Antibody-dependent Cell-mediated Cytotoxicity Activity Research Articles

Abstract 619: ALT-803 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NEO-201 against human carcinoma cells

Abstract Background: ALT-803 is a novel IL-15 superagonist complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α/IgG1 Fc fusion protein. It has been reported that ALT-803 exerted a significant anti-tumor activity in murine myeloma, breast, colon carcinomas, in rat bladder cancer, and in human bladder, ovarian carcinomas in vivo. ALT-803 was also found to enhance ADCC against wide range of human carcinoma cells in vitro. NEO-201 is a novel humanized IgG1 monoclonal antibody (mAb) which was found to react against a variety of cultured human carcinoma cell lines and tumor tissues. NEO-201 can mediate ADCC against human carcinoma cells. Methodology: We investigate the ability of ALT-803 to modulate the ADCC mediated by NE0-201, employing NK cells isolated from normal donors as effectors. Human NK cells were treated with ALT-803 at different concentrations for 48h prior to be used as effector cells, and human carcinoma cell lines expressing the NE0-201 antigen were utilized as targets in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and to modulate NK cells gene expression was evaluated by flow cytometry and by using the Nanostring analysis respectively. Results: The treatment with ALT-803 significantly enhanced the ADCC activity mediated by NEO-201 against NEO-201 positive carcinoma cells. The effect ALT-803 was dose-dependent and achieved statistical significance at all doses tested compared to vehicle control treatment. Treatment of NK cells with ALT-803 enhanced ADCC activity also from donors with minimal ADCC activity and lowered the effective dose of NEO-201 required to initiate the ADCC response compared to untreated NK cells. ADCC activity can be blocked by using anti-CD16 and anti-TIM3 blocking antibody. Phenotypic analysis of NK cells treated with 25 ng/ml of ALT-803 for 48h demonstrated that ALT-803 can enhance the expression of TIM3 and NKG2D and the mean fluorescence intensity (MFI) of granzyme B and CD107a in CD16/CD56 positive NK cells. Nanostring analysis of human NK cells treated with ALT-803 at different concentrations for 48h, showed that ALT-803 was able to modulate mRNA expression of 62 genes (1.6 log2 fold change compared to vehicle control was considered significant). ALT-803 up-regulated the mRNA expression of 43 genes, including NK activating receptors, factors involved in the NK cytotoxicity, cytokines and their receptors, and down-regulated the mRNA expression of 19 genes, including NK inhibiting receptors and factors involved in the activation of apoptosis. Conclusions: ALT-803 can enhance ADCC activity mediated by NEO-201 against human carcinoma cells. The enhancement of the ADCC activity may be in part due to the increase in the expression of TIM3, NKG2D, granzyme B, and CD107a positive NK cells, as well as to the modulation of transcripts that are involved in the NK activation and cytotoxicity. Citation Format: Massimo Fantini, Justin M. David, Hing C. Wong, Christina M. Annunziata, Philip M. Arlen, Kwong Yok Tsang. ALT-803 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NEO-201 against human carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 619.

A novel Ebola virus antibody-dependent cell-mediated cytotoxicity (Ebola ADCC) assay

Ebolaviruses are highly virulent pathogens that cause Ebola viral disease (EVD). Data from non-human primate (NHP) models and from human survivors of EVD suggest that anti-Ebola antibodies play an integral role in protection. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a potential mechanism through which anti-Ebola antibodies may mediate protection. We developed a robust Ebola-specific ADCC assay for use in ongoing trials of Ebola vaccines. Stable cell lines for inducible Zaire ebolavirus glycoprotein (EBOV GP) expression were developed to provide a uniform source of target cells in the assay, and were combined with an existing human natural killer (NK) cell line as the effector cell. When applied to commercially available anti-EBOV GP monoclonal antibodies, the assay clearly differentiated antibody with high ADCC activity from those with low or no ADCC activity. Anti-EBOV ADCC activity was also detected in plasma samples from rhesus macaques immunized with a candidate Ebola vaccine. The Ebola ADCC assay reported here will be a useful tool in studying the functionality of anti-EBOV GP antibodies elicited by Ebola vaccines in ongoing and future clinical trials.

Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody

Photostability testing of therapeutic proteins is a critical requirement in the development of biologics. Upon exposure to light, pharmaceutical proteins may undergo a change in structure, stability, and functional properties that could have a potential impact on safety and efficacy. In this work, we studied how exposure to light, according to ICH guidelines, leads to photo-oxidation of a therapeutic IgG1 mAb. We also tested the ability of five different excipients to prevent such oxidation. In samples that were exposed to light, we found that the CH2 domain was considerably destabilized but there were no major changes in the overall structure of the protein. Aggregation of the protein was observed because of light exposure. Mass spectrometry identified that light exposure oxidizes two key methionine residues in the Fc region of the protein. In terms of function, a loss in binding to the neonatal Fc receptor, decreased antibody-dependent cell-mediated cytotoxicity and cell proliferation activities of the protein were seen. Combined analysis of the photo-oxidation effects on the structure, stability, aggregation, and function of the mAb has identified the underlying unifying mechanism. Among the sugars and amino acids tested, methionine was the most effective in protecting mAb against photo-oxidation.

ECD4-Ig Variants That More Potently Neutralize HIV-1.

The human immunodeficiency virus type 1 (HIV-1) entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralized all HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates that it has been tested against, suggesting that it may be useful in clinical settings, where antibody escape is a concern. Here, we characterize three new eCD4-Ig variants, each with a different architecture and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as the original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented the promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications.IMPORTANCE HIV-1 bNAbs have properties different from those of antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral life cycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the utility of antibodies as a treatment for HIV-1 infection or as part of an effort to eradicate latently infected cells. eCD4-Ig is an antibody-like entry inhibitor that closely mimics HIV-1's obligate receptors. eCD4-Ig appears to be qualitatively different from antibodies, since it neutralizes all HIV-1, HIV-2, and SIV isolates. Here, we characterize three new structurally distinct eCD4-Ig variants and show that each excels in a key property useful to prevent, treat, or cure an HIV-1 infection. For example, one variant neutralized HIV-1 most efficiently, while others best enlisted natural killer cells to eliminate infected cells. These observations will help generate eCD4-Ig variants optimized for different clinical applications.

Anticancer activity and antibody-dependent cell-mediated cytotoxicity of novel anti-nucleolin antibodies

Nucleolin arises as a relevant target for cancer therapy, as it is overexpressed at the surface of cancer and angiogenic endothelial cells thus enabling a dual cellular targeting strategy. Immunotherapeutic strategies, albeit of proven therapeutic relevance, have been scarcely explored against this target. Therefore, this work aimed at engineering an anti-nucleolin VHH-based antibody capable of triggering anticancer immune responses. Herein, anti-nucleolin VHHs have been generated upon grafting F3 peptide-derived nucleolin-binding sequences onto a VHH CDR1 or CDR3. One of these nucleolin-binding CDR3-grafted VHH was subsequently fused to a human IgG1 Fc region, enabling a significant antibody-dependent cell-mediated cytotoxicity (ADCC). The generated anti-nucleolin VHH revealed increased binding and antiproliferative effects against cancer cells, relative to the parental VHH, while the VHH-Fc counterpart presented increased cytotoxicity relative to the corresponding VHH. This VHH-Fc also triggered an ADCC effect, in the nanomolar range, against a nucleolin-overexpressing cancer cell line. This effect was evidenced by a 2 or 1.7-fold increase of cell death, in the presence of PBMCs, relative to the parental VHH-Fc or the VHH counterpart, respectively. Overall, these formats represent the first anti-nucleolin VHHs and the first anti-nucleolin antibody with ADCC activity that have been successfully developed.

Frontline Science: Plasma and immunoglobulin G galactosylation associate with HIV persistence during antiretroviral therapy.

Global antibody glycosylation is dynamic and plays critical roles in shaping different immunological outcomes and direct antibody functionality during HIV infection. However, the relevance of global antibody or plasma glycosylation patterns to HIV persistence after antiretroviral therapy (ART) has not been characterized. First, we compared glycomes of total plasma and isolated immunoglobulin G (IgG) from HIV+ ART-suppressed, HIV+ viremic, and HIV-negative individuals. Second, in ART-suppressed individuals, we examined the associations between glycomes and (1) levels of cell-associated HIV DNA and RNA in PBMCs and isolated CD4+ T cells, (2) CD4 count and CD4%, and (3) expression of CD4+ T-cell activation markers. HIV infection is associated with persistent alterations in the IgG glycome including decreased levels of disialylated glycans, which is associated with a lower anti-inflammatory activity, and increased levels of fucosylated glycans, which is associated with lower antibody-dependent cell-mediated cytotoxicity (ADCC). We also show that levels of certain mono- and digalactosylated nonfucosylated glycomic traits (A2G1, A2G2, and A2BG2), which have been reported to be associated with higher ADCC and higher anti-inflammatory activities, exhibit significant negative correlations with levels of cell-associated total HIV DNA and HIV RNA in ART-suppressed individuals. Finally, levels of certain circulating anti-inflammatory glycans are associated with higher levels of CD4 T cells and lower levels of T-cell activation. Our findings represent the first proof-of-concept evidence that glycomic alterations, known to be associated with differential states of inflammation and ADCC activities, are also associated with levels of HIV persistence in the setting of ART suppression.

Abstract P3-05-04: An IL-15 superagonist enhances antibody-dependent cell-mediated cytotoxicity against breast cancer cells regardless of FCGR3A (CD16) genotype and rescues NK cell from TGF-β1-induced immunosuppression

Abstract It has been reported that the Natural killer (NK) cell with FCGR3A (CD16a) V genotype is associated with enhanced clinical response to IgG1 monoclonal Ab (mAb) therapy such as trastuzumab, rituximab and cetuximab (1,2), suggesting a role of antibody-dependent cell-mediated cytotoxicity (ADCC) induced by NK cells. NK cells express three types of polymorphism of CD16; FcγRIIIa-158 VV, VF, FF, which are derived from the genotype of FCGR3A. It is a clinical challenge to improve the outcome in patients with FCGR3A 158FF genotype whose NK cells have lower affinity to mAb and mediate poor ADCC activity. The IL-15 superagonist/IL-15Rα-Fc fusion complex (ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, inducing their expansion, cytotoxity, and ADCC against B cell lymphoma (3, 4, 5). Here, we examined the effect of ALT803 on NK cell-mediated ADCC activity by the the anti-HER2 IgG1 mAb trastuzumab in HER2+ cell lines (SKBR3, BT474, MDA-MB-453). In addition, we used the anti-epidermal growth factor receptor(EGFR) IgG1 mAb cetuximab in EGFR positive TNBC cell lines (MDA-MB-231, SUM149, BT549). Finally, we examined the anti-PD-L1 IgG1 mAb avelumab was used for PD-L1 positive breast cancer cell lines (MDA-MB-231, BT549). Trastuzumab, cetuximab, and avelumab all significantly increased NK cell-induced lysis via ADCC. ALT803 significantly further increased both NK induced lysis and ADCC activity in all the cell lines. There was a significant positive correlation for the mean of ADCC lysis induced by NK cells from three FF (21%), three VF (33%), three VV (45%) donors. ALT803 significantly increased the mean of ADCC lysis by NK cells from all donors of each genotype to the same extent. ALT803 increased the expression of NK cell-activating receptors and cytotoxic granules regardless of the genotype of NK cell FCGR3A in terms VV, VF, or FF. We further examined the potential of ALT803 for NK cell-cytotoxicity suppressed by TGF-β1 which is one of the main barriers to immunity in the tumor microenvionment (TME). NK cells treated with TGF-β1 showed lower expression of activating receptors and cytotoxic granules, culminating in decreased lysis of MDA-MB231. ALT803 inhibited TGF-β1 from down-regulating the expression of NK cell-activating receptors and cytotoxic granules, and from suppressing the cytotoxicity of NK cells to MDA-MB231. In conclusion, the IL-15 superagonist ALT803 can potentially increase the clinical benefit of ADCC-based mAb therapy for breast cancer patients, regardless of the genotype of FCGR3A. Moreover, ALT803 prevented NK cell-cytotoxity from TGF-β1-induced suppression, providing a rationale for ALT803 therapy to overcome TME-mediated immunosuppression. References (1) Gavin et. al. JAMA Oncol.2017;3(3) (2) Musolino et. al. J Clin Oncol.2008;26(33) (3) Xu et. al. Cancer Res.2013;73(10) (4) Kim et. al. Oncotarget.2016;7(13) (5) Rosario et. al. Clin. Cancer Res. 2016; 22(3) Citation Format: Fujii R, Wong HC, Schlom J, Hodge JW. An IL-15 superagonist enhances antibody-dependent cell-mediated cytotoxicity against breast cancer cells regardless of FCGR3A (CD16) genotype and rescues NK cell from TGF-β1-induced immunosuppression [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-04.

Characterization of glycoengineered anti-HER2 monoclonal antibodies produced by using a silkworm-baculovirus expression system.

Silkworm-baculovirus expression systems are efficient means for the production of recombinant proteins that provide high expression levels and post-translational modifications. Here, we characterized the stability, glycosylation pattern and antibody-dependent cell-mediated cytotoxicity activity of anti-HER2 monoclonal antibodies containing native or glycoengineered mammalian-like N-glycans that were produced by using a silkworm-baculovirus expression system. Compared with a monoclonal antibody produced by using a Chinese hamster ovary cell expression system, the glycoengineered monoclonal antibody had comparable thermal stability and a higher antibody-dependent cell-mediated cytotoxicity activity. These results suggest that silkworm-baculovirus expression systems are next-generation expression systems potentially useful for the cost-effective production of therapeutic antibodies.

Intracellular cytokine staining (ICS) for detection of NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC)

Objective To analyze the possibility of using intracellular cytokine staining (ICS) to evaluate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and to detect the changes in ADCC activity among patients with chronic HIV and/or HCV infection. Methods Flow cytometry was performed to determine the percentages of NK cells and the expression of NK cell receptors. ImageStreamX MarkⅡ system was used to identify the expression of CD3, CD56, CD16 and CD32 on CD56brightNK and CD56dimNK subsets. Degranulation process and cytokine production in NK cells were detected using an antigen-antibody complex model of P815/Ab in combination with ICS. Differences in NK cell-mediated ADCC were evaluated among patients infected with HIV and/or HIV and healthy subjects by flow cytometry. Results The percentages of CD107a+ and IFN-γ+ NK cells were positively correlated with the decrease of mean fluorescence intensity (MFI) of CD16. ICS assay revealed a positive correlation between the secretion of CD107a and IFN-γ by NK cells. CD16 was highly expressed in CD56dimNK cells. The ADCC mediated by CD56dimNK cells was stronger than that mediated by CD56brightNK cells. The rate of target cell lysis detected by rapid fluorescence assay was positively correlated with the percentage of CD107a+ /IFN-γ+ NK cells measured by ICS. NK cell-mediated ADCC was suppressed in patients with chronic HIV and/or HCV infection. Conclusion This study suggests that ICS assay could be used to evaluate NK cell-mediated ADCC. It also reveals that NK cell-mediated ADCC is suppressed in patients with chronic HIV and/or HCV infection. Key words: NK cell; ADCC; HIV; HCV; CD107a; IFN-γ

Targeting of embryonic annexin A2 expressed on ovarian and breast cancer by the novel monoclonal antibody 2448.

Monoclonal antibodies (mAbs) play an increasingly important role in cancer therapy. To address the wide heterogeneity of the disease, the identification of novel antigen targets and the development of mAbs against them are needed. Our lab previously generated a panel of mAbs against human embryonic stem cells (hESC) using a whole cell immunization approach in mice. These mAbs can potentially target oncofetal antigens and be repurposed for antibody or antibody drug conjugate (ADC) therapy. From this panel, the novel IgG1 2448 was found to bind surface antigens on hESC and multiple cancer cell lines. Here, we show 2448 targets a unique glycan epitope on annexin A2 (ANXA2) and can potentially monitor the Epithelial-Mesenchymal Transition (EMT) in ovarian and breast cancer. To evaluate 2448 as a potential drug, 2448 was engineered and expressed as a chimeric IgG1. Chimeric 2448 (ch2448) demonstrated efficient and specific killing when conjugated to cytotoxic payloads as an ADC. In addition, ch2448 elicited potent antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro and in vivo. Further engineering of ch2448 to remove fucose in the Fc domain enhanced ADCC. Overall, these findings indicate that embryonic ANXA2 is an attractive target and suggest that ch2448 is a promising candidate for further therapeutic development.

Fc Glyco- and Fc Protein-Engineering: Design of Antibody Variants with Improved ADCC and CDC Activity.

Monoclonal antibodies are established treatment options in cancer therapy. However, not all patients benefit from antibody therapy. Basic research and findings from clinical trials revealed that certain Fc-mediated effector mechanisms triggered by monoclonal antibodies are essential for efficient antitumor activity. Today, next-generation monoclonal antibodies can be designed displaying tailor-made improved effector functions. The introduction of Fc-engineering technologies offers the potential to fine-tune Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDC). Fc-engineered antibodies hopefully will overcome some limitations of current forms of antibody therapy.

Expansion of allogeneic NK cells with efficient antibody-dependent cell cytotoxicity against multiple tumors.

Monoclonal antibodies (mAbs) have significantly improved the treatment of certain cancers. However, in general mAbs alone have limited therapeutic activity. One of their main mechanisms of action is to induce antibody-dependent cell-mediated cytotoxicity (ADCC), which is mediated by natural killer (NK) cells. Unfortunately, most cancer patients have severe immune dysfunctions affecting NK activity. This can be circumvented by the injection of allogeneic, expanded NK cells, which is safe. Nevertheless, despite their strong cytolytic potential against different tumors, clinical results have been poor.Methods: We combined allogeneic NK cells and mAbs to improve cancer treatment. We generated expanded NK cells (e-NK) with strong in vitro and in vivo ADCC responses against different tumors and using different therapeutic mAbs, namely rituximab, obinutuzumab, daratumumab, cetuximab and trastuzumab.Results: Remarkably, e-NK cells can be stored frozen and, after thawing, armed with mAbs. They mediate ADCC through degranulation-dependent and -independent mechanisms. Furthermore, they overcome certain anti-apoptotic mechanisms found in leukemic cells.Conclusion: We have established a new protocol for activation/expansion of NK cells with high ADCC activity. The use of mAbs in combination with e-NK cells could potentially improve cancer treatment.

Histone Deacetylase Inhibitors Downregulate CCR4 Expression and Decrease Mogamulizumab Efficacy in CCR4-Positive Mature T-Cell Lymphomas

Background: Histone deacetylase inhibitors (HDACis) are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and adult T-cell lymphoma/leukemia (ATLL). CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against CTCL, PTCL, and ATLL. However, their combined effects and interactions have not been examined thus far. We previously showed that CCR6, a chemokine receptor, is overexpressed in cutaneous T-cell lymphomas (Ito et al., 2014 Blood). Moreover, we recently demonstrated that HDACis downregulate CCR6 expression in advanced cutaneous T-cell lymphomas (Abe et al., 2017 Oncotarget). These reports lead us to hypothesize that HDACis might also downregulate CCR4 in various T-cell lymphomas. In this study, we clarify the effect of the combined use of mogamulizumab and HDACis on various T-cell and NK-cell lymphomas. Based on our findings, we discuss what benefits or adverse effects might be assumed for patients if these molecular targeting agents are used in clinical practice.Methods: We evaluated changes in CCR4 expression and antibody-dependent cell-mediated cytotoxicity (ADCC) activities against mogamulizumab- and HDACi-treated T-cell and NK-cell lymphoma lines and primary cases. To determine which HDAC mainly regulated CCR4 expression, we used isoform-specific HDACis and induced knockdown of respective HDACs for T-cell lymphoma cell lines. To examine the effect of CCR4 downregulation by HDACis in clinical cases, we examined the CCR4 expression of CTCL skin samples, which were obtained from the same patients before and after HDACi treatment (n = 6).Results: We first examined the expression of CCR4 for 15 T-cell and NK-cell lymphoma cell lines and a peripheral blood mononuclear cell (PBMC) sample derived from healthy donors to investigate the effect of vorinostat, a pan-HDACi, on CCR4 expression. The expression of CCR4 was mostly expressed in the (11 out of 15) cell lines: ATLL (MT-1, MT-2, MT-4, and TL-Su), CTCL (My-La, HH, and MJ), and NK/T-cell lymphoma cell lines (Kai3, SNK6, HANK1, and SNK10). We found that vorinostat decreases mRNA expression and surface expression of CCR4 except for the cell lines without CCR4 expression. Next, we used isoform-specific HDACis to examine which isoform of HDAC is involved in the regulation of CCR4. We used the following class-specific HDACis: romidepsin as a class I selective HDACi, CI-994 as an HDAC1/HDAC2-selective inhibitor, RGFP966 as an HDAC3-selective inhibitor, ricolinostat as an HDAC6-selective inhibitor, and PCI-34051 as an HDAC8-selective inhibitor. When these drugs were exposed to T-cell lymphoma cells, romidepsin and CI-994 strongly suppressed CCR4 expression. These results suggest that class I HDACs might controls CCR4 expression. We further performed knockdown experiments using siRNAs against HDAC1, HDAC2, and HDAC3. When we compared the expression change of CCR4 in HDAC-knockdown cells, HDAC2 knockdown cells showed the most significantly decreased expression of CCR4. These results suggest that class I HDACs, especially HDAC2, might be deeply involved in CCR4 expression regulation. When we examined the CCR4 expression in skin samples from primary CTCL, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after vorinostat treatment. Finally, when we conducted an ADCC assay with mogamulizumab by using various lymphoma cell lines and primary T-cell lymphoma samples, we found that the efficacy of mogamulizumab was significantly reduced by pre-treatment with vorinostat.Conclusion: Our results suggest that the primary use of HDACis before treatment of mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results provide potential implications for optimal therapeutic sequences in various CCR4 positive T-cell and NK-cell lymphomas. DisclosuresKitadate:Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Eisai: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Toyama kagaku: Research Funding. Abe:Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa:TaNeDS (Daiichi Sankyo): Research Funding.

Non-neutralizing Antibodies Targeting the V1V2 Domain of HIV Exhibit Strong Antibody-Dependent Cell-mediated Cytotoxic Activity

The development of an effective vaccine against HIV-1 has proven to be challenging. Broadly neutralizing antibodies (bNAbs), whilst exhibiting neutralization breadth and potency, are elicited only in a small subset of infected individuals and have yet to be induced by vaccination. Case-control studies of RV144 identified an inverse correlation of HIV-1 infection risk with antibodies (Abs) to the V1V2 region of gp120 with high antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing activity of Abs was not found to contribute to this protective outcome. Using primary effector and target cells and primary virus isolates, we studied the ADCC profile of different monoclonal Abs targeting the V1V2 loop of gp120 that had low or no neutralizing activity. We compared their ADCC activity to some bNAbs targeting different regions of gp120. We found that mAbs targeting the V1V2 domain induce up to 60% NK cell mediated lysis of HIV-1 infected PBMCs in a physiologically relevant ADCC model, highlighting the interest in inducing such Abs in future HIV vaccine trials. Our data also suggest that in addition to neutralization, lysis of infected cells by Abs can effectively participate in HIV protection, as suggested by the RV144 immune correlate analysis.

Superior in vitro and in vivo activity of trastuzumab-emtansine (T-DM1) in comparison to trastuzumab, pertuzumab and their combination in epithelial ovarian carcinoma with high HER2/neu expression

BackgroundEpithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. The objective of this study was to compare the anti-tumor activity of HER2/neu-targeting monoclonal antibodies, trastuzumab (T), pertuzumab (P), combination of trastuzumab and pertuzumab (T+P) and trastuzumab-emtansine (T-DM1) in EOC with high HER2/neu expression. MethodsPrimary EOC cell lines were established and cell blocks were analyzed for HER2/neu expression. Cytostatic, apoptotic and antibody-dependent cell-mediated cytotoxicity (ADCC) activities of T, P, T+P and T-DM1 were evaluated in vitro. The in vivo antitumor activity was tested in xenograft models with 3+ HER2/neu expression. ResultsHigh (3+) HER2/neu expression was detected in 40% of the primary EOC cell lines. T, P, T+P, and T-DM1 were similarly effective in inducing strong ADCC against primary EOC cell lines expressing 3+ HER2/neu. The combination of T and P was more cytostatic when compared with that of T or P used alone (p<0.0001 and p<0.0001, respectively). T-DM1 induced significantly more apoptosis when compared with T+P (p<0.0001). Finally, T-DM1 was significantly more effective in tumor growth inhibition in vivo in EOC xenografts overexpressing HER2/neu when compared to T alone, P alone and T+P (p=0.04). ConclusionIn vitro and in vivo experiments with 3+ HER2/neu expressing EOC revealed limited anti-tumor activity of T or P. T-DM1 showed superior anti-tumor activity to T and P as single agents and as a combination. Our preclinical data support the design of clinical studies with T-DM1 for the treatment of chemotherapy-resistant EOC overexpressing HER2/neu.

Abstract 5611: Use of ecto-domain vimentin to target brain cancers

Abstract Pritumumab is a natural human IgG1 kappa antibody derived from a regional draining lymph node of a patient with cervical carcinoma. The recognized antigen is an altered form of vimentin, called ecto-domain vimentin (EDV), that is expressed on the cell surface of epithelial tumor cells. For brain cancers, immunohistological analysis showed the binding of pritumumab to EDV to be restricted to tumor cells and not normal cells and tissues suggesting EDV may be a useful biomarker. A recombinant version of the mAb was made using the GPEx® system in CHO cells that showed comparable binding activity by flow cytometry, immunohistochemical analysis, Western blot analysis, and Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC; EC50 of 39.6ng/ml) activity. Further studies include establishing xenograft models with both SCID and athymic nude mice in which pritumumab was effective in preventing tumor growth of both brain cancer stem cells as well as pancreatic cancer in nude mice but not in SCID mice suggesting an active immune response is necessary for optimal treatment. Analysis of a blood brain barrier model suggests pritumumab shows minimal distribution in normal brain tissues and significant binding in tumor areas of brain tissues indicating the mAb crosses the tumor brain barrier. Overall, these data together suggest that EDV is a suitable target and biomarker for brain cancers. Citation Format: Ivan Babic, Rajesh Mukthavaram, Pengfei Jiang, Eric Glassy, Natsuko Nomura, Sandeep Pingle, Mark C. Glassy, Santosh Kesari. Use of ecto-domain vimentin to target brain cancers [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 5611. doi:10.1158/1538-7445.AM2017-5611

Abstract 3025: Preclinical characterization of a novel monoclonal antibody targeting a neo-antigen expressed in ovarian and GI malignancies

Abstract Objectives: We have developed a novel humanized IgG1 monocolonal antibody, NEO-201, which targets a neoantigen expressed on ovarian and GI malignancies. This antigen has homology to CEACAM; however, it has specificity to tumor tissues and not normal tissues. Our aims were as follows: (1) to evaluate the expression of this antigen on tumor cell lines derived from various ovarian and GI cancers; and (2) to determine the functionality of NEO-201. Methods: Expression of the antigen was identified by western blot analysis and flow cytometry. Functionality was determined by an antibody dependent cell-mediated cytotoxicity (ADCC) assay using the antigen-expressing cell lines identified by western blot analysis. Cell lines not expressing the antigen were used as negative controls. The effector cells used in the ADCC assay were natural killer (NK) cells isolated from PBMCs of normal donors and high-affinity activated NK (haNK) cells. haNK cells are NK-92 cells genetically engineered to express the high-affinity variant of human CD16 (FcgRIIIA-V158) and the human IL-2 gene. haNK cells have little or no inhibitory expression of killer cell immunoglobulin-like receptors, a unique feature that may optimize their highly cytotoxic activity against a broad range of malignancies. Results: Using western blot analysis, 2 of 5 ovarian cancer cell lines (OV90 and PE01) and 1 of 2 colorectal cancer cell lines (LS174T) stained positively for NEO-201. Flow cytometry analysis on ovarian, colorectal, and pancreatic cancer cell lines confirmed the expression of the NEO-201 neoantigen on 1 of 2 ovarian cancer cell lines (OV90), 1 of 2 colorectal cancer cell lines (LS174T), and 2 of 2 pancreatic cancer cell lines (ASPC-1 and CFPAC-1). ADCC assay was performed using OV90 and ASPC-1 tumor cell lines. Cell lines not expressing the neoantigen were used as negative controls. Killing of tumor cells expressing the neoantigen was also observed with NEO-201 alone; however, the lysis was significantly augmented when NK cells from the PBMCs of normal donors or haNK cells were included in the ADCC assay. Conclusions: These finding demonstrate that the NEO-201 neoantigen is expressed on several tumor cell lines representing ovarian and GI malignancies. NEO-201 can mediate ADCC activity in the presence of NK cells and NK cell lines. In addition, NEO-201 can induce apoptosis in tumor cell lines expressing the NEO-201 neoantigen. Furthermore, preliminary studies demonstrated that this neoantigen is expressed in numerous cancer types (not shown). In vivo studies as well as IND-related studies are planned. Citation Format: Kristen Zeligs, Philip M. Arlen, Kwong Tsang, Lidia Hernandez, Massimo Fantini, Christina M. Annunziata. Preclinical characterization of a novel monoclonal antibody targeting a neo-antigen expressed in ovarian and GI malignancies [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 3025. doi:10.1158/1538-7445.AM2017-3025

First-in-human phase 1/2 study of MCLA-128, a full length IgG1 bispecific antibody targeting HER2 and HER3: Final phase 1 data and preliminary activity in HER2+ metastatic breast cancer (MBC).

2522 Background: MCLA-128 is a novel IgG1 bispecific antibody with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity targeting HER2 and HER3 receptors. We report final phase 1 single agent escalation data, and safety and preliminary activity at the recommended phase 2 dose (RP2D). Methods: In the phase 1 part, patients (pts) with advanced solid tumors received MCLA-128 every 3 weeks (q3w) IV over 1-2 hr from 40 to 900 mg. In the phase 2 part, pts with selected metastatic indications were treated at the RP2D. Antitumor activity was assessed as per RECIST 1.1. Clinical benefit rate (CBR) was defined as CR + PR + SD ≥12 weeks. Results: As of January 2017, 28 advanced solid tumor pts were treated in the escalation part. No dose limiting toxicities were seen. The RP2D was established as 750 mg q3w (flat dose, corticosteroid premedication) based on safety and PK data. Fifteen pts with HER2 amplified tumors were treated at the RP2D (8 MBC, 4 gastric, 2 ovarian, 1 colorectal). Median age was 52 years (range 33-71), ECOG PS 0/1: 3/12, all ≥2 metastatic sites. The safety profile at the RP2D confirmed dose escalation data; the most common AEs were infusion related reactions in 6 pts (40%; G1-2 in 5 pts, G4 in 1 pt), and G1-2 diarrhea, rash, fatigue in 2 pts each (13%). No congestive heart failure or significant LVEF decreases occurred. The 8 MBC pts had a median 5.5 prior lines of metastatic therapy (range 4-14), all had progressed on 3 prior Her2 inhibitor therapies and received a median of 4.5 MCLA-128 cycles (range 2-12); 1 had a confirmed PR, 5 had SD (including 2 sustained, 11 and 12 cycles). SD was also seen in 2 evaluable MBC pts treated at 480 mg in the phase 1 part (7 and 4 cycles). Overall, CBR in these 10 MBC pts was 70%. Evaluation of other indications is ongoing. Conclusions: MCLA-128 showed a well tolerated safety profile. Consistent antitumor activity was seen in heavily pretreated MBC patients progressing on HER2 therapies. Further exploration of MCLA-128 based combinations with chemotherapy or trastuzumab in less pretreated MBC patients progressing after ≥2 prior Her2 inhibitors including TDM-1 is planned. Clinical trial information: NCT02912949.

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia

BackgroundThe CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL.MethodsPatient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model.ResultsPF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine.ConclusionsWe show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

Humoral responses against HIV in male genital tract: role in sexual transmission and perspectives for preventive strategies.

: Most new HIV infections occur via sexual routes. The induction of protective anti-HIV antibodies in genital mucosa is an important step toward reducing HIV transmission. Mucosal anti-HIV antibodies may play a dual role by either protecting against HIV transmission or facilitating it. Protective properties against HIV of mucosal IgGs and IgAs exhibiting neutralizing or antibody-dependent cell-mediated cytotoxicity activities have been described in highly exposed seronegative individuals. Conversely, some IgGs may facilitate the crossing of HIV free-particles through epithelial barriers by transcytosis. Hence knowledge of the mechanisms underlying anti-HIV antibody production in the genital tract and their exact role in sexual transmission may help to develop appropriate preventive strategies based on passive immunization or mucosal vaccination approaches. Our review focuses on the characteristics of the humoral immune responses against HIV in the male genital tract and related prevention strategies.