CD4 Mimic-Neutralizing Antibody Conjugates Synthesized by Site-Specific Modification Methods as HIV-1 Entry Inhibitors.

The characteristics of antibody-dependent cellular cytotoxicity (ADCC) directed by a panel of human and chimpanzee antienvelope (anti-Env) monoclonal antibodies (MAbs) of different epitope specificities were studied; this was accomplished by using target cells expressing human immunodeficiency virus type 1 (HIV-1) Envs of either primary or laboratory-adapted strains. Human MAbs of similar apparent affinities (1 x 10(9) to 2 x 10(9) liters/mol) against either a "cluster II"-overlapping epitope of gp41 or against the CD4 binding site, V3 loop, or C5 domain of gp120 directed substantial and comparable levels of specific lysis against targets infected with laboratory-adapted strains of HIV-1. As expected, those MAbs specific for relatively conserved regions of Env generally exhibited ADCC activity against a broader range of HIV-1 strains than those directed against variable epitopes. Significant ADCC activities of selected MAbs against primary isolate Env-expressing cells were demonstrated. In addition, a new ADCC epitope in the V2 domain of gp120 was defined. CD56+ cells were demonstrated to be the effector cells in these studies by fluorescence-activated cell sorting followed by ADCC assays. Notably, all anti-Env MAbs tested in this study, including MAbs directed against each of the known neutralization epitope clusters in gp120, directed significant levels of ADCC against targets expressing Env of one or more HIV-1 strains. These results imply that many, if not most, HIV-1-neutralizing human Abs of high affinity (> or = 3 x 10(8) liters/mol in these studies) and of the immunoglobulin G1 (IgG1) subclass (i.e., the predominate IgG subclass) are capable of directing ADCC. Since neutralizing Abs have been associated with long-term survival following HIV-1 infection, this suggests that ADCC activity may be beneficial in vivo.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.

Effector cells mediating natural killer (NK) cell activity and antibody‐dependent cellular cytotoxicity (ADCC) have been receiving considerable interest recently as they may represent an additional cellular defense mechanism against maglinancy. Spleen cells from normal young C3H (MTV+) female mice were found to have relatively high natural killer (NK) activities toward RL 1 and YAC‐1 tumor cell lines and high antibody‐dependent cell‐mediated cytotoxicity (ADCC) activities toward chicken RBC and SB tumor targets. Serum inhibition of NK and ADCC activities was either not demonstrable or only seen at low levels in the young mice. Following injection of a transplantable mammary adenocarcinoma that grew progressively in these mice, both NK and ADCC activities decreased and serummediated inhibition of both NK and ADCC increased as the tumors grew. NK and ADCC activities declined with age in C3H (MTV+) mice, and the serum inhibition of NK and ADCC activities of young mice increased with age. Aging mice, however, which had developed spontaneous mammary adenocarcinoma, although having increased levels of inhibitor against both NK and ADCC activities and decreased NK and ADCC activities of their spleen cells as compared to young normal mice, did not have different levels from those of aging mice which had not developed the mammary tumor. Correlation was poor between the amount of serum inhibitor(s) on NK and ADCC activities. NK versus RL 1 and ADCC for chicken RBC were not correlated, but NK versus YAC‐1 and ADCC for the tumor cell line SB appeared to be correlated. Decreasing NK and ADCC and increasing serum inhibitory activity toward these functions appeared to correlate with tumor progression. Sera from transplantable tumor‐bearing young C3H mice contained increased amounts of circulating immune complexes, but the amount of circulating immune complexes was only weakly correlated with the serum inhibition of NK activity of spleen cells from young animals by sera from tumor‐bearing young mice. These findings indicate that a serum blocking factor(s) that can inhibit natural cell‐mediated cytotoxicity or antibody‐mediated cytotoxicity in vitro must be considered in analyzing host resistance to tumor growth.

Serum-free culture supernatants of unstimulated normal human peripheral blood mononuclear cells contain soluble suppressor factor(s) (SSF) that significantly inhibit natural (NK) and antibody-dependent cellular cytotoxic (ADCC) activities of allogenic lymphocytes against a variety of target cells. Lymphocytes precultured with increasing concentrations of SSF showed a dose-dependent suppressive effect on these cytotoxic functions that was optimal at a concentration of 20% volume/volume. Adherent cells were not required for the production of SSF. Suppression was evident even at higher effector: target cell ratios and the inhibition was not reversed by washing lymphocytes. SSF was not itself cytotoxic, was stable at 56 degrees C, and its suppressive effect was maximal after 72 hr of incubation with effector lymphocytes. Initial estimate of the molecular weight of SSF by ultra-filtration was less than 20,000 daltons. Gel filtration of SSF on Sephacryl S-200 resulted in the elution of two peaks of activity; one in the region between markers of 13,700 and 25,000 daltons, and the other less than 13,700 daltons. Both fractions demonstrated significant suppressive activity on NK and ADCC functions of allogenic lymphocytes. SSF inhibition of NK activity could be partially reversed by incubating lymphocytes for 1 hr with human leukocyte interferon (IF) and almost completely reversed after 24 hr of IF treatment. A few selected monosaccharides (alpha-methyl-d-mannoside, l-fucose and l-rhamnose) showed a dose-dependent blocking effect on SSF activity, which suggests that SSF may act via receptor sites recognized by these sugars. As demonstrated for other lymphocyte functions, NK and ADCC activities may also be modulated by SSF elaborated by normal PBL.

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.

Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic monoclonal antibodies

NK cell activity is impaired in HIV-infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post-binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV-infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K-562 and U-937 cell lines using a 51Cr release assay; (ii) bind and kill K-562 and U-937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); (iv) kill anti-IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV-infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K-562 and U-937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF-alpha and IFN-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post-binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 is in agreement with the hypothesis of a 'general anergic process' during HIV infection.

Therapeutic mAbs are used to manage a wide range of cancers and autoimmune disorders. However, mAb-based treatments are not always successful, highlighting the need for a better understanding of the factors influencing mAb efficacy. Increased levels of oxidative stress associated with several diseases are counteracted by the activities of various oxidoreductase enzymes, such as thioredoxin (Trx), which also reduces allosteric disulfide bonds in proteins, including mAbs. Here, using an array of in vitro assays, we explored the functional effects of Trx-mediated reduction on the mechanisms of action of six therapeutic mAbs. We found that Trx reduces the interchain disulfide bonds of the mAbs, after which they remain intact but have altered function. In general, this reduction increased antigen-binding capacity, resulting in, for example, enhanced tumor necrosis factor (TNF) neutralization by two anti-TNF mAbs. Conversely, Trx reduction decreased the antiproliferative activity of an anti-tyrosine kinase-type cell-surface receptor HER2 mAb. In all of the mAbs, Fc receptor binding was abrogated by Trx activity, with significant loss in both complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) activity of the mAbs tested. We also confirmed that without alkylation, Trx-reduced interchain disulfide bonds reoxidize, and ADCC activity is restored. In summary, Trx-mediated reduction has a substantial impact on the functional effects of an mAb, including variable effects on antigen binding and Fc function, with the potential to significantly impact mAb efficacy in vivo.

ERBB2 is a proto-oncogene of multiple cancers including breast and gastric cancers with HER2 protein overexpression or gene amplification and has been proven clinically as a valid target for these cancers. HER2-targeting agents such as Herceptin®, Kadcyla® and ENHERTU® have been approved by the FDA for the treatment of breast cancer, but these drugs still face the challenge of acquired resistance and/or severe adverse reactions in clinical use. Therefore, there is significant unmet medical need for developing new agents that are more effective and safer for patients with advanced HER2-positive solid tumors including breast and gastric cancers. We report here the making of MRG002, a novel HER2-targeted antibody drug conjugate (ADC), and preclinical characterization including pharmacology, pharmacodynamics and toxicology and discuss its potential as a novel agent for treating patients with HER2-positive solid tumors. MRG002 exhibited similar antigen binding affinity but much reduced antibody-dependent cellular cytotoxicity (ADCC) activity compared to trastuzumab. In addition to potent in vitro cytotoxicity, MRG002 showed tumor regression in both high- and medium-to-low HER2 expressing in vivo xenograft models. Furthermore, MRG002 showed enhanced antitumor activity when used in combination with an anti-PD-1 antibody. Main findings from toxicology studies are related to the payload and are consistent with literature report of other ADCs with monomethyl auristatinE. MRG002 has demonstrated a favorable toxicity profile and potent antitumor activities in the breast and gastric PDX models with varying levels of HER2 expression, and/or resistance to trastuzumab or T-DM1. A phase I clinical study of MRG002 in patients with HER2-positive solid tumors is ongoing (CTR20181778).

Natural (or “spontaneous”) cytotoxicity against the K-562 myeloid cell line and antibody-dependent cellular cytotoxicity (ADCC) against sensitized Chang liver cells were monitored simultaneously in a 4 hr 51Cr release assay, using the same preparations of normal human peripheral blood lymphocytes as effector cells. Both types of cytotoxic activity were expressed by lymphocytes bearing receptors for sheep erythrocytes and for the Fc portion of IgG. Up to 80% of the total natural killer (NK) cell and K cell (ADCC) activities were recovered in E-rosette pellets separated under optimal conditions. Since the ability to form rosettes with sheep erythrocytes is primarily a marker for human T cells, it seems likely that both NK and K cells are in the T cell lineage. Further, both NK and K cell cytolytic activities were severely depressed after brief exposure of lymphocyte suspensions or rosette pellets to ammonium chloride solutions. Both activities returned to normal values after 24-hr incubation at 24°C. Such sensitivity of human lymphocytes to ammonium chloride solutions may explain why other investigators have failed to observe NK or ADCC activity by E-RFC. When lymphocytes bearing Fc receptors were adsorbed onto plastic surfaces coated with a monolayer of immobilized antigen-antibody complexes made up of TNP and rabbit anti-TNP serum, both NK and ADCC activities were simultaneously depleted. Such depletion was completely blocked when Staphylococcal protein A, which binds selectively to the Fc portion of IgG, was incubated on the antigen-antibody monolayers before adding the lymphocytes. This confirmed that both NK and ADCC activities were mediated by effector lymphocytes bearing Fc receptors. When protein A at concentrations ranging from 0.01 to 100 µg/ml was added to the 51Cr release assays, ADCC was significantly inhibited, presumably due to binding of protein A to available Fc sites of the sensitizing antibody. However, protein A had no effect on NK reactivity at any of the concentrations tested. When lymphocytes were treated for 30 min at 37°C with purified trypsin or α-chymotrypsin at concentrations from 0.01 to 10 mg/ml, NK activity was significantly inhibited whereas ADCC was unaffected. Other enzymes depressed (pronase, subtilisin) had no effect except at high concentrations (lipase), or stimulated (neuraminidase) both NK and K cell activity similarly, and therefore failed to distinguish between the effector cell types. Thus, our data indicate that effector cells in NK and ADCC are in overlapping, if not identical, subpopulations of Fc receptor-bearing lymphocytes of probable T cell lineage. The cytolytic mechanisms in NK and ADCC appear to be distinct, since we have been unable to find evidence for an “arming” antibody involved in this NK system.

Synthesis, antiviral activity and resistance of a novel small molecule HIV-1 entry inhibitor

Background: Human cancers employ a number of mechanisms to evade host immune responses. Here we report the effects of the tumor-shed antigen CA125 (MUC16) on suppressing IgG1-mediated antibody-dependent cellular cytotoxicity (ADCC). This evidence stems from prespecified subgroup analysis of an 1100 patient Phase 3 clinical trial testing the investigational agent farletuzumab, a monoclonal antibody (mAb) to folate receptor alpha, plus standard-of-care in patients with recurrent ovarian cancer. In this study, patients with low levels of CA125 [no greater than 3X the upper limit of normal (< 3X ULN)] treated with farletuzumab compared to placebo demonstrated improvements in both progression free survival (HR 0.49, p = 0.0028) and overall survival (HR 0.44, p = 0.0108). Farletuzumab’s pharmacologic activity is mediated in part through ADCC. Here we show that CA125 inhibits farletuzumab’s ADCC activity by suppressing antibody interaction with the CD16a Fc-Receptor on effector cells. Methods: Functional assays employing Jurkat-hCD16 reporter cells as well as primary NK cells were used to measure ADCC activity. CA125 isolates from various patients and cell lines were used in ADCC assays to monitor CA125 effects on immune-effector function. Multiple isogenic tumor cells expressing endogenous CA125 and their CA125-null counterparts were also used to monitor CD16a-mediated ADCC activity. CD16a+ cell binding assays were used to measure the effects of CA125 inhibition on CD16a Fc-Receptor interaction and activation. Recombinant CD16a and humanized mAbs were used in ELISA format to measure molecular binding. Results: Functional assays showed that exogenously added tumor-shed CA125 was able to suppress ADCC mediated by IgG1-type mAbs against several target cell lines. This CA125-mediated ADCC suppression was also observed in cells naturally expressing membrane-bound CA125 but not in isogenic CA125-null cells. Molecular studies suggest that CA125-mediated immunosuppression is caused by the perturbation of antibody interaction with the CD16a Fc-Receptor and that this effect is specific to IgG-type mAbs. Conclusions: Here we demonstrate that the tumor-shed antigen CA125 can elicit immunosuppression by blocking the interaction of tumor-targeting mAbs and CD16a Fc-Receptor on immune effector cells. This effect appears to be through a direct interaction of CA125 with antibody-CD16a proteins. These findings support a biological mechanism for the clinical observation that relapsed ovarian cancer patients with low CA125 blood levels have improved clinical responses to the experimental agent farletuzumab. These findings also provide new insights on the potential biological mechanisms for which tumors produce tumor-shed antigens such as CA125 to suppress host anti-tumor immune responses. Citation Format: J. Bradford Kline, Rina Kennedy, Shawn Fernando, Jennifer McDonough, Earl Albone, Elizabeth B. Somers, Wenquan Wang, Charles Schweizer, Luigi Grasso, Nicholas C. Nicolaides. CA125/MUC16 suppresses antibody-dependent cellular cytotoxicity of IgG1-based therapies via perturbation of antibody Fc-Receptor engagement on immune effector cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2017-644

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays an important role in HIV-1 entry and severs as an attractive target for development of HIV-1 entry inhibitors, a new class of anti-HIV drugs. Triggered by gp120 binding to CD4 and a coreceptor, gp41 undergoes a conformation shift from a native prefusogenic state to a fusogenic state, in which the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) associate to form a six-helix bundle, representing the fusion-active gp41 core. Any compound that disrupts the gp41 six-helix bundle formation may inhibit the gp41-mediated membrane fusion, thereby blocking HIV-1 entry into target cells. Peptides derived from the gp41 NHR and CHR regions, designated N- and C-peptides, can interact with the counterpart regions in gp41 and interfere with the six-helix bundle formation between the viral NHR and CHR region, thus inhibiting fusion of the virus with the target cell. One of the C-peptides, T-20 (brand name: Fuzeon), was recently approved by the US FDA as the first HIV entry inhibitor which can be used for treatment of AIDS patients who fail to respond to the current antiretroviral drugs, e.g., the reverse transcriptase inhibitors and protease inhibitors. The limitations of T-20 include lack of oral availability and high cost of production. Thus it is essential to develop small molecule HIV-1 entry inhibitors targeting gp41. This review summarizes the newly developed techniques for high throughput screening (HTS) and characterization of the HIV-1 entry inhibitors targeting gp41. The theories behind these techniques are also discussed. It is expected that the "drug-like" compounds with potent HIV-1 fusion inhibitory activity will be identified in the near future and used as leads for development of the low molecular weight HIV-1 entry inhibitors for the chemotherapy of HIV-1 infection and AIDS.

Two chimeric mouse-human antibodies, ING-1 (IgG1, kappa) and ING-2 (IgG1, lambda), have been constructed starting from anticarcinoma mouse hybridomas. These antibodies bind to different tumor-associated antigens which are present on human breast carcinoma cell lines at 10(5)-10(6) antigens/cell; ING-1 binds to a 40-kD membrane glycoprotein, while ING-2 binds to a glycoprotein of greater than 300 kD. In competitive binding experiments, both chimeric antibodies have identical binding activity to the parental mouse antibodies. The antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytolysis (CDC) activities of these antibodies were studied on carcinoma target cell lines. ING-1 mediates potent ADCC, but ING-2 had undetectable or very weak ADCC activity. ING-2 ADCC activity was significantly reduced by the addition of human serum, but ING-1 ADCC was unaffected. Neither ING-1 nor ING-2 mediated CDC of breast carcinoma cell lines, but ING-1 mediated CDC of a colon carcinoma cell line. ING-1 antibody-antigen complexes are stable on the target cell surface for at least 2 hours, while much of bound ING-2 is lost from the surface of cells due to internalization or shedding. The activities of these antibodies confirm that the target antigen plays an important role in the biological effector functions triggered by cell-surface-bound antibodies. Both of these chimeric antibodies are candidates for further study as immunoconjugates for cancer diagnosis or therapy, and the unconjugated ING-1 antibody has promise for cancer therapy due to its potent activation of ADCC.

In the peripheral blood of patients with chronic B-cell lymphocytic leukemia (B-CLL) absolute numbers of E-rosetting lymphocytes were increased. The proportions of TG and TM cell subsets were analyzed, as were their effects on the pokeweed mitogen (PWM)-dependent differentiation of normal allogenic B cells or of autologous leukemic cells. The TG lymphocyte subset was further studied for its cytotoxic activity in antibody-dependent cellular cytotoxicity (ADCC). A marked increase both in percentages and in absolute numbers of TG cells was found. TM lymphocytes percentages were normal, but because of the T lymphocytosis occurring in all patients, the absolute numbers of TM were increased. TM and TG subsets showed helper and suppressor activity, respectively, in PWM-induced B-cell differentiation. TG cells displayed effector cell activity in ADCC. The results provide further evidence that T lymphocytes from patients with B-CLL are functionally normal. However, a noticeable increase of the T-cell subset having suppressor and cytotoxic activity in ADCC was observed. This may be the consequence of a normal immune reaction to the leukemic population.