Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells

Despite the clinical success of CD20-specific antibody rituximab, malignancies of B-cell origin continue to present a major clinical challenge, in part due to an inability of the antibody to activate antibody-dependent cell-mediated cytotoxicity (ADCC) in some patients, and development of resistance in others. Expression of chimeric antigen receptors in effector cells operative in ADCC might allow to bypass insufficient activation via FcγRIII and other resistance mechanisms that limit natural killer (NK)-cell activity. Here we have generated genetically modified NK cells carrying a chimeric antigen receptor that consists of a CD20-specific scFv antibody fragment, via a flexible hinge region connected to the CD3ζ chain as a signaling moiety. As effector cells we employed continuously growing, clinically applicable human NK-92 cells. While activity of the retargeted NK-92 against CD20-negative targets remained unchanged, the gene modified NK cells displayed markedly enhanced cytotoxicity toward NK-sensitive CD20 expressing cells. Importantly, in contrast to parental NK-92, CD20-specific NK cells efficiently lysed CD20 expressing but otherwise NK-resistant established and primary lymphoma and leukemia cells, demonstrating that this strategy can overcome NK-cell resistance and might be suitable for the development of effective cell-based therapeutics for the treatment of B-cell malignancies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The human natural killer cytotoxic cell line NK-92, once armed with a murine CD16 receptor,represents a convenient cellular tool for the screening of mouse mAbs according to their ADCC potential

To take advantage of the large number of well-characterized mouse immunoglobulins (IgGs) for the study of antibody-dependent cell-mediated cytotoxicity (ADCC) in human cells, we armed human cytotoxic lymphocytes with a mouse receptor for the Fc portion of IgG antibodies. The human ΝΚ−92 natural killer cell line was transduced with a mouse receptor gene (mCD16), which was stably expressed on the cell surface (referred to as NK-92^mCD16). When tested against a B-lymphoblastoid cell line (BLCL) coated with mouse anti-CD20 IgG1, IgG2a or IgG2b monoclonal antibodies (mAbs), the newly expressed mouse Fc receptor enabled the NK-92^mCD16 cells to kill the BLCL by ADCC. Next, using the NK-92^mCD16 we compared mouse mAbs directed at B lineage specific CD antigens for their ability to induce ADCC against human Epstein-Barr virus- infected B lymphoblastoid (for anti-CD19, -CD20 and -CD21) or against myeloma (for anti-CD38 and –CD138) target cells. Our results demonstrated that the “NK-92^mCD16 assay” allows convenient and sensitive discrimination of mouse mAbs for their ability to mediate ADCC in a human cellular system. In addition, our results provide examples of dissociation between opsonization and target cell killing through ADCC. These “murinized” human effector cells thus represent a convenient cellular tool for the study of ADCC.     

Resolution of adhesion- and activation-associated components of monoclonal antibody-dependent human NK cell-mediated cytotoxicity.

Flow cytometry was used to investigate two functional parameters of human natural-killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC): (i) the frequency of NK cells which formed conjugates (NKC) with autologous monoclonal antibody (mAb)-coated lymphocyte target cells, a measure of the avidity of CD16-dependent cell-cell adhesion, and (ii) the rise in the intracellular concentration of ionized calcium ([Ca2+]i) elicited in NKC by contact with target cells, a measure of CD16-dependent NK cell activation. For each of four rat IgG2b mAb directed against target cell antigens CDw52, CD5, CD45, and class I HLA, there existed quantitatively similar relationships between ADCC and rise in NKC[Ca2+]i but significant inter-mAb differences with respect to the ADCC vs the NKC frequency relationship. Cytolytic efficiencies of mAb appeared to be determined at the level of the NK cell, dependent upon CD16 and LFA-1, but restricted with respect to quantitative levels of NKC[Ca2+]i. In concert with this notion, targets coated with an IgG1 isotype-switch variant alpha CDw52 mAb promoted significant conjugate formation but failed to elicit a rise in NKC[Ca2+]i or ADCC. Thus, Fc regions of antibodies make contacts with NK cell CD16 which may strengthen cell-cell adhesion without eliciting an activation stimulus, a finding which supports a complexity of CD16 functional regulation of probable significance in the clinical consequences of antibody responses or therapeutic mAb manipulations.     

Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.     

Compromised NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Chronic SIV/SHIV Infection

Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to the control of HIV/SIV infection. However, little is known about the ADCC function of natural killer (NK) cells in non-human primate model. Here we demonstrated that ADCC function of NK cells was significantly compromised in chronic SIV/SHIV infection, correlating closely with the expression of FcγRIIIa receptor (CD16) on NK cells. CD32, another class of IgG Fc receptors, was identified on NK cells with higher expression in the infected macaques and the blockade of CD32 impacted the ability of NK cells to respond to antibody-coated target cells. The inhibition of matrix metalloproteases (MMPs), a group of enzymes normally involved in tissue/receptor remodeling, could restore NK cell-mediated ADCC with increased CD16 expression on macaque NK cells. These data offer a clearer understanding of NK cell-mediated ADCC in rhesus macaques, which will allow us to evaluate the ADCC repertoire arising from preclinical vaccination studies in non-human primates and inform us in the future design of effective HIV vaccination strategies.     

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.     

Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner

Integrin engagement on lymphocytes initiates “outside-in” signaling that is required for cytoskeleton remodeling and the formation of the synaptic interface. However, the mechanism by which the “outside-in” signal contributes to receptor-mediated intracellular signaling that regulates the kinetics of granule delivery and efficiency of cytolytic activity is not well understood. We have found that variations in ICAM-1 expression on tumor cells influence killing kinetics of these cells by CD16.NK-92 cytolytic effectors suggesting that changes in integrin ligation on the effector cells regulate the kinetics of cytolytic activity by the effector cells. To understand how variations of the integrin receptor ligation may alter cytolytic activity of CD16.NK-92 cells, we analyzed molecular events at the contact area of these cells exposed to planar lipid bilayers that display integrin ligands at different densities and activating CD16-specific antibodies. Changes in the extent of integrin ligation on CD16.NK-92 cells at the cell/bilayer interface revealed that the integrin signal influences the size and the dynamics of activating receptor microclusters in a Pyk2-dependent manner. Integrin-mediated changes of the intracellular signaling significantly affected the kinetics of degranulation of CD16.NK-92 cells providing evidence that integrins regulate the rate of target cell destruction in antibody-dependent cell cytotoxicity (ADCC).     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Killing of human tumor cell lines by human monocytes and murine monoclonal antibodies

We evaluated the capacity of freshly isolated blood monocytes to mediate antibody-dependent cellular-mediated cytotoxicity (ADCC) in cooperation with murine anti-tumor monoclonal antibodies (MAbs). Blood monocytes isolated from most donors by adherence selection to fibronectin-coated plastic surfaces and subsequently depleted of natural killer/killer (NK/K) cells exhibited significant ADCC activity against tumor cell lines in combination with an IgG3 antitumor MAb (BR55-2). However, significant variation in ADCC competence was observed among donors. Culture parameters influencing monocyte ADCC activity were evaluated and optimized. The influence of MAb isotype on ADCC capacity of anti-tumor MAbs was also evaluated using anti-tumor class-switch variant hybridoma proteins and a panel of anti-tumor MAbs. MAbs of the IgG2a and IgG3 subclasses exhibited high ADCC potential, whereas MAbs of the IgG2b subclass exhibited no ADCC activity. One of two IgG1 MAbs tested exhibited high ADCC activity with monocyte effectors. The role of monocytes or macrophages in tumor remission of cancer patients undergoing MAb immunotherapy is not known. However, correlative studies of monocyte ADCC capacity and responsiveness of cancer patients to MAb immunotherapy may help to establish the role of these effectors in MAb-mediated tumor remissions.     

HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART)

Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4⁺ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.     

The NK-92 cell line—30 years later: its impact on natural killer cell research and treatment of cancer

The NK-92 cell line, established in 1992, mirrors all the characteristics of highly active blood natural killer (NK) cells but with much broader and greater cytotoxicity. The cell line was established from the blood cells of a patient with lymphoma and has been made widely available for research since it was deposited into the American Type Culture Collection in 1998. The worldwide distribution of NK-92 cells has led to a plethora of scientific discoveries that have greatly increased the understanding of NK-cell biology. NK-92 cells also have been developed for clinical use, overcoming the challenges of obtaining and expanding NK cells from donor or patient blood. More than 100 patients with cancer have now been treated all over the world with unmodified or genetically engineered NK-92 cells. Modified cells include high-affinity Fc-receptor expressing NK-92 cells (haNK^R) and various chimeric antigen receptor targeted haNK cells (t-haNK^TM). Infusions of either unmodified or modified NK-92 cells have been reported to be safe and efficacious, leading in some cases to disease remission even in patients who had failed multiple previous lines of therapy. It is the purpose of this review to distill the plethora of scientific data on NK-92 cells and its genetic variants, highlighting relevant experimental findings that have contributed to a better understanding of NK cell biology and summarize the therapeutic potential of these cells for treatment of cancer and infections.     

Bispecific minibodies targeting HER2/neu and CD16 exhibit improved tumor lysis when placed in a divalent tumor antigen binding format

Unconjugated monoclonal antibodies have emerged as important therapeutic agents for selected malignancies. One mechanism by which antibodies can exert cytotoxic effects is antibody-dependent cellular cytotoxicity (ADCC). In an effort to increase the efficiency of ADCC at tumor sites, we have focused on the construction of bispecific antibodies specific for the tumor antigen HER2/neu and the Fc gamma RIII-activating receptor (CD16) found on NK cells, mononuclear phagocytes, and neutrophils. Here, we describe the production of bispecific minibodies in two distinct binding formats. The parent minibody was constructed such that the IgG1 C(H)3 constant domain serves as the oligomerization domain and is attached to an anti-CD16 and an anti-HER2/ neu single-chain Fv via 19- and 29-amino acid linkers, respectively. This molecule can be expressed in mammalian cells from a dicistronic vector and has been purified using sequential affinity purification techniques. Analysis by surface plasmon resonance shows that the bispecific minibody can bind to HER2/neu and CD16, both individually and simultaneously. Furthermore, cytotoxicity studies show that the minibody can induce significant tumor cell lysis at a concentration as low as 20 nm. A trimeric, bispecific minibody (TriBi) that binds dimerically to HER2/neu and monomerically to CD16 induces equivalent cytotoxicity at lower antibody concentrations than either the parent minibody or the corresponding single-chain dimer. Both minibody constructs are stable in mouse and human serum for up to 72 h at 37 degrees C. These minibodies have the potential to target solid tumors and promote tumor lysis by natural killer cells and mononuclear phagocytes.     

CD86 Is an Activation Receptor for NK Cell Cytotoxicity against Tumor Cells

CTLA4Ig has been successfully used in the clinic for suppression of T cell activation. However, patients treated with CTLA4Ig experienced reduced incidence of tumors than predicted, but the underlying mechanism remains unknown. In this paper, we showed that brief administration of CTLA4Ig significantly reduced tumor metastasis and prolonged the survival of host mice bearing B16 melanoma. Depletion of NK cells prior to CTLA4Ig administration eliminated the CTLA4Ig-mediated anti-tumor activity. CTLA4Ig enhanced NK cell cytotoxicity to tumor cells via up-regulation of NK cell effecter molecules CD107a and perforin in vivo. In addition, we demonstrated that, upon activation, NK cells could significantly increase the expression of CD86 both in vitro and in vivo, and ligation of CD86 with CTLA4Ig significantly increased the ability of NK cells to kill tumor cells. Furthermore, a human NK cell line that expressed high level of CD86 was directly activated by CTLA4Ig so that killing of tumor targets was enhanced; this enhanced killing could be inhibited by blocking CD86. Our findings uncover a novel function of CTLA4Ig in tumor immunity and suggest that CD86 on NK cells is an activating receptor and closely involved in the CTLA4Ig-mediated anti-tumor response.     

Irradiated chimeric antigen receptor engineered NK-92MI cells show effective cytotoxicity against CD19+ malignancy in a mouse model

Background aimsAnti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells.MethodsNK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19⁺ malignant cells was assessed.ResultsCAR NK exhibited specific cytotoxicity against CD19⁺ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19⁺ malignancy capacity similar to that of unirradiated CAR NK.ConclusionsFive Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.     

Lenalidomide enhances antibody-dependent cellular cytotoxicity of solid tumor cells in vitro: influence of host immune and tumor markers

We evaluated the effect of combining lenalidomide with therapeutic antibodies on antibody-dependant cell-mediated cytotoxicity (ADCC) of solid tumor cells, and the requirement for expression of natural killer (NK) cell-activating receptors and their solid tumor surface ligands. Twenty-three human tumor cell lines (colon, breast, lung, head and neck, ovary, and bone sarcoma) were analyzed. NK effector cells were isolated from healthy donors, pre-treated with and without lenalidomide, and incubated with antibody-coated tumor cells to determine ADCC. In blocking experiments, NK cells were pre-incubated with anti-DNAM-1 or anti-NKG2D antibodies, and target colorectal cells were pre-incubated with anti-CD155 (PVR), anti-MIC-A/B, or anti-ULBP 3 antibodies. Differences between groups were assessed using unpaired and paired Student’s t test and one-way ANOVA. Lenalidomide enhanced NK cell-mediated ADCC of trastuzumab- and cetuximab-coated tumor cells. Activity against colorectal cancer cells was dependent on target antigen expression, but independent of KRAS status and FcγRIIIa genotype. The extent of ADCC and its enhancement by lenalidomide correlated with NK cell expression of NKG2D and DNAM-1, and tumor cell expression of PVR and MIC-A. Blocking of NKG2D and, to a lesser extent, DNAM-1 inhibited ADCC. Anti-MIC-A/B monoclonal antibody blocked natural cytotoxicity, but not ADCC. Lenalidomide enhances the ability of IgG1-isotype antibodies to mediate ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D–NKG2D ligand interactions, but appears to be independent of MIC-A/B. This provides a rationale for exploratory clinical studies and an assessment of potential biomarkers predictive of clinical benefit.     

Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface

The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM(+) (ovarian) and GD2(+) (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.     

Adoptive transfer of gene-modified primary NK cells can specifically inhibit tumor progression in vivo

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-zeta signaling domains (scFv-CD28-zeta). In this study we demonstrated that mouse NK cells gene-modified with this chimera could specifically mediate enhanced killing of an erbB2(+) MHC class I(+) 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-erbB2(+) lymphoma. In summary, these data suggest that use of genetically modified NK cells could broaden the scope of cancer immunotherapy for patients.     

Natural killer (NK) cell subsets in the mouse. NK-1.1+/LGL-1+ cells restricted to lysing NK targets, whereas NK-1.1+/LGL-1- cells generate lymphokine-activated killer cells

Our laboratory has recently identified a novel Ag, LGL-1, that is expressed on a major population of mouse NK cells. Two color immunofluorescence analysis has demonstrated that spleen cells consist of two major subsets of NK cells. We have identified an NK-1.1+/LGL-1+ subset that consists of 50% of the total NK cells and an NK-1.1+/LGL-1- subset comprising the remaining 50%. Because numerous reports have identified NK cells as the major cell type mediating lymphokine-activated killing (LAK), the NK-1.1+/LGL-1+ and NK-1.1+/LGL-1- subsets were examined for their contribution toward LAK generation, as defined by their ability to lyse P815 tumor targets. Antibody plus C depletion experiments with the use of anti-LGL-1 indicated that LGL-1+ cells were not found on LAK precursor or effector cells. Two-color cell sorting experiments were also performed to separate freshly isolated NK-1.1+/LGL-1+ spleen cells from the NK-1.1+/LGL-1- subset. It was found that the vast majority of LAK activity (greater than 95%) is derived from the NK-1.1+/LGL-1- cells. Cell sorting of LAK effectors also demonstrated that the NK-1.1+/LGL-1- cells mediated the vast majority of lysis against P815 targets. Similar results were obtained when NK cell subsets were analyzed for their contribution toward ADCC. These findings may prove important in understanding and further elucidating the contribution of NK cells to the LAK phenomenon. Our data also indicates that subsets of NK cells exist that may function differently in response to stimulation by various lymphokines and cytokines.     

Enhancement of the anti-melanoma response of Hu14.18K322A by αCD40 + CpG

Targeted monoclonal antibodies (mAb) can be used therapeutically for tumors with identifiable antigens such as disialoganglioside GD2, expressed on neuroblastoma and melanoma tumors. Anti-GD2 mAbs (αGD2) can provide clinical benefit in patients with neuroblastoma. An important mechanism of mAb therapy is antibody-dependent cellular cytotoxicity (ADCC). Combinatorial therapeutic strategies can dramatically increase the anti-tumor response elicited by mAbs. We combined a novel αGD2 mAb, hu14.18K322A, with an immunostimulatory regimen of agonist CD40 mAb and class B CpG-ODN 1826 (CpG). Combination immunotherapy was more effective than the single therapeutic components in a syngeneic model of GD2-expressing B16 melanoma with minimal tumor burden. NK cell depletion in B6 mice showed that NK cells were required for the anti-tumor effect; however, anti-tumor responses were also observed in tumor-bearing SCID/beige mice. Thus, NK cell cytotoxicity did not appear to be essential. Peritoneal macrophages from anti-CD40 + CpG-treated mice inhibited tumor cells in vitro in an hu14.18K322A antibody-dependent manner. These data highlight the importance of myeloid cells as potential effectors in immunotherapy regimens utilizing tumor-specific mAb and suggest that further studies are needed to investigate the therapeutic potential of activated myeloid cells and their interaction with NK cells.