MAbs.
`v. 2, no. 1 (Jan—Feb 2010)
`General Collection
`W1 MA144C
`2010-06-16 07:05:54
`
`V°'Ume 2 ° |ssue1 - JanuaryIFebruary 2010
`w<w~«w-
`‘«Vw4v1'r|\'1|‘|l'vl um .-... ..—..- . .1.........
`"*"-~«~-s~wm.«..»m.».¢...‘..........».,..-wa-«»»»~...w..1 ....a..m..—..mW--1;. mm.~1V"‘1‘\
`
`Editor in-Chief
`_
`.
`Janice M. Relchert
`
`PROPERTY OF THE
`§_ A_:;_
`§ NATIONAL
`LIBRARY or
`umouu lvtnlnllu or uvutl
`E MEDICINE
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`LILLY EX. 1040 - 1/11
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`LILLY EX. 1040 - 1/11
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`

`

`
`
`About the cover. The cover illustra-
`arrangt'e<'>:eSrl‘1tow:a sketch of possible
`Single Ch .5 o oligomeric forms of
`forms 3”‘ '95. Higher molecular
`are found in size exclusion
`chromatograms of an |gG variant
`thVe"':e‘;l/lelifiht chain connected to
`linker It car): beam by a 34 amino acid
`~
`speculated that these
`forms result from a crossover
`assembll’ Process similar to diabody
`Or diFabody formation. For
`additional information see
`
`Schirrmann et al., pp. 73-6,
`
`VOLUME 2 ISSUE 1
`JANUARY/FEBRUARY 2010
`
`Eniioit-iN—Ciiiiai:
`JANICE M. RE|CllliR'I‘
`
`
`
`L”1'_"“_"] EDITORIAL
`1
`mAb therapeutic products and risk management
`Janice M Ri’:‘lCl’l£‘-ll.
`
`O MINI-REVIEW
`3
`Canakinumab
`Eugen Dliimolea
`
`14
`
`REVIEW
`Evolution of anti-CD20 monoclonal antibody therapeut
`E‘/o§}s>lii'i C)i'la;/oglu and l auruii P Aiidoly
`
`ics in oncology
`
`1 REPORTS
`20
`A dua|—targeting PDGFRB/VEGF-A molecule assembled from stable antibody fragments
`demonstrates anti-angiogenic activity in vitro and in vivo
`Rol.‘i€=iT l/iabry, Debra Ci Gilbrsitsoii, /xniaiida liaiik, 'li,iyeii ‘/ii, Dai'i Ardourel, (_ wig ()5i,;md¢.,'
`Brenda Siei/ei"i_<., Siisari Jiilieri, Secil lrarike, Bieiit Mi’;‘(‘| i-gin, lei ll lll:'3l Brody, Scoii Prcgngill‘
`l’i()l)c‘=r1 llcsl-.’)r, ( iiidy Yigii,
`l"lamarjl iei, lvlegaiil aniry, Aiiitia Wolf, Tom lfii_il<.<>wr~,l<i,
`Nels ll
`Monica /\i ldi:>r<.oii»l~la|ey, Keiiiicili lligiscl, Oi l’ai‘i, llaiik l iiiiikliri, l’<‘-iiiiy ll’iorn,r;i<,oii, Mike D(_)(_ld','
`Sara Ui'ideiw::iod, S<oii Pei-erson, Pallavui V 3|‘/£)l’\lJlIltil zll id Marl. ‘,,i‘iav:>ly
`
`35
`
`42
`
`53
`
`n of the CD4*CD25*Foxp3‘ T regulatory cells
`Differential effects of IL-2 and IL-21 on expansioted antibody dependent cellular cytotoxicity
`with redundant roles in natural killer cell media
`in chronic lymphocytic leukemia
`l>i_iili:>i‘ie llr)./z;iwi,l«i, ‘fi/z‘Iyii.rj= Kii ii:l‘.vr>ili‘l
`l‘yi‘i (,lie=ii-73/,
`/iiiiria Ciovvda, /‘islia Ramai iui iiii, Caro
`Jolin ( Byrd illifl l\li"1l_0lE}l£'Il”I l\/llJIlil.l',<llTiy
`Amy l elirriari,
`David l€jiJOUl£l,
`l~/iicliael i’aligii,iri,
`
`Cold denaturation of monoclonal antibodies
`Kristi L Lazar, Thomas W. Paiapoff and ‘i/ikas K ‘sl’iaini«;i
`
`Neutralizing epitopes of the SARS-CoV S-protein cluster independent of repertoire, antigen
`structure or mAb technology
`,|l"IIiI Waiig, Fiaiicis A Pli_mim.ui,
`Jody D. Berry, Kevin Hay, James M. Rini, li-/leng Yu, l
`Cindi R. Corbett and Anton /\.r'idoi“iov
`
`1 BRIEF REPORTS
`
`Target mediated dispo
`Application in the detection 0
`Slivveta R Uiva and Jo?/;=p|‘i P Balthazar
`
`sition ofT84.66, a monoclonal anti—CEA antibody:
`fcolorectal cancer xenografts
`
`67
`
`73
`
`s of single chain immunoglobulin (sc|gG)
`Oligomeric form
`l\/ienzel, l\/iirilmel l'lLl3l,
`l{?3L|’ a Prilop, 'l l ioinas Jostorl
`Thomas Schirrm.:—ii"in, Cliristian
`and S1&‘I'ai‘i Di.iliel
`
`O POINT OF VIEW
`77
`Antibody fragments: Hope and hype
`/\aroi'iL Nelson
`
`This material was csopiesj
`at the N LM a rid may be
`Subject LIE {in-pyright‘ Laws
`
`LILLY EX. 1040 - 2/11
`
`LILLY EX. 1040 - 2/11
`
`

`

`L‘::‘::] EDITOR’S CORNER
`84
`Antibodies to watch in 2010
`Jamie M Rev h-art
`
`Note of appreciation to reviewers
`Jamie M F’.E‘i(i’IE*rl,
`
`Upcoming meetings
`Jarur M R=‘wCrwrr
`
`LILLY EX. 1040 - 3/11
`
`LILLY EX. 1040 - 3/11
`
`

`

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`
`./ill’) l.«')l'l{l{"-r i2'l')’,4,l-.’*li(,v'
`riir—-Jr. j’. l.
`r‘'/ 9.1: lriiiii.ri"y'l~lriiiai_v ./l)l’7,'
`L
`
`POINT OF VIEW
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Antibody fragments
`
`Hope and hype
`
`Aaron L. Nelson
`
`Tufts University School ofMedicinc; Boston, MA USA
`
`he antibody molecule is modular and
`separate domains can be extracted
`through biochemical or genetic means.
`It is clear from review of the literature
`
`safety and efficacy of antibody products.
`It is tempting to further speculate that the
`properties of designed antibodies could
`confer additional benefits for bionianufac—
`
`that a wave of novel, antigen—specific
`molecular forms may soon enter clini—
`cal evaluation. This report examines the
`developmental histories of therapeutics
`derived from antigen-specific fragments
`of antibodies produced by recombinant
`processes. Three general
`types of frag-
`ments were observed, antigen—binding
`fragments (Fab), single chain variable
`fragments (scFv) and “third generation”
`(3G), each representing a successive wave
`of antibody fragment
`technology.
`In
`parallel, drug developers have explored
`inulti—specificity and conjugation with
`exogenous
`functional moieties
`in all
`three fragment types. Despite high hopes
`and an active pipeline, enthusiasm for
`differentiating performance of fragments
`should, perhaps, be tempered as there are
`yet few data that suggest these molecules
`have distinct clinical properties due only
`to their size.
`
`Introduction
`
`long
`have
`designers
`drug
`Antibody
`h
`othesized that the modular nature of
`VP
`
`iminunoglobulins could be exploited to
`engineer “customized” therapeutics, with
`pharinacologic properties optimized for
`specific applications. Thus, half—life and
`distribution, valency, affinity and avid-
`ity,
`tissue penetration and bioactivities
`could each be controlled b
`selection of
`Y
`
`appropriate molecular domains or defined
`genetic
`features,
`thereby theoretically
`allowing developers control over both
`
`This material W‘aS—Efl~|3lErd
`at: the N LM a rird may be
`Eu bjecglfifio-py‘right Laws
`
`turing, such as improved purity, quality
`and quantity of goods, and homogene—
`ity of molecular species produced. This
`is a remarkable vision, and one might be
`tempted to see the beginnings ofthis revo-
`lution in antibody fragment products such
`the marketed therapeutic certolizumab
`pegol (Cimzia).
`focuses on therapeutics
`This report
`derived from antigen-specific fragments
`of antibodies produced by recombinant
`processes of any derivation and discusses
`5/i ofthese molecules that entered clinical
`
`study sponsored by a commercial firm, as
`well as candidates in preclinical develop-
`ment. Fusion proteins such as etanerccpt
`(Enbrel), which is composed ofthe ligand
`binding portion of TNFOL receptor fused
`to an antibody Fc domain, were therefore
`excluded. Due to the extensive literature
`
`describing the technologies and specific
`antibody drug candidates, only selected
`references are provided.
`Three technologies——antigen—binding
`fragments (Fab), single chain variable frag-
`ments (scFv), and “third generation" (SG)
`moleciiles——represent
`successive waves
`of antibody fragment
`technologies (Fig.
`1). Fabs are clearly the most thoroughly
`explored; knowledge and experience was
`generated during the development ofthree
`US Food and Drug Administration (FDA)
`approved therapeutics (Table 1), six agents
`in active clinical development, and 20 dis-
`continued programs, which collectively
`account for 49% of 54 identified antibody
`fragments that entered the commercial
`
`LILLY EX. 1040 - 5/11
`
`77
`
`Key words: atitibody fragments, scFv,
`Fab, technology developtneiit, aiitibody—
`drug conjugate
`Submitted: 11/10/09
`
`Accepted: 11/27/09
`
`Previously published online:
`www.landesbioscience.coin /journals/
`mabs/article/10786
`
`Correspondence to: Aaron L. Nelson;
`Email: aaron.lowell.nelson@gmarl.com
`
`www.|andesbioscience.com
`
`LILLY EX. 1040 - 5/11
`
`

`

`
`
`Fv
`
`-1
`(fd + LC) i_
`
` Fab
`
`1 CH1
`
`CL1
`
`VHl-l/ VH ;
`
`ypes. Depiction of a full size antibody and various antibody fragment
`j types CH constant heavy chain; CL, constant light chain: |gG. immunoglobulin; Fab, antigen bind-
`. mg fragment; SCFV, single chain variable fragment, VH, variable heavy chain; l/L, variablelight chain.
`
`j I University of Braunschweig. Figure used with permission.
`
`bispecific and multimeric molecules will
`expand, creating additional opportunities
`for clinical benefit. Given the coming diver-
`sification of fragment types, it is possible
`that antibody fragments, or modular ther-
`apeutics incorporating antibody domains,
`with efficacy and safety profiles superior
`to full-sized mAbs will be approved in the
`coming decade. Alternatively, fragments,
`as a class, may never empirically out—per—
`form full—si7.ed mAbs generally, but drug
`developers and clinicians might
`identify
`select applications for which fragment-
`derived molecules have unique utility.
`
`Pros and Cons
`
`in
`Fragmentation of antibodies results
`altered physiochemical features of these
`therapeutic molecules. For instance,
`the
`smaller size of fragments permits penetra-
`tion into tissues inaccessible to full-size
`
`mAbs.5"‘ As previously noted, antibody
`fragments may prove easier and less costly
`to manufacture due to the lack ofglycosy—
`lation and relatively small size, which per-
`mits use ofprokaryotic expression systems.
`However, fragments lack the Fc domain
`that serves to both stabilize full—size anti-
`
`bodies and allow FcR—mediated recycling.
`As a consequence, fragments are rapidly
`degraded in humans and have short cir-
`culating half—lives.5 Several strategies have
`been developed to extend the half—life of
`fragments, including conjugation to pro-
`teins such as albumin“ and l’EGylation,7
`which was applied to the FDA approved
`anti-TNFOL Fab,
`certolizumab
`pegol.
`However, biomanufacturing advantages
`of fragment production may be lost
`if
`l’EGylation is required because the pro-
`cess can prove expensive and technically
`challenging.
`Lack ofan Fc domain and the absence
`
`of in vivo processes that select against
`B cell production of unstable antibody
`species can increase the risk of aggrega-
`tion during production or purification of
`antibody fragments, which in turn might
`increase the possibility of iinmunogenic—
`ity in patients. The lack of an Fc domain
`has additional consequences because frag-
`ments can effect therapeutic action only
`by binding either ligand or receptor; they
`do not
`induce Fc—n1ediated
`functions
`
`\
`
`clinical pipeline.‘ scFvs are a less mature,
`but significantly progressed set of tech-
`nologies, with multiple agents in Phase
`3 testing and a growing diversity of mar.
`phologies. The antibody fragment pipeline
`is expanding, with 10 scFvs accounting
`for 40% of the active clinical pipeline,
`and many in preclinical research.‘ Least
`mature are the “3C}” fragment technolo-
`gies, including single domain and “minia-
`turized" antibody therapeutic molecules.
`These classes have few representatives in
`clinical study (6%), but account
`for at
`least lialfofthe identified preclinical pipe-
`line.‘ Moreover, strong interest in explor-
`ing multi-specificity and conjugation with
`exogenous functional moieties continues.
`Therefore, it is clear that a wave of novel,
`antigen-specific molecular forms is now
`entering clinical evaluation; various trends
`in their development are considered here.
`Enthusiasm for differentiating per-
`formance of fragments should, perhaps,
`be tempered as there are yet
`few data
`that suggest
`these molecules have dis-
`tinct clinical properties due only to their
`size. Of the three fragments approved by
`the FDA, only certolizuinab pegol has
`competitors for the same clinical
`indica-
`tions (Crolm disease, rheumatoid arthri-
`tis, ankylosing spondilitis). Although no
`head-to—head comparative trials have yet
`
`78
`
`been conducted, certolizumab pegol offers
`no clear advantages with regard to efficacy
`or safety over infliximab, adalimumab or
`golimumab? Perhaps the most direct, con-
`clusive data regarding comparison of an
`antibody fragment with a full—length form
`will come from trials directly comparing
`ranibizumab (Lucentis) with bevaci7.umab
`
`(Avastin) for the treatment of age-related
`macular
`degeneration. Concern
`over
`the high price of ranibizumab and the
`molecular similarity between the antigen-
`binding domains of both molecules have
`been used to justify several
`trials (clini-
`caltrials.gov identifiers NCT00593450,
`NCT00710229), despite the fact that the
`FDA has not approved bevacizumab for
`this indication. Only in biomanufactui'—
`ing may there be significant differences
`between fragments and full-sized mol-
`ecules, e.g., the smaller size of fragments
`may permit cheaper, faster production in
`microbial systems, although tests of this
`contention have not yet been described in
`the public literature.
`Nonetheless, drug developers continue
`to tinker with an increasingly diverse series
`ofmolecular modules, permitting the cre-
`ation of ever smaller highly—specific bind-
`ing domains and the selection of diverse
`functional modifications.
`In addition,
`it
`
`is likely that clinical evaluation of many
`
`such as antibody-dependent cell-inediated
`
`This i'l'lat‘.E-l'lanl1§§‘g§-°EiEl~|:}lEtd
`at the N LM 5! I'l- may be
`Subject US {lo-piyright Laws
`
`LILLY EX. 1040 - 6/‘I1
`Volume 2 issue 1
`
`LILLY EX. 1040 - 6/11
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`

`

`
`
`Table 1. Monoclonal antibody fragments approved in the US
`Generic (brand) names
`Description
`Anti—GPllb/Illa chimeric Fab
`
`abciximab (Reopro)
`
`ranibizumab (Lucentis)
`
`certolizumab pegol (Cimzia)
`
`Anti-VEGF-A humanized Fab
`
`Anti-TNFalpha pegylated
`humanized Fab
`
`Indication of 1“ approval
`
`Clot prevention in angioplasty
`Macular degeneration
`Moderate to severe Crohn
`disease
`
`Date of 1" approval
`l2/22/94
`
`06/30/06
`
`04/22/08
`
`Sponsor
`Centocor
`
`Genentech
`
`UCB
`
`Fab, antigen binding fragment; GP, glycoprotein; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
`
`complement-dependent
`or
`cytotoxicty
`cytotoxicity unless specifically conjugated
`to an effector moiety.“
`
`Overview of Fragments in
`Development
`
`As part of ongoing studies of therapeutic
`antibody development, Tufts Center for
`the Study of Drug Development recently
`reviewed the progress of 54 fragment
`the1'apeutic candidates in clinical devel-
`opment and 38 molecules in preclinical
`research described in public literature.‘
`Fragments have,
`to date,
`collectively
`performed similarly to their
`full—si7.ed
`brethren in the clinic;
`the two types
`of molecules demonstrate comparable
`cumulative success rates and both must
`
`overcome a primary barrier to progression
`at Phase 2 (Fig. 2). Moreover, most frag-
`ments have been developed with the intent
`to treat neoplastic and immunological
`conditions, matching broader trends also
`observed in full—si7.ed mAbs. A high frac-
`tion of fragments in clinical development
`were conjugated to functional moieties,
`an unsurprising finding given that most
`fragments lack Fc effector domains. That
`said, a subtle, but notable, shift in thera-
`
`peutic indications selected for fragment
`development was
`identified,
`indicating
`that drug developers may be increasingly
`interested in immunomodulation, rather
`
`than antineoplastic activities. Finally,
`successive waves of fragment therapeutics
`that entered clinical development were
`observed, with Fabs followed by second
`generation scFvs, and the initiai represen-
`tatives of a third generation of fragment
`therapeutics,
`including “rniniaturized”
`mAbs and single antigen-binding domain
`molecules.
`
`Although Fabs are discussed, the focus
`ofthis article is on second— and third-gen-
`eration fragment
`technologies,
`including
`scFvs, “miniaturi'/.ed” and single domain
`
`antibodies, as these approaches are the cur-
`rent focus of the industry. Development
`trends that are woven through diverse
`fragment classes, such as the advent of
`multi-specific and functionally conjugated
`molecules, are additionally considered,
`and highlighted molecules therefore may
`be discussed in multiple sections.
`
`Antigen Binding Fragments
`
`Fab agents are the oldest class of motto-
`clonal antibody (tnAb)
`fragment
`thera-
`peutics, demonstrated by the fact that all
`eight fragment therapeutics that entered
`clinical development before 1995 were
`Fabs.‘ This class of fragments is also argu-
`ably the most successful, accounting for
`/19% of fragments to have entered clini-
`cal development and three FDA approved
`clinical applications. Abciximab (ReoPro,
`Centocor/_lohnson 8: Johnson) is a Fab
`fragtnent of a chimeric antibody against
`platelet glycoprotein IIb/IIIa, approved in
`1994 as an adjunct to prevent thrombosis
`during to coronary artery catheteri7.ation
`for ST-elevation myocardial
`infarction.
`Ranibizumab (Lucentis, Genentech) is a
`humanized Fab directed against vascular
`endothelial growth factor A, approved
`in 2006 as a treatment for neovascular
`
`(wet) age-related macular degeneration.
`Certoliztnnab pegol
`(UCB)
`is a peg)!-
`lated anti-TNFOL Fab approved in 2008
`for treatment of Crohn disease. Beyond
`these monoclonal
`fragments, polyclonal
`Fab agents are also marketed,
`including
`CroFab, DigiFab and Digibind. The cor-
`ollary to successful experiences with Fab
`technology is the large number of failed
`projects. Failure and success alike are
`learning experiences, and provide knowl-
`edge that can be applied in pharmacology,
`regulatory concerns and biomanufactur-
`ing, which may explain why numerous
`companies continue to test Fabs in clinical
`development.
`
`Single-Chain Variable Fragments
`
`Single-chain variable fragments (scFvs)
`are recombinant molecules in which the
`
`variable regions oflighr and heavy imrnu—
`noglobulin chains encoding antigen-bind-
`ing domains are engineered into a single
`polypeptide. Generally,
`the VH and VL
`sequences are joined by a flexible linker
`sequence, and a series of variants are gen-
`erated for optitnizing binding affinity and
`stability." Molecular engineers have con-
`tinued to diversity the fundamental scFv
`molecule, resulting in paired scFvs that
`bind to one another through complemen-
`tary regions to form bivalent
`tnolecules
`(diabodies), complementary scFvs them-
`selves produced as a single chain (tandem
`scFvs or tascFvs), and bispecific tandem
`scFvs (bis—scFvs), among others. Nearly
`as many scFv therapeutics have entered
`clinical development
`(19) as Fabs (23),
`accounting for 40% of clinically evalu-
`ated fragments.‘ These candidates include
`12 active and nine discontinued agents,
`most in early development, with all hav-
`ing entered clinical study after 1995. \X/ith
`at least 11 scFvs publicly described in pre-
`clinical research,‘ interest in this technol-
`
`ogy remains strong. From these data, scFvs
`appear to be a promising technology that,
`while
`yet unproven, will have many
`opportunities to achieve clinical success in
`the future.
`
`Five monovalent, monospecihc scFvs
`were developed, only two ofwhich remain
`active projects
`in
`early development.
`ESBA-105 is an anti—TNFOL scFv in Phase
`
`1 development by ESBATech for ophthal-
`mic indications. Efungumab (Mycograb),
`an scFv that binds to the heat shock pro-
`tein of Crznz/it//1 /z//Iiams,
`is in Phase 2
`
`development by NeuTec, a wholly-owned
`subsidiary of Novartis. Novomab-G2 is
`an anti-cancer scFv discontinued in Phase
`
`2 by Viventia after the company decided
`to pursue a formulation that
`included a
`
`www.|andesbioscience.com
`
`This material wastcsn-pied
`at the Nrlllfilgitcl may be
`in bject LIE {fie-pyright Laws
`
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`

`
`
`cytoxic conjugate. Pexelizumab is an anti-
`C5 scFv discontinued in 2007 by Alexion
`Pharmaceuticals and Proctor 8C Gamble
`
`after primary outcomes were not met in
`two Phase 3 trials. Aurograb, an scFv that
`binds to a surface protein of metliicilliti-
`resistant Stzzp/1}//ococczts /zurezts, was discori-
`tinued in 2008 after Phase 2 trials failed to
`
`show efficacy.
`Diabodies, composed of iioii—covalent
`dimers ofscFvs, are bivalent aritigen—bind-
`irig molecules. Few, ifaiiy, diabodies have
`entered clinical development, and, to my
`knowledge, none have commercial spon-
`sorship. An iodine-123-labeled diabody
`versioti ofthe anti-CEA chimeric antibody
`cT8/L66 is being evaluated for pre-surgical
`iminunoscintigraphic detection of col-
`orectal cancer in a study sponsored by the
`Beckman Research Institute ofthe City of
`Hope (Clinicaltrials.gov NCTOO647153).
`Using molecular genetics,
`two scFvs
`can be engineered in tandem itito a single
`polypeptide, separated by a linker domain,
`called a “tandem scFv” (tascFv). TascFvs
`
`have been found to be poorly soluble“’
`and require refolding“ when produced in
`bacteria, or they may be manufactured in
`mammalian cell culture systems, which
`avoids refolding requirements but may
`result in poor yields.” Construction of a
`rascFv with genes for two different scFvs
`yields a “bispecific sirigle-chain variable
`fragments” (bis-scFvs). Only two tascFvs
`have been developed clinically by C()II1—
`mercial firms; both are bispecific agents
`in active early phase development by
`Microniet for oncologic indications, and
`are described as “Bispecific T-cell Engagers
`(l3iTE)”." Blinatumomab is
`an ariti-
`CD19/anti-CD3 bispecific
`tascFv that
`poteritiates T-cell responses to B—cell non-
`Hodgkin lyniplioma in Phase 2.” MT110
`is an anti-EP-CAM/anti-CD3 bispecific
`tascFv that poteiitiates T-cell responses to
`solid tumors in Phase 1.” Bispecific, tet-
`ravalent “TandAbs” are being researched
`by Affinied, but none have yet reached
`clinical development.
`
`Third Generation Molecules
`
`Another approach to reducing the size of
`antigen—binding immuiioglobuliii-derived
`recombinant proteins has been to “min-
`iaturize” full—si7.ed mAbs by removing
`
`domains deemed iiori-essential for func-
`tion."’ Miniaturized mAbs were included
`
`in the “3G” fragment category due to the
`relatively recent advent of this approach,
`in contrast to scFvs, which were described
`
`as early as 1988. Only a handful of“min-
`iaturized” antibodies have entered clinical
`
`development.
`Among the best examples ofmAb min-
`iaturization are the small modular immu-
`
`nopharmaceuticals (SMIPS) from Triibioii
`Pharmaceuticals. These molecules, which
`can be motiovaleiit or bivalent, are recom—
`
`binant siiigle-cliain molecules containing
`one VL, one V” antigen-binding domain,
`and one or
`two
`constant
`“effector”
`
`domains, all connected by linker domains.
`Presumably, such a molecule might offer
`the advantages ofincreased tissue or tumor
`penetration claimed by fragments while
`retaining the immune effector functions
`conferred by constant domains. At
`least
`three “miniaturized” SMIPs have entered
`
`clinical development. TRU-015, an anti-
`CD20 SMIP developed in collaboration
`with \X/yeth, is the most advanced project,
`having progressed to Phase 2 for rheurna-
`toid arthritis (RA). Earlier attempts in
`systemic lupus erythrernatosus (SLE) and
`B cell lymphomas were ultimately discon-
`tinued. Trubion and Facet Biotechnology
`are collaborating in the development of
`TRU-016, an anti-CD37 SMIP, for the
`
`treatment ofCLL and other lymphoid neo-
`plasias, a project that has reached Phase 2.
`Wyetli has licensed the anti-CD20 SMIP
`SBI-087 for the treatment ofautoimmuiie
`
`diseases, including RA, SLE and possibly
`multiple sclerosis, although these projects
`remain in the earliest stages of clinical
`testing.
`
`Genmab is researching application of
`their “Unibody" technology,
`in which
`the hinge region has been removed from
`IgG4 molecules.
`\X/hile IgG4 molecules
`are unstable and can exchange light-lieavy
`chain heterodimers with one another,
`
`deletion of the hinge region prevents
`heavy ehaiii—heavy chain pairing entirely,
`leaving highly specific monovalent light/
`heavy heterodimers, while retaining the
`Fc region to ensure stability and half-life
`in vivo. This configuration may minimi7.e
`the risk ofirnmurie activation or oiicogeiiic
`growth, as lgG4 interacts poorly with FcRs
`and monovalent unibodies fail to promote
`
`80
`
`‘.\
`
`This material ijéxabstee-pied
`at: the N Llvl a fill méiy‘ be
`Eu bjeet LIE {lo-pyright Laws
`
`intracellular signaling complex formation.
`These c()titentions are, however,
`largely
`supported by laboratory, rather than clini-
`cal, evidence. Biotecnol is also developing
`a “miniaturized" niAb, CAB051, which is
`
`a “compacted” 100 kDa anti-HER2 anti—
`body in preclinical research.
`Recombinant therapeutics composed of
`single antigen—binding domains have also
`been developed, although they currently
`account for only 4% of the clinical pipe-
`line.‘ These molecules are extremely small,
`with molecular weights approximately
`one-tenth of those observed for full—si7.ed
`
`mAbs. Arana and Domantis engineer
`molecules composed of antigen—binding
`domains of human immuiioglobulin light
`or heavy chains, although only Arana has
`a candidate in clinical testing, ART-621,
`an anti-TNPOL molecule in Phase 2 study
`for the treatment of psoriasis and rheu-
`matoid arthritis. Ablynx produces “nano-
`bodies” derived from the antigen—binding
`variable heavy chain regions (Vmris) of
`heavy chain antibodies found in camels
`and llamas, which lack light chains.“’ Two
`Ablynx anti-von \X/illebraiid Factor nano-
`bodies have advanced to clinical develop-
`ment,
`including ALX-0081,
`in Phase 2
`development as an intravenous therapy to
`prevent thrombosis in patients utidergo-
`ing percutaiieous coronary intervention
`for acute coronary syndrome, and ALX-
`0681, a Phase 1 molecule for subcutaneous
`
`administration intended for both patients
`with acute coronary syndrome and throm-
`botic thrombocytopenic purpura.
`In addition, at
`least 16 domain inol-
`
`ecules are being researched in preclini-
`cal studies, accounting for nearly half of
`the identified preclinical
`research pipe-
`line, with Ablynx responsible for 5 and
`Domantis responsible for the remaining
`11 candidate. Domantis, a wholly—owned
`subsidiary ofGlaxoSmithKline since 2007,
`produces single-doinain molecules based
`on human iriirnunoglobulin sequences,
`which may or may prove advantageous in
`the clitiic.
`These data are consistent with the rela-
`
`tive novelty ofindividual antibody domain
`molecules, and demonstrate how little is
`
`known about the clinical efficacy ofagents
`derived from these binding domains,
`despite encouraging pre-cliiiical research
`and great commercial interest.
`
`LILLY EX. 1040 - 8/11
`Volume 2 Issue 1
`
`LILLY EX. 1040 - 8/11
`
`

`

`
`
`Multi-Specificity
`
`Although multi-specific molecules were
`described in the previous sections,
`this
`approach is sufficiently important to war-
`rant separate, parallel consideration when
`evaluating trends in the development of
`antibody fragment therapeutics.
`Natural antibodies express two identical
`antigen—binding domains, and are therefore
`mono-specific, but bivalent.
`In contrast,
`bispecific molecules possess two different
`antigen-l)inding domains, each with differ-
`ent target specificity. Bispecific antibodies
`were initially described in the mid 1980s,
`and were generated by the fusion of two
`antibody-producing cells, each with dis-
`tinct specificity.” These “quadromas” pro-
`duced multiple molecular species, as the
`two distinct light chains and two distinct
`heavy chains were free to recombine in
`the quadromas in multiple configurations.
`Since these early efforts, bispecific Fabs,
`scFvs and full—si7.e mAbs have been gener-
`ated and tested using a variety of technolo-
`gies. Six bispecific fragments have entered
`development, including four discontinued
`bispecific Fabs, three of which were from
`Medarex, and two bispecific tascFvs in
`active development by Microtnet. At least
`11
`bispecific fragments
`therapeutics in
`preclinical
`research have been described
`in public documents, including two Fabs
`from Imrnunomedics, five scFvs including
`four from Afhrned and one from Targa, as
`well as four bispecific human “domain”
`antibodies from Domantis. Nine of those
`
`fragments are intended for development
`in oncologic indications, and the remain-
`ing two for immunological disorders. The
`Imtnunomedic compounds are each tri-
`meric, with two arms specific for one tar-
`get, and a third arm specific to a second
`target. These molecules function for the
`most part by potentiating B cell, T cell or
`natural killer cell responses to tumor cells.
`Although not clearly an antibody frag-
`ment, Trion Pharma l1as sought to develop
`“trispecific” molecules by expressing bispe-
`cific antibodies with two distinct Fabs and
`
`an FC demonstrating highly potent mac-
`rophage activation properties. This was
`first described fora mouse lgG2a anti-Ep-
`CAM,
`rat
`lgG2b anti-CD3 quadroma,
`called BiUll.'“ The authors propose that
`BiUll permits the co-localization oftumor
`
`cells expressing Ep-CAM, T cells express-
`ing CD3, and macrophages expressing
`FCYRI, potentiating the costimulatory and
`anti-tumor functions ofthe immune cells.
`
`Trion has three trispecific full-let1gtl1 mAbs
`that have entered clinical development,
`including catumaxomab (Removab), an
`anti-EpCAM rat-human hybrid molecule
`approved in the EU, and in Phase 3 study
`in the US, for malignant ascites.
`
`Conjugation
`
`the conjuga-
`Similar to tnulti-specificity,
`tion ofexogenous effector moieties to anti-
`body ftagtnent molecules is an important
`approach taken by drug designers. For this
`reason, although conjugated molecules
`were described in previous sections, sepa-
`rate consideration is given to this topic.
`Full-length antibodies have biologi-
`cal functions conferred by both antigen
`binding and the complement
`fixation
`and cellular activation capacity of the FC
`domain. The biological function of the
`three approved Fab fragment therapeutics,
`however, is strictly limited to the capacity
`of the single binding domain molecule to
`bind the target. Nearly half of fragments
`ir1 clinical development were conjugated
`to exogenous functional moieties and all
`ofthese were intended for the treatment of
`
`cancer. This constitutes a higher propor-
`tion than that observed for all anti-cancer
`
`mAbs, of which approximately 44% are
`innnunoconjugates.” These data demon-
`strate that drug developers are well aware
`of the functional limitation of fragments
`that
`lack effector domains or see frag-
`ments as an opportunity to more precisely
`control the functional properties of thera-
`peutic candidates.
`A total of 24 conjugated antibody frag-
`ments have entered clinical study, with
`eight active projects and one molecule
`approved
`in China. Metuximab-I131
`(Licartin) is a murine lgG1 anti-CD1/17
`F(ab)2 conjugated to iodine-131 produced
`by Chengdu Hoist I-litech and approved
`by the Chinese State FDA for the treat-
`ment of liver cancer;
`this molecule has
`
`not met regulatory approval in any other
`country. The remaining eight projects in
`active development are well-distributed
`throughout the phases of clinical devel-
`opment, with two in Phase 3 and two in
`
`Phase 2. Ofthe 24 conjugated fragments,
`12 are fragments conjugated to cellular tox-
`ins, seven are conjugated to radioisotopes,
`three to eytolcines and one to an enzyme
`to target
`toxic metabolites;
`the type of
`moiety conjugated to 1
`is unknown.
`Twelve Fab conjugates have entered eliti-
`ical development,
`including metuxin1ab
`I-131 and an active Phase 3 Can