Levels Of Antibody-dependent Cellular Cytotoxicity Research Articles

SARS-CoV-2 infection triggers more potent antibody-dependent cellular cytotoxicity (ADCC) responses than mRNA-, vector-, and inactivated virus-based COVID-19 vaccines.

Neutralizing antibodies (NAbs) are elicited after infection and vaccination and have been well studied. However, their antibody-dependent cellular cytotoxicity (ADCC) functionality is still poorly characterized. Here, we investigated ADCC activity in convalescent sera from infected patients with wild-type (WT) severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or omicron variant compared with three coronavirus disease 2019 (COVID-19) vaccine platforms and postvaccination breakthrough infection (BTI). We analyzed ADCC activity targeting SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in convalescent sera following WT SARS-CoV-2-infection (n = 91), including symptomatic and asymptomatic infections, omicron-infection (n = 8), COVID-19 vaccination with messenger RNA- (mRNA)- (BNT162b2 or mRNA-1273, n = 77), adenovirus vector- (n = 41), and inactivated virus- (n = 46) based vaccines, as well as post-mRNA vaccination BTI caused by omicron (n = 28). Correlations between ADCC, binding, and NAb titers were reported. ADCC was elicited within the first month postinfection and -vaccination and remained detectable for ≥3 months. WT-infected symptomatic patients had higher S-specific ADCC levels than asymptomatic and vaccinated individuals. Also, no difference in N-specific ADCC activity was seen between symptomatic and asymptomatic patients, but the levels were higher than the inactivated vaccine. Notably, omicron infection showed reduced overall ADCC activity compared to WT SARS-CoV-2 infection. Although post-mRNA vaccination BTI elicited high levels of binding and NAbs, ADCC activity was significantly reduced. Also, there was no difference in ADCC levels across the four vaccines, although NAbs and binding antibody titers were significantly higher in mRNA-vaccinated individuals. All evaluated vaccine platforms are inferior in inducing ADCC compared to natural infection with WT SARS-CoV-2. The inactivated virus-based vaccine can induce N-specific ADCC activity, but its relevance to clinical outcomes requires further investigation. Our data suggest that ADCC could be used to estimate the extra-neutralization level against COVID-19 and provides evidence that vaccination should focus on other Fc-effector functions besides NAbs. Also, the decreased susceptibility of the omicron variant to ADCC offers valuable guidance for forthcoming efforts to identify the specific targets of antibodies facilitating ADCC.

Fc-mediated functions of nirsevimab complement direct respiratory syncytial virus neutralization but are not required for optimal prophylactic protection.

Nirsevimab is an extended half-life (M252Y/S254T/T256E [YTE]-modified) monoclonal antibody to the pre-fusion conformation of the respiratory syncytial virus (RSV) Fusion protein, with established efficacy in preventing RSV-associated lower respiratory tract infection in infants for the duration of a typical RSV season. Previous studies suggest that nirsevimab confers protection via direct virus neutralization. Here we use preclinical models to explore whether fragment crystallizable (Fc)-mediated effector functions contribute to nirsevimab-mediated protection. Nirsevimab, MEDI8897* (i.e., nirsevimab without the YTE modification), and MEDI8897*-TM (i.e., MEDI8897* without Fc effector functions) binding to Fc γ receptors (FcγRs) was evaluated using surface plasmon resonance. Antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent cellular cytotoxicity (ADCC) were assessed through in vitro and ex vivo serological analyses. A cotton rat challenge study was performed with MEDI8897* and MEDI8897*-TM to explore whether Fc effector functions contribute to protection from RSV. Nirsevimab and MEDI8897* exhibited binding to a range of FcγRs, with expected reductions in FcγR binding affinities observed for MEDI8897*-TM. Nirsevimab exhibited in vitro ADNP, ADCP, ADCD, and ADCC activity above background levels, and similar ADNP, ADCP, and ADCD activity to palivizumab. Nirsevimab administration increased ex vivo ADNP, ADCP, and ADCD activity in participant serum from the MELODY study (NCT03979313). However, ADCC levels remained similar between nirsevimab and placebo. MEDI8897* and MEDI8897*-TM exhibited similar dose-dependent reduction in lung and nasal turbinate RSV titers in the cotton rat model. Nirsevimab possesses Fc effector activity comparable with the current standard of care, palivizumab. However, despite possessing the capacity for Fc effector activity, data from RSV challenge experiments illustrate that nirsevimab-mediated protection is primarily dependent on direct virus neutralization.

Elevated binding and functional antibody responses to SARS-CoV-2 in infants versus mothers

Infant antibody responses to viral infection can differ from those in adults. However, data on the specificity and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in infants, and direct comparisons between infants and adults are limited. Here, we characterize antibody binding and functionality against Wuhan-Hu-1 (B lineage) strain SARS-CoV-2 in convalescent plasma from 36 postpartum women and 14 of their infants infected with SARS-CoV-2 from a vaccine-naïve prospective cohort in Nairobi, Kenya. We find significantly higher antibody titers against SARS-CoV-2 Spike, receptor binding domain and N-terminal domain, and Spike-expressing cell-surface staining levels in infants versus mothers. Plasma antibodies from mothers and infants bind to similar regions of the Spike S2 subunit, including the fusion peptide (FP) and stem helix-heptad repeat 2. However, infants display higher antibody levels and more consistent antibody escape pathways in the FP region compared to mothers. Finally, infants have significantly higher levels of antibody-dependent cellular cytotoxicity (ADCC), though, surprisingly, Spike pseudovirus neutralization titers between infants and mothers are similar. These results suggest infants develop distinct SARS-CoV-2 binding and functional antibody activities and reveal age-related differences in humoral immunity to SARS-CoV-2 infection that could be relevant to protection and COVID-19 disease outcomes.

Abstract 6300: Therapeutic potential of EO-3021/SYSA1801, a Claudin18.2 antibody-drug conjugate, for the treatment of CLDN18.2-expressing cancers

Abstract Background: Claudin18.2 (CLDN18.2), a tight junction protein normally expressed only on gastric mucosa, is overexpressed in gastric, pancreatic, esophageal, ovarian, lung and other solid tumors. Unlike in normal tissue, CLDN18.2 is exposed on epithelial surfaces in malignancy. There are no approved targeted therapies for CLDN18.2-expressing cancers. An antibody-drug conjugate (ADC) composed of a monoclonal antibody (mAb) targeting CLDN18.2 with a monomethyl auristatin E (MMAE) payload site-specifically conjugated via a cleavable linker at a drug-antibody ratio (DAR) of 2, EO-3021/SYSA1801, was developed to target CLDN18.2-expressing cancer cells, minimize toxicities and maximize therapeutic index. Methods: HEK293-CLDN18.2 cells were used to evaluate CLDN18.2-dependent EO-3021 endocytosis, MMAE payload release, inhibition of proliferation and antibody dependent cellular cytotoxicity (ADCC). Cell cycle distribution and caspase-3/7 activity were measured 24 hours after treating BxPC3-CLDN18.2 pancreatic cells with EO-3021 or the unconjugated antibody, EO-3021 mAb. Inhibition of proliferation by EO-3021 was measured on cell lines ectopically expressing medium to high CLDN18.2 and on cell lines with endogenous low to medium CLDN18.2 expression. Xenograft studies evaluating tumor growth inhibition by EO-3021, EO3021 mAb, and cisplatin or gemcitabine were done in gastric (NUGC4; NUGC4-CLDN18.2), pancreatic (Patu8988S; BxPC3-CLDN18.2), and lung (NCI-H460) cancer models. Results: EO-3021 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on CLDN18.2 expression. EO-3021 (EC50: 172 ng/ml) and EO-3021 mAb (EC50: 130 ng/ml) demonstrated similar levels of ADCC. EO-3021 but not EO-3021 mAb promoted G2/M cell cycle arrest and apoptosis and exhibited potent activity across cell lines with low, medium, and high CLDN18.2 expression (IC50: 7-456 ng/mL). EO-3021 induced tumor regressions with a single dose across low, medium, and high CLDN18.2-expressing in vivo models derived from pancreatic (2-10 mg/kg), gastric (0.5-10 mg/kg), and lung cancers (4 mg/kg), respectively. In contrast, standard of care (SOC) chemotherapies and EO-3021 mAb did not induce tumor regressions across in vivo models. Conclusions: EO-3021 demonstrated profound in vivo antitumor activity and outperformed SOC in gastric, pancreatic, and lung cancer models. Results from in vitro and in vivo studies highlight the promising therapeutic potential of EO-3021/SYSA1801 for patients with CLDN18.2-expressing cancers. A Phase 1 study is ongoing to evaluate the safety, tolerability, pharmacokinetics and preliminary anti-tumor activity of SYSA1801 in patients with CLDN18.2 positive advanced solid tumors (NCT05009966). Citation Format: Mo Dan, Xiwu Hui, Yancui Wang, Can Yuan, Thomas O'Hare, Valerie Malyvanh Jansen, Shawn M. Leland, Yang Zhang, David Dornan, Xiaoyan Wang. Therapeutic potential of EO-3021/SYSA1801, a Claudin18.2 antibody-drug conjugate, for the treatment of CLDN18.2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6300.

Potential of Antibody-Dependent Cellular Cytotoxicity in Acute and Recovery Phases of SARS-CoV-2 Infection

Abstract Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a global pandemic that has resulted in millions of casualties. Although researchers have reported the existence of neutralizing antibodies and viral T cell immunity against SARS-CoV-2, little is known about the presence of antibody-dependent cellular cytotoxicity (ADCC) and its role in combating SARS-CoV-2 infection. Methods: Nineteen acute COVID-19 patients at the First Affiliated Hospital of Guangzhou Medical University from January to February, 2020 and 55 recovery COVID-19 patients at the Second Peoples Hospital of Changde City from February, 2020 to February, 2021 were recruited in this study. Longitudinal plasma samples were collected. A virus-specific ADCC assay was performed to study the COVID-19 plasma samples. The correlations between ADCC and total IgG titer, including anti-RBD, anti-N, and neutralizing antibody titer were analyzed. Results: A high level of ADCC with 0.86% of IFN-γ+CD107a+ NK cells induced by anti RBD antibodies and with 0.54% of IFN-γ+CD107a+ NK cells induced by anti N antibodies was observed. This activity peaked at 3 weeks after disease onset with 1.16% and 0.63% of IFN-γ+CD107a+ NK cells induced by anti RBD and anti N antibodies respectively, declined to 0.32% and 0.32% of IFN-γ+CD107a+ NK cells respectively after more than 2 months, and persisted for 12 months after disease onset. The ADCC did not aggravate the severity of COVID-19 in terms of sequential organ failure assessment, although ADCC decreased with the age of COVID-19 patients. Interestingly, ADCC response is not correlated with neutralizing antibody titer or total IgG titers against S protein RBD and N protein in acute patients. ADCC in recovered patients showed a significant correlation with anti RBD IgG titer (R 2 = 0.33, P < 0.001). Conclusion: Antibodies from COVID-19 patients against the N protein and S protein RBD domains could stimulate high levels of ADCC response. Our results provide evidence that vaccination should not only focus on neutralizing antibodies but also binding antibodies that may facilitate the antiviral function of ADCC, especially in the elderly.

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

An Anti-HER2 Monoclonal Antibody H2Mab-41 Exerts Antitumor Activities in Mouse Xenograft Model Using Dog HER2-Overexpressed Cells.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in a variety of cancer types, including breast, lung, gastric, pancreatic, and colorectal cancers. Trastuzumab, a humanized anti-HER2 monoclonal antibody (mAb), has been shown to provide significant survival benefits in HER2-overexpressing breast cancer and gastric cancer patients. Previously, an anti-HER2 mAb, H2Mab-41 (IgG2b, kappa), was developed in our laboratory and its antitumor activity was demonstrated in mouse xenograft models of human colon cancer. The present study aimed to investigate the ability of H2Mab-41 to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dog HER2 (dHER2)-overexpressed cell lines, and thus exert its antitumor activity against dHER2-overexpressed tumors in vivo. Flow cytometry results demonstrated the cross-reactivity of H2Mab-41 with dHER2. Further evaluation of interaction between H2Mab-41 and dHER2-overexpressed CHO-K1 (CHO/dHER2) cells indicated moderate binding affinity of H2Mab-41 toward dHER2, with a dissociation constant (KD) of 2.6 × 10-8 M. In vitro analysis revealed that the administration of H2Mab-41 induced high levels of ADCC and CDC in CHO/dHER2 cells. Furthermore, intraperitoneal administration of H2Mab-41 in mouse xenograft models of CHO/dHER2 resulted in significant inhibition of tumor development compared to the control mouse IgG. Thus, the findings of the present study demonstrated the in vivo safety and efficacy of H2Mab-41, highlighting its suitability to be included as a part of a therapeutic regimen for dHER2-expressing canine cancers.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 134-mG2a-f Exerts Antitumor Activities in Mouse Xenograft Models of Dog Epidermal Growth Factor Receptor-Overexpressed Cells

The epidermal growth factor receptor (EGFR) is a type I transmembrane protein, which is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR is a crucial mediator of cell growth and differentiation and forms homodimers or heterodimers with other HER family members to activate downstream signaling cascades. We previously established an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1), by immunizing mice with the ectodomain of hEGFR. In this study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) and further defucosylated (134-mG2a-f) to facilitate antibody-dependent cellular cytotoxicity (ADCC). Although 134-mG2a-f was developed against hEGFR, it was shown to cross-react with dog EGFR (dEGFR) using flow cytometry. The dissociation constant (KD) of 134-mG2a-f against dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells was determined by flow cytometry to be 3.3 × 10-9 M, indicating that 134-mG2a-f possesses a high binding affinity to dEGFR. Analysis in vitro revealed that 134-mG2a-f contributed to high levels of ADCC and complement-dependent cytotoxicity (CDC) in experiments targeting CHO/dEGFR cells. Furthermore, the in vivo administration of 134-mG2a-f significantly inhibited the development of CHO/dEGFR in comparison with the results observed in response to control mouse IgG. Taken together, the findings of this study demonstrate that 134-mG2a-f could be useful as part of a therapeutic regimen for dEGFR-expressing canine cancers.

Abstract 1856: Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry

Abstract Tumor-specific monoclonal antibodies (mAb) which induce NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) have proven to be successful against several types of cancer. Evaluating the potency of a new, tumor-specific mAb requires testing with multiple sources of donor cells since the degree of NK cell-mediated ADCC is known to be variable between patients. Thus, in addition to evaluating whether a new tumor-specific mAb can effectively induce NK cell-mediated ADCC, it is also important to characterize the donor NK cells utilized in order to provide insight into potential treatment efficacy. In the current study, we investigated the application of a novel, high throughput assay in assessing the ADCC potency of different anti-hCD20 mAbs against a Burkitt's lymphoma cell line using effector cells obtained from multiple donors. This multiplexed assay incorporates a cells and beads format to enable the simultaneous measurement of target cell killing, along with enumeration and phenotyping of the NK cells, and quantification of up to 8 secreted effector proteins and cytokines. The assay requires a sample volume of just 10 µl and can be performed in a single well of a 96 or 384 well plate using the iQue® platform for advanced flow cytometry. Data analysis and visualization was done using the integrated iQue Forecyt® software package. The results showed a mAb concentration-dependent increase in tumor cell killing, along with corresponding increases in secretion of Granzyme B and CD178 (Fas Ligand), and production of IFNγ, TNF, and RANTES. The degree of NK cell-mediated ADCC and effector protein secretion levels were dependent on both the anti-hCD20 mAb and donor cells used, with higher levels of ADCC and effector protein secretion observed using a non-fucosylated anti-hCD20 mAb as compared to a conventional, fucosylated anti-hCD20 mAb. This method enabled the rapid assessment of the potency of different mAbs in ADCC mediated by NK cells, and also effectively distinguished potential differences in mAb efficacy between donors. Citation Format: Julie Lovchik, Mark Carter. Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1856.

Anti‑EGFR monoclonal antibody 134‑mG2a exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma.

The epidermal growth factor receptor (EGFR), a transmembrane receptor and member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo- or heterodimers with other HER family members to activate downstream signaling cascades in a number of cancer cells. In a previous study, the authors established an anti-EGFR monoclonal antibody (mAb), EMab-134, by immunizing mice with the ectodomain of human EGFR. EMab-134 binds specifically to endogenous EGFR and can be used to detect receptor on oral cancer cell lines by flow cytometry and western blot analysis; this antibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) to facilitate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab-134 and 134-mG2a against EGFR-expressing CHO-K1 (CHO/EGFR) cells were deter-mined by flow cytometry to be 3.2×10−9 M and 2.1×10−9 M, respectively; these results indicate that 134-mG2a has a higher binding affinity than EMab-134. The 134-mG2a antibody was more sensitive than EMab-134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134-mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC-2, and SAS cells. Moreover, the in vivo administration of 134-mG2a significantly inhibited the development of CHO/EGFR, HSC-2, and SAS mouse xenografts in comparison to the results observed in response to EMab-134. Taken together, the findings of the present study demonstrate that the newly-formulated 134-mG2a is useful for detecting EGFR by flow cytometry, western blot analysis and immunohistochemistry. Furthermore, the in vivo results suggested that it may also be useful as part of a therapeutic regimen for patients with EGFR-expressing oral cancer.

FcγRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils.

The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface FcγRIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.

The multi-functionality of N-809, a novel fusion protein encompassing anti-PD-L1 and the IL-15 superagonist fusion complex

ABSTRACTHere we describe a novel bifunctional fusion protein, designated N-809. This molecule comprises the IL-15/IL15Rα superagonist complex containing the Fc-domain of IgG1 (N-803, formerly designated as ALT-803) fused to two single chain anti-PD-L1 domains. The fully human IgG1 portion of the N-809 molecule was designed to potentially mediate antibody dependent cellular cytotoxicity (ADCC). The studies reported here show that N-809 has the same ability to bind PD-L1 as an anti-PD-L1 monoclonal antibody. RNAseq studies show the ability of N-809 to alter the expression of an array of genes of both CD4+ and CD8+ human T cells, and to enhance their proliferation; CD8+ T cells exposed to N-809 also have enhanced ability to lyse human tumor cells. An array of genes was differentially expressed in human natural killer (NK) cells following N-809 treatment, and there was increased expression of several surface activating receptors; there was, however, no increase in the expression of inhibitory receptors known to be upregulated in exhausted NK cells. N-809 also increased the cytotoxic potential of NK cells, as shown by increased expression of granzyme B and perforin. The lysis of several tumor cell types was increased when either NK cells or tumor cells were exposed to N-809. Similarly, the highest level of ADCC was seen when both NK cells (from donors or cancer patients) and tumor cells were exposed to N-809. These studies thus demonstrate the multi-functionality of this novel agent.

HLA I shield tumor skin T lymphocytes from NK-cell-mediated elimination

Mycosis fungoides is the most common type of cutaneous T cell lymphoma and is associated with bad prognosis in advanced stage of the disease. We investigated the mechanisms of resistance to therapeutic monoclonal antibodies directed against cell surface receptors. Given the fact that natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is a key mode of action of those therapies, we addressed the question if patients with CTCL possess a fully functional ADCC. We isolated NK cells from skin and blood of patients with MF stage I–IV, Sézary Syndrome (SS) patients and healthy individuals. An aCella-TOX GAPDH assay was used to detect the amount of endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the level of ADCC in each individual patient. Functional ADCC in CTCL patients was severely abrogated. The percentage of NK cells in the blood of CTCL patients was within normal limits. Trogocytosis, a mechanism of cellular communication that can hamper ADCC by cleaving the surface of the tumor cells from the targeted molecule, did not play an essential role in CTCL. However, overexpression of MHC I on the skin tumor cells in CTCL was important factor in helping them escape NK-cell activity. Enhancement of NK cell activity in CTCL may be a promising strategy to overcome resistance to treatment and improve efficacy of therapeutic monoclonal antibodies directed against cell surface receptors.

SYD985, a Novel Duocarmycin-Based HER2-Targeting Antibody-Drug Conjugate, Shows Antitumor Activity in Uterine and Ovarian Carcinosarcoma with HER2/Neu Expression.

Purpose: Carcinosarcomas (CS) are highly aggressive gynecologic malignancies containing both carcinomatous and sarcomatous elements with heterogeneous HER2/neu expression. We compared the efficacy of SYD985 (Synthon Biopharmaceuticals BV), a novel HER2-targeting antibody-drug conjugate (ADC), to trastuzumab emtansine (T-DM1, Genentech-Roche) against primary uterine and ovarian CS.Experimental Design: Eight primary CS cell lines were evaluated for HER2/neu surface expression by IHC and gene amplification by FISH assays. The in vitro experiments included cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability, and bystander killing. In vivo activity was studied in mouse xenograft and patient-derived xenograft (PDX) models.Results: SYD985 and T-DM1 induced similar levels of ADCC against CS cell lines with low and high HER2/neu expression when challanged in the presence of effector cells. In contrast, SYD985 was 7- to 54-fold more potent than T-DM1 in the absence of effector cells. SYD985, unlike T-DM1, was active against CS demonstrating low or heterogeneous HER2/neu expression. Specifically, the mean IC50 values were 0.060 μg/mL and 3.221 μg/mL (P < 0.0001) against HER2/neu 0/1+ cell lines and 0.013 μg/mL and 0.096 μg/mL (P < 0.0001) against HER2/neu 3+ cell lines for SYD985 versus T-DM1, respectively. Importantly, unlike T-DM1, SYD985 induced efficient bystander killing of HER2/neu 0/1+ tumor cells admixed with HER2/neu 3+ cells. In vivo studies confirmed that SYD985 is more active than T-DM1 in CS and highly effective against HER2/neu expressing xenografts and PDX.Conclusions: SYD985 may represent a novel and highly effective ADC against HER2-expressing CS. Clinical studies with SYD985 in patients harboring chemotherapy-resistant CS with low/moderate and high HER2 expression are warranted. Clin Cancer Res; 23(19); 5836-45. ©2017 AACR.

Humoral Antibody Responses to HIV Viral Proteins and to CD4 Among HIV Controllers, Rapid and Typical Progressors in an HIV-Positive Patient Cohort.

The purpose of this study was to assess humoral antibody responses as a function of disease progression (DP) in a well-defined HIV+ cohort. We quantified antibodies to HIV-1 gp120, Gag, and CD4 receptor by enzyme-linked immunosorbent assay in sera from a cohort of 97 HIV+ subjects at defined stages of DP. We also measured antibody-dependent cellular cytotoxicity (ADCC) as a function of the clinical status of the patients. We purified antibodies to CD4 and gp120 and assessed them for specificity, ability to block gp120 binding to target cells, ability to block virus infection, and ability to facilitate ADCC. All of the HIV+ patient samples were positive for antibodies to HIV gp120 and p24 and 80% showed evidence of hypergammaglobulinemia. Approximately 10% of cohort members were positive for antibodies to CD4, but we noted no significant correlation relevant to DP. There were statistically significant differences between the groups concerning the level of humoral response to gp120 and Gag. However, we observed no distinction in ability of anti-gp120 antibodies purified from each group to neutralize infection. In addition, there was a statistically significant difference in ADCC, with elite controllers exhibiting significantly lower levels of ADCC than the other five groups. We detected IgA anti-gp120 antibodies, but did not correlate their presence with either DP or ADCC levels. The results are consistent with the interpretation that the humoral antibody response to the antigens assessed here represents a signature of the level of viremia but does not correlate with clinical status of HIV infection.

Abstract 2358: KHK2805, a novel ADCC- and CDC-enhanced anti-FOLR1 antibody with AccretaMab® technology, shows a potent anti-tumor activity in combination with pemetrexed

Abstract Introduction: Folate receptor 1 (FOLR1, FR alpha) is a folate transporter which is expressed in many cancers including ovarian cancer (OvC) and non-small cell lung cancer (NSCLC), and which is an attractive target for cancer therapy, is currently the subject of ongoing studies. We established KHK2805, a novel anti-FOLR1 monoclonal antibody with AccretaMab® technology to enhance both the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. It was demonstrated that KHK2805 exhibits markedly high ADCC and CDC activity levels in clinical samples from ovarian cancer patients, while a tolerable safety profile has been observed in preclinical models using cynomolgus monkeys (100 mg/kg weekly for 4 weeks, intravenously). A greater understanding of the biology of FOLR1 is important for providing a novel therapeutic option for KHK2805 for patients with cancer. Pemetrexed (PEM), a second-generation anti-folate which inhibits thymidylate synthase, glycinamide ribonucleotide transformylase and dihydrofolate reductase, is used as a standard therapy for patients with cancers such as NSCLC. It is not fully understood how PEM treatment affects the expression of FOLR1 on cancer cells. We therefore examined the level of FOLR1 expression in cancer cells after PEM treatment, and the anti-tumor activity of KHK2805 in combination with PEM. Materials and Methods: The FOLR1 expression levels after PEM treatment were examined in OvC, NSCLC, and endometrial cancer cells by flow cytometry. The ADCC activity of KHK2805 against PEM-treated cells was evaluated. The anti-tumor activity of KHK2805 in combination with PEM was investigated in SCID mice. Results: Flow cytometry showed that PEM treatment increased the FOLR1 expression of various cancers such as OvC (IGROV1, SKOV3, MCAS), NSCLC (NCI-H1437, NCI-H2228), and endometrial cancer (MESSA, HEC1A, HEC1B) cells. In addition, PEM treatment enhanced the ADCC activity of KHK2805 against MCAS, NCI-H1437, and NCI-H2228, in comparison to cells that did not receive PEM treatment. Furthermore, the anti-tumor activity of KHK2805 in SCID mice bearing subcutaneous MCAS tumors was enhanced by PEM treatment. Conclusions: PEM induced further FOLR1 expression in OvC, NSCLC, and endometrial cancer, resulting in the enhancement of the ADCC activity of KHK2805. The use of KHK2805 with PEM might therefore be a potent therapeutic option. Citation Format: Munetoshi Ando, Keiko Nagata, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. KHK2805, a novel ADCC- and CDC-enhanced anti-FOLR1 antibody with AccretaMab® technology, shows a potent anti-tumor activity in combination with pemetrexed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2358.

MiR-143 or miR-145 overexpression increases cetuximab-mediated antibody-dependent cellular cytotoxicity in human colon cancer cells.

miR-143 and miR-145 are downregulated in colon cancer. Here, we tested the effect of restoring these miRNAs on sensitization to cetuximab in mutant KRAS (HCT116 and SW480) and wild-type KRAS (SW48) colon cancer cells. We evaluated cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the modulation of signaling pathways involved in immune effector cell-mediated elimination of cancer cells. Stable miR-143 or miR-145 overexpression increased cell sensitivity to cetuximab, resulting in a significant increase of cetuximab-mediated ADCC independently of KRAS status. Importantly, HCT116 cells overexpressing these miRNAs triggered apoptosis in result of cetuximab-mediated ADCC, effected by peripheral blood mononuclear cells (p < 0.01). This was associated with increased apoptosis and caspase-3/7 activity, and reduced Bcl-2 protein expression (p < 0.01). In addition, caspase inhibition abrogated cetuximab-mediated ADCC in HCT116 cells overexpressing either miR-143 or miR-145 (p < 0.01). Furthermore, Bcl-2 silencing led to high level of cetuximab-mediated ADCC, compared to control siRNA (p < 0.05). Importantly, granzyme B inhibition, abrogated cetuximab-mediated ADCC, reducing caspase-3/7 activity (p < 0.01). Collectively, our data suggests that re-introduction of miR-143 or miR-145 may provide a new approach for development of therapeutic strategies to re-sensitize colon cancer cells to cetuximab by stimulating cetuximab-dependent ADCC to induce cell death.

CC-122 Exhibits Potent Anti-Lymphoma Activity in Combination with Obinutuzumab through Cell Autonomous and Antibody Dependent Cell Mediated Cytotoxicity

Background: CC-122 modulates the CRL4 E3 ligase complex, which results in recruitment and degradation of Aiolos and Ikaros. This results in cell autonomous anti-lymphoma and immunomodulatory effects on T and NK-cell function; such as increased expression of activation markers and cytokine production such as IFN-γ and TNF-α (Gandhi, ASH 2012). Single agent CC-122 is currently in a Phase I clinical trial for diffuse large B-cell lymphoma (DLBCL), multiple myeloma, and solid tumors (NCT01421524) and in combination with obinutuzumab (GA101) in DLBCL and indolent non-Hodgkin lymphoma (iNHL) (NCT02417285). We sought to understand the effects of combining CC-122 with anti-CD20 monoclonal antibody obinutuzumab (GA101) in in vitro and in vivo models of DLBCL and follicular lymphoma (FL).Methods: Apoptosis and cell toxicity was measured by Annexin V/ToPro-3 flow cytometry and CellTiter Glo luciferase assays, respectively. Antibody dependent cellular cytotoxicity (ADCC) was measured by anti-CD3 stimulated peripheral blood mononuclear cells (PBMCs) treated with vehicle or 100 nM CC-122 for 3 days. Prior to co-culture, target tumor cells were incubated with vehicle or GA101 for 1 hour, washed and incubated with treated PBMCs for an additional 4 hours followed by an apoptosis assay. DOHH2 and OCI-LY10 xenograft models were analyzed in C.B-17/Icr-Prkdcscid/IcoIcr mice, which retain NK cells, and CC-122 was dosed at 30 mg/kg, qd days (days 1-5 out of a 7 day schedule).Results: In the DOHH2 FL cell line, the percentage of Annexin V and ToPro-3 positive DOHH2 cells treated with 40 nΜ CC-122 or 1000 ng/mL GA101 was 35% and 40%, respectively. The combination of CC-122 and GA101 resulted in a synergistic enhancement with 82% cell death (theoretical additivity = 61%). Treatment of RL FL cells with 40 nΜ CC-122 or 2000 ng/mL GA101 resulted in 14% and 12% apoptotic cells, respectively. CC-122 combined with GA101 showed a synergistic enhancement of 44% cell death (theoretical additivity = 24.2%). In ADCC assays, DOHH2 cells incubated with vehicle treated PBMCs demonstrated 14% apoptosis. DOHH2 cells treated with 1000 ng/ml GA101 and vehicle treated PBMCs exhibited 42% apoptosis. The addition of CC-122 (100 nM) treated PBMCs to GA101 treated DOHH2 cells increased the level of ADCC to 57%. Similarly, CC-122 treatment of PBMCs co-cultured with GA101 treated RL cells led to increased levels of apoptosis by ADCC (75.7%) compared to DMSO treated PBMC with GA101 treated RL cells (62.8%) and vehicle treated co-cultures (33.9%).In a DOHH2 xenograft model, the mean tumor volume (MTV) for vehicle treated controls was 2818 mm3 at the conclusion of the study, compared to CC-122 MTV of 1568 mm3 (44% tumor growth inhibition (TGI)). GA101 monotherapies at 0.5 and 3 mg/kg once weekly yielded dose-related MTVs of 1576 mm3 (44% TGI) and 826 mm3 (71% TGI), respectively. CC-122 in combination with GA101 at 0.5 mg/kg resulted in a MTV of 694 mm3 (75% TGI), whereas the combination of CC-122 with GA101 at 3 mg/kg yielded a MTV of 493 mm3 (83% TGI). All regimens produced TGI that was statistically different from vehicle treated controls (p≤0.001).In 2 DLBCL cell lines (OCI-LY10, ABC-DLBCL and WSU-DLCL2, GCB-DLBCL), CC-122 treatment induced apoptosis with an IC50 ranging from 100 to 300 nΜ. Cell toxicity of either single agent CC-122, GA101 or a combination of the two was measured. OCI-LY10 and WSU-DLCL2 cells treated with 40 nΜ CC-122 reduced viability by 20% and 15%, respectively. OCI-LY10 and WSU-DLCL2 cells treated with 2000 ng/mL GA101 reduced viability by 46% and 62%, respectively. The combination of CC-122 with GA101 in either cell line resulted in additive effects of 58% and 68%, respectively.In the OCI-LY10 xenograft model, CC-122 demonstrated no significant reduction in MTV compared to vehicle. Single agent GA101 at 0.5 and 2.5 mg/kg once weekly yielded dose-related reduction in TGI's of 30.5% and 49.2%, respectively. CC-122 in combination with GA101 at 0.5 mg/kg resulted in 47% TGI), whereas the combination of CC-122 with GA101 at 2.5 mg/kg yielded a 94.5% TGI.Conclusion: Together, these data demonstrate that CC-122 has potent cell autonomous and immunomodulatory activities and that the addition of GA101 provides a synergistic effect in FL and is additive in DLBCL when compared to either as single agents. These data provide pre-clinical proof of concept that a combination of CC-122 and GA101 in the treatment of DLBCL and iNHL may be clinically beneficial. DisclosuresChiu:Celgene: Employment, Equity Ownership. Hagner:Celgene: Employment, Equity Ownership. Pourdehnad:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Klippel:Celgene Corporation: Employment, Equity Ownership. Klein:Roche: Employment. Thakurta:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene: Employment, Equity Ownership.

Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy

Background aimsEx vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell–based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process. MethodsEnriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus–transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression. ResultsAutomated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (±0.9) × 109 activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-γ and tumor necrosis factor-α and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell–activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential. ConclusionsWe established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.

Anti-Leukemic Activity of Daratumumab in Acute Myeloid Leukemia Cells and Patient-Derived Xenografts

Daratumumab is a human antibody that binds to CD38 on the cell surface and induces cell killing by multiple mechanisms including complement mediated cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and apoptosis. In pre-clinical and clinical studies, daratumumab has been shown to effectively kill multiple myeloma (MM) cells and to enhance the potency of other treatments against MM. The purpose of the study was to investigate in vitro and in vivo efficacy of daratumumab against 9 acute myeloid leukemia (AML) cell lines and patient-derived samples.First, we evaluated the expression of CD38, complement inhibitory proteins (CIP) CD46, CD55, CD59, and FcgR1 (CD64) on AML cell lines (n=9), AML patient cells (n=10) and healthy donor bone marrow using flow cytometry. CD38 enumeration showed a substantial variation between cell lines (12,827±19, 320 molecules/cell) and between AML patients (11,560±8, 175 molecules/cell), while CD38 expression was more consistent in bone marrow (BM) from healthy donors (1,176±355 molecules/cell). The daratumumab-induced apoptosis observed in cell lines (MOLM-13, MOLM-16, MV-4-11, NB4) in vitro was not correlated with CD38 expression levels. Daratumumab induced minimal ADCC (5-20%) and low levels of (2-5%) CDC mediated cell killing in 6 AML cell lines tested. We did not observe a direct correlation between CD38 expression and ADCC, CDC, nor between CDC and CIP expression. Interestingly, treatment of two human Acute Promyelocytic Leukemia (M3) cell lines HL-60 and NB-4 with all-trans retinoic acid (ATRA) induced a 10-30-fold increase in CD38 expression, suggesting that ATRA could be used in combination with daratumumab.While we, and others, have shown that pre-incubation of primary AML cells with anti-CD38 antibodies inhibits engraftment in NSG mice, we aimed at evaluating the anti-leukemic activity of daratumumab in a therapeutic xenograft model using 3 different AML patients. NSG mice (10/group/patient) were transplanted with T cell-depleted AML cells and BM aspirates were collected 4-6 weeks later to assess leukemia burden in each mouse prior to treatment. Animals were untreated (Ctrl) or received daratumumab (10 mg/kg), or IgG1 isotype once a week for five weeks. We assessed AML burden (% huCD45+ CD33+) in BM, spleen (SPL) and peripheral blood (PB) within 5 days after the last treatment. First, we evaluated an AML (#3406, FLT3-ITD, see figure) with high expression of CD38 (13,445 molecules/cell) and low CD64 (489/cell) was evaluated. Daratumumab significantly reduced leukemia burden in SPL and PB, but had no effect in BM. The same daratumumab-induced reduction in peripheral blasts and lack of effect in BM was observed in 2 other AML patient xenografts (#7577, M1 IDH mutant/FLT3-ITD with 6,529 CD38 molecules/cell; #8096, M2 with 335 CD38 molecules/cell). Interestingly, we observed that daratumumab treatment led to a drastic reduction in CD38 surface expression in AML blasts including in BM, indicating that daratumumab efficiently targeted CD38 in bone marrow blasts. Our results suggest that the bone marrow microenvironment can impair the anti-leukemic activity of daratumumab observed in other tissues. Ongoing xenograft studies are testing whether induction with chemotherapy (Ara-C+doxorubicin), or with other agents disrupting the bone marrow microenvironment, can enhance the anti-leukemic activity of daratumumab. [Display omitted] DisclosuresDos Santos:Janssen R&D: Research Funding. Xiaochuan:Janssen R&D: Research Funding. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. Danet-Desnoyers:Janssen R&D: Research Funding.