CDC Assays Research Articles

14. Monkeypox viral detection from different body sites. Northern California, June–July 2022

Abstract Background Unlike prior outbreaks of monkeypox, the 2022 outbreak is unique because lesions have been predominantly noted in the anogenital area, especially among persons identifying as gay, bisexual, or other men who have sex with men. The role of various body sites in monkeypox transmission is currently unclear. We assessed monkeypox PCR positivity from various body sites. Methods We collected demographic, clinical, and laboratory data from patients with confirmed monkeypox at Stanford during June–July 2022. We obtained samples from skin lesions, oropharynx, saliva, conjunctiva, urine, semen, and rectum. Monkeypox DNA analysis was performed on site using a quantitative PCR modified from published CDC assays. Results During June–July 2022, 10 patients were confirmed to have monkeypox. All were men; median age was 40 (range 24–46). Four patients identified as Hispanic, three as Caucasian, two as African American, and one as Asian. Additionally, three had HIV infection and the remaining seven were on HIV pre-exposure prophylaxis. At the time of diagnosis, eight patients (80%) described a prodrome of fever and malaise. Seven (70%) had genital lesions; the remaining three (30%) presented with only extra-genital lesions. Complications included two patients with proctitis (20%), one with phimosis (10%) and one with tonsillar involvement and impending airway compromise (10%). A total of seven (70%) received tecovirimat, and one was hospitalized. Median time from symptom onset to sample collection was seven days (range 3–14). All patients had at least one skin lesion sample positive for monkeypox virus DNA (Table 1). Six rectal samples (100%), Eight oropharyngeal samples (89%) and two (66%) urine samples were also positive. Conclusion We found monkeypox PCR positivity from different body sites. Public health messaging should consider targeting behaviors that involve body sites beyond skin, with particular consideration to mucosal surfaces. Disclosures All Authors: No reported disclosures.

Potent immunomodulatory and antitumor effect of anti-CD20-IL2no-alpha tri-functional immunocytokine for cancer therapy.

The anti-CD20 antibody rituximab (RTX) has substantially improved outcomes of patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing lymphomas. An approach to increase the clinical effectiveness of anti-tumor therapy is the use of antibody-cytokine fusion proteins (immunocytokines (ICKs)) to deliver at the tumor site the antibody effector functions and cytokines that trigger anti-tumor activities. In particular, IL-2-based ICKs have shown significant results in preclinical studies but not in clinical trials due to the toxicity profile associated to high doses IL-2 and the undesired expansion of Tregs. To improve the efficacy of RTX therapy, we fused a murine (mIgG2a) or a human (hIgG1) version of RTX to a mutated IL-2 (no-alpha mutein), which has a disrupted affinity for the high affinity IL-2 receptor (IL-2R) to prevent the stimulation of Tregs and reduce the binding to endothelial cells expressing CD25, the α chain of high affinity IL-2R. Characterization of anti-CD20-IL2no-alpha ICKs was performed by SDS-PAGE, Western-blotting and SEC-HPLC and also by several functional in vitro techniques like T-cell proliferation assays, apoptosis, CDC and ADCC assays. The in vivo activity was assessed by using murine tumor cells expressing huCD20 in C57/Bl6 mice. Both ICKs exhibited similar in vitro specific activity of their IL2no-alpha mutein moieties and kept CD20-binding capacity. Anti-CD20-IL2no-alpha (hIgG1) retained antibody effector functions as complement-dependent cytotoxicity and enhanced direct apoptosis, NK cell activation and antibody-dependent cellular cytotoxicity relative to RTX. In addition, both ICKs demonstrated a higher antitumor efficacy than parental molecules or their combination in an EL4-huCD20 tumor model in immunocompetent mice. Anti-CD20-IL2no-alpha (hIgG1) strongly expanded NK and CD8+ T cells but not Tregs in tumor-bearing mice. These findings suggest that anti-CD20-IL2no-alpha could represent an alternative treatment for B cell lymphoma patients, mainly those refractory to RTX therapy.

P848: TINOSTAMUSTINE (EDO-S101), AN ALKYLATOR AND HISTONE DEACETYLASE INHIBITOR, ENHANCES THE EFFICACY OF DARATUMUMAB IN PRECLINICAL MODELS OF MULTIPLE MYELOMA

Background: Tinostamustine (EDO-S101) is a novel alkylating and deacetylase inhibiting molecule designed to improve drug access to DNA strands, induce DNA damage and counteract its repair in cancer cells. It has shown anti-myeloma (MM) activity in different preclinical models both in monotherapy and in combination with bortezomib. Aims: To evaluate whether tinostamustine enhances the anti-myeloma effect of daratumumab. Methods: Tinostamustine was provided by Mundipharma and daratumumab was obtained from pharmacy surplus of the University Hospital of Salamanca. Mechanisms of action of daratumumab were studied by flow cytometry (FCM) in MM cell lines pretreated with tinostamustine and cultured as specified: 1) with F(ab)2 fragments (direct apoptosis via crosslinking); 2) with 10% human serum (CDC assays); 3) co-cultures of MM and NK cells (ADCC assays). Expression of CD38 and NKG2D ligands (MICA and MICB) after tinostamustine treatment was evaluated by FCM, WB and qPCR. The effects of tinostamustine + daratumumab combination were also studied ex vivo in bone marrow (BM) samples from MM patients, and in vivo in two plasmacytoma models developed in CB17-SCID and NK-cell-humanized NSG mice. Results: Pretreatment of U266, JJN3, MM.1S, NCI-H929, RPMI-8226 and MOLP-8 cell lines with tinostamustine (1-2.5 μM) for 48 h increased CD38 expression. Tinostamustine-pretreated myeloma cells showed a higher level of daratumumab binding, as demonstrated by anti-human-IgG1-AF488 staining. Also, tinostamustine (1-2.5 μM) increased MICA and MICB expression in MM cell lines. In ex vivo cultures, tinostamustine increased expression of CD38 in 4 out of 10 patients and MICA in 3 out of 5 patients. Next, the influence of tinostamustine pretreatment (2.5 μM) on several daratumumab mechanisms was evaluated. In apoptosis via crosslinking, the percentage of apoptotic cells in presence of daratumumab was higher in tinostamustine-pretreated MOLP-8 cells vsDMSO-pretreated cells (90.57%±7.35 vs 60.36%±2.86; p<0.001). Likewise, pretreatment with tinostamustine increased the percentage of Annexin-V+/7AAD+ MOLP-8 cells in CDC assays with daratumumab, although not reaching statistical significance (93.22%±1.05 vs73.39%±11.93). In MM-NK co-cultures in absence of daratumumab (NK cell-mediated direct cytotoxicity), the percentages of apoptotic cells in tinostamustine-pretreated vs DMSO-pretreated cells were: 83.64%±5.28 vs 61.58%±6.81 in MOLP-8 cells (p<0.05); 54.27%±5.99 vs 34.24%±7.37 for RPMI-8226 (p<0.05); and 26.15%±4 vs 12.32%±1.97 in MM.1S (p<0.05). However, in the presence of daratumumab (ADCC) these percentages showed a similar tendency although not significant: 88.57%±3.77 vs 78.89%±5.57 in MOLP-8; 70.75%±5.69 vs 58.28%±7.52 for RPMI-8226; and 56.74%±7.25 vs 45.68%±7.1 in MM.1S. Ex vivo experiments with patients’ BM samples (n = 10) showed that simultaneous combination of daratumumab + tinostamustine significantly increased the percentage of eliminated MM cells compared to individual treatments, with an acceptable toxicity on healthy lymphocytes (Figure 1a). Finally, in the CB17-SCID model, tinostamustine treatment (24 h) followed by daratumumab administration controlled tumor growth significantly better than daratumumab alone (Figure 1b). The combination prolonged the median survival as compared to vehicle, and monotherapy with tinostamustine or daratumumab (Figure 1b). Similar results were obtained in the NSG model. Image:Summary/Conclusion: Our preclinical data demonstrate that tinostamustine increases the anti-myeloma effect of daratumumab presumably due to enhanced expression of CD38 and MICA.

Abstract 5523: LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model

Abstract Objective: Although immunotherapies, e.g., anti-PD-(L)1 has achieved great success in treating cancers, a large portion of patients still cannot benefit from it, due largely to primary refractory or acquired resistance leading to unsatisfied ORR or relapse. TNFR2 is a receptor for TNF-α, expressed highly selective in immune cells, and some tumor cells, transducing survival and immunostimulatory signal to cells expressing this receptor. Targeting TNFR2 may modulate this signaling, therefore a humanized anti-TNFR2 antibody, LBL-019, was developed for monotherapy or in combination with other anti-tumor therapies. Methods: LBL-019 is a monoclonal antibody of IgG1 subtype. The binding affinity to TNFR2 was determined by Fortebio. The epitope of TNFR2 was identified by differential binding to a serial human/mouse chimeric TNFR2 proteins. The binding of TNF-α to either TNFR2 protein or TNFR2 expressed in cells was examined by using ELISA and flow cytometry. The in vitro activity of LBL-019 was measured using several in vitro assays, e.g., cell proliferation and cytokine release in T cells, ADCC and CDC assays. The anti-tumor activity of LBL-019 was investigated in a model of human TNFR2 transgenic mice (Shanghai Model Organisms) implanted with MC38-OVA cells. Results: The binding affinity of LBL-019 was measured as 5.32 nM to human and 3.62 nM to cynomolgus TNFR2. It was found that TNFR2 was expressed in immune cells including T cells and monocytes examined in human PBMC, which was upregulated when T cells were activated. TNFR2 was also found highly expressed in some tumor cell lines and tumor TILs (from in vivo studies described below). The epitope of TNFR2 that LBL-019 binds was identified and mapped out CRD1 domain. The binding of TNF-α with either TNFR2 protein or TNFR2 expressed in immune cells was blocked by LBL-019. The growth of CD8+ T cells and CD4+ T cells was significantly enhanced, when incubated with LBL-019, along with elevated IFN-γ release. In a model of human TNFR2 transgenic mice implanted with MC38-OVA cells, LBL-019 was shown significant tumor growth inhibition (TGI) in a dose dependent manner as compared with vehicle treated mice (p&amp;lt;0.01). It appeared that the antibody with wild type Fc showed a better anti-tumor activity than that with silenced Fc, suggesting that Fc mediated NK killing of tumor cells or Treg may have contributed in part to the observed anti-tumor efficacy in this model. In addition, LBL-019 was also exhibited a synergy of anti-tumor activity, when dosed with anti-PD-1 (TGI reaching 79.51%). Conclusions: LBL-019, a humanized anti-TNFR2 antibody, shows great anti-tumor efficacy in a mouse tumor model, likely due to the mechanisms involving enhanced proliferation of Teff, elevated IFN-γ release, and possibly killing of tumor cells and Treg. The data support further development of LBL-019 as a novel therapeutic antibody for cancer, and clinical trial is expected to launch early 2022. Citation Format: Huan Lin, Xiao Huang, Yurong Qin, Yujia Dang, Peng Zhang, Jianming Sun, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-019, a novel anti-TNFR2 antibody, shows a potent anti-tumor efficacy in a mouse MC38 model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5523.

Interlaboratory comparison of SARS-CoV2 molecular detection assays in use by U.S. veterinary diagnostic laboratories.

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results.

Abstract 3361: Discovery and preclinical characterizations of a humanized anti-claudin 18.2 antibody SPX-101

Abstract Claudin 18.2 (CLDN 18.2) is a major component of the tight junctions and is highly expressed in several cancers including gastric (80%), esophageal (80%), pancreatic (60%) and lung cancers, but only expressed at very low levels in healthy tissues. The chimeric IgG1 CLDN 18.2 antibody zolbetuximab significantly improved PFS and OS in gastric cancer patients in a CLDN 18.2-dependent manner on the background of EOX. This research aimed to produce humanized anti-claudin18.2 antibodies with enhanced activities and improved pharmaceutical characteristics compared to known CLDN 18.2 antibodies. We used several immunization methods to produce mouse anti-CLDN18.2 antibodies with high affinity and excellent selectivity against CLDN 18.1. Humanization of a selected clone led to a humanized anti-CLDN18.2 antibody (h533o) with significantly improved binding affinity and cellular activity in ADCC and CDC assays compared to zolbetuximab. The binding affinity of h533o was relatively insensitive to elevated temperatures or acidity, suggesting the robustness of target engaging ability in tumor microenvironment. Maturation and selection by the off-rate approach, using phage libraries that systematically modified the CDR regions, identified multiple antibodies with comparable affinity and selectivity. Mutational scan of the Fc region in h533o identified SPX-101 with reduced ADA effect in mice while retaining other favorable properties. SPX-101 suppressed tumor growth in several xenograft mouse models including NUGC4 and HEK293 cells that stably expressed CLDN18.2. In summary, SPX-101 represents a humanized anti-CLDN18.2 antibody with high affinity and specificity, high effector cell-mediated cytotoxicity, low immunogenicity, and in vivo tumor control efficacy in xenograft rodent models. Citation Format: Guidong Zhu, Jichun Ma, Jingdong Ye, Jingdong Qin, Yi Cai. Discovery and preclinical characterizations of a humanized anti-claudin 18.2 antibody SPX-101 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3361.

Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in the city of Wuhan, Hubei Province, China, has spread worldwide and is threatening human life. The detection of SARS-CoV-2 is critical for preventing new outbreaks, curbing disease spread, and managing patients. Currently, a reverse-transcription polymerase chain reaction (RT-PCR) assay is used to detect the virus in clinical laboratories. However, although this assay is considered to have high specificity, its sensitivity is reportedly as low as 60–70 %. Improved sensitivity is, therefore, urgently required. MethodsWe used the primers and single-quencher probes recommended by the CDC (N1, N2 and N3) in the USA and the NIID (N1 and N2) in Japan. In addition, we designed double-quencher probes according to the virus sequence provided by the NIID to develop a further assay (termed the YCH assay [N1 and N2]). Using these assays, we conducted RT-PCR with serially diluted DNA positive controls to assess and compare the detection sensitivity of the three assays. Furthermore, 66 nasopharyngeal swabs were tested to determine the diagnostic performances. ResultsThe threshold cycle (Ct) value of the RT-PCR was relatively low for the CDC and YCH assays compared with the NIID assay. Serial dilution assays showed that both the CDC and YCH assays could detect low copy numbers of the DNA positive control. The background fluorescence signal at the baseline was lower for the YCH assay compared with the NIID assay. We assessed the diagnostic performance between single- (NIID) and double-quencher (YCH) probes using 66 nasopharyngeal swabs. When the results of YCH-N2 assay were used as a reference, each assay detected SARS-CoV-2 with positive percent agreements of 56 % for NIID-N1, 61 % for YCH-N1, and 94 % for NIID-N2, and 100 % negative percent agreements for NIID-N1, YCH-N1 and NIID-N2. ConclusionDouble-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2.

Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories.

A reliable diagnostic assay is crucial to early detect new COVID-19 cases and limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Since the onset of the COVID-19 pandemic, the World Health Organization has published several diagnostic molecular approaches developed by referral laboratories, including Charité (Germany), HKU (Hong Kong), China CDC (China), US CDC (United States), and Institut Pasteur, Paris (France). We aimed to compare the sensitivity and specificity of these different RT-PCR assays using SARS-CoV-2 cell culture supernatants and clinical respiratory samples. Overall, the different RT-PCR assays performed well for SARS-CoV-2 detection and were all specific except the N Charité (Germany), and N2 US CDC (United States) assays. RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive assays. The data presented herein are of prime importance to facilitate the equipment choice of diagnostic laboratories, as well as for the development of marketed tests.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Indirect mechanisms of action of a novel IgG1 monoclonal antibody, NEO-201, to enhance immune killing of tumor.

17 Background: NEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 that demonstrates tumor sensitivity and specificity. Functional analysis revealed that NEO-201 is capable of engaging innate immune effector mechanism including ADCC and CDC to directly kill tumor cells expressing its target. Previous studies demonstrated safety/tolerability in non-human primates, and an on going clinical trial at the NCI is currently exploring its dosing and safety. We have explored indirect mechanisms of its action that may enhance immune tumor killing. NEO-201 has the ability to enhance NK activity by blocking the binding of CEACAM5 on tumor cells to CEACAM1 on NK cells. In addition NEO-201 can target and eliminate regulatory T cells (Tregs). Methods: In vitro functional assays, using various human tumor cell lines as target cells and NK-92 cells (CEACAM1+/CD16−) as effectors, were conducted to assess the ability of NEO-201to enhance antitumor cytotoxicity of NK-92 cells by blocking the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells. In addition, flow cytometry analsyis and CDC assays were performed to evaluate the ability of NEO-201 to target and eliminate human Tregs in vitro. Results: Addition of NEO-201 significantly enhanced NK-92 cell cytotoxicity against highly CEACAM5+/NEO-201+ cells, suggesting that its activity is correlated with the level of CEACAM5+/NEO-201+ tumor cells. Furthermore, NEO-201 targets human Tregs and mediates killing of opsonized T-reg cells via CDC. Conclusions: Previously we have demonstrated the ability of NEO-201 as an IgG1 mab to destroy tumor cells expressing its target directly through both ADCC and CDC. This study suggests that in addition, NEO-201may also mediate immune killing through additional mechanisms including blocking the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells to reverse CEACAM1-dependent inhibition of NK cytotoxicity. In addition, we have shown that NEO-201 is also able to recognize and eliminate human Tregs. Ongoing studies are looking at leveraging this phenomenon by combining NEO-201 with checkpoint inhibitors.

Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus

BackgroundThe recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources.MethodsWe developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively.ResultsThese assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/μl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83).ConclusionsThe ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas.

P259 Comparison of two cell preparation methods: Standard Ficoll hypaque vs. Easysep for flow and CDC crossmatching

Aim Background: Preparation of lymphocytes from living related and deceased donor for flow and CDC crossmatching is labor intensive and occasionally difficult. It is not uncommon that the number of donor cells and cell viability are inadequate, which makes it difficult to obtain valid crossmatch results prior to transplant. Aim: The aim of our study was to determine efficacy of cell isolation methods using Ficoll hypaque vs. Easy Sep technique. Methods Lymphocytes were isolated from living and/or deceased donor peripheral blood, spleen and lymph node samples using Ficoll hypaque density gradient method and EasySep. The volume of initial samples used for each method was same. We compared cell yield, purity, initial/ final viability, we also evaluated reaction pattern and strength of CDC and flow crossmatches tests using lymphocytes isolated using these two methods. Results The cell yield from peripheral blood using Ficoll hypaque method was twofold higher but less pure by 30–40%. The cell yield and purity from spleen and lymph node were the same for both techniques. The number of T and B cells separated by Dyna Beads using cells prepared by both methods for CDC crossmatch were similar. The strength of reaction for cells prepared by Easy Sep was the same as the current method. However, the reactions were easier to read using cells isolated by the EasySep method due to reduction in number of granulocytes. The initial and final cell viability were the same at 95–98% for both techniques. EasySep enrichment procedure provided more concentrated T and B cells population for flow crossmatch testing. This resulted in better gating of T and B cells for analysis and faster acquiring of the 20,000 events required per well. Conclusions EasySep method is a faster and less labor-intensive method to isolate lymphocytes for the flow and CDC crossmatch assays. The high quality of lymphocyte preparation, with less platelets and granulocytes, makes results easy to interpret. The EasySep method uses negative selection technique to deplete unwanted cells from the cell suspension resulting in no injury or manipulation of the lymphocytes required by the CDC and Flow crossmatch assays. This process can be automated by EasySep bio-robot.

Asymmetric Fc Engineering for Bispecific Antibodies with Reduced Effector Function.

Asymmetric bispecific antibodies are a rapidly expanding therapeutic antibody class, designed to recognize two different target epitopes concurrently to achieve novel functions not available with normal antibodies. Many therapeutic designs require antibodies with reduced or silenced effector function. Although many solutions have been described in the literature to knockout effector function, to date all of them have involved the use of a specific antibody subtype (e.g., IgG2 or IgG4), or symmetric mutations in the lower hinge or CH2 domain of traditional homodimeric monospecific antibodies. In the context of a heterodimeric Fc, we describe novel asymmetric Fc mutations with reduced or silenced effector function in this article. These heteromultimeric designs contain asymmetric charged mutations in the lower hinge and the CH2 domain of the Fc. Surface plasmon resonance showed that the designed mutations display much reduced binding to all of the Fc gamma receptors and C1q. Ex vivo ADCC and CDC assays showed a consistent reduction in activity. Differential scanning calorimetry showed increased thermal stability for some of the designs. Finally, the asymmetric nature of the introduced charged mutations allowed for separation of homodimeric impurities by ion exchange chromatography, providing, as an added benefit, a purification strategy for the production of bispecific antibodies with reduced or silenced effector function.

Abstract 3518: Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies

Abstract The unique ability of flow cytometry to simultaneously examine intricate details of multiple cell subsets is unparalleled and the platform is now a dominant tool for measuring antibody mediated effector functions on cancer cells. The objective of this study is to illustrate the utility of flow cytometry as a single platform for concurrently measuring the multiple mechanisms of actions of therapeutic antibodies such as ADCC, ADCP, Apoptosis, CDC and Trogocytosis on human cancer cells and immune functioning cells. Rituximab® was tested with the Burkett's Lymphoma cell line (Daudi) for all assays between the ranges of 0.01 and 100ug/mL. Apoptosis was evaluated by measuring Caspase 3/7 staining. ADCC assays were established with Rituximab®/CFSE labeled Daudi cells and human PBMC’s. ADCP was assessed with Rituximab®/ CFSE labeled Daudi cells and CD14+/CD11b+ in vitro generated phagocytes using a 4 hour exposure. CDC assays were established with Daudi cells/ 10% pooled human serum using 7-AAD as the cytotoxic indicator in a 4 hour assay. Trogocytosis was measured up to 4 hours with Rituximab®/ Daudi and PBMC's and CD19/20 and CD14. All assays were analyzed using a FACS ARIA III flow cytometer and replicates of 3 were evaluated for statistical relevance. Results demonstrated that Rituximab® induced effects were observed in all 5 assays using flow cytometry. Apoptosis was induced when 0.01ug/mL Rituximab® was present. ADCP occurred at levels as low as 0.01ug/mL Rituximab®. ADCC and CDC occurred at levels as low as 0.1ug/mL Rituximab®. Trogocytosis, indicated by the transfer of CD19+ B cells to CD14+ monocytes, occurred at 0.1ug/ml Rituximab®. In summary, we demonstrated the clinical applicability of flow cytometry as a single platform to simultaneously evaluate five different mechanisms of actions that can occur when therapeutic antibodies are used to treat cancer cells. Citation Format: Antony R. Chadderton, Shane Harvey, Brandy Strake, Jill Giles-Komar, Renold J. Capocasale, T. Shantha Raju. Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies. [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 3518.

Abstract 2955: Herceptarg, a novel heterodimeric biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab, exerts potent anti-tumoral activity

Abstract Introduction: Trastuzumab and pertuzumab are humanized antibodies recognizing different functional HER2 epitopes. Preclinical data demonstrated strong anti-tumoral efficacy when combining them (Scheuer et al., Canc Res, 2009). The CLEOPATRA trial showed that the addition of pertuzumab to trastuzumab + docetaxel strongly improves overall survival of HER2+ metastatic breast cancer patients (Swain et al., NEJM, 2015). Herceptarg is a novel heterodimeric 1+1 biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab. Methods: Using consensus light chains, pertuzumab heavy chain affinity maturation via phage display and knob-into-holes technology, a heterodimeric biparatopic common light chain antibody based on trastuzumab and pertuzumab was generated (Figure 1A). This bispecific antibody was characterized in direct comparison to the respective parental antibodies and their combination in vitro by surface plasmon resonance, proliferation, ADCC and CDC assays and in vivo using the orthotopic KPL-4 breast cancer xenograft model. KPL-4 cells were provided by Prof. Kurebayashi (Kawasaki Medical School, Kurashiki, Japan). Results: In vitro, Herceptarg has the highest binding affinity for HER2 on cells, mediates potent ADCC activity, comparable or superior growth inhibition activity of breast and gastric cancer cells and CDC superior to the combination of pertuzumab and trastuzumab. In vivo, Herceptarg (10 mg/kg, q1w) mediates anti-tumoral efficacy in the orthotopic KPL-4 breast cancer xenograft model resulting in tumor regression comparable to the combination of trastuzumab and pertuzumab (10 mg/kg, q1w, each), and superior to the respective single agent therapies. Conclusions: Taken together, these data demonstrate that Herceptarg, as a single IgG1 bispecific antibody is superior (or at least comparable) to the combination of trastuzumab and pertuzumab in vitro and in vivo and may ultimately improve outcome of breast and gastric cancer patients. Citation Format: Ekkehard Moessner, Thomas Hofer, Inja Waldhauer, Werner Scheuer, Ralf Hosse, Lydia Duerner, Mi He, Karlheinz Zick, Jens Fischer, Claudio Sustmann, Tina Weinzierl, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. Herceptarg, a novel heterodimeric biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab, exerts potent anti-tumoral activity. [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 2955.

Abstract 2460: Dissecting the in vitro and in vivo mechanism of action of obinutuzumab (GA101) in preclinical models using an immune effector-dead version of obinutuzumab

Abstract Introduction: Obinutuzumab (GA101) is a glycoengineered type II CD20 antibody mediating superior direct cell death induction, ADCC and ADCP compared to rituximab while CDC is strongly reduced. Obinutuzumab is approved for 1st line treatment of CLL in combination with chlorambucil in the US and Europe. The contribution of these mechanisms of action (MOA) to the efficacy of obinutuzumab in patients is unclear. In order to further dissect the MOA GA101 and rituximab and variants with abolished FcγR and C1q binding were assessed in vitro and in vivo. Methods: Glycoengineered GA101, non-glycoengineered wild type (wt) GA101, effector-dead GA101 and rituximab with P329G LALA mutations in the Fc were generated and characterized i) for binding to FcγRs and CD20 positive cells by FACS, ii) for cell death induction by AnnexinV/PI staining, iii) in ADCC, ADCP and CDC assays, and iv) for depletion of CD19 positive B cells in whole blood from healthy volunteers. Anti-tumoral efficacy was assessed using established 300 mm2 s.c. SU-DHL4 xenograft tumors in Scid beige mice (30 mg/kg, q7d x 4, ip). Results: Variants of GA101 and rituximab retain CD20 binding and cell death inducing properties of the respective parental antibody. The introduction of P329G LALA in the Fc part abolishes binding to Fcγ receptors and C1q, so that they do not mediate ADCC, ADCP or CDC any longer, while direct cell death induction is not affected as compared to GA101 and rituximab, respectively. In whole blood assays GA101 showed the best B cell depletion in terms of potency and absolute B cell depletion followed by wtGA101 and rituximab. Notably, GA101 P329G LALA retained significant B cell depletion, while rituximab P329G LALA did not mediate B cell depletion. Thus, in whole blood B cell depletion, ADCC/ADCP plays an important role, but direct cell death induction is equally important for the MOA of GA101, whereas it does not contribute to B cell depletion of rituximab. In the SU-DHL4 xenograft model GA101 (TGI &amp;gt; 100%, non-parametric TCR (npTCR) 0 with 95% CI 0-0) and wtGA101 (TGI &amp;gt; 100%, npTCR 0, 95% CI 0-0.006) showed equivalent efficacy with complete tumor remission in 9/10 mice for GA101 and 8/10 mice for wtGA101. GA101 P329G LALA showed anti-tumoral efficacy (TGI 62%, npTCR 0.43, 95% CI 0.30-0.60) comparable to rituximab (TGI 71%, npTCR 0.37, 95% CI 0.29-0.50), while rituximab P329G LALA showed only residual anti-tumoral activity (TGI 26%, npTCR 0.73, 95% CI 0.44-0.92). Summary: These data indicate that in whole blood B cell depletion and xenograft models, direct cell death and ADCC/ADCP through the glycoengineered Fc part are required for anti-tumoral efficacy of obinutuzumab. Most notably, the effector dead version of obinutuzumab that completely lacks ADCC, ADCP and CDC activity, but has retained direct cell death induction, is as efficacious as rituximab in vivo supporting the role of direct effects for the efficacy of obinutuzumab. Citation Format: Sylvia Herter, Frank Herting, Gunter Muth, Erwin van Puijenbroek, Claudia Ferrara, Sabine Lang, Marina Bacac, Ekkehard Moessner, Pablo Umana, Christian Klein. Dissecting the in vitro and in vivo mechanism of action of obinutuzumab (GA101) in preclinical models using an immune effector-dead version of obinutuzumab. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2015-2460

Pan HLA I Chimeric Human IgG Reveal Subclass Differences in Complement Fixing Assays.

Purpose: The purpose of this study was to compare hIgG1 and hIgG2 HLA I antibodies in their capacity to activate complement in in vitro assays, and to determine whether any correlation existed between antibody strength (MFI in single antigen assay) and ability to fix C1q. Methods: We generated chimeric pan HLA I antibodies by fusing W6/32 variable regions to human IgG1 and human IgG2 constant regions. Binding to HLA I and activation of complement was assessed by solid phase (SAB Labscreen and C1qScreen (OneLambda); C3d bead assay (Immucor/Lifecodes) and cell-based assays (flow cytometry, cell-based ELISA). Results: HLA I hIgG1 and hIgG2 bound highly to all alleles and loci of HLA I on single antigen (SAB) beads. While the distribution differed among alleles, depending on antigen density (with Cw15 having the highest, and Cw4 the lowest, MFI), there was no significant difference between subclasses (R2=0.967). Both chimers also bound to native antigen on endothelial cells (EC). HLA I hIgG1 caused C1q deposition on all LSA1 beads in a concentration dependent manner. There was an association between SAB MFI and C1q binding that varied depending on HLA locus; on B locus beads (R2>0.9) but not C locus beads. C1q positivity (MFI>1000) correlated with MFI in SAB tests of >12000 MFI. C1q was also deposited on all HLA I beads in response to high dose HLA I hIgG2, although hIgG2 was significantly less potent than hIgG1 at fixing C1q. The degree of C1q deposition by HLA I hIgG2 varied significantly (MFI >25000 to <1000) from bead to bead at the same concentration of antibody, particularly on C locus beads. hIgG2 showed poor correlation between SAB MFI and C1q-fixing capacity. Both HLA I hIgG1 and hIgG2 also resulted in functional complement activation, as measured by C3d deposition on single antigen beads, C3b deposition on EC, and CDC-XM positivity in a concentration-dependent manner. hIgG1 was significantly more effective at complement activation than hIgG2. Conclusions: hIgG2, while less potent than hIgG1, fixed complement in C1q, C3b and CDC assays at high concentrations, challenging the notion that hIgG2 DSA is less pathogenic. Moreover, C1q deposition in solid phase assays was quite variable from bead to bead, at the same concentration of pan HLA I antibody, suggesting that antigen density or denatured state of antigen significantly influences sensitivity in complement assays. In general, HLA-C locus beads were the most variable in assays measuring hIgG and C1q binding. DISCLOSURE:Reed, E.: Grant/Research Support, Pfizer, Novartis, True North Therapeutics, Immucor.

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Aim The presence of C1q antibodies may be important for assessing the risk of antibody mediated rejection (AMR) in solid organ transplants. We studied the correlation between the C1q-single antigen bead assay (C1q-SAB) with the long-incubation complement-dependent lymphocytotoxicity assay (CDC) and the IgG-single antigen bead assay (IgG-SAB). Methods 60 reference sera (38 Class I and 22 Class II) from the UCLA serum exchange program were characterized using the CDC, Luminex PRA (Gen-probe Inc.) and IgG-SAB (One Lambda Inc.) assays. We also tested pre- and post-transplant sera from 16 renal allograft recipients. All patients had persistent posttransplant DSA as demonstrated by the IgG-SAB assay. Results The C1q-SAB assay showed 85% concordance with the CDC assay as 15% of the CDC positive antibodies were negative in the C1q test. Correlation between the SAB MFI and C1q positivity showed that 90% of sera with the MFI of >18,000 in the IgG-SAB assay were C1q binding. A retrospective study of 16 renal transplant recipients developing post-transplant donor specific antibodies (DSA) showed 5 of 9 patients diagnosed as C4d+ AMR displayed C1q positive DSA (P = 0.145). One out of 7 patients without AMR displayed C1q binding DSA. Conclusions Our data indicate that antibody strength correlates with the capacity to bind C1q (Fig. 1). However we identified a subset of antibodies that displayed high MFI values and CDC positivity, that failed to bind C1q. The development of C1q positive DSA was not significantly associated with positive C4d staining on the graft. Additional studies are required using a larger cohort of patients to verify these findings [Fig. 1]. Download : Download full-size image

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Aim Donor directed cytotoxic positive HLA antibodies have been associated with hyperacute rejection in solid organ transplantation. A potentially more sensitive new solid phase luminex single antigen –C1q assay is used as an adjunct to the CDC assay in detecting these cytotoxic antibodies. The aim of our study was to compare the binding of HLA antibodies in several assays: Luminex single antigen (LSA), LSA with diluted sera, C1q, enhanced C1q, and CDC assays to determine their accuracy, significance and relevance to Flow and CDC crossmatches Methods Sera from 39 recipients with multiple HLA class I and /or II antibodies by LSA were tested by LSA-C1q and CDC frozen panel assay. Enhanced LSA-C1Q assay using 4X serum volume and longer incubation time as well as flow and CDC crossmatch assays were performed on 5 samples that were CDC and/or flow positive and LSA-C1q negative. Results 72% of the time, the antibodies detected by the CDC assay was positive by LSA, LSA-dil tests, while 28% were positive by LSA and LSA-dil but negative by C1q. For two sera, negative LSA-C1q results were contributed by high background. The binding increased from negative/low positive by LSA-C1q to moderate/strong binding with enhanced LSA-C1q assay for 5 sera. The detection level of antibodies to DR15, DR16 and DR4 seemed to be problematic and had a low detection level by LSA-C1q assay but increased to positive by enhanced LSA-C1q assay. Conclusions sIn most cases LSA-C1q assay is efficient in identifying CDC+ antibodies. However there appear to be cases where the enhanced LSA-C1q and CDC assay detect antibodies missed by the C1q assay. Several test methods are required to determine the presence and function of HLA antibodies.

Pyrethroid resistance inAedes aegyptiandAedes albopictusfrom Port-au-Prince, Haiti

In Port-au-Prince, Haiti, the status of insecticide resistance has not recently been evaluated for Aedes aegypti (L) and Aedes albopictus (Skuse) populations. No prophylactics exist for dengue, so prevention is only through vector control methods. An earthquake occurred in Haiti on January 12, 2010, with a magnitude of 7.0 Mw that devastated the area. Dengue became a major concern for the humanitarian relief workers that entered the country. Bottle bioassays were conducted in the field on adult mosquitoes reared from larvae collected from the grounds of the U.S. Embassy and from an adjacent neighborhood in eastern Port-au-Prince, Haiti. At the CDC, Fort Collins, CO, bioassays, molecular, and biochemical assays were performed on mosquitoes reared from field-collected eggs. A small percentage of the population was able to survive the diagnostic dose in bioassays run in Haiti. Mosquitoes tested at the CDC demonstrated no phenotypic resistance. A variety of factors could be responsible for the discrepancies between the field and lab data, but temperature and larval nutrition are probably most important. Knowledge of localized resistance and underlying mechanisms helps in making rational decisions in selection of appropriate and effective insecticides in the event of a dengue outbreak.