Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting.

BACKGROUND: On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors. METHODS: Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. RESULTS: Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals. CONCLUSIONS: These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance.

NK-92: an 'off-the-shelf therapeutic' for adoptive natural killer cell-based cancer immunotherapy.

Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-versus-host disease. Efficacy and clinical in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clinical NK cell lots to allow repeated clinical applications remains an additional challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufacturing practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purification methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a number of tumor types tested, which was confirmed in preclinical mouse studies. Subsequent clinical testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a number of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.

Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.

Treatment of patients with advanced cancer with the natural killer cell line NK-92.

BACKGROUND AIMS: Natural killer (NK) cells, either naive or genetically engineered, are increasingly considered for cellular therapy of patients with malignancies. When using NK cells from peripheral blood, the number of expanded NK cells can be highly variable and the need for NK cell enrichment can make the process expensive. The NK-92 cell line (CD56+/CD3-) that was isolated from a patient with lymphoma has predictable high cytotoxic activity and can be expanded under good manufacturing practice conditions in recombinant interleukin-2. METHODS: Fifteen patients (age, 9-71 years) with advanced, treatment-resistant malignancies, either solid tumors/sarcomas (n = 13) or leukemia/lymphoma (n = 2), received two infusions of NK-92 cells, given 48 h apart. Three cohorts of patients were treated with escalating doses of NK-92 cells (n = 7 at 1 × 10(9), n = 6 at 3 × 10(9) and n = 2 at 1 × 10(10) cells/m(2)). RESULTS: No infusion-related or long-term side effects were observed. The dose of 10(10) cells/m(2) was considered the maximum expandable cell dose with the use of an established culture bag system. Three fourths of patients with lung cancer had some anti-tumor response. Only one patient of seven had development of human leukocyte antigen antibodies. The persistence of NK-92 cells (male origin) in the circulation was confirmed by Y chromosome-specific polymerase chain reaction in two female patients. CONCLUSIONS: Infusions of NK-92 cells up to 10(10) cells/m(2) were well tolerated. Despite the allogeneic nature of NK-92, development of human leukocyte antigen antibodies in these patients with cancer appears to be rare. The cells can persist in the recipient's circulation for at least 48 h. Some encouraging responses were seen in patients with advanced lung cancer.

Cellular therapy of cancer with natural killer cells-where do we stand?

Although T-lymphocytes have received most of the attention in immunotherapy trials, new discoveries around natural killer (NK) cells suggest that they also should be suitable effector cells for cellular therapy of cancer. In addition to direct cytotoxicity, NK cells produce an array of immune-active cytokines, among them interferons and granulocyte-macrophage colony-stimulating factor, which places them at the crossroads of innate and adaptive immunity. They also augment monoclonal antibody activity through antibody-mediated cellular cytotoxicity and can be transfected with chimeric antigen receptors. One of the stumbling blocks for NK cell-based therapies has been the inability to predictably obtain and expand larger numbers from donors, but also to achieve sufficiently high transfection efficiency of target genes. The first clinical trials with NK cells suggest some benefit, but more definite evidence is needed to justify this relatively expensive treatment.

Detection and characterization of syndecan-1-associated heparan sulfate 6-O-sulfated motifs overexpressed in multiple myeloma cells using single chain antibody variable fragments.

This study was undertaken to generate human single chain variable antibody fragments (scFvs) reacting specifically against multiple myeloma (MM) cells using the phage display technique. To isolate myeloma-specific scFvs, we used a simple subtractive strategy by adsorbing the Griffin #1 antibody phage library against myeloma cells in the presence of excess decoy biotinylated HL60 cells, and then removing the unwanted decoy cells using streptavidin coated plates. From eleven scFvs that were isolated, two antibodies, D4A4 and D6B10 stained MM cell lines and patient MM cells with higher intensity than normal plasma cells. Both D4A4 and D6B10 scFvs immunoprecipitated syndecan-1 from myeloma cells and recognized sulfated motifs on syndecan-1-associated heparan sulfate (HS) chains. ScFv D4A4 competed with D6B10 for binding to MM cells. However, they differed in their fine specificities. ScFv D6B10 recognized HS 2,6-O-, N-sulfated motifs and, in contrast, binding of scFv D4A4 required N-sulfation combined with either 2-O- or 6-O-sulfation. Increased D6B10 binding on MM cells suggests that their HS chains contain a greater number of 2,6-O-, N-sulfated motifs than normal plasma cells. Since these highly sulfated motifs bind various angiogenic and growth factors and present them to their respective receptors, they could be instrumental for MM cell survival, proliferation and metastasis. Therefore, scFvs D4A4 and D6B10 provide a means to easily monitor changes in sulfation patterns of heparan sulfate during myeloma tumor progression.

Clinicopathological significance of major histocompatibility complex class I-related chain a and B expression in thyroid cancer.

CONTEXT: Major histocompatibility complex class I-related chains A and B (MICA/B) are two stress-inducible ligands for the immunoreceptor NKG2D that is expressed on cytotoxic T cells and natural killer (NK) cells. It is not known whether MICA/B expression is up-regulated in thyroid cancer as a result of oncogene activation. OBJECTIVE: The objective of the investigation was to study MICA/B expression and regulation in thyroid cancer and its role in mediating the cytotoxicity of NK cells. METHODS: MICA/B expression in thyroid cancer was analyzed by immunohistochemical staining. Cell surface MICA/B levels in thyroid tumor cell lines and fresh tumor cells were analyzed by flow cytometric analysis. The susceptibility of thyroid tumor cells to NK cell killing was tested by using (51)Cr release assay. RESULTS: MICA/B was expressed at moderate or high levels in 18 of 39 papillary thyroid carcinomas and four of eight anaplastic thyroid carcinomas. MICA/B expression was confirmed by flow cytometric analysis in three fresh thyroid neoplasms. MICA/B expression was detected in eight of 10 thyroid tumor cell lines and correlated with their sensitivity to killing by the NKG2/D-positive NK-92 cells. Blocking of NKG2D and MICA/B interaction by specific antibodies partially led to the inhibition of NK-92 cell-mediated cytotoxicity. The MAPK inhibitors were able to block MICA/B expression in MRO87 and HeLa cells. Transient transfection of mutant BRAF and RAS oncogenes led to increased MICA/B expression in 293 cells and WRO82 cells. CONCLUSION: MICA/B expression is up-regulated in thyroid cancer, probably due to the activation of the MAPK pathway. MICA/B in thyroid cancer plays an important role in NK-92 cell-mediated cytotoxicity.

Natural killer cells: can they be useful as adoptive immunotherapy for cancer?

As part of the innate immune system, natural killer (NK) cells form the first line of defence against pathogens or transformed/cancerous host cells. Recent experimental and clinical data show the possibility of exploiting NK activity as a cell-based immunotherapy to treat cancer. This review discusses the recent knowledge on NK cell biology that has impacted on its development as a treatment for cancer.

Identification of CD19 and CD20 peptides for induction of antigen-specific CTLs against B-cell malignancies.

The purpose of these studies was to develop immunogenic peptides derived from the CD19 and CD20 self-antigens for the induction of antigen-specific CTLs against B-cell malignancies. A total of seven peptides were designed and examined for their HLA-A2.1 affinity and immunogenicity. Of these peptides, we identified two highly immunogenic HLA-A2.1-specific peptides, CD19(150-158) (KLMSPKLYV) and CD20(188-196) (SLFLGILSV), which were capable of inducing peptide-specific CTLs. The CTLs displayed HLA-A2.1-restricted and antigen-specific cytotoxicity against Burkitt's lymphoma, chronic B cell leukemia, and multiple myeloma cell lines. The CD19 or CD20 peptide-specific CTL cytotoxicity was confirmed using HLA-A2.1(+) T2 cells presenting the appropriate peptide. No cytotoxic activity was observed against T2 cells presenting the irrelevant MAGE-3 peptide or T2 cells alone. In addition, the CTLs displayed a significant (P < 0.05) increase in cell proliferation and IFN-gamma secretion (>830 ng/mL) following restimulation with HLA-A2.1(+)/CD19(+)/CD20(+) tumor cells. The CTLs also displayed a distinct phenotype consisting of a high percentage of CD69(+)/CD45RO(+) and a low percentage of CD45RA(+)/CCR7(+) CD4(+) or CD8(+) T cells characteristic of effector memory cell population. Cyclic guanosine 3',5'-monophosphate culture conditions using serum-free AIM-V medium containing human AB serum, recombinant human interleukin 2 (Proleukin) and CD3/CD28 Dynabeads were developed resulting in a 35-fold expansion of CD20 peptide-specific CTLs. The expanded CD20-CTLs retained their cytotoxic activity (28-49%) against the Burkitt's lymphoma cell line. In conclusion, we report here on the identification of novel immunogenic CD19(150-158) (KLMSPKLYV) and CD20(188-196) (SLFLGILSV) peptides that have immunotherapeutic potentials as peptide vaccines or targeted T-cell therapies for treating B-cell malignancies.

Allogeneic hematopoietic stem-cell transplantation with reduced-intensity conditioning in intermediate- or high-risk patients with myelofibrosis with myeloid metaplasia.

A total of 21 patients with myelofibrosis with myeloid metaplasia (MMM), with a median age of 54 years (range, 27-68 years), were prepared with a reduced-intensity conditioning (RIC) regimen. The patients received an allogeneic marrow (n = 3) or peripheral blood stem-cell (n = 18) transplant from HLA-matched related (n = 18) or unrelated (n = 2), or 1 Ag-mismatched related (n = 1), donors. RIC regimens included fludarabine/total body irradiation 200 cGy (n = 5) or 450 cGy (n = 1), fludarabine/melphalan (n = 7), thiotepa/cyclophosphamide (n = 7), and thiotepa/fludarabine (n = 1). At the time of transplantation, all of the patients were at intermediate (n = 13) or high (n = 8) risk, according to the Dupriez classification. Of the patients, 19 had grade III or IV marrow fibrosis. All of the patients achieved full engraftment but one. Posttransplantation chimerism analysis showed more than 95% donor cells in 18 patients, while 2 patients achieved complete donor chimerism after donor leukocyte infusion (DLI). Acute graft-versus-host disease (GVHD) grades II to IV was observed in 7 patients, grades III to IV in 2, and extensive chronic GVHD in 8 of 18 evaluable patients. There were 3 patients who died from acute GVHD, infection, and relapse. There are 18 patients alive 12 to 122 months (median, 31 months) after transplantation, and 17 are in remission (1 after a second transplantation). The use of RIC regimens in allogeneic stem cell transplantation results in prolonged survival in intermediate/high-risk MMM patients.

Heteroclitic CD33 peptide with enhanced anti-acute myeloid leukemic immunogenicity.

The goal of these studies was to engineer a synthetic CD33 peptide with enhanced immunogenicity for the induction of acute myeloid leukemia (AML)-specific CTLs. Eight modified CD33 peptides YLISGDSPV, YIGSGDSPV, YIIIGDSPV, YIILGDSPV, YIISGISPV, YIISGDLPV, YIISGDSWV and YIISGDSPL were designed for increased HLA-A2.1 or T cell receptor affinity and compared with the native CD33(65-73) peptide, AIISGDSPV, for enhanced immunogenicity. The YLISGDSPV peptide was found to be the most immunogenic epitope producing highly cytolytic CTLs against AML target cells. The CTLs generated withYLISGDSPV peptide showed CD33 peptide-specificity through targeting of both native (AIISGDSPV) and modified (YLISGDSPV) peptide presenting EBV-BLCL. The CTL cultures displayed a distinct phenotype consisting of a high percentage of activated memory (CD69(+)/CD45RO(+))-CD8(+)and a low percentage of naive (CD45RA(+)/CCR7(+))-CD8(+)cells. In addition, T-cell clones specific to the YLISGDSPV peptide were isolated and characterized to target AML cells. The clones exhibited both HLA-A2.1-restricted and AML cell-specific cytotoxicity that was mediated through a granule-dependent pathway. More importantly, the CTL clones did not lyse or inhibit the proliferation of normal CD34(+) progenitor cells. In conclusion, we report on the identification of a highly immunogenic heteroclitic YLISGDSPV CD33 epitope that is a promising candidate for immunotherapy targeting AML.

The effect of LIGHT in inducing maturation of monocyte-derived dendritic cells from MDS patients.

LIGHT is a recently cloned novel cytokine belonging to the TNF family that is selectively expressed on immature dendritic cells (iDCs) generated from monocytes isolated from human PBMCs. In these studies, we demonstrate that exogenous soluble LIGHT or soluble CD40 ligand (CD40L) can promote monocyte-derived dendritic cell maturation in vitro by the up-regulation of CD86, CD80, CD83, and HLA-DR antigen expression. Immature dendritic cells differentiated from monocytes of MDS patients displayed lower levels of costimulatory and HLA-DR molecules compared with iDCs differentiated from monocytes of normal subjects. However, upon induction of maturation by LIGHT or CD40L, the expression of costimulatory and HLA-DR molecules is comparable between DCs from MDS and normal subjects. Exogenous LIGHT- and CD40L-stimulated mature DCs (mDCs) also displayed increased antigen presentation to autologous T lymphocytes (tetanus toxin) or allogeneic T lymphocytes in mixed lymphocyte reactions. DCs matured by LIGHT showed increased secretion of IL-6, IL-12p75, and TNF-alpha, but not IL-1beta. We conclude that both LIGHT and CD40L are immunoregulating factors that induce monocyte-derived iDCs from MDS patients to undergo maturation resulting in increased antigen presentation and T-cell activation. Monocyte-derived DCs can be stimulated to undergo phenotypic and functional changes with LIGHT that might be applied in the development of a DC-based vaccine for MDS treatment.

Identification of novel CD33 antigen-specific peptides for the generation of cytotoxic T lymphocytes against acute myeloid leukemia.

Identification of immunogenic peptides for the generation of cytotoxic T lymphocytes (CTLs) may lead to the development of novel cellular therapies to treat disease relapse in acute myeloid leukemia (AML) patients. The objective of these studies was to evaluate the ability of unique HLA-A2.1-specific nonameric peptides derived from CD33 antigen to generate AML-specific CTLs ex vivo. We present data here on the identification of an immunogeneic HLA-A2.1-specific CD33(65-73) peptide (AIISGDSPV) that was capable of inducing CTLs targeted to AML cells. The CD33-CTLs displayed HLA-A2.1-restricted cytotoxicity against both mononuclear cells from AML patients and the AML cell line. The peptide- as well as AML cell-specificity of CD33-CTLs was demonstrated and the secretion of IFN-gamma by the CTLs was detected in response to CD33(65-73) peptide stimulation. The cultures contained a distinct CD33(65-73) peptide-tetramer(+)/CD8(+) population. Alteration of the native CD33(65-73) peptide at the first amino acid residue from alanine (A) to tyrosine (Y) enhanced the HLA-A2.1 affinity/stability of the modified CD33 peptide (YIISGDSPV) and induced CTLs with increased cytotoxicity against AML cells. These data therefore demonstrate the potential of using immunogenic HLA-A2.1-specific CD33 peptides in developing a cellular immunotherapy for the treatment of AML patients.

Use of the anti-idiotype breast cancer vaccine 11D10 in conjunction with autologous stem cell transplantation in patients with metastatic breast cancer.

The results of cytotoxic therapy, including dose-intensive therapy requiring autologous stem cell transplantation (ASCT), have been disappointing in patients with metastatic breast cancer, as almost all patients eventually experience disease progression. There has been a renewed interest in immunotherapeutic strategies in this disease, including evaluation of several breast cancer vaccines. In the current study, we describe the results of a program in which the anti-idiotype breast cancer vaccine 11D10 (TriAb) was administered before and after ASCT in patients with metastatic breast cancer chemosensitive to previous conventional therapy. The toxicity of this approach was acceptable, and idiotype-specific humoral and T-cell proliferative responses were observed in the majority of patients within a few weeks post-ASCT. The actuarial 3-year overall survival rate was 48% (95% CI, 32%-64%), while the progression-free survival rate was 32% (95% CI, 19%-45%). Multivariate analysis identified achievement of a strong antibody and cellular immune response to the vaccine as the only significant prognostic factors for outcome. The ability to reliably produce robust immune responses after ASCT is encouraging. Further studies are required to determine if the immune response mediates an antitumor benefit in these patients.

Role of immunotherapy in stem cell transplantation.

Relapse of the underlying malignancy continues to be a major problem after both autologous and allogeneic stem cell transplantation. Over the years, it has been recognized that immune-mediated graft-versus-tumor effects are crucially involved in eliminating minimal disease and controlling its recurrence after stem cell transplantation. This recognition has led to a number of studies that have attempted to stimulate a cellular immune response in the recipient, especially after allogeneic transplantation. Immunotherapy after autologous transplantation has to take into consideration the fact that patients' immune cells frequently are compromised and tolerance to the host tumor may have developed. Hence, trials involving the administration of cytokines (such as with interleukin and interferon) have shown limited benefits. This situation is different for allogeneic transplantation for which the infusion of donor lymphocytes has shown disease regression, especially in patients with chronic leukemias. However, such treatment is effective only if the patient has limited disease, and severe graft-versus-host disease frequently has to be accepted as a complication. This fact has led investigators to pursue the generation of specific lymphocytes that can recognize tumor antigens but not necessarily induce graft-versus-host disease. Such studies are in the early stages, and although some promising results have been observed, it is unclear at this point if the antitumor effect can be separated sufficiently from the graft-versus-host disease mediated by allogeneic lymphocytes. More recently, it has been shown that natural killer (NK) cells can have an antitumor effect in myeloid malignancies, particularly if the cells are allogeneic and do not recognize self-HLA antigens. At this point, it appears that engineered T-lymphocytes and allogeneic NK cells may be useful in preventing or treating relapse after allogeneic transplantation. It remains to be seen if such novel cellular therapies can also be implemented after autologous transplantation via the use of engineered allogeneic immune cells.

Hematopoietic stem cell transplantation: bone marrow vs. mobilized peripheral blood.

Peripheral blood stem cells have largely replaced bone marrow as the source of cells in autologous transplantation because of more rapid neutrophil and platelet recovery and faster immune reconstitution. Allogeneic peripheral blood stem cells similarly lead to faster hematologic recovery: however, their effects on graft-vs.-host disease, relapse, survival, and immune reconstitution have been less certain. Eight randomized trials have been published to date comparing the clinical outcomes of allogeneic-related donor bone marrow transplantation (BMT) vs. PBSCT and will be reviewed. In addition, comparisons between the two stem cell sources in unrelated donor transplantation and the increasingly utilized nonmyeloablative transplantation will be discussed.