cells. The targeted transport of immunotoxins toward disease area leads to subsequent elimination of the target cells. The formation and application of immunotoxins specific for different antigens have been described many times [ 98,991, but problems that hinder wide application of such preparations still remain. Conjugates of whole toxin molecules and cellspecific antibodies are highly active immunotoxins, but they also possess nonspecific toxicity which restricts their application in vivo [ 1001. Therefore, fragments of toxins which are devoid of receptor-recognizing sites (which alone are inactive with respect to intact cells) are currently used as toxic components of immunotoxins. Such hybrid molecules are highly specific [ 1011. However, their activity is much lower than that of immunotoxins which contain intact toxins and is completely dependent on the efficiency of target antigen endocytosis [ 991. In many cases (e.g., in some carcinomas), interaction with surface antigens does not lead at all to intracellular translocation of immunotoxins [ 1021. Therefore, in such instances, the treatment with immunotoxins is not beneficial. many tumour-specific antigenic Moreover, markers are not only located on the cell membrane, but are also secreted into intracellular medium. The immunotoxin efficacy is therefore considerably reduced because of the blocking effect of secreted antigens. Recently, a new class of immunotoxins termed ‘respecrins’ (receptor-specific screened toxins) which are free of the above described disadvantages has been proposed [ 103,104]. The essence of respecrin construction (Fig. 7 ) is that a toxin is covalently bound with a target antigen (or its epitope-containing fragment) in such a way that the conjugate retains its biological activity; interaction of the hybrid molecule with antibodies to the target antigen leads to formation of an immunocomplex in which the toxic component is inactive (screened by antibodies).

29

Fig. 7. The structure and mechanism of action of respecrin. (I) Cells containing the target antigen. (II) Nontarget cells. F, inactive factor; F+, conjugate of the active factor with the epitope; Ab, antibody masking (screening) the active factor [931.

In order to prepare the respecrin species, the conjugate solution is mixed with the solution of antibodies to the target antigen. The respecrin interaction with the cells which contain or secrete the target antigen is accompanied by a substitution reaction which results in the release of the toxic component. No substitution reaction proceeds when the respecrin meets nontarget cells: the toxic component remains masked by antibodies. Hence, the antibodies in respecrin combine the functions of target recognition and protective screening of the physiologically active factor. Possibilities of the above formulated approach were demonstrated using several in vitro models [ 104,105 1. Here, we will illustrate these results by an example of respecrin, containing staphylococcal enterotoxin A (SEA) as an active component. This bacterial toxin displays at least two types of biological activity. On the one hand, SEA is one of the most potent T-cell mitogens [ 106 1. Its activity can be seen, for example, when SEA is added to mononuclear cells. It completely depends on the presence in the culture of cells displaying class II main histocompatibility complex (MHC ) molecules, for example, monocytes or B-cells, which fullil accessory functions [ 107 1. In this case, MHC molecules serve as SEA pri-

mary receptor. As a result of interaction with this receptor on the cell surface, SEA acquires an ability to bind with the variable region of /3 subunit of T-cell receptor (V,TCR), thus triggering a cascade of reactions necessary to activate T-cell proliferation [ IO8 1. The second type of SEA activity is mediated by its interaction with another receptor, presented mostly on proliferating lymphoid and neuronal cells [ 1091, and is connected with the presence in the toxin structure of a polypeptide that is capable of Ca*+ -independent activation of calmodulin-dependent enzymes and, once inside a cell, of blocking its proliferative activity. SEA-containing respecrin specific for IgG was prepared. The role of epitope-containing fragment which is covalently attached to SEA was fulfilled by IgG, while IgG-specific rabbit Mabs acted as a screening component. Conjugation of SEA with IgG did not lead to any significant loss of either its mitogenic or proliferative activity. To demonstrate possibility of reversibly screening both mitogenic and antiproliferative activity of SEA in respecrin, two cellular models were used. The antiproliferative action of respecrin was evaluated by suppression of DNA biosynthesis in ConA-prestimulated human mononuclear cells [ 1031. When the respecrin (i.e., the conjugate preincubated with a IO-fold excess of antibodies to IgG) was added to the cells, the antiproliferative effect was not observed. At the same time, antibodies to IgG did not influence the antiproliferative activity of free SEA and did not produce any effect when added to the cells alone. When free IgG were present in the cell culture medium, the addition of respecrin led to inhibition of cell proliferation. In other words, when the target antigen (in our case IgG) was present in the system, target-dependent dissociation of respecrin took place, accompanied by release of its active part, which in its turn led to manifestation of the effect. Thus, using the above described cellular model, it was has been shown that it is possible to create a supramacromolecular complex in which SEA displays its antiproliferative activity only in the presence of the tar-

30

get to which this complex is addressed. In the above described experimental model, IgG added to the culture were used as a target antigen. Below, we consider the action of respecrin during its interaction with the cell surface antigen and the antigen secreted by the cell. The latter case is most unfavourable for regular immunotoxin application, since interaction with secretory antigens leads to neutralization of immunotoxins and a decrease in their efficiency. On the contrary, in the case of respecrin, there was essential difference between membrane-bound and secretory antigens, because interaction of the former with the target led to release of the biologically active component, whose action was mediated by its receptor, and not by the target. It has been already mentioned that activation of T-cell proliferation by SEA is mediated by high affinity interaction of the toxin with class II MHC antigens and subsequent presentation of SEA to VBTCR. Isolated T-cells are sensitive to the action of SEA (or SEA-IgG conjugate) only in the presence of accessory cells. Both monocytes and B-cells may act as accessory cells presenting SEA to T-cells. The extent of SEA mitogenic effect does not depend on the type of accessory cells used (monocytes or B-cells). In contrast to monocytes, B-cells display and secrete IgG. This makes possible the use of the above system as a model for investigating the action of IgG-dependent respecrin. Table 4 shows the effect of monospecific human antibodies to

IgG on the mitogenic activity of SEA-IgG conjugate. When monocytes were used as SEA-presenting cells, preincubation of the conjugate with antibodies to IgG led to disappearance of the mitogenic effect. In other words, the IgG-dependent respecrin did not possess activity in the cell system containing no IgG. When B-cells were used as accessory ones, the respecrin was active and stimulated T-cell proliferation. In this case, the activation of respecrin resulted from the presence in the cell system of B-cell IgG which competed with SEA-IgG conjugate for the binding of screening antibodies (Fig. 8 ). It is therefore possible to construct a respecrin whose biological action is displayed only upon interaction with endogenous antigens specific to this or that type of cells. In contrast to immunotoxins, whose action is realized as a result of di-

A

I3

A

@4@ Y

1

EXlti-IgG

1

T-cell proliferation

TABLE 4 Effect of SEA-containing respecrin on T-cell proliferation in the presence of B-cells or monocytes or in the absence of accessory cells (from Ref. [ 2 ] ) Experimental conditions”

[‘H ]Thymidine incorporation

Without accessory cells In presence of monocytes In presence of B-cells

3000 5000 16000

(cpm)

“The respecrin is obtained by mixing the SEA-&G conjugate with 1O-fold molar excess of antibodies to IgG. The accessory cells are preincubated with the respecrin for 1 h, thoroughly washed and then added to isolated T-cells. The proliferation of the T-cells was monitored by [‘HI thymidine incorporation.

T-cell proliferation Fig. 8. Mitogenic effect of IgG-dependent respecrin on T-cell proliferation in the presence of accessory cells. (A) The IgGSEA conjugate binds with MHC II receptors of both monocytes and B-cells. The complexes obtained then interact with VBTCR thus activating T-cell proliferation. (B) MHC II binding sites are masked in the respecrin species. Therefore, the respecrin does not interact with monocytes and the mitogenic effect is not observed in this case. Meanwhile, in the case of B-cells expressing IgG molecules, the respecrin is activated due to substitution of the IgG-SEA conjugate by free IgG. Under these conditions, the activation of T-cell proliferation is observed.

31

rect interaction with the target cell, respecrins are compartment-specific compounds. This means that delivery of respecrin to the region with an elevated content of the target antigen is accompanied by release of its biologically active component, which may affect not only antigen-positive but other cells in this region. However, it is clear that the highest concentration of secreted antigens should be near the secretory site and, therefore, antigen-positive cells are the most likely targets for respecrins. This applies with even greater force to membrane antigens. In our opinion, respecrins have great potential for the treatment of local pathological processes, for example solid tumours. Acknowledgments The authors would like to thank their colleagues in Moscow Institute of Biotechnology and Research Center of Molecular Diagnostics and Therapy who contributed to the above discussed research. Among others, we are especially grateful to Drs. Nikolai Melik-Nubarov, Elizaveta Moskaleva and Vladimir Slepnev who made a significant impact on our studies. The planning of this work and analysis of its results were strongly influenced by Professor Gerard Buttin (Laboratory of Somatic Genetics, Pasteur Institute), Professor Eugenii Severin (Research Center of Molecular Diagnostics and Therapy) and Academician Victor Kabanov (Polymer Department, M.V.Lomonosov Moscow State University ), to whom we are also very grateful. References 1 A.V. Kabanov, A.V. Levashov and V.Yu. Alakhov, Lipid modification of proteins and their membrane transport, Prot. Engin., 3 (1989) 39-42. 2 A.V. Kabanov, V.Yu. Alakhov and V.P. Chekhonin, Enhancement of macromolecule penetration into cells and nontraditional drug delivery systems, In: V.P. Skulachev (Ed.) Sov. Sci. Rev., D., Physicochem. Biol., Harwood Academic Publishers, New York, 1992, Vol. 11, part 2, pp. l-75. 3 B.M. Sefton and J.E. Buss, The covalent modification of enkaryotic proteins with lipid, J. Cell Biol., 104 (1987)1449-1453.

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