The Journal of Immunology
`
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`
`Adoptive Transfer of Gene-Modified Primary NK Cells Can
`Specifically Inhibit Tumor Progression In Vivo1
`
`Hollie J. Pegram,* Jacob T. Jackson,* Mark J. Smyth,*† Michael H. Kershaw,2*†
`and Phillip K. Darcy2,3*†
`
`NK cells hold great potential for improving the immunotherapy of cancer. Nevertheless, tumor cells can effectively escape NK
`cell-mediated apoptosis through interaction of MHC molecules with NK cell inhibitory receptors. Thus, to harness NK cell effector
`function against tumors, we used Amaxa gene transfer technology to gene-modify primary mouse NK cells with a chimeric
`single-chain variable fragment (scFv) receptor specific for the human erbB2 tumor-associated Ag. The chimeric receptor was
`composed of the extracellular scFv anti-erbB2 Ab linked to the transmembrane and cytoplasmic CD28 and TCR-␨ signaling
`domains (scFv-CD28-␨). In this study we demonstrated that mouse NK cells gene-modified with this chimera could specifically
`mediate enhanced killing of an erbB2ⴙ MHC class Iⴙ lymphoma in a perforin-dependent manner. Expression of the chimera did
`not interfere with NK cell-mediated cytotoxicity mediated by endogenous NK receptors. Furthermore, adoptive transfer of gene-
`modified NK cells significantly enhanced the survival of RAG mice bearing established i.p. RMA-erbB2ⴙ lymphoma. In summary,
`these data suggest that use of genetically modified NK cells could broaden the scope of cancer immunotherapy for patients. The
`Journal of Immunology, 2008, 181: 3449 –3455.
`
`N atural killer cells comprise 5–10% of PBLs and play an
`
`important role in the body’s first line of defense against
`pathogen invasion and malignant transformation (1, 2).
`Unlike the exquisite Ag specificity observed for T and B lympho-
`cytes, NK cells instead express a number of different activation
`and inhibition receptors which provide a balance of signals that
`dictate their overall response (3). This recognition system used by
`NK cells and the fact that they do not require prior sensitization
`provides a degree of flexibility to rapidly recognize different
`pathogens.
`Several elegant studies have demonstrated that NK cells can
`effectively control tumor growth in mice mediated through release
`of perforin and cytokines (4 – 6). Their importance in anti-tumor
`immunity is further illustrated by the increased incidence of leu-
`kemia in patients with dysfunctional NK cells (7). The observation
`that NK cells can effectively respond to tumor cells exhibiting
`defective or altered MHC class I has made them promising effec-
`tors for immunotherapeutic strategies that target tumor escape
`
`*Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne,
`Victoria, Australia; and †Department of Pathology, University of Melbourne, Mel-
`bourne, Australia
`
`Received for publication April 17, 2008. Accepted for publication July 3, 2008.
`
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance
`with 18 U.S.C. Section 1734 solely to indicate this fact.
`1 This work was supported by a National Health and Medical Research Council Pro-
`gram Grant and a Cancer Council of Victoria research grant. M.H.K. and P.K.D. were
`supported by National Health and Medical Research Council of Australia R.D. Wright
`Research Fellowships. M.J.S. was supported by a National Health and Medical Re-
`search Council Senior Principal Research Fellowship.
`
`H.J.P. performed the research, analyzed the data, and wrote first draft of the paper.
`J.T.J. performed the research. M.J.S. analyzed the data. M.H.K. designed the research
`and analyzed the data. P.K.D. designed the research, analyzed the data, and wrote the
`manuscript.
`2 M.H.K. and P.K.D. contributed equally as senior authors.
`3 Address correspondence and reprint requests to Dr. Phillip K. Darcy and Dr. Mi-
`chael H. Kershaw, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett
`Street, Victoria, 8006, Australia. E-mail addresses: phil.darcy@petermac.org and
`michael.kershaw@petermac.org
`
`www.jimmunol.org
`
`variants (8). Nevertheless, tumor cells have developed several
`mechanisms to impede NK cell function, which include the ex-
`pression of ligands that interact with NK cell inhibitory recep-
`tors (9).
`Immunotherapeutic strategies to enhance NK cell anti-tumor ac-
`tivity have included the use of specific cytokines (10) or adoptive
`transfer of autologous ex vivo IL-2-activated lymphokine killer
`cells (LAK4; Ref.11). However, these approaches have only re-
`sulted in moderate success in restricted numbers of patients (12).
`More promising results have been recently achieved in the trans-
`plant setting with the use of allogeneic NK cells against acute
`myeloid leukemia (13, 14). Another emerging approach to address
`the problem of NK cell-mediated inhibition by tumors involves the
`genetic modification of NK cells with chimeric single-chain vari-
`able fragment (scFv) receptors that directly target tumor-associ-
`ated Ags (TAA). This approach has successfully been used to en-
`hance tumor recognition by primary T cells (15–24), and several
`studies have demonstrated specific killing of tumor target cells
`following redirection of NK cell lines (25–27) or primary human
`NK cells (28) with chimeric receptors. Nevertheless, investigation
`of whether these genetically engineered primary NK cells can spe-
`cifically reject tumor in vivo has never been reported and has been
`hampered by lack of an efficient method for expressing transgenes
`in mouse NK cells.
`In this study we have used Amaxa Nucleofector technology, an
`electroporation-based procedure, to genetically engineer primary
`mouse NK cells with an scFv anti-erbB2-CD28-␨ chimeric recep-
`tor. We and others have shown that this novel receptor design
`incorporating both costimulatory CD28 and TCR-␨domains linked
`in the one intracellular domain could optimally trigger activation
`of transduced PBMC after Ag stimulation (15, 21, 29, 30). The
`Amaxa system uses optimized electrical parameters to enhance
`delivery of DNA to the cell nucleus, which increases transfection
`
`4 Abbreviations used in this paper: LAK, lymphokine killer cells; scFv, single-chain
`variable fragment; TAA, tumor-associated Ags; FasL, Fas ligand; WT, wild type.
`
`Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
`
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`ENHANCED TUMOR INHIBITION BY GENE-MODIFIED NK CELLS
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`FIGURE 1. Gene modification of primary murine NK cells with the
`anti-erbB2 chimeric receptor. A, Schematic representation of the scFv-anti-
`erbB2-CD28-␨receptor. The chimeric receptor consisted of the VH and VL
`regions of the anti-erbB2 mAb joined by a flexible linker, a CD8-␣ mem-
`brane-proximal hinge region (MP), and the transmembrane (TM) and cy-
`toplasmic regions of the mouse CD28 signaling chain fused to the intra-
`cellular domain of human TCR-␨. B, The expression of the chimeric scFv
`anti-erbB2 receptor in mouse NK cells was analyzed following staining
`with an anti-tag Ab mAb and PE-labeled sheep anti-mouse Ig (thick line)
`or a secondary PE-conjugated Ab (thin line). C, Expression of GFP in
`transfected (thick line) vs nontransfected (thin line) NK cells was analyzed
`by flow cytometry. Results shown are representative of seven experiments
`performed.
`
`51Cr-release assay. In brief, NK cells were incubated with 1 ⫻ 105 51Cr-
`labeled tumor targets at various E:T ratios in triplicate wells of a 96-well
`round-bottom plate (in 200 ␮l of complete DMEM). The percentage of
`specific release of 51Cr into the supernatant was assessed as described
`previously (31).
`
`Adoptive transfer
`
`The ability of gene-modified NK cells expressing the ␣-erbB2-CD28-␨
`receptor to enhance the survival of tumor bearing mice was investigated in
`the following model. C57BL/6 RAG-1⫺/⫺ mice were injected i.p. with 2 ⫻
`105 RMA-parental or RMA-erbB2 tumor cells. Mice were then treated on
`days 0, 1, days 0, 1, 2, 3 (early model), or days 3, 4 (delayed model) with
`2 ⫻ 106 (per injection) of ␣-erbB2 NK cells or GFP-NK control cells
`delivered i.p. In some experiments gene-modified NK cells were coadmin-
`istered with high dose IL-2 (200,000 IU/ml) injected i.p. on days 0, 1, and
`2. To investigate the persistence of NK cells in vivo, 2 ⫻ 106 donor gene-
`modified NK cells from congenic C57BL/6- ptprca (CD45.1⫹) mice were
`transferred into RMA-erbB2 tumor-bearing RAG-1⫺/⫺ recipient mice
`(CD45.2⫹) cell on days 0 and 1. Three mice at each time point were then
`sacrificed on days 1, 2, and 5 following tumor injection, and spleens were
`harvested and i.p. washes were performed to determine the number of
`CD45.1⫹ cells present.
`
`Statistical analysis
`The Mann-Whitney U test was used for statistical analysis. Values of p ⬍
`0.05 were considered significant.
`
`Results
`Expression of the chimeric anti-erbB2 receptor in primary
`mouse NK cells
`
`The genetic modification of primary mouse NK cells with scFv
`chimeric receptors using retroviral-based transduction methods has
`proven difficult. To address this, we used the Amaxa Nucleofector
`system to gene modify mouse NK cells with the scFv ␣-erbB2-
`CD28-␨chimeric receptor (Fig. 1A). Using this method, high level
`expression of the ␣-erbB2 receptor was achieved in mouse NK
`
`efficiency and gene expression levels. NK cells expressing the chi-
`meric receptor were demonstrated to enhance target cell killing
`following receptor ligation. Furthermore, adoptive transfer of
`scFv-receptor gene-modified NK cells led to significant growth
`inhibition of erbB2⫹ T cell lymphomas in mice. These data sug-
`gest that gene-modified NK cells may have significant potential as
`an effective immunotherapy for cancer.
`
`Materials and Methods
`Cell lines
`
`The C57BL/6 murine lymphoma cell lines RMA and RMA-S are T cell
`lymphomas derived from the Rauscher murine leukemia virus-induced
`RBL-5 cell line (8). The murine melanoma cell line B16-F10 was obtained
`from American Type Culture Collection. The erbB2-expressing cell lines
`RMA-erbB2 and B16-F10-erbB2 were generated by transduction with a
`retroviral vector (murine stem cell vector) encoding the cDNA for human
`erbB2. All cell lines were maintained in complete DMEM medium con-
`taining 10% (v/v) FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100
`␮g/ml streptomycin (Invitrogen). The murine lymphoma cell line YAC-1
`was maintained in RPMI 1640 medium (Invitrogen), with 10% FCS (v/v),
`2 mM L-glutamine, 0.1 mM non-essential amino acids (Life Technologies),
`1 mM sodium pyruvate (Life Technologies), 100 U/ml penicillin, 100
`␮g/ml streptomycin (Life Technologies), and 5 ⫻ 10⫺5 mM 2ME.
`
`Mice
`C57BL/6, C57BL/6-ptprca, and C57BL/6 RAG-1-deficient (RAG-1⫺/⫺)
`mice were purchased from the Walter and Eliza Hall Institute of Medical
`(C57BL/6-pfp⫺/⫺) and
`Research. C57BL/6 perforin (pfp)-deficient
`C57BL/6 gld (Fas ligand (FasL) mutant) were bred at the Peter MacCallum
`Cancer Centre. All mice were housed in specific pathogen free conditions
`at the Peter MacCallum Cancer Centre and mice 6 –12 wk of age were used
`in all experiments.
`
`Isolation of NK cells
`
`Dissected spleens from C57BL/6 mice were crushed into hypotonic lysis
`buffer and filtered to create a single cell suspension. NK cells were then
`selected using anti-DX5 Microbeads or an NK cell isolation beading kit
`(Miltenyi Biotec) according to manufacturer’s specifications. The cells
`were then grown in RPMI 1640 medium containing 10% (v/v) FCS, 2 mM
`L-glutamine, 5 ⫻ 10⫺5 mM 2ME, 100 U/ml penicillin, 100 ␮g/ml strep-
`tomycin (Life Technologies), 2 mM HEPES, and 1000 IU/ml recombinant
`human IL-2 (Biological Resources Branch Preclinical Repository, National
`Cancer Institute).
`
`Gene modification of NK cells
`
`Seven-day IL-2-activated mouse NK cells were gene modified by electro-
`poration using the Amaxa Nucleofector system (Amaxa Biosystems). In
`brief, NK cells were placed in 0.1 ml electroporation solution with either
`4 ␮g pMAX plasmid DNA encoding the scFv ␣-erbB2-CD28-␨ chimeric
`receptor or GFP. Following electroporation, the cells were placed into 2 ml
`Amaxa recovery medium with 600 IU/ml recombinant human IL-2 for 24 h
`before being used in experiments.
`
`Flow cytometry
`
`Expression of the chimeric scFv receptor on the surface of NK cells was
`determined by indirect immunofluorescence with a primary c-myc tag Ab
`(Cell Signaling Technology), followed by staining with a secondary PE-
`labeled anti-mouse Ig mAb (BD Biosciences). Background fluorescence
`was determined by staining cells with an isotype control Ab followed by a
`secondary PE-conjugated anti-mouse Ig mAb. Direct detection of GFP by
`flow cytometry was examined in transfected vs non-transfected NK cells.
`Phenotyping of cell surface marker expression on NK cells was determined
`by staining cells with allophycocyanin-conjugated Abs specific for NK1.1,
`DX5, CD11b, and CD27 (eBioscience) and biotin-conjugated KLRG1,
`NKG2D (eBioscience), and biotin- conjugated CD94, CD25, and CD69
`(BD Pharmingen). This was the followed by staining with a PerCPCy5.5-
`streptavidin (BD Pharmingen) Ab. MHC class I expression on tumor cells
`was determined by staining cells with a PE-conjugated Ab specific for
`mouse H2kb (BD Biosciences).
`
`Cytotoxicity
`
`The ability of gene-modified mouse NK cells from wild-type (WT) and/or
`gene-targeted mice to specifically kill tumor targets was assessed in a 4 h
`
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`3451
`
`RMA-erbB2
`RMA-S-erbB2
`
`*
`
`20:1
`
`10:1
`E:T ratio
`
`5:1
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`Specificlysis(%)
`
`FIGURE 3. MHC class I inhibition of NK cell cytotoxicity. GFP-NK
`effector cells were used in a 4 h 51Cr release assay against the MHC class
`I⫹ RMA-erbB2 cell line or class I deficient RMA-S-erbB2 tumor cell line.
`GFP-NK cells were able to more effectively kill the RMA-S-erbB2 cell line
`(open squares) compared with the RMA-erbB2 cell line (closed squares; ⴱ,
`p ⬍ 0.05, as determined by a Mann-Whitney U test). Results are repre-
`sentative of three independent experiments.
`
`etry to compare expression of a number of molecules expressed by
`␣-erbB2-NK and control GFP-NK cells, including activation and
`inhibition receptors. In three independently performed experi-
`ments, we observed no difference in the expression of NK cell
`markers NK1.1 or DX5 between GFP-NK and ␣-erbB2-NK cells
`(Fig. 2, A and B). There was also no difference in the level of
`expression of the CD11b marker between transfected NK cells
`(Fig. 2C). In addition, comparable expression of inhibitory recep-
`tors Ly49A, KLRG1, CD94, and the activatory receptor NKG2D,
`was observed between ␣-erbB2-NK and GFP-NK cells (Fig. 2,
`D–F). The levels of expression of the costimulatory receptor CD27
`and activation markers CD69 and CD25 were also expressed at
`similar levels between the transfected NK cell types (Fig. 2, H–J).
`These data indicated that transfection of mouse NK cells with the
`scFv chimeric receptor has not phenotypically altered expression
`of a number of important NK cell-associated markers.
`
`Ag-specific cytotoxicity mediated by anti-erbB2-NK cells
`
`Although NK cells can mediate effective killing of target cells,
`they are often inhibited by recognition of MHC class I molecule.
`Indeed, this is supported by our data demonstrating low killing by
`GFP-NK effector cells of the MHC class I⫹ lymphoma cell line
`RMA-erbB2 compared with the class I-deficient RMA-S-erbB2
`cell line (Fig. 3). To determine whether our gene-modified NK
`cells expressing the ␣-erbB2 chimeric receptor could overcome
`MHC-class I inhibition we assessed their ability to kill RMA cells,
`either expressing the erbB2 TAA (RMA-erbB2) or not. Impor-
`tantly, the level of MHC class I expression on RMA-erbB2 and
`RMA cells was equivalent (Fig. 4). We demonstrated at least a two
`fold increase in the level of killing of RMA-erbB2 cells by
`␣-erbB2-NK cells compared with control GFP-NK cells (Fig. 5A).
`This enhanced killing was erbB2 Ag-specific because ␣-erbB2-NK
`and GFP-NK cells mediated comparable lysis of RMA parental
`cells (Fig. 5B). We next determined whether anti-erbB2-NK cells
`could increase killing of another erbB2⫹ cell line. In this experi-
`ment we demonstrated enhanced killing by anti-erbB2-NK cells of
`a mouse melanoma cell line B16 expressing erbB2 Ag (B16-F10-
`erbB2) compared with control GFP-NK cells (Fig. 5C). Again this
`was erbB2-specific because equivalent killing of parental B16-F10
`cells by anti-erbB2 or GFP-NK cells was observed (Fig. 5D). We
`also showed that expression of the scFv receptor or GFP had no
`impact on the endogenous cytotoxic ability of NK cells. We dem-
`onstrated comparable cytotoxicity of a NK cell-sensitive target cell
`line, YAC-1, by either non-transfected 7-day IL-2-activated NK
`
`The Journal of Immunology
`
`A
`
`NK1.1
`
`B
`
`DX5
`
`C
`
`C 11bD
`
`D
`
`Ly49A
`
`E
`
`KLRG1
`
`F
`
`NKG2D
`
`G
`
`CD94
`
`H
`
`CD27
`
`I
`
`CD69
`
`J
`
`CD25
`
`100
`
`100
`101
`102
`103
`104
`101
`102
`103
`Mean fluorescence intensity
`
`104
`
`120
`
`90
`
`60
`
`30
`
`0
`120
`
`90
`
`60
`
`30
`
`0
`120
`
`90
`
`60
`
`30
`
`0
`120
`
`90
`
`60
`
`30
`
`0
`120
`
`90
`
`60
`
`30
`
`0
`
`Relativecellcount
`
`FIGURE 2. Phenotypic characterization of gene-modified primary
`mouse NK cells. The surface expression of various NK cell markers, ac-
`tivation markers, and activation/inhibition receptors was analyzed by flow
`cytometry following staining with appropriate Abs. Cells used for analysis
`were gated on either anti-tag positive cells (representing anti-erbB2 recep-
`tor expressing cells; thick line) or gated on GFP positive cells (representing
`control NK cells; thin line), or on unstained anti-erbB2-NK cells (dotted
`line). There was no significant difference in expression of the following
`molecules between anti-erbB2 or GFP transfected NK cells; NK1.1 (A),
`DX5 (B), CD11b (C), Ly49A (D), KLRG1 (E), NKG2D (F), CD94 (G),
`CD27 (H), CD69 (I), or CD25 (J). Results shown are representative of
`three independent experiments.
`
`cells following staining with a c-myc tag mAb specifically recog-
`nizing a c-myc tag epitope incorporated into the extracellular do-
`main of the chimeric receptor (46 ⫾ 10%, n ⫽ 7; Fig. 1B). Equiv-
`alent levels of expression of autonomous GFP were also observed
`in control mouse NK cells (49 ⫾ 11%, n ⫽ 7; Fig. 1C). Impor-
`tantly, the transfected NK cell populations were TCR␤ negative
`(data not shown). Cell viability ranged between 60 and 90% fol-
`lowing electroporation.
`
`Phenotypic characterization of gene-modified primary mouse
`NK cells
`
`We next investigated whether expression of the chimeric scFv re-
`ceptor had any affect on NK cell phenotype. We used flow cytom-
`
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`ENHANCED TUMOR INHIBITION BY GENE-MODIFIED NK CELLS
`
`Unstained
`RMA-erbB2
`RMA-parental
`
`100
`
`75
`
`50
`
`25
`
`3452
`
`Relativecellcount
`
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`
`**
`
`*
`
`10:1
`
`5:1
`
`1:1
`
`WT NK
`gld NK
`pfp NK
`
`10:1
`
`5:1
`
`1:1
`
`B
`60
`
`40
`
`20
`
`0
`
`D
`60
`
`40
`
`20
`
`10:1
`
`5:1
`
`1:1
`
`0
`
`10:1
`E:T Ratio
`
`5:1
`
`1:1
`
`A
`60
`
`40
`
`20
`
`0
`
`C
`60
`
`40
`
`20
`
`0
`
`Specificlysis(%)
`
`FIGURE 6. Cytotoxicity of erbB2⫹ tumor cells by anti-erbB2 NK cells
`is perforin-dependent. Gene-modified NK cells derived from WT, perforin-
`deficient (pfp⫺/⫺), or gld (Fas ligand mutant) mice were used in a 4 h 51Cr
`release assay. A, Killing of RMA-erbB2 target cells by anti-erbB2 NK cells
`derived from pfp⫺/⫺, but not WT or gld mice, was completely abrogated.
`Equivalent background killing of RMA-erbB2 tumor cells (B) and RMA
`parental cells (C) by GFP-NK cells or RMA-parental cells by ␣-erbB2 NK
`cells (D) derived from either WT or gld mice. (ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬎ 0.05
`as determined by a Mann-Whitney U test). Results shown are representa-
`tive of two independent experiments.
`
`gld (mutant FasL) mice. Importantly, the level of expression of the
`scFv chimeric receptor was comparable in NK cells derived from
`WT and gene-targeted mice (data not shown). It is also important
`to note that previous studies have shown that other functional path-
`ways of NK cells from perforin-deficient mice (i.e., FasL-mediated
`killing) are intact (33). In cytotoxicity assays, we demonstrated no
`killing of RMA-erbB2 target cells by ␣-erbB2 NK cells derived
`from pfp⫺/⫺ mice (Fig. 6A). In contrast, the sensitivity of RMA-
`erbB2 cells to ␣-erbB2 NK cells derived from gld mice or WT
`mice was similar (Fig. 6A). As further specificity controls we ob-
`served comparable background killing of RMA-erbB2 by GFP-NK
`cells and RMA-parental cells by either ␣-erbB2 or GFP-NK cells
`derived from WT and gld mice (Fig. 6, B–D). These data demon-
`strated that gene-modified primary mouse NK cells mediated Ag-
`specific cytotoxicity through a perforin-dependent mechanism.
`
`Ag-specific inhibition of tumor growth mediated by
`anti-erbB2-NK cells
`
`We next assessed the ability of gene-modified mouse NK cells
`expressing the ␣-erbB2 chimeric receptor to mediate Ag-specific
`inhibition of tumor growth in vivo. Tumor cells (RMA-parental or
`RMA-erbB2) were injected i.p. into RAG-1⫺/⫺ mice that then
`received early transfer (days 0, 1) or delayed transfer (days 3, 4) of
`2 ⫻ 106 gene-modified NK cells (␣-erbB2-NK or GFP-NK cells).
`In these experiments, we demonstrated significantly increased sur-
`vival of mice with RMA-erbB2 tumor that received ␣-erbB2 gene-
`modified NK cells delivered at early or at later time points (Fig. 7,
`A and B). This effect was Ag-specific because there was no sig-
`nificant increase in survival of mice with RMA-erbB2 that re-
`ceived control GFP-NK cells. Furthermore, ␣-erbB2 NK cells had
`no anti-tumor effect in mice injected with RMA parental tumor. In
`another experiment we demonstrated that coadministration of high
`dose IL-2 (200,000 IU/ml) with gene-modified NK cells did not
`improve the anti-tumor effect in mice (data not shown). We also
`investigated the persistence of our adoptively transferred gene-
`modified NK cells by using donor NK cells from congenic C57BL/
`
`0
`
`100
`
`101
`
`103
`
`104
`
`102
`H2Kb
`FIGURE 4. Expression of MHC class I on RMA tumor cells. MHC
`class I expression on RMA parental (thick line) and RMA-erbB2 (thin line)
`tumor cells was determined by staining cells with a PE-conjugated Ab
`specific for mouse H2kb. Unstained RMA tumor cells (dotted line) were
`used as a control.
`
`cells or gene-modified NK cells (Fig. 5E). These data demon-
`strated that expression of the scFv receptor targeting TAA could
`endow primary mouse NK cells with the ability to overcome MHC
`class I-mediated inhibition and kill NK cell-sensitive tumors.
`
`Target cell lysis mediated by gene-modified NK cells was
`perforin dependent
`
`It has been reported that NK cells lyse their targets predominantly
`via the granule exocytosis pathway involving perforin; however,
`they can also mediate apoptotic activity through FasL or TRAIL
`pathways (5, 32). To determine the mechanism of killing used by
`our gene-modified primary mouse NK cells, we genetically mod-
`ified NK cells from C57BL/6 WT, perforin-deficient (pfp⫺/⫺), and
`
`Anti-erbB2-NK
`GFP-NK
`
`*
`
`10:1
`
`5:1
`
`1:1
`
`Anti-erbB2-NK
`GFP-NK
`
`*
`
`20:1
`
`10:1
`
`1:1
`
`B
`60
`50
`40
`30
`20
`10
`0
`D
`50
`40
`30
`20
`10
`0
`
`Anti-erbB2-NK
`GFP-NK
`
`10:1
`
`5:1
`
`1:1
`
`Anti-erbB2-NK
`GFP-NK
`
`20:1
`
`10:1
`
`1:1
`
`Untransfected
`Anti-erbB2-NK
`GFP-NK
`10:1
`
`5:1
`E:T ratio
`
`1:1
`
`A
`60
`50
`40
`30
`20
`10
`0
`C
`50
`40
`30
`20
`10
`0
`E
`100
`80
`60
`40
`20
`0
`
`Specificlysis(%)
`
`FIGURE 5. Enhanced cytotoxicity of erbB2⫹ tumor cells by anti-erbB2
`NK cells. A, Gene modification of primary mouse NK cells with the scFv
`chimeric anti-erbB2 receptor enhanced killing of RMA-erbB2 target cells
`compared with GFP-NK cells. B, Anti-erbB2 NK and GFP-NK cells equiv-
`alently killed RMA parental cells. C, Gene modification of primary mouse
`NK cells with the scFv chimeric anti-erbB2 receptor enhanced killing of
`B16-F10-erbB2 target cells compared with GFP-NK cells. D, Anti-erbB2
`NK and GFP-NK cells equivalently killed B16-F10 parental cells. E, Anti-
`erbB2 NK, GFP-NK, or untransfected cells equivalently killed the NK cell
`sensitive target YAC-1. (ⴱ, p ⬍ 0.05, as determined by a Mann-Whitney U
`test). Results are expressed as average ⫾ SEM of triplicates from three
`independent experiments.
`
`UPenn Ex. 2067
`Miltenyi v. UPenn
`IPR2022-00855
`Page 3452
`
`

`

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`
`The Journal of Immunology
`
`3453
`
`doses of anti-erbB2-NK significantly enhanced the survival of
`mice (⬃35% mice tumor-free) compared with previous experi-
`ments involving two injections of gene-modified NK cells (Fig. 8).
`Mice that received control GFP-NK cells or RMA-erbB2 tumor
`alone rapidly succumbed to disease. Collectively, this data dem-
`onstrated for the first time that adoptive transfer of gene-modified
`primary mouse NK cells could mediate an effective Ag-specific
`tumor response in vivo.
`
`Discussion
`The use of NK cells for cancer immunotherapy is gaining much
`attention. The most promising developments have come from the
`transfer of allogeneic NK cells in the allogeneic transplant stem
`cell setting (34). Recent results have demonstrated that treatment
`of acute myeloid leukemia patients with alloreactive NK cells
`could substantially increase their survival without associated graft-
`vs-host effects (14). Nevertheless, improvements in the use of al-
`loreactive NK cells are required given that these cells had no ap-
`parent effect in patients with acute lymphoid leukemia (14). Other
`therapies involving the transfer of IL-2-activated LAK cells have
`shown only modest anti-tumor effects in patients. This is due in
`part to their nonspecific nature and to HLA-mediated inhibitory
`signals induced by interaction with NK cell inhibitory receptors
`(9). A novel way to overcome these problems and enhance NK cell
`anti-tumor activity involves their genetic modification with scFv
`chimeric receptors that can specifically recognize TAA. To test
`this we used the Amaxa transfection system to genetically engineer
`primary mouse NK cells with a chimeric scFv receptor with spe-
`cific recognition for the erbB2 TAA. Importantly, the expression
`of the chimeric receptor in mouse NK cells did not interfere with
`their natural cytotoxic capability against NK-sensitive target cells.
`We demonstrated that NK cells engineered with the scFv anti-
`erbB2 receptor could significantly enhance killing of an essentially
`NK-insensitive lymphoma cell line in an erbB2⫹ Ag-specific man-
`ner. Furthermore, for the first time, we demonstrated that adoptive
`transfer of receptor-modified primary mouse NK cells could spe-
`cifically enhance the survival of tumor-bearing mice.
`A number of studies have shown that gene modification of var-
`ious mouse and human NK cell lines with scFv chimeric receptors
`could specifically enhance their anti-tumor activity in vitro (25, 27,
`35, 36). Another report demonstrated that human primary NK cells
`expressing an anti-CD19 scFv receptor could specifically kill
`CD19⫹ leukemic cells (28). Nevertheless, the ability of primary
`NK cells to mediate Ag-specific anti-tumor effects in vivo has not
`been formally tested. This has been due to difficulties in using
`retroviral-based approaches to efficiently express chimeric scFv
`receptors in NK cells, which is particularly the case for primary
`mouse NK cells. In our study, we were able to demonstrate proof
`of principle that adoptively transferred gene-modified primary
`mouse NK cells could specifically mediate anti-tumor inhibition in
`vivo. Indeed, mice treated with four doses of anti-erbB2-NK cells
`resulted in ⬃35% long term survivors. Nevertheless, these exper-
`iments were performed in RAG-1⫺/⫺ mice where the presence of
`endogenous NK cells may have competed for important growth
`factors and cytokines limiting both the persistence and activity of
`gene-modified NK cells. Persistence could potentially be improved
`by a non-myeloablative conditioning regimen before adoptive NK
`cell transfer to produce a conducive cytokine environment. This
`type of approach has been demonstrated to enhance the therapeutic
`efficacy of adoptively transferred T cells in both mouse models and
`in patients (37– 41). Alternatively, in future experiments, the use of
`⫺/⫺ recipient mice (that also lack NK cells) may overcome
`RAG␥
`c
`this problem in an experimental setting and result in increased
`
`RMA-erbB2 +
`Anti-erbB2 NK
`RMA-erbB2
`alone
`RMA +
`Anti-erbB2-NK
`RMA-erbB2 +
`GFP-NK
`5
`
`10
`
`*
`
`15
`
`20
`
`25
`
`30
`
`*
`
`20
`15
`10
`5
`Days after tumor inoculation
`
`25
`
`A
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`B
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`0
`
`Survival(%)
`
`FIGURE 7. Enhanced survival of tumor-bearing mice after adoptive
`transfer of ␣-erbB2 NK cells. Groups of 5–10 mice were injected i.p. with
`2 ⫻ 105 RMA-erbB2 tumor alone (closed squares) or injected with RMA-
`erbB2 tumor and treated with two doses of 2 ⫻ 106 ␣-erbB2-NK cells
`(closed triangles) or GFP-NK cells (open triangles) on days 0 and 1 (A) or
`days 3 and 4 (B). Mice bearing RMA tumor were treated with 2 ⫻ 106
`␣-erbB2-NK cells (open squares). Mice bearing RMA-erbB2 tumor and
`treated with ␣-erbB2 NK cells showed significantly increased survival
`compared with mice treated with control GFP NK cells (ⴱ, p ⬍ 0.05, as
`determined by a Mann-Whitney U test). Results shown are representative
`of two experiments performed. Arrows indicated days of NK cell transfer.
`
`6-PTPRCa mice. In these experiments we could not detect signif-
`icant persistence of these cells 7 days post transfer in recipient
`mice (data not shown). To determine whether increasing the num-
`ber of doses of anti-erbB2-NK cells could enhance the anti-tumor
`response, RAG-1⫺/⫺ recipient mice bearing RMA-erbB2 tumor
`were injected i.p with 2 ⫻ 106 anti-erbB2-NK or GFP-NK cells on
`days 0, 1, 2, 3. We demonstrated that increasing the number of
`
`RMA-erbB2+ Anti-
`erbB2-NK
`RMA-erbB2+ GFP-NK
`RMA-erbB2 alone
`
`*
`
` 40
`30
`20
`10
`
`Days after tumor inoculation
`
`50
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`Survival(%)
`
`Increased transfer of ␣-erbB2 NK cells leads to tumor free
`FIGURE 8.
`survival of mice. Groups of six mice were injected i.p. with 2 ⫻ 105
`RMA-erbB2 tumor alone (closed squares) or injected with RMA-erbB2
`tumor and treated with four doses of 2 ⫻ 106 ␣-erbB2-NK cells (closed
`triangles) or GFP-NK cells (open triangles) on days 0, 1, 2, and 3. Mice
`bearing RMA-erbB2 tumor and treated with four doses of ␣-erbB2 NK
`cells showed significantly increased survival compared with mice treated
`with control GFP NK cells (ⴱ, p ⬍ 0.05, as determined by a Mann-Whitney
`U test). Arrows indicated days of NK cell transfer.
`
`UPenn Ex. 2067
`Miltenyi v. UPenn
`IPR2022-00855
`Page 3453
`
`

`

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`
`3454
`
`ENHANCED TUMOR INHIBITION BY GENE-MODIFIED NK CELLS
`
`persistence of gene-modified NK cells. The combination of lym-
`phodepletion and CD34⫹ hematopoietic stem cells has shown to
`further enhance the activity of transferred T cells (42). Thus, it
`would be of interest in future studies to test whether these types of
`regimens can similarly increase persistence and anti-tumor effects
`of transferred gene-modified NK cells.
`The anti-tumor effects observed with adoptive transfer of LAK
`cells in melanoma patients have been dependent on coadministra-
`tion of high dose IL-2 (43). However, in our mouse model coad-
`ministration of high dose IL-2 did not improve the anti-tumor ef-
`fects by gene-modified NK cells. There have been reports that
`other cytokines such as IL-15 are required for the persistence of
`NK cells (44, 45). Thus, it would be interesting to test in our model
`whether cytokines such as IL-15 may be of some benefit.
`To achieve receptor expression in this study we used a non-viral
`vector system, which has attractive safety aspects compared with
`viral-based systems, particularly when considering clinical appli-
`cations. In addition, expression levels using this method, although
`high, were largely transient, lasting ⬃72 h. This provides added
`safety by reducing the risk of long-term autoimmunity associated
`with prolonged presence of potentially autoreactive cells. Never-
`theless, improvements in safety and efficiency of gene transfer
`technology for primary mouse NK cells may lead to increased
`anti-tumor effects. A recent report using lentivirus transduction
`demonstrated long term and stable expression of GFP in mouse
`NK cells in vitro without affecting NK cell phenotype and function
`(46). It will be of interest in future experiments to test whether the
`use of lentiviral vectors can maintain stable