Dual checkpoint blockade of CD47 and LILRB1 enhances CD20 antibody-dependent phagocytosis of lymphoma cells by macrophages

<jats:p>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.</jats:p>

section_content: Therapeutic antibodies are well established in the treatment of cancer (1). In B-cell Non-Hodgkin lymphomas (B-NHL), tumor targeting CD20 antibodies such as rituximab and obinutuzumab have considerably improved the patient outcome (2). However, individual patients fail to respond and relapsed or refractory disease remains challenging. Progress was made by the approval of chimeric antigen receptor T cell therapies, tailor-made fragment crystallizable (Fc)-engineered antibodies, bispecific antibodies and T cell immune checkpoint inhibitors (1, (3) (4) (5) (6). Besides, antibodies targeting checkpoints in innate immune cells such as natural killer (NK) cells or myeloid cells have gained increasing attention (7). In particular, they may hold the potential to boost key functions of typical tumor targeting antibodies such as antibody dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP) (1, 7, 8). 

Several studies highlighted that macrophages, which in humans express the activating immunoglobulin g Fc region receptors (FcgR) FcgRI, FcgRIIA and FcgRIIIA as well as inhibitory FcgRIIB, represent major effector cells for rituximab and other therapeutic antibodies (2, 9, 10). According to their functional polarization, macrophages were roughly categorized into classically activated, pro-inflammatory M1 macrophages and alternatively activated, anti-inflammatory M2 macrophages. This classification was later refined by grouping the latter into M2a, M2b, M2c and M2d subtypes to take account of distinct stimuli and diverse functional properties (11, 12). However, macrophages exert a high level of plasticity and M1 and the different M2 states represent the extreme ends in a broad spectrum of different functional states. Also, subsets with intermediate phenotypes exist (12). Macrophages are among the most frequent normal cells in the tumor microenvironment (9). High numbers of tumor-associated macrophages (TAM), which often become tumor-edited into a pro-tumorigenic M2like state, correlated with tumor progression and poor prognosis (13) (14) (15) (16) ). Yet, during antibody therapy, macrophages may contribute to tumor eradication by eliminating malignant cells directly by ADCC and ADCP or by promoting adaptive immune responses by presenting tumor antigens to T cells (8, 10). Thus, studies in B-NHL patients receiving rituximab suggested that a high content of TAM correlated with improved survival and that rituximab therapy abrogated the correlation between high numbers of TAM and poor prognosis (15, 17, 18). The antitumoral functions of macrophages during antibody therapy may be further promoted by certain chemotherapeutic agents, as demonstrated for example for cyclophosphamide in a murine model of B cell leukemia (19). 

In regard of the important role of macrophages, strategies were developed to improve their recruitment and ADCP function. These approaches include Fc engineering to enhance the antibody`s affinity to activating FcgR (20, 21) as well as the blockade of inhibitory checkpoints that interfere with FcgR signaling (10). Signal-regulatory protein (SIRP) a is one of the best characterized myeloid inhibitory receptors. SIRPa recognizes the ubiquitously expressed 'Don`t Eat Me!' signal molecule CD47, and antibody blockade of either CD47 or SIRPa strongly enhanced ADCP by macrophages (22, 23). For example, the combination of rituximab with a CD47 antibody increased ADCP of lymphoma cells by macrophages in vitro and improved the therapeutic efficacy in xenograft models of B-NHL (24). Moreover, accompanying CD38 antibody therapy with CD47 checkpoint blockade demonstrated efficacy in patient-derived T cell acute lymphoblastic leukemia xenograft models (25). At present, monoclonal CD47 antibodies, anti-SIRPa antibodies and SIRPa-Fc fusion proteins, as well as small molecule inhibitors are in different stages of pre-clinical or clinical development (23, (26) (27) (28) (29) (30). Promising results were obtained with the combination of rituximab plus the CD47 antibody hu5F9-G4 (magrolimab) in a clinical phase Ib study in B-NHL patients (28). Although the IgG4 antibody magrolimab was well tolerated by the patients, safety of CD47 targeting remains a serious concern due to the ubiquitous CD47 expression by normal cells, which may cause on-target toxicity. In particular, CD47 expressing red blood cells (RBC), which display cell surface "Eat Me!" signals and downregulate CD47 during their lifespan, are vulnerable to CD47 antibody therapy. This may result in enhanced RBC clearance during CD47 antibody therapy, even when antibody formats with diminished Fcmediated functions such as IgG4 isotypes are employed (10, 28). 

Leukocyte immunoglobulin-like receptor (LILR) B 1 may represent another target for immune checkpoint blockade in monocytes and macrophages (10, 31). LILRB1 and other members of the inhibitory subfamily B of LILR share structural similarities with human killer cell immunoglobulinlike receptors (KIR) and are characterized by cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIM) (32). In addition to several pathogen-derived ligands, LILRB1 binds classical human leukocyte antigen (HLA) class I as well as nonclassical HLA-G and HLA-F molecules by interaction with the a3 domain and b2-microglobulin (33) (34) (35). Recently, it has been shown that antibody blockade of either LILRB1 or HLA-class I promotes phagocytosis of solid tumor cells, that genetic ablation of cell surface expression of HLA class I and CD47 augmented ADCP by anti-EpCAM or anti-EGFR antibodies and that MHC class I expression confers protection from macrophages in a murine tumor model (36). In addition, macrophages express LILRB2, another inhibitory receptor for HLA class I molecules that interferes with FcgR signaling (37, 38). Interestingly, LILRB2 antagonism was suggested to reprogram tumorassociated myeloid cells, to enhance pro-inflammatory responses and to promote antitumor effects of T cell immune checkpoint inhibitors (39). However, whether LILRB2 directly impairs phagocytosis is currently not known (10). 

Here, the impact of HLA class I expression on macrophagemediated ADCP of lymphoma cells and the potential of antibodies blocking the cognate inhibitory receptors LILRB1 and LILRB2 was analyzed. To prevent the induction of any Fcmediated effects by these blocking antibodies, which may cause difficulties in unraveling effects mediated by FcgR engagement or receptor blockade, Fc-silent IgGs variants were used, in which both FcgR and complement binding was abrogated by Fc engineering (40, 41). Clinically, such Fc-silent immune checkpoint blocking antibodies may cause less on-target side effects, as they do not induce ADCC, ADCP or CDC on their own and avoid cross-linkage of antibody bound receptors by effector cells through Fc-FcgR interactions. The anti-LILRB1 antibody was found to significantly enhance ADCP when combined with CD20 and CD47 antibodies by analyzing different B-NHL cell lines and tumor cells from chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL) patients.
section_name: Introduction

section_name: Materials and methods

section_content: The antibodies rituximab, obinutuzumab and trastuzumab (Hoffmann-La Roche AG, Basel, Switzerland) were provided by the institutional pharmacy. The allophycocyanin (APC)-labeled CD11b antibody (clone M1/70) as well as phycoerythrin (PE)conjugated antibodies specific for CD80 (clone REA661), CD163 (clone REA812), LILRB1 (clone REA998) or LILRB2 (clone REA184) and the corresponding isotype antibody (clone REA293) were from Miltenyi Biotec (Bergisch Gladbach, Germany). The APC-conjugate of the anti-LILRB1 antibody clone GHI/75 as well as Brilliant Violet 421-conjugated CD163 (GHI/61) and Brilliant Violet 510-coupled CD15 (W6D3) antibodies were purchased from BioLegend (San Diego, CA, USA). Murine antibodies against cell surface antigens CD20 (clone S1815E), HLA-A,-B,-C (clone W6/32), HLA-G (clone 87G), SIRPa (clone 15-414), LILRB1 (clone GHI/75), CD47 (clone B6H12) and an IgG2a isotype (clone MOPC-173) were purchased from BioLegend. The anti-LILRB2 antibody (clone 287219) and an IgG1 istotype antibody (clone 11711) were obtained from R&D systems, Inc. (Minneapolis, MN, USA). Secondary PE-conjugated F(ab') 2 fragments of goat anti-human Fcg region antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Human recombinant macrophage colony stimulating factor (M-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-g and interleukin-10 (IL-10) were purchased from PeproTech (Cranbury, NJ, USA). Lipopolysaccharide (LPS) O127:B8 was obtained from Sigma-Aldrich (St. Louis, MO, USA).
section_name: Antibodies and reagents

section_content: Carnaval, DG-75 and SU-DHL-4 (German Collection of Microorganisms and Cell Cultures GmbH, DSMZ, Braunschweig, Germany) cells were cultured in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS, Thermo Fisher Scientific) and 1% penicillin (Pen)/ streptomycin (Strep) solution (Lonza, Basel, Switzerland). Granta 519 (DSMZ), Chinese hamster ovary (CHO)-K1 (DSMZ) and Lenti-X ™ 293T cells (Clontech, Saint-Germainen-Laye, France) were kept in Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific) containing 10% FCS and 1% Pen/Strep. MEC2 cells (DSMZ) were maintained in Iscove's Modified Dulbecco's Medium (Thermo Fisher Scientific) supplemented with 20% FCS and 1% Pen/Strep. Cells were cultured in a humidified atmosphere at 37°C and 6% CO 2.
section_name: Cell culture

section_content: Experiments were approved by the Ethics Committee of the faculty of medicine, LMU Munich (18-821 and 21-0816), in accordance with the Declaration of Helsinki. Blood samples were collected after receiving the donors`written informed consents. MNC were isolated from peripheral blood or leukoreduction system chambers by density gradient centrifugation using Ficoll ® Paque Plus (Cytiva, Marlborough, MA, USA). Monocytes were isolated by plastic adherence using monocyte attachment medium (PromoCell GmbH, Heidelberg, Germany) following the manufacturer`s protocols. To generate nonpolarized M0 macrophages adherent monocytes were cultivated in X-Vivo ™ 15 (Lonza) medium supplemented with 0. 5% Pen/Strep and, unless otherwise indicated, M-CSF at a concentration of 50 ng/ml for 7 days. In individual experiments, M-CSF was replaced by GM-CSF (10 ng/ml). If not specified, polarized M1 macrophages were obtained by culturing cells in the presence of GM-CSF (10 ng/ml) for 6 days to drive macrophage differentiation towards an M1 phenotype, before stimulation with IFN-g (10 ng/ml) and LPS (100 ng/ml) for additional 48 h. M2 macrophages (M2c subtype) were polarized with M-CSF (50 ng/ml) for 6 days, before IL-10 was added at a concentration of 10 ng/ml for additional 48 h. Macrophages were harvested by accutase (Thermo Fisher Scientific) treatment following the manufacturer`s recommendations.
section_name: Isolation of mononuclear cells and generation of macrophages

section_content: Macrophages, defined as CD163-positive and CD15negative cells, were isolated from cryo-preserved BM-derived samples from diffuse large B cell lymphoma (DLBCL) patients by density-gradient centrifugation and fluorescence activated cell sorting as described previously (42). Bone marrow samples from DLBCL patients with BM infiltration served as a source for lymphoma associated macrophages (LAM). The purity was greater than 95%. Each patient gave informed consent prior to surgery or bone marrow biopsy, and the institutional ethics committee approved the study (Erlangen: Ref. number 21-403-Br).
section_name: Isolation of bone marrow macrophages from lymphoma patients

section_content: Anti-LILRB1 and anti-LILRB2 antibodies were derived from the sequences from the antibody clones GHI/75 and 19. h1, respectively (43, 44). As a murine IgG1 antibody GHI/75 has been demonstrated to block HLA class I/b2M binding to LILRB1, thereby promoting phagocytosis of cancer cells (36). The antibody clone 19. h1 is a humanized version of antibody 19. 1, which had been selected for its abilities to block interactions between HLA class I and LILRB2 and to shift macrophage polarization towards an M1 phenotype (44). For construction of the Fc-silent anti-LILRB1 antibody (LILRB1-IgGs), DNA fragments encoding the variable light (VL) and heavy (VH) chains of antibody GHI/75 as well as the constant human k light (LC) and IgG2s heavy chain (HC) regions (amino acid substitutions: V234A/G237A/P238S/H268A/ V309L/A330S/P331S) were synthesized de novo (Thermo Fisher Scientific) according to published sequences (40, 45). LC and HC sequences were cloned into vector pSecTag2/Hygro C (Thermo Fisher Scientific) using NheI/PmeI restriction sites. To generate an IgG1s (amino acid substitutions L234A/L235A/ G237A/P238S/H268A/A330S/P331S) variant (41) of the anti-LILRB2 antibody 19. h1 (LILRB2-IgGs), VL and VH sequences were synthesized de novo (Thermo Fisher Scientific) conforming to published sequences (44) and ligated into vectors pSecTag2-LC (20) and pSecTag2-HC-IgG1s (C Kellner, unpublished) as NheI/HindIII and NheI/PpuMI cassettes, respectively. For generation of antibody CD47-IgGs, VL and VH regions of antibody hu5F9 (46) were synthesized de novo and cloned into vectors pSec-CD3-HC-IgG2s (M. Peipp, unpublished) and pSecTag2-LC (20) as NheI/HindIII and NheI/PpuMI cassettes, respectively. The expression vector for the HC of an Fcengineered version of rituximab (RTX-DE; amino acid substitutions S239D/I332E) was generated by excising rituximab VH regions from vector pSecTag2-CD20-HC (47) and ligation into vector pSecTag2-HC-DE (20) using NheI/ PpuMI restriction sites. The vectors encoding rituximab LC, trastuzumab LC or an Fc-engineered HC of trastuzumab (HER2-DE; amino acid substitutions S239D/I332E) have been described previously (20, 47). The vector encoding an Fc-silent IgG2s HC variant of trastuzumab (HER2-IgGs) was obtained by ligation of trastuzumab VH chain sequences in vector pSec-CD3-HC-IgG2s using NheI/PpuMI restriction sites. For expression, Lenti-X ™ 293T cells were co-transfected with HC and LC expression vectors by calcium phosphate transfection with chloroquine following standard protocols. Cell culture supernatants were collected for six days. Antibodies were purified by affinity chromatography using CaptureSelect ™ IgG-CH1 affinity matrix (Thermo Fisher Scientific) as described earlier (47) and dialyzed against PBS (Thermo Fisher Scientific). The IgA2 isotype variants of rituximab (RTX-IgA2) and the anti-epidermal growth factor receptor (EGFR) antibody cetuximab have been described previously and were expressed in CHO-S cells (Thermo Fisher Scientific) by transient transfection following published procedures (48) (49) (50).
section_name: Cloning, expression and purification of recombinant antibodies

section_content: Purity, integrity and concentrations of the purified an tibodies wer e det erm in ed by micro fl uidic c hip electrophoresis under reducing and non-reducing conditions. Four microliters of antibody preparations were analyzed using the Agilent Protein 230 Kit and the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer's recommendations.
section_name: Microfluidic chip electrophoresis

section_content: SDS-PAGE and Western Transfer experiments were performed using standard procedures as described elsewhere (47). Antibody LC and HC were detected with horseradish peroxidase (HRP)-conjugated goat anti human k light chain (Bio-Rad Laboratories, Inc. ) and goat anti-human IgG/Fc specific (Merck, Darmstadt, Germany) antibodies, respectively.
section_name: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western Transfer experiments

section_content: Gel filtration was performed using the ÄKTApure protein purification system (Cytiva). For analysis, 100 -300 µg of antibodies were loaded on a Superdex ™ 200 Increase 10/300 GL column (Cytiva) and analyzed at a flow speed of 0. 75 ml/min using PBS as running buffer.
section_name: Gel filtration

section_content: DNA sequences encoding human full-length LILRB1 and LILRB2 proteins (UniProtKB accession numbers Q8NHL6 and Q8N423, respectively) were synthesized de novo (Thermo Fisher Scientific) and cloned into expression vector pcDNA 3. 1 (+) (Thermo Fisher Scientific). CHO-K1 cells were transfected using Lipofectamine ® LTX and Plus ™ Reagent (Thermo Fisher Scientific) according to the manufacturer's recommendation. After 48 h, the transfected cells (i. e. CHO-LILRB1 and CHO-LILRB2, respectively) were used in functional analysis.
section_name: Expression of cell surface antigens by transient transfection

section_content: Flow cytometry experiments were performed on a FACSCalibur ™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with the exception of multi-colour analysis, which were performed on a FACSCanto II cytometer (BD Biosciences). Three hundred thousand cells were washed once with 2 ml PBS containing 1% bovine serum albumin (FACS buffer). In direct immunofluorescence assays, cells were incubated with fluorescent dye-conjugated antibodies at dilutions recommended by the manufacturer in FACS buffer at 4°C for 60 min. Expression of cell surface antigens was quantified by calibrated indirect flow cytometry using Qifikit ® (Dako, Glostrup, Denmark) according to the manufacturer's recommendations. Murine antibodies were applied at a concentration of 20 µg/ml in FACS buffer supplemented with 1 mg/ml pooled human immunoglobulin (Gamunex ® 10%; Grifols, Barcelona, Spain) to block FcgR. Binding of chimeric or humanized antibodies was analyzed by incubation of cells with antibodies at a concentration of 50 µg/ml at 4°C for 60 min. Cells were washed once with 2 ml of FACS buffer. F(ab') 2 fragments of goat anti-human IgG, Fcg fragment specific antibodies were used for detection. To analyze the ability of anti-LILRB1 and anti-LILRB2 antibodies to block receptor binding by HLA class I molecules, 0. 3 × 10 6 CHO-LILRB1 or CHO-LILRB2 cells were incubated with the antibodies at a concentration of 50 µg/ml in 20 µl FACS buffer for 1 h at 4°C. Then, 3 µl of PE-conjugated MHC I Dextramer ® of CMV pp65 peptide (NLVPMVATV)-loaded HLA-A*0201 (Immudex, Kopenhagen, Denmark) were added. Cells were incubated for 30 min, washed with FACS buffer, and analyzed. Mean fluorescence intensity (MFI) values were normalized to the control reaction and residual binding of HLA molecules was calculated. In all experiments, appropriate scatter gates were applied to exclude debris or dead cells and 10,000 events were counted. Data were analyzed using FlowJo v10. 7. 2 software (Becton Dickinson).
section_name: Flow cytometry

section_content: Twenty thousand macrophages per well were plated in M0, M1 or M2c macrophage differentiation media on 8 well µ-slides (Ibidi GmbH, Graefelfing, Germany) and incubated overnight. Lymphoma cells were labeled using carboxyfluorescein succinimidyl ester (CFSE) Cell Division Tracker Kit (BioLegend) according to the manufacturer's recommendations. Forty thousand lymphoma cells were added to each well in a final volume of 300 µl. Antibodies were applied and cells were incubated at 37°C with 6% CO 2 for 2 h. Non-phagocytosed target cells were removed by exchanging the supernatant by fresh medium. ADCP was determined by counting the number of phagocytosed tumor cells per individual macrophages using the fluorescence microscope Axio Observer D1 (Carl Zeiss AG, Oberkochen, Germany), unless otherwise indicated. Fifty to 100 macrophages were analyzed. The phagocytic index was calculated by the equation: Phagocytic index = (number of engulfed target cells/number of macrophages) × 100. In individual experiments, cytoplasmic membranes of macrophages and nuclei were stained with CellBrite ™ Orange Cytoplasmic Membrane Labeling Dye (Biotium, Inc., Fremont, CA, USA) and NucBlue ™ Live ReadyProbes ™ (Thermo Fisher Scientific), respectively, according to the manufacturer's recommendations. 

The phagocytic activity of LAM was analyzed by plating purified LAM in 8-chamber slides (Thermo Fisher Scientific). LAM were co-incubated for 2 h with Cytolight Rapid Green (Sartorius, Göttingen, Germany) labeled Carnaval cells (E:T cell ratio: 1:1) in the presence of the indicated antibodies at a concentration of 1 µg/ml. The adherent cells were washed with PBS, stained with an APC-conjugated CD11b antibody, and were fixed with 4% paraformaldehyde in PBS. The slide was then overlaid with DAPI medium and covered with a glass cover slide. Slides were analyzed using a confocal microscope (LSM700, Carl Zeiss AG).
section_name: Analysis of ADCP by fluorescence microscopy

section_content: To analyze the phagocytic uptake of target cells, 4 × 10 4 macrophages were plated in 50 µl of M0, M1 or M2c macrophage differentiation media in 96-well cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and allowed to settle at 37°C with 6% CO 2 for at least 1 h. Antibodies were added at the indicated concentrations. Target cells were labeled with the pH-sensitive labeling dye pHrodo ® (Sartorius AG) at 500 ng/ml according to the manufacturer's recommendations. Per well, 8 × 10 4 target cells were added in 50 µl X-Vivo ™ 15 medium and live cell imaging was initiated using the IncuCyte ® system (Sartorious AG). Four images per well were recorded every 30 minutes and red object counts per image were determined. 

To analyze the depletion of target cells, CFSE-labeled target cells were co-cultured with macrophages in the presence of antibodies in 8 well µ-slides (Ibidi GmbH) as described above for ADCP analysis by fluorescence microscopy. After 2 h, the supernatant was carefully resuspended without disturbing adherent macrophages. For quantitation, 100 µl were transferred to a 96-well plate, pelleted by centrifugation and green fluorescent cells were counted using the Incucyte ® system by analyzing nine images per well. Relative residual numbers of target cells were determined by normalizing data to the control reaction performed in the absence of antibodies and the percentage of target cell depletion was calculated.
section_name: Live cell imaging

section_content: Statistical and graphical analyses were performed using the GraphPad Prism 8. 0. 2 software (GraphPad Software, La Jolla, CA, USA). Statistically significant differences between treatment groups were assessed using two-sided Student´s t-test, one-way or two-way ANOVA and S ̌idàk´s, Tukey´s or Fisher´s LSD posttests, as indicated. The correlation between the ratio of CD20 to HLA class I molecules per cell and susceptibility to ADCP was calculated using the Pearson correlation test. P-values ≤ 0. 05 were considered statistically significant.
section_name: Data processing and statistical analyses

section_content: In an effort to define critical determinants of ADCP of lymphoma cells, we analyzed the sensitivity of the lymphoma cell lines Granta 519 (MCL), Carnaval (DLBCL), DG-75 (Burkitt lymphoma), MEC2 (CLL) and SU-DHL-4 (DLBCL) using human, non-polarized M0 macrophages. To induce ADCP, cells were treated with the CD20 antibody rituximab or a combination of rituximab and a variant of the CD47 antibody magrolimab with abrogated FcgR binding (referred to as CD47-IgGs; Supplementary Figure 1 ). Each antibody was analyzed at the saturating concentration of 10 µg/ml. The analysis by fluorescence microscopy revealed that the extent of ADCP induced by rituximab or by the combination of rituximab plus CD47-IgGs differed considerably between the cell lines. The CD47 antibody, which was unable to trigger ADCP on its own due to abrogated FcgR binding, enhanced ADCP by rituximab in Carnaval, MEC2 and Granta 519 cells significantly. Minor addon effects from the CD47 antibody were observed with DG-75 cells, which were hardly engulfed even in the presence of both antibodies, and SU-DHL-4 cells, which were extraordinarily sensitive to rituximab-mediated ADCP (Figure 1A ). Regarding the 'Don´t Eat Me!' function described for HLA class I molecules by interaction with the inhibitory receptor LILRB1, we hypothesized that their expression interfered with ADCP of lymphoma cells and contributed to the observed differences. Therefore, the cell surface expression levels of HLA class I molecules, the rituximab target antigen CD20 and CD47 were determined (Figure 1B ). The cell lines showed diverging expression of these antigens, with high variation in the expression of classical HLA class I molecules (HLA-A,-B,-C) and CD20, while moderate differences in CD47 expression were observed. HLA-G expression was not detectable with the exception of Granta 519 cells. Although a correlation between the expression of either HLA class I or CD20 and ADCP by combination treatment with rituximab and the CD47 antibody was not found (Supplementary Figure 2 ), a correlation between ADCP and the expression ratio of CD20 to HLA class I molecules was observed (Figure 1C ; Supplementary Table 1 ). Thus, the number of displayed binding sites for rituximab and inhibitory HLA class I molecules contributed to determining the sensitivity to ADCP. 

MCSF-generated M0 macrophages expressed significant amounts of the HLA class I receptors LILRB1 and LILRB2 (Figure 1D ). In an attempt to further enhance ADCP by blocking the anticipated inhibitory function of HLA class I receptors, Fc-silent antibodies against LILRB1 and LILRB2 (LILRB1-IgGs and LILRB2-IgGs, respectively) were generated (Supplementary Figures 1A, B ). Gel filtration revealed that the antibodies were monomeric and no significant multimers or higher molecular weight aggregates were detectable (Supplementary Figure 1C ). The antigen specific binding of both antibodies was confirmed by flow cytometry using CHO-K1 cells transiently transfected with either LILRB1 or LILRB2 cDNA expression constructs, showing that the two antibodies exerted the expected binding profile (Supplementary Figure 3 ). Moreover, both LILRB1-IgGs and LILRB2-IgGs were able to block binding of soluble HLA class I molecules to LILRB1 and LILRB2 transfected CHO cells, respectively (Figure 2A ). To analyze, whether the antibody blockade of LILRB1 and

The ratio of CD20 to HLA-A,-B,-C antigen expression determines the efficacy of the combination of rituximab and a CD47 blocking antibody in initiating ADCP. LILRB2 translated in enhanced ADCP, initial experiments with M0 macrophages and Carnaval target cells were performed. As a result, although not being effective alone or when combined with rituximab only, the LILRB1-IgGs antibody significantly enhanced ADCP when applied together with both rituximab and CD47-IgGs (Figure 2B ). In contrast, no improvements in ADCP were observed by LILRB2 blockade, neither when LILRB2-IgGs was applied alone nor in combination (Figure 2B ). To demonstrate the antigen-specific mode of action of LILRB1-IgGs, an Fc-silent control antibody, HER2-IgGs, was combined with rituximab and CD47-IgGs in ADCP reactions (Figure 2C ). Whereas again LILRB1-IgGs significantly improved ADCP, the HER2-IgGs control antibody did not mediate any effects. Phagocytosis was also not observed when the LILRB1-IgGs and CD47-IgGs antibodies were applied in the absence of rituximab (Figure 2C ), indicating that disruption of both signaling pathways was not sufficient in the absence of an activating signal. In addition, the direct comparison of the two triple combinations consisting of rituximab and CD47-IgGs plus either LILRB1-IgGs or LILRB2-IgGs revealed significant differences between the two treatment groups, further demonstrating the benefits of the antigen-specific blockade of LILRB1 (Supplementary Figure 4A ). In agreement with these findings, the application of the triple antibody combination consisting of rituximab, CD47-IgGs plus LILRB1-IgGs resulted in an enhanced depletion of Carnaval target cells, as analyzed by determining the residual remaining target cells using the IncuCyte ® live cell imaging system (Figure 2D ). 

LILRB1-IgGs revealed the potential also to enhance ADCP of other lymphoma cell lines. In experiments with both Granta 519 and MEC2 cells LILRB1-IgGs was effective when applied in a triple combination with rituximab and CD47-IgGs (Figure 2E ). Remarkably, rituximab plus the dual checkpoint blockade of LILRB1 and CD47 resulted in profound phagocytosis of DG-75 cells, which hardly responded to the treatment with rituximab plus CD47-IgGs. Yet, ADCP of SU-DHL-4 cells was not significantly improved further, which might be explained by their high susceptibility to ADCP (Figure 2E ). Thus, among the analyzed cell lines, the highest fold improvement in ADCP by inclusion of LILRB1-IgGs was achieved with DG-75 cells (Figure 2F ), which had the lowest CD20 expression levels (Figure 1B ). In contrast, LILRB2 blockade was not effective in enhancing ADCP of MEC2 and DG-75 cells (Supplementary Figure 4B ). Using DG-75 target cells, the potency of LILRB1 blockade relative to LILRB2 blockade was further demonstrated in a direct comparison of the two respective antibodies in combination with rituximab and CD47-IgGs (Supplementary Figure 4C ). Therefore, we focused on the LILRB1-IgGs antibody in subsequent experiments. To analyze the effects of the dual checkpoint blockade with a different CD20 antibody, ADCP was investigated in combinations with the Fc glyco-engineered CD20 antibody obinutuzumab using Granta 519 target cells. As observed in experiments with rituximab, LILRB1-IgGs demonstrated efficacy and enhanced ADCP when applied together with obinutuzumab and CD47-IgGs (Figure 2G ). Next, the efficacy of LILRB1 blockade was analyzed in combinations with an IgA2 isotype switch variant of rituximab (RTX-IgA2; Figure 2H ). Using Carnaval target cells, RTX-IgA2 induced ADCP, and its efficacy was enhanced by combination with CD47-IgGs. Importantly, LILRB1-IgGs further potentiated ADCP when added to the RTX-IgA2 and CD47-IgGs combination. 

Individual macrophages are able to engulf multiple tumor target cells. Indeed, the analysis by fluorescence microscopy demonstrated that serial ADCP of Carnaval cells by M0 macrophages occurred in particular when rituximab was combined with CD47 and anti-LILRB1 antibodies (Figure 3A ). To quantify the relative contribution, phagocytic events were assigned to uptake of the first lymphoma cell (initial phagocytosis) or to engulfment of subsequent cells (serial phagocytosis; Figure 3B ). Serial phagocytosis was observed with Carnaval, Granta 519, MEC2 and, to a lesser extent, with DG-75 cells, and was promoted by co-treatment with CD47-IgGs and LILRB1-IgGs antibodies. Especially with more ADCP sensitive Carnaval and Granta 519 cells, increases in extent of ADCP by co-blockade of LILRB1 and CD47 were attributed to enhanced phagocytic activities of individual macrophages engulfing multiple target cells. The differences in the occurrence of serial phagocytosis of Carnaval and DG-75 cells was further evidenced by grouping macrophages according to the numbers of engulfed target cells (Supplementary Figure 5 ). 

To analyze kinetics of ADCP induction, live cell imaging experiments were performed using DG-75 cells as targets (Figures 3C, D ). Again, ADCP by rituximab was enhanced by dual checkpoint blockade of CD47 and LILRB1. ADCP occurred rapidly and reached a peak after 2 h. To analyze the dose dependent mode of action of LILRB1-IgGs, rituximab and CD47-IgGs were complemented with varying concentrations of either LILRB1-IgGs or the control antibody HER2-IgGs (Figure 3E ). As a result, ADCP augmented with increasing concentrations of LILRB1-IgGs while HER2-IgGs was not effective. 

Improving FcgR engagement by Fc engineering has been shown to enhance ADCP (21). To analyze, whether ADCP could be further potentiated, an Fc-engineered variant of rituximab (RTX-DE) was generated by introducing the amino acid substitutions S239D/I332E (Supplementary Figure 1 ). This modification enhances the antibody`s affinity to activating FcgRI, FcgRIIA and FcgIIIA and improves ADCP and ADCC (51). RTX-DE was compared with rituximab in the absence or presence of immune checkpoint inhibitors in ADCP assays using DG-75 cells by live cell imaging (Figure 3F ). As a result, rituximab and RTX-DE were only marginally effective, in agreement with previous findings for this cell line. By combination of both antibodies with CD47-IgGs ADCP was enhanced. Importantly, the triple antibody combination consisting of RTX-DE, LILRB1-IgGs and CD47-IgGs was more effective than the triple antibody combination containing rituximab. This demonstrates the impact of efficient FcgR engagement and indicates that ADCP can be promoted further by improving the affinity of the tumor targeting antibody to activating FcgR. 

To analyze the impact of HLA class I receptors on ADCP by polarized macrophages, macrophages were differentiated towards M1 and M2c phenotypes using GM-CSF, LPS and IFN-g or M-CSF and IL-10, respectively. Macrophage polarization was verified by determining expression levels of the M1 and M2 marker antigens CD80 and CD163, respectively (Figure 4A ) The analysis of LILRB1 and LILRB2 cell surface expression revealed that M1 and M2c macrophages expressed both receptors at similar levels as M0 macrophages (Figure 4A ). However, the expression of SIRPa was reduced in M1 macrophages. M1 and M2c macrophages were then analyzed as effector cells for combinations of rituximab, CD47-IgGs and LILRB1-IgGs in ADCP assays with Carnaval cells and compared to non-polarized, M-CSF differentiated M0 macrophages from the same donors (Figure 4B ). As a result, rituximab was effective in inducing ADCP with different macrophage populations (Figure 4B ). In experiments with M0 and M2c macrophages, CD47-IgGs augmented rituximabmediated ADCP significantly. With M1 macrophages a similar trend was observed, but statistical significance was not reached. Importantly, LILRB1-IgGs enhanced ADCP not only by M0 macrophages, but also by polarized M1 or M2c macrophages when applied in addition to rituximab and CD47-IgGs. As observed with M0 macrophages, LILRB1 blockade alone was not sufficient to enhance ADCP by rituximab with both M1 and M2c macrophages (Figure 4B ). Moreover, ADCP by differentially polarized macrophages was analyzed by live cell imaging using DG-75 as target cells (Figure 4C ). These experiments demonstrated improved ADCP of DG-75 cells by rituximab when combined with both LILRB1-IgGs and CD47-IgGs antibodies irrespective of the macrophage polarization status. In agreement with results obtained with M0 macrophages, LILRB2-IgGs was not effective when M1 or M2c macrophages were analyzed (Supplementary Figures 4D, E ). Furthermore, M0 macrophages were differentiated from monocytes in the presence of either M-CSF or GM-CSF and then polarized to M1 macrophages using LPS and IFN-g. Interestingly, cell surface expression of both LILRB1 and LILRB2 was upregulated upon polarization with LPS and IFN-g, while SIRPa expression was reduced (Figure 4D ). In ADCP experiments using fluorescence microscopy, non-activated M0 macrophages differentiated with GM-CSF were not effective in comparison with macrophages differentiated with M-CSF (Figure 4E ). Pre-treatment with LPS/ IFN-g improved ADCP by GM-CSF macrophages, in particular when the triple combination consisting of RTX, CD47-IgGs and LILRB1-IgGs was applied. LPS/IFN-g stimulation also slightly enhanced the ADCP activity of M-CSF macrophages, which were superior to GM-CSF macrophages for each antibody treatment also upon M1 polarization with LPS/IFN-g. Of note, the triple combination consisting of RTX, LILRB1-IgGs and CD47-IgGs was most efficacious in experiments with both macrophage populations. 

In an effort to analyze the potential of LILRB1 and CD47 coblockade to enhance the ADCP activity also of lymphomaassociated macrophages (LAM), MNC were prepared from BM samples from DLBCL patients with BM infiltration (Figure 5 ). Of note, flow cytometry analysis revealed a strong expression of LILRB1 by LAM, which were defined as CD163-positive/CD15negative cells (Figures 5A, B ). For comparison, also LILRB1 expression by BM macrophages from DLBCL patients without BM infiltration was assessed (Figures 5A, B ). A trend towards a higher LILRB1 expression in the mean was found in LAM, although the observed differences were not statistically significant. For subsequent ADCP analysis, CD163-positive/ CD15-negative LAM were purified by fluorescence activated cell sorting and analyzed without further manipulation as effector cells for rituximab and the antibody combinations using Carnaval target cells (Figures 5C, D ). Importantly, a considerable further improvement in ADCP was observed when the antibody triple combination consisting of rituximab, CD47-IGgs and LILRB1-IgGs was applied relative to the combination treatment with RTX and CD47-IgGs only. 

Because cell lines do not reflect the clinical heterogeneity of patients, the dual checkpoint blockade of CD47 and LILRB1 was investigated using tumor cells from patients. CLL cells were enriched from the peripheral blood of twelve patients. The analysis of cell surface antigen expression levels revealed pronounced expression of CD20, CD47 and classical HLA class I molecules in all CLL patient samples, in contrast to HLA-G, which was hardly detected (Figure 6A ). Patient cells were then analyzed in ADCP experiments using M0 macrophages from healthy donors and fluorescence microscopy (Figure 6B ). CD47-IgGs enhanced ADCP by rituximab in the majority of individual patient samples, with exception of samples CLL_04 and CLL_10, in which CD47 blockade did not improve ADCP despite considerable CD20 and CD47 expression (Supplementary Figure 6 ). Of note, dual checkpoint blockade of CD47 and LILRB1 enhanced ADCP by rituximab further and LILRB1-IgGs amplified the degree of ADCP when combined with rituximab and CD47-IgGs. Thus, in all cases analyzed the triple antibody combination of rituximab, LILRB1-IgGs and CD47-IgGs was more efficacious than the double antibody combination consisting of rituximab and CD47-IgGs (Figure 6C ; Supplementary Figure 6 ). Sample group analysis revealed statistical significance of the observed differences between different treatment groups (Figure 6D ). The application of LILRB1-IgGs with rituximab in the absence of the CD47 antibody did not translate into higher ADCP (Figure 6D ). With all CLL cell samples tested, LILRB2 blockade was not effective (unpublished data). Further analysis revealed that, as also observed with cell line experiments, serial ADCP events occurred and were observed more frequently upon treatment with the antibody triple combination (Figure 6E ). Finally, dual checkpoint blockade of CD47 and LILRB1 was tested with MCL tumor cells isolated from two patients (Figure 6F ). Similar to CLL cells, rituximab-mediated ADCP was augmented by coblockade of CD47 and LILRB1 with LILRB1-IgGs providing an amplifying effect. Serial ADCP, kinetics, dose-dependency and further enhanced ADCP by improved FcgR engagement. (A) CFSE-labeled Carnaval cells were incubated with Cell Brite™ Orange labeled human M0 macrophages in the presence of rituximab (RTX), CD47-IgGs and LILRB1-IgGs (E:T cell ratio: 1:2). Shown are three macrophages, having engulfed one or three Carnaval cells, or none. Nuclei were stained with NucBlue™. The bottom image represents a microscope composite image of the same cells viewed separately with the red, green and blue channels as indicated. (B) LILRB1-IgGs (LILRB1) and CD47-IgGs (CD47) antibodies promote serial ADCP of Carnaval (n = 9), Granta 519 (n = 8), MEC2 (n = 6) and DG-75 (n = 6) B-NHL cells. Phagocytic events by treatment with RTX, RTX + CD47-IgGs, the triple combination of RTX + CD47-IgGs + LILRB1-IgGs or trastuzumab (IgG1) were assigned to uptake of the first lymphoma cell (initial phagocytosis) or to engulfment of subsequent cells (serial phagocytosis) and are presented as a proportion of phagocytic index values. Bars indicate mean values ± SEM. Statistically significant differences between treatment groups in initial (*) and serial (#) phagocytosis event values are indicated (P ≤ 0. 05, two-way ANOVA with Tukey`s post hoc test). (C) ADCP induced by treatment with different antibodies (10 µg/ml) alone or in combination was analyzed by live cell imaging using pHrodo ® labeled DG-75 cells and human M0 macrophages (E:T cell ratio: 1:2). For analysis, cells were imaged at different time points and red fluorescent objects were counted. Shown are representative results for the control reaction without added antibodies (w/o), treatment with RTX, RTX + CD47-IgGs (CD47) and RTX + CD47-IgGs + LILRB1-IgGs (LILRB1). The images were taken after 1. 5 h (n = 9). (D) The kinetics of ADCP was analyzed by live cell imaging as described before. M0 macrophages from different donors were used as effector cells and DG-75 cells were applied as targets. Data points represent means ± SD of red object counts per image (n = 9; w/o, without added antibody). Statistically significant (P ≤ 0. 05) differences between treatment groups RTX + CD47-IgGs vs. RTX + CD47-IgGs + LILRB1-IgGs are indicated (*P ≤ 0. 05; two-way ANOVA with Fisher´s LSD test). (E) PHrodo ® labeled DG-75 lymphoma cells were incubated with human M0 macrophages from different donors (n = 4) in the presence of RTX and CD47-IgGs (each at a constant concentration of 10 µg/ml) plus LILRB1-IgGs at varying concentrations. HER2-IgG2s (IgGs) was used as an isotype control. ADCP was analyzed over 4 h by live cell imaging. Data points represent means ± SD of red object count per image. Statistically significant differences between LILRB1-IgGs and HER2-IgGs treatments are indicated (*P ≤ 0. 05, two-way ANOVA with Fisher´s LSD test). (F) RTX and an Fc-engineered RTX variant with enhanced FcgR binding (RTX-DE) were analyzed either alone or in combination with CD47-IgGs and LILRB1-IgGs as indicated using pHrodo ® labeled DG-75 cells and human M0 macrophages. Trastuzumab (IgG1) and its Fc-engineered version HER2-DE served as controls. ADCP was determined by live cell imaging analysis. Data points represent means of red object counts per image ± SD (n = 4). *, statistically significant differences between RTX-DE + CD47-IgGs + LILRB1-IgGs vs. RTX-DE + CD47-IgGs; #, statistically significant differences between RTX-DE + CD47-IgGs + LILRB1-IgGs vs. RTX + CD47-IgGs + LILRB1-IgGs; P ≤ 0. 05, two-way ANOVA with Fisher´s LSD test.
section_name: Results

section_content: Analysis of different B-NHL cell lines revealed that the expression ratio of CD20 to HLA class I molecules determined the sensitivity to ADCP by the combination of rituximab and a CD47 blocking antibody. We thus investigated the impact of blocking the HLA class I receptors LILRB1 and LILRB2 on CD20 antibody-mediated ADCP with or without concomitant masking of CD47 using Fc-silent antibodies. While the anti-LILRB2 antibody was not effective, the anti-LILRB1 antibody enhanced ADCP considerably, but strictly required simultaneous CD47 blockade and the presence of a tumor targeting CD20 antibody to become effective. Thus, the dual checkpoint blockade of CD47 and LILRB1 enhanced ADCP by rituximab, obinutuzumab, an Fc-engineered variant of rituximab and an IgA2 version of rituximab. The LILRB1 coblockade promoted serial uptake of lymphoma cells, and demonstrated efficacy in both B-NHL cell lines and freshly isolated MCL or CLL cells from patients. 

In recent years, impressive clinical results were obtained with the application of adaptive immune checkpoint inhibitors to establish T cell tumor immunity (6, 52). Regarding B cell lymphomas, promising results were observed with immune checkpoint inhibitors targeting the PD-1/PD-L1 axis in the treatment of classical Hodgkin lymphoma with response rates exceeding 70% (53). Yet, response rates with immune checkpoint monotherapies in the majority of B-NHL types including DLBCL, follicular lymphoma and CLL were unsatisfactory (53, 54). For example, a phase II trial with the anti-PD-1 antibody nivolumab in patients with relapsed or refractory DLBCL revealed response rates of only 10%, and no objective responses were observed in relapsed CLL patients upon treatment with the anti-PD-1 antibody pembrolizumab (55, 56). The application of immune checkpoint inhibitors in combination with other therapies such as R-CHOP chemoimmunotherapy may hold promise (53, 57), but will require further investigation. Limitations arise when tumors create an immune hostile microenvironment and exert insufficient immunogenicity. In this situation, the recruitment of innate immune cells, which in the tumor microenvironment contribute to tumor immunity, may be an alternative (7, 58). Particularly the immune checkpoint blockade in myeloid cells has gained increasing attention and encouraging clinical results were obtained by the combination treatment with rituximab and the CD47 antibody magrolimab in B-NHL patients (28). 

As demonstrated here, the blockade of LILRB1 in addition to CD47 may offer a possibility further enhancing rituximabmediated ADCP of lymphoma cells. However, even when both antigens were blocked, a considerable variation between different target cells in susceptibility to ADCP was observed. This may reflect the complex regulation of phagocytosis, which is governed by an interplay between activating and inhibitory receptors. Thus, 'Don´t Eat Me!' functions have been demonstrated for several antigens including programmed death ligand 1, CD24, adipocyte plasma membrane-associated protein, and signaling lymphocyte activation molecule (SLAM) family members (10, (59) (60) (61). Cognate receptors may cooperate with LILRB1 and SIRPa, and the expression of ligands for such receptors may contribute to less ADCP sensitive phenotypes of for example DG-75 cells or CLL cells from certain patients in our Efficacy of dual checkpoint blockade of CD47 and LILRB1 with differentially polarized macrophages. (A) Left panel: Monocytes were differentiated with M-CSF (M0 macrophages), GM-CSF, LPS and IFN-g (M1 macrophages) or M-CSF and IL-10 (M2c macrophages) and stained with PE-conjugated antibodies against CD163, CD80 (blue shaded peaks) or an isotype control antibody (black outlined peaks). Cell surface expression was analyzed by flow cytometry. One representative experiment is shown (n = 3). Right panel: Cell surface expression of LILRB1, LILRB2 and SIRPa by M0, M1 and M2c macrophages was analyzed by calibrated flow cytometry. Data points indicate specific antibody binding capacity (SABC) for macrophage preparations from individual donors. Horizontal lines represent mean values ± SD (n = 7). *P ≤ 0. 05; ns, not significant; two way ANOVA and Tukey`s post hoc test. (B) Macrophages were differentiated in parallel from monocytes from seven donors towards M0, M1 or M2c phenotypes and analyzed in fluorescence microscopy-based ADCP assays using CFSE-labeled Carnaval cells (E:T cell ratio: 1:2). Efficacy was determined for rituximab (RTX) vs. phagocytosis in the absence of an antibody (w/o, first graph), RTX + CD47-IgGs (CD47) vs. RTX (second graph), and RTX + CD47-IgGs + LILRB1-IgGs (LILRB1) vs. RTX + CD47-IgGs (third graph) and RTX + LILRB1-IgGs vs. RTX (fourth graph). Antibodies were applied at a concentration of 10 µg/ml. Statistically significant differences are indicated (*P ≤ 0. 05; **P ≤ 0. 01; ***P ≤ 0. 001; ns, not significant; two-way ANOVA with S ̌idàk´s multiple comparisons test). (C) Live-cell imaging analysis of ADCP by M0, M1 and M2c macrophages in the presence of the indicated antibodies (each at a concentration of 10 µg/ml). HER2-IgGs (IgGs) was used in control reactions. Target cells were pHrodo ® labeled DG-75 cells (E:T cell ratio: 1:2). Data points represent means ± SD of the red object count per image of independent experiments using macrophages from six different donors that were polarized in parallel towards M0, M1 or M2c phenotypes (w/o, without antibody; #, statistically significant differences between RTX + CD47-IgGs vs. RTX; *, statistically significant differences between RTX + CD47-IgGs + LILRB1-IgGs vs. RTX + CD47-IgGs, two-way ANOVA and Fisher´s LSD test; P ≤ 0. 05). (D) Macrophages were differentiated from peripheral monocytes in the presence of either M-CSF (left graph) or GM-CSF (right graph) for six days. Cells were left untreated or stimulated with IFN-g and LPS for additional 48 h and analyzed for cell surface expression of LILRB1, LILRB2 and SIRPa by calibrated flow cytometry. Data points represent the specific antibody binding capacities (SABC) that were determined for individual macrophage preparations. Statistically significant differences between groups treated with IFN-g and LPS and the control groups are indicated (*P ≤ 0. 05; **P ≤ 0. 01; ***P ≤ 0. 001; two-way ANOVA with Fisher´s LSD test). (E) Macrophages were differentiated with M-CSF or GM-CSF and analyzed without further stimulation (M0 macrophages; left graph; n = 5) or after polarization with LPS and IFN-g (M1 macrophages; right graph; n = 4) in 2 h ADCP assays using CFSE-labeled Carnaval cells and the different antibodies as indicated. ADCP was analyzed by fluorescence microscopy. Data points indicate phagocytic index values for macrophages from individual donors. Horizontal lines represent mean values ± SD (*P ≤ 0. 05; **P ≤ 0. 01; ***P ≤ 0. 001; ns, not significant; two-way ANOVA and Fisher´s LSD test). 

study. In addition, the engagement of pro-phagocytic receptors such as prolow-density lipoprotein receptor-related protein 1, CD137, CD11b and the currently discussed SLAMF7, as well as additional target cell characteristics such cell size, shape or rigidity may be important (10, (62) (63) (64) (65). 

The observation that the anti-LILRB1 antibody required simultaneous blockade of the CD47-SIRPa axis suggests that SIRPa exerts a dominant inhibitory role in the regulation of ADCP. Whether differences between receptors in signaling pathways exist, leading to impairment of phagocytosis at different stages of phagocytosis initiation, still needs to be investigated. Both receptors signal via ITIM in their intracellular domains. Initiation of SIRPa signaling suppresses phagocytosis by reducing contacts between macrophages and target cells through inhibition of integrin activation, inhibition of cytoskeleton rearrangement by dephosphorylation of myosin IIA, and inactivation of neighboring FcgR through dephosphorylation of ITAM (66) (67) (68). The molecular pathways by which LILRB1 regulates phagocytosis by macrophages have not been clarified, yet impaired tyrosine phosphorylation of FcgR chain and inhibition of intracellular calcium mobilization was demonstrated upon co-ligation of FcgRI and LILRB1 (38). Of note, the dual checkpoint blockade of CD47 and LILRB1 alone was not sufficient to trigger phagocytosis and the presence of an FcR engaging CD20 antibody was required. Thus, in this approach, the specificity for phagocytic target cell elimination is maintained and is pre-defined by the tumor targeting antibody bearing a functional Fc domain to provide an activating signal. 

In contrast to LILRB1, no benefits were obtained by blockade of LILRB2 -though the receptor was expressed by monocytederived macrophages at similar levels as LILRB1, the antibody used in this study blocked receptor binding of HLA molecules in agreement with previous findings (44), and interference with FcgR signaling by LILRB2 signaling has been demonstrated previously (38). However, structural differences in binding exist in that LILRB1 binds only b2-microglobulin associated HLA molecules, while LILRB2 also binds free forms (35, 69). In addition, the receptors differ in their affinity to individual HLA alleles (69). We assume that the quality of receptor engagement, the affinity to HLA molecules or the initiation and strength of the individual receptor's intracellular signaling pathway may contribute to the observed differences. In addition, it cannot be fully excluded that the particular antibody clone employed in these experiments exerted agonistic functions and activated LILRB2 signaling in parallel to inhibition of ligand binding. Enhanced ADCP by co-blockade of LILRB1 and CD47 was demonstrated for combinations with CD20-specific IgG antibodies. Improved ADCP was observed not only in combinations with the native IgG1 molecule rituximab, but also with the Fc glyco-engineered antibody obinutuzumab and the Fc-protein engineered rituximab variant RTX-DE. Moreover, co-inhibition of LILRB1 and CD47 was effective, when the antibodies were combined with an IgA2 variant of rituximab. IgA antibodies may hold potential for cancer immunotherapy. They are able to trigger myeloid effector cells including neutrophils, monocytes and macrophages by engagement of the IgA Fc receptor FcaRI. Previously it has been demonstrated that CD47 blockade enhances macrophagemediated ADCP by CD20 IgA antibodies (49). As demonstrated in the present study, the efficacy can be further improved by coblockade of LILRB1, suggesting that LILRB1 also regulates signaling by FcaRI. 

Besides promoting ADCP, anti-LILRB1 antibodies may exert additional effector functions. Both antibody blockade of LILRB2 and genetic deletion of LILRB1 were shown to drive macrophage polarization towards an inflammatory M1 phenotype (36, 39). Therefore, the blockade of these receptors may facilitate to relieve immune suppression in the tumor microenvironment by shaping TAM or myeloid derived suppressor cells (58). In addition, enhanced engulfment of tumor cells may increase antigen presentation and promote T cell responses (10). In a murine tumor model, adaptive T cell responses were observed after treatment with CD47 antibodies (70). However, to unravel a role for LILRB1 in this context will require further investigation. In addition, LILRB1 and LILRB2 are expressed by other immune cell populations. For example, LILRB1 is expressed by T cells and a subpopulation of NK cells (32). Therefore, manipulation of LILRB1 may also promote cytotoxic functions by lymphocytes (71, 72). Regarding that NK cells may express SIRPa in certain situations even coblockade of LILRB1 and CD47 may be effective in enhancing NK cell-mediated ADCC (73). In neutrophils, conflicting results on the expression of LILRB1 were published, but LILRB2 is displayed (32, 74). Albeit LILRB2-IgGs lacked efficacy in enhancing ADCP by macrophages, it may be worth testing this antibody with neutrophils, in which blockade of the CD47-SIRPa axis was shown to enhance ADCC and trogoptosis (75). 

To further advance the proposed concept animal studies will be required. However, this is a challenging issue, because LILRB1 is not expressed in mice and the murine receptor orthologue paired immunoglobulin-like receptor B does not react with HLA/b2M complexes (36). The generation of LILRB1 transgenic mice could offer an opportunity to study this. In a previous study, LILRB1 knock-in mice were established in the background of immune competent mice (76), but here immune deficient mice will be required to facilitate the engraftment of human lymphoma cells. Humanized mice in which human immune cells are established by transplantation of human CD34-positive progenitor cells could offer another option (77). However, the presence of human immune cells may hamper the co-engraftment of tumor cells and partial HLA matching may be required. In addition, co-existing murine macrophages, which respond to CD47 but not to anti-LILRB1 antibody blockade (36), may distort results, and an accompanying depletion of murine macrophages may be necessary. Yet, our in vitro results provide a rationale to undertake these efforts to realize such xenograft in vivo studies. 

In conclusion, our preclinical in vitro results suggest potential of combining CD20, CD47 and anti-LIRB1 antibodies. LILRB1 blockade complemented CD47 inhibition and thus a dual checkpoint blockade of CD47-SIRPa and LILRB1-HLA class I interactions may have the potential to improve antibody therapy of lymphomas further by enhancing ADCP by macrophages. Therefore, combinations of tumor targeting antibodies with LILRB1 and either CD47 or SIRPa immune checkpoint inhibitors deserve further evaluation in animal models towards clinical application. AM and CK. Investigation, TZ, SL, IM, RW, PH, TR, NB, CF, HB and CK. Resources, NT, JB, TH, OW, CW, MB-B, MP, HB, DS and AH. Writing -original draft preparation, TZ and CK. Writing -review and editing, SL, RW, TH, OW, MB-B, CW, TV, DS, HB and AH. Visualization, TZ, SL, HB and CK. Supervision, CK. All authors have read and agreed to the published version of the manuscript.
section_name: Discussion

section_name: Acknowledgments

section_content: Irene Pauls, Lena Halang, and Fiona E. Rosmus are kindly acknowledged for expert technical assistance. The Deutsche JoseĆ arreras Leukämie-Stifung and the Deutsche Gesellschaft für Hämatologie und Medizinische Onkologie are gratefully acknowledged for the JoséCarreras-DGHO scholarship for doctoral research to TZ ( 05 PSD/2021 ). We also kindly thank the study participants.
section_name: Acknowledgments

section_name: Funding

section_content: This work was funded by research grants by the Deutsche Krebshilfe ( 70113524 and 70113533, to DS and CK), the Verein zur Förderung von Wissenschaft und Forschung an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München (to CK) and the Deutsche JoséCarreras Leukämie-Stiftung to TH ( DJCLS 10 R/2021 ).
section_name: Funding

section_name: Data availability statement

section_content: The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
section_name: Data availability statement

section_name: Ethics statement

section_content: The studies involving human participants were reviewed and approved by Ethics Committee of the faculty of medicine, LMU Munich. The patients/participants provided their written informed consent to participate in this study.
section_name: Ethics statement

section_content: Conceptualization, CK. Methodology, TZ, SL, MP, HB, AM and CK. Validation, TZ, SL and CK. Formal analysis, TZ, SL, IM,
section_name: Author contributions

section_content: 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.
section_name: Conflict of interest

section_content: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
section_name: Publisher's note

section_content: The Supplementary Material for this article can be found online at: https://www. frontiersin. org/articles/10. 3389/ fimmu. 2022. 929339/full#supplementary-material
section_name: Supplementary material