oceedings of the National Academyof Sciences of the United States of America
`
`www.pnas.org -
`
`Plant organogenesis
`
`PNA
`
`i0
`
`“pat
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`Legged robots on granular media
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`Improving nitrogen management
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`Kelp genes hold seaice history
`
`Protecting neurons from Alzheimer’s
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`Miltenyi Ex. 1015 Page 1
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`

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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Control of large, established tumor xenografts
`with genetically retargeted humanT cells
`containing CD28 and CD137 domains
`Carmine Carpenito*, Michael C, Milone*®, Raffit Hassan‘, Jacqueline C. Simonet, Mehdi Lakhal*, Megan M.Suhoskia,
`n¢, Steven M. Albelda®, Richard G. Carroll*®,
`Angel Varela-Rohena®, Kathleen M. Haines*, Daniel F. Heitja
`JamesL. Riley, Ira Pastan®’, and Carl H. June”?
`Abramson Family Cancer ResearchInstitute, "Department of Pathology and Laboratory Medicine, 4DepartmentofBiostatistics and Epidemiology, and
`Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and ‘Laboratory of Molecular Biology,
`National Cancer Institute, Center for Cancer Research, National Institutes of Health, Bethesda, MD 20892-4264
`
`Contributed by Ira Pastan, December 23, 2008 (sent for review October 14, 2008)
`Because mesothelin has recently been reported to bind tg
`Mesothelin is a cell-surface molecule over-expressed on a large
`CA125/MUCI16, one possible biological role of this membrane
`fraction of carcinomas, and thusis an attractive target of immu-
`protein may be linked to heterotypic cell adhesion, which may
`notherapy. A molecularly targeted therapy for these cancers was
`facilitate the metastatic spread of mesothelin-bearing cancer
`created by engineeringTcells to express a chimeric receptor with
`cells (10). In earlier studies, tumor cells expressing mesothelin
`high affinity for human mesothelin. Lentiviral vectors were used to
`have been shownto be killed by MHC Class I-restricted T cells
`express a single-chain variable fragmentthat binds mesothelin and
`(11). In addition, mesothelin-specific antibodies and antibody.
`that is fused to signaling domainsderived from T-cell receptorzeta,
`based immunotoxins have been shown to cause tumorregression
`CD28, and CD137 (4-1BB). When stimulated by mesothelin, lenti-
`in preclinical and clinical studies (12, 13). Here we reportthat
`virally transduced T cells were induced to proliferate, express the
`lentiviral vectors can efficiently generate T cells that specifically
`antiapoptotic gene Bcl-X,, and secrete multiple cytokines, all fea-
`target mesothelin. The mesothelin-retargeted T cells eradicate
`tures characteristic of central memory T cells. When transferred
`large pre-established mesothelioma xenografts in NODjseid/
`intratumorally or intravenously into NOD/scid/IL2ry~/~ mice en-
`IL2ry’ mice at
`low effector-to-target (E:T) ratios, and the
`grafted with large pre-established tumors, the engineeredT cells
`incorporation of costimulatory domains enables the prolonged
`reduced the tumor burden, and in somecasesresulted in complete
`persistence of the engineered T cells in tumor-bearing mice
`eradication of the tumors at low effector-to-target ratios. Incor-
`following intratumoralor i.v. administration.
`poration of the CD137 signaling domain specifically reprogrammed
`cells for multifunctional cytokine secretion and enhanced persis-
`tence of T cells. These findings have important implications for
`adoptive immunotherapy of cancer, especially in the context
`of poorly immunogenic tumors. Genetically redirected T cells
`have promise of targeting T lymphocytes to tumor antigens,
`confer resistance to the tumor microenvironment, and providing
`immunosurveillance.
`
`adoptive immunotherapy| chimeric receptor | mesothelin
`
`A principal limitation of cancer immunotherapy is that most
`solid tumors are poorly antigenic, expression of MHC Class
`I molecules is very low, no T cells are available that have high
`avidity for tumor specific antigens, or no T cells that have the
`desired specificities remain in the patient after chemotherapy.
`To address these problems, tumor antigen-specific T cells have
`been engineered by introduction of chimeric immunoreceptors
`(CIR)—or “T bodies”—that have antibody-based external re-
`ceptor structures and cytosolic domains that encode signal
`transduction modules ofthe T-cell receptor (1). These constructs
`can function to retarget T cells in vitro in an MHC-unrestricted
`manner. Several pilot clinical trials to test this concept have
`recently been reported (2-4). In all of the trials, safety has been
`documented. However, the principalissue revealed in theinitial
`trials has been poorin vivo persistence and expression of the
`transgene. Approaches to remedy these shortcomings currently
`involve improved receptor design, the incorporation of costimu-
`latory signaling domainsinto the signaling module, and reducing
`the immunogenicity of the T-body construct (5).
`Mesothelin is a glycosyl-phosphatidyl
`inositol-linked mem-
`brane glycoprotein expressed in a variety of cancers (6). Immu-
`nohistochemistry studies have shown that mesothelin is over-
`expressed in virtually all pancreatic ductal adenocarcinomas and
`mesotheliomas(7), and in a high percentage ofepithelial ovarian
`cancers, as well as non-small cell carcinomas of the lung (8, 9).
`
`3360-3365 |
`
`PNAS
`
`| March 3, 2009
`
`|
`
`vol. 106
`
`| no.9
`
`www.pnas.org/cgi/dei/10.1073/pnas.0813i _
`
`Results
`Chimeric receptors were designed that contain the SS1 scFv that
`recognizes human mesothelin (Fig. 14). SS1 was chosen because
`it has a high binding affinity to mesothelin (Ky = 0.7 nM)(14),
`and because it has been found to be safe in patients whem
`administered as a recombinant immunotoxin (13). We createda
`series of SS1 scFv-based chimeric receptors that contain the
`TCR-£ signal-transduction domain with the CD28 and CDI37
`(4-1BB) intracellular domains in tandem. Chimeric receptors”
`that contained a truncated form of the TCR-¢ intracellular
`domain (SS1-Az) or an anti-CD19 scFv (anti-CD19-z) were
`designed as controls for signal transduction and binding speck
`ficity, respectively.
`|
`Because of the increased efficiency of transduction and
`pression, and the possible decreased potential for adverse oh
`fects, such as insertional mutagenesis compared to retroviruses:
`we used lentiviral-vector technology to express the fusion come
`structs in primary human T cells using clinically validated
`techniques (15). The cDNA sequences containing the vane’
`fusion constructions were cloned into a third-generation lemty
`viral vector in which the CMV promoter was replaced with
`EF-1a promoter (16). Lentiviral vector supernatants transduced
`primary T cells with high efficiency (Fig. 1B). In preliminary
`
`Author contributions: C.C., M.C.M., J.L.R., and C.HJ. designed research; C.C., M.CM.,HOS
`M.L., M.M.S., A.V.-R., and K.M.H. performed research; R.H. and |.P. contributed
`reagents/analytic tools; C.C., D-F.H., S.M.A., and R.G.C. analyzed data; and GC. am
`wrote the paper.
`The authors declare no conflict of interest.
`‘To whom correspondence may be addressed. E-mail: or pastani@mail.nih.gov ~
`cjune@exchange.upenn.edu.
`This article contains supporting information online at www.pnas.org/cgi/content™
`0813101106/DCSupplemental.
`© 2009 by The National Academy ofSciences of the USA
`
`Miltenyi Ex. 1015 Page 2
`
`Miltenyi Ex. 1015 Page 2
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`

`

`CD8a
`Leader:
`
`SS1 scFv expression
`
`M08 _ GFP
`—— Zeta
`—— BBz
`—1— 282
`— 28BBz
`—as Az
`+ CD19-z
`
`E:T ratio
`
`
`
`B eenerer ZetaTo
`10:1 E:T ratio during a 4-h assay, and at <1:1 E:T ratio during
`j
`/
` a aT
`
`a 48-h culture [supporting information (SI) Fig. $1], suggesting
`|
`(3160)||!
`|
`that the redirected T cells were capable ofserial killing. More-
`28z
`|
`over, the lysis was specific because T cells transduced with GFP
`ng ||
`cp28 TM: 4 \
`or an irrelevant anti-CD19 scFv chimeric receptor showed no
`(981) |
`z
`(2467)
`\|
`i
`14
`3 st
`cytotoxic activity against the sametargetcells, excluding allo-
`—_—__—_—_——
`—“nesotheln
`\
`o
`ae;
`
`signaling domains
`:
`a
`=|"ww 6 ae
`reactivity or nonspecific lysis. Furthermore, T cells expressing a
`binding domain | seBBz| faza]
`
`
`truncated TCR-¢intracellular domain (S$1-Az)alsofailed to kill
`89% |
`94% ||
`
`tkb
`2kb
`(695) |
`|
`(2367) |
`|
`mesothelin-expressing targets, demonstrating the requirement
`
`for an intact TCR-¢ signaling domain.
`Studies in mouse tumor models indicate that antitumoreffects
`JO PAK
`of redirected CD8T cells will likely be sustained by CD4 cells
`(23). Therefore, we transduced primary human CD4 and CD8 T
`cells and measured cytokine secretion after exposure to me-
`aaoo
`sotheliomacells (Fig. S24). The release of inflammatory cyto-
`%\ys
`kines wastriggered by mesothelin, and both CD4 and CD8T cells
`secreted Th1 cytokines. In contrast, the T cells secreted low or
`undetectable amounts of IL-4, IL-5, IL-10, and IL-17 (data not
`shown), consistent with a predominant Th1 cytokine bias under
`fig. 1. Generation and cytolytic activity of antimesothelin lentiviral vector-
`these culture conditions. The pattern of cytokine release ob-
`
`er gineered T cells. (A) Schematic representation of the mesothelin-binding
`served was consistent with the knownsignal transduction prop-
`chimeric receptors. A binding-control chimeric receptor with a truncated TCR¢
`erties of the various signaling domains, confirming the modular
`domain and a specificity control receptor with an anti-CD19 scFv were also
`nature of the domains and that they can function in cis on the
`constructed. (B) Expression of the $$1 scFv fusion proteins was examined on
`fusion proteins, For example, IL-2 and TNF-a were only secreted
`human primary CD4T cells. Transduction efficiencies are indicated with mean
`by CD4Tcells, and this was dependent on the CD28 domain.
`fluorescence intensity of the transduced populationsin parentheses. (C) Cell
`Both CD4 and CD8T cells released [FN-y and IL-6, and either
`‘sul ace expression of mesothelin on K562, K562.meso, OvCa61.4, OvCa68.4,
`the CD28 or the CD137signaling domain wassufficient. There-
`and M108 wasdeterminedby flow cytometry (Top). Cells were incubated with
`fore, in contrast to cytotoxicity, where TCR-¢ was sufficient,
`aither the mouse anti-human mesothelin antibody CAK1 (solid black line) or
`al
`isotype control (dotted fine) followed by staining with a PE-conjugated
`cytokine secretion was more pronounced in primary T cells
`‘goat anti-mouse Ig, The cytolytic activity of the chimeric receptors on primary
`expressing a costimulatory signaling domain. Mesothelin-
`‘human CD8 + T cells targetingcell lines expressing mesothelin was determined
`independent cytokine secretion was not observed.
`“us
`ig a 4h *'Crrelease assay (Bottom). Results are expressed as a mean and SD
`An emerging concept in the vaccine field indicates that the
`of triplicate wells from 1 of at least 3 separate experiments.
`presence of multifunctional T cells is associated with improved
`control of chronicviralinfections, and that this may be important
`for T-cell-based cancer therapies (24). Transduced T cells were
`experiments, the SS1-transduced T cells were found to have
`
`stimulated with mesothelin-expressing tumorcells and the CD4
`stained proliferation in the presence of various cell lines that
`and CD8T-cell subsets stained for intracellular IFN-y, TNF-a,
`press mesothelin, while culture in the absence of mesothelin-
`
`IL-2, and GM-CSF (Fig. $2B). Cytokine production was re-
`pressing feedercellsfailed to sustain T-cell proliferation (data
`stricted to the fraction of T cells expressing chimeric receptors
`t shown). All constructs were brightly expressed on the surface
`
`(data not shown). The incorporation of the CD28signaling
`CD4 and CD8T cells, and expression was stable for at least
`
`domain increased the fraction of cytokine secreting CD4 and
`Months in culture (not shown). T-cell expansion during culture
`CD8Tcells early in culture; however, after 20 h of culture, the
`with mesothelin-expressing tumorcells was higherfor T cells that
`fraction of responding cytokine-secreting cells was similar forall
`mtained SS1 scFv fused with either costimulatory domain than
`signaling constructs. However, the fraction of polyfunctional T
`T cells expressing the SS1 TCR-é signaling domain only (data
`cells was highly dependent on the presence of costimulatory
`t shown), consistent with previous reports that have tested T
`domains,as nearlyall cytokine-secretingcells expressing only the
`S$ with other chimeric receptors in vitro (17-22). Further-
`TCR-¢ domain were monofunctional. In contrast, highly multi-
`ore, the SS1 scFv fused with costimulatory domains enriched
`functional T cells secreting all 4 cytokines were primarily ob-
`
`€ population of T cells to near purity during culture on
`served in the cells expressing the BB:TCR-¢ or 28:BB:TCR-¢
`Mesothelin-expressing tumors (data not shown).
`signaling domains. Importantly, the CD137 signaling domain was
`To investigate the antitumor potential of the transduced T
`notable for supporting multifunctional CD4 and CD8T cells, a
`tells, effector function was measured in standard 5'Cr release
`feature that might prolong the functionof the T cells in the tumor
`
`says using mesothelin-negative K562 cells, K562.meso (a de-
`microenvironment.
`Hvative engineered to express mesothelin), and primary tumor
`The expressionof antiapoptotic genes can confer resistance to
`ines isolated from patients with ovarian cancer and malignant
`the toxic effects of the tumor microenvironment (25). Most of
`
`ssothelioma (Fig. 1C). T cells transduced with SS1 scFv
`the T cells expressing CD28, and to a lesser extent the CD28:BB-
`*Hiciently lysed K562.meso but did notkill parental K562cells.
`signaling domains, expressed Bel-X, after culture with the M108
`Importantly, the SS1-transduced T cells were also highly cyto-
`mesotheliomacell line (Fig. S2C). In contrast, T cells expressing
`Oxic for carcinoma cells that express mesothelin, killing
`BB:TCR-¢, or a truncated TCR-¢ had lower or undetectable
`YvCa68.4 and M108cell lines derived from patients with ovarian
`Cd
`levels of Bel-X,. The expression of Bel-X;, in T cells expressing
`cer and mesothelioma,while failing to kill ovarian tumorcells
`
`CD28 and CD137 signaling domains was not constitutive, as T
`t did not express mesothelin (OvCa61.4). Transduction of
`cells cultured in media (see Fig. $2C) or with mesothelin-
`~YCa61.4 cells with a mesothelin-expressing lentiviral vector
`negative tumorcells (data not shown) did not express detectable
`Fendered them susceptible to SS1 scFv lysis (data not shown).
`levels of Bcl-X,.
`¢ inclusion of CD28 and CD137 costimulatory domainsin
`Asan initial test of in vivo specificity of the SS1 constructs, a
`“dem or in triplicate with TCR-¢ generally failed to increase
`modified Winn assay was done using A431 cells transduced with
`MVitro cytotoxicity above that of T cells expressing $S1-TCR-¢
`mesothelin (26). Rag2y~~ mice were injected s.c. on opposite
`wily. The killing was efficient, with plateau lysis occurring at a
`flanks with either A431.meso or A431 parental tumorcells.
`
`is
`
`
`
`
`
`MEDICALSCIENCES
`
`Ha
`
`“MPenito etal,
`
`Miltenyi Ex. 1015 Page 3
`| March 3,2009 |
`vol. 106
`| no.9 |
`3361
`
`PNAS
`
`Miltenyi Ex. 1015 Page 3
`
`

`

`e
`1000
`o
`5
`g
`5
`5 500
`
`a”E
`—
`vo
`5
`$
`°o
`E
`
`90
`
`
`T cells transduced with SS1 chimeric receptors were coinjected
`with the tumorcells at a 1:2 E:T ratio (Fig. $3). SS1 receptors
`containing the CD28 and CD137 domains were able to inhibit
`tumor growth in a mesothelin-specific manner. However, the
`TCR-¢ group was only able to delay A431.meso tumor growth.
`In contrast, T cells transduced with the truncated TCR¢ or with
`GFP had no effect on tumor growth. Even at a high E:T ratio
`(8:1),
`the control T cells had no effect on tumor growth,
`confirming an allogeneic effect was not contributing to the
`antitumor effects (data not shown).
`To further explore the potential antitumorefficacy of the SS1
`scFv constructs, we developed a rigorous xenograft model using
`M108 tumor cells. When implanted into the flanks of NOD/
`seid/IL2ry~ (NOG) mice, M108 cells proliferated and continued
`to express mesothelin (Fig. $4). Groups of NOG mice were
`injected in the flanks with serially passaged primary M108 tumor
`cells (Fig. 24). On weeks 6 and 7 after establishing the tumor,
`when the tumor burden was ~500 mm, the mice were treated
`with intratumoral injections of 15 < 10° T cells (~70%-80%
`transgene-positive). A potent antitumoreffect was observed(see
`Fig. 2A). Statistical modeling of the tumor growth curves
`revealed significant differences among the treatment groups
`(P < 0.0001 by a Wald test that all groups gave equal log-tumor
`volume curves). The 7 curves separated themselves into 4
`groups: the 3 control treatments (saline, GFP, and Az) were not
`significantly different from each other and showed continued
`growth beyondthe time of T cell transfer; the TCR-¢ treatment
`showed a mixed pattern of continued growth in some animals but
`slowed growth or tumor decline in others (Fig. 2B); the BBz
`treatment showed relatively slow tumordecline; and the 28z and
`28BBz treatments showed rapid tumor decline and werestatis-
`tically indistinguishable from each other.
`The plots of tumor burdenforthe individual mice (see Fig. 2B)
`indicate the magnitude and reproducibility of the antitumor
`
`Fig.2. Mesothelin retargetedTcells eradicate large pre-established tumo
`effects of the T cells endowed with chimeric receptors containing
`in vivo: effect of costimulatory signaling domains and route of administ
`costimulatory domains, with 1 mouse reaching a tumor volume
`
`(A) Human primary M108 tumors were established in the flanks of NOG
`of 1,000 mmbefore tumorregression to subpalpablelevels. The
`After 6 weeks, when the tumors reached a volume of ~500 mm3, then
`
`mice werekilled when tumors reached volumes >2,000 mm? and
`were treated with 2 intratumoral injections of 15 x 10° T cells (~70%-
`tumors were photographed (Fig. $5). Tumors from mice treated
`chimeric receptor-positive) on days 46 and 53 (arrows). Results are exp
`
`with control T cells were highly vascularized, while many of the
`as a mean tumor volume (mm? + SEM) with n = 8 forall groups, a
`
`small tissue masses harvested from the 28z or 28BBz mice and
`representative of 2 experiments. The plots for BBz, CD28z, and CD28B
`several of the BBz mice were fibrotic or necrotic masses.
`significantly different from the control treatment groups (P < 0.000
`
`However, small regions of tumor with abnormal morphology
`log tumor volume curves). (
`Wald test that all groups gave equal
`
`were still evident, as determined by histology. Thus, T cells
`tumor-growth curves of the individual mice in each group are sho
`
`M108-bearing NOG mice were treated with T lymphocytes expressil
`expressing mesothelin-specific chimeric receptors containing
`
`28BBz chimeric receptors (against mesothelin or CD19) via intratumor
`CD28 and 4-1BB domains were able to control or eradicate
`i.p. (IP), andi.v. (IV) routes and the effect on tumor growth wasassessed.
`
`large established tumorsafter intratumoral injections, There was
`tumors reached a mean volume of ~500 mm},2 injections of 10 x 10°
`
`no significant contribution of alloreactivity to the antitumor
`(>90% CIR-positive) on days 43 and 49 (arrows) were performed. The
`effects because mice treated with T cells expressing GFP or a
`are expressed as a mean tumor volume (mm? + SEM)with n = 8 for the
`
`truncated TCR-¢ receptor had tumor growth that was equivalent
`and intratumoral injection groups, and n = 7 for the i.v. and i.p. group
`to mice injected with saline.
`
`In the previous experiment, vigorous and specific antitumor
`effects were noted in mice treated with intratumoral injections
`the ranking (lowest to highest) was intratumoral $S1-28BB4,&
`of the engineered T cells, directly assessing their potential
`intratumoral anti-CD19-28
`SS1-28BBz,
`i.p. SS1-28BBz,
`
`antitumor effects in the vascularized tumor microenvironment.
`and saline. The overall F test for comparison of the mean
`
`We next tested whether the T cells would be effective using
`significant at P < 0.0001, and in pairwise comparisons
`
`several routes of administration (Fig. 2C). Tumors were estab-
`treatment wasnotsignificantly different (at P = 0.05) frot
`lished with M108 cells, and after the tumors reached a mean
`adjacent treatments, but was significantly different from?
`
`volume of ~500 mm*, injections of 10 x 10° T cells transduced
`others. Thus, we conclude that SS1-28BBz eradication of } a
`with SS1-28:BB:TCR-¢ (>90% positive) on weeks 6 and 7 were
`tumoris antigen-specific because the anti-CD19-28BBz g
`
`given by Lv., i.p., or intratumoral routes. For these experiments,
`displayed no antitumoractivity, and that intratumoral appea®
`
`the CD28:BB:TCR-¢ construct was chosen because it has dis-
`be the superior route of administration, marginally faster ™
`played the most potent and reproducible antitumoreffects, as
`iv. (~7 day delay in response) butsignificantly better tha ie
`well as enhanced engraftment properties (see below). Following
`
`iv. and intratumoral injections, a potent antitumor effect was
`served in the pilot clinicaltrials testing chimeric receptors(=~
`again observed, with a more rapid reduction in tumor mass
`and because long-term engraftment of adoptively transfet
`following the intratumoral route of administration. We analyzed
`
`cells correlates with antitumor effects (27, 28), we next
`tumor volumeson the natural log scale. For log-tumor volume,
`Miltenyi Ex. 1015 Page 4
`
`
`—?— saline
`--O--CD19-28BBz IT
`
`—O—$$1-28BBzIP
`
`—¥—$81-28BBz Iv
`Jl’
`1000) —~ss1-2eBBz IT /*
`
`500
`
`
`
`
`
`tumorvolume(mm?)
`
`days post tumorinjection
`
`3362 | www.pnas.org/cgi/doi/10.1073/pnas.0813101106
`
`
`
`Miltenyi Ex. 1015 Page 4
`
`

`

`scFy-transduced T cells on days 46 and 53 after tumorinjection
`was obtained on day 73: that is, 20 days after the last adoptive
`T cell transfer, and quantified for the presence of CD4 and CD8
`T cells (see Fig. 34). For all conditions tested, the CD8 T cell
`counts were higher than the CD4 T cell counts. Analysis of
`variance of the CD4 data indicates significant differences among
`the treatment arms (P < 0.0001). In pairwise comparisons of the
`treatment groups, adjusted for multiplicity by the Tukey method,
`cell counts in mice endowed with 28BBz weresignificantly higher
`than BBz and GFP,while the BBz, 28z, Zeta, and Az groups were
`indistinguishable. Analysis of the CD8 data also revealed sig-
`nificant differences among the treatment arms (P < 0.0001). In
`Tukey-adjusted pairwise comparisons, the cell counts in mice
`engrafted with cells expressing 28BBz and BBz were significantly
`higher (P < 0.05) than GFP, and the 28BBz, BBz, Zeta, 28z, and
`Az groups were indistinguishable. It was unexpected that the
`T-cell counts in the mice given T cells transduced with the 28z
`were not higher than T cells expressing TCR-¢ only, given their
`superior IL-2 production and increased Bel-X;, expression shown
`in Fig. $2). Furthermore, the persistence of T cells in peripheral
`blood did not have a direct correlation with tumor burden
`(compare Fig. 2.4 and B with Fig. 3A).
`Finally, we measured the persistence of T cells in vivo in the
`mice given 28BBz as a function of the route of administration.
`In the experiment shown in Fig. 2C, mice were injected with T
`cells on days 43 and 49, and were bled on day 65 to measure T-cell
`engraftment. Tumor volumes and peripheral blood persistence
`on day 65 are plotted for each individual mouse in each group
`(see Fig. 3B). By inspection of the plots,
`the presence of
`SS1-28:BB:z T cells generally correlates inversely with tumor
`burden in that the groups of mice given T-cell injections by
`intratumoralor iv. routes all had substantial levels of T cells in
`peripheral blood and the lowest tumor burdens, while mice given
`SS1-28:BB:z by ip. or anti-CD19-28:BB:z by intratumoral
`administration, had high-tumor burdens andlow levels of T-cell
`engraftment. The overall F test comparing the mean T-cell
`counts across groups wassignificant (P < 0.0001). The group
`with the highest mean T-cell count was iv. SSI-28BBZ. In
`Tukey-adjusted pairwise comparisons of the means,
`iv. SS1
`administration gave a significantly higher (P < 0.05) count than
`i.p. SS1 administration and intratumoral anti-CD19 scFv admin-
`istration. Mice treated with intratumoral SS1 also had signifi-
`2000
`a
`200
`cantly higher (P < 0.05) T-cell counts than the intratumoral
`1500
`2 s~ 1500
`1500
`anti-CD19 group, suggesting that
`tumor antigen drives the
`
`.E1000 1000
`1000
`expansion of the adoptively transferred T cells in vivo. Further-
`5
`500
`500
`500
`more, in addition to a low-level T-cell engraftment, mice injected
`with T cells expressing a nonbinding anti-CD19 scFv 28:BB:z
`receptor had tumor burdens equivalent to mice injected with
`saline, indicating that the specific triggering of the T cell by the
`M108 cells rather than allogeneic or xenogeneiceffects are required
`for high-level engraftment and antitumor effects observed for the
`T cells expressing the SS1 scFv 28:BB:z receptor. Thus, long-term
`systemic persistence of SS1 scFv 28:BB:z engineered T cells is
`driven by tumor antigen and occurs in tumor-bearing mice follow-
`ing either intratumoral or iv. administration, with i.v. statistically
`equivalent to intratumoral but significantly better than i.p.
`
`(cellsperuL)eRoa@s868$5ooOSooo
`
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`5 ; 7500
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`
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`2001
`
`100
`
`$S1-28BBz IT
`10099 10000
`7500
`5
`2501
`
`_
`
`;
`
`$S1-28BBz IP 100
`
`$S1-28BBz IV
`
` 2000
`
`
`
`
`
`
`
`CD28 and 4-1BBsignals enhance the persistence of human T lympho-
`Fig.3.
`yy es after treatment of M108 tumor. (A) Peripheral blood from M108-bearing
`OG mice treated with intratumoral injections of S$1 chimeric receptor-
`ansduced T cells was obtained on day 73 and quantified for the presence of CD4
`id CD8 T cells by a FACS Trucountassay. Analysis of variance of the CD4 and CD8
`indicates significant differences among the treatment arms (P < 0.0001).
`sults are expressed as a mean absolute count perjl of peripheral blood + SD
`n = 8 for all groups. (B) Persistence ofT cells in vivo was assessedafteri.v.(IV),
`vith
`intratumoral(IT), or i.p. (IP) route of administration. Mice from the experimentin
`19. 2C were bled and analyzed for peripheralT cell persistence on day 65 by a
`FACS Trucountassay. Absolute T-cell count per jL of peripheral bloodis shown
`Tor
`individual mice in each group with corresponding tumor volumesdirectly
`Delow at day 65. The overall Ftest comparing the mean T-cell counts across groups
`Was
`significant (P < 0.0001).
`In Tukey-adjusted pairwise comparisons of the
`Means (at P = 0.05), i.v. and intratumoral $51 administration gave a significantly
`higher T-cell count than i.p. $$1 administration.
`
`i ined the persistence of the lentiviral vector-engineered T cells
`M tumor-bearing mice. Wefirst examined the mice from the
`“xperiment shown in Fig. 24. Peripheral blood from M108-
`*earing NOG mice treated with intratumoral injections of SS1
`
`“*Penito et al.
`
`
`
`MEDICALSCIENCES
`
`Discussion
`
`Retargeted T cells are particularly attractive for the therapy of
`carcinomas, where the available endogenous T-cell receptor
`repertoire may be limiting because of tolerance and previous
`cytotoxic chemotherapy, and MHCClass I expression may be
`decreased. However, while initial clinical studies demonstrate
`feasibility with the retargeted T cells, poor in vivo persistence
`and expression of the transgene have been documented, and the
`therapy has hadless clinical activity than expected (2-4). Our
`studies have addressed all of these issues. Mesothelin proved to
`be an attractive target as the retargeted T cells efficiently and
`Miltenyi Ex. 1015 Page 5
`| March3,2009 |
`vol. 106
`| no.9 |
`3363
`
`PNAS
`
`Miltenyi Ex. 1015 Page 5
`
`

`

`specifically killed a variety of tumors that express mesothelin.
`Previous studies have shown that T cells in the tumor migga
`
`The engineered T cells could kill tumorcells at ~1:10 E:T ratio
`environmenthave a numberofsignaling perturbations (39), a
`in vitro, and at an unprecedented 1:40 E:T ratio in vivo (see
`study has shown that chimeric receptors with a CD28 signa}j
`
`domain enhances the resistance of T cells to regulatory T cells
`below). It was unexpected that expression of the CD137 signaling
`domain was most correlated with persistence of the T cells in
`(40). While we have not yet directly tested the effects ge
`
`vivo, as the CD28 domain was predicted to be mostefficient by
`regulatory T cells on T-effector cells expressing our chime
`
`the preponderanceofin vitro measures of cytokine function and
`receptors, our data would suggest
`that
`the combination
`
`Bel-X,_ expression.
`CD137 and CD28 are moreeffective than either CD137 or
`
`The eradication of large, long-term pre-established tumors by
`alone, based on the antitumoreffects and persistence in tumor.
`
`immunotherapy has rarely been reported. Most preclinical mod-
`bearing mice. Although CD28is sufficient for in vivo antitumy,
`
`els in a therapeutic setting have tested tumors that have been
`activity and the addition of CD137is required for engraftmep
`implanted for less than 2 weeks beforeinitiation of therapy (29).
`the combination of the 2 signals may provide the most effective:
`
`Wehavepresented evidence that T cells expressing SS1 fused to
`therapy and long-term protection.
`1
`
`CD28 and CD 137 costimulatory domains wereable to reject very
`Wetested several routes of administration, and found that
`large, well-established tumors of 500 to 1,000 mm*. Given that
`intratumoraland i.v. routes were nearly equivalent and distip
`the experimentsin Fig. 24 used 2 injections of T cells containing
`superior tothe i.p. route. The delay in tumor regression with
`
`a total of 20 to 24 x 10° T cells transduced with the SS1
`iv. route compared to the intratumoral route may be at Je
`
`construct, and assuming that a 1,000-mm* tumor mass contains
`partially explained by soluble mesothelin in the plasma of
`
`at least 1 X 10° cells, we estimate that the T cells are able to
`mice (data not shown), reflecting that patients with mesothe
`
`eradicate tumors at an initial E:T ratio of ~1:40 in vivo.
`expressing tumors have been documented to have circulatip
`Therefore, our preclinical data would support treating patients
`mesothelin (41). We expect to primarily test the i.v.
`routej
`
`with tumor burdens ofat least 1 * 10!* cells, because our current
`phase I clinical studies; however, the intratumoral route ma
`large-scale manufacturing can routinely produce | x 10!' trans-
`have certain advantages. First, trafficking of T cells into soliq
`
`duced T cells during a 10-day culture (15). Tumorcells of 1 x
`tumors can be rate-limiting (42) and direct injection eliminates
`10" is a clinically relevant tumor burden, representing 1 kg of
`trafficking as a variable. Second, the intratumoral route p
`tumorbulk. Further studies will be required to determine to what
`increase the therapeutic index in situations where therejg ;
`
`extent this tumor eradication can be attributed to serial killing
`potential for on-target, off-organ toxicity.
`In specific,
`
`of tumorcells by the infused T cells and to the tumor-induced
`mesothelin-redirected T cells may cause peritonitis or ple
`proliferation of the retargeted T cells and the cytotoxic effects
`pericarditis, because ofpatternsof tissue-specific expression (6)
`
`of the daughter cells.
`We do not expect that this will be a major problem beca’
`It is likely that several mechanisms account for the enhanced
`previous clinical studies with mesothelin antibodies and imn
`
`efficiency of the redirected T cells observed in the presentstudy.
`noconjugates have not yet revealed unmanageable toxicity
`
`First, previous studies have generally used T cells transduced
`13). Finally, recent studies have shownthe safety and feasibi
`
`with retroviruses. The high efficiencies associated with lentivi-
`of intratumoral injection of T cells (43). In summary, chime
`ral-mediated transduction, combined with robust in vitro cell-
`T-cells targeted to mesothelin and possessing intracytoplasm
`
`expansion methods, make the rapid expansion of large numbers
`signaling domains from TCR¢, CD28, and CD137 appear h
`of therapeutically relevant numberfeasible. In the present study
`effective at
`treating large, well-established tumors in mi
`
`we have usedlentiviral vectors, which have a higher transduction
`Clinical translation of this approach to tumors,such as mesotl
`efficiency, thus shortening the in vitro culture to 7 to 10 days, and
`lioma and ovarian cancer, is currently underway.
`permitting the use of the T cells early at a time when we have
`
`shown previously that the average telomere length of the cul-
`Methods
`tured T cells is actually longer than at the start of culture (30).
`Generation of Antimesothelin T-Body Molecules. Chimeric antimesothelin sch
`We attribute this to the previous demonstration