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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.)

Patients with cancer frequently receive immune-checkpoint inhibitors (ICIs), which may modulate immune responses to COVID-19 vaccines. Recently, cytokine release syndrome (CRS) was observed in a patient with cancer who received BTN162b2 vaccination under ICI treatment. Here, we analyzed adverse events and serum cytokines in patients with 23 different tumors undergoing (n = 64) or not undergoing (n = 26) COVID-19 vaccination under ICI therapy in a prospectively planned German single-center cohort study (n = 220). We did not observe clinically relevant CRS (≥grade 2) after vaccination (95% CI 0-5.6%; Common Terminology of Adverse Events v.5.0) in this small cohort. Within 4 weeks after vaccination, serious adverse events occurred in eight patients (12.5% 95% CI 5.6-23%): six patients were hospitalized due to events common under cancer therapy including immune related adverse events and two patients died due to conditions present before vaccination. Despite absence of CRS symptoms, a set of pairwise-correlated CRS-associated cytokines, including CXCL8 and interleukin-6 was >1.5-fold upregulated in 40% (95% CI 23.9-57.9%) of patients after vaccination. Hence, elevated cytokine levels are common and not sufficient to establish CRS diagnosis., (© 2022. The Author(s).)

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.)

Acute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2-like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease-like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients., (© 2022 by The American Society of Hematology.)

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.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent malignancy in children and also occurs in adulthood. Despite high cure rates, BCP-ALL chemotherapy can be highly toxic. This type of toxicity can most likely be reduced by antibody-based immunotherapy targeting the CD19 antigen which is commonly expressed on BCP-ALL cells. In this study, we generated a novel Fc-engineered CD19-targeting IgG1 antibody fused to a single chain tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) domain (CD19-TRAIL). As TRAIL induces apoptosis in tumor cells but not in healthy cells, we hypothesized that CD19-TRAIL would show efficient killing of BCP-ALL cells. CD19-TRAIL showed selective binding capacity and pronounced apoptosis induction in CD19-positive (CD19 + ) BCP-ALL cell lines in vitro and in vivo. Additionally, CD19-TRAIL significantly prolonged survival of mice transplanted with BCP-ALL patient-derived xenograft (PDX) cells of different cytogenetic backgrounds. Moreover, simultaneous treatment with CD19-TRAIL and Venetoclax (VTX), an inhibitor of the anti-apoptotic protein BCL-2, promoted synergistic apoptosis induction in CD19 + BCP-ALL cells in vitro and prolonged survival of NSG-mice bearing the BCP-ALL cell line REH. Therefore, IgG1-based CD19-TRAIL fusion proteins represent a new potential immunotherapeutic agent against BCP-ALL.

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy., 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 © 2020 Macarrón Palacios, Grzeschik, Deweid, Krah, Zielonka, Rösner, Peipp, Valerius and Kolmar.)

Background: Native cluster of differentiation (CD) 19 targeting antibodies are poorly effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are crucial effector functions of therapeutic antibodies in cancer immunotherapy. Both functions can be enhanced by engineering the antibody's Fc region by altering the amino acid sequence (Fc protein-engineering) or the Fc-linked glycan (Fc glyco-engineering). We hypothesized that combining Fc glyco-engineering with Fc protein-engineering will rescue ADCC and CDC in CD19 antibodies., Results: Four versions of a CD19 antibody based on tafasitamab's V-regions were generated: a native IgG1, an Fc protein-engineered version with amino acid exchanges S267E/H268F/S324T/G236A/I332E (EFTAE modification) to enhance CDC, and afucosylated, Fc glyco-engineered versions of both to promote ADCC. Irrespective of fucosylation, antibodies carrying the EFTAE modification had enhanced C1q binding and were superior in inducing CDC. In contrast, afucosylated versions exerted an enhanced affinity to Fcγ receptor IIIA and had increased ADCC activity. Of note, the double-engineered antibody harboring the EFTAE modification and lacking fucose triggered both CDC and ADCC more efficiently., Conclusions: Fc glyco-engineering and protein-engineering could be combined to enhance ADCC and CDC in CD19 antibodies and may allow the generation of antibodies with higher therapeutic efficacy by promoting two key functions simultaneously.

The complement system represents a powerful part of the innate immune system capable of removing pathogens and damaged host cells. Nevertheless, only a subset of therapeutic antibodies are capable of inducing complement dependent cytotoxicity, which has fuelled the search for new strategies to potentiate complement activation. Properdin (FP) functions as a positive complement regulator by stabilizing the alternative pathway C3 convertase. Here, we explore a novel strategy for direct activation of the alternative pathway of complement using bi-specific single domain antibodies (nanobodies) that recruit endogenous FP to a cell surface. As a proof-of-principle, we generated bi-specific nanobodies with specificity toward FP and the validated cancer antigen epidermal growth factor receptor (EGFR) and tested their ability to activate complement onto cancer cell lines expressing EGFR. Treatment led to recruitment of FP, complement activation and significant deposition of C3 fragments on the cells in a manner sensitive to the geometry of FP recruitment. The bi-specific nanobodies induced complement dependent lysis of baby hamster kidney cells expressing human EGFR but were unable to lyse human tumour cells due to the presence of complement regulators. Our results confirm that FP can function as a surface bound focal point for initiation of complement activation independent of prior C3b deposition. However, recruitment of FP by bi-specific nanobodies appears insufficient for overcoming the inhibitory action of the negative complement regulators overexpressed by many human tumour cell lines. Our data provide general information on the efficacy of properdin as an initiator of complement but suggest that properdin recruitment on its own may have limited utility as a platform for potent complement activation on regulated cell surfaces., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Therapeutic monoclonal antibodies (mAb), directed toward either tumor antigens or inhibitory checkpoints on immune cells, are effective in cancer therapy. Increasing evidence suggests that the therapeutic efficacy of these tumor antigen-targeting mAbs is mediated-at least partially-by myeloid effector cells, which are controlled by the innate immune-checkpoint interaction between CD47 and SIRPα. We and others have previously demonstrated that inhibiting CD47-SIRPα interactions can substantially potentiate antibody-dependent cellular phagocytosis and cytotoxicity of tumor cells by IgG antibodies both in vivo and in vitro IgA antibodies are superior in killing cancer cells by neutrophils compared with IgG antibodies with the same variable regions, but the impact of CD47-SIRPα on IgA-mediated killing has not been investigated. Here, we show that checkpoint inhibition of CD47-SIRPα interactions further enhances destruction of IgA antibody-opsonized cancer cells by human neutrophils. This was shown for multiple tumor types and IgA antibodies against different antigens, i.e., HER2/neu and EGFR. Consequently, combining IgA antibodies against HER2/neu or EGFR with SIRPα inhibition proved to be effective in eradicating cancer cells in vivo In a syngeneic in vivo model, the eradication of cancer cells was predominantly mediated by granulocytes, which were actively recruited to the tumor site by SIRPα blockade. We conclude that IgA-mediated tumor cell destruction can be further enhanced by CD47-SIRPα checkpoint inhibition. These findings provide a basis for targeting CD47-SIRPα interactions in combination with IgA therapeutic antibodies to improve their potential clinical efficacy in tumor patients., (©2019 American Association for Cancer Research.)

Current combination therapies elicit high response rates in B cell malignancies, often using CD20 antibodies as the backbone of therapy. However, many patients eventually relapse or develop progressive disease. Therefore, novel CD20 antibodies combining multiple effector mechanisms were generated. To study whether neutrophil-mediated destruction of B cell malignancies can be added to the arsenal of effector mechanisms, we chimerized a panel of five previously described murine CD20 antibodies to the human IgG1, IgA1 and IgA2 isotype. Of this panel, we assessed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct cell death induction capacity and studied the efficacy in two different in vivo mouse models. IgA antibodies outperformed IgG1 antibodies in neutrophil-mediated killing in vitro , both against CD20-expressing cell lines and primary patient material. In these assays, we observed loss of CD19 with both IgA and IgG antibodies. Therefore, we established a novel method to improve the assessment of B-cell depletion by CD20 antibodies by including CD24 as a stable cell marker. Subsequently, we demonstrated that only IgA antibodies were able to reduce B cell numbers in this context. Additionally, IgA antibodies showed efficacy in both an intraperitoneal tumor model with EL4 cells expressing huCD20 and in an adoptive transfer model with huCD20-expressing B cells. Taken together, we show that IgA, like IgG, can induce ADCC and CDC, but additionally triggers neutrophils to kill (malignant) B cells. We conclude that antibodies of the IgA isotype offer an attractive repertoire of effector mechanisms for the treatment of CD20-expressing malignancies.

Antibody therapy of cancer is increasingly used in the clinic and has improved patient's life expectancy. Except for immune checkpoint inhibition, the mode of action of many antibodies is to recognize overexpressed or specific tumor antigens and initiate either direct F(ab') 2 -mediated tumor cell killing, or Fc-mediated effects such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/P) after binding to activating Fc receptors. All antibodies used in the clinic are of the IgG isotype. The IgA isotype can, however, also elicit powerful anti-tumor responses through engagement of the activating Fc receptor for monomeric IgA (FcαRI). In addition to monocytes, macrophages and eosinophils as FcαRI expressing immune cells, neutrophils are especially vigorous in eliminating IgA opsonized tumor cells. However, with IgG as single agent it appears almost impossible to activate neutrophils efficiently, as we have visualized by live cell imaging of tumor cell killing. In this study, we investigated Fc receptor expression, binding and signaling to clarify why triggering of neutrophils by IgA is more efficient than by IgG. FcαRI expression on neutrophils is ~2 times and ~20 times lower than that of Fcγ receptors FcγRIIa and FcγRIIIb, but still, binding of neutrophils to IgA- or IgG-coated surfaces was similar. In addition, our data suggest that IgA-mediated binding of neutrophils is more stable compared to IgG. IgA engagement of neutrophils elicited stronger Fc receptor signaling than IgG as indicated by measuring the p-ERK signaling molecule. We propose that the higher stoichiometry of IgA to the FcαR/FcRγ-chain complex, activating four ITAMs (Immunoreceptor Tyrosine-based Activating Motifs) compared to a single ITAM for FcγRIIa, combined with a possible decoy role of the highly expressed FcγRIIIb, explains why IgA is much better than IgG at triggering tumor cell killing by neutrophils. We anticipate that harnessing the vast population of neutrophils by the use of IgA monoclonal antibodies can be a valuable addition to the growing arsenal of antibody-based therapeutics for cancer treatment.

Epidermal growth factor receptor (EGFR) ectodomain variants mediating primary resistance or secondary treatment failure in cancer patients treated with cetuximab or panitumumab support the need for more resistance-preventive or personalized ways of targeting this essential pathway. Here, we tested the hypothesis that the EGFR nanobody 7D12 fused to an IgG1 Fc portion (7D12-hcAb) would overcome EGFR ectodomain-mediated resistance because it targets a very small binding epitope within domain III of EGFR. Indeed, we found that 7D12-hcAb bound and inhibited all tested cell lines expressing common resistance-mediating EGFR ectodomain variants. Moreover, we assessed receptor functionality and binding properties in synthetic mutants of the 7D12-hcAb epitope to model resistance to 7D12-hcAb. Because the 7D12-hcAb epitope almost completely overlaps with the EGF-binding site, only position R377 could be mutated without simultaneous loss of receptor functionality, suggesting a low risk of developing secondary resistance toward 7D12-hcAb. Our binding data indicated that if 7D12-hcAb resistance mutations occurred in position R377, which is located within the cetuximab and panitumumab epitope, cells expressing these receptor variants would retain sensitivity to these antibodies. However, 7D12-hcAb was equally ineffective as cetuximab in killing cells expressing the cetuximab/panitumumab-resistant aberrantly N-glycosylated EGFR R521K variant. Yet, this resistance could be overcome by introducing mutations into the Fc portion of 7D12-hcAb, which enhanced immune effector functions and thereby allowed killing of cells expressing this variant. Taken together, our data demonstrate a broad range of activity of 7D12-hcAb across cells expressing different EGFR variants involved in primary and secondary EGFR antibody resistance., (©2019 American Association for Cancer Research.)

Three FDA-approved epidermal growth factor receptor (EGFR) antibodies (cetuximab, panitumumab, necitumumab) are clinically available to treat patients with different types of cancers. Interestingly, panitumumab is of human IgG2 isotype, which is often considered to have limited immune effector functions. Unexpectedly, our studies unraveled that human IgG2 antibodies against EGFR mediated effective CDC when combined with another noncross-blocking EGFR antibody. This second antibody could be of human IgG1 or IgG2 isotype. Furthermore, EGFR antibodies of human IgG2 isotype were highly potent in recruiting myeloid effector cells such as M1 macrophages and PMN for tumor cell killing by ADCC. Tumor cell killing by PMN was more effective with IgG2 than with IgG1 antibodies if tumor cells expressed lower levels of EGFR. Additionally, lower expression levels of the "don't eat me" molecule CD47 on tumor cells enabled ADCC also by M2 macrophages, and improved PMN and macrophage-mediated ADCC. A TCGA enquiry revealed broadly varying CD47 expression levels across different solid tumor types. Together, these results demonstrate that human IgG2 antibodies against EGFR can promote significant Fc-mediated effector functions, which may contribute to their clinical efficacy. The future challenge will be to identify clinical situations in which myeloid effector cells can optimally contribute to antibody efficacy., (©2018 American Association for Cancer Research.)

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.

Compared to the evolutionary diversity of antibody isotypes, the spectrum of currently approved therapeutic antibodies is biased to the human IgG1 isotype. Detailed studies into the different structures and functions of human isotypes have suggested that other isotypes than IgG1 may be advantageous for specific indications - depending on the complex interplay between the targeted antigen or epitope, the desired mode of action, the pharmacokinetic properties, and the biopharmaceutical considerations. Thus, it may be speculated that with the increasing number of antibodies becoming available against a broadening spectrum of target antigens, identification of the optimal antibody isotype for particular therapeutic applications may become critical for the therapeutic success of individual antibodies. Thus, investments into this rather unexplored area of antibody immunotherapy may provide opportunities for distinction in the increasingly busy 'antibody space'. Therefore, IgG, IgA, IgE as well as IgM isotypes will be discussed in this review.

Background: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies., Methods and Results: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody., Conclusion: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

Triggering of the complement cascade induces tumor cell lysis via complement-dependent cytotoxicity (CDC) and attracts and activates cytotoxic cells. It therefore represents an attractive mechanism for mAb in cancer immunotherapy development. The classical complement pathway is initiated by IgG molecules that have assembled into ordered hexamers after binding their Ag on the tumor cell surface. The requirements for CDC are further impacted by factors such as Ab epitope, valency, and affinity. Thus, mAb against well-validated solid tumor targets, such as the epidermal growth factor receptor (EGFR) that effectively induces complement activation and CDC, are highly sought after. The potency of complement activation by IgG Abs can be increased via several strategies. We identified single-point mutations in the Fc domain (e.g., E345K or E430G) enhancing Fc:Fc interactions, hexamer formation, and CDC after Ab binds cell-surface Ag. We show that EGFR Abs directed against clinically relevant epitopes can be converted into mAb with unprecedented CDC activity. Alternative strategies rely on increasing the affinity of monomeric IgG for C1q by introduction of a quadruple mutation at the C1q binding site or via generation of an IgG1/IgG3 chimera. In this study we show that selective enhancement of C1q binding via avidity modulation is superior to the unattended increase in C1q binding via affinity approaches, particularly for target cells with reduced EGFR expression levels. Improving Fc:Fc interactions of Ag-bound IgG therefore represents a highly promising and novel approach for potentiating the anti-tumor activity of therapeutic mAb against EGFR and potentially other tumor targets., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Antibodies of IgA isotype effectively engage myeloid effector cells for cancer immunotherapy. Here, we describe preclinical studies with an Fc engineered IgA2m(1) antibody containing the variable regions of the EGFR antibody cetuximab. Compared with wild-type IgA2m(1), the engineered molecule lacked two N-glycosylation sites (N166 and N337), two free cysteines (C311 and C472), and contained a stabilized heavy and light chain linkage (P221R mutation). This novel molecule displayed improved production rates and biochemical properties compared with wild-type IgA. In vitro, Fab- and Fc-mediated effector functions, such as inhibition of ligand binding, receptor modulation, and engagement of myeloid effector cells for antibody-dependent cell-mediated cytotoxicity, were similar between wild-type and engineered IgA2. The engineered antibody displayed lower levels of terminal galactosylation leading to reduced asialoglycoprotein-receptor binding and to improved pharmacokinetic properties. In a long-term in vivo model against EGFR-positive cancer cells, improved serum half-life translated into higher efficacy of the engineered molecule, which required myeloid cells expressing human FcαRI for its full efficacy. However, Fab-mediated effector functions contributed to the in vivo efficacy because the novel IgA antibody demonstrated therapeutic activity also in non-FcαRI transgenic mice. Together, these results demonstrate that engineering of an IgA antibody can significantly improve its pharmacokinetics and its therapeutic efficacy to inhibit tumor growth in vivo., (©2015 American Association for Cancer Research.)

Complement-dependent cytotoxicity (CDC) has been suggested to be an important mechanism of action of tumor-targeting Abs. However, single unmodified epidermal growth factor receptor (EGFR)-targeting IgG1 Abs fail to trigger efficient CDC. For the current study, we generated a CDC-optimized variant of the EGFR Ab matuzumab (H425 wt) by introducing amino acid substitutions K326A/E333A (H425 mt). This Ab was then used to elucidate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert Ab-dependent cell-mediated cytotoxicity (ADCC). H425 mt, but not H425 wt, significantly induced complement deposition, release of anaphylatoxins, and CDC against distinct tumor cell lines, whereas no differences in ADCC by MNC or PMN were detected. Notably, stronger cytotoxicity was induced by H425 mt than by H425 wt in whole blood assays and in experiments in which MNC or PMN were combined with serum. Although MNC-ADCC was not affected by C5 cleavage, the cytotoxic activity of PMN in the presence of serum strongly depended on C5 cleavage, pointing to a direct interaction between complement and PMN. Strong cell surface expression of C5a receptors was detected on PMN, whereas NK cells completely lacked expression. Stimulation of PMN with C5a led to upregulation of activated complement receptor 3, resulting in enhanced complement receptor 3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR Abs may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in Ab-based tumor therapy., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Binding of C1q to target-bound IgG initiates complement-mediated lysis (CML) of pathogens, as well as of malignant or apoptotic cells, and thus constitutes an integral part of the innate immune system. Despite its prominent molecular flexibility and higher C1q binding affinity compared with human IgG1, IgG3 does not consistently promote superior CML. Hence the aim of this study was to investigate underlying molecular mechanisms of IgG1- and IgG3-driven complement activation using isotype variants of the therapeutic epidermal growth factor receptor (EGFR) Ab cetuximab. Both IgG1 and IgG3 Abs demonstrated similar EGFR binding and similar efficiency in Fab-mediated effector mechanisms. Whereas anti-EGFR-IgG1 did not promote CML of investigated target cells, anti-EGFR-IgG3 triggered significant CML of some, but not all tested cell lines. CML triggered by anti-EGFR-IgG3 negatively correlated with expression levels of the membrane-bound complement regulatory proteins CD55 and CD59, but not CD46. Notably, anti-EGFR-IgG3 promoted strong C1q and C3b, but relatively low C4b and C5b-9 deposition on analyzed cell lines. Furthermore, anti-EGFR-IgG3 triggered C4a release on all cells but failed to induce C3a and C5a release on CD55/CD59 highly expressing cells. RNA interference-induced knockdown or overexpression of membrane-bound complement regulatory proteins revealed CD55 expression to be a pivotal determinant of anti-EGFR-IgG3-triggered CML and to force a switch from classical complement pathway activation to C1q-dependent alternative pathway amplification. Together, these data suggest human anti-EGFR-IgG3, although highly reactive with C1q, to weakly promote assembly of the classical C3 convertase that is further suppressed in the presence of CD55, forcing human IgG3 to act mainly through the alternative pathway., (Copyright © 2014 by The American Association of Immunologists, Inc.)

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.

Isolated rabbit hearts were perfused aerobically (45 min) and hypoxically (105 min), using a modified Langendorff technique. The mean functional diameter of capillaries (MFDC) was calculated from the perfusion rate per minute and the inflow resistance by the model of Hagen-Poiseuille. The MFDC expresses the mean lumen of all capillaries of the heart, regardless of the different behaviour of the capillaries in the different regions of the myocardium. The MFDC decreased very rapidly after the onset of a hypoxia from 3.5 +/- 0.3 micron to about 70% of the initial diameter within 5-10 min (p less than 0.01) and then in a slower range within the following 70 min to about 44% of the initial diameter. The release of lactate dehydrogenase (LDH) from the hypoxic myocardium was detectable after 45 min of hypoxia and rose drastically after 60 min of hypoxia in our model. The decrease of the MFDC as well as the release of LDH from the hypoxic myocardium can be diminished by application of O-(beta-hydroxyethyl)-rutoside or 10 mM mannitol to the hypoxic perfusion medium. Both substances have antioxidant activities. It is discussed that the injury of the microvasculature is an early process during hypoxia, which can potentiate the hypoxic changes of the myocardial cells by additional diminution of the supply with oxygen and substrates. The protecting activities of substances with antioxidant actions to hypoxic myocardium were supported.

A method is proposed which yields an approximate solution of the Free-Wilson model very rapidly without using a computer. Although the resulting group contributions are numerically somewhat different from the exact Free-Wilson solution they correctly reflect the relative order of the substituents with respect to their effect on biological activity within each position as well as the relative importance of different positions. Thus, the approximate results can well be used to select the most promising candidates for further synthesis and testing.