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Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcγRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, co-targeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential., Competing Interests: AF, MH, SD and TS are co-founders and shareholders of YUMAB GmbH. SL, CK, MG, MP and YUMAB GmbH submitted a patent application related to the described antibodies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lutz, Klausz, Albici, Ebinger, Sellmer, Teipel, Frenzel, Langner, Winterberg, Krohn, Hust, Schirrmann, Dübel, Scherließ, Humpe, Gramatzki, Kellner and Peipp.)

Immune checkpoint blockade is a compelling approach in tumor immunotherapy. Blocking inhibitory pathways in T cells has demonstrated clinical efficacy in different types of cancer and may hold potential to also stimulate innate immune responses. A novel emerging potential target for immune checkpoint therapy is leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1). LILRB1 belongs to the superfamily of leukocyte immunoglobulin-like receptors and exerts inhibitory functions. The receptor is expressed by a variety of immune cells including macrophages as well as certain cytotoxic lymphocytes and contributes to the regulation of different immune responses by interaction with classical as well as non-classical human leukocyte antigen (HLA) class I molecules. LILRB1 has gained increasing attention as it has been demonstrated to function as a phagocytosis checkpoint on macrophages by recognizing HLA class I, which represents a 'Don't Eat Me!' signal that impairs phagocytic uptake of cancer cells, similar to CD47. The specific blockade of the HLA class I:LILRB1 axis may provide an option to promote phagocytosis by macrophages and also to enhance cytotoxic functions of T cells and natural killer (NK) cells. Currently, LILRB1 specific antibodies are in different stages of pre-clinical and clinical development. In this review, we introduce LILRB1 and highlight the features that make this immune checkpoint a promising target for cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zeller, Münnich, Windisch, Hilger, Schewe, Humpe and Kellner.)

Introduction: Primary human blood cells represent an essential model system to study physiology and disease. However, human blood is a limited resource. During healthy donor plateletpheresis, the leukoreduction system chamber (LRSC) reduces the leukocyte amount within the subsequent platelet concentrate through saturated, fluidized, particle bed filtration technology. Normally, the LRSC is discarded after apheresis is completed. Compared to peripheral blood, LRSC yields 10-fold mononuclear cell concentration., Methods: To explore if those retained leukocytes are attractive for research purposes, we isolated CD3+ T cells from the usually discarded LRSCs via density gradient centrifugation in order to manufacture CD19-targeted chimeric antigen receptor (CAR) T cells., Results: Immunophenotypic characterization revealed viable and normal CD4+ and CD8+ T-cell populations within LRSC, with low CD19+ B cell counts. Magnetic-activated cell sorting (MACS) purified CD3+ T cells were transduced with CD19 CAR-encoding lentiviral self-inactivating vectors using concentrated viral supernatants. Robust CD19 CAR cell surface expression on transduced T cells was confirmed by flow cytometry. CD19 CAR T cells were further enriched through anti-CAR MACS, yielding 80% CAR+ T-cell populations. In vitro CAR T cell expansion to clinically relevant numbers was achieved. To prove functionality, CAR T cells were co-incubated with the human CD19+ B cell precursor leukemia cell line Nalm6. Compared to unmodified T cells, CD19 CAR T cells effectively eradicated Nalm6 cells., Conclusion: Taken together, we can show that lymphocytes isolated from LRSCs of plateletpheresis sets can be efficiently used for the generation of functional CAR T cells for experimental purposes., Competing Interests: The authors confirm that none of the material has been published or is under consideration elsewhere. S.K. has received honoraria from TCR2 Inc., Novartis, BMS, and GSK SK and is inventor of several patents in the field of immuno-oncology. S.K. received license fees from TCR2 Inc. and Carina Biotech. S.K. received research support from TCR2 Inc., Tabby Therapeutics, Plectonic GmbH, and Arcus Bioscience for work unrelated to the manuscript. A.G. received research support from Tabby Therapeutics. The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Antibody-dependent cellular phagocytosis (ADCP) by macrophages, an important effector function of tumor targeting antibodies, is hampered by 'Don´t Eat Me!' signals such as CD47 expressed by cancer cells. Yet, human leukocyte antigen (HLA) class I expression may also impair ADCP by engaging leukocyte immunoglobulin-like receptor subfamily B (LILRB) member 1 (LILRB1) or LILRB2. Analysis of different lymphoma cell lines revealed that the ratio of CD20 to HLA class I cell surface molecules determined the sensitivity to ADCP by the combination of rituximab and an Fc-silent variant of the CD47 antibody magrolimab (CD47-IgGσ). To boost ADCP, Fc-silent antibodies against LILRB1 and LILRB2 were generated (LILRB1-IgGσ and LILRB2-IgGσ, respectively). While LILRB2-IgGσ was not effective, LILRB1-IgGσ significantly enhanced ADCP of lymphoma cell lines when combined with both rituximab and CD47-IgGσ. LILRB1-IgGσ promoted serial engulfment of lymphoma cells and potentiated ADCP by non-polarized M0 as well as polarized M1 and M2 macrophages, but required CD47 co-blockade and the presence of the CD20 antibody. Importantly, complementing rituximab and CD47-IgGσ, LILRB1-IgGσ increased ADCP of chronic lymphocytic leukemia (CLL) or lymphoma cells isolated from patients. Thus, dual checkpoint blockade of CD47 and LILRB1 may be promising to improve antibody therapy of CLL and lymphomas through enhancing ADCP by macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zeller, Lutz, Münnich, Windisch, Hilger, Herold, Tahiri, Banck, Weigert, Moosmann, von Bergwelt-Baildon, Flamann, Bruns, Wichmann, Baumann, Valerius, Schewe, Peipp, Rösner, Humpe and Kellner.)

Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies' effector functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gehlert, Rahmati, Boje, Winterberg, Krohn, Theocharis, Cappuzzello, Lux, Nimmerjahn, Ludwig, Lustig, Rösner, Valerius, Schewe, Kellner, Klausz and Peipp.)

Immunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with "double-hit" diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all- trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Baumann, Arndt, Petersen, Lustig, Rösner, Klausz, Kellner, Bultmann, Bastian, Vogiatzi, Leusen, Burger, Schewe, Peipp and Valerius.)

Natural killer (NK) cells exert an important role in cancer immune surveillance. Recognition of malignant cells and controlled activation of effector functions are facilitated by the expression of activating and inhibitory receptors, which is a complex interplay that allows NK cells to discriminate malignant cells from healthy tissues. Due to their unique profile of effector functions, the recruitment of NK cells is attractive in cancer treatment and a key function of NK cells in antibody therapy is widely appreciated. In recent years, besides the low-affinity fragment crystallizable receptor for immunoglobulin G (FcγRIIIA), the activating natural killer receptors p30 (NKp30) and p46 (NKp46), as well as natural killer group 2 member D (NKG2D), have gained increasing attention as potential targets for bispecific antibody-derivatives to redirect NK cell cytotoxicity against tumors. Beyond modulation of the receptor activity on NK cells, therapeutic targeting of the respective ligands represents an attractive approach. Here, novel therapeutic approaches to unleash NK cells by engagement of activating NK-cell receptors and alternative strategies targeting their tumor-expressed ligands in cancer therapy are summarized., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. 
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P8-D6 is a novel dual inhibitor of human topoisomerase I (TOP1) and II (TOP2) with broad pro-apoptotic antitumor activity. NCI-60 screening revealed markedly improved cytotoxicity of P8-D6 against solid and leukemia cell lines compared with other single and dual topoisomerase inhibitors, for example, irinotecan, doxorubicin, or pyrazoloacridine. In this study, we investigated the capacity of P8-D6 to inhibit myeloma cell growth in vitro and in vivo Growth inhibition assays demonstrated significant anti-myeloma effects against different myeloma cell lines with IC 50 values in the low nanomolar range. Freshly isolated plasma cells of patients with multiple myeloma were killed by P8-D6 with similar doses. P8-D6 activated caspase 3/7 and induced significant apoptosis of myeloma cells. Supportive effects of bone marrow stromal cells on IL6-dependent INA-6 myeloma cells were abrogated by P8-D6 and apoptosis occurred in a time- and dose-dependent manner. Of note, healthy donor peripheral blood mononuclear cells and human umbilical vein endothelial cells were not affected at concentrations toxic for malignant plasma cells. Treatment of myeloma xenografts in immunodeficient SCID/beige mice by intravenous and, notably, also oral application of P8-D6 markedly inhibited tumor growths, and significantly prolonged survival of tumor-bearing mice., (©2021 American Association for Cancer Research.)

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Blockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab, and obinutuzumab are of human immunoglobulin G1 (IgG1) isotype. We investigated the impact of the variable regions of these 3 CD20 antibodies when expressed as human IgA2 isotype variants. All 3 IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear cells. Both effector mechanisms were significantly enhanced in the presence of a CD47-blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, an IgA2 variant of obinutuzumab (OBI-IgA2) was consistently more potent than an IgA2 variant of rituximab (RTX-IgA2) or an IgA2 variant of ofatumumab (OFA-IgA2) in triggering ADCC. Furthermore, we observed more effective direct tumor cell killing by OBI-IgA2 compared with RTX-IgA2 and OFA-IgA2, which was caspase independent and required a functional cytoskeleton. IgA2 variants of all 3 antibodies triggered complement-dependent cytotoxicity, with OBI-IgA2 being less effective than RTX-IgA2 and OFA-IgA2. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX-IgA2 or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which may be relevant in the context of CD47-SIRPα blockade., (© 2021 by The American Society of Hematology.)

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Despite several therapeutic advances, patients with multiple myeloma (MM) require additional treatment options since no curative therapy exists yet. In search of a novel therapeutic antibody, we previously applied phage display with myeloma cell screening and developed TP15, a scFv targeting intercellular adhesion molecule 1 (ICAM-1/CD54). To more precisely evaluate the antibody's modes of action, fully human IgG1 antibody variants were generated bearing wild-type (MSH-TP15) or mutated Fc to either enhance (MSH-TP15 Fc-eng.) or prevent (MSH-TP15 Fc k.o.) Fc gamma receptor binding. Especially MSH-TP15 Fc-eng. induced potent antibody-dependent cell-mediated cytotoxicity (ADCC) against malignant plasma cells by efficiently recruiting NK cells and engaged macrophages for antibody-dependent cellular phagocytosis (ADCP) of tumor cells. Binding studies with truncated ICAM-1 demonstrated MSH-TP15 binding to ICAM-1 domain 1-2. Importantly, MSH-TP15 and MSH-TP15 Fc-eng. both prevented myeloma cell engraftment and significantly prolonged survival of mice in an intraperitoneal xenograft model. In the subcutaneous model MSH-TP15 Fc-eng. was superior to MSH-TP15, whereas MSH-TP15 Fc k.o. was not effective in both models - reflecting the importance of Fc-dependent mechanisms of action also in vivo. The efficient recruitment of immune cells and the potent anti-tumor activity of the Fc-engineered MSH-TP15 antibody hold significant potential for myeloma immunotherapy.

BACKGROUND Organ donation-rates using deceased donors and organizational approaches to organ donation differ drastically between countries at a similar level of health care as measured by the Euro Health Consumer Index (EHCI). MATERIAL AND METHODS Expert opinions from intensive care nurses, physicians, transplant coordinators and transplant surgeons from Austria, Germany, Spain, and the U.K. were obtained in semi-structured interviews followed by qualitative content analysis. Results were reported back to all interview partners to identify potential controversies and consensus recommendations. RESULTS No controversies could be detected. On a variety of beneficial factors an interprofessional consensus between interview partners could be reached: A) The relevance of standardization of the screening for potential donors, the family approach and training; B) standards and best-practice procedures should be regulated and supervised by state authorities; C) full transparency and the prevention of scandals is essential; D) overburdened intensive care unit (ICU) doctors need to be supported by full-time in-house special nurses who organize donor evaluation, transport logistics and pastoral care, if required; E) public awareness campaigns are helpful; F) a broad public consensus on the concept of donation after brain and cardiac death is essential; G) incentives for the reporting of potential organ donors are inappropriate; H) an opt-out system alone is not sufficient. CONCLUSIONS Expert opinions from different professional backgrounds from different European health care systems reach a broad consensus on the most relevant issues for the improvement of organ donation.

An enhanced expression of human epidermal growth factor receptor 2 (HER2, ErbB2) often occurs in an advanced stage of breast, ovarian, gastric or esophageal cancer, and pancreatic ductal adenocarcinoma (PDAC). Commonly, HER2 expression is associated with poor clinical outcome or chemoresistance in ovarian and breast cancer patients. Treatment with humanized anti-HER2 monoclonal antibodies, such as trastuzumab or pertuzumab, has improved the outcome of patients with HER2-positive metastatic gastric or breast cancer, but not all patients benefit. In this study, the bispecific antibody [(HER2) 2 xCD16] in the tribody format was employed to re-direct CD16-expressing γδ T lymphocytes as well as natural killer (NK) cells to the tumor-associated cell surface antigen HER2 to enhance their cytotoxic anti-tumor activity. Tribody [(HER2) 2 xCD16] comprises two HER2-specific single chain fragment variable fused to a fragment antigen binding directed to the CD16 (FcγRIII) antigen expressed on γδ T cells and NK cells. Our results revealed the superiority of tribody [(HER2) 2 xCD16] compared to trastuzumab in triggering γδ T cell and NK cell-mediated lysis of HER2-expressing tumor cells, such as PDAC, breast cancer, and autologous primary ovarian tumors. The increased efficacy of [(HER2) 2 xCD16] can be explained by an enhanced degranulation of immune cells. Although CD16 expression was decreased on γδ T cells in several PDAC patients and the number of tumor-infiltrating NK cells and γδ T cells was impaired in ovarian cancer patients, [(HER2) 2 xCD16] selectively enhanced cytotoxicity of cells from these patients. Here, unique anti-tumor properties of tribody [(HER2) 2 xCD16] are identified which beyond addressing HER2 overexpressing solid tumors may allow to treat with similar immunoconstructs combined with the adoptive transfer of γδ T cells and NK cells refractory hematological malignancies. A major advantage of γδ T cells and NK cells in the transplant situation of refractory hematological malignancies is given by their HLA-independent killing and a reduced graft- versus -host disease.

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.

Background/objectives: Pancreatic ductal adenocarcinoma (PDAC) still has a poor prognosis and current treatments including immunotherapy often fail. This might be due to the pronounced immunosuppressive milieu impairing infiltration and function of immune effector cells. This study aimed at a comprehensive analysis of immune cells in PDAC patients by determining absolute and relative peripheral blood cell numbers of immune cell subsets along with their functional capacity., Methods: Whole blood cells or isolated peripheral blood mononuclear cells were characterized by flow cytometry. PDAC tissues were analyzed by immunohistochemistry. Anti-tumor activity of immune effector cells was determined by RTCA system., Results: Our data demonstrate that relative CD4 + memory- and regulatory T cell numbers were enhanced, whereas determination of absolute cell numbers revealed generally lower immune cell numbers in PDAC patients compared to healthy controls. γδ T cells accumulated at higher numbers compared to αβ T cells in the malignant ductal epithelium of PDAC tissues indicating that γδ T cells infiltrate into the tumor. Cytotoxicity against tumor cells of even small numbers of T- and NK cells could be induced by a bispecific antibody targeting CD3 + T cells to human epidermal growth factor receptor (HER)2 expressing PDAC cells or Trastuzumab. Importantly, a critical number of γδ T cells was required for significant tumor cell killing by a bispecific antibody engaging the γδ T cell receptor on γδ T cells and HER2 on tumor cells., Conclusion: Monitoring immune cells along with the determination of their functional capacity provides a comprehensive assessment as a prerequisite for a personalized immunotherapeutic PDAC treatment., (Copyright © 2016 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

The mammalian target of rapamycin plays an important role in multiple myeloma. The allosteric mammalian target of rapamycin inhibitor everolimus has long been approved for immunosuppression and has shown activity in certain cancers. This investigator-initiated phase I trial explored the use of everolimus in relapsed and/or refractory multiple myeloma patients who had received two or more lines of prior treatment. Following a dose-escalation design, it called for a fixed dose of oral everolimus. Blood drug levels were monitored and the biological activity of everolimus was evaluated in bone marrow. Seventeen patients were enrolled (age range, 52 to 76 years). All had been previously treated with stem cell transplantation and proteasome inhibitors and almost all with immunomodulatory drugs. No dose-limiting toxicity was observed and the intended final daily dose of 10 mg was reached. Only one severe adverse event was assessed as possibly related to the study drug, namely atypical pneumonia. Remarkably few infections were observed. Although the trial was mainly designed to evaluate feasibility, anti-myeloma activity, defined as clinical benefit, was documented in ten of 15 evaluable patients at every dose level including eight patients with stable disease, one patient with minor remission and one with partial remission. However, the median time to progression was 90 days (range, 13 to 278 days). The biomarker study documented on-target activity of everolimus in malignant plasma cells as well as the microenvironment. The observed responses are promising and allow further studies to be considered, including those testing combination strategies addressing escape pathways. This trial is registered with EudraCT number 2006-002675-41., (Copyright© Ferrata Storti Foundation.)

The success of γδ T cell-based immunotherapy, where the cytotoxic activity of circulating γδ T lymphocytes is activated by nitrogen-containing bisphosphonates (n-BP), or possibly by bispecific antibodies or the combination of both, requires a profound knowledge of patients' γδ T cells. A possible influence of radio- or chemotherapy on γδ T cells as well as their reported exhaustion after repetitive treatment with n-BP or their lack of response to various cancers can be easily determined by the monitoring assays described in this perspective article. Monitoring the absolute cell numbers of circulating γδ T cell subpopulations in small volumes of whole blood from cancer patients and determining γδ T cell cytotoxicity using the Real-Time Cell Analyzer can give a more comprehensive assessment of a personalized tumor treatment. Possible future directions such as the combined usage of n-BP or phosphorylated antigens together with bispecific antibodies that selectively target γδ T cells to tumor-associated antigens, will be discussed. Such strategies induce expansion and enhance γδ T cell cytotoxicity and might possibly avoid their exhaustion and overcome the immunosuppressive tumor microenvironment.

Therapeutic antibodies used in the treatment of cancer patients are able to mediate diverse effector mechanisms. Dependent on tumor entity, localization, and tumor burden different effector mechanisms may contribute to the in vivo antitumor activity to a variable degree. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) have been suggested as being important for the in vivo activity of therapeutic antibodies like rituximab or trastuzumab. In recent years, several strategies have been pursued to further optimize the cytotoxic potential of monoclonal antibodies by modifying their Fc part (Fc engineering) with the ultimate goal to enhance antibody therapy.Since Fc engineering approaches are applicable to any Fc-containing molecule, strategies to enhance CDC or ADCC activity of full antibodies or scFv-Fc fusion proteins by altering the primary Fc sequence are described.

Antibodies of human IgA isotype are critical components of the mucosal immune system, but little is known about their immunotherapeutic potential. Compared with IgG antibodies, IgA molecules carry a C-terminal tail piece extension of 18 amino acids with a free cysteine at position 471. This cysteine is required for the formation of dimeric IgA antibodies, but may impair molecular characteristics of monomeric IgA antibodies as therapeutic reagents. Thus, we generated and characterized a d471-mutated antibody against the epidermal growth factor receptor (EGFR) and compared it to its respective IgA2m(1) wild type antibody. Both wild type and mutated IgA antibodies demonstrated similar EGFR binding and were similarly efficient in inhibiting EGF binding and in blocking EGF-mediated cell proliferation. Recruitment of Fc-mediated effector functions like antibody-dependent cell-mediated cytotoxicity by monocytes, macrophages or PMN was similar, but the d471-mutated IgA exhibited different biochemical properties compared with wild type antibody. As expected, mutated IgA did not form stable dimers in the presence of human joining (J)-chain, but we also observed reduced levels of dimeric aggregates in the absence of J-chain. Furthermore, glycoprofiling revealed different glycosylation patterns for both antibodies, including considerably less mannosylation of d471-mutated antibodies. Overall, our results demonstrate that the deletion of the C-terminal cysteine of IgA2 did not affect the investigated effector functions compared with wild type antibody, but it improved biochemical properties of an IgA2m(1) antibody against EGFR, and may thereby assist in exploring the immunotherapeutic potential of recombinant IgA antibodies.

Today monoclonal antibodies are widely used in cancer therapy. However, clinical experience as well as translational research into antibodies' pharmacology and effector mechanisms has identified limitations of antibody therapy, including inefficient effector cell recruitment or initiation of complement-dependent cytotoxicity (CDC). These insights opened alleys for further improvement of antibodies' immunomodulatory functions. While second generation antibodies were predominantly engineered to reduce immunogenicity, progress in antibody engineering now enables the generation of antibodies with novel interesting features. The introduction of Fc engineering technologies offers the potential to tailor Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), CDC or phagocytosis. Approaches to improve Fc-mediated effector mechanisms by Fc-engineering allow for the design of so-called "fit-for-purpose" antibodies or antibody-derivatives, hopefully overcoming some limitations of current forms of antibody therapy.