Manfred Schwab
`Editor
`
`Miltenyi Ne Kh 1
`
`Encyclopedia of
`Serle
`
`VOLUME 1
`
`A-C
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`Y) Springer
`
`Miltenyi Ex. 1013 Page 1
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`

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`Manfred Schwab
`Professor for Genetics
`Director Division of Tumour Genetics (B030)
`German Cancer Research Center (DKFZ)
`Im NeuenheimerFeld 280
`69120 Heidelberg
`Germany
`
`A CLP. Catalog record for this book is available from the Library of Congress
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`ISBN: 978-3-540-36847-2
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`Miltenyi Ex. 1013 Page 2
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`transformation by dysregulating the expression of key
`
`> Epigenetic Therapy
`>» Fusion Genes
`
`Chimeric T Cell Receptors
`
`HinricH ABKEN
`Tumor Genetics, Clinic I Internal Medicine and Center
`for Molecular Medicine Cologne, University
`of Cologne, Cologne, Germany
`
`Chimeric T Cell Receptors
`
`649
`
`The TCR Format
`Based on the similarity of the primary structure and
`the spatial conformation of the variable regions of
`immunoglobulin (Ig) and TCR a and B chain molecules,
`antibody derived binding regions Vj, and V_ for antigen
`were grafted on to the constant domain ofthe TCR o and
`B chains, respectively (Fig. 1). Thereby, the TCR Va
`and VB domainsare replacedresulting in chimeric TCR
`molecules with antibody directed T cell recognition.
`The strategy, however, requires simultaneous expres-
`sion of two modified TCR chainsin engineered T cells.
`Moreover,
`the transfected TCR o and § chains fre-
`quently heterodimerize with the corresponding chains
`of the endogenous TCRresulting in chain mispairing
`and reduced expression of the recombinant TCR.
`
`The Single Chain Format
`The alternative format of a chimeric TCR (immuno-
`receptor, T-body) consists of one polypeptide chain
`which is composed ofan extracellular binding and an
`RecombinantTcell receptor; Immunoreceptor; T-body
`intracellular signaling domain(Fig. 1). The single chain
`format avoids a numberofdifficulties that rise with
`the TCR formatincluding simultaneous expression ofthe
`two recombinant TCR chains and mispairing with
`the endogenous TCR. Theantigen binding domainis
`derived from a single chain fragmentofvariable regions
`(> scFv)antibody,the signaling moiety is preferentially
`derived from the CD3¢ chain ofthe TCR/CD3 complex
`or, alternatively, from the y chain ofthe high affinity IgE
`Fc receptor (FceRI). The scFv moiety used for binding
`is generated from an antibody molecule by genetically
`joining the Vy and V, immunoglobulin regions via a
`flexible peptide linker, e.g., (Gly,Ser)3, resulting in a
`continuous polypeptide chain of the Vy-linker-V; or
`V_-linker-Vy type. »Phage display techniques are
`applied to isolate scFv antibodies with high binding
`affinity. Due to the antibody derived binding domain,
`chimeric immunoreceptors can be generated that bind
`antigen of any chemical composition or conformation
`includingclassical and nonclassical T cell targets like
`carbohydrates, as far as an antibody exists. Thereby, a
`broad variety of chimeric TCRs were generated against
`tumorassociated antigens like » HER-2/neu, or > can-
`cer germline antigens. Instead of an scFv domain,any
`polypeptide with specific binding properties, e.g.,
`receptor ligands, may be suitable as targeting domain
`as well. The majority of chimeric TCRs of the
`immunoreceptor type harbor antibody derived binding
`domainsthat mediate T cell activation independently of
`MHC.However,antibodiesare available that recognize
`the processed peptide in the context of MHC. MHC/
`
`Chimeric T cell receptors are recombinant transmem-
`brane receptor molecules, which are derived from the
`receptor
`(TCR) complex and consist of
`an extracellular binding domain, a transmembrane
`domain, and an intracellular domain to initiate T cell
`activation upon engagement of the specific receptor
`
`Characteristics
`Recombinant TCRsprovidethebasis to engineer T cells
`with predefined specificity in order to redirect
`the
`»T cell response toward defined target cells and to
`break tolerance for use in adoptive immunotherapy.
`Moreover, chimeric TCRs are valuable tools
`for
`the analysis of receptor driven activation of immune
`cells including T and NKcells. Several formats of
`recombinant TCRs have been designed during the last
`years, most of them are chimeric molecules com-
`posed of an extracellular binding and anintracellular
`signaling domain.
`T cells exhibit their binding specificity via the TCR
`complex. For a numberofapplications in > molecular
`therapy, T cells with predefined specificity would
`be desirable. By » viral vector-mediated gene transfer,
`T cells can be grafted with a recombinant TCR of
`predefined specificity, which allows to redirect engi-
`neered T cells toward defined target cells. The approach,
`
` Miltenyi Ex. 1013, Page 3
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` ee oeeeeeeeOTTTT
`
`RICCTtReLLTCieLLLIL
`ee eeerrr ITTRUST ReeLL
`
`
`
`
`
`
`
`LyefrJL 4 Loc L3Lt
`
`scFv
`
`Tn
`
`scFv
`
`TM/ \
`
`FceRI y
`
`syk
`
`scFv
`
`Am
`
`CD3E
`
`scFv
`
`Spacer
`
`CD3t
`
`scFv
`
`Spacer
`Iam
`cp28
`
`b
`
`CD3t
`
`Chimeric T Cell Receptors. Figure 1 Modular composition of chimeric T cell receptors (TCRs). The physiological
`TCR complex consists of the a and B chain for antigen recognition and the CD3 complexto initiate intracellular
`signaling. Fusion of the antibody derived binding domains V,, and V, to the a and B chains of the TCR creates a
`chimeric TCR.In an alternative format the chimeric TCR consists of one polypeptide chain with the single chain
`fragmentof variable region (scFv) antibody as antigen binding domain whichis fused via spacer and
`transmembrane (TM) domainto theintracellular signaling domain which is preferentially derived from the CD3¢
`chain or the FceRI y chain, oralternatively from Ick, syk, or CD3e. Chimeric TCRsof the second generation
`harborin addition to the CD3Z chain a costimulatory domain, e.g., the CD28 domain.
`
`superiorin redirecting T cells in vivo. Some advantages
`of MHC independent chimeric receptors, however, are
`obvious, including targeting of unconventional T cell
`antigens like carbohydrates and targeting of cells even
`when their target antigen is not properly processed or
`presented in the MHC. Both CD8° and CD4° T cells can
`be redirected by the same immunoreceptor. Thelatter is
`of clinical relevance since redirected T cells of both
`CD4 and CD8"
`subpopulations execute granule-
`dependent cytolysis of target cells when stimulated
`via a chimeric TCR that circumvents MHCrestriction.
`The intracellular signaling domain of the chimeric
`TCR is most frequently derived from the CD3¢ or the
`FceRI y chain. Both signaling chains contain immuno-
`receptor tyrosine activation motifs (»ITAMs),
`the
`CD3¢€ chain three, the FceRI y chain one ITAM,which
`become phosphorylated upon receptor crosslinking.
`The signaling domains
`thereby serve as
`specific
`adaptors for downstream signaling proteins of the
`TCR complex. Alternatively, signaling moieties of
`
`downstream kinases like Ick can be used asactivation
`domains in chimeric TCRs.
`Expressed in cytotoxic T lymphocytes (»CTLs),
`crosslinking of the chimeric receptor by antigen
`engagement
`initiates T cell activation resulting in
`proliferation, cytokine secretion, and specific cytolysis
`of antigen expressing target cells. Chimeric TCRs
`define a new specificity of the grafted effector cell. This
`is indicated by the fact that (i) T cells with immuno-
`receptor are activated upon coincubation with target
`cells that express the defined antigen on the cell surface
`whereas antigen-negative target cells do not initiate
`receptorsignaling in receptorgrafted T cells,(ii) T cells
`without immunoreceptor or with immunoreceptor of
`other specificity are not activated upon coincubation
`with antigen expressing target cells, and (ili) receptor
`triggered activation can be blocked by antibodies
`directed toward the scFv domain of the immunorecep-
`tor. The efficacy ofT cell activation depends on various
`parameters
`including.theyf pression, Se gt the
`
`Miltenyi Ex. 1013 Page 4
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`Chimeric T Cell Receptors_651
`
`the density of
`receptor on the effector cell surface,
`antigen on the targetcell, the targeted epitope and the
`position of the epitope within the antigen.
`
`Thestrategy of redirecting T cells by chimeric TCRs
`has advantages over antibody-based therapies.
`In
`particular, the strategy makes use of the autologous
`cellular defense system represented by immunological
`effector cells that actively penetrate tissues. In contrast
`Chimeric T Cell Receptors with Primary
`to antibodies, the engineered effector cells persist and
`and Costimulatory Signal
`circulate over long periods of time,
`i.e., until up to
`NaiveTcells, in contrast to preactivated T cells, are not
`1 year or even longer, amplify by proliferation upon
`fully activated when stimulated via CD3¢ but require
`antigen encounter and interact with numerous target
`>costimulation via CD28. When T cells encounter
`cells by executing their cytolytic attack. Thereby, the
`antigen but lack costimulatory signals antigen-specific
`chimeric TCR strategy is anticipated to be most
`tolerance or anergyis induced. Current chimeric TCR
`powerful
`in eliminating (micro-) »metastasis and
`strategies therefore aim to provide appropriate costi-
`> circulating tumorcells. Since T cells in patients with
`mulatory signals on effector T cells in order to avoid
`advancedstagesofthe disease are frequently defective
`induction of anergy. This is done by simultaneous
`in TCRsignaling, the chimeric receptor approach will
`expression of two recombinant immunoreceptors, one
`require repeated administration of “freshly” grafted
`with the CD3¢ and the other with the CD28 signaling
`T cells to substitute burn-out or anergized CTLs. Once
`domain or, technically more convenient, by combining
`initiated the immunereaction is thought to be spe-
`the intracellular CD3¢ chain together with the CD28
`cifically sustained by chimeric receptor signaling upon
`signaling domain into one polypeptide chain receptor
`repetitive stimulation with antigen. Havingtarget cells
`molecule (Fig. 1). Different costimulatory domains
`eliminated, the immunereaction is supposedto beself-
`result in different activation profiles. T cells triggered
`limiting since T cells that do not furthermore interact
`by the combined CD28-CD3¢ signaling chimeric
`with their antigen enter apoptosis. It
`is furthermore
`receptor exhibit increased IFN-y secretion andsecretion
`anticipated that some engineered T cells convert into
`ofsubstantial amounts ofIL-2 whereasTcells equipped
`memory cellsthat will be active in > immunoprevention
`with the CD3¢ chimeric receptor secrete lower amounts
`oftumorrelapse.
`of IFN-y and do notsecrete IL-2. The specific cytolytic
`Clinical trials have been and are currently initiated
`capacity, noteworthy, is not dramatically altered when
`using engineeredT cells equipped with chimeric TCRs
`preactivated T cells are stimulated via the CD28-CD3¢
`in order to eliminate tumorcells, e.g., of »ovarian
`compared to the CD3¢ signaling receptor.
`carcinoma,renal cell carcinoma, lymphoma, and mel-
`anoma. Thesafety of the immunotherapeutic strategies
`to redirect T cells by chimeric TCRs is currently
`The major application of chimeric TCR strategy is to
`explored. Oneof the major risks of redirecting T cells
`redirect cytotoxic T cells (CTLs) toward defined tumor
`with chimeric TCRs, however, is the risk of autono-
`cells or virus infected cells. T cells equipped with a
`mous amplification ofengineeredTcells independently
`tumorspecific, chimeric TCR break tolerance toward
`of antigen. Secondly, there is the risk of an unwanted
`autologous tumor cells as demonstrated in mouse
`autoimmune reaction toward healthy tissues that ex-
`models in vivo. Moreover, patient’s T cells from the
`press the same antigen as the target cell, however at
`peripheral blood can be redirected in vitro toward the
`lowerlevels. Costimulatory chimeric TCRs may harbor
`autologous tumorcells isolated from a tumor biopsy of
`a higher risk of autoaggression compared to CD3¢
`the samepatient. The approach is universal since by
`signaling receptors. Crucial questions in the treatment
`inserting the appropriate binding domain into the
`of malignant diseases, however, are whether effector
`chimeric TCR,T cells can be redirected toward a broad
`cells with chimeric TCRs haveretained their homing
`variety of tumor associated antigens expressed on
`capabilities after genetic manipulation in order to
`tumorcells of different entities, including » gastroin-
`accumulate at
`the tumorsite, whether they execute
`testinal tumors and ®pancreas carcinoma (CEA, CA19-
`their effector functionsat the tumorsite and areresistant
`9, B72-4), »melanoma (HMW-MAA, p97, GD3,
`toward suppression by » T regulatory cells, and whether
`Mage-1), breast carcinoma and Pepithelial
`tumors
`they remain silent upon contact with healthy tissues
`(>Her2/neu ErbB2, Muc1), renalcell carcinoma (G250),
`with physiological expression oftarget antigen.
`> prostate carcinoma (PSMA), > ovarian cancer (FBP),
`>hematological malignancies
`including
`»multiple
`myeloma and »Hodgkin’s lymphoma (CD19, CD20,
`
`References
`
`Miltenyi Ex. 1013 Page 5
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`

`

`single chains consisting of antibody-binding domains and
`the gammaor zeta subunits of the immunoglobulin and
`T-cell receptors. Proc Natl Acad Sci USA 90:720-724
`3, Goverman J, Gomez SM, Segesman KD et al. (1990)
`Chimeric immunoglobulin-T cell receptor proteins form
`functional
`receptors:
`implications for T cell
`receptor
`complex formation and activation. Cell 60:929-939
`4. Hombach A, Heuser C, Abken H (2002) The recombinant
`Tcell receptorstrategy: insights into structure and function
`of recombinant immunoreceptors on the way towards an
`optimal receptor design for cellular immunotherapy. Curr
`Gene Ther 2:211-226
`(1987)
`5. Kuwana Y, Asakura Y, Utsunomiya N et al.
`Expression of chimeric receptor composed of immuno-
`globulin-derived V regions and T-cell receptor-derived C
`regions. Biochem Biophys Res Commun 149:960-968
`
`Chimeric Transcripts
`
`> Fusion Genes
`
`——
`Chinese Medicine, Traditional Chinese
`Medicine, Oriental Medicine
`
`> Chinese versus Western Medicine
`
`Chinese versus Western Medicine
`
`WILLIAM CHI-SHING CHO
`Department of Clinical Oncology, Queen Elizabeth
`Hospital, Kowloon, Hong Kong
`Synonyms
`Chinese medicine, Traditional Chinese medicine, Orien-
`tal medicine; Western medicine, Modern medicine, Con-
`ventional medicine, Mainstream medicine, Orthodox
`medicine, Biomedicine, Allopathic medicine
`
`Definition
`Chinese medicine is a patient-oriented medical system
`that treats the » cancer patients instead of the malignant
`diseases. It is believed that » qi (the Chinese term for
`
`system of channels within the human body, through
`which the vital energy and blood circulate, and by
`which the internal organs are connected with superficial
`organs and tissues, and the body is made an organic
`whole. Using these holistic and harmonic approaches,
`Chinese medicine emphasizes to strengthen the body
`resistance. It attaches importance to the self-healing
`ability ofhuman body to remove pathogenic factors and
`recover health. Many of its cancer therapies, such as
`Chinese medication (including medicinal decoction,
`patent medicine, and proprietary medicine), medicated
`diet, acupuncture, and moxibustion,as well as >» qigong
`and massage, are employed for enhancing this power.
`Western medicine is an evidence-based medical
`system,
`in which authorized medical and healthcare
`professionals struggle to avert the destruction of cancer
`patients by the unrelenting displacement of normal
`homeostasis accompanying neoplastic growth. Theclini-
`cal problems faced by oncologists include overcoming
`the inherent or acquiredresistance of the malignantcell
`to therapy, ameliorating the toxicities of aggressively
`applied therapies, as well as exploiting the synergistic
`potency of surgery, radiotherapy, and chemotherapy.
`In the last few decades,
`the limited clinical skills
`demandtheuse ofdestructive agent for cancer therapy.
`In the post-genomic era, > targeted therapy and novel
`therapeutic strategy are applied to complement
`conventional treatment for an achievement of optimal
`anticancerresults.
`
`Characteristics
`Developing History
`The Yellow Emperor’s Canon of Internal Medicine
`is believed to be the earliest medical monograph in
`China, which appeared during the Warring States period
`(475-221 BC),first defined the etiology oftumor. Since
`the ancient times, Chinese medicine has madea great
`contribution to the health of the Asian people. Based on
`empiricalandclinical experience, Chinese medicine has
`been systematized and theorized in complex practice.
`Manysafe and effective methods have been developed
`to diagnose and treat cancer over the past thousands
`of years.
`Hippocrates (460-375 BC), the father of Western
`medicine, first attributed the origin of cancer to natu-
`ral causes. Improved microscopes, stimulated cancer
`researches, and important discoveries in human and
`animalstudies haveresulted in a better understanding of
`neoplasia. The last few decades has witnessed spectac-
`ular progress in describing the fundamental molecular
`basis of cancer following the advent of molecular bio-
`logy and genetics, which allowsthe device of advanced
`or targeted therapy fayfREAGTEx. 1013 Page 6
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