`European Peptide Symposium
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`•
`I
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`Editor: Hernani L.S. Maia
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`ESCOM
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`Celltrion, Inc., Exhibit 1017
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`W:l PF:528
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`1994
`v . 23
`C. Ol--------SEQ : P07580000
`TI: PEPTIDES
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`02/HJ/9 7
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`@) PROP E R T Y OF T HE
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`NATIONA.L
`LIBRARY OF
`MEDICINE
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`Peptides 1994
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`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·
`Ill~
`II
`· 1111111111 lll\
`ESCOM • Leiden • 1995
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`CIP-data Koni nklijke Bibliotheek, Den Haag
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`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 90-72 199-21-9 (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 otherwise, without prior permission in writing from the copyright holder.
`
`Printed in The Netherlands
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`Contents in brief
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`Abbreviations
`The Fifth Josef Rudinger Memorial Lecture
`Lectures
`
`l
`11
`nr
`IV
`
`v
`
`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 Immunol(cid:173)
`ogy or Peptides
`Receptors, other Binding Proteins and Biological Aspects of
`Peptides
`
`Posters
`
`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
`
`VI
`VII
`VJll
`
`IX x
`XJ
`XII
`XIII
`XIV
`xv
`xvr
`xvu
`xvrn
`Author Index
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`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
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`923
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`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 high(cid:173)
`affinity 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 maleimidc(cid:173)
`containing 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
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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.
`
`Co ncl us io ns
`
`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
`143( 1989) 1650.
`
`lmmunol.,
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`820
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