Proc. NatL Acad. Sci. USA
`Vol. 79, pp. 626-629, January 1982
`Medical Sciences
`
`A covalent linkage between daunorubicin and proteins that is
`stable in serum and reversible by lysosomal hydrolases, as
`required for a lysosomotropic drug-carrier conjugate:
`In vitro and in vivo studies
`(cancer chemotherapy/anthracyclines/lysosomes/peptidic spacer arm/L1210 leukemia)
`ANDRE' TROUET, MICHtLE MASQUELIER, ROGER BAURAIN, AND DANIELLE DEPREZ-DE CAMPENEERE
`International Institute of Cellular and Molecular Pathology and Universit6 Catholique de Louvain, B-1200 Brussels, Belgium
`Communicated by Christian de Duve, September 22, 1981
`
`Daunorubicin (DNR) has been conjugated to suc-
`ABSTRACT
`cinylated serum albumin by an amide bond joining the amino
`group of the drug and a carboxyl side chain of the protein either
`directly or with the intercalation of a peptide spacer arm varying
`from one to four amino acids. During in vitro incubation with ly-
`sosomal hydrolases, intact DNR could be released extensively only
`from conjugates prepared with a tri- or tetrapeptide spacer arm.
`These latter conjugates remained very stable in the presence of
`serum. When tested in vivo against the intraperitoneal form of
`L1210 leukemia, the conjugates in which DNR was linked to serum
`albumin directly or via one amino acid were completely inactive
`but the conjugate with a dipeptide spacer arm was not more active
`than free DNR. In parallel with the in vitro studies, the best ther-
`apeutic results were obtained with the conjugates formed with tri-
`and tetrapeptidic spacer arms; they were much more active than
`DNR, inducing a high percentage of long-term survivors. Thus,
`use of a tri- or tetrapeptide spacer arm is essential to obtain
`DNR-protein conjugates that remain stable in serum and from
`which DNR can be released through the action of lysosomal hy-
`drolases. The in vivo results suggest, moreover, that these con-
`jugates are endocytosed by L1210 cells and that DNR is released
`intracellularly after digestion by lysosomal enzymes. This conju-
`gation method can be applied to other drugs possessing a free
`amino group and to various potential carriers, such as antibodies,
`polypeptide hormones, and glycoproteins, that have amino or car-
`boxyl side chains.
`
`During the past decade, the use ofcarriers for the selective tar-
`geting of anti-tumor drugs has been advocated with increasing
`frequency and has led to numerous reports on the association
`of drugs such as anthracyclines, methotrexate, bleomycin,
`chlorambucil, and 1-f3-D-arabinofuranosyl cytosine (cytosine
`arabinoside) with carriers such as DNA (1, 2), liposomes (3, 4),
`immunoglobulins (5, 6), hormones (7, 8), and other proteins
`(9, 10) or polypeptides (11).
`However, insufficient attention has been paid to the nature
`ofthe link between the drug and the carrier. For a drug-carrier
`conjugate to be effective, the link between drug and carrier
`must remain stable in the bloodstream and withstand the action
`of serine hydrolases. On the other hand, unless the drug is able
`to act in conjugated form at the cell surface, it has to be released
`from the carrier after interaction ofthe conjugate with the target
`cell, and its mode of release must be such as to allow the drug
`to reach its biochemical target-usually situated intracellu-
`larly-and to interact effectively with it. Because the most gen-
`eral fate of molecules bound by surface receptors is to be in-
`teriorized by endocytosis and conveyed to the lysosomes for
`
`The publication costs ofthis article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertise-
`ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
`
`digestion, an obvious way ofensuring appropriate release ofthe
`drug is to rely on lysosomal hydrolysis. This approach is evi-
`dently limited to drugs that are not inactivated in the lysosomes
`and that can reach their biochemical target from the lysosomal
`compartment. The principles governing this "lysosomotropic"
`chemotherapy have been developed in greater detail elsewhere
`(12, 13).
`We have developed and tested, both in vitro and in vivo, a
`bond meeting the above requirements between daunorubicin
`(DNR) and bovine serum albumin. DNR was chosen because,
`like doxorubicin (adriamycin), it is a potent drug having, on its
`daunosamine moiety, a primary amino group suitable for an
`amide type linkage (Fig. 1) and because we know from previous
`work that it has the properties of lysosome resistance (1) and
`transmembrane diffusibility (14) needed for effective action
`after intralysosomal release. Albumin was selected as a model
`carrier because of its protein nature and ready availability. Pro-
`teins and polypeptides, (for instance, antibodies, hormones,
`glycoproteins, and lectins), are good candidates as carriers for
`antitumoral drugs.
`We describe in this paper how a suitable albumin-DNR con-
`jugate can be prepared, provided that an oligopeptidic spacer
`arm is intercalated between the drug and the carrier. This con-
`jugation method was tested in vitro by measuring the release
`of DNR in the presence of serum and lysosomal hydrolases and
`in vivo by evaluating the chemotherapeutic activity of the con-
`jugate on the L1210 murine leukemia.
`
`MATERIALS AND METHODS
`Amino Acid and Peptide Derivatives of DNR. DNR HCl was
`obtained from Rhone-Poulenc, S.A. (France). N-L-Leucyl-
`DNR (Leu-DNR) was synthesized by reaction of the N-carbox-
`yanhydride derivative ofL-leucine with DNR as described (15).
`N-L-alanyl-L-leucyl-DNR (Ala-Leu-DNR) was prepared by re-
`action of Leu-DNR with the N-trityl alaninate of N-hydroxy-
`succinimide (16, 17). N-L-leucyl-L-alanyl-L-leucyl-DNR (Leu-
`Ala-Leu-DNR) and N-L-alanyl-L-leucyl-L-alanyl-L-leucyl-DNR
`(Ala-Leu-Ala-Leu-DNR) were synthesized as described for Ala-
`Leu-DNR by successive condensation of Ala-Leu-DNR and
`Leu-Ala-Leu-DNR with the appropriate amino acid.
`In an alternative procedure, the tri- and tetrapeptides were
`first synthesized by the solid-phase method of Merrifield (18)
`and subsequently linked to DNR in the presence of dicyclo-
`hexylcarbodiimide and N-hydroxysuccinimide.
`Conjugation of DNR and Derivatives to Bovine Serum Al-
`bumin. The protein carrier was first succinylated. Bovine serum
`
`Abbreviations: DNR, daunorubicin; HPLC, high-pressure liquid chro-
`matography; ECD, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide'HCl.
`
`626
`
`IMMUNOGEN 2077, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Medical Sciences: Trouet et al.
`
`Proc. Natl. Acad. Sci. USA 79 (1982)
`
`627
`
`and from the protein content as measured by the method of
`Lowry et al. (21). The conjugates were finally sterilized by fil-
`tration on Millipore GS filter (0.22 ,um) and stored in the dark
`at 40C.
`Digestion of the Drug-Protein Conjugates by Lysosomal
`Enzymes. Drug-protein conjugates at a final anthracycline con-
`centration of 17.8 ,uM were incubated at 370C in the presence
`of a purified lysosomal fraction (0.5 mg of protein per ml) in 0.1
`M citrate, pH 5.5/5 mM cysteine. The lysosomes were isolated
`from the livers ofrats treated with Triton WR-1339 (22). At var-
`ious times the amount of intact DNR released was determined
`by high-pressure liquid chromatography (HPLC) on 100-,ul ali-
`quots as described (23).
`Incubation in the Presence of Serum. Drug-protein conju-
`gates were incubated at 370C at a final anthracycline concen-
`tration of 17.8 ,u M in the presence of 95% calf serum. Aliquots
`were analyzed for release of intact DNR by HPLC as above.
`Chemotherapeutic Tests. Female DBA2 mice (Charles
`River, France) were inoculated intraperitoneally with 104
`L1210 leukemic cells on day 0 and with the drugs on days 1 and
`2. Mice were weighed daily, and the weight change on day 8
`was taken as an index ofoverall toxicity. The percentage increase
`in life-span and the number of long-term survivors on day 30
`were used as chemotherapeutic indices.
`
`RESULTS
`DNR, Leu-DNR, Ala-Leu-DNR, Leu-Ala-Leu-DNR, and Ala-
`Leu-Ala-Leu-DNR were conjugated to succinylated albumin
`(Fig. 1) and the sensitivities of the conjugates to serum and ly-
`sosomal hydrolases in vitro and their chemotherapeutic activ-
`ities in vivo were studied in parallel.
`Conjugation of DNR and Its Derivatives to Succinylated
`Serum Albumin. Irrespective of whether DNR or its peptide
`derivatives were used, a conjugation yield in anthracycline
`varying between 56% and 76% was observed. The use of suc-
`cinylated albumin decreased the formation of albumin poly-
`mers, and chromatography of the conjugates on Sepharose 6B
`(Pharmacia, Fine Chemicals, Uppsala, Sweden) indicated that
`more than 70% of the conjugates behaved like monomeric al-
`bumin and less than 5% was excluded owing to a molecular
`weight higher than 1,000,000.
`The number of anthracycline molecules (DNR or peptide
`derivatives) linked per molecule of albumin varied between 10
`and 21. Analysis by HPLC after chloroform/methanol extrac-
`tion (23) of the conjugates treated by Porapak chromatography
`showed that a maximum of 5% of the DNR or derivative bound
`to albumin was not covalently linked and could be removed by
`extraction in organic solvents. Without Porapak chromatogra-
`phy, this proportion could be as much as 20% or more, in spite
`of the Bio-Gel filtration step.
`Digestion by Lysosomal Enzymes. Fig. 2 illustrates the re-
`lease of free DNR observed during incubation of the various
`DNR conjugates with purified lysosomal enzymes. No DNR
`was released from albumin-DNR, and very little was released
`from albumin-Leu-DNR or albumin-Ala-Leu-DNR. The rate
`of DNR release increased markedly when the peptide spacer
`arm was lengthened to three or four amino acids. About 60%
`ofthe bound drug was released as free DNR from albumin-Leu-
`Ala-Leu-DNR and 75%, from albumin-Ala-Leu-Ala-Leu-DNR
`after 10 hr of incubation.
`A pH optimum of5.5 was observed for the enzymatic release
`of DNR from albumin-Ala-Leu-Ala-Leu-DNR in the presence
`of lysosomal hydrolases.
`Stability in Presence of Serum. Albumin-Leu-Ala-Leu-
`DNR and albumin-Ala-Leu-Ala-Leu-DNR were stable in pres-
`
`NH
`C=O
`CH-R
`IN
`
`0n=04
`C=O
`CH2
`2
`C=O
`NH
`CH2
`CH2
`CH2
`CH2
`
`I,
`
`I
`
`0
`
`DAUNORUBICIN
`
`:) ACID (S)
`AMIN(
`
`SUCC
`
`:INYL
`
`SERUM
`
`ALBUMIN
`
`" / OOH
`
`Structure of the daunorubicin-albumin conjugates. Dau-
`FIG. 1.
`norubicin was linked to succinylated serum albumin either directly
`(n = 0) or via an oligopeptidic spacer arm composed of one to four amino
`acids (n = 1-4).
`albumin (Armour, Eastbourne, England) was dissolved in water
`at 100 mg/ml, and the pH was adjusted to 7.5 with 0.5 M
`NaOH. Succinic anhydride (0.68 mmol; Aldrich, Beerse, Bel-
`gium) was then added stepwise while the pH was maintained
`at 7.5 with 0.5 M NaOH. Another 0.68 mmol of succinic an-
`hydride was added subsequently, and the preparation was ex-
`tensively dialyzed against phosphate-buffered saline (NaCl, 137
`mM; KCl, 3 mM; Na2HPO4, 8 mM; KH2PO4, 1.5 mM), steril-
`ized by filtration on Millipore GS filters (0.22 ,um), and kept
`at 4°C. The yield varied between 88% and 95% as determined
`from the number ofremaining free amino groups measured with
`trinitrobenzenesulfonate (19).
`For the conjugation step, 20 ,mol ofDNR or ofits derivatives
`were added to 50 mg of succinylated albumin (5 ml of solution
`at 10 mg/ml). Then, 7.5 mg of 1-ethyl-3-(3-dimethylamino-
`propyl)carbodiimide HCl (ECD) (Sigma) were added, and the
`solution was kept in the dark at 4°C without stirring. Another
`3.75 mg of ECD was then added, and the solution was kept
`overnight at 25°C.
`The drug-protein conjugate was separated from the remain-
`ing free drug and reagents by filtration on Bio-Gel P-100
`(100-200 mesh; Bio-Rad). The elution profiles of proteins and
`anthracyclines were monitored by measuring absorbance at 280
`and 475 nm, respectively. Free drug adsorbed on the protein
`was eliminated by adsorption chromatography on Porapak Q
`(Waters Associates). The Porapak used was first suspended in
`ethanol for 15 min and then washed extensively with H20 and
`phosphate-buffered saline, which served also as eluant (20).
`The drug/protein molar ratio was computed from the absorb-
`ance of the solution at 475 nm (E'm = 165 assumed for DNR)
`
`IMMUNOGEN 2077, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`628
`
`Medical Sciences: Trouet et aL
`
`Proc. Nad Acad. Sci. USA 79 (1982)
`
`Chemotherapeutic Results. The therapeutic effects of DNR
`and its various conjugates on the intraperitoneal form of L1210
`leukemia are summarized in Table 1. DNR exerted a moderate
`activity, with an increase in life-span of 39% at a dose of 2 mg/
`kg; the 5 mg/kg dose is toxic. It induced a weight loss of more
`than 10% on day 8 and the death of some animals before the
`controls.
`Albumin-DNR and albumin-Leu-DNR had no chemother-
`apeutic effect at 5 and 7.5 mg/kg and seemed to have little tox-
`icity because no significant weight loss was observed on day 8
`at the highest dose. The effects of albumin-Ala-Leu-DNR at 5
`and 7.5 mg/kg were similar to the effect of DNR at a lower
`dosage; albumin-Leu-Ala-Leu-DNR and albumin-Ala-Leu-Ala-
`Leu-DNR had markedly higher therapeutic effects, with an
`average of 65% survivors on day 30. Moreover, at a dose of 7.5
`mg/kg, these conjugates induced a distinctly lower weight loss
`than did DNR at 5 mg/kg. Therefore, they seem to be less toxic
`as well. Succinylated albumin had neither therapeutic nor toxic
`effects, even at doses higher than those used with the
`conjugates.
`
`DISCUSSION
`DNR has been linked covalently to succinylated serum albumin
`either directly or with the intercalation of a spacer arm con-
`sisting of one to four amino acids. The condensation of the
`aminosugar moiety of DNR and the carboxylic side chains of
`succinylated albumin was realized with the aid ofwater-soluble
`carbodiimide.
`The direct conjugate between DNR and succinyl albumin
`was entirely resistant to hydrolysis by lysosomal enzymes. This
`could be related either to an intrinsic resistance of the succi-
`nyl-daunosamine linkage to lysosomal hydrolases or to steric
`hindrance by the bulky protein molecule.
`
`50
`
`z
`
`Time, hr
`
`Influence of length of oligopeptidic spacer arm an the re-
`FIG. 2.
`lease of DNR linked to succinylated serum albumin during incubation
`for up to 10 hr at 370C and pH 5.5 in the presence of purified rat liver
`lysosomes. The release of free intact DNR was followed by HPLC and
`fluorometry. a, From albumin-DNR; o, albumin-Leu-DNR; m, albu-
`min-Ala-Leu-DNR; e, albumin-Leu-Ala-Leu-DNR; *, albumin-Ala-
`Leu-Ala-Leu-DNR.
`
`ence of serum. After 24-hr incubation in the presence of 95%
`calf serum, the maximal release of DNR amounted to only 2.5%
`of the bound drug.
`
`Table 1.
`
`DNR
`
`Drug
`
`Albumin-DNR
`
`Albumin-Leu-DNR
`
`Albumin-Ala-Leu-DNR
`
`Albumin-Leu-Ala-Leu-DNR
`
`DNR/protein,
`mol/mol
`-
`-
`-
`14.9
`11.7
`11.6
`11.6
`121
`12.1
`14.5
`14.4
`20.5
`17.0
`20.7
`17.1
`15.2
`14.9
`13.8
`15.1
`15.1
`15.1
`13.9
`-
`
`Chemotherapeutic activity of DNR-albumin conjugates
`Dose, mg/kg/day
`ILS,*
`Survivors
`Weight variation,
`on day 30t
`%*
`As DNR
`As protein
`%
`5/91
`+0.4
`39
`2
`-
`-12.3
`0/52
`-
`6
`5
`-11.0
`0/8
`7.5
`7
`-
`0/7
`+6.5
`-2
`40
`5
`+2.2
`0/9
`9
`51
`5
`0/10
`+0.9
`7.5
`8
`77
`0/6
`-0.9
`7.5
`9
`77
`0/10
`+3.5
`6
`49
`5
`0/10
`-3.4
`6
`7.5
`74
`0/10
`-4.7
`30
`5
`41
`0/10
`33
`-1.3
`62
`7.5
`8/10
`+0.4
`>211
`29
`5
`6/10
`+0.5
`>200
`5
`35
`8/10
`-7.9
`7.5
`>211
`43
`7/10
`-1.7
`>200
`52
`7.5
`4/10
`-3.0
`107
`39
`5
`7/10
`-3.9
`>211
`5
`40
`5/9
`+0.9
`>189
`5
`43
`6/10
`-9.8
`>211
`7.5
`59
`10/10
`-2.5
`>211
`59
`7.5
`4/8
`-4.4
`>200
`7.5
`59
`7/9
`-0.4
`7.5
`>189
`64
`0/16
`+9.1
`-6
`59
`-
`0/8
`-1
`+2.4
`89
`-
`L1210 cells (104) were injected intraperitoneally on day 0 into DBA2 mice. Drugs were given intraperitoneally on days 1
`and 2.
`* Increase in life-span relative to untreated controls.
`tNumber of survivors on day 30/total number of mice.
`* Mean percentage increment in weight of the animals between days 0 and 8.
`
`Albumin-Ala-Leu-Ala-Leu-DNR
`
`Albumin
`
`IMMUNOGEN 2077, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Medical Sciences: Trouet et al
`
`Proc. Natl. Acad. Sci. USA 79 (1982)
`
`629
`
`In order to overcome this problem, we intercalated an oli-
`gopeptidic spacer arm, varying from one to four amino acids,
`between DNR and the succinylated protein. Leucine was se-
`lected as the amino acid adjacent to DNR because, among sev-
`eral DNR derivatives tested, Leu-DNR is the most rapidly and
`extensively hydrolyzed by lysosomal enzymes (15). Ala-Leu-
`DNR, Leu-Ala-Leu-DNR, and Ala-Leu-Ala-Leu-DNR were
`chosen as intermediates because Ala-Leu-DNR was found to be
`the best substrate for acid hydrolases among three dipeptide
`DNR derivatives examined (15) and because the alternation-of
`alanine and leucine provided tri- and tetrapeptide derivatives
`that are sufficiently water soluble as well as sensitive to lyso-
`somal hydrolysis.
`The approach chosen proved successful. Some degree of hy-
`drolysis was observed even with a single amino acid in the
`spacer arm, but it was very slow. Only a slight improvement
`was obtained when a second amino acid was intercalated. How-
`ever, when a third amino acid was inserted, the extent of hy-
`drolysis by lysosomal enzymes increased from 8% to 60% in 10
`hr. It reached 75% with a tetrapeptide spacer arm. These tri-
`and tetrapeptide conjugates remained perfectly stable in the
`presence of serum, as required for authentic lysosomotropic
`drug-carrier complexes.
`The chemotherapeutic efficiency ofthe conjugates paralleled
`closely their sensitivity to lysosomal hydrolysis, even to the
`point of becoming significant with a dipeptide spacer arm and
`of increasing dramatically when the number of intercalated
`amino acids was increased from two to three. This does not in
`itself prove that the conjugates act according to the theoretical
`lysosomotropic model. But it certainly supports such a conclu-
`sion strongly, especially since an alternative explanation cannot
`readily be proposed on the basis of what is known of the pro-
`cessing of proteins by cells.
`If it seems likely, therefore, that the therapeutic activity of
`the conjugates depends on lysosomal release of the drug; their
`relatively lower toxicity, and consequently improved therapeu-
`tic index compared to free DNR, remains to be explained. Rel-
`ative cosegregation of both the target cells and the drug con-
`jugates in the peritoneal cavity provides the simplest explanation.
`But it is possible that the use of succinylated albumin as carrier
`may have fostered selective uptake because we have found re-
`cently that succinylation of albumin enhances its endocytosis
`by L1210 cells in vitro-and that conjugates ofLeu-Ala-Leu DNR
`orAla-Leu-Ala-Leu-DNR with nonsuccinylated albumin have
`only a small chemotherapeutic effect on L1210 leukemia.
`Prior succinylation of the carrier protein was originally
`adopted to increase the yield of drug conjugation and to de-
`crease the amount ofprotein polymerization in the presence of
`carbodiimide. Although it turned out to be advantageous in the
`present case, it is-undesirable in a general conjugation proce-
`dure to be used with proteins selected for their ability to bind
`specifically to surface receptors ofthe target cells. Such binding
`properties are likely to be altered drastically by succinylation
`in many cases.
`Whatever improvement may be made in the actual coupling
`procedure, it is clear that the tri- and tetrapeptide arms de-
`scribed in this paper allow the linking of DNR to a protein by
`acovalent bond that, although being stable in serum, is sensitive
`to lysosomal hydrolases, and that they yield conjugates active
`in vivo. These three criteria, especially the in vivo activity, were
`
`not entirely met in the previously published procedures for
`linking DNR directly to proteins by means ofcarbodiimide (6),
`glutaraldehyde (6), or periodate oxidation (24) or indirectly with
`a.leucylarginylglucopyranosyl spacer arm (10).
`Our spacer arms are likely to find many applications. In prin-
`ciple, they can serve to link DNR to any proteins, as well. as to
`many other potential carrier molecules that possess, or can be
`fitted with an appropriate amino or carboxyl group. Conversely,
`other drugs besides DNR can be modified by this procedure,
`provided they have a free amino group or some other conve-
`nient attachment point, and have the ability to reach their in-
`tracellular target in active form ifreleased inside lysosomes. Our
`results thus open exciting prospects ofusing antibodies, peptide
`hormones, glycoproteins, and other substances that can be rec-
`ognized by cell-surface receptors as carriers not only for anti-
`tumoral drugs.but also for other drugs-for instance, chemo-
`therapeutic agents against intracellular parasites (25).
`
`We thank Prof. C. de Duve for helpful criticism and discussion in
`the course of this work and Dr. R. B. Merrifield for initiating one of us
`(M.M.) to the solid-phase method of peptide synthesis. This work was
`supported by the Caisse Generale d'Epargne et de Retraite (Brussels,
`Belgium) and by Rhone-Poulenc, S.A. (Paris, France).
`
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`
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