Preclinical Evaluation of Chimeric Antigen Receptors...

Cancer Therapy: Preclinical

Preclinical Evaluation of Chimeric AntigenReceptors Targeting CD70-Expressing CancersQiong J.Wang, Zhiya Yu, Ken-ichi Hanada, Krishna Patel, David Kleiner,Nicholas P. Restifo, and James C. Yang

Abstract

Purpose: CD70 expression in normal tissues is restricted toactivated lymphoid tissues. Targeting CD70 on CD70-expressingtumors could mediate "on-target, off-tumor" toxicity. This studywas to evaluate the feasibility and safety of using anti-humanCD70 CARs to treat cancer patients whose tumors express CD70.

Experimental Design: Seven anti-human CD70 CARs withbinding moieties from human CD27 combined with CD3-zetaand different costimulatory domains from CD28 and/or 41BBwere constructed. In vitro functionality of these receptors wascompared and in vivo treatment efficacy was evaluated in axenograft mouse model. A homologous, all murine anti-CD70CAR model was also used to assess treatment-related toxicities.

Results: The CAR consisting of the extracellular bindingportion of CD27 fused with 41BB and CD3-zeta (trCD27-

41BB-zeta) conferred the highest IFNg production againstCD70-expressing tumors in vitro, and NSG mice bearing estab-lished CD70-expressing human tumors could be cured byhuman lymphocytes transduced with this CAR. In the murineCD27-CD3-zeta CAR model, significant reduction of estab-lished tumors and prolonged survival were achieved usingCAR-transduced splenocytes in a dose-dependent manner.Host preirradiation enhanced treatment efficacy but increasedtreatment-related toxicities such as transient weight loss andhematopoetic suppression. The treatment did not appear toblock adaptive host immune responses.

Conclusions:Preclinical testing supports the safety and efficacyof a CD27-containing CAR targeting CD70-expressing tumors.Clin Cancer Res; 23(9); 2267–76. �2016 AACR.

IntroductionCell-based immunotherapies have shown promising clinical

outcomes in recent years. Adoptive transfer of tumor-infiltratinglymphocytes (TIL) can achieve durable complete regression ofmetastatic melanoma (1). Alternatively, redirecting autologous Tcells with chimeric antigen receptors (CAR) or alpha-beta T cellreceptors (TCR) against tumor-associated antigens also mediatedlong-term durable remissions of late-stage cancers refractory tostandard therapies (2–4). The advantage of CARs is that they cantarget antigens expressed on the cell surface without MHC restric-tion, therefore making them more widely applicable. However,treating solid cancers using CAR immunotherapies remains chal-lenging due to the paucity of safe, clinically effective tumor-associated cell-surface antigens identified to date (5, 6).

CD70 was initially identified as the ligand for CD27, acostimulatory receptor involved in T-cell proliferation andsurvival (7, 8). Studies on CD70 expression revealed the samerestricted expression profile in both humans and mice (9, 10),with CD70 only found on a small percentage of activated T cells

and antigen presenting cells in draining lymph nodes duringviral infection (10). Interestingly, a number of human tumorscan also express CD70, including solid cancers such as clear cellrenal cancer (RCC), glioblastoma, and hematological malig-nancies (11–14). The mechanism of overexpression of CD70on tumors remains unknown, but a recent study suggested thedysregulated pVHL/HIF pathway may be involved in RCC (15).Nonetheless, due to its restricted expression pattern on normaltissues and overexpression in cancers, CD70 may be an attrac-tive therapeutic target. Approaches using antibody–drug con-jugates directed against CD70 have shown some antitumoractivity in vitro, and demonstrated some clinical responsesagainst RCC and non-Hodgkin's lymphoma in a phase I clinicaltrial (13, 16). In addition, T cells genetically engineered with aCAR consisting of full-length CD27 coupled with the CD3-zetasignaling domain (CD3-zeta) has shown CD70-specific tumorrecognition (17). These studies suggested that CD70 could be apotential immunotherapeutic target. However, due to CD70expression by activated lymphoid tissues, the potential for"on-target, off-tumor" toxicity may hinder potential clinicalapplications, as has been observed when targeting CD19 onmalignant and normal B cells with a CAR (2, 18). Therefore, inthis study, we have compared the antitumor reactivities of anti-human CD70 CARs in which different portions of CD27 werefused with various costimulatory signaling domains from 41BBand/or CD28, and CD3-zeta, and established a murine modelto test the potential for on-target, off-tumor toxicities. Ourstudy demonstrates that anti-human and anti-murine CD70CARs were effective in vitro and in vivo, respectively. However,some treatment-related toxicities were also observed, althoughthey appeared to be reversible and tolerable in the murinemodel. Among the anti-human CD70 CAR constructs, CD27

Surgery Branch and Laboratory of Pathology, Center for Cancer Research, NCI,NIH, Bethesda, Maryland.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthors:Qiong J.Wang, NCI, NIH, 9000 Rockville Pike, Building10/CRC, Room 3W-3840, Bethesda, MD 20892. Phone: 301-435-6264; Fax: 301-496-0011; E-mail: [email protected]; and James C. Yang, E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-16-1421

�2016 American Association for Cancer Research.

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without its intracellular signaling domain, fused with thecostimulatory of 41BB and then CD3-zeta, designated asCD27-41BB-zeta, appeared to be most active in vitro. Therefore,plans are under way to test this anti-CD70 CAR in patients withadvanced, refractory tumors expressing CD70.

Materials and MethodsMice, tumor lines, and antibodies

C57BL/6J and NSGmice (The Jackson Laboratory) were main-tained per protocols in the NIH animal facility. Murine tumorlines B16 and B16/mCD70 (retrovirally transduced with murineCD70) were maintained in RPMI 1640 (Life Technologies) with10% fetal bovine serum (FBS; Life Technologies). All mousestudies were approved by the National Cancer Institute AnimalCare and Use Committee.

Tumor lines from RCC patients were established and main-tained in DMEM (Life Technologies), including 10% FBS, 10%tryptose phosphate (Sigma), 1� insulin-transferrin-selenium(Life Technologies) and 1 � serum pyruvate (Life Technologies).Melanoma tumor lines were maintained in RPMI 1640 (LifeTechnologies) with 10% FBS. All cell lines included in the studywere generated at Surgery Branch, NCI, and tested and identitiesconfirmed by HLA genotyping. The cell lines were maintained inthe cell culture only when they were needed in the experimentsand usually kept in culture for approximately a month, andmycoplasma testing were done routinely using mycoplasmadetection kit (Lonza). The cell lines were reassessed for HLA andantigen expression by flow cytometry and coculture assays whenthey were thawed for each experiment.

Monoclonal antibodies (mAb), including FITC-labeled anti-mouse CD3, anti-mouse CD45.1, and anti-human CD8 Abs,PE-labeled anti-mouse and anti-human CD70 Abs, PE-cy7-labeled anti-human CD3 Ab, APC-labeled anti-mouse CD27Ab, APC-cy7-labeled anti-mouse CD8, and purified anti-mouseCD3 and anti-mouse CD28 Abs were purchased from BDPharmingen. APC-labeled anti-human CD27 Ab was purchasedfrom eBiosciences.

Constructionof humananti-CD70CARs, retroviral production,retroviral transduction of human PBL, and in vitro reactivity oftransduced cells

Genes encoding seven human anti-CD70 CARs, includingcDNAs for full-length CD27 (flCD27) or truncated CD27(trCD27; including extracellular and transmembrane portions ofCD27, aa1-211) fusedwithCD28and/or 41BB signaling domainsand CD3-zeta were constructed and cloned into the pMSGV1plasmid (Fig. 1A). Retroviral production and transduction werethe same as described previously (19). Briefly, 293gp cells weretransfected with 9 mg of anti-CD70 CARs and 4.5 mg of plasmidRD114using Lipofectamine 2000 (Life Technologies; 60mL). Twodays later, the supernatants were harvested and used to transduceanti-CD3 stimulated PBL. PBL from allogeneic donors were sti-mulated with soluble OKT-3 (50 ng/mL) and rhIL2 (300 IU/mL)for 2 days before transduction was performed. The stimulatedcells were added to 24-well plates initially coated with Retro-Nectin (Takara) and subsequently precoated with retrovirus byspinoculation (2,000� g, 32�C,2hours) at 5�105/mL. Theplateswere then centrifuged at 1,000� g for 10 minutes, and incubatedovernight at 37�C in a 5% CO2 incubator. This procedure wasrepeated the next day and cells were split as necessary tomaintaincell density between 0.5 and 1 � 106 cells/mL. Transductionefficiency was estimated by analyzing human CD27 expressionon retrovirally transduced cells and comparing this to mock-transduced T cells. To test their reactivity, retrovirally transducedcells (1 � 105) were cocultured with 5 � 104 human tumor lineswith or without CD70 expression at 37�C, 5%CO2 overnight. Thesupernatants were harvested and tested for IFNg secretion byELISA (Thermo Fisher Scientific).

Mouse xenograft studiesNSG mice were injected subcutaneously with 1.5 � 105

2654R human renal cancer cells. Eighteen days after inocula-tion, when tumors were approximately 5 mm in diameter, micereceived 6 � 106 intravenous human T cells retrovirally trans-duced with anti-CD70 CARs or control T cells, followed byintraperitoneal administration of 200,000 IU of rhIL2 per dayfor 3 days. Each group included 5 randomly assigned tumorbearing mice, and all tumor measurements were performed bya blinded impartial observer.

Construction of murine anti-CD70 CAR, retroviral production,transduction of murine CD3 T cells, and in vitro functionalanalysis of transduced cells

cDNA encoding murine CD27 fused with murine CD3-zetasignaling domain (mCD27-zeta) was constructed in thepMSGV1 plasmid. To produce retrovirus, 293gp cells weretransfected with 9 mg of pMSGV1-mCD27-zeta and 4.5 mg ofplasmid pCL-Eco (Addgene) using Lipofectamine 2000 (LifeTechnologies; 60 mL). Two days later, the supernatants wereharvested and used to transduce activated mouse T cells. Sple-nocytes from C57BL/6J mice were harvested and CD3þ T cellswere isolated by negative selection using a mouse pan–T-cellisolation kit II (Miltenyi Biotec). Murine CD3 T cells were thenstimulated with plate-bound anti-mouse CD3 (1 mg/mL), andsoluble anti-mouse CD28 (1 mg/mL) and rhIL2 (30 IU/mL) for2 days before transduction was performed. Transduction ofstimulated cells was performed similarly as described above byspinoculation. Transduction efficiency was determining byanalyzing mouse CD27 expression on retrovirally transduced

Translational Relevance

CD70 has been identified as a biomarker for clear cell renalcell cancer (RCC) as well as several hematological malignan-cies, such as non-Hodgkin's lymphoma. In this study, we firstevaluated the in vitro function of anti-human CD70 chimericantigen receptor (CAR) constructs by fusing different portionsof CD27 with CD3-zeta in combination with costimulatorysignaling domains such as CD28 and/or 41BB. Amongthem, the extracellular binding portion of CD27 fused with41BB and CD3-zeta appeared to be the best against CD70-expressing tumors, and curative effects and long-term survivalwere observed when CAR-expressing T cells were adoptivelytransferred to tumor-bearing NSG mice. We then testedin vivo toxicity and efficacy using a homologous, completelymurine model. This showed only transient cytokine toxicity atcell doses 100-fold higher than needed to show efficacy. Aphase I/II clinical trial using the anti-human CD70-41BB-CD3zeta CAR against RCC and other CD70-expressing tumorsis under way.

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Clin Cancer Res; 23(9) May 1, 2017 Clinical Cancer Research2268




T cells, compared with cells just stimulated with anti-CD3,anti-CD28 and rhIL2. To assess the reactivity of mCD27-zeta,retrovirally transduced murine T cells (1� 105) were coculturedwith B16/mCD70 or B16 (5 � 104) at 37�C, 5% CO2 overnightand supernatant was harvested and tested for mouse IFNgsecretion by ELISA (R&D Systems).

Treatment efficacy ofmurine T cells retrovirally transducedwithmurine anti-CD70 CAR in vivo

C57BL/6J mice were injected subcutaneously (s.c.) with 0.5 �106 B16 or B16/mCD70 tumors. Ten days after inoculation, whentumors were approximately 5 mm in diameter, mice received upto 107 intravenous (i.v.) murine T cells retrovirally transducedwithmCD27-zeta or stimulated, untransduced T cells as a control,

followed by intraperitoneal (i.p.) administration of 200,000 IUofrhIL2 per day for 3 days. Splenocytes from pmel-1 TCR transgenicmice, that are reactive to gp100 in both B16 and B16/mCD70tumors, were activated in vitro in the presence of 1 mmol/Lhgp10025–33 peptide and 30 IU/mL rhIL2 for 7 days. As a positiveadoptive cell therapy control, a total of 106 activated pmel T cellswere given to mice i.v. along with recombinant vaccinia virusencoding hgp100 (2 � 107 pfu) and the regimen of i.p. rhIL2 asabove. Where specified, sublethal total body irradiation (TBI;500 cGy) was given to mice immediately prior to cell transfer.Mice were given from 104 to 107 retrovirally transduced murineT cells when assessing minimal effective treatment dosage. Alltreatment groups were randomly assigned, and all tumor mea-surements were performed by a blinded impartial observer.

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Figure 1.

The reactivity of anti-human CD70 CARs against CD70-expressing tumors in vitro. A, Schematic of anti-human CD70 CAR constructs. Seven constructs were madeusing different portions of CD27 in combination with CD28 or 41BB costimulatory domain and CD3 zeta signaling domain. B, Comparison of CD27 expressionon T cells transduced with anti-human CD70 CARs. Allogeneic PBL were stimulated with anti-CD3 and retrovirally transduced with 7 anti-human CD70 CARs.Three days after transduction, cells were labeled with anti-CD3-FITC and anti-CD27-APCmAb, and analyzed on FACS CantoII. Data shownwere gated on propidiumiodide–negative cells. UT: untransduced controls. An arbitrary CD27-negative gate for untransduced cells was used to compare the transduction efficiency betweendifferent CARs. C, Comparison of antitumor reactivity of T cells transduced with anti-human CD70 CARs. Allogeneic PBL were stimulated with anti-CD3 andretrovirally transduced with 7 anti-human CD70 CARs, respectively. Three days after transduction, transduced T cells were cocultured with a panel of tumor linesovernight, and tested for IFNg production by ELISA the next day.

Chimeric Antigen Receptors Targeting CD70

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In vivo persistence of transferred T cells transducedwithmurineanti-CD70 CAR and assessment of treatment-related toxicity

T cells from Ly5.1 congenic mice were stimulated with plate-bound anti-murine CD3 and soluble anti-mouse CD28 Abs for2 days, and retrovirally transduced with mCD27-zeta. Four daysafter transduction, T cells (107) were injected i.v. to C57BL/6Jmice, followed by i.p. administration of 200,000 IU of rhIL2 perday for 3 days. Splenocytes were harvested from day 3 to day 12,and the presence of transferred cellswas determinedby expressionofCD45.1-positive cells.Measurements ofweight, absolute bloodcell counts, blood chemistry, and the number of splenocytes andserum cytokines were determined in both tumor-bearing andnon–tumor-bearing mice.

Assessment of host immune responses after adoptive celltransfer

C57BL/6Jmicewere injected i.v. withmurine T cells retrovirallytransduced with mCD27-zeta or untransduced control T cells,followed by i.p. administration of 200,000 IU of rhIL2 per dayfor 3 days. Thirty-two days after transfer, mice were immunizedwith vaccinia virus encoding either OVA or gp100. Splenocytesfrom treated and control animals were harvested 7 days afterimmunization, and cultured in vitro in the presence of 1 mmol/LOVA257–264 or gp10025–33 peptides for 7 days, and then tested forIFNg production of by coculturing T cells with LPS-stimulatedlymphoblasts pulsed with either peptide.

Statistical analysisWilcoxon rank-sum test was used to compare tumor slopes

between each treatment groups, and log-rank test was used toanalyze survival.

ResultsThe effectiveness of anti-human CD70 CAR in vitro and in vivo

Seven anti-human CD70 CAR retroviral vectors were con-structed and transduced into anti-CD3 stimulated normal donorperipheral blood lymphocytes (PBL) to evaluate their expressionand antitumor activities. As shown in Fig. 1A, full-length CD27(flCD27) fused with the CD3 zeta signaling domain (CD3-zeta)with or without CD28 and/or 41BB signaling domains wereconstructed, designated as flCD27-zeta, flCD27-CD28-zeta,flCD27-41BB-zeta, and flCD27-CD28-41BB-zeta, as several stud-ies demonstrated CD28 and 41BB signaling domains couldaugment antitumor reactivities and in vivo persistence (20–24).Similarly, three anti-human CD70 CARs using truncated CD27(trCD27, aa 1–211; i.e., CD27 without its intracellular domain)were also constructed, shown as trCD27-CD28-zeta, trCD27-41BB-zeta, and trCD27-CD28-41BB-zeta. PBL retrovirally trans-duced with each of the seven vectors showed various degrees ofCD27 expression (Fig. 1B). Asmight be expected, PBL successfullytransduced with these CAR were devoid of CD3þCD70þ cells.flCD27-zeta appeared to be the bestwith�92%of cells expressingCD27 in comparison with �70% of CD27 expression onT cells transduced with trCD27-41BB-zeta, flCD27-41BB-zeta, orflCD27-28-41BB-zeta (and 0.26% of stimulated but untrans-duced PBL). Because mock-transduced or untransduced T cellsstill expressed certain level ofCD27, positivity ofCD27expressionin these anti-CD70 CAR-expressed T cells was only counted whenexpression level was above the level of mock-transduced oruntransduced T cells. This may lead to underestimate transduc-

tion efficiency of anti-CD70 CARs. Downregulation of CD3 onthese transduced T cells was also observed, and consistently seenwith high transduction efficiency, suggesting that CD3 internal-ization may occur with introduced CD3-zeta chain signalingdomain. Surprisingly, only 45%, 16%, and 3% were CD27positive when T cells were transduced with trCD27-28-zeta,trCD27-28-41BB-zeta, and flCD27-28-zeta, respectively. To testantigen-dependent tumor recognition by these anti-humanCD70CARs, a panel of CD70-negative tumor lines (624mel and938mel), and their stably transduced CD70 expressing counter-parts (624/CD70 and 938/CD70), and RCC tumor lines naturallyexpressing high to low levels of CD70 (RCC HC, RCC RO, RCCDS, andRCCMW, respectively; Supplementary Fig. S1 and ref. 25)were included. All of the CARs, except for flCD27-28-zeta, dem-onstrated specific anti-CD70 reactivity, as they only recognizedCD70-positive tumor lines, but not CD70-negative tumor lines(Fig. 1C). The control vector did not show any reactivity againstthese tumors. Among the candidate CARs, trCD27-41BB-zetaappeared to possess the highest antitumor reactivity by IFNgproduction. In addition to flCD27-28-zeta, lower anti-CD70reactivity was also observed in T cells transduced with trCD27-28-zeta, suggesting that signaling through CD28 may haveadverse effects on these constructs. Based on transduction effi-ciency and antitumor reactivity of these CARs, we decided tocompare the efficacy offlCD27-zeta and trCD27-41BB-zeta in vivo.The CD70-positive human renal tumor line, 2654R, was injectedsubcutaneously into NSG mice and once palpable, treated withhuman T cells transduced with either flCD27-zeta or trCD27-41BB-zeta. A curative effect was observed in both groups as shownin Fig. 2A and B. None of the control groups showed any delayedtumor growth. Our results suggest that either CAR could beeffective in treating CD70-positive tumors in vivo. However,because normal tissues ofNSGmice donot express humanCD70,we could not assess the treatment-related toxicities using thismodel. Therefore, a mouse tumor model with tumors expressingmurine CD70 was generated to address these issues.

Treatment efficacy of murine anti-CD70 CARTo generate a mouse model, an anti-murine CD70 CAR was

constructed by fusing full-lengthmurineCD27withmurine CD3-signaling domain (mCD27-zeta). Murine T cells expressed highlevels of CD27 after retroviral transduction with the anti-murineCD70 CAR compared with mock-transduced or untransducedT cells (Fig. 3A). Meanwhile, a stably transfected cell line, B16/mCD70, was generated by transducing B16melanoma (a murineCD70-negative tumor line recognized by pmel T cells; Fig. 3B;ref. 26), with murine CD70. As shown in Fig. 3C, pmel T cellsproduced mouse IFNg when cocultured with either parental B16tumor, or B16/mCD70. However, mCD27-zeta could conferspecific, anti-CD70 reactivity only against B16/mCD70. To testthe antitumor efficacy ofmCD27-zeta in vivo, C57BL/6Jmice wereimplanted with either B16 or B16/mCD70 subcutaneously, andthen treated with mouse T cells retrovirally transduced withmCD27-zeta or other controls when tumors became palpable.While pmel T cells (given with concomitant vaccination and IL2)could reduce tumor burdens in irradiatedmice carrying either B16or B16/mCD70, mCD27-zeta-transduced T cells (given only withIL2) delayed tumor growth only in irradiated mice carrying B16/mCD70, in an antigen-specific manner (Fig. 3D). Mice that weretreated with untransduced T cells or left untreated did not showany delay of tumor growth. In addition, treatment efficacy was

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dose dependent, with 1� 105 the lowest effective T-cell numberin irradiated mice (Fig. 3E). In nonirradiated mice, however,treatment effects could only be observed when 1 � 107 cellswere transferred. This may be attributable to superior persis-tence of transferred cells seen in irradiated mice compared withnonirradiated mice (Supplementary Fig. S2A). Furthermore,irradiated B16/mCD70-bearing mice treated with 1 � 105

to 1 � 107 CAR T cells had significantly better survival thanmice treated with no cells or 1 � 107 mock-transduced T cells(Fig. 3F). Although curative effects could be achieved in micethat were treated with high cell doses (1 of 5 given either 1 �106 or 1 � 107 cells), the majority of mice showed tumorgrowth inhibition rather than durable regressions. To delineatepossible mechanisms of immune escape, we analyzed mCD70expression on tumors progressing in treated mice (Fig. 3G).While tumors from control animals remained mCD70 positive,tumors from mCD27-zeta–treated mice completely lackedmCD70 expression, implicating antigen loss as a cause ofimmune escape. While efficacy data were interesting, thismouse model was primarily developed to analyze the in vivotoxicity of using a CD27-zeta CAR to target CD70.

Short-term treatment-related toxicities of anti-murine CD70CAR

To assess treatment-related toxicities, C57BL/6J mice wereimplanted with either B16 or B16/mCD70, treated withmCD27-zeta–transduced or control T cells, and body weightevaluated as an indicator of systemic cytokine toxicity. IrradiatedB16/mCD70 mice receiving mCD27-zeta–transduced T cellsshowed significant lower body weight than those receivinguntransduced T cells, pmel T cells, or left untreated from day 6today 10, but recovered 2weeks after the cell transfer (Fig. 4A).Onthe other hand, no significant differences were observed in non-irradiated recipients of T-cell transfers. Interestingly, lower bodyweight was also observed in irradiated,mCD27-zeta–treatedmicethat were implanted with antigen-negative B16 tumors, whichsuggested that this transient toxicity results primarily frommCD27-zeta–transduced cells interacting with normal endoge-nous host cells.

To further define the toxicity of mCD27-zeta CAR cells inde-pendent of the effects of tumor growth, we conducted experi-ments in non–tumor-bearing C57BL/6J mice. Body weight,peripheral white blood cell (WBC) and lymphocyte counts,splenocyte counts, blood chemistries, and serum cytokines wereevaluated for 2 weeks after treatment. Similar to tumor-bearingmice, irradiated mice that were treated with mCD27-zeta–trans-duced T cells showed significant lower bodyweight at days 6 and 8than those that were treated with mock-transduced or untrans-duced T cells, but recovered approximately 2 weeks after celltransfer (Supplementary Fig. S2B). This effect was not observedin nonirradiated mice. As would be predicted, whole body irra-diation, with or without CAR T-cell transfer dramaticallydecreased absolute WBC and lymphocyte counts as well as sple-nocyte counts compared with all nonirradiated groups (Fig. 4Band Supplementary Fig. S2C). Although blood chemistry did notshow any differences among groups, we could detect IFNg inmouse serum fromday 3 to day 5 only in irradiatedmice thatweretreated with mCD27-zeta CAR T cells (Fig. 4C). However, nodifferences could be detected in the very low levels of CD70expression on splenocytes among these groups (Fig. 4D). Takentogether, our data suggest that transient cytokine toxicity, asindicated by weight loss and elevated serum IFNg levels, couldbe detected in mice undergoing whole body irradiation followedby mCD27-zeta CAR T-cell transfer, and this resulted primarilyfrom reactivity against endogenous host cells.

Immune competence of mice treated with anti-murine CD70CAR

Because successfully targeting CD70 could affect some T cellsand APCs, we tested immune responses in treated mice. Onemonth after cell transfer, treated mice were immunized withvaccinia-OVA or vaccinia-gp100. Splenocytes from treated micewere stimulated and tested for reactivity against OVA or gp100peptides. As shown in Fig. 5A, 7 days after in vitro stimulation withOT-I257–264, splenocytes from mice treated with mCD27-zeta ormock-transduced cells (with or without recipient preirradiation)were reactive to OT-I257–264. Similar results were observed withmice immunized with vaccinia-gp100 (Fig. 5B), as T cells frommice immunizedwith vaccinia-gp100were reactive to gp10025–33,regardless which treatment the mice had received. Interestingly,T cells from mice given mCD27-zeta CAR T cells with irradia-tion appeared to bemore reactive than other groups. Nonetheless,our data show that mice treated with anti-murine CD70 CAR

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Figure 2.

Adoptive cell transfer of antihuman CD70 CAR-transduced cells treatinghuman cancers in NSGmice.A, The human renal tumor line, 2654R, was injectedinto NSG mice subcutaneously, and 6� 106 T cells transduced with flCD27-zetaor trCD27-41BB-zeta were injected intravenously when tumors becamepalpable. Mice given no treatment, the same number of untransduced T cells(UT) or mock-transduced T cells served as controls. Serial tumor measurementswere obtained and tumor area calculated. Five mice were included in eachgroup. Center bar, mean; error bars, SEM. B, Kaplan–Meier analysis of survival intumor-bearing mice receiving adoptive transferred T cells transduced withanti-human CD70 CARs versus controls (P < 0.0001). Four of 5 mice fromeach treatment group are cured. "ACT" represents "adoptive cell transfer."






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T cells could still mount immune responses against exogenousantigens.

DiscussionWe have demonstrated in this study that using CD27, the

natural receptor that engages CD70, we could construct CARs totarget CD70-expressing tumors. CD70 can be expressed on acti-vated normal lymphocytes, but we did not encounter in vitro"fratricide" by CD27-zeta CAR expressing T cells as an impedi-ment to activating, transducing, and expanding populations ofreactive, CAR-transduced T cells. We then compared 7 differentanti-human CD70 CARs introduced in a replication-defectivegamma-retrovirus for in vitro CD70-specific antitumor reactivity.These 7 CARs could all be categorized as second-generation CARs(flCD27-zeta, trCD27-28-zeta, and trCD27-41BB-zeta) or third-generation CARs (flCD27-28-zeta, flCD27-41BB-zeta, trCD27-28-41BB-zeta, and fCD27-28-41BB-zeta) given the costimulatorynature of the full-length CD27 receptor. Significant differenceswere observed when different costimulatory signaling domainswere included in the constructs. T cells transduced with receptorscontaining only the 41BB cosignaling domain, i.e., trCD27-41BB-zeta and flCD27-41BB-zeta, showed high transduction effi-ciency and anti-CD70–specific reactivity, while T cells includingonly a CD28 cosignaling domain, such as trCD27-28-zeta andflCD27-28-zeta, showed poor receptor expression and lower anti-CD70 reactivity. Interestingly, adding the 41BB signaling domain(constructs trCD27-28-41BB-zeta andflCD27-28-41BB-zeta com-pared with trCD27-28-zeta and flCD27-28-41BB-zeta, respective-ly) could partially compensate for the poor performance of CD28alone in these constructs. Our findings coincide with a recentpublication in which the authors demonstrate that 41BB costi-mulation reduces, but CD28 costimulation induces, exhaustionofCAR-transduced T cells (27). Although the intracellular domainof CD27may augment antitumor reactivity in vivo (28), includingthe full-length CD27 receptor in the CAR did not reverse thedeleterious effect of the CD28 costimulatory domain alone in ourin vitro study. Overall, the second-generation construct, trCD27-41BB-zeta, appeared to be the best among the constructs we testedin vitro. Comparing trCD27-41BB-zeta with flCD27-zeta in axenograft experiment, both constructs showed similar curativeeffects against a naturally expressingCD70þ tumor. Recent studies

using CD19 CAR treating multiple hematologic malignancieshave shown dramatic clinical responses (2, 3, 29), and the in vivoexpansion of CAR-transduced T cells correlated with clinicalresponses (29). In fact, multiple preclinical studies have demon-strated that the 41BB costimulatory domain can enhance in vivopersistence and survival of CAR-transduced T cells (21, 24). Arecent study further suggests that 41BB promotes the growth ofcentral memory T cells with enhanced fatty acid oxidation andmitochondrial biogenesis (30). Therefore, we have chosentrCD27-41BB-zeta for our future clinical studies.

One of the biggest drawbacks with T cells targeting overexpres-sing tumor antigens is the "on-target" toxicity against normaltissues (2, 31–34). The severity of these treatment-related toxi-cities is largely dependent on which normal tissues express thetargeted antigen. T cells engineered with an anti-CD19 CAR, forinstance, can effectively treat late-stage cancer patients withCD19þ B-cell malignancies. However, it can also cause a transientacute cytokine release syndrome as well as long-term eradicationof normalCD19þB cells in somepatients (2, 35). These effects canbe managed medically in nearly all patients and are consideredtolerable in view of the efficacy of this cell transfer. Thus, analysisof antigen expression patterns and toxicity in an appropriate all-murine model can be valuable when vetting a potential tumor-associated antigen targeted by adoptive T-cell therapy. Similar toprevious studies, we could only detect CD70 expression on a verysmall subpopulation of human peripheral blood cells, andmurine lymphoid tissues, such as splenocytes, lymphnodes, bonemarrow, and peripheral blood. An all murine model targetingmCD70 with a CAR using the mCD27 binding domain couldelucidate the in vivo consequences of depleting this subpopulationof normal lymphocytes and possibly reveal other unsuspectedCD70 expression on normal tissues, but further strict quantitativetranslation of toxicities may not be possible when homologousreceptor components and host species also have to be changed.Therefore, for our in vivo murine toxicity studies, we used a basicCD27-zeta CAR that not only depleted CD70 expressing immunecells but also had demonstrated efficacy against tumor. Using thistherapeutically effective anti-murine CD70 CAR, we demonstrat-ed that acute toxicities such as weight loss and low lymphocytecounts occurred in the irradiated treatment group at the highestcell doses but resolved within 2 weeks after cell transfer. Inaddition, low levels of serum IFNg could be detected in the

Figure 3.The effectiveness of an anti-murine CD70 CAR in treating murine CD70-expressing tumor. A, Expression of CD27 in retrovirally transduced murine T cells. MurineT cells were stimulated with anti-murine CD3 and CD28 Abs and retrovirally transduced with mCD27-zeta. Mock or untransduced murine T cells were included.Three days after transduction, T cellswere stainedwith anti-murine CD3-FITC and anti-murine CD27-APC and analyzed on FACSCantoII. An arbitrary CD27-negativegate of untransduced T cells were used to determine the percentage of CD27 expression. B, Expression of murine CD70 in murine tumor lines. B16, a murinemelanoma line; B16/mCD70, B16 tumor retrovirally transduced with murine CD70. C, Reactivity of anti-murine CD70 CAR. Splenocytes were stimulated with anti-mouse CD3 and CD28, and transduced with anti-murine CD70 CAR (mCD27-zeta). Three days after transduction, 1 � 105 transduced cells were coculturedwith 5� 104 targets, and tested formouse IFNg secretion. pmel T cells (from an anti-gp100 TCR transgenicmouse strain), untransduced andmock-transduced T cellsserved as controls. D, Treatment efficacy of anti-murine CD70 CAR in vivo. B16 or B16/mCD70 was injected s.c. into C57BL/6J mice and 500 cGy of whole bodyradiation followed by 1 � 107 murine T cells transduced with mCD27-zeta were injected i.v. 11 days after tumors were implanted. Mice given 1 � 106 pmelT cells along with recombinant vaccinia virus encoding hgp100 (rVVhgp100), 1 � 107 untransduced T cells, or no treatment served as controls. E, Delayed tumorgrowth by anti-CD70 CAR was dose- and irradiation-dependent. B16/mCD70 was injected s.c. into C57BL/6J mice. Eleven days later, when tumors were palpable,mCD27-zeta–transduced cells were adoptively transferred with or without total body irradiation (TBI) at doses from 1 � 104 to 1 � 107, again with PBS, 1 � 107

mock-transduced T cells, or 1� 106 pmel T cells with rVVhgp100 as controls. Serial tumor measurements were obtained and tumor area calculated. Five mice wereincluded in each group. Center bar, mean; error bars, SEM. F, Kaplan–Meier analysis of survival in tumor-bearing mice receiving 1� 104 to 1� 107 T cells transducedwith anti-murine CD70 CARs. Survival was significantly prolonged for mice receiving 1 � 105, 1 � 106, or 1 � 107 CAR T cells compared with all controlgroups (P < 0.001). G, CD70 expression in tumors from mice with progressive disease after treatment. B16/mCD70 tumor–bearing mice were treated as described.Twenty days after adoptive cell transfer, mice were sacrificed, tumors were taken from each group, labeled with anti-mouse CD70 mAb and analyzedon FACS Canto II.






Figure 4.

Short-term impact of anti-murine CD70CAR in themurinemodel.A,Bodyweight ofmice treatedwith anti-murine CD70CAR (mCD27-zeta). B16 or B16/mCD70wasinjected s.c. into C57BL/6J mice, and 11 days after tumors were implanted, some groups were given 500 cGy of TBI or left untreated. Then, 1 � 107 murineT cells transduced with mCD27-zeta or control T cells were injected i.v. B–D, Total WBC and lymphocyte counts (B), IFNg secretion in mouse serum (C), and CD70expression on splenocytes (D), from B16/mCD70 tumor–bearing mice treated with anti-murine CD70 CAR. C57BL/6J mice, either exposed to 500 cGy or leftuntreated, were given 1 � 107 murine T cells transduced with mCD27-zeta or control T cells.


Wang et al.




irradiated treatment group during the first week after cell transfer.Our data suggest endogenous CD70-expressing cells may inducethese short-term, self-limited toxicities. Analyses of blood chem-istry and sequential histological studies of multiple tissues alsodid not show any consistent differences associated with the

transfer of these CAR T cells. Moreover, the immune competenceof the treated mice appeared to remain intact, because they couldrespond normally to immunization using vaccinia virus encodingOVA or gp100. This would imply that CD70 expression is not anobligate step in T-cell activation or APC function. Any impact onthis small normal population of immune cells appears similarand analogous to that seenwhen clinically targeting the normal B-cell antigen CD19 with CAR T cells. No unacceptable toxicitieswere encountered in this preclinical evaluation of an anti-CD70CAR using CD27 as the binding domain. The potential foradditional toxicities with more complex receptor structuresalways exists, but this will be best addressed by careful phase Idose-escalation trials. Such a clinical trial (NCT02830724) withescalating cell doses of CAR-transduced autologous PBL inpatients with late-stage CD70þ cancers is under way.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Q.J. Wang, J.C. YangDevelopmentofmethodology:Q.J.Wang, Z. Yu,K. Patel,N.P. Restifo, J.C. YangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Q.J. Wang, Z. Yu, K.-i. Hanada, K. Patel, D. KleinerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):Q.J. Wang, K. Patel, D. Kleiner, N.P. Restifo, J.C. YangWriting, review, and/or revision of the manuscript:Q.J. Wang, Z. Yu, K. Patel,D. Kleiner, N.P. Restifo, J.C. YangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Q.J. Wang, N.P. Restifo, J.C. YangStudy supervision: Q.J. Wang, J.C. Yang

AcknowledgmentsThe authors thank Drs. Steven A Rosenberg and Paul Robbins for thoughtful

discussions.

Grant SupportThis research was supported by the Intramural Research Program of the NIH,

National Cancer Institute, Center for Cancer Research.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 2, 2016; revised October 5, 2016; accepted October 23, 2016;published OnlineFirst November 1, 2016.

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The immune competence of C57BL/6Jmice treatedwith anti-murine CD70CAR.Non–tumor-bearing C57BL/6J mice, either exposed to 500 cGy or leftuntreated, were given 1 � 107 i.v. murine T cells retrovirally transduced withmCD27-zeta or mock transduced. After 32 days, the mice were injected i.v. withvaccinia-OVA or vaccinia-gp100. Spleenswere taken 7 days after immunization,and cultured with OVA257–264 or gp10025–33 peptides for 1 week, and tested fortheir reactivity against either peptide. A, Reactivity of splenocytes from miceimmunized with vaccinia-OVA against OVA257–264. B, Reactivity of splenocytesfrom mice immunized with vaccina-gp100 against gp10025–33.






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2017;23:2267-2276. Published OnlineFirst November 1, 2016.Clin Cancer Res Qiong J. Wang, Zhiya Yu, Ken-ichi Hanada, et al. CD70-Expressing CancersPreclinical Evaluation of Chimeric Antigen Receptors Targeting

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