Direct and antibody-dependent cell-mediated cytotoxicity of head and neck squamous cell carcinoma cells by high-affinity natural killer cells.

High affinity natural killer cells (haNKs) are a cell therapy product capable of mediating both direct and antibody-dependent cell-mediated cytotoxicity (ADCC). These cells may be particularly useful in tumors that escape T-cell anti-tumor immunity by harboring antigen processing and presentation defects. Here, we demonstrated that haNKs directly kill both HPV-positive and negative head and neck squamous cell carcinoma cells. Variable tumor cell sensitivity to haNK direct cytotoxicity did not correlated with MHC class I chain-related protein A or B (MICA or MICB) expression. Importantly, haNK killing was significantly enhanced via ADCC mediated by cetuximab or avelumab in cells with higher baseline EGFR or PD-L1 expression, respectively. The ability of IFNγ to induce tumor cell PD-L1 expression correlated with enhanced PD-L1-specific ADCC. IFNγ induced neither tumor cell EGFR expression nor EGFR-specific ADCC. Although a single dose of 8 Gy IR did not appear to directly enhance susceptibility to haNK killing alone, enhanced PD-L1- and EGFR-mediated ADCC after IR correlated with increased PD-L1 and EGFR expression in one of four models. This pre-clinical evidence supports the investigation of haNK cellular therapy in combination with ADCC-mediating mAbs, with or without IR, in the clinical trial setting for patients with advanced HNSCCs. Given the MHC-unrestricted nature of this treatment, it may represent an opportunity to treat patients with non-T-cell inflamed tumors.

Expanding the use of monoclonal antibody therapy of cancer by using ionising radiation to upregulate antibody targets.

BACKGROUND: Monoclonal antibody (mAb) therapy for the treatment of solid and haematologic malignancies has shown poor response rates as a monotherapy. Furthermore, its use is limited to tumours expressing certain molecular targets. It has been shown that single-dose radiation can induce immunogenic modulation that is characterised by cell-surface phenotypic changes leading to augmented tumour cell/cytotoxic T-cell interaction. METHODS: We examined radiation's ability to upregulate mAb therapy targets. We also used radiation to sensitise tumour cells to antibody-dependent cell-mediated cytotoxicity (ADCC). RESULTS: Radiation significantly increased cell-surface and total protein expression of mAb targets HER2, EGFR, and CD20. Focusing on HER2, targeted by trastuzumab, we observed significant upregulation of HER2 following radiation of 3 out of 3 breast cancer cell lines, one of which was triple negative, as well as in residential stem-cell populations. HER2 upregulation was sustained up to 96 h following radiation exposure and was largely dependent on intracellular reactive oxygen species. Improved ADCC and sensitisation to the antiproliferative effects of trastuzumab demonstrated the functional significance of radiation-induced HER2 upregulation. CONCLUSIONS: We show that single-dose radiation enhances mAb therapy. These findings highlight a mechanism for combining radiation with immunotherapy and expand the patient population that can be treated with targeted therapy.

Ataxia telangiectasia and Rad3-related overexpressing cancer cells induce prolonged G2 arrest and develop resistance to ionizing radiation.

We investigated whether ataxia telangiectasia and rad3-related (ATR) kinases regulate prolongation of ionizing radiation (IR) induced-G2 arrest and radioresistance in ataxia telangiectasia mutated-intact cancer cells. ATR overexpressing cancer cells showed prolonged-G2 arrest after IR exposure and were significantly resistant to DNA damaging stresses. The phosphorylation of p-Ser¹5-p53, p-Ser³45-Chk1, and p-Tyr¹5-Cdk1 phosphorylation was increased until 36 h after IR exposure in ATR-overexpressing cells, whereas p-Ser¹°-histone H3 decreased. ATR-overexpressing cells also showed rapid attenuation of increased γ-H2AX foci after IR exposure compared with control cells. In contrast, ATR knockdown cells had limited clearance of γ-H2AX foci after IR exposure. In conclusion, ATR overexpression seems to primarily induce prolonged G2 arrest after IR exposure, which increases IR resistance by enhancing DNA damage repair. These results may provide useful clues for understanding the function of ATR in controlling IR-induced G2 arrest and radiation response.

The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators.

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C(H)3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcgamma receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcgamma receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.

Keratinocytes from patients with lupus erythematosus show enhanced cytotoxicity to ultraviolet radiation and to antibody-mediated cytotoxicity.

Keratinocyte cytotoxicity is an important component of the immunopathology of photosensitive lupus erythematosus, and antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to be an important mechanism by which autoantibodies, especially those specific for SS-A/Ro, can induce keratinocyte damage in models of photosensitive lupus. We provide further evidence that keratinocytes from patients with photosensitive lupus show significantly greater ultraviolet radiation (UVR)-induced cytotoxicity, and that ADCC of these targets is especially enhanced by autologous patient's serum or by anti-SS-A/Ro+ sera. Keratinocytes from normal uninvolved skin of 29 patients with cutaneous lupus erythematosus (LE) were grown in cell culture and tested as targets in cytotoxicity experiments in vitro. Cultured keratinocytes from patients with systemic lupus erythematosus (SLE) and subacute cutaneous lupus erythematosus (SCLE) showed significantly greater cytotoxicity following UVR treatment than did keratinocytes from normal adult controls or from neonatal foreskins (P < 0.01). The same cultures also showed greater UVR-induced binding of IgG from fractionated anti-SS-A/Ro+ preparations. ADCC experiments were also performed using keratinocytes cultured from patients with SLE, SCLE, discoid lupus erythematosus (DLE), and normal controls. When keratinocytes were incubated in autologous serum plus a standard mononuclear cell effector population, the percentage of ADCC observed was significantly greater in cultures containing keratinocytes and sera from the SLE and SCLE patients (P < 0.001). When cultured keratinocytes were added to different IgG antibody probes, plus standard mononuclear effector populations, greater ADCC was seen using the anti-SS-A/Ro probe and keratinocytes from patients with SLE or SCLE. With normal human neonatal keratinocyte targets, the anti-SS-A/Ro probe induced greater ADCC than that seen with anti-ssDNA or normal human serum. We have shown that keratinocytes from patients with some forms of lupus erythematosus (SLE and SCLE) show greater cytotoxicity in vitro when irradiated with UVR, and greater susceptibility to ADCC whether the antibody source is their own serum or an anti-SS-A/Ro probe.

[The effect of sodium nucleinate on the precursors of antigen-dependent suppressors following irradiation]. / Vliianie nukleinata natriia na predshestvenniki antigenzavisimykh supressorov posle oblucheniia.

Sodium nucleinate was administered perorally to mice in the dose of 30 mg/kg. After the treatment the suppression activity of spleen cells decreased. The decrease was more pronounced in mice exposed to the doses of 0.5 and 1 Gy than in intact mice. The dose of 240 mg/kg did not affect suppression activity.

[Immune reaction in monkeys to single and fractionated low-dose gamma-irradiation]. / Reaktsiia immunnoi sistemy obezían na odnokratnoe i fraktsionirovannoe gamma-obluchenie v malykh dozakh.

A study was made of total and local immunity of two groups of Papio hamadryads subjected to single and ten-fold external gamma-irradiation at a cumulative dose of 5 cGy. It has been shown that with equal dosages changes in the relative and absolute number of lymphocytes in the peripheral blood, the number of circulating T-cells and their functional activity are more pronounced in animals subjected to fractionated irradiation. Both groups exhibited similar disturbances in the functional activity of antibody-dependent killers and in local immunity of stomatopharynx. Analysis of the immunological data and the results of hydrocortisone content determinations in blood serum of exposed animals has demonstrated the presence of both direct effect of low-level radiation on the immune system and indirect effect that is particularly pronounced in case of multifraction long-term irradiation.

[The biological effects in animals in relation to the accident at the Chernobyl Atomic Electric Power Station. 8. The status of cellular immunity in different generations of rats]. / Biologicheskie éffekty u zhivotnykh v sviazi s avariei na Chernobyl'skoi AES. Soobshchenie 8. Sostoianie keltochnogo immuniteta u raznykh pokolenii krys.

In studying immunity in laboratory rats of different generations (P, F1 and F2) brought up in Chernobyl in 1989-1990 the authors have revealed the development of leuko- and lymphopenia; decrease in the absolute content of immunocompetent cells bearing Fc receptors to IgG; stable and long-lasting suppression of blood NK cell activity; reduction of antibody-dependent cytotoxicity; and changed ability of blood lymphocytes to interact contactly with allogenic mast cells. The most considerable disorders have been found in 6- and 9-month-old F1 rats and in 3- and 6-month-old F2 rats.

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to gamma-irradiation.

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. We have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to gamma-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-gamma (rmIFN-gamma) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by gamma-irradiation. Concomitant priming of gamma-irradiated J774 M phi with rmIFN-gamma increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC. Irradiated J774 cells will also provide a homogenous, stably primed cell type in which to examine the mechanism(s) of cytotoxicity employed by tumoricidal M phi.

X-linked codominant genes control all types of non-major histocompatibility complex-restricted cytotoxicity.

In most individuals, natural killer (NK) activity is abolished after lymphocyte irradiation with 3,000 cGy, while lymphocytes from a minority of males retain 100% NK activity and lymphocytes from some females retain 50% NK activity after this dose. Radiation sensitivity of NK activity is controlled by X-linked codominant genes. The frequency of the allele that imparts resistance is 7%. We studied a unique family in which both parents have the resistant allele such that the father is completely resistant and the mother is partially resistant. The three offspring of this couple were one sensitive male, one partially resistant female, and one completely resistant female. The radiation sensitivity of nonspecific cytotoxic functions mediated by various types of effector cells from all five family members were evaluated in order to determine whether other cytotoxic functions were controlled by the same set of genes. The cytotoxic functions investigated were: NK and lymphokine-activated killing, anomalous killing and lectin-dependent cellular cytotoxicity, and antibody-dependent cell-mediated cytotoxicity. Our data indicate that the radiation sensitivity of all types of nonspecific cytotoxic cells is under the same genetic control.

Radiation and surgical stress induce a significant impairment in cellular immunity in patients with esophageal cancer.

The effects of preoperative radiation plus surgical stress on immunity were examined in 29 patients with esophageal cancer, including 14 patients who experienced radiation therapy and 15 who did not, as well as 15 age-, sex- and body weight-matched control subjects. Absolute numbers of the total lymphocytes and OKT3 (all T cells), OKT4 (helper/inducer T cells) and OKT8 (suppressor/cytotoxic T cells) positive lymphocytes were almost the same in both patient groups before treatment. Both the in vitro response to phytohemagglutinin (PHA) and antibody dependent cell-mediated cytotoxicity (ADCC) were depressed in the patients when compared to the controls before treatment. Dual treatment of radiation and surgery led to a marked reduction of lymphocytes in the numbers and activities of PHA and ADCC, when compared to findings in the non-radiation group. Especially, the number of OKT4 positive lymphocytes and the OKT4 to OKT8 ratio decreased most and recovery was slow. While ADCC activity in the non-radiation group recovered at 28 postoperative days (POD), the response to PHA did not return to the pretreatment levels. Serum levels of IgG, IgM and IgA were within normal limits throughout the course of treatment. The B1 (all B cells) positive lymphocytes significantly decreased after the treatments. These results suggest that radiation plus surgery shifts the host immunity toward immunosuppression and induces a significant impairment of cellular immunity in patients with esophageal cancer.

Effect of microwaves on the activity of murine macrophages in vitro.

The effect of nonionizing electromagnetic radiation in the microwave range was studied on the adherence, opsonization, phagocytosis, nitroblue tetrazolium reduction and antibody-dependent cell cytotoxicity of peritoneal macrophages from BALB/c mice. After 15 min of irradiation with microwaves modulated in pulses at a frequency of 2,450 MHz and a power density of 32 mW/cm2, an increase in all the parameters studied was observed. The use of microwaves as a possible tool for the stimulation of the immune system is discussed.

Studies of natural killer activity and augmentation by OK-432 in patients with gynecological malignancies.

The natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities were determined in peripheral blood lymphocytes of patients with gynecologic malignancy. The effects of a preparation of Streptococcus pyogenes (OK-432) and of interferon were determined. Patients with advanced cancer exhibited lower natural cytotoxicity than normal women. NK and ADCC activities were decreased following surgery and NK activity was decreased following chemotherapy. Radiation reduced the percentage of Leu-7-positive cells in the circulation of the patients. OK-432 augmented the NK activity of both healthy women and patients with early malignancy. Interferon was less effective than OK-432 in augmenting NK activity.

Morphologic and phenotypic analysis of canine natural killer cells: evidence for T-cell lineage.

Canine natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity were studied utilizing a canine thyroid adenocarcinoma cell line and a lymphoblastoid cell line (CT-45S), respectively, as cell targets. Fractionation of peripheral blood mononuclear cells by Percoll discontinuous-gradient centrifugation resulted in a six- to sevenfold enrichment in large granular lymphocytes (LGL) in parallel with a twofold increase in NK activity (%specific lysis) in low-density fractions. Further enrichment in LGL (78 +/- 6%) and NK activity (threefold increase) was obtained by lytic treatment of low-density fractions 2 and 3 with monoclonal antibody WIG4. By means of cytolytic treatment with additional monoclonal antibodies the phenotype of canine NK cells was determined as Dly-1+, Dly-6+, 1A1+, E-11+, DT-2-, WIG4-. Some NK cells were also Ia+. NK activity was relatively radioresistant with 40% specific lysis even after irradiation with 40 Gy. Among the populations examined, the highest NK activity was found in peripheral blood mononuclear cells, followed by splenic mononuclear cells and bone marrow mononuclear cells. These results indicate that canine NK cells have the morphology of LGL, are relatively radioresistant, and express cell surface antigens suggesting a T-cell lineage.

Post-partum antibody-dependent cell-mediated immunity in the rat following perinatal exposure to iodine-131.

A study was recently completed which indicated the first generation of adult rats that had been exposed perinatally to iodine-131 possessed peripheral blood lymphoid-cells capable of expressing cytotoxicity towards cultured small bowel adenocarcinoma target cells, i.e., active antitumor cell-mediated immunity (CMI). The results gathered during the current investigation suggest that such animals similarly express anti-tumor antibody-dependent cell-mediated immunity (ADCC). The animal model employed consisted of Fisher F344 inbred rats exposed to iodine-131 (sodium) during their 16th to 18th day of gestation, and at an interval of two months post-partum when the offsprings had matured into adults, they and their mothers were evaluated for the presence of serum components capable of expressing ADCC activities toward X-ray induced small bowel adenocarcinoma target cells. Significant ADCC activities were found to be expressed by the offspring while no analogous immunological responses could be detected in the serum of the mothers. This lack of maternal ADCC activity suggests the existence of a biological block developing during pregnancy resulting in the mother being immunological nonresponsive to carcinogenic insults. One serum component present in the offspring identified as being responsible for initiating ADCC was an immunoglobulin of the IgG class as based upon its physical characteristics: solubility, molecular weight, and reactivity with anti-immunoglobulins, pepsin, and protein A. The interpretation of these findings is that perinatal exposure to radioiodine results in the development of cells having foreign-like properties in the offspring which are recognized by the animal's immune system, thus resulting in detectable antitumor CMI and ADCC immune responses.

Mechanism of inhibition of human natural killer activity by ultraviolet radiation.

Ultraviolet radiation (UVR) has been shown to inhibit various immune functions in vivo and in vitro. We have confirmed that UVR inhibits human natural killer (NK) activity in vitro and have shown that UVR inhibits human ADCC. In this report, the mechanism by which UVR inhibits NK function was investigated by analyzing the stage at which the inhibiting activity occurs and the ability of the NK cells to release cytotoxic factors previously shown to be involved in CMC. Single cell assays in Agarose revealed that inhibition of NK activity was localized at the postbinding lethal hit stage rather than the initial recognition or binding stage of lysis. We then examined whether UV-treated cells were able to release cytotoxic factors after stimulation with target cells. As expected, stimulated cells released cytotoxic factors, yet, surprisingly, these factors were also released in the absence of stimulator cells. The spontaneous release was detectable in the supernatants as early as 30 min after UV irradiation. The lytic material examined in 48- to 72-hr viability assays was not NK specific, because lysis was obtained with a wide range of NK sensitive and resistant target cells. These results demonstrate that UVR does not alter the capacity of the cells to secrete cytotoxic material, but in fact enhances its release. Several possible mechanisms are proposed to explain the UVR-induced NK inhibition.

Hyperthermic enhancement of antibody-complement cytotoxicity against normal mouse B lymphocytes and its relation to capping.

Normal mouse B lymphocytes were exposed to water-bath hyperthermia in vitro and examined for susceptibility to antibody-complement (Ab-C) cytotoxicity. Enhancement of Ab-C cytotoxicity was observed during heat treatment at 42 or 43 degrees C. Sensitivity to Ab-C cytotoxicity returned to normal levels by 2-3 hr post exposure to 42 degrees C. No such recovery was observed when cells were preheated at 43 degrees C for 40 min. The mechanism responsible for heat-induced enhancement of Ab-C cytotoxicity may be related to the way heat affects the redistribution of membrane-bound antigen-antibody (Ag-Ab) complexes. To investigate this possibility, cells were preheated at 37, 42, or 43 degrees C. The Ab-C assay was then performed at 37 degrees C immediately or 2.5 hr after hyperthermia. The distribution of Ag-Ab complexes was evaluated by immunofluorescence. A direct correlation was found between the hyperthermic enhancement of Ab-C cytotoxicity and the hyperthermic inhibition of capping, a process where membrane-bound Ag-Ab complexes coalesce into a polar cap on the cell surface. Sensitivity to Ab-C cytotoxicity returned to normal levels when cells restored the ability to cap Ag-Ab complexes following 42 degrees C hyperthermia. Cells heated at 43 degrees C were still sensitive to Ab-C cytotoxicity and did not recover the capping ability even 2.5 hr after heat treatment.

Radioresistance of culture-induced augmented natural killer-like activity.

Human NK activity is radiosensitive under the control of X-linked genes. We have evaluated the expression of these genes in other forms of cellular cytotoxicity. The NK radioresistant and radiosensitive phenotype is expressed in ADCC. Specific cellular cytotoxicity, generated in a MLC with a radiosensitive donor as responder, was radioresistant. NK-like activity recruited from nonadherent cells of radiosensitive subjects stimulated with allogenic cells, mitogens (PHA, Con A or PWM), or recall antigens (TT or PPD) was radioresistant. The acquisition of radioresistance was relatively rapid, beginning within 24 hr after exposure to PHA, prior to detectable proliferation. Radioresistance of MLR augmented NK-like activity was maximal 3 days after initiation of the culture. MLR augmented NK-like activity was spared by the immunosuppressive polypeptide antibiotic CsA at doses up to 1 micrometer/ml. CsA did, however, reduce acquisition of radioresistance by the NK-like activity at doses above 0.01 mu gm/ml, a concentration which does not inhibit uptake of 3H-thymidine but does reduce the level of specific CML. These data suggest that mitogens and antigens, including allogeneic cells, are recruiting radioresistant NK-like activity which can be distinguished from the radiosensitive spontaneous NK activity of the cell donor. Further, in the MLR, both radiosensitive and radioresistant NK-like activity may be recruited.