Preclinical Evaluation of Chimeric Antigen Receptors Targeting CD70-Expressing Cancers Qiong J

Published OnlineFirst November 1, 2016; DOI: 10.1158/1078-0432.CCR-16-1421

Cancer Therapy: Preclinical Clinical Cancer Research Preclinical Evaluation of Chimeric Antigen Receptors Targeting CD70-Expressing Cancers Qiong 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 to 41BB-zeta) conferred the highest IFNg production against activated lymphoid tissues. Targeting CD70 on CD70-expressing CD70-expressing tumors in vitro, and NSG mice bearing estab- tumors could mediate "on-target, off-tumor" toxicity. This study lished CD70-expressing human tumors could be cured by was to evaluate the feasibility and safety of using anti-human human lymphocytes transduced with this CAR. In the murine CD70 CARs to treat cancer patients whose tumors express CD70. CD27-CD3-zeta CAR model, significant reduction of estab- Experimental Design: Seven anti-human CD70 CARs with lished tumors and prolonged survival were achieved using binding moieties from human CD27 combined with CD3-zeta CAR-transduced splenocytes in a dose-dependent manner. and different costimulatory domains from CD28 and/or 41BB Host preirradiation enhanced treatment efficacy but increased were constructed. In vitro functionality of these receptors was treatment-related toxicities such as transient weight loss and compared and in vivo treatment efficacy was evaluated in a hematopoetic suppression. The treatment did not appear to xenograft mouse model. A homologous, all murine anti-CD70 block adaptive host immune responses. CAR model was also used to assess treatment-related toxicities. Conclusions: Preclinical testing supports the safety and efficacy Results: The CAR consisting of the extracellular binding of a CD27-containing CAR targeting CD70-expressing tumors. portion of CD27 fused with 41BB and CD3-zeta (trCD27- Clin Cancer Res; 23(9); 2267–76. 2016 AACR.

Introduction and antigen presenting cells in draining lymph nodes during viral infection (10). Interestingly, a number of human tumors Cell-based immunotherapies have shown promising clinical can also express CD70, including solid cancers such as clear cell outcomes in recent years. Adoptive transfer of tumor-infiltrating renal cancer (RCC), glioblastoma, and hematological malig- lymphocytes (TIL) can achieve durable complete regression of nancies (11–14). The mechanism of overexpression of CD70 metastatic melanoma (1). Alternatively, redirecting autologous T on tumors remains unknown, but a recent study suggested the cells with chimeric antigen receptors (CAR) or alpha-beta T cell dysregulated pVHL/HIF pathway may be involved in RCC (15). receptors (TCR) against tumor-associated antigens also mediated Nonetheless, due to its restricted expression pattern on normal long-term durable remissions of late-stage cancers refractory to tissues and overexpression in cancers, CD70 may be an attrac- standard therapies (2–4). The advantage of CARs is that they can tive therapeutic target. Approaches using antibody–drug con- target antigens expressed on the cell surface without MHC restric- jugates directed against CD70 have shown some antitumor tion, therefore making them more widely applicable. However, activity in vitro, and demonstrated some clinical responses treating solid cancers using CAR immunotherapies remains chal- against RCC and non-Hodgkin's lymphoma in a phase I clinical lenging due to the paucity of safe, clinically effective tumor- trial (13, 16). In addition, T cells genetically engineered with a associated cell-surface antigens identified to date (5, 6). CAR consisting of full-length CD27 coupled with the CD3-zeta CD70 was initially identified as the ligand for CD27, a signaling domain (CD3-zeta) has shown CD70-specifictumor costimulatory receptor involved in T-cell proliferation and recognition (17). These studies suggested that CD70 could be a survival (7, 8). Studies on CD70 expression revealed the same potential immunotherapeutic target. However, due to CD70 restricted expression profile in both humans and mice (9, 10), expression by activated lymphoid tissues, the potential for with CD70 only found on a small percentage of activated T cells "on-target, off-tumor" toxicity may hinder potential clinical applications, as has been observed when targeting CD19 on malignant and normal B cells with a CAR (2, 18). Therefore, in Surgery Branch and Laboratory of Pathology, Center for Cancer Research, NCI, this study, we have compared the antitumor reactivities of anti- NIH, Bethesda, Maryland. human CD70 CARs in which different portions of CD27 were Note: Supplementary data for this article are available at Clinical Cancer fused with various costimulatory signaling domains from 41BB Research Online (http://clincancerres.aacrjournals.org/). and/or CD28, and CD3-zeta, and established a murine model Corresponding Authors: Qiong J. Wang, NCI, NIH, 9000 Rockville Pike, Building to test the potential for on-target, off-tumor toxicities. Our 10/CRC, Room 3W-3840, Bethesda, MD 20892. Phone: 301-435-6264; Fax: 301- study demonstrates that anti-human and anti-murine CD70 496-0011; E-mail: [email protected]; and James C. Yang, E-mail: CARs were effective in vitro and in vivo, respectively. However, [email protected] some treatment-related toxicities were also observed, although doi: 10.1158/1078-0432.CCR-16-1421 they appeared to be reversible and tolerable in the murine 2016 American Association for Cancer Research. model. Among the anti-human CD70 CAR constructs, CD27

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Construction of human anti-CD70 CARs, retroviral production, Translational Relevance retroviral transduction of human PBL, and in vitro reactivity of CD70 has been identified as a biomarker for clear cell renal transduced cells cell cancer (RCC) as well as several hematological malignan- Genes encoding seven human anti-CD70 CARs, including cies, such as non-Hodgkin's lymphoma. In this study, we first cDNAs for full-length CD27 (flCD27) or truncated CD27 evaluated the in vitro function of anti-human CD70 chimeric (trCD27; including extracellular and transmembrane portions of antigen receptor (CAR) constructs by fusing different portions CD27, aa1-211) fused with CD28 and/or 41BB signaling domains of CD27 with CD3-zeta in combination with costimulatory and CD3-zeta were constructed and cloned into the pMSGV1 signaling domains such as CD28 and/or 41BB. Among plasmid (Fig. 1A). Retroviral production and transduction were them, the extracellular binding portion of CD27 fused with the same as described previously (19). Briefly, 293gp cells were 41BB and CD3-zeta appeared to be the best against CD70- transfected with 9 mg of anti-CD70 CARs and 4.5 mg of plasmid expressing tumors, and curative effects and long-term survival RD114 using Lipofectamine 2000 (Life Technologies; 60 mL). Two were observed when CAR-expressing T cells were adoptively days later, the supernatants were harvested and used to transduce transferred to tumor-bearing NSG mice. We then tested anti-CD3 stimulated PBL. PBL from allogeneic donors were sti- in vivo toxicity and efficacy using a homologous, completely mulated with soluble OKT-3 (50 ng/mL) and rhIL2 (300 IU/mL) murine model. This showed only transient cytokine toxicity at for 2 days before transduction was performed. The stimulated cell doses 100-fold higher than needed to show efficacy. A cells were added to 24-well plates initially coated with Retro- phase I/II clinical trial using the anti-human CD70-41BB- Nectin (Takara) and subsequently precoated with retrovirus by CD3zeta CAR against RCC and other CD70-expressing tumors spinoculation (2,000 g,32C, 2 hours) at 5 105/mL. The plates is under way. were then centrifuged at 1,000 g for 10 minutes, and incubated overnight at 37 Cina5%CO2 incubator. This procedure was repeated the next day and cells were split as necessary to maintain cell density between 0.5 and 1 106 cells/mL. Transduction fi without its intracellular signaling domain, fused with the ef ciency was estimated by analyzing human CD27 expression costimulatory of 41BB and then CD3-zeta, designated as on retrovirally transduced cells and comparing this to mock- CD27-41BB-zeta, appeared to be most active in vitro. Therefore, transduced T cells. To test their reactivity, retrovirally transduced cells (1 105) were cocultured with 5 104 human tumor lines plans are under way to test this anti-CD70 CAR in patients with advanced, refractory tumors expressing CD70. with or without CD70 expression at 37 C, 5% CO2 overnight. The supernatants were harvested and tested for IFNg secretion by fi Materials and Methods ELISA (Thermo Fisher Scienti c). Mice, tumor lines, and antibodies Mouse xenograft studies C57BL/6J and NSG mice (The Jackson Laboratory) were main- NSG mice were injected subcutaneously with 1.5 105 tained per protocols in the NIH animal facility. Murine tumor 2654R human renal cancer cells. Eighteen days after inocula- lines B16 and B16/mCD70 (retrovirally transduced with murine tion, when tumors were approximately 5 mm in diameter, mice CD70) were maintained in RPMI 1640 (Life Technologies) with received 6 106 intravenous human T cells retrovirally trans- 10% fetal bovine serum (FBS; Life Technologies). All mouse duced with anti-CD70 CARs or control T cells, followed by studies were approved by the National Cancer Institute Animal intraperitoneal administration of 200,000 IU of rhIL2 per day Care and Use Committee. for 3 days. Each group included 5 randomly assigned tumor Tumor lines from RCC patients were established and main- bearing mice, and all tumor measurements were performed by tained in DMEM (Life Technologies), including 10% FBS, 10% a blinded impartial observer. tryptose phosphate (Sigma), 1 insulin-transferrin-selenium (Life Technologies) and 1 serum pyruvate (Life Technologies). Construction of murine anti-CD70 CAR, retroviral production, Melanoma tumor lines were maintained in RPMI 1640 (Life transduction of murine CD3 T cells, and in vitro functional Technologies) with 10% FBS. All cell lines included in the study analysis of transduced cells were generated at Surgery Branch, NCI, and tested and identities cDNA encoding murine CD27 fused with murine CD3-zeta confirmed by HLA genotyping. The cell lines were maintained in signaling domain (mCD27-zeta) was constructed in the the cell culture only when they were needed in the experiments pMSGV1 plasmid. To produce retrovirus, 293gp cells were and usually kept in culture for approximately a month, and transfected with 9 mg of pMSGV1-mCD27-zeta and 4.5 mgof mycoplasma testing were done routinely using mycoplasma plasmid pCL-Eco (Addgene) using Lipofectamine 2000 (Life detection kit (Lonza). The cell lines were reassessed for HLA and Technologies; 60 mL). Two days later, the supernatants were antigen expression by flow cytometry and coculture assays when harvested and used to transduce activated mouse T cells. Sple- þ they were thawed for each experiment. nocytes from C57BL/6J mice were harvested and CD3 T cells Monoclonal antibodies (mAb), including FITC-labeled anti- were isolated by negative selection using a mouse pan–T-cell mouse CD3, anti-mouse CD45.1, and anti-human CD8 Abs, isolation kit II (Miltenyi Biotec). Murine CD3 T cells were then PE-labeled anti-mouse and anti-human CD70 Abs, PE-cy7- stimulated with plate-bound anti-mouse CD3 (1 mg/mL), and labeled anti-human CD3 Ab, APC-labeled anti-mouse CD27 soluble anti-mouse CD28 (1 mg/mL) and rhIL2 (30 IU/mL) for Ab, APC-cy7-labeled anti-mouse CD8, and purified anti-mouse 2 days before transduction was performed. Transduction of CD3 and anti-mouse CD28 Abs were purchased from BD stimulated cells was performed similarly as described above by Pharmingen. APC-labeled anti-human CD27 Ab was purchased spinoculation. Transduction efficiency was determining by from eBiosciences. analyzing mouse CD27 expression on retrovirally transduced

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A flCD27-zeta LTR CD27 (full-length) CD3zeta LTR

trCD27-28-zeta LTR CD27 (truncated) CD28 CD3zeta LTR

trCD27-41BB-zeta LTR CD27 (truncated) 41BB CD3zeta LTR

trCD27-28-41BB-zeta LTR CD27 (truncated) CD28 41BB CD3zeta LTR

flCD27-28-zeta LTR CD27 (full-length) CD28 CD3zeta LTR

flCD27-41BB-zeta LTR CD27 (full-length) 41BB CD3zeta LTR

flCD27-28-41BB-zeta LTR CD27 (full-length) CD28 41BB CD3zeta LTR

B flCD27-zeta trCD27-28-zeta trCD27-41BB-zeta trCD27-28-41BB-zeta flCD27-28-zeta flCD27-41BB-zeta flCD27-28-41BB-zeta UT CD3

CD27 C 60,000 624 mel 624/CD70 938 mel 40,000 938/CD70 RCC HC

g (pg/mL) RCC RO 20,000 IFN RCC DS RCC MW Medium 0

a ta ta ta ta ta e e e e ck eta z z ze z z o - - - M 8-z B B B- BB B -28- B 1B -41 -41 27 -4 -41 flCD27-zet 7 D 7 8 2 -28 lC 2 -2 trCD27-2 D f 7 C 27 2 tr D flCD D C tr flC

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 made using different portions of CD27 in combination with CD28 or 41BB costimulatory domain and CD3 zeta signaling domain. B, Comparison of CD27 expression on 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-APC mAb, and analyzed on FACS CantoII. Data shown were gated on propidium iodide–negative cells. UT: untransduced controls. An arbitrary CD27-negative gate for untransduced cells was used to compare the transduction efﬁciency between different CARs. C, Comparison of antitumor reactivity of T cells transduced with anti-human CD70 CARs. Allogeneic PBL were stimulated with anti-CD3 and retrovirally transduced with 7 anti-human CD70 CARs, respectively. Three days after transduction, transduced T cells were cocultured with a panel of tumor lines overnight, and tested for IFNg production by ELISA the next day.

T cells, compared with cells just stimulated with anti-CD3, followed by intraperitoneal (i.p.) administration of 200,000 IU of anti-CD28 and rhIL2. To assess the reactivity of mCD27-zeta, rhIL2 per day for 3 days. Splenocytes from pmel-1 TCR transgenic retrovirally transduced murine T cells (1 105)werecocultured mice, that are reactive to gp100 in both B16 and B16/mCD70 4 with B16/mCD70 or B16 (5 10 )at37 C, 5% CO2 overnight tumors, were activated in vitro in the presence of 1 mmol/L and supernatant was harvested and tested for mouse IFNg hgp10025–33 peptide and 30 IU/mL rhIL2 for 7 days. As a positive secretion by ELISA (R&D Systems). adoptive cell therapy control, a total of 106 activated pmel T cells were given to mice i.v. along with recombinant vaccinia virus Treatment efﬁcacy of murine T cells retrovirally transduced with encoding hgp100 (2 107 pfu) and the regimen of i.p. rhIL2 as murine anti-CD70 CAR in vivo above. Where speciﬁed, sublethal total body irradiation (TBI; C57BL/6J mice were injected subcutaneously (s.c.) with 0.5 500 cGy) was given to mice immediately prior to cell transfer. 106 B16 or B16/mCD70 tumors. Ten days after inoculation, when Mice were given from 104 to 107 retrovirally transduced murine tumors were approximately 5 mm in diameter, mice received up T cells when assessing minimal effective treatment dosage. All to 107 intravenous (i.v.) murine T cells retrovirally transduced treatment groups were randomly assigned, and all tumor mea- with mCD27-zeta or stimulated, untransduced T cells as a control, surements were performed by a blinded impartial observer.

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In vivo persistence of transferred T cells transduced with murine tion efficiency of anti-CD70 CARs. Downregulation of CD3 on anti-CD70 CAR and assessment of treatment-related toxicity these transduced T cells was also observed, and consistently seen T cells from Ly5.1 congenic mice were stimulated with plate- with high transduction efficiency, suggesting that CD3 internal- bound anti-murine CD3 and soluble anti-mouse CD28 Abs for ization may occur with introduced CD3-zeta chain signaling 2 days, and retrovirally transduced with mCD27-zeta. Four days domain. Surprisingly, only 45%, 16%, and 3% were CD27 after transduction, T cells (107) were injected i.v. to C57BL/6J positive when T cells were transduced with trCD27-28-zeta, mice, followed by i.p. administration of 200,000 IU of rhIL2 per trCD27-28-41BB-zeta, and flCD27-28-zeta, respectively. To test day for 3 days. Splenocytes were harvested from day 3 to day 12, antigen-dependent tumor recognition by these anti-human CD70 and the presence of transferred cells was determined by expression CARs, a panel of CD70-negative tumor lines (624mel and of CD45.1-positive cells. Measurements of weight, absolute blood 938mel), and their stably transduced CD70 expressing counter- cell counts, blood chemistry, and the number of splenocytes and parts (624/CD70 and 938/CD70), and RCC tumor lines naturally serum cytokines were determined in both tumor-bearing and expressing high to low levels of CD70 (RCC HC, RCC RO, RCC non–tumor-bearing mice. DS, and RCC MW, respectively; Supplementary Fig. S1 and ref. 25) were included. All of the CARs, except for flCD27-28-zeta, dem- Assessment of host immune responses after adoptive cell onstrated specific anti-CD70 reactivity, as they only recognized transfer CD70-positive tumor lines, but not CD70-negative tumor lines C57BL/6J mice were injected i.v. with murine T cells retrovirally (Fig. 1C). The control vector did not show any reactivity against transduced with mCD27-zeta or untransduced control T cells, these tumors. Among the candidate CARs, trCD27-41BB-zeta followed by i.p. administration of 200,000 IU of rhIL2 per day appeared to possess the highest antitumor reactivity by IFNg for 3 days. Thirty-two days after transfer, mice were immunized production. In addition to flCD27-28-zeta, lower anti-CD70 with vaccinia virus encoding either OVA or gp100. Splenocytes reactivity was also observed in T cells transduced with trCD27- from treated and control animals were harvested 7 days after 28-zeta, suggesting that signaling through CD28 may have immunization, and cultured in vitro in the presence of 1 mmol/L adverse effects on these constructs. Based on transduction effi- OVA257–264 or gp10025–33 peptides for 7 days, and then tested for ciency and antitumor reactivity of these CARs, we decided to IFNg production of by coculturing T cells with LPS-stimulated compare the efficacy of flCD27-zeta and trCD27-41BB-zeta in vivo. lymphoblasts pulsed with either peptide. The CD70-positive human renal tumor line, 2654R, was injected subcutaneously into NSG mice and once palpable, treated with Statistical analysis human T cells transduced with either flCD27-zeta or trCD27- Wilcoxon rank-sum test was used to compare tumor slopes 41BB-zeta. A curative effect was observed in both groups as shown between each treatment groups, and log-rank test was used to in Fig. 2A and B. None of the control groups showed any delayed analyze survival. tumor growth. Our results suggest that either CAR could be effective in treating CD70-positive tumors in vivo. However, because normal tissues of NSG mice do not express human CD70, Results we could not assess the treatment-related toxicities using this The effectiveness of anti-human CD70 CAR in vitro and in vivo model. Therefore, a mouse tumor model with tumors expressing Seven anti-human CD70 CAR retroviral vectors were con- murine CD70 was generated to address these issues. structed and transduced into anti-CD3 stimulated normal donor peripheral blood lymphocytes (PBL) to evaluate their expression Treatment efficacy of murine anti-CD70 CAR and antitumor activities. As shown in Fig. 1A, full-length CD27 To generate a mouse model, an anti-murine CD70 CAR was (flCD27) fused with the CD3 zeta signaling domain (CD3-zeta) constructed by fusing full-length murine CD27 with murine CD3- with or without CD28 and/or 41BB signaling domains were signaling domain (mCD27-zeta). Murine T cells expressed high constructed, designated as flCD27-zeta, flCD27-CD28-zeta, levels of CD27 after retroviral transduction with the anti-murine flCD27-41BB-zeta, and flCD27-CD28-41BB-zeta, as several stud- CD70 CAR compared with mock-transduced or untransduced ies demonstrated CD28 and 41BB signaling domains could T cells (Fig. 3A). Meanwhile, a stably transfected cell line, B16/ augment antitumor reactivities and in vivo persistence (20–24). mCD70, was generated by transducing B16 melanoma (a murine Similarly, three anti-human CD70 CARs using truncated CD27 CD70-negative tumor line recognized by pmel T cells; Fig. 3B; (trCD27, aa 1–211; i.e., CD27 without its intracellular domain) ref. 26), with murine CD70. As shown in Fig. 3C, pmel T cells were also constructed, shown as trCD27-CD28-zeta, trCD27- produced mouse IFNg when cocultured with either parental B16 41BB-zeta, and trCD27-CD28-41BB-zeta. PBL retrovirally trans- tumor, or B16/mCD70. However, mCD27-zeta could confer duced with each of the seven vectors showed various degrees of specific, anti-CD70 reactivity only against B16/mCD70. To test CD27 expression (Fig. 1B). As might be expected, PBL successfully the antitumor efficacy of mCD27-zeta in vivo, C57BL/6J mice were þ þ transduced with these CAR were devoid of CD3 CD70 cells. implanted with either B16 or B16/mCD70 subcutaneously, and flCD27-zeta appeared to be the best with 92% of cells expressing then treated with mouse T cells retrovirally transduced with CD27 in comparison with 70% of CD27 expression on mCD27-zeta or other controls when tumors became palpable. T cells transduced with trCD27-41BB-zeta, flCD27-41BB-zeta, or While pmel T cells (given with concomitant vaccination and IL2) flCD27-28-41BB-zeta (and 0.26% of stimulated but untrans- could reduce tumor burdens in irradiated mice carrying either B16 duced PBL). Because mock-transduced or untransduced T cells or B16/mCD70, mCD27-zeta-transduced T cells (given only with still expressed certain level of CD27, positivity of CD27 expression IL2) delayed tumor growth only in irradiated mice carrying B16/ in these anti-CD70 CAR-expressed T cells was only counted when mCD70, in an antigen-specific manner (Fig. 3D). Mice that were expression level was above the level of mock-transduced or treated with untransduced T cells or left untreated did not show untransduced T cells. This may lead to underestimate transduc- any delay of tumor growth. In addition, treatment efficacy was

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A Short-term treatment-related toxicities of anti-murine CD70 400 CAR To assess treatment-related toxicities, C57BL/6J mice were ) 2 300 flCD27-zeta implanted with either B16 or B16/mCD70, treated with mCD27-zeta–transduced or control T cells, and body weight trCD27-41BB-zeta 200 evaluated as an indicator of systemic cytokine toxicity. Irradiated Mock B16/mCD70 mice receiving mCD27-zeta–transduced T cells UT showed signiﬁcant lower body weight than those receiving 100

Tumor size (mm PBS untransduced T cells, pmel T cells, or left untreated from day 6 to day 10, but recovered 2 weeks after the cell transfer (Fig. 4A). On 0 the other hand, no signiﬁcant differences were observed in non- 0 1020304050150300 irradiated recipients of T-cell transfers. Interestingly, lower body Day post ACT weight was also observed in irradiated, mCD27-zeta–treated mice B that were implanted with antigen-negative B16 tumors, which 100 suggested that this transient toxicity results primarily from mCD27-zeta–transduced cells interacting with normal endoge- 80 PBS nous host cells. ﬁ Untransduced To further de ne the toxicity of mCD27-zeta CAR cells inde- 60 Mock pendent of the effects of tumor growth, we conducted experi- – 40 flCD27-zeta ments in non tumor-bearing C57BL/6J mice. Body weight, trCD27-41BB-zeta peripheral white blood cell (WBC) and lymphocyte counts,

Percent survival Percent 20 splenocyte counts, blood chemistries, and serum cytokines were evaluated for 2 weeks after treatment. Similar to tumor-bearing 0 mice, irradiated mice that were treated with mCD27-zeta–trans- 0 50 100 150 200 250 300 duced T cells showed significant lower body weight at days 6 and 8 Day post ACT than those that were treated with mock-transduced or untransduced T cells, but recovered approximately 2 weeks after cell Figure 2. transfer (Supplementary Fig. S2B). This effect was not observed Adoptive cell transfer of antihuman CD70 CAR-transduced cells treating in nonirradiated mice. As would be predicted, whole body irra- human cancers in NSG mice. A, The human renal tumor line, 2654R, was injected into NSG mice subcutaneously, and 6 106 T cells transduced with flCD27-zeta diation, with or without CAR T-cell transfer dramatically or trCD27-41BB-zeta were injected intravenously when tumors became decreased absolute WBC and lymphocyte counts as well as sple- palpable. Mice given no treatment, the same number of untransduced T cells nocyte counts compared with all nonirradiated groups (Fig. 4B (UT) or mock-transduced T cells served as controls. Serial tumor measurements and Supplementary Fig. S2C). Although blood chemistry did not were obtained and tumor area calculated. Five mice were included in each show any differences among groups, we could detect IFNg in – group. Center bar, mean; error bars, SEM. B, Kaplan Meier analysis of survival in mouse serum from day 3 to day 5 only in irradiated mice that were tumor-bearing mice receiving adoptive transferred T cells transduced with anti-human CD70 CARs versus controls (P < 0.0001). Four of 5 mice from treated with mCD27-zeta CAR T cells (Fig. 4C). However, no each treatment group are cured. "ACT" represents "adoptive cell transfer." differences could be detected in the very low levels of CD70 expression on splenocytes among these groups (Fig. 4D). Taken together, our data suggest that transient cytokine toxicity, as indicated by weight loss and elevated serum IFNg levels, could dose dependent, with 1 105 the lowest effective T-cell number be detected in mice undergoing whole body irradiation followed in irradiated mice (Fig. 3E). In nonirradiated mice, however, by mCD27-zeta CAR T-cell transfer, and this resulted primarily treatment effects could only be observed when 1 107 cells from reactivity against endogenous host cells. were transferred. This may be attributable to superior persistence of transferred cells seen in irradiated mice compared with Immune competence of mice treated with anti-murine CD70 nonirradiated mice (Supplementary Fig. S2A). Furthermore, CAR irradiated B16/mCD70-bearing mice treated with 1 105 Because successfully targeting CD70 could affect some T cells to 1 107 CAR T cells had significantly better survival than and APCs, we tested immune responses in treated mice. One mice treated with no cells or 1 107 mock-transduced T cells month after cell transfer, treated mice were immunized with (Fig. 3F). Although curative effects could be achieved in mice vaccinia-OVA or vaccinia-gp100. Splenocytes from treated mice that were treated with high cell doses (1 of 5 given either 1 were stimulated and tested for reactivity against OVA or gp100 106 or 1 107 cells), the majority of mice showed tumor peptides. As shown in Fig. 5A, 7 days after in vitro stimulation with growth inhibition rather than durable regressions. To delineate OT-I257–264, splenocytes from mice treated with mCD27-zeta or possible mechanisms of immune escape, we analyzed mCD70 mock-transduced cells (with or without recipient preirradiation) expression on tumors progressing in treated mice (Fig. 3G). were reactive to OT-I257–264. Similar results were observed with While tumors from control animals remained mCD70 positive, mice immunized with vaccinia-gp100 (Fig. 5B), as T cells from tumors from mCD27-zeta–treated mice completely lacked mice immunized with vaccinia-gp100 were reactive to gp10025–33, mCD70 expression, implicating antigen loss as a cause of regardless which treatment the mice had received. Interestingly, immune escape. While efficacy data were interesting, this T cells from mice given mCD27-zeta CAR T cells with irradia- mouse model was primarily developed to analyze the in vivo tion appeared to be more reactive than other groups. Nonetheless, toxicity of using a CD27-zeta CAR to target CD70. our data show that mice treated with anti-murine CD70 CAR

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T cells could still mount immune responses against exogenous using CD19 CAR treating multiple hematologic malignancies antigens. have shown dramatic clinical responses (2, 3, 29), and the in vivo expansion of CAR-transduced T cells correlated with clinical responses (29). In fact, multiple preclinical studies have demon- Discussion strated that the 41BB costimulatory domain can enhance in vivo We have demonstrated in this study that using CD27, the persistence and survival of CAR-transduced T cells (21, 24). A natural receptor that engages CD70, we could construct CARs to recent study further suggests that 41BB promotes the growth of target CD70-expressing tumors. CD70 can be expressed on acti- central memory T cells with enhanced fatty acid oxidation and vated normal lymphocytes, but we did not encounter in vitro mitochondrial biogenesis (30). Therefore, we have chosen "fratricide" by CD27-zeta CAR expressing T cells as an impedi- trCD27-41BB-zeta for our future clinical studies. ment to activating, transducing, and expanding populations of One of the biggest drawbacks with T cells targeting overexpres- reactive, CAR-transduced T cells. We then compared 7 different sing tumor antigens is the "on-target" toxicity against normal anti-human CD70 CARs introduced in a replication-defective tissues (2, 31–34). The severity of these treatment-related toxi- gamma-retrovirus for in vitro CD70-specific antitumor reactivity. cities is largely dependent on which normal tissues express the These 7 CARs could all be categorized as second-generation CARs targeted antigen. T cells engineered with an anti-CD19 CAR, for (flCD27-zeta, trCD27-28-zeta, and trCD27-41BB-zeta) or third- instance, can effectively treat late-stage cancer patients with þ generation CARs (flCD27-28-zeta, flCD27-41BB-zeta, trCD27- CD19 B-cell malignancies. However, it can also cause a transient 28-41BB-zeta, and fCD27-28-41BB-zeta) given the costimulatory acute cytokine release syndrome as well as long-term eradication þ nature of the full-length CD27 receptor. Significant differences of normal CD19 B cells in some patients (2, 35). These effects can were observed when different costimulatory signaling domains be managed medically in nearly all patients and are considered were included in the constructs. T cells transduced with receptors tolerable in view of the efficacy of this cell transfer. Thus, analysis containing only the 41BB cosignaling domain, i.e., trCD27- of antigen expression patterns and toxicity in an appropriate all- 41BB-zeta and flCD27-41BB-zeta, showed high transduction effi- murine model can be valuable when vetting a potential tumor- ciency and anti-CD70–specific reactivity, while T cells including associated antigen targeted by adoptive T-cell therapy. Similar to only a CD28 cosignaling domain, such as trCD27-28-zeta and previous studies, we could only detect CD70 expression on a very flCD27-28-zeta, showed poor receptor expression and lower anti- small subpopulation of human peripheral blood cells, and CD70 reactivity. Interestingly, adding the 41BB signaling domain murine lymphoid tissues, such as splenocytes, lymph nodes, bone (constructs trCD27-28-41BB-zeta and flCD27-28-41BB-zeta com- marrow, and peripheral blood. An all murine model targeting pared with trCD27-28-zeta and flCD27-28-41BB-zeta, respective- mCD70 with a CAR using the mCD27 binding domain could ly) could partially compensate for the poor performance of CD28 elucidate the in vivo consequences of depleting this subpopulation alone in these constructs. Our findings coincide with a recent of normal lymphocytes and possibly reveal other unsuspected publication in which the authors demonstrate that 41BB costi- CD70 expression on normal tissues, but further strict quantitative mulation reduces, but CD28 costimulation induces, exhaustion translation of toxicities may not be possible when homologous of CAR-transduced T cells (27). Although the intracellular domain receptor components and host species also have to be changed. of CD27 may augment antitumor reactivity in vivo (28), including Therefore, for our in vivo murine toxicity studies, we used a basic the full-length CD27 receptor in the CAR did not reverse the CD27-zeta CAR that not only depleted CD70 expressing immune deleterious effect of the CD28 costimulatory domain alone in our cells but also had demonstrated efficacy against tumor. Using this in vitro study. Overall, the second-generation construct, trCD27- therapeutically effective anti-murine CD70 CAR, we demonstrat- 41BB-zeta, appeared to be the best among the constructs we tested ed that acute toxicities such as weight loss and low lymphocyte in vitro. Comparing trCD27-41BB-zeta with flCD27-zeta in a counts occurred in the irradiated treatment group at the highest xenograft experiment, both constructs showed similar curative cell doses but resolved within 2 weeks after cell transfer. In þ effects against a naturally expressing CD70 tumor. Recent studies addition, 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. Murine T 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 cells were stained with anti-murine CD3-FITC and anti-murine CD27-APC and analyzed on FACS CantoII. An arbitrary CD27-negative gate of untransduced T cells were used to determine the percentage of CD27 expression. B, Expression of murine CD70 in murine tumor lines. B16, a murine melanoma 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 cocultured with 5 104 targets, and tested for mouse IFNg secretion. pmel T cells (from an anti-gp100 TCR transgenic mouse strain), untransduced and mock-transduced T cells served 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 body radiation 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 pmel T cells along with recombinant vaccinia virus encoding hgp100 (rVVhgp100), 1 107 untransduced T cells, or no treatment served as controls. E, Delayed tumor growth 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 were included 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 transduced with anti-murine CD70 CARs. Survival was significantly prolonged for mice receiving 1 105,1 106,or1 107 CAR T cells compared with all control groups (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 analyzed on FACS Canto II.

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Figure 4. Short-term impact of anti-murine CD70 CAR in the murine model. A, Body weight of mice treated with anti-murine CD70 CAR (mCD27-zeta). B16 or B16/mCD70 was injected 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 murine T 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 CD70 expression on splenocytes (D), from B16/mCD70 tumor–bearing mice treated with anti-murine CD70 CAR. C57BL/6J mice, either exposed to 500 cGy or left untreated, were given 1 107 murine T cells transduced with mCD27-zeta or control T cells.

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Chimeric Antigen Receptors Targeting CD70

transfer of these CAR T cells. Moreover, the immune competence of the treated mice appeared to remain intact, because they could respond normally to immunization using vaccinia virus encoding OVA or gp100. This would imply that CD70 expression is not an obligate step in T-cell activation or APC function. Any impact on this small normal population of immune cells appears similar and analogous to that seen when clinically targeting the normal B- cell antigen CD19 with CAR T cells. No unacceptable toxicities were encountered in this preclinical evaluation of an anti-CD70 CAR using CD27 as the binding domain. The potential for additional toxicities with more complex receptor structures always exists, but this will be best addressed by careful phase I dose-escalation trials. Such a clinical trial (NCT02830724) with escalating cell doses of CAR-transduced autologous PBL in þ patients with late-stage CD70 cancers is under way.

Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed.

Authors' Contributions Conception and design: Q.J. Wang, J.C. Yang Development of methodology: Q.J. Wang, Z. Yu, K. Patel, N.P. Restifo, J.C. Yang Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Q.J. Wang, Z. Yu, K.-i. Hanada, K. Patel, D. Kleiner Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Q.J. Wang, K. Patel, D. Kleiner, N.P. Restifo, J.C. Yang Writing, review, and/or revision of the manuscript: Q.J. Wang, Z. Yu, K. Patel, D. Kleiner, N.P. Restifo, J.C. Yang Administrative, technical, or material support (i.e., reporting or organizing Figure 5. data, constructing databases): Q.J. Wang, N.P. Restifo, J.C. Yang The immune competence of C57BL/6J mice treated with anti-murine CD70 CAR. Study supervision: Q.J. Wang, J.C. Yang Non–tumor-bearing C57BL/6J mice, either exposed to 500 cGy or left untreated, were given 1 107 i.v. murine T cells retrovirally transduced with mCD27-zeta or mock transduced. After 32 days, the mice were injected i.v. with Acknowledgments vaccinia-OVA or vaccinia-gp100. Spleens were taken 7 days after immunization, The authors thank Drs. Steven A Rosenberg and Paul Robbins for thoughtful discussions. and cultured with OVA257–264 or gp10025–33 peptides for 1 week, and tested for their reactivity against either peptide. A, Reactivity of splenocytes from mice

immunized with vaccinia-OVA against OVA257–264. B, Reactivity of splenocytes Grant Support from mice immunized with vaccina-gp100 against gp10025–33. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The costs of publication of this article were defrayed in part by the payment of ﬁ irradiated treatment group during the rst week after cell transfer. page charges. This article must therefore be hereby marked advertisement in Our data suggest endogenous CD70-expressing cells may induce accordance with 18 U.S.C. Section 1734 solely to indicate this fact. these short-term, self-limited toxicities. Analyses of blood chemistry and sequential histological studies of multiple tissues also Received June 2, 2016; revised October 5, 2016; accepted October 23, 2016; did not show any consistent differences associated with the published OnlineFirst November 1, 2016.

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Preclinical Evaluation of Chimeric Antigen Receptors Targeting CD70-Expressing Cancers

Qiong J. Wang, Zhiya Yu, Ken-ichi Hanada, et al.

Clin Cancer Res 2017;23:2267-2276. Published OnlineFirst November 1, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-1421

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