Absence Of Target Cells Research Articles

Abstract 5677: MB101, a novel PD-L1 x CD3 targeted bispecific antibody, exhibits potent tumor-killing efficacy with minimal off-tumor toxicity

Abstract Bispecific antibody (bsAb) which targets tumor-associated antigens and simultaneously induces CD3-mediated T cell activation, is one of the promising therapeutic approaches for cancer treatment. However, despite numerous CD3-based bsAb have been developed, the clinical application faces pronounced hurdles, such as increased on-target off-tumor toxicity and cytokine release syndrome (CRS) by using high-affinity anti-CD3 moieties. Accordingly, there are still needs for the development of CD3-based bsAb with more favorable properties, which are tumor-specific targeting and potent tumor killing activity without excessive cytokine release. Thus, we developed MB101, a novel PD-L1 x CD3 bsAb with low affinity to CD3, which is uncoupled potent tumor cytotoxicity from severe cytokine release. MB101 showed a strong binding affinity to PD-L1 (KD = 0.4 nM) but a weak binding affinity to CD3 (KD = 1.0 μM), indicating preferentially targeting to PD-L1+ tumor cells. MB101 showed an effective tumor cell killing activity with higher Emax compared to avelumab mono- or combination treatment with CD3 mAb in PD-L1+ MDA-MB-231 cells. T cell-mediated cytotoxicity stimulated by MB101 in co-culture with target cells was accompanied by lower secretion of pro-inflammatory cytokines than CD3 mAb and bsAb comparator with high-affinity anti-CD3 arm. Additionally, in the absence of target cells, MB101 treatment to PBMC induced a very low level of pro-inflammatory cytokines, suggesting the possibility of reduced CRS risk. Furthermore, we proved that T cell-mediated cytotoxicity by MB101 was increased in a PD-L1 expression-dependent manner of tumor cells, but not normal cells, indicating minimal on-target off-tumor toxicity. Consistently with in vitro results, MB101 effectively regressed tumor volume (TGI=83%) without induction of excessive cytokine release in human CD3 knock-in mouse model. In addition, MB101 showed superior tumor growth inhibition (TGI=70%) than that of avelumab (TGI=44%) in humanized mouse xenograft model. In summary, MB101 selectively triggers robust PD-L1-dependent tumor cytotoxicity while inducing very low cytokine release, suggesting that MB101 might be a promising therapeutic option as CD3-based bsAb for cancer treatment. Citation Format: Suna Kim, Hyojoo Bang, Yongjun Jung, Sunjung Cho, Youngsam Kim, Sang-hyun Park, Seok Chan Kang, Youngjin Park, Sungyoub Jung. MB101, a novel PD-L1 x CD3 targeted bispecific antibody, exhibits potent tumor-killing efficacy with minimal off-tumor toxicity. [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 5677.

Abstract 2865: CC-3, an IgG-based B7-H3xCD3 bispecific antibody for targeting of gastrointestinal cancers

Abstract Despite substantial improvements over the last decades, survival rates in metastatic gastrointestinal cancer are still far from satisfactory, with an accordingly high medical need for new treatment strategies. B7-H3 (CD276) is a member of the B7 immune checkpoint family. Initially thought to act as co-stimulator, recent studies revealed that B7-H3 rather has an inhibitory role for T cells and contributes to tumor immune evasion. Clinically, its overexpression has been linked to invasive and metastatic potential as well as poor prognosis. Due to its expression on both, tumor cells and tumor vasculature, in a variety of cancer entities including colorectal cancer, B7-H3 attracted our interest as therapeutic target for T cell-recruiting bispecific antibodies (bsAbs). We postulate that “dual targeting” of both, the cancer cells and the tumor vasculature may support the influx of T cells into the tumor site, a critical prerequisite for successful immunotherapy of solid tumors allowing for subsequent destruction of antigen-positive malignant cells. We generated a panel of monoclonal antibodies directed to different epitopes of the B7-H3 molecule. After biochemical characterization, we selected two antibodies with distinct binding proprieties and subsequently used them for the construction of Tcell-recruiting B7-H3xCD3 bsAbs in an IgG-based (IgGsc) format. To reduce side effects, constructs were cloned using a UCHT-1 derived low affinity anti-CD3 sequence. In vitro characterization using colorectal and other carcinoma cells allowed for selection of a construct with optimal functional properties (thereafter termed CC-3) as revealed by target cell-restricted induction of T cell activation, proliferation and tumor cell killing. In vivo, potent efficacy of CC-3 was documented in a lung metastasis model and by its ability to eliminate large established flank tumors using immunocompromised NSG mice adoptively transferred with human effector cells. Despite the high therapeutic efficacy of CC-3, no toxicity was observed in the absence of target cells. In summary, CC-3 is a bsAb with promising therapeutic activity against B7-H3 positive tumors. GMP compliant production of CC-3 is presently ongoing to enable evaluation in a clinical “first in human” study in patients with colorectal cancer. Citation Format: Latifa Zekri, Martina S. Lutz, Ilona Hagelstein, Timo Manz, Monika Engel, Boris Klimovich, Nisha Prakash, Anna Chashchina, Sebastian Hörner, Stefanie Müller, Melanie Märklin, Martin Pflügler, Gundram Jung, Helmut R. Salih. CC-3, an IgG-based B7-H3xCD3 bispecific antibody for targeting of gastrointestinal cancers [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 2865.

Abstract P191: The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR+ tumor cells

Abstract The epidermal growth factor receptor (EGFR) is an overexpressed antigen on several solid cancers resulting in uncontrolled cell proliferation. Current EGFR-targeting agents such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) mainly rely on EGFR signaling inhibition which is challenged by limited efficacy due to dose limiting toxicity, and/or intrinsic or acquired resistance of the tumor by e.g., KRAS or BRAF mutations. To overcome these limitations and to address the high medical need across EGFR-positive cancers, we investigate a unique approach of combining AFM24, a tetravalent bispecific EGFR- and CD16A-binding innate cell engager (ICE), with the patient-specific NK cell product SNK01. AFM24 mediates high affinity targeting and ADCC of EGFR+ tumor cells by NK cells, independent of EGFR signaling inhibition and irrespective of the mutational status of the tumor. AFM24 is currently being tested as monotherapy in a Phase 1/2a study in patients with EGFR-positive tumors. SNK01 is a first-in-kind, autologous non genetically modified NK cell product with significant anti-tumor cytotoxicity and high and homogeneous expression of CD16A, NKG2D, NKp46 and DNAM-1 , that can be consistently produced even from heavily pre-treated cancer patients. SNK01 showed activity against numerous solid tumors in preclinical studies and in the phase I trials. In order to investigate the synergistic potential of this unique combination, SNK01 cells were generated from PBMCs of healthy individuals and their functional activation by AFM24 in response to EGFR-positive A-431 cells was assessed in cytotoxicity assays and flow cytometry-based CD107a (marker for NK cell degranulation) and IFN-γ mobilization assays. AFM24 binding to and CD16A expression on SNK01 cells was measured by flow cytometry. Both, binding of AFM24 and expression of CD16A were detected on nearly 100% of SNK01 cells, indicating saturation of CD16A-positive SNK01 cells with AFM24. In the presence of AFM24, lysis of EGFR-positive A-431 cells by SNK01 cells was significantly enhanced. Likewise, in response to AFM24, upregulation of CD107a and IFN-γ expression by SNK01 cells was significantly increased upon cross-linking to A-431 cells. By contrast, AFM24 alone did not lead to significant NK cell activation in the absence of target cells or vice versa. Hence, activation of SNK01 cells was strictly dependent on AFM24-mediated cross-linking of SNK01 cells with target cells. In conclusion, this combination holds promise to direct the AFM24-augmented anti-tumor activity of SNK01 cells to EGFR-positive tumor cells independent of EGFR signaling pathway mutations thereby providing a potential new treatment option for underserved patient populations. An IND for a Phase I/II combination trial of AFM24 and SNK01 has been cleared and first-patient-in is on track for the second half of 2021. Citation Format: Jens Pahl, Ting-Ying (Hazel) Cheng, Uwe Reusch, Arndt Schottelius, Wolfgang Fischer, Yong Man Kim, Stephen Chen, Joachim Koch. The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR+ tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P191.

CD18 Antibody Application Blocks Unwanted Off-Target T Cell Activation Caused by Bispecific Antibodies.

Simple SummaryBispecific antibodies are a very effective immunotherapy against different types of cancer since they activate T cells in the presence of tumor cells. However, they can cause severe side effects, such as a systemic inflammation called cytokine release syndrome. We aimed to clarify an important mechanism that causes cytokine release syndrome. In cocultures of T cells with endothelial cells or lymphoid cells, application of bispecific antibodies can induce T cell activation and cytokine release in the absence of tumor cells. By blocking the adhesion molecule CD18, this interaction is interrupted and the unwanted T cell activation is diminished. CD18 blockade, however, does not interfere with T cell activation when tumor cells are present. Therefore, CD18 blockade could prevent side effects of bispecific antibodies without decreasing the anti-tumor effect.T cell-recruiting bispecific antibodies (bsAbs) are successfully used for the treatment of cancer. However, effective treatment with bsAbs is so far hampered by severe side effects, i.e., potentially life-threatening cytokine release syndrome. Off-target T cell activation due to binding of bispecific CD3 antibodies to T cells in the absence of target cells may contribute to excessive cytokine release. We report here, in an in vitro setting, that off-target T cell activation is induced by bsAbs with high CD3 binding affinity and increased by endothelial- or lymphoid cells that act as stimulating bystander cells. Blocking antibodies directed against the adhesion molecules CD18/CD54 or CD2/CD58 markedly reduced this type of off-target T cell activation. CD18 blockade—in contrast to CD2—did not affect the therapeutic activity of various bsAbs. Since CD18 antibodies have been shown to be safely applicable in patients, blockade of this integrin holds promise as a potential target for the prevention of unwanted off-target T cell activation and allows the application of truly effective bsAb doses.

Synergistic Effect by Combining a gp120-Binding Protein and a gp41-Binding Antibody to Inactivate HIV-1 Virions and Inhibit HIV-1 Infection

Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.

Mechanistic Studies of Lassa Fever Virus Glycoprotein-Mediated Membrane Fusion

Lassa Fever Virus (LASV) GPC glycoprotein binds to α-dystroglycan on the cell surface, allowing virus uptake and transport to acidic late endosomes. GPC then engages the LAMP1 receptor and mediates fusion of viral and endosomal membranes. To overcome difficulties associated with mechanistic studies of virus-endosome fusion, we investigated LASV GPC-function using a cell-cell fusion model. We found that cells expressing LASV GPC fuse to mammalian cells upon exposure to mildly acidic pH 5.5-6.2 and that this fusion is enhanced by ectopic expression of LAMP1. Consistent with the role of LAMP1 as the LASV receptor, avian cells expressing a divergent LAMP1 orthologue did not support GPC-mediated fusion at mildly acidic pH, but ectopic expression of human LAMP1 or exposure to pH below 5.0 rescued fusion. These findings demonstrate that human LAMP1 enhances fusion efficiency and shifts the fusion optimum to less acidic pH, but it is dispensable for GPC function. Consistent with this notion, we show that LASV GPC is irreversibly inactivated when exposed to pH below 5.0 in the absence of target cells. Further mechanistic experiments show that, similar to other viral glycoproteins, LASV GPC-mediated fusion proceeds through a hemifusion intermediate, which can be arrested by exposure to low pH in the cold, and that this intermediate can be converted to full fusion by chlorpromazine treatment. Incorporation of lyso-phosphatidylcholine into the plasma membrane inhibits GPC-induced hemifusion, consistent with fusion proceeding through a highly curved, negative curvature lipid intermediate (stalk). Real-time measurements of transfer of fluorescent cytoplasmic markers shows that LASV GP-mediated fusion pores did not enlarge as readily as pores formed by other viral glycoproteins, likely due to a less optimal composition of the plasma membrane compared to the limiting membrane of late endosomes where LASV fusion occurs.

Abstract 3239: SMIC CAR T cells: CAR T with temporally-controlled, programmable cytokine signaling outputs

Abstract CAR T cell therapy has attained unprecedented success in the treatment of hematological malignancies. However, clinical benefit in solid tumor indications has been limited in part due to suppressive solid tumor microenvironments (TME) that inhibit T cell effector function and persistence. While the provision of cytokine support can help CAR T cells overcome suppressive TMEs, conventional approaches, such as combining CAR T therapy with systemically-administered cytokines/cytokine mimetics, or engineering CAR T cells to constitutively secrete cytokines can result in toxicities and adverse events. To mitigate these safety concerns, we designed Small Molecule-Inducible Cytotails (SMIC) that can be specifically activated by the clinically validated dimerizer drug, AP1903. SMIC is a chimeric fusion receptor comprising the FKBP(F36V) ectodomain, the transmembrane and JAK-activating domains of TpoR and one or more intracellular STAT-binding domains derived from the cytokine receptor (Cytotail) of interest. In response to AP1903, different SMIC designs resulted in downstream STAT activation that mimicked signaling of the parental cytokine receptor. Furthermore, two Cytotails could be fused in tandem to generate dual signaling outputs from a single receptor. Using 2nd generation CARs directed towards solid tumor targets, we demonstrated that SMIC CAR T cells bearing different Cytotail fusions could be programmed towards divergent phenotypes, such as those promoting expansion (IL7Rα Cytotail), memory maintenance (EGFR Cytotail) or effector function (IL12Rβ Cytotail). RNASeq analysis of SMIC CAR T cells bearing an IL7Rα.IL12Rβ tandem Cytotail (SMIC7.12) revealed near-identical gene expression profiles to control CAR T cells treated with exogenously supplemented IL-7 and IL-12, demonstrating that SMIC closely mimicked the signaling of native cytokine receptors. In the absence of target cells, AP1903 treatment alone was sufficient to expand SMIC7.12 CAR T cells more efficiently than exogenously supplemented IL-7 and IL-12, while preserving a juvenile memory phenotype. Compared to control CAR T cells, AP1903-activated SMIC7.12 enhanced CAR T cell cytotoxicity in vitro, and promoted CAR T cell activity in a subcutaneous tumor model. In conclusion, SMIC represents a novel chimeric cytokine receptor that can improve the potency and persistence of CAR T cells, while minimizing safety risks associated with cytokine co-therapy or constitutive cytokine secretion. Furthermore, fusion of different/combinatorial Cytotails to SMIC offers the flexibility for user-programmable signaling outputs, permitting control over CAR T cell phenotype and function. Citation Format: Regina Lin, Andrew Nager, Spencer Park, Janette Sutton, Zea Melton, Yi Zhang, Bijan Boldajipour, Tom Van Blarcom, Siler Panowski, Javier Chaparro-Riggers, Barbra Sasu. SMIC CAR T cells: CAR T with temporally-controlled, programmable cytokine signaling outputs [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 3239.

Development of a bioassay to detect T-cell-activating impurities for T-cell-dependent bispecific antibodies

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit a patient’s T cells to kill cancer cells. There are increasing numbers of TBDs in clinical trials, demonstrating their widely recognized therapeutic potential. Due to the fact that TDBs engage and activate T cells via an anti-CD3 (aCD3) arm, aCD3 homodimer (aCD3 HD) and high-molecular-weight species (HMWS) are product-related impurities that pose a potential safety risk by triggering off-target T-cell activation through bivalent engagement and dimerization of T-cell receptors (TCRs). To monitor and control the level of unspecific T-cell activation, we developed a sensitive and quantitative T-cell-activation assay, which can detect aCD3 HD in TDB drug product by exploiting its ability to activate T cells in the absence of target cells. This assay provides in-vivo-relevant off-target T-cell-activation readout. Furthermore, we have demonstrated that this assay can serve as a platform assay for detecting T-cell-activating impurities across a broad spectrum of aCD3 bispecific molecules. It therefore has the potential to significantly benefit many T-cell-recruiting bispecific programs.

Immunocytochemistry Based on a Cell-Type-Specific Aptamer for Rapid Immunostaining of Adenocarcinoma Cells in Clinical Serosal Fluids

All too often, conventional immunocytochemistry (ICC) via an antibody on cytological samples is limited to a few smears due to scant cellularity. To circumvent these limitations, this study employed a cell-type-specific aptamer as the core tool in ICC protocols for a timely and highly specific ICC diagnosis. S6, an aptamer against A549 lung carcinoma cells, was adopted instead of antibodies in this study for differentiating cancer cells in serosal fluids. Here, we developed three different strategies for discriminating the adenocarcinoma cells in effusion cytology specimens using the S6 aptamer in ICC. These strategies included a biotin-labeled S6 aptamer, an FAM-labeled S6 aptamer, and an activatable S6 aptamer. A total of 112 serosal fluid specimens with known diagnoses were evaluated by all three modes of use of the S6 aptamer. ICC procedures based on biotin-labeled or FAM-labeled S6 aptamers required time-consuming washing to avoid interference from nonspecific adsorption. ICC procedures based on an activatable S6 aptamer probe showed a weak fluorescence signal in the absence of target cells, but the procedures showed a strong fluorescence signal due to alteration of the conformation without any complicated washing steps, in the presence of targets. The specificity and sensitivity are higher in all three different ICC protocols based on the S6 aptamer than those for antibody protocols for differentiating adenocarcinoma cells in clinical effusion cytology. ICC based on cell-type-specific aptamers, instead of on a panel of a set of antibodies, is promising as an auxiliary method for the diagnosis of cancer.

Development of a BiTE®-mediated CD8+ cytotoxic T-lymphocyte activity assay to assess immunomodulatory potential of drug candidates in Cynomolgus macaque

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of studies and endpoints. While the functional assessment of CD8+ cytotoxic T-lymphocytes (CTL) is well-characterized in the clinic, the lack of a robust macaque CTL functional assay has been an important hurdle in evaluating and accurately quantifying cell-mediated CD8+ T-cell effector responses in the nonclinical setting. This paper describes the development of an assay to measure CTL activity in peripheral blood mononuclear cells (PBMC) isolated from Cynomolgus macaques. A human EGFR/CD3 Bispecific T-cell Engager (BiTE®) was used to mount a robust CD8+ T-cell response in the presence of target-expressing cells. Upon target engagement, degranulation of CD107a and production of interferon (IFN)-γ both reliably indicated a robust functional response in CD8+ T-cells. The BiTE®-mediated stimulation method proved to be favorable when compared to other methods of stimulation in the absence of target cells. These studies demonstrated acceptable longitudinal variability of the functional assay and sensitivity to dexamethasone-mediated immunosuppression. Taken together, the results indicated an assay leveraging CD3-bispecific antibodies and target-expressing cells can provide a robust approach to the in vitro or ex vivo assessment of CTL function in Cynomolgus macaques. Because the impairment of CTL activity by immunomodulators is recognized to be an important contributor to decreased antiviral defense and increased carcinogenicity risk, we believe that this novel assay to be a valuable addition to the immunotoxicology assessment of therapeutic drug candidates.

Abstract B26: Immunomodulatory activity of Isatuximab

Abstract Isatuximab, previously known as SAR650984 is a naked chimeric IgG1 anti-CD38 monoclonal antibody. It is currently in clinical trials demonstrating single-agent activity in heavily pretreated relapsed/refractory multiple myeloma (RRMM). Isatuximab induces killing of CD38 positive tumor cells by triggering Fc dependent effector functions such as ADCC, ADCP and CDC, as well as Fc independent direct apoptotic effect. However, its effects in immune cells that express CD38 are emerging and are not yet well defined. The aim of this study was to investigate the effects of Isatuximab on immune effector cells, to better understand its anti-tumor mechanisms underlying the clinical benefit. Among immune sub-populations, Natural Killer (NK) cells and monocytes have the highest CD38 receptor density. Further, NK cells and monocytes play critical roles in immunity against cancer. We investigated Isatuximab's effect on these immune effector cells including their expansion, cytokine secretion and activation. Treatment (8 days at 1µg/ml) of 106 whole human peripheral blood mononuclear cells with Isatuximab induced expansion of NK and monocytes. In addition to enhance expansion, Isatuximab was able to induce the release of IFN-γ (~4000-7000 pg/ml) and TNF-α (~150-200 pg/ml) in NK cells in the absence of target cells. Incubation of Isatuximab (1µg/ml) for an hour also enhanced cytotoxic activity of human NK cells, both NK-92 cell line and isolated human NK cells, against CD38 negative K562 and JHH6 tumor cell lines. In the case of monocytes, Isatuximab activates phagocytosis by human monocytic cell line THP-1 and purified human monocytes. Furthermore, Isatuximab-activated NK cells can induce monocyte to M1 macrophage polarization through IFN-γ release in vitro. These findings collectively show evidence of Isatuximab being an immune-modulatory agent able to induce NK cell and monocyte activation. Further study is needed to determine whether Isatuximab's immunomodulatory activity could be utilized for the treatment of both CD38-positive and negative cancers by enhancing its anti-tumor function. Citation Format: Srimathi S. Srinivasan, Anlai Wang, Zhili Song, Francisco Adrián. Immunomodulatory activity of Isatuximab. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B26.

A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification

A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 105 cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis.

Abstract 1489: Development of a humanized ROR1 x CD3 bispecific DART® molecule for the treatment of solid and liquid tumors

Abstract Introduction: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in chronic lymphocytic leukemia and a subset of solid tumors, including lung, breast, ovarian, colon, and pancreatic cancers, as well as sarcoma. Limited adult tissue expression and its absence in normal leukocytes makes ROR1 a promising cancer therapeutic target. We have developed a Dual-Affinity Re-Targeting (DART®) protein for redirecting T lymphocytes to lyse tumor cells via monovalent recognition of ROR1 on tumors and CD3 on T cells. ROR1 x CD3 DART protein was engineered for improved half-life with the incorporation of a modified Fc domain, lacking effector function. Methods: The ROR1 x CD3 DART protein was stably expressed in CHO cells and purified to homogeneity by a standard antibody platform. Bispecific binding was evaluated by ELISA and SPR analysis. In vitro functional studies were performed with lymphoma and solid tumor cell lines in the presence of primary human T cells. Tumor growth inhibition was evaluated in NOD/SCID/IL-2 gamma chain KO (NOG) mice coimplanted with human T cells and either mantle cell lymphoma (MCL) or lung cancer cell lines (5:1 effector : target cell ratio) followed by treatment with ROR1 x CD3 DART protein by intravenous (IV) administration. In vivo activity was also evaluated in human PBMC-reconstituted NOG/B2m deficient mice bearing established intradermal tumor xenografts following IV treatment with ROR1 x CD3 DART molecule. Pharmacokinetic analysis of the DART molecule was performed in human neonatal Fc receptor (hFcRn) transgenic mice. Results: The ROR1 x CD3 DART protein displayed the expected bispecific binding for ROR1 and CD3 antigens and retained the affinity and specificity of the parent mAbs. The DART molecule mediated dose-dependent lysis of ROR1-positive MCL and solid tumor (breast, lung, and osteosarcoma) cell lines through recruitment of human T cells. DART molecule-mediated killing of ROR1-expressing target cells was accompanied by target-dependent T-cell activation and cytokine release; however, no activity was observed in the absence of target cells and no cytokine release was observed with human PBMCs alone. The ROR1 x CD3 DART protein displayed extended circulating half-life after administration to hFcRn-transgenic mice. In mouse efficacy studies, the growth of HBL-2 (MCL), HOP-92 (lung cancer), or NIH-1975 (a lung cancer line resistant to erlotinib) cells co-implanted with human T cells in NOG mice was inhibited by treatment with the ROR1 x CD3 DART protein at doses in the mcg/kg range. The ROR1 x CD3 DART molecule also demonstrated antitumor activity with high complete response rates in human PBMC-reconstituted mice bearing established HBL-2 cell xenografts. Conclusion: The promising in vitro and in vivo activity of the Fc-bearing ROR1 x CD3 DART molecule supports further investigation as a potential candidate for the cancer treatment. Citation Format: Bhaswati Barat, Gurunadh Chichili, Valentina Ciccarone, James Tamura, Sergey Gorlatov, Michael Spliedt, Francine Chen, Scott Koenig, Paul Moore, Ezio Bonvini, Ralph Alderson, Syd Johnson. Development of a humanized ROR1 x CD3 bispecific DART® molecule for the treatment of solid and liquid tumors. [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 1489.

In Vitro and In Vivo Characterization of CD19/CD3 Tandab AFM11 and CD19/CD16A Tandab AFM12 Targeting NHL

Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12.Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed.Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described.Conclusions:Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated. DisclosuresNo relevant conflicts of interest to declare.

Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization

A simple but highly sensitive colorimetric method was developed to detect cancer cells based on aptamer–cell interaction. Cancer cells were able to capture nucleolin aptamers (AS 1411) through affinity interaction between AS 1411 and nucleolin receptors that are over expressed in cancer cells, The specific binding of AS 1411 to the target cells triggered the removal of aptamers from the solution. Therefore no aptamer remained in the solution to hybridize with complementary ssDNA-AuNP probes as a result the solution color is red. In the absence of target cells or the presence of normal cells, ssDNA-AuNP probes and aptamers were coexisted in solution and the aptamers assembled DNA-AuNPs, produced a purple solution. UV–vis spectrometry demonstrated that this hybridization-based method exhibited selective colorimetric responses to the presence or absence of target cells, which is detectable with naked eye. The linear response for MCF-7 cells in a concentration range from 10 to 105 cells was obtained with a detection limit of 10 cells. The proposed method could be extended to detect other cells and showed potential applications in cancer cell detection and early cancer diagnosis.

Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet-aptamer nanoprobe.

A novel dual-activatable fluorescence/MRI bimodal platform is designed for tumor cell imaging by using a redoxable manganese dioxide (MnO2) nanosheet-aptamer nanoprobe. The redoxable MnO2 nanosheet acts as a DNA nanocarrier, fluorescence quencher, and intracellular glutathione (GSH)-activated MRI contrast agent. In the absence of target cells, neither fluorescence signaling nor MRI contrast of the nanoprobe is activated. In the presence of target cells, the binding of aptamers to their targets weakens the adsorption of aptamers on the MnO2 nanosheets, causing partial fluorescence recovery, illuminating the target cells, and also facilitating the endocytosis of nanoprobes into target cells. After endocytosis, the reduction of MnO2 nanosheets by GSH further activates the fluorescence signals and generates large amounts of Mn(2+) ions suitable for MRI. This platform should facilitate the development of various dual-activatable fluorescence/MRI bimodalities for use in cells or in vivo.

Visual and highly sensitive detection of cancer cells by a colorimetric aptasensor based on cell-triggered cyclic enzymatic signal amplification.

Rapid and efficient detection of cancer cells at their earliest stages is one of the central challenges in cancer diagnostics. We developed a simple, cost-effective, and highly sensitive colorimetric method for visually detecting rare cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and linker DNAs stably coexist in solution, and the linker DNA assembles DNA-AuNPs, producing a purple solution. In the presence of target cells, the specific binding of HAPs to the target cells triggers a conformational switch that results in linker DNA hybridization and cleavage by nicking endonuclease-strand scission cycles. Consequently, the cleaved fragments of linker DNA can no longer assemble into DNA-AuNPs, resulting in a red color. UV-vis spectrometry and photograph analyses demonstrated that this CTCESA-based method exhibited selective and sensitive colorimetric responses to the presence of target CCRF-CEM cells, which could be detected by the naked eye. The linear response for CCRF-CEM cells in a concentration range from 10(2) to 10(4) cells was obtained with a detection limit of 40 cells, which is approximately 20 times lower than the detection limit of normal AuNP-based methods without amplification. Given the high specificity and sensitivity of CTCESA, this colorimetric method provides a sensitive, label-free, and cost-effective approach for early cancer diagnosis and point-to-care applications.

Specific gene delivery to liver sinusoidal and artery endothelial cells

AbstractDifferent types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

Abstract 4572: The CD30/CD16A RECRUIT TandAb AFM13 activates patient NK cells resulting in specific tumor cell killing.

Abstract AFM13 represents a new class of bispecific antibodies, based on the RECRUIT-TandAb platform, which enables the generation of therapeutics with improved efficacy, convenient administration regimens, and excellent safety profiles. AFM13 was developed for the treatment of Hodgkin Lymphoma (HL) and other CD30+ malignancies. AFM13 targets CD30 on HL tumor cells and recruits NK cells via CD16A. Preclinical data demonstrate specific and highly efficient anti-tumor activity by the selectively recruited NK cell immune response, which addresses key deficiencies of monoclonal antibodies: (i) reduced binding to the 158F allotype of CD16A and (ii) non-selective binding to immune effector cells via both activating CD16A and non-signalling CD16B receptors. In vitro assays demonstrate that AFM13 binds with high affinity to both CD30 and CD16A antigens and rapidly induces lysis of CD30+ cells at picomolar concentrations. Further, AFM13 activates CD16A only in the presence of tumor cells: there is no systemic activation of NK-cells in the absence of target cells. A robust GMP process in mammalian cells and a lyophilized formulation with excellent stability are established. Finally, no toxicity is observed in Cynomolgus monkeys. AFM13 was investigated in an open-label single-arm phase I dose escalation trial in heavily pre-treated patients with relapsed/refractory HL. Each patient received 4 weekly doses of AFM13. Seven dose levels from 0.01 to 7.0 mg/kg were escalated in cohorts of 3 patients. In addition, one cohort of 4 patients received AFM13 twice a week at 4.5 mg/kg for 4 weeks. All dose levels of AFM13 proved to be well tolerated and safe and clear signs of anti-tumor activity were demonstrated. We have performed an analysis of the activation of NK cells and clearance of soluble CD30 in humans since these are key parameters for the mode of action of AFM13. We confirmed a correlation between anti-tumor activity parameters and biomarkers based on a statistically significant dose-dependent increase in the activation of NK cells and reduction in soluble CD30 levels. Since the activation of NK cells has been reported to facilitate low-level expression of CD30 on NK cells, which we also observed in several patients in the highest AFM13 dose groups, we investigated whether this may affect NK cell cytotoxicity. Our ex vivo evaluation of samples from patients that received AFM13 and in vitro evaluation of activated NK cells demonstrated no inhibition of NK cell-mediated cytotoxic activity. Furthermore, AFM13 exhibits a remarkable half-life of 1 day which represents a substantial increase relative to that of diabody-like formats currently being evaluated in the clinic for haematological malignancies. Thus, AFM13 has demonstrated encouraging biologic activity and represents a new targeted therapy for heavily pre-treated patients with HL. Citation Format: Achim Rothe, Max S. Topp, Anas Younes, Uwe Reusch, Stefan Knackmuss, Erich Rajkovic, Dennis A. Eichenauer, Horst Hummel, Katrin S. Reiners, Markus Dietlein, Joerg Kessler, Miroslav Ravic, Christian Hucke, Eugene Zhukovsky, Elke Pogge von Strandmann, Andreas Engert. The CD30/CD16A RECRUIT TandAb AFM13 activates patient NK cells resulting in specific tumor cell killing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2013-4572

Modeling Killer Cell Migration and Search Strategies

During the immune response, cytotoxic T lymphocytes (CTL) and Natural Killer cells (NK cells) patrol the human body. Upon recognition of virus-infected or cancerous target cells, the killer cells release lytic granules at the immunological synapse and kill the targets. For optimal immune function, the migration machinery has to be adjusted in order to maximize the search efficiency.We used in vitro time lapse microscopy on primary human CTL and NK cells to study their migration in a two-dimensional geometry. In the absence of target cells, CTL alternate between a mobile and a stationary state. The preference for each state depends on the external calcium concentration. On a short time scale (seconds/minutes), killer cell migration is characterized by directional persistence, while migration on a longer time scale (minutes/hours) is random. A mathematical model of a persistent random walker can accurately describe killer cell migration and reveals an optimum for target search efficiency. This optimum depends on the length of persistent motion in one direction and the size of the search space. We believe that this model is very useful to understand how regulation of killer cell migration can serve to optimize the immune response.