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.

Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. I. Characterization of natural killer cell induction.

Lymphocytic choriomeningitis virus (LCMV) infection of C3H/St, nude (BALB/c background), and other mice induced high levels of natural killer (NK) cell activity in the spleen and peritoneum. L-929 cells were used as targetsand were not lysed by spleen or peritoneal cells from uninfected mice. The cytotoxic cells were characterized as NK cells because they were nonadherent, nonphagocytic lymphocytes lacking theta and immunoglobulin antigens on their plasma membranes. Their activity was sensitive to 6 mM EDTA and to heating for 5 h at 37 degrees C, but resisted treatment with 0.5 percent trypsin. No role for antibody could be demonstrated in these assays. Relative to cytotoxic T-cell activity, the induction of NK cell activity was resistant to X-irradiation of mice with 1,000 rads but was sensitive if mice were first treated with Strontium-89, a bone-seeking isotope. NK cells were induced by LCMV in all tested strains of mice. In C3H/St mice NK cell activity was detected as early as 1 day and peaked at 3 days postinfection. Maximum activity in C3H/St mice was observed in mice 5-10 wk of age, but significant NK activity was also induced in newborns, which subsequently carried virus in their tissues for the duration of their lives. Older LCMV-carriers did not have detectable spleen NK cell activity. No memory oranamnestic response could be demonstrated for NK cell induction. NK cell activity was not induced by LCMV challenge of LCMV-immune mice, but was induced in those mice by infection with Pichinde virus, a closely related virus. The advent of NK cell activity correlated with the synthesis of interferon in LCMV-infected mice. Culture fluids lacking virus infectivity but containing interferon induced cytotoxic cell activity in nude and C3H/St mice. These experiments suggest that LCMV induced NK cells via an interferon-dependent mechanism. When studied in several strains of mice, the continued expression of NK cell activity did not seem to directly correlate with spleen interferon levels, suggesting that additional factors may play a role as well in maintaining the activity of the NK cell in vivo.

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.

Depression of natural antitumor resistance of C57BL/6 mice by leukemogenic doses of radiation and restoration of resistance by transfer of bone marrow or spleen cells from normal, but not beige, syngeneic mice.

Small fractionated doses of radiation (4 weekly doses of 179 rad) induced leukemia in 93% of C57BL/6J mice. These leukemogenic doses of radiation were associated with suppression of natural killer (NK) cell activity as assessed in vitro by cytotoxic activity of spleen cells or in vivo by the ability of irradiated mice to eliminate iv inoculated radiolabeled YAC-1 lymphoma cells. This suppressive effect of irradiation was profound and prolonged, being detectable for at least 7 weeks after the last dose of radiation. NK reactivity of irradiated C57BL/6 +/+ or C57BL/6 beige mice could be reconstituted by iv transfer of 10 X 10(6) bone marrow cells or 50 x 10(6) spleen cells from normal C57BL/6 mice. In contrast, transfer of bone marrow or spleen cells from beige donors did not change the NK reactivity of irradiated C57BL/6 +/+ or C57BL/6 bg/bg recipients. It was also shown that the irradiation protocol did not eliminate potentially reactive NK cells, because following interferon incubation (10(3) U/ml), the NK activity of spleen cells of irradiated mice was substantially increased. This study supports the hypothesis of an important role for NK cells in radiation-induced leukemogenesis and forms the basis for exploration of the needed, more direct data on the effects of reconstitution of NK activity on subsequent development of tumors.

Antibody-dependent lysis of tumor cells in vivo. II. Elimination of chromium-51 as a measurement of cytolysis.

The effect of antibody on tumor cells was studied in vivo by measurement of the rate of elimination of 51Cr in A/J mice inoculated with labeled Ehrlich tumor cells. Mice receiving ip injections of tumor cells and normal serum eliminated 51Cr rapidly during the first 24 hours and at a much slower rate thereafter. This biphasic pattern of elimination was due to the fact that 51Cr predominantly labels small (less than 13,000 mol wt) intracellular molecules, which the mouse rapidly eliminates, whereas larger labeled molecules are eliminated slowly. Antibody to tumor cells significantly accelerated the elimination of 51Cr at concentrations that regularly suppressed tumor growth. Antibody also induced a faster elimination rate in mice treated with cobra venom factor but not in mice treated with silica or inoculated sc with the tumor cells. Unlabeled tumor cells inhibited antibody-induced 51Cr clearance in normal mice but not in proteose peptone-treated mice. These results suggested that peritoneal cells are required in the induction of antibody-dependent cytolysis in vivo. In addition, actively or passively alloimmunized mice exhibited a similar accelerated 51Cr elimination rate when inoculated with the appropriate labeled target cells.

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.

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.

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.

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.

Effect of radiation therapy and in vitro x-ray exposure on lymphocyte subpopulations and their functions.

Radiation treatment of breast cancer patients (45.0 Gy) profoundly affected the peripheral blood lymphocytes. The number of these cells was markedly reduced with non-T-cells being more extensively depleted than T-cells immediately after radiation. The long-lasting lymphopenia, on the other hand, was mainly due to reduced number of T-cells. Antigen and mitogen stimulability, MLC reactivity, pokeweed (PWM)-induced immunoglobulin (Ig) production in vitro, and different cytotoxic functions decreased. Depletion of lymphocytes largely restored the radiation-depressed lymphocyte reactivity. The effects of in vitro exposure of blood lymphocytes to x-rays were similar to those seen after radiotherapy. Non-T-cells and T-cells with Fc-receptors for IgG were relatively radiosensitive. This latter observation agreed well with demonstrated increase of PWM-induced Ig synthesis after in vitro exposure to x-rays. T-suppressor cells defined by monoclonal antibodies were, however, radioresistant. The cytotoxic functions were reduced. No correlations were found between the pretreatment immunological status or the extent of radiation-induced immunological suppression, respectively, and prognosis.

[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 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.

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.

Antibody-dependent cellular cytotoxicity-mediated serotherapy against murine neuroblastoma. II. In vitro and in vivo treatment using effector cells from normal and X-irradiated humans.

Human peripheral lymphocytes (HLc) have been studied in vitro as possible effector cells in an antibody-dependent cellular cytotoxicity (ADCC) reaction. HLc were found to be active against murine neuroblastoma cells (MNB) inoculated into the flank of syngeneic mice. Both the time of onset of tumor appearance and the mean survival time of tumor-bearing host mice were beneficially influenced. Occasional animals could be cured of up to 10(5) tumor cells (1--10 cells of MNB are lethal). This level of tumor cytotoxicity approaches that of tolerance-dose chemotherapy and is without demonstrable side-effects. HLc from patients who had just received = 3,000 rads fractionated therapeutic X-irradiation were equally effective as HLc from control non-irradiated donors when assayed at equivalent HLc : tumor cell ratios. HLc could also inhibit MNB tumor cell growth in the ascitic form, confirming in vivo activity. Overall, HLc appeared almost as active as rat spleen cells in mediating a useful anti-tumor ADCC. This approach may ultimately prove useful in man, especially in the peritoneal cavity, and is currently limited only by the need to develop appropriate antisera. It is proposed and emphasized that such antisera need not necessarily be directed at tumor-specific antigens. Organ-specific antibodies such are already known to develop spontaneously in some human auto-immune diseases might be equally useful and are a naturally occurring potential source of appropriately expressed genetic material.

The effect of in vitro irradiation on PHA-mediated cytotoxicity and lymphocytes with receptors for the Fc part of Ig.

In vitro irradiation of purified human lymphocytes reduced PHA-mediated cytotoxicity (MICC) against chicken erythrocytes in a dose-dependent fashion. Lymphocytes with receptors for IgG present in both unfractionated and T-cell enriched cell preparations were depleted after X-ray exposure. T-cells with IgM-receptors, however, appeared to be more resistant. Different lymphocyte subpopulations seem to cooperate in MICC, and shifts of their proportions may be one explanation of the reduction of MICC after X-ray exposure.

The effect of ultraviolet irradiation on the localization of syngeneic and allogeneic lymphoid cells and on alloimmunogenicity.

Ultraviolet (u.v.) irradiation was found to have only a modest inhibitory effect on the alloimmunogenecity of murine lymphoid cells in vivo when the response was assessed by the primary cytotoxic antibody response. Ultraviolet irradiation had no effect on the initial (1 hr) organ localization of chromium-labelled lymphoid cells in syngeneic or allogeneic recipients using either CBA or DBA/2 cells. However the 24 hr localization in peripheral lymph nodes was considerably and similarly depressed in syngeneic and allogeneic recipients. This effect was shown, by alloantibody treatment of recipients of allogeneic cells, to be due mainly to lack of entry into lymph nodes after 1 hr. In agreement with much of the published data, control data for these experiments indicated little difference in lymph node localization of lymph node cells in allogeneic and syngeneic recipients using both CBA and DBA/2 cells in both CBA and DBA/2 recipients. The localization of spleen cells in lymph nodes was found to be rather more sensitive to the allogeneic environment though not as much as has been previously described for spleen cells in contrast to the long established data on lymph node cells. A marked difference in the degree of lymph node localization of CBA and CBA/2 cells was found regardless of the recipient strain.

Cellular suppression of murine ADCC and NK activities induced by Corynebacterium parvum.

Administration of a single dose of C. parvum (CP) induces depression of splenic NK activity in mice after a lag period of 3-5 days and this depression lasts about 2 weeks. The depressed levels of NK activity noted in this study depended on time of CP administration and were associated with the induction of suppressor cell activity. Neonatally thymectomized or sublethally irradiated mice had unimpaired ability to generate suppressor cells following CP treatment. Depletion of adherent/phagocytic cells by carbonyl iron plus magnetism, Sephadex G-10 filtration, or both neither enriched NK activity nor removed suppressor activity from the spleens of CP-treated mice. Antibody-dependent cellular cytotoxicity (ADCC) against lymphoma targets was also depressed in CP-treated mice, accompanied by a concomitant appearance of suppressor cells that interfere with ADCC at the effector level.

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.