NOVA ACTA LEOPOLDINA
`Ahhandlungen der Deutscben Akademie der Naturforecher Leopolilima
`1w Auftrage des Pxsidinms herau8gegeben von
`JOACIHM-HERMANN SCHARF
`Director Ephemeridum der Akademie
`
`NEUE FOLGE
`
`NUMMER 269
`
`BAND 61
`
`Leopoldina-Symposium
`Functional and Regulatory
`Aspects of Enzyme Action
`May 25 to 28, 1988
`in Halle (Saale)
`
`Organized and edited by:
`Ernst J. M. HELMREICH (Wiirzburg)
`Mitglied der Akademie
`Helmut HOLZER (Freiburg)
`Mitglied der Akademie
`
`Alfred SCHELLENBERGER (Halle)
`Mitglied des Senates der Akademie
`
`With 148 Figures and 37 Tables
`
`Deutsche Akademie der Naturforscher Leopoldina. !{aHe (Sflak) 19R9
`EXHIBIT 1193 Jefferson Foote, Ph.D.
`2/4/18 Planet Depos-T Rosate, RDR. CRR, CSR 10891
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`Redaktion: Prof Dr .nEd.flr,rer.nat .Dr .h. C. Joachin-Hermann SCJARF als
`Director henerithmi,
`Prof.Dr. sc .nat. Alfred SCMZZNBffCM und
`D:ipl.-Phys. Rainer-M. JAcOBI,
`wissenach. Assistent der AIdnie
`
`Die Zeitathrift erscheint im KzmxLssicnaverlag Johann Aithrosius Barth,
`DM-7010 Leipzig, PostschlieBfach 09, Ruf 70131.
`Jedes Heft let einze]n käuflich!
`
`Lizenztrr: Deutsche Pdiadeinie der Naturforscher Leopoldina, Halls (Saale)
`Chefredakteur und Herausber: Prof .Dr.Dr.Dr.h.c. J.-H. SC}MRF,
`1R-010 Halle (Saale), Postschliefach 302
`Veröffentlicht unter der Lizenznunir 1393 des Presseamtes beini Vorsitzen-
`den des Ninisterrates der L
`Printed in the German Democratic Republic
`Gesamthersteflung: Drucldiaus "Freiheit", Halle (Saale), BT !rseburg
`
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`
`CONTENTS
`
`Preface
`
`Chapter 1: Enzyme Catalysis - Basic Principles;
`
`S1N, R. L.: 1rEition-state Structure and Its Bole in Enzyme
`Catalysis and Enzyme Regulation
`
`HUHER, R.: Flexibility and Rigidity in Proteins and Protein Pig-
`ment Complexes
`
`FISGJER, G.: Slow Conforciatiorial Charges and Their Enzymology
`
`Chapter 2: Enzyme Catalysis - Theoretical Aspects
`
`BLUNLEIL, T.L.: TheThree-dimensional Structures of Aspartic
`Proteinases and Their Inhibitors - Lessons for 1ug Design
`
`KELETI, T.: Kinetic Power, a Key Parameter of Metabolic Coiitro].
`in Homogeneous and Heterogeneous Systems
`
`HESS, B., NARXS, N., HOLIER9 S.C., and PLESSER, T.: Nonlinsar
`Dynamics in Chemistry and Biology
`
`tDO]E, J.: Humanized Antibodies
`
`Chapter 3: Enzyme Catalysis - Special Mechanisms
`
`IHAIJER, R. K.: Structure and Fumtion of ?thyl-06M Reductase,
`a Nickel-Porphirx,id Containing Enzyme in ?Thamgenic
`Axaebactera
`
`(1IRIS1E}49
`IT)BLER, H.P., NEBT, P.,
` P., WIRING, H., KOCEIAR, S .,
`and HALE, T.: Enzymic Vitamin B6-Catalysis: Medbardstic and
`Evolutionary Aspects
`
`9
`
`13
`
`31
`
`35
`
`57
`
`59
`
`79
`
`103
`
`113
`
`123
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`
`
`PAU19
` ID., and SG1I4L, R.: The Mechanism of a-G]ncan P1csorylases
`Studied by Substrate Analogs and Site-directed l4itagenesis
`
`143
`
`Chapter 4: Enzyme Catalysis and Regulation - Role of Domain
`Siucture and Subunit IritematiorE
`
`NAADOVA, N.K.: Furtional Aspects of Protein-Protein Interactior
`in Oligairc Enzymes. NAD
`dent Othydrogeriases
`KLEINKAUF, H., and v. D}1REN, H.: Interacting .?tzl1ienzyu
`
`LANE, A.L, SIReSSER, A., and KIRSC1NER, K.: Catalysis of Coupled
`Reactions. Ccmamicatton Between the Two Active Sites of
`Tryptophare Synthase
`
`Chapter 5: Enzyme Regulation - F\iixtiona]. and ,Kinetic Aspects
`
`SCHAQ!WtN, H.K.: Effects of Amino Acid Substitutions on the
`Catalytic and. Regulatory Properties of 'E. Coli Aspaxvtate Frere-
`oa±anylase (A2ase)
`
`HERVf, G.: Mechanism of Allos-teric Heterotropic Interactions in
`Edierithiä Coli Aspartate Trarscarbamylase
`
`SCHELLENR(R, A., HOBNER, C., K5NIG, S., FLATA.U, S., and
`NEEF, H.: Substxte Activation of Pyruvate Decartoxylase -
`Mechanistic Aspects
`
`HOBNER, G., and WOLNA, P.: Oscillations in Regulator Enzyme
`Systerm at Constant Substrate Input
`
`Chapter 6: Enzyme Regulation - Special Systems
`
`HOLR, H.: Regulatory Protein Phosorylation in Yeast
`
`HOFMANN, E., KELS(fR, M., FREN7EL, J., and SQ1EUENBERR, W.:
`Regulation of 'the Fructose 6-Phosphate/Fructose 2,6-Bisphos-
`phate Cycle
`
`HEKMN, H., PEUFER, T., and HEUIREIai, E.J.M.: Protein-Protein
`Interactions in Hormonal Signal Transmission
`
`169
`
`189
`
`209
`
`213
`
`217
`
`225
`
`23
`
`253
`
`269
`
`289
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`
`
`
`
`WALTER, U., B1XiLER9 W., FEINEQ(E, N., NIE&RDING, N., WALtM'NM, R.,
`sseier-regulated Protein Kineses
`and LOHMANN, S.M.: Second
`and Their Role in Hepatocyte and Platelet Function
`
`SEVERIN, E.S., and AIAXHOV, V.Yu.: Role of Ca
`Regulation of Incellular Sys t
`
`and cAMP in the
`
`Chapter 7: Protein Turrxyver and Intracellular Proteolysis
`
`LEVINE, R.L., BIVETT, A.J., and (IRVERA, J.: Oxidative Modifica-
`tion of Proteins: Potential Physiologic and Pathologic Roles
`
`SWITR, R. L.: Regulation of Selective Intracellular Proteolysis
`in &ZlZZW eubtilia
`
`XkIUN, N., and KNANI, E.: Mechanisms and Regulation of
`Protein Degradation in I.ysosame
`
`Chapter 8: Enzymes - IntraoeThilaD Traffic
`
`BWL, C.: Intace].lular Protein 'Ibpogenssis
`
`RAPOPORT, TA., WIEFM4NN, M. KURZa1ALIA, T.V., and HAR'IMANN,. E.:
`Signal Recognition in Protein Trarslooation Across the Endo-
`plasmic Reticulum Nenbrere
`
`VEST!.*&R, D., and Sa!ATZ, G.: Blockage and Quantification of
`Nitochorxlrial Protein Import Sites by an Internally Cross-
`lirked Hybrid Precursor Protein
`
`311
`
`329
`
`355
`
`367
`
`369
`
`385
`
`399
`
`409
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`Nova acta Leopoldina NF 61 Nr. 269, 103-110 (1989)
`
`Humanized Antibodies
`
`by Jefferson FOOTE(Cambrldge)
`
`With 2 Figures and 2 Tables
`
`Summary "Humanized* antibodies are created by trnsplant1ng, via recombinant
`IA techniques, the antigen binding site of a rodent monoclonal antibody onto a
`man immunoglobulin. This process has been applied three limes in our laboratory,
`Irting with antibodies to the hapten 3-nitro, 4-hydroxy-phenacetyl, to the protein
`tigen tysozyme, and with a therapeutic antibody, Campath-1, recognizing a human
`nphocyte marker. Findings obtained with each of the three examples are discussed.
`
`Physicians have long faced the dilemma that whereas antibodies of specificity
`against any pathogen can be raised in animals, the use of such antibodies in human
`patients is accompanied by potentially lethal allergic reactions, conversely, human
`antisera are more safely administered, but very few specificities are obtainable from
`human volunteers Monoclonal human antibodies made by in vitro operations would
`seem to present a way out of this dilemma, but currently, the human-based analog of
`the munne hybridoma technology Is beset with methodological problems (CARSON
`and FREIMARK 1986)
`As an alternative to a frontal assault on the activation, selection, and propagation
`of human lymphocytes, several groups at the Laboratory of Molecular Biology have
`attempted to convert mouse monoclonal antibodies to human form by the methods of
`protein engineering. Michael Neuberger developed a system for stably re-introducing,
`and expressing, cloned immunoglobulin genes in cultured mouse plasmacytoma cells
`(NEUBERGER 1983) The gene structure of immunoglobulins is such that the variable
`domains, which determine antigen specificity, and constant domains are encoded on
`separate exons This makes It very easy to create a 'chimeric molecule with human
`constant dotnains replacing their mouse counterparts This was achieved without
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`apparent effect on antigenic specificity (NEUBERGER et al. 1985). However, even in a
`chimeric antibody, retaining the variable domains dictates that fully a third of the
`molecule will remain "mouse".
`Homology studies (WU and KABAT 1970, KABAT et al, 1987) demonstrated that
`the variable domain of an immunoglobulin was composed of four "framework" regions
`of highly conserved sequence, sandwiching three regions of extreme sequence
`vanabllhty. Biochemical studies and X-ray crystallography confirmed that residues in
`these positions were almost exclusively the ones which interacted directly with antigen,
`hence the name, "complementarity determining regions (CDR's). Several years ago,
`Greg Winter proposed that the CDR's of a mouse monoclonal antibody could be
`combined, via gene synthesis, with human framework sequences, to yield a
`"humanized'* variable domain. Such constructs In turn could be joined with human
`constant regions, as in a chimeric, to constitute a molecule indistinguishable from a
`human antibody.
`Two questions may be asked of a humanized antibody. Does it continue to bind
`antigen? Does it function in vivo, finding its target white escaping the surveillance of
`the patients immune system? The former question, a problem in structural chemistry,
`has been easier to answer.
`
`A genomic clone was obtained of the heavy chain of a mouse hybridoma line
`specific for this hapten (NEUBERGER 1983). (The hybridoma was of the lambda type.
`A peculiarity of this system is that there is very little heterogeneity in mouse lambda
`sequences, hence antigen specificity is largely a function of the heavy chain
`sequence.) A gene was synthesized from oligonucleotides, with a corresponding
`protein sequence Identical to the mouse sequence in the region of the CDR's, and a
`framework sequence identical to that of the human myeloma protein NEWM (SAUL et
`al. 1978). The mouse and humanized sequences are compared in Fig. 1. Shifting to
`the NEWM frameworks has the effect of putting 37 point mutations in the mouse gene.
`This construction, and the unmodified mouse variable domain, were each joined with
`human IgE constant regions, and transfected into a cell line which ordinarily secretes
`only a mouse lambda light chain (JONES et al. 1986). Clones were selected which
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`secreted complete antibodies into the culture medium, consisting of the mouse lambda
`chain in combination with the humanized heavy chain or the parallel mouse control.
`
`Mouse
`Humanized
`
`
`
`framework 1
`
`30
`I
`QUQLQQPGRELUPSRSUKLSCKRSSYTFT
`SVIII1H
`QUQLQESGPOLURPSOTLSLTCTUSOSTFS
`.••s•••..
`u..
`•
`•
`
`framework 2
`
`49
`36
`IWKQRP5RGLEUIB
`UUROPPSRGLEII I G
`.•
`framework 3
`
`RI DPI$SGGTKYHEKFKS
`
`95
`67
`KRTLTUDKPSSTRYIIQLSSLTSEDSRUYYCRR
`RU 11* UDT SK N QF SLRL S SUTRA OTR UY V CRR
`• 55
`.. S. S••SS•••S
`
`VDVYGSSVFDY
`
`framework 4
`120
`110
`IIGQGTTLTUSS
`U QG S LUTUSS
`S..
`
`Fig. 1 CDR grafting in the heavy chain of anti-NP
`
`The antigen binding properties of the two antibodies were tested by the
`fluorescence quench method (EISEN 1964), using the 5-amino-caproic amide of NP.
`As seen In Table I, the humanized molecule bound the hapten with an affinity less than
`a factor of 2 weaker than the mouse control. A second peculiarity of this system is that
`the anti-NP hybridoma binds the 5-lodo derivative of NP, NIP, more tightly than NP
`itself, even though the latter had been the original immunogen. Accordingly, a second
`measurement was made, of the affinity for NIP. Again the humanized molecule showed
`an affinity just slightly weaker than the mouse construct (Table 1). Thus not only was
`antigen binding retained through humanization, but also specificity In distinguishing
`between an Iodine and a Hydrogen atom at the same ring position. In structural terms,
`the hapten binding site on the humanized construct must be virtually identical to that of
`the mouse antibody, despite the 37 point mutations in the frameworks.
`
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`Table 1 Hapten Dissociation Constants from Mouse and Humanized anti-NP
`
`Mouse
`Humanized
`
`NP
`
`1200 n
`1900
`
`NIP
`
`20 n
`70
`
`Although grafting the CDAs onto the human frameworks was clearly a success in
`the anti-NP case, it must be kept in mind that only the heavy chain was humanized.
`Furthermore, the haptens were small molecules, and bound to a small pocket on the
`heavy chain, whereas true antigens would be macromolecular, and would bind to a
`much larger surface on the variable domain
`A more realistic target for humanization was the mouse anti-lysozyme whose
`crystal structure has been solved In Roberto Poijak's laboratory (AMIT at at. 1986)
`cONA clones were obtained for both chains A humanized anti-lysozyme heavy chain
`was made directly from the anti-NP construct, using three synthetic oligonucleotldes
`which spanned the CDRs. A human IgG2 constant region was added to complete the
`heavy chain The heavy chain was expressed as before, and then assembled In vitro
`with the mouse anti-lysozyme light chain (VERHOEVEN at al. 1988). This
`half-humanized molecule also bound lysozyme, again with somewhat reduced affinity
`relative to the original hybridoma, thus extending the findings In the humanized
`anti-NP experiment to a macriomolecular antigen
`A completely humanized antl-lysozyme was made The CDR sequences from the
`kappa tight chain were combined with consensus human kappa frameworks (The
`frameworks were very similar to those of the human kappa light chain dimer REI (EPP 4
`at al 1974), of known three-dimensional structure) A human kappa constant region
`was added, and the light and heavy chain constructs were co-transfected into a
`Previously non-secreting mouse myeloma cell One. Complete immunoglobulin was
`subsequently purified in sufficient quantity for physical studies comparing the mouse
`and humanized antibodies In their interaction with lysozyme
`The fluorescence emission spectrum of the complex of anti-lysozyme and
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`lysozyme is considerably quenched relative to the sum of the independently obtained
`emission spectra of the two molecules. In the case of the mouse antibody, the
`quenching is so extreme that the addition of lysozyme, though itself fluorescent,
`actually causes the overall fluorescence of an antibody sample to decline. This is
`shown In Figure 2, in which the mouse anti-tysozyme and a control antibody directed
`against an entirely different antigen are titrated with lysozyme.
`
`650
`
`600
`
`I
`j550
`
`p500
`
`450
`
`400
`
`350(cid:9)
`
`U
`
`1
`
`lysozyme/anUbody
`
`Fig. 2 Stoichlometry of lysozymeanti-lysozyme spectral change.
`The curvature in Figure 2 in the vicinity of the titration breakpoint is a function of
`the equilibrium constant for the formation of the antibody-antigen complex. The value
`of this equilibrium constant can be calculated by performing titratlons at several
`antibody concentrations and fitting the resulting data to a simple binding equation This
`method indicates a dissociation constant of 2 nM for the mouse, and 70 nM for the
`humanized antibody.
`Rapid kinetics measurements, using a stopped-flow apparatus, and exploiting the
`same spectral change, show very similar association rates for the mouse and
`humanized antibodies The difference in the equilibrium constants is due to a half-life
`on the order of 10 minutes for the lysozyme complex with the mouse antibody, and on
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`41,11,
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`the order of 10 seconds in the case of the humanized molecule.
`The anti-lysozyme system has been developed for answering questions of
`structural chemistry concerning the interaction of humanized antibodies with antigen.
`Given the known three-dimensional structure and the physical techniques which have
`been applied, the effects of mutational alteration on the thermodynamic and kinetic
`parameters of this system can be interpreted in a meaningful way.
`
`4. Humanized Campath4
`In our approach to second question posed of a humanized antibody, whether it
`functions as intended In the human body, we have attempted to develop a
`therapeutically useful molecule.
`Several years ago, Herman Waldmann and his colleagues developed a rat
`monoclonal antibody, Campath-1 (HALE at al 1983) LIttle is known about the
`function of the human antigen It recognizes, but what is significant Is that this antigen
`is found on the surfaces of only a few differentiated cells of the immune system, B and
`T lymphocytes, and monocytes, not on the undifferentiated stem cells, or on any other
`cell type. The rat antibody has been used clinically for treating graft-versus-host
`disease following bone marrow transplants, suppressing tissue rejection after organ
`transplants, and for the direct elimination of leukemic coils. Its drawbacks are the
`occurence of severe side effects, including anaphylactic shock, and that its
`effectiveness is compromised after approximately ten days by the induction of an
`anti-rat response.
`As Its limitations seemed attributable entirely to its rat origin, Campath-1 seemed
`a good target for humanization. cDNA clones were made of the heavy and light chains.
`A humanized light chain gene was converted from the anti-lysozyme construct. A
`humanized heavy chain gene was converted from the anti-NP. In the latter
`construction, the change Ser-27 -> Phe was introduced Modeling had suggested that
`this would Improve affinity; many human sequences occur with Phe In this position, so
`this change would not be expected to create an immuno1Oglcal inconsistency. Human
`kappa and IgGi constant regions were used to complete the construction
`(RIECHMANN at al. 1988). (A number of other rat and human heavy chain isotypes
`were employed In constructions used as controls.)
`
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`It
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`The humanized Campath-1 obtained after transfection was subjected to a
`number of tests to determine its suitability for use in vivo, and the results of these are
`compiled in Table 2. In an enzyme linked Immunosorbent assays (EUSA), comparing
`the relative affinity for the Campath-1 antigen, a humanized construction (with a rat
`light chain and heavy chain constant region, necessary for a valid comparison) was
`only slightly less active than the rat antibody. In the complement lysis assay, human
`lymphocytes were isolated, and the separate antibodies bound. Human serum was
`added, and complement reactions initiated by the presence of the bound antibodies
`caused the lysis of the cells. In this test, the humanized and rat molecules were almost
`identically effective. In antibody-dependent cell mediated cytotoxicity (ADCC) tests,
`human lymphocytes are again coated with antibody, and their lysis is aóhieved by the
`addition of human effector K cells. In this case the humanized antibody proved more
`effective than the rat.
`
`Table 2 Properties of "Carnpath" Antibodies
`
`ELISA
`Complement Lysis .
`ADCC
`
`Rat
`
`0.7
`2
`0.008
`
`Humanized
`
`2.1
`3
`0.001
`
`The complement lysis and ADCC reactions are realistic mimics of the type of
`reactions expected in vivo to eliminate the Campath-1 target cells. The evidence from
`these in vitro tests indicates that the humanized antibody should work as well as the rat
`molecule as a therapeutic agent. Clinical trials starting now should show whether the
`immunogenicity of the humanized Campath-1 has been reduced to the basal level
`expected of a truly human antibody.
`
`Acknowledgement: J. F. is a Fellow of the Jane Coffin Childs Memorial Fund
`for Medical Research.
`
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`p
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`IF
`
`References
`
`AMIT, A. G., MARIUZZA, R A., PHILLIPS, S. E. V., and POLJAK, R. J. (1986) Science
`233,747-753
`CARSON, D. A., and FREIMARK, B. D. (1986) Adv. Immunol. 38,275-311
`EISEN, H. N. (1964) Methods Med. Res 10, 115-121
`EPP, 0., COLMAN, P. , FEHLHAMMER, H., BODE, W., SCHIFFER, M., HUBER, R., and
`PALM, W. (1974) Eur. J. Blochem. 45,513-524
`HALE, G., CLARK, M., and WALDMANN, H. (I 9W) J. Immunol. 134,3056-3061
`HALE, G., BRIGHT, S., CHUMBLEY, G., HOANG, T., METCALF, D., MUNRO, A. J., and
`WALDMANN, H. (1983) Blood 62,873-882
`JONES, P. T.., DEAR, P. H., FOOTE, J., NEUBERGER, M. S., and WINTER, G. (1986)
`Nature 321,522-525
`KABAT, E. A., WU, T. T., REID-MILLER, M., PERRY, H. M., and GOTrESMAN, K. S.
`(1987) Sequences of proteins of Immunological interest, 2nd ed. (Bethesda:
`Dept. of Health and Human Services)
`NEUBERGER, M. S. (1983) EMBOJ. 2,1373-1378
`NEUBERGER, M. S., WILLIAMS, G. T., MITCHELL, E. B., JOUHAL, S. S., FLANAGAN,
`J. G., and RABBITS, T. H. (I 9W) Nature 314,268-270
`RIECHMANN, L, CLARK, M., WALDMANN, H., and WINTER, G. (1988) Nature 332,
`323-327
`SAUL, F. A., AMZEL, M., and POUAK, A. J. (1978) J. 8101. Chem. 253, 585-597
`VERHOEVEN, M., MILSTEIN, C., and WINTER, G. (1988) Science 239,1534-1536
`WU, T. T., and KABAT, E. A. (1970)J. Exp. Med. 132,211-250
`
`Address correspondence to:
`
`Jefferson Foote
`MRC Laboratory of Molecular Biology
`Hills Road
`Cambridge CB2 20H
`England
`
`110
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