IMMUNOLOGY
`
`Engineering antigen-specific primary human NK cells
`against HER-2 positive carcinomas
`
`Anna Kruschinskia, Andreas Moosmannb, Isabel Poschkec, Håkan Norellc,d, Markus Chmielewskie, Barbara Seligerf,
`Rolf Kiesslingc, Thomas Blankensteina,g, Hinrich Abkene, and Jehad Charoa,1
`
`aMax-Delbru¨ ck Center for Molecular Medicine, D-13092 Berlin, Germany; bClinical Cooperation Group Molecular Oncology, University of Munich,
`Marchioninistrasse 25, 81377 Munich, Germany; cCancer Centrum Karolinska, Karolinska Institutet, 171 76 Stockholm, Sweden; dDepartment of Surgery,
`Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425; eDepartment I of Internal Medicine, Tumor Genetics, and Center for
`Molecular Medicine Cologne, University of Cologne, D-50931 Cologne, Germany; fMartin Luther University Halle, Wittenberg, Institute of Medical
`Immunology, 06112 Halle, Germany; and gInstitute of Immunology, Charite Campus Benjamin Franklin, D-12203 Berlin, Germany
`
`Edited by George Klein, Karolinska Institutet, Stockholm, Sweden, and approved September 19, 2008 (received for review May 16, 2008)
`
`NK cells are promising effectors for tumor adoptive immunother-
`apy, particularly when considering the targeting of MHC class I low
`or negative tumors. Yet, NK cells cannot respond to many tumors,
`which is particularly the case for nonhematopoietic tumors such as
`carcinomas or melanoma even when these cells lose MHC class I
`surface expression. Therefore, we targeted primary human NK cells
`by gene transfer of an activating chimeric receptor specific for
`HER-2, which is frequently overexpressed on carcinomas. We found
`that these targeted NK cells were specifically activated upon
`recognition of all evaluated HER-2 positive tumor cells, including
`autologous targets, as indicated by high levels of cytokine secre-
`tion as well as degranulation. The magnitude of this specific
`response correlated with the level of HER-2 expression on the
`tumor cells. Finally, these receptor transduced NK cells, but not
`their mock transduced counterpart, efficiently eradicated tumor
`cells in RAG2 knockout mice as visualized by in vivo imaging. Taken
`together, these results indicate that the expression of this activat-
`ing receptor overrides inhibitory signals in primary human NK cells
`and directs them specifically toward HER-2 expressing tumor cells
`both in vitro and in vivo.
`
`immunotherapy 兩 in vivo imaging 兩 tumor biology 兩 tolerance 兩
`retroviral transduction
`
`Tumor cells with defined antigens have been successfully tar-
`
`geted with adoptively transferred T cells (1, 2). This therapy is
`frequently associated with tumor regression as well as the devel-
`opment of MHC class I (MHC-I) negative or low tumor-cell
`variants (2–4). Unlike T cells, which express their own antigen
`specific receptor, the TCR, NK cells are devoid from the expression
`of such a receptor. In the periphery, self-reactive T cells are
`inactivated by various tolerance mechanisms. Therefore, expressing
`a second antigen-specific receptor in T cells (5) might render these
`cells self-reactive if the introduced targeting receptor activates the
`recognition through an endogenous self-specific TCR. The poten-
`tial use of targeted NK cells for adoptive immunotherapy provides
`an effector cell type with the capacity of recognizing antigen
`positive as well as MHC-I negative or low tumor cells. NK cells
`account for ⬇10–15% of human blood lymphocytes and constitute
`an important component of the innate immune system (6). They are
`characterized by a CD56⫹ CD3⫺ surface phenotype as well as by
`the expression of various activating and inhibitory receptors (6, 7).
`NK cells recognize and kill virus-infected cells and transformed cell
`lines (6–8). Unlike cytotoxic T lymphocytes, NK cells mediate
`cytolysis without the need for prior sensitization (6). According to
`the missing self recognition model suggested by Klas Ka¨rre, NK
`cells can sense the absence of self MHC-I on other cells and respond
`by killing these cells (7, 9). Killing is inhibited by the expression of
`inhibitory receptors that recognize self MHC-I molecules on target
`cells, which results in transmitting an inhibitory signal that prevents
`cytotoxic action triggered by activating receptors (7, 10, 11). In the
`absence of dominant inhibitory signal (7, 8, 12), cytolytic granules
`are released and various cytokines critical to the immune response,
`
`such as IFN-␥, are produced (6–8). In man and mouse, NK cells
`attain a state of unresponsiveness to self by tolerance mechanisms
`as recently reviewed (7, 12).
`Here we investigated the potential of genetically engineered
`primary human NK cells to specifically target HER-2⫹ tumors
`independently of MHC restriction by an Ab-based chimeric recep-
`tor (CR). HER-2 is ubiquitously expressed in many epithelial
`tumors and overexpressed in a variety of carcinomas, including
`ovarian and breast cancers (13–15). HER-2 overexpression corre-
`lates with increased aggressiveness of malignancy and poor prog-
`nosis (13, 15). The first approved immunological treatment of
`HER-2⫹ metastatic breast cancer was trastuzumab, a humanized
`HER-2-specific Ab (13, 14, 16, 17). We here show that primary
`human NK cells are amenable for specific targeting toward NK-
`cell-resistant HER-2⫹ tumor cells by the expression of a specific
`CR. Hence, these engineered NK cells acquired a new tumor
`specificity without losing their capacity to recognize MHC-Ilow
`target cells. Thus, engineered HER-2 specific NK cells can use 2
`non-MHC-I-restricted recognition systems to target tumor cells.
`
`Results
`Expansion and Transduction of Primary Human NK Cells. To efficiently
`transduce NK cells, we optimized the culture conditions that
`primarily favor the proliferation of NK cells (11, 18). This procedure
`resulted in NK cell populations of high purity (80–90% CD56⫹
`CD3⫺ NK cells). Using the pMIG vector that expresses GFP as a
`reporter, we tested 2 retroviral transduction protocols. The first
`protocol was based on spinoculation, and the second protocol was
`based on RetroNectin-assisted transduction [supporting informa-
`tion (SI) Fig. S1].
`
`Genetically Engineered NK Cells Express the Transduced Receptor.
`The CR construct specific for HER-2 was cloned into the pMIG
`vector (Fig. 1A). PBL-derived primary human NK cells of 10
`different healthy donors were transduced to express the CR by
`using the spinoculation protocol. Flow cytometry analysis revealed
`that PBL-derived NK cells represented 81% of the cell population
`(Fig. 1 B–D), 61% of which expressed the CR (Fig. 1 E and F)
`as detected by antibodies specific for human Ig recognizing the
`extracellular domain of the CR. The average of transduction of
`the 10 different donors was 55 ⫾ 11%, and the geometric mean
`of the fluorescence signal was 31 ⫾ 10 (Fig. 1G). The CR
`
`Author contributions: J.C. designed research; A.K., A.M., I.P., and H.N. performed research;
`A.M., I.P., H.N., M.C., B.S., and R.K. contributed new reagents/analytic tools; A.K., T.B., H.A.,
`and J.C. analyzed data; and A.K., T.B., H.A., and J.C. wrote the paper.
`
`The authors declare no conflict of interest.
`
`This article is a PNAS Direct Submission.
`1To whom correspondence should be addressed. E-mail: j.charo@mdc-berlin.de.
`
`This article contains supporting information online at www.pnas.org/cgi/content/full/
`0804788105/DCSupplemental.
`
`© 2008 by The National Academy of Sciences of the USA
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.0804788105
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`PNAS 兩 November 11, 2008 兩 vol. 105 兩 no. 45 兩 17481–17486
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`Fig. 1. Her-2 -specific CR is efficiently expressed on transduced NK cells. (A)
`Schematic representation of the CR used in this study. The construct was
`cloned into pMIG replacing IRES and GFP. The resulting construct is designated
`pMSCV-CR. scFv, single-chain fragment variable; hIgG1-Fc, human IgG1 crys-
`tallizable fragment. (B–F) Staining of CR-transduced primary human NK cells
`from 1 representative donor. Cells were stained with a mouse anti human
`CD56 Ab conjugated to APC, a mouse anti-human CD3 Ab conjugated to Cy-5,
`and a goat anti-human Ig Ab conjugated to PE. (B) CD3 and CD56 expression
`on CR-transduced cells. (C) Isotype control and (D) mock-transduced cells
`stained for CR. Cells were gated on the CD56⫹ population (E) and were then
`analyzed for the expression of CR and CD3 (F). (G) Percent and geometric mean
`of the expression of the CR on primary NK cells from 10 different donors
`analyzed by flow cytometry.
`
`expression on NK cells remained stable for more than 1 month
`from the transduction date (data not shown).
`
`NK Cells Genetically Engineered to Express a HER-2-Specific Receptor
`Recognize HER-2-Expressing Target Cells. To assess the function and
`specificity of CR-NK cells, we analyzed the ability of these cells to
`recognize HER-2-expressing target cells in an IFN-␥release assay.
`CR-transduced or control-transduced PBLs were CD3 depleted to
`remove residual T and NKT cells resulting in 95–99% of the cells
`displaying the CD56⫹ CD3⫺ NK cell phenotype (data not shown).
`CR or control engineered NK cells were cocultured with either the
`HER-2-negative tumor cell line C1R/A2 or the HER-2-expressing
`transfectant of this cell
`line, C1R/A2HER2. K562 cells were
`included as control target cells. Fig. 2 summarizes the results
`obtained from NK cells of 10 healthy donors included in this assay.
`CR-NK as well as mock-NK cells were induced by C1R/A2 cells to
`produce on average 1,300 pg/ml of IFN-␥ (Fig. 2). Significantly
`higher levels of IFN-␥ were produced by CR-NK cells, but not by
`mock-NK cells, in response to stimulation by C1R/A2HER2 cells
`with an average of 6,400 pg/ml (Fig. 2). As expected, when cultured
`with K562 cells, all gene-engineered NK cells produced significant
`levels of IFN-␥ (1,600 pg/ml) that were similar to those produced
`by nonengineered NK cells (Fig. 2 and data not shown). Neither
`CR- nor mock-NK cells produced IFN-␥ spontaneously (Fig. 2).
`To determine the ability of CR-NK cells to specifically recognize
`the endogenously expressed HER-2 on cancer cells, we tested a
`panel of breast- and ovarian-carcinoma cells lines (Fig. 3A), 4 of
`which are HER-2⫹ and 1 of which is HER-2⫺, for their ability to
`induce cytokine production by CR-NK cells. CR-NK cells from 5
`donors were able to recognize all HER-2⫹ cell lines and produced
`significantly higher levels of IFN-␥ and IL-2 than mock-NK cells
`(Fig. 3 B and D). Interestingly, IL-2 production by CD3⫺ CR-NK
`cells was confined to a small subset of these cells representing
`10–14% of the CD3⫺ CD56⫹-transduced cells as determined by
`
`Fig. 2. CR-NK cells are specifically stimulated by HER-2-expressing cells. IFN-␥
`was measured in the supernatant of stimulated NK cells from 10 donors.
`Values shown represent mean values of triplicates obtained from IFN-␥-
`specific ELISA. Effector cells (5 ⫻ 104) were mock-transduced or CR-transduced
`NK cells. They were either cultured without target cells (none) or cocultured
`with equal number of K562 cells (4 donors), C1R/A2 cells, or C1R/A2HER2 cells
`(10 donors). P values were calculated by using the Wilcoxon–Mann–Whitney
`test and indicate the difference between the groups.
`
`IL-2 secretion assay (Fig. S2C). There was a good correlation
`between the level of IFN-␥or IL-2 production by the CR-NK cells
`and HER-2 expression on the cancer cells (Fig. 3 C and E). While
`the recognition of cancer cell lines expressing higher levels of
`HER-2 was better than that of cells with lower HER-2 expression
`level, those cells that did not express HER-2 were not recognized,
`confirming the specificity of the recognition pattern of CR-NK
`cells. Furthermore, additional HER-2⫹ breast carcinoma cell lines
`were tested for the ability of inducing IFN-␥or IL-2 production by
`CR-NK cells derived from different donors and were found to
`induce high levels of IFN-␥ and IL-2 production by CR- but not
`mock-NK cells (Fig. S2). MHC-I expression on the different tumor
`cell lines varied but did not correlate with the level of CR-NK cell
`response (Fig. S3).
`In addition to cytokine release, HER-2-specific NK-mediated
`cytotoxicity was induced by HER-2⫹ target cells as indicated by the
`degranulation assay. In this assay, mock-NK cells significantly
`degranulated in response to mitogenic (75%) and K562 cell stim-
`ulation (37–53%) and to some extent to the carcinoma lines MDA
`MB 453 and Cal 51 (22% and 38%, respectively) (Fig. 4A and Fig.
`S4). CR-NK cells, while degranulated in response to mitogenic
`(78%) and K562 (36–39%) cell stimulation, also responded well to
`all HER-2 expressing targets to a much higher extent (up to 64%)
`than mock-NK cells. Notably, CR-NK cells did not degranulate in
`response to the HER-2 negative targets C1R/A2 or MDA MB 468.
`The level of degranulation by CR-NK cells correlated with the level
`of HER-2 expression on the target carcinoma cell lines, which was
`in accordance with the cytokine release data. We also analyzed the
`ability of CR-NK cells to lyse HER-2⫹ tumor cells. Therefore, a
`51Cr release assay was performed by using SKOV3 cell line as
`target. CR-NK cells but not mock-NK cells from 3 different donors
`specifically lysed HER-2⫹ SKOV3 cells (Fig. 4B).
`
`CR-NK Cells Recognize Autologous HER-2 Expressing Target Cells as
`well as Freshly Isolated Ovarian Carcinomas ex Vivo. Paired mini LCL
`and HER-2 mini LCL from 1 healthy donor and 1 ovarian cancer
`patient were produced. These LCLs were obtained by transforming
`B cells from the same donor with either mini Epstein–Barr virus
`(miniEBV) construct or a mini-EBV construct that additionally
`expresses HER-2. HER-2 expression on these cells was confirmed
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`Fig. 3. CR-NK cells recognize HER-2⫹ but not HER-2⫺ carcinoma. Effector cells (5 ⫻ 104) were pMIG (mock)-transduced or CR-transduced NK cells. They were cocultured
`with equal numbers of the indicated carcinoma cell line. Supernatants were harvested and measured for IFN-␥or IL-2 using specific ELISA. (A) Five different carcinoma
`cell lines were selected based on the increasing level of HER-2 expression on their surface. IFN-␥(B) and IL-2 (D) production by CR-NK cells derived from 5 different donors
`in response to stimulation and correlation between HER-2 expression levels on the different carcinoma lines and IFN-␥(C) or IL-2 (E) production levels by CR-NK cells
`in response to stimulation by these lines are shown. P values were calculated by using the Wilcoxon–Mann–Whitney test and indicate the difference between the groups.
`
`by FACS (Fig. S5). CR-NK cells from the same donors were able
`to specifically recognize the HER-2, but not control mini LCL (Fig.
`5A). Similarly, autologous and allogenic CR-NK cells obtained
`from ovarian carcinoma patients or healthy donors, but not
`mock-NK cells, were able to recognize freshly isolated ovarian
`tumor cells ex vivo (Fig. 5B and Fig. S6).
`
`Tumor Challenge Model and in Vivo Imaging. To evaluate the in vivo
`potential of these antigen-specific primary human NK cells, we
`
`chose the SKOV3 cell line because of its ability to grow in RAG2
`knockout mice (Fig. 6B and data not shown) and transduced it to
`express the luciferase gene to follow its survival and outgrowth.
`After inoculating RAG2 knockout mice s.c. with either SKOV/
`CBG cells alone (control) or together with mock- or CR-NK cells
`from 2 different donors, the animals were imaged for biolumines-
`cence. This bioluminescence imaging model allows monitoring of
`tumor cell fate during the first few days after injection, at the time
`that tumor formation cannot be detected by palpation. In Fig. 6A,
`
`Fig. 4. CR-NK degranulate in response to HER-2-specific stimulation and lyse HER-2⫹ target cells. (A) Effector cells identified by size and granularity were pMIG
`(mock)-transduced or CR-transduced NK cells from 1 donor. They were either cultured without target cells (none), or with PMA and ionomycin (⫹PMA/I), or
`cocultured with the indicated cell line for 5 h while being stained for CD107a conjugated with PE. One representative experiment of five performed is depicted.
`(B) Effector cells were pMIG (mock)-transduced or CR-transduced NK cells from 3 donors. They were cocultured with 51Cr-labeled SKOV3 cells in different E:T ratios
`for 4 h, and supernatants were analyzed for 51Cr release.
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`CR-NK cells specifically recognize autologous HER-2 mini-LCLs and ovarian tumor cells ex vivo. (A) Control or HER-2 mini-LCLs were cocultured with
`Fig. 5.
`autologous CR-NK cells. (B) Freshly isolated ovarian tumor cells were cocultured with autologous or allogenic CR-NK cells. Supernatants were harvested and
`measured for IFN-␥by using specific ELISA. HER-2 status was estimated by measuring the percentage of tumor cells in samples and the level of HER-2 expression
`by FACS. P values were calculated by using the Wilcoxon–Mann–Whitney test and indicate the difference between the groups.
`
`1 mouse of each group is shown. If SKOV/CBG cells were injected
`alone, light signal decreased during the first 6 days but recovered on
`day 9 and increased over time, which was followed by increase in
`mean tumor diameter (Fig. 6 A Top and B and Fig. S7). A similar
`signal kinetic was seen in most of the mice that were coinjected with
`
`SKOV/CBG cells together with mock-NK cells. In these mice, light
`signal decreased more dramatically but nonetheless started to
`recover on day 9 and increased over time, followed by an increase
`in the mean tumor diameter (Fig. 6 A Middle and C and Fig. S7).
`All mice that have received coinjection with mock-NK cells from
`
`Fig. 6. CR-NK cells efficiently eradicate HER-2⫹ carcinoma in a RAG2⫺/⫺ mouse model. RAG2⫺/⫺ mice were inoculated s.c. with 5 ⫻ 106 SKOV/CBG cells alone or together
`with the same number of CR⫹-NK cells or mock-NK cells from 2 different donors. (A) In vivo imaging of tumor cell outgrowth or death in 1 representative mouse from
`each group. Because of increase in signal intensity correlating with tumor outgrowth, acquisition time and binning were changed as indicated, and 2 pseudocolor scales
`corresponding to signal intensity are depicted with a minimum of 2 ⫻ 104 for days 1–9 and 1 ⫻ 106 for days 21–65. Exact quantification of signal intensity is provided
`in Fig. S7. (B–D) Mean tumor diameter. Tumors were measured 1–2 times per week by using a digital caliper. Survival comparison was performed by using the
`Wilcoxon–Mann–Whitney test. The P values for the difference between the groups are P ⬍ 0.001 and P ⫽ 0.02 for differences between B and D and between C and
`D, respectively. The difference between B and C is not statistically significant. Numbers of live mice in each group are described above the abscissa.
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`IMMUNOLOGY
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`donor 2 succumbed to tumor outgrowth. Two of four mice that
`received similar coinjection but with mock-NK cells from donor 1
`also succumbed, whereas the other 2 mice survived the tumor
`challenge (Fig. 6C and Fig. S7). When SKOV/CBG cells were
`injected together with CR-NK cells, the light signal decreased
`initially to the same extent as the group with mock-NK coinjection
`but by day 4, no signal above background (Fig. S7) was detectable
`from the SKOV/CBG cells despite prolonged imaging, and no
`tumor could be palpated in these mice (Fig. 6 A Lower and D). All
`mice that have received the coinjection of SKOV/CBG cells to-
`gether with CR-NK cells survived and remained tumor-free.
`
`Discussion
`Adoptively transferred T cells are regarded today as the most
`effective treatment for patients with metastatic melanoma (2).
`Earlier studies with this type of therapy have reported a subsequent
`frequent loss of MHC-I expression (3, 4) on melanoma tissue
`sections as well as on melanoma cell lines, which were later shown
`to be recognized and killed by IL-2-activated NK cell lines and
`clones (19). NK cells initially identified by their ability to recognize
`transformed cells are limited by their recognition mechanisms that
`depend on sensing the fine-tuned expression of activation versus
`inhibitory ligands on their target cells (6, 7, 12, 20–22). Although
`MHC-I loss or down-regulation is often associated with NK rec-
`ognition, some tumors that lack MHC-I are resistant to NK killing.
`Various approaches were evaluated to overcome tumor-cell resis-
`tance to NK cells. These included the use of allo NK cells (23),
`blocking the inhibitory signal (24), or triggering the activation
`receptors by using monoclonal antibodies or by genetic modifica-
`tion of tumor cells (11, 25, 26).
`Here we tested a genetic approach to enhance specific tumor
`recognition by NK cells based on the recognition of the tumor-
`associated antigen HER-2 via a CR (27). HER-2-specific CR-NK
`cells recognized C1R/A2HER2 cells in a HER-2-dependent fash-
`ion. However, unlike transformed cells from hematopoietic origin
`expressing several activation ligands (7, 11, 28), cells derived from
`solid tumors are rather resistant to NK recognition. Nonetheless, all
`HER-2-expressing breast carcinomas that we have tested were
`recognized by CR-NK cells, and the extent of recognition corre-
`lated with the level of HER-2 expression on these tumor cells.
`Taken together, the data indicate that CR-NK cells have the
`capacity to respond specifically to HER-2-expressing target by
`IFN-␥ and IL-2 production, the 2 cytokines essential for tumor
`rejection and NK cell survival, as well as degranulation and specific
`lysis of HER-2⫹ target cells. These responses occurred while
`CR-NK cells maintained their classical NK specificity as indicated
`by the response to K562 cells. Hence, these cells use 2 independent
`mechanisms, which are non-MHC-I restricted, to recognize their
`target cells.
`Remarkably, CR-NK cells recognized autologous HER-2
`miniLCLs but not their HER-2-negative counterpart, indicating
`that the activation provided by the CR signaling was sufficient to
`override the collective inhibitory signals provided by the corre-
`sponding inhibitory ligands on the autologous targets. Additionally,
`HER-2-positive ovarian carcinoma explanted from 5 patients ex-
`pressed HER-2 at levels that permitted recognition by CR-NK cells
`ex vivo.
`This universal recognition of HER-2⫹ target cells may be ex-
`plained by the level of activation provided by the CR used in our
`study, which may have overridden the sum of inhibitory signals
`provided by any of the tested cell lines. Apart from the qualitative
`difference in signaling, the affinity of the binding domain (Kd ⫽
`10⫺8 M) of the CR is much higher than that of the reported
`interactions between known inhibitory NK-receptors and their
`ligands (29). Therefore, it will be interesting to study whether NK
`cells engineered with CRs with lower affinity would also be able to
`overcome NK inhibition.
`
`HER-2 is an attractive target for immunotherapy, and Ab
`(trastuzumab)-based therapy targeting HER-2 is clinically ap-
`proved (16). Trastuzumab is believed to manifest its effect through
`direct Ab binding, NK-mediated Ab-dependent cell cytotoxicity,
`and by blocking angiogenesis through inhibition of VEGF expres-
`sion (13, 14). Distinguishing the CR-NK cell approach from the use
`of trastuzumab or other strategies targeting HER-2 is that not all
`HER-2⫹ tumors or cell lines are responsive to this Ab-based
`therapy (13, 14, 16, 30, 31) or to this extent to siRNA-mediated
`HER-2 targeting that we have earlier evaluated (31). Only tumor
`cells that have gene amplified HER-2, accounting for 1/3 of the
`HER-2⫹ tumors, represent good targets for these Ab and siRNA
`treatments. In contrast, the CR-NK cells were able to recognize all
`tested HER-2⫹ carcinomas regardless of their gene amplification
`status. Interestingly, Neve et al. (32) have recently reported that
`breast cancer cell lines have molecular features that mirror primary
`breast tumors, which permitted prediction of response to targeted
`therapy by trastuzumab. Based on this study, the majority of breast
`carcinoma lines that we have tested and shown to be sensitive to
`CR-NK effector function would not be responsive to trastuzumab.
`Indeed, both MCF-7 and MDA MB 453 cell lines that were shown
`to be resistant to trastuzumab treatment (32) did induce a robust
`CR-NK cell response. Furthermore, cell lines expressing lower
`levels of HER-2, such as MCF-7, cannot be specifically recognized
`by trastuzumab-directed NK cells via Ab-dependent cell cytotox-
`icity (33, 34), unless transfected to overexpress HER-2 (35). No-
`tably, MCF-7 was efficiently recognized by CR-NK cells.
`Haploidentical hematopoietic transplantation for the treatment
`of leukemia was reported to exploit alloreactive NK cells to increase
`the probability of survival of high-risk acute myeloid leukemia,
`representing a positive precedent for adoptive NK cell therapy (23).
`Additionally, acute lymphoblastic leukemia cells were shown to be
`a good target for NK cells transduced with CD19 specific receptor
`(36). Few reports have investigated the potential of using genetically
`engineered NK-like lymphoma cells in adoptive immunotherapy
`targeting solid tumors such as breast cancer. Transduction of an
`NK-like lymphoma line (NK-92) targeted these cells to HER-2-
`expressing tumor cells. These attempts demonstrated that tumor
`outgrowth was transiently delayed in mice coinjected with a HER-
`2-positive tumor cells and targeted NK-92 cells, but mice protection
`could not be achieved (37, 38). Moreover, these NK-92 cells are
`transformed and therefore not ideal for immunotherapy and do not
`reflect the biology of primary NK cells expressing a CR. Adoptively
`transferred primary NK cells have the potential of long-term
`persistence and proliferation in the recipient and do compete well
`for the utilization of homeostasis growth factors (39), a prerequisite
`for successful adoptive immunotherapy (1, 2). Our initial in vivo
`study shows that CR-engineered NK cells can overcome their
`intrinsic inhibition, which limited their function in most of the
`animals that received mock-transduced NK cells, and these CR-NK
`cells did prevent tumor outgrowth in all mice that have received this
`treatment.
`The ability to genetically engineer primary NK cells, apart from
`providing an opportunity to further the analysis of NK cell biology,
`can represent an effective alternative or a complement to the
`currently used approaches in cancer immunotherapy.
`
`Materials and Methods
`Retroviral Vectors. The CR construct specific for HER-2, C6.5-scFv-Fc-CD3␨-CD28,
`was earlier described (40, 41). The CR was cloned into the modified retroviral
`vector pMIG described elsewhere (1, 42) replacing the GFP and IRES fragment. The
`resulting construct was designated pMSCV-CR. The pMIG vector encoding GFP, or
`the pMSCVred vector encoding red fluorescent protein, which was generated by
`replacing the IRES and the GFP encoding region of pMIG by the RFP encoding
`region from the DSred-Express plasmid (Clontech, BD Biosciences), were used as
`controls. The click beetle green (CBG) luciferase encoding retroviral vector was
`constructed by cloning the luciferase cDNA obtained from the pCBG99 plasmid
`(Promega) and shuttled through the phRL-null plasmid into the pMIG vector. The
`resulting plasmid was designated pMIG-CBG.
`
`Kruschinski et al.
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`PNAS 兩 November 11, 2008 兩 vol. 105 兩 no. 45 兩 17485
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`UPenn Ex. 2061
`Miltenyi v. UPenn
`IPR2022-00855
`Page 17485
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`Cloning of the various constructs was confirmed by restriction mapping,
`partial sequencing, and transient transfection into 293T cells. The 10A1-
`pseudotyped retrovirus was generated by cotransfection of 293T cells with the
`above mentioned plasmids and the gag, pol, and env encoding pCL-10A1 vector
`by using Lipofectamine 2000 (Invitrogen). Briefly, 1 ⫻ 106 293T cells were seeded
`into a T 25flask 1 day before transfection. The next day, medium was replaced
`with 4 mL of fresh medium. Ten microliters of Lipofectamine 2000 and 3 ␮g of
`DNA of each plasmid were used (6 ␮g of total DNA) diluted in 1 mL of Opti-MEM
`for transfection of 1 T 25flask of 293T cells according to the manufacturer’s
`manual. The next day, medium was changed to 5 mL of RPMI medium 1640, 10%
`(vol/vol) FBS, and the cells were moved to a 32 °C, 5% CO2 humidified incubator.
`Virus supernatant was collected on 3 successive days and filtered through a
`0.45-␮m sterile filter before use.
`
`Expansion of NK Cells and Retroviral Transduction. NK cells were expanded as
`previously described (11, 18) with the following modification: a 30-min plastic
`adherence step was used and cells were cultured in RPMI medium 1640, 10%
`(vol/vol) FBS in 6-well tissue culture plates. Transduction was performed on day 6
`and was repeated on 2 subsequent days using either of the 2 following protocols:
`a RetroNectin-assessed protocol and a spinoculation-assessed protocol. For Ret-
`roNectin-based transduction, 6-well tissue culture plates were coated with 50 ␮g
`per well RetroNectin (TaKaRa) as recommended by the manufacturer. One day
`later, 4 mL of virus supernatant was added to each well and plates were incubated
`for 30 min at 32 °C and then for an additional 24 h at 4 °C (43). Virus supernatant
`was removed and replaced by cells (1 ⫻ 106 cells/mL) in RPMI medium 1640, 10%
`(vol/vol) FBS, containing 200 IU/mL IL-2 (kindly provided by O. Krieter, Chiron
`GmbH, Marburg, Germany). A half mililiter of fresh virus supernatant was added
`to each well, and cells were incubated at 32 °C for 24 h. Transduction was
`repeated on 2 successive days. After the third transduction, cells were maintained
`in RPMI medium 1640, 10% (vol/vol) FBS, and 200 IU/ml IL-2 at 37 °C.
`Spinoculation-based transduction was performed in 24-well tissue culture
`plates (1) using 2 ⫻ 105 cells per well in a total volume of 2 mL of virus supernatant
`diluted 1:1 in culture medium in the presence of 8 ␮g/ml polybrene (Sigma–
`Aldrich) and 200 IU/mL IL-2. Cells were centrifuged at 805 ⫻ g at 32 °C for 90 min.
`
`1. Charo J, et al. (2005) Bcl-2 overexpression enhances tumor-specific T-cell survival.
`Cancer Res 65:2001–2008.
`2. Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME (2008) Adoptive cell
`transfer: A clinical path to effective cancer immunotherapy. Nat Rev Cancer 8:299 –308.
`3. Khong HT, Restifo NP (2002) Natural selection of tumor variants in the generation of
`‘‘tumor escape’’ phenotypes. Nat Immun 3:999 –1005.
`4. Restifo NP, et al. (1996) Loss of functional ␤2-microglobulin in metastatic melanomas
`from five patients receiving immunotherapy. J Natl Cancer Inst 88:100 –108.
`5. Gladow M, Uckert W, Blankenstein T (2004) Dual T cell receptor T cells with two defined
`specificities mediate tumor suppression via both receptors. Eur J Immunol 34:1882–1891.
`6. Trinchieri G (1989) Biology of natural killer cells. Adv Immunol 47:187–376.
`7. Lanier LL (2005) NK cell recognition. Annu Rev Immunol 23:225–274.
`8. Yokoyama WM, Kim S (2006) Licensing of natural killer cells by self-major histocom-
`patibility complex class I. Immunol Rev 214:143–154.
`9. Karre K, Ljunggren HG, Piontek G, Kiessling R (1986) Selective rejection of H-2-deficient
`lymphoma variants suggests alternative immune defence strategy. Nature 319:675–678.
`10. Moretta A, Moretta L (1997) HLA class I specific inhibitory receptors. Curr Opin
`Immunol 9:694 –701.
`11. Wilson JL, et al. (1999) NK cell triggering by the human costimulatory molecules CD80
`and CD86. J Immunol 163:4207– 4212.
`12. Lanier LL (2006) Natural killer cells: Roundup. Immunol Rev 214:5– 8.
`13. Menard S, Pupa SM, Campiglio M, Tagliabue E (2003) Biologic and therapeutic role of
`HER2 in cancer. Oncogene 22:6570 – 6578.
`14. Meric-Bernstam F, Hung MC (2006) Advances in targeting human epidermal growth
`factor receptor-2 signaling for cancer therapy. Clin Cancer Res 12:6326 – 6330.
`15. Slamon DJ, et al. (1989) Studies of the HER-2/neu proto-oncogene in human breast and
`ovarian cancer. Science 244:707–712.
`16. Finn RS, Slamon DJ (2003) Monoclonal antibody therapy for breast cancer: Herceptin.
`Cancer Chemother Biol Response Modif 21:223–233.
`17. Slamon DJ, et