Proceedings of the Twenty-Third European Peptide Symposium

• I

Editor: Hernani L.S. Maia

ESCOM

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v . 23 1994 C. Ol--------SEQ : P07580000 TI: PEPTIDES

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@)PROP ERT Y OF T HE

NATIONA.L LIBRARY OF MEDICINE

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Peptides 1994 Proceedings of the Twenty-Third European Peptide Symposium September 4-10, 1994, Braga, Portugal

Edited by

H.L.S. Maia . Department of Chemistry

University of Minho Guallar P-4710Braga Portugal

I// 1111111111·

II Ill~ · 1111111111 lll\ ESCOM • Leiden • 1995

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CIP-data Koni nklijke Bibliotheek, Den Haag

Peptides

Peptides 1994: Proceedings of the Twenty-Third European Peptide Symposium, September 4-10, 1994, Braga, Portugal I ed. by H.L.S. Maia. - Leiden : ESCOM - lit. With index, ref. Subject headings: Peptides I Proteins.

ISBN 90-72199-21-9 (hardbound)

Published by:

ESCOM Science Publishers B.V. P.O. Box 214 2300 AE Leiden The Netherlands

©Copyright 1995 by ESCOM Science Publishers 8.V.

All righls reserved. No part or this publication may be reproduced, stored in a retrieval system or transmilled in any form or by any means, electronic, mechanical, photocopy ing, recording or o therwise, without prior permission in writing from the copyright holder.

Printed in The Netherlands

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Contents in brief

Abbreviations

The Fifth Josef Rudinger Memorial Lecture

Lectures

l 11

nr IV

v

Posters

VI VII VJll IX x XJ XII XIII XIV xv xvr xvu xvrn

Synthesis and Analysis of Amino Acids and Peptides Isolation, Purification, Conformation and Physical Chemistry of Peptides

Peptide Mimetics, de novo Design and Pept ide Libraries Structure-Activity Relationships, Peptide Vaccines and Immunol­ogy or Peptides

Receptors, other Binding Proteins and Biological Aspects of Peptides

Am ino Acid and Peptide Synthesis Synthesis of Iliologically Active Peptides Analytical Methods for Peptides Isolation and Purification of Peptides de Novo Design and Peptide Libraries Conformation and Physical Chemistry of Peptides Structure-Activity Relationships Glyco-, Lipo-, Phospho- and Nucleopcptides 13iology and Mechanisms of Action of Peptides Receptors and other J3inding Proteins Peptide Vaccines and Immunology Peptide Pharmacology Miscellaneous

Author Index

Subject Index

xx iii

19 45

67 99

123

143 31 1 395 425 44 1 501 585 717 761 783 809 875 887

903

923

xx ix

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Monocyte- and neutrophil-mediated lysis of SCCL by a bispecific molecule comprised of Lys3-BN and mAb22

J. Chen•, M. Mokotoffb, J.-H. Zhoua, M.W. Fangerc and E.D. Balla "Hematology/Bone Marrow Tra11splcmtation, U11iversity of Pi{lsburgh Medical Center and

Pi11sb11rgh Cancer lnstit111e, Pi{lsburgh. PA 15261. U.S.A. b Department of Plwrmace111ical Sciences. School of Pharmacy, U11ii•ersity of Pi11sb11rgh.

Pi11sb11rgh. PA 15261. U.S.A. '" Department of Microbiology. Dartmouth Medical School. Leba11011. NH 03756, U.S.A.

r o tr odu ction

Small cell carcinoma of the lung (SCCL) grows rapidly, metastasizes early and eventually causes the death of about 30,000 people per year in the U.S.A. Various combinations of chemotherapy have been used with liule improvement in the long-term survival rate. For these reasons recent efforts have focused on developing other therapeutic strategics for the treatment of SCCL, such as immunological and hormonal therapy [I], and intensive chemotherapy with autologous bone marrow or peripheral stem cell support (2]. Most human SCCL cell lines produce gastrin releasing peptide (GRP), which is similar in action and sequence to bombesin (BN), a peptide from frog skin. These SCCL cell lines produce GRP and express a single class of high-affinity receptors for BN/GRP (3].

As a result of BN/GRP receptors having limited distribution in the body, these receptors could serve as targets for directing specific immune reactions. We have developed a novel approach of immunotherapy using the BN/GRP receptors expressed on SCCL cells as targets. We have made a bispccific molecule (immunoconjugatc, IC) between Lys3-BN and a monoclonal antibody (mAb22) [4] against the human high­affinity Fe gamma receptor (FcyRI, CD64), which is expressed on the surface of human monocytes (Mo) and IFNy-activated polymorphonuclcar neutrophils (PMN). We hypothesized that this IC should be able to redirect these immune effector cells towards SCCL cells and, thus, elicit specific antibody-dependent cell-mediated cytotoxicity (ADCC) of the carcinoma cells.

Results a nd Discussion

The IC, mAb22-Lys-BN, was obtained by the reaction of Lys3-BN with N-suecinimidyl S-acetylthioacetate (SAT A). The Lys3-BN-SA TA conjugate was purified by reversed phase HPLC. Deacetylation to the free sulfhydryl, Lys3-BN-SH, was accomplished with hydroxylamine (NH20 H) and purification was performed by HPLC. The antibody mAb22, or its F(ab'h fragment (Medarcx, Inc.), was reacted with sulfosuccinimidyl 4-(N-maleimidomcthyl)-eyclohexanc- l-carboxylatc (Sulfo-SMCC, Pierce) lo produce a malcimide-containing Ab. The final conjugation of Lys3-BN-SH with the maleimidc­containing Ab was effected by mixing equimolar amounts overnight at RT.

The ability of two immunoconjugatcs, mAb22-Lys-BN and F(ab'h-Lys-BN, LO bind to four SCCL cell lines (NCI-H69, H345. SHP-77, and DMS273) was determined by now cytometric analysis, using the indirect staining method. The binding was directly proportional to the amount of IC used to stain the cells. This was manifested both by an increase in the absolute percentage of cells stained positively and by an augmentation

819

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of the mean nuorescence intensity (MR) of the entire cell population. In general. the IC prepared between the whole antibody. mAb22, and Lys ' -BN had a higher MR than that prepared between the F(ab'h fragment and Lys'-BN.

AOCC assays used target (T) SCCL cells that were incubated with 51 Cr. The effector (E) cells, Mo or PMN. were obtained from different donors and activated by prior incubation with rlFNy. The cytotoxic potencies varied among donors. In all four SCCL cell lines. more than 80% of the cells were lysed by Mo from different donors. Tumor cell lysis was primarily dependent on the EfT ratio used for each donor. The greatest lysis, with either Mo or PMN, was consistently achieved at an EfT ratio of 100:1. About 80% of SHP-77 cells were lysed by PMN from two different donors, while with the H69 cells the lysis was about 45%. Since PMN do not express FCyR I on their cell surface without rfFNy stimulation, non-stimulated PMN had much less cytotoxicity on target cells.

Mo-mediated cytotoxicity was studied under different assay conditions in each of the four SCCL cell lines. When the target cells were incubated with activated effector (Mo) cells in the absence of IC, about 15-60% tumor cell lysis was observed, depending upon donor. The addition of mAb22 did not cause any further cell lysis. However, the addition of IC (mAb22-Lys-BN) resulted in an incrense in tumor cell lysis to about 50-95%. The IC-induced SCCL cell lysis could be significantly (p < 0.05) blocked by adding an excess amount of either mAb22 or Lys3-BN. Similarly, with two SCCL lines, PMN IC-induced tumor cell Iris could be significantly blocked by adding an excess amount of either mAb22 or Lys -BN.

Concl us ions

We have produced a novel IC between mAb22 and Lys3-BN. which binds to BN/GRP receptors on the cell surface of four SCCL cell lines and to FcyRJ on both human Mo and PMN. As hypothesized, IC mAb22-Lys-BN can direct immune effector cells towards target tumor cells and elicit a specific AOCC, which leads to the lysis of target SCCL cells. This cytotoxicity is dependent on Elf-cell ratios. can be induced in a wide range of IC concentrations, and can be blocked by the addition of an excess amount of either parental molecule, mAb22 or Lys3-BN.

Refe r ences

Avis. 1.L .• Kovacs. T.0 .G .. Kasprzyk. PG. Trc,ion. A.M. Banholorncw. R .• Wobh. J.H .. Cuuiua. F. and Mulshinc. J.L .. J. N:ul. Cancer ln,L. 83(1991)1470

2 Vredenburgh. JJ. nnd Ball. E.D. Cancer Res .. 50( 1990)7216 3 Moody. T.W. and Cuni11a, F .. Life Sci. 52( 1993)1161 4 Guyrc. P.M .. Graziano. R.F .• Vance. B.A .. Morgancllt. P.M and Fangcr. M.W. J lmmunol.,

143( 1989) 1650.

820

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