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`43d
`
`Biothemital Society Transactions (2003) Volume 31, part 2
`
`currently in clinical development have been generated using
`this approach; one example is an anti—(transfon'ning growth
`factor ($2) mAb for the treatment of fibrotic disorders [6].
`Finally, transgenic animals have been generated [7,8] in which
`natural lg genes are deleted and replaced with the human
`loci necessary for the production of lgG/IgM. This in nine
`approach has been used to generate several mAbs, including
`ABX-EGF (Abgenix Corp), a high—affinity anti—[epidermal
`growth factor (EGF) receptor] antibody for use in the
`treatment of EGP—responsive tumours [9]. In summary, these
`techniques have reduced the immunogcnicity of mAbs to
`an extent where repeated doses can be administered without
`impaired efficacy.
`
`Antibodies by design: the right tools
`for the job
`A sound understanding of the biochemical pathway being
`targeted is central to the ‘wish list’ for a mAb therapeutic. In
`many applications the recruitment of host effector functions
`through Fc receptors (including complement fixation and
`antibodysdependent cell—mediated cytotoxicity) is essential.
`In humans the IgGI isotypc is the preferred therapeutic
`choice for triggering effector cascades. An approved antibody
`with the potential to trigger effector function is Herceptin,
`an an ti-I-IERZ/neu antibody used in the treatment of breast
`cancer. In addition to the well-described ability to block
`HerZ-depcndent growth, some of the clinical benefit from
`Herccptin treatment is thought to arise from an ability
`to promore antibody—dependent cell-mediated cytotoxicity
`[10]. Conversely, when target neutralization is the only goal,
`it may be preferable to use the IgG4 isotype that is incapable
`of triggering these cascades [11]. Irrespective of isotype there
`are several additional benefits to the IgG format. Divalent IgG
`molecules have the potential to cross-link cell—surface antigen
`in the absence of macrophage/natural killer cell Fc receptors.
`For example, in some CDZO—positive cell lines cross-linking
`has been shown to induce apoptosis [12]. An alternative
`to using this is to use antibody fragments such as Fab or
`scFv. Both of these formats are unable to trigger effector
`function and because of a reduced size have an increased
`
`tumour penetration. These fragments are especially suited to
`the delivery of ‘payload' (see below). One obvious drawback
`of Fabs/scFvs is a reduced half—life (hours compared with 2—
`3 weeks with IgG). It is, hOcher, possible to improve the
`serum longevity of these proteins by conjugating to inert
`polymers such as poly(cthylene)glycols [13]. Examples of
`cancer targets where neutralization is the primary goal include
`matrix metaIIOproteascs (‘MMPs'}, urokinasc plasminogen
`activator ('uPA’} and vascular endothelial growth factor
`(‘VEGF’), which are all secreted proteins associated with
`tumour progression.
`
`Antibody conjugates: adding insult
`to Inflll’V
`I: was Paul Erlich in 1900 who first coined the phrase ‘magic
`bullet’, accurately predicting the value of antibodies as smart
`
`r2003 Biochemical SocietyI
`
`2
`
`weapons in the delivery of destructive payload [14]. A
`number of approaches have been developed, which in the
`context of anti—cancer protocols provide measurable im-
`provements
`in cell killing. The major categories
`are
`radioisotopes, protein toxin—enzyme fusions and small-
`molecule conjugates.
`Antibodies are routinely used to concentrate doses of
`radiation in tissues for both therapeutic and diagnostic pur—
`poses. Common isotopes used to this end include iodine-I31,
`yttrium-90, indium-111 and technicium-99. Tumour killing
`by unlabelled mAbs is
`limited by the degree of antigen
`density on the tumour cell and the ability to penetrate
`tumours adequately. Although radiolabelled niAbs may be
`less restricted by antigen density in their efficacy they
`Can gain an advantage by a ‘bystander’ effect
`in killing
`antigen—negative tumour cells. Conversely, this phenomenon
`would also be responsible for non—Specific toxicity. The two
`most extensively studied radiolahelled mAbs are chalin
`(”Y—labelled anti—CD20) and Bexxar
`(ml—labelled anti—
`CDZO), the former receiving recent US Food and Drug
`Administration (FDA) approval for the treatment of non-
`Hodgkins lymphoma. For therapeutic purposes “oY—labellcd
`miAbs may be better debulking agents for larger tumours
`because of the increased path length of the emission compared
`with mI—labelled mAbs, which may be preferable for
`targeting post-therapy minimal disease [15].
`Protein toxins are a large group derived from a variety of
`microbial, plant and human sources. Above all other mAb
`conjugates, these toxins can be engineered directly into the
`antibody-constant regions, significantly reducing the mantl-
`facturing costs. Examples include Pscedomonas exotoxin,
`which when conjugated to anti—CD22 has been shoWn to
`dramatically increase cell killing [16]. For plant toxins, de-
`glycosylated ricin (it-Chain has the longeSt and most successful
`history. In one recent report a ricin—CDI‘) conjugate was
`shown to confer a large increase in the ability of antibody to
`kill malignant B-cells [17]. The major foreseeable problem
`with protein toxins
`is
`their potential
`immunogenicity,
`especially when considering microbial toxins to which an
`individual may already have been sensitized. An alternative
`would be to use cytotoxic human proteins. Angiogenin, .1
`human RNase, has been shown to induce apoptosis when
`delivered into the cytoplasm. In one recent publication:
`bacterially expressed CDJOL—angiogcnin fusion protein was
`found to be capable of killing a wide range of CD30+
`Hodgkin-derived cell lines [18]. This toxin may represent
`a clever way to avoid host immune responses.
`The third payload group comprises toxic small molecules
`which are usually DNA-complexing agents or inhibitDrS
`of the cell cycle. In this situation the antibody conjugfltc
`is internalized and the toxic drug liberated after cleavage
`of a pH- or enzyme-sensitive linker. As mAbs can tar;cc
`chemotherapy exclusively to cancer cells, more pow"t
`chemotherapy can be used when attached to mAbs than
`when administered systemically, for example maytansil‘lc
`conjugates [19]. Small toxic drug molecules have petenrifll
`advantages: in general they have a negligible immunogenicitl'
`
`

`

`r135
`Drug Discovery and Design
`
`phase I clinical trials. Mechanisms of action for conjugated
`and naked antibodies are illustrated in Figure 1.
`
`Problems, solutions and evolution
`We can infer from the previous discussion that the modern
`antibody investigator has the technological tool kit necessary
`to ensure the smooth transition of a project from antigen
`discovery to therapeutic mAb. What
`then is the major
`bottleneck that still exists in the discovery process? The
`completion of the human genome combined with advances
`in proteomics technologies have helped to enhance our
`understanding of the complex interplay between genetic,
`transcriptional and translational alterations in human cancers.
`Although bioinformaticians have made strides in identifying
`potentially interesting novel cancer targets there is a central
`bottleneck at the point when cancer biologists must inves tw
`igate each individual gene for therapeutic potential. These
`assays are time—coasurning and labour—intensive. Looking at
`the current crop of approved antibody targets, they have
`all benefited from around 10u20years of detailed academic
`research. The future goal will be to design high-throughput
`biology modes to move novel cancer targets quickly towards
`clinical development.
`Insight
`into the future of antibody therapeutics can
`probably be glimpsed through technologies such as ribosome
`display. Here, antibody-fragment libraries are not displayed
`on bacteriophage but are produced entirely in ot’tro and
`there is also the potential to introduce mutagenesis steps
`into the antibody~encoding RNA sequences. This system
`has the advantages of greater library size (2—10”), potentially
`higher mAb affinity and improved speed compared with
`standard phage display [21]. If this technique tells us anything
`about the future of antibody discovery it suggests that soon
`the generation of humanized/human mAbs may become as
`routine as PCR is today: We await the release of the first
`human antibody-generation kit!
`
`Conclusions
`
`Kohlcr and Milsteins’ dream of mAbs as exquisitely sensitive
`therapeutics has finally been realized. Two antibodies,
`I-Ierccptin and Rituxan, have proved that this class of drugs
`can be effective (and highly lucrative) anti—cancer agents
`for those companies brave enough to enter this research
`'graveyard’. From a stalled start, mAbs now represent
`around 25% of the novel biological entities entering clinical
`trials, indicating that the biotechnology community at large
`has finally recognized the speed and efficiency of
`the
`antibody platform. It is also becoming clear (and it is ironic)
`that the classical monoclonal antibody may yet become
`the commercial saviour of the l’llgl'lvtccl‘l proteomics and
`genomics revolutions.
`
`References
`‘I Shawler, D L, Bartholomew, RM, Smith, L M and Dillman, R 0. (1985)
`J. Immunol 135, 1530-1535
`
`3
`3
`
`s 2003 Bioc hemicat Society
`
`figul'e 1| Mechanisms of action for therapeutic antibodies
`The therapeutic potential at man resides in an ability to modulate
`several dilleterit pathways (A) For a receptor involved in a growth-
`
`pra-noting pathway. mnh binding may prevent ligand binding and signal
`transduction tag. mobs against EGF receptors and HerZ/c-erb-BZ). In a
`SllllllEll fashion. mnbs may target cell-surface growth factor receptors
`fo| degradation as opposed to a natural recycling process (Hetcepttn
`anti-Herz).
`(B) Cell~suttace cross-linking may initiate a cascade that
`kills
`luntour
`cells
`(cg. Rituxamnediated cross-linking of
`(020).
`
`(c) Several mat) formats have the tapacrtv to activate the classical (Clo)
`{fltflplemEfil pathway. (D) Macrophages and natural killer cells express
`cell
`.iillfafli' Fc receptors which, when bound to immobilized rnnb,
`become activated and secrete cytotoxic mediators. {E} Finally] mAbS
`can no engineered to deliver toxic pavloads including radionuclides,
`
`protein toxins and small molecules (Bexxar, mI-labelleci anti-C020, and
`Mylotarg Zogamicin anti-C033)
`
`A
`
`B
`
`C
`0 O
`
`0A A 0A0
`J a
`
`+ Y
`
`YYY
`
`_
`
`sk-
`
`
`
`
`
`and compared with radionuclides are relatively easy to
`_ hal'tclle. Mylotarg is
`a humanized anti-CD33 linked to
`Caliclteamicin. This useful therapy for acute myelogenous
`[What-mitt ('AML’) was the first toxin coniugate ever to be
`alitrarnvecl, showing that thc small-molecule payload group
`has iust as much validity as other regimes. Recruitment
`of effector cells using bispecific antibodies (one specificity
`Elirected towards the tumour, the other to the effector cell) has
`“'50 been reported widely [20]. These reagents are expected to
`be difficult to manufacture in large quantities but have shown
`Promise in eitro, in animal models and now also in some
`
`

`

`436
`
`alod'remical Society Itanmtlons (2003) Volume 31, part 2 -
`
`2 Queen, C, Schneider, WP. and Selick, H1 (1989) Proc. Natl. Mad.
`Sci. USA. 86, 10029—10033
`3 Winter, 6., Grilliths, All, Hawkins, RE. and Hoogenboom, HR. (1994)
`Annu. Rev. rmmunol. 12, 433—455
`4 Marks, J.D., Hoogenboom, HR, Bonnert, LP, McCaflerly, J, Griffiths, AD.
`-and Winter, 5. (1991) J. Mol. Biol. 222, 581-597
`5 Griffiths,A.D.(1993)Curr1Opin. Immunol. 5, 263-267
`5 Thompson, J.E., Vaughan, TJ. and Williams, AJ. [1999}
`J. Immunol. Methods 227, 1H9
`7 Mendez, M], Green, LL, Corvalan, 1R, 11am, Maynard-Currie, CE,
`Yang, X.D., Gallo, ML, Louie, BM, Lee, 0.9., Erickson, KL. et al. {199?}
`Nat. Genet. 15, 146-156
`3 Ishida, I, Tornizuka, K, Yoshida, H., Tahara, r, Takahashi, N, ohguma, A,
`Tanaka, 5., Umehashi, M, Maeda, H., Nozaltl, C. et al. (2002) Cloning
`Stem Cells 4, 91—102
`9 Yang, it.D., Jla, XL, Corvalan, JR, Wang, P. and Davis, (.6. (2001)
`Crit. Rev. Oncol. Hematoi. 38, 17—23
`10 Baselga, l. and Albanell, 1. (2001) Ann. Oncol. suppl. 1,
`SSS-S41
`11 Antoniw, P, Farnsworth, AP, Turner, &, Haines, AM, Mountain, PL,
`Mackintosh, J., Shochat, o, Humm, J, Welt, 5., old, L]. et al. (1996)
`Br. J. Cancer 74, 513—524
`
`12 Shan, D, Ledbetter, M. and Press, ow. (2000) Cancer Irnrnunol.
`Immunoiher. 48, 623—683
`'
`13 Lee, LS, Eonoirer, C, Shi, C, Whitlow, M. and Filpula, D. (1999)
`Bioconj. Chem. '10, 9?3-981
`14 Erlich, P. (1900] Proc. R. Soc. London. 66, 424
`15 Eelenetz, AD. {1999) Curr. Opin. Oncol. 11, 375-380
`16 Mansfield, E, Ar'nlol, P, Pastan, J. and FlrzGeraIrJ, DJ. {1997) Blood 9o,
`2020—2026
`17 Conry, RM, Khataeli, MB, Saleh, M.N., Ghelie, V, Vltella, ES, Liu, T
`and LoBLrgiio, M. (1995) J. immunmher. Emphasis Tumor immunol
`18, 231211
`18 Huhn, M, Sasse, S, Tur, MK, Matthey, B, Schinkolhe, T., Rybak, SM,
`Barth, S. and Engert, A. (2001) Cancer Res. 61, 3737-8242
`19 Smilh, S. (2001) Curr. Opin. Mol. Ther. 3, 198-203
`2|) Peipp, M. and Valerius, T. (2002) Biochem. Soc Trans. 3|},
`507-511
`21 Hanes, J, Schaitiizel, C, Knappik, A. and Pluckihun, A. {2000)
`Nat. Biotechnol. 18, 1282—1292
`
`
`Received 20 November 2002
`
`92003 Biochemifil Society
`
`4
`
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`n Cover illustration | THP—I
`macrophages treated with
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`,’ nttrans.o;g
`
`HSL
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`transactions
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`iochemital Society
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`BSCTBS 31(2) 399-435
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`iSSN: 0300-512?
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`April 2003
`
`Volume 3! part 2
`
`;
`
`Genes in food—why the furore?
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`Poiyamines and their role in human disease
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`Drug discovety and design
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`Human ageing: from the bench to the clinic -
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