Toshiyuki Suzawa

Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugate via a poly(ethylene glycol)-based cleavable linker

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.

Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation.

Novel anti-tumor agent, duocarmycin derivative DU-257, was designed and synthesized to prepare immunoconjugate in order to confirm the feasibility of enzymatically cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine. Oxyethylamine arm was introduced at 4-methoxy position of segment B of DU-86 to form DU-257 and evaluated its property. DU-257 retained similar stability and potency with DU-86 while enhanced hydrophilicity suggested. DU-257 was condensed to the PEG-dipeptidyl linker through carboxyl terminal of dipeptide, and enzymatic release of DU-257 using a model enzyme, thermolysin, similar enzyme of which was shown to be overexpressed at various tumor sites, was evaluated by HPLC analysis. Cleavage between the linker amino acids by the model protease and release of DU-257 as valine conjugated form was confirmed. The enzymatically released form of DU-257 expressed its cytotoxicity without loss of the potency for HeLaS3 and SW1116 tumor cell lines, although the efficacy was different in individual cells. DU-257 was then conjugated through the linker to KM231 monoclonal antibody specifically reactive to GD3 antigen which was shown to be expressed on the surface of many malignant tumors such as SW1116. The conjugate retained its binding specificity for SW1116 cell with a similar activity with KM231. Furthermore, the conjugate showed significant growth inhibition on SW1116 cell at a concentration of 75 microg/mL while no effect on antigen negative cell, HeLaS3. These results suggest that the conjugate retained its anti-tumor effect only when it bound on and was activated at the target cell, simultaneously. DU-257 will be one of the candidate of anti-tumor agent for application to immunoconjugate and its conjugate with KM231 via PEG-dipeptidyl linker will be a useful entity for cancer therapy related to sLe(a) expression.

Synthesis and HPLC analysis of enzymatically cleavable linker consisting of poly(ethylene glycol) and dipeptide for the development of immunoconjugate.

A model compound of anti-tumor agent, segment B of duocarmycin derivative DU-86, was conjugated to tumor-specific antibody via a cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine (Ala-Val), to confirm the feasibility of the linker for application to immunoconjugate. The release of segment B from the linker was evaluated by HPLC analysis. When segment B was derivatized to have an amino residue and then linked to PEG through a dipeptide, segment B was cleaved at the peptide bond by a particular enzyme, thermolysin (EC 3.4.24.4), but not by plasmin (EC 3.4.2 1.7.), indicating that certain protease specifically expressed at the tumor site would be capable of peptide-specific digestion and release of anti-tumor agent since a thermolysin-like enzyme has been reported to be expressed at many tumor cells. Furthermore, the results showing that cell extract from G361 human melanoma had an ability to digest the linker peptide while the linker was stable in normal human serum suggested the tumor-specific activation of the conjugated agent. Segment B was conjugated via the linker to murine monoclonal antibody KM641 reactive to GD3 ganglioside to form immunoconjugate and the quantitative release of segment B under the treatment with the enzyme was also confirmed. These results indicate the possibility of double targeting based on both the recognition ability of tumor specific antibody and tumor specific activation of the anti-tumor agents to enhance tumor treatment efficacy and to decrease unwanted side effects.

Improvement of biological activity and proteolytic stability of peptides by coupling with a cyclic peptide

The cyclic moiety of an endothelin antagonist peptide RES-701-1, composed of 10 amino acids with an amide bond between alpha-NH(2) of Gly1 and beta-COOH of Asp9, was coupled to some biologically active peptides aiming to improve their activities and stabilities against proteolytic degradation. Coupling of the cyclic peptide to the N-terminal of RGD-peptides, maximally 4-fold improvement of in vitro activity compared to the original peptide has been achieved. Coupling of it to protein farnesyltransferase inhibiting peptides resulted to improve in vitro activity maximally 3-fold. These peptides coupled with the cyclic peptide also showed enhanced stability against some typical proteases. These results indicate that this cyclic peptide can stabilize the conformations of the peptides coupled to its C-terminus. Coupling of our cyclic peptide is anticipated to be a novel conformational stabilizing method for biologically active peptides, results to improve their activity and stability.

New PEG2 type polyethylene glycol derivatives for protein modification

Although proteins with 2,4-bis (o-methoxypolyethylene glycol)-6-chloro-s-triazine (PEG2-Cl) as a divalent PEG modification have some advantages compared to proteins with the linear PEG modification, PEG2Cl cannot react with amino groups at neutral pH. Therefore, we have prepared new PEG2 derivatives that have an activated ester as the functional group. We confirmed that these derivatives are useful for the divalent modification of proteins, such as bSOD and rhG-CSF.

Preparation and properties of branched oligoglycerol modifiers for stabilization of liposomes.

We examined the effect on drug delivery of liposomes with surfaces that were modified with branched oligoglycerols (BGLs) and explored possible formulation advantages to increase drug exposure. BGL012 is a branched oligoglycerol derivative with a cascade-like structure of 12 glycerol units, characterized as a widely spread structure in aqueous solution. We prepared BGL-phospholipid derivatives (BGL-PEs), including BGL012, by coupling 1,2-distearoylphosphatidylethanolamine to BGLs. BGL012-PE modification of the liposomes (BGL012L) achieved a long circulation time after intravenous injection in rats. The circulation lifetime of BGL012L was almost the same as that of polyethylene glycol (PEG)-modified liposomes. The surface of BGL012L induced the formation of a fixed aqueous layer and reduced protein adsorption on the liposome surface, without strong interference with the binding reaction on the liposome. Thus, the newly synthesized branched oligoglycerol derivatives are considered to have useful hydrophilic and physical properties for modifying the liposome surface to increase drug exposure.

A Novel Liposome Surface Modification Agent that Prolongs Blood Circulation and Retains Surface Ligand Reactivity

Abstract Liposomes are recognized as potentially useful drug carriers but many problems preclude practical medical application. Liposomes bind with serum proteins (opsonization) and are captured by the reticuloendothelial system cells in the liver and spleen, which limits their ability to deliver drugs to other target sites. Modification of lipids with flexible, hydrophilic polymers such as poly(ethylene glycol) (PEG) to yield sterically stabilized liposomes is one approach to improve liposome blood circulation and tissue distribution properties. In this study, we examined liposomes prepared using lipids modified with a new branched oligoglycerol (BGL) moiety for steric stabilization. This novel BGL comprised 14 glycerol units (termed BGL014) connected with flexible ether linkages, resulting in a branched cascade-like structure that is highly expanded in aqueous solution. BGL014 was coupled to 1,2-distearoylphosphatidylethanolamine to yield BGL014-modified lipids. Incorporation of BGL014 into liposomes (BGL014L) resulted in long blood circulation times, despite a much thinner fixed aqueous layer thickness compared to PEG formulations. BGL014 produced a liposome surface coating that appears to function through steric inhibition of non-specific protein binding without strong interference of specific protein-binding reactions. Liposome structure and functionality was maintained following BGL014-modification, as the incorporation ratio of drug remained high. These results suggest that the BGL014 modification of liposomes is a promising approach to produce stable and long circulating drug carriers capable of selective binding to specific proteins.

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity.

Hybrid peptides were constructed from endothelin B receptor (ET(B)) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ETB as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ETB but for ETA. Although all analogues had antagonistic effects on ETA, some analogues had proved to function as agonist on ETB confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ETB-selective agonist with weak ETA antagonism (3, KT7421); (2) ETB-selective antagonist with weak ETA antagonism (29, KT7539); (3) ETB agonist with potent ETA antagonism (27, KT7538); and (4) non-selective ETA/ETB antagonist (26, KT7540).

RES-701-1/endothelin-1 hybrid peptide having a potent binding activity for type B receptor

Abstract N-Terminal cyclic peptide of RES-701-1, an endothelin (ET) type B receptor selective antagonist, was chemically combined to C-terminal peptide of ET family. Among a series of the hybrid peptide synthesized, RES-701-1(1–10)/[A15]ET-1(12–21) hybrid peptide possessed both a potent binding activity with an IC50 value of 0.24nM and a selectivity for type B receptor.

Advantage of Alagln as an Additive to Cell Culture Medium

Advantage of dipeptides, L-alanyl-L-glutamine (AlaGln) and L-alanyl-L-tyrosine (AlaTyr), for the use of cell culture was tested in this study. Addition of the dipeptide to cell culture medium attenuated viability decline of cells and enhanced production titer of monoclonal antibody (Mab). Differences in stability between amino acids and dipeptides may significantly impact on cell culture performance. For example, medium containing glutamine (Gln) produced more ammonia than that of AlaGln. Accumulation of ammonia resulted in reduction of Mab titer, showing the advantage of using AlaGln.