Blockade of the CD47/SIRPα checkpoint axis potentiates the macrophage-mediated anti-tumor efficacy of tafasitamab.

Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.

CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS.

ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

Activity of tafasitamab in combination with rituximab in subtypes of aggressive lymphoma.

Background: Despite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects. Methods: Antibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Results: Three different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model. Conclusion: This study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo.

Tafasitamab mediates killing of B-cell non-Hodgkin's lymphoma in combination with γδ T cell or allogeneic NK cell therapy.

Tafasitamab is an Fc-modified monoclonal antibody that binds to CD19, a cell-surface antigen that is broadly expressed on various types of B-cell non-Hodgkin's lymphoma (NHL). Antibody-dependent cellular cytotoxicity (ADCC), a key mode of action of tafasitamab, is mediated through the binding of tafasitamab's Fc region to FcγRIIIa receptors on immune effector cells and results in antitumor activity. Despite the proven clinical activity of tafasitamab in combination with lenalidomide in the treatment of diffuse large B-cell lymphoma (DLBCL), a higher number of immune cells in cancer patients may improve the activity of tafasitamab. Here, we characterized two ex vivo-expanded FcγRIIIa receptor-expressing cell types-γδ T and MG4101 natural killer (NK) cells-as effector cells for tafasitamab in vitro, and found that in the presence of these cells tafasitamab was able to induce ADCC against a range of NHL cell lines and patient-derived cells. We also explored the concept of effector cell supplementation during tafasitamab treatment in vivo by coadministering MG4101 NK cells in Raji and Ramos xenograft models of NHL. Combination treatment of tafasitamab and allogeneic MG4101 NK cells in these models demonstrated a survival benefit compared with tafasitamab or MG4101 monotherapy (Raji: 1.7- to 1.9-fold increase in lifespan; Ramos: 2.0- to 4.1-fold increase in lifespan). In conclusion, adoptive cell transfer of ex vivo-expanded allogeneic NK or autologous γδ T cells in combination with tafasitamab treatment may potentially be a promising novel approach to increase the number of immune effector cells and enhance the antitumor effect of tafasitamab.

Neutralization of IL-17C Reduces Skin Inflammation in Mouse Models of Psoriasis and Atopic Dermatitis.

IL-17C is a functionally distinct member of the IL-17 family that was believed to play a role in the pathogenesis of psoriasis. Here we confirmed that IL-17C is involved in psoriasis and explored potential roles for IL-17C in atopic dermatitis (AD). An anti-IL-17C antibody, MOR106, was generated that potently and selectively binds to human and mouse IL-17C, thereby inhibiting the binding of IL-17C to its IL-17RE receptor. The antibody inhibited cutaneous inflammation in an IL-23-induced psoriatic-like skin inflammation model. In lesional skin of patients with AD, IL-17C expression levels were increased and localized to keratinocytes and infiltrating immune cells. To determine the contribution of IL-17C to AD pathogenesis, MOR106 was tested in two distinct in vivo models. In the calcipotriol-induced AD model, ear skin inflammation, TSLP, and IL-33 protein production in ears was suppressed by MOR106. Consistently, in the flaky tail strain mouse model, spontaneous development of AD-like skin inflammation was reduced by MOR106. Moreover, serum IgE levels, number of mast cells in skin and T helper type 2-related cytokines IL-4 and CCL17 in serum were all reduced. Overall, our results indicate that IL-17C is a central mediator of skin inflammation beyond psoriasis and is relevant in particular in AD.

Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody.

X-ray structure analysis of 4 antibody Fab fragments, each in complex with human granulocyte macrophage colony stimulating factor (GM-CSF), was performed to investigate the changes at the protein-protein binding interface during the course of in vitro affinity maturation by phage display selection. The parental antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized), MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All antibodies bind to a conformational epitope that can be divided into 3 sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF N-terminus (residues 11-24), a short second sub-epitope (residues 83-89) and a third at the C-terminus (residues 112-123). Modifications introduced during affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional hydrogen bonds and van der Waals contacts, respectively, providing a rationale for the observed improvement in binding affinity and neutralization potency. Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash with GM-CSF binding to GM-CSF receptor α and ß chain. This predicted mutually exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding inhibition assay. Finally, high throughput sequencing of clones obtained after affinity maturation phage display pannings revealed highly selected consensus sequences for CDR-H2 as well for CDR-L3, which are in accordance with the sequence of the highest affinity antibody MOR04357. The resolved crystal structures highlight the criticality of these strongly selected residues for high affinity interaction with GM-CSF.

Using the low-pass monogenic signal framework for cell/background classification on multiple cell lines in bright-field microscope images.

PURPOSE: Several cell detection approaches which deal with bright-field microscope images utilize defocusing to increase image contrast. The latter is related to the physical light phase through the transport of intensity equation (TIE). Recently, it was shown that it is possible to approximate the solution of the TIE using a low-pass monogenic signal framework. The purpose of this paper is to show that using the local phase of the aforementioned monogenic signal instead of the defocused image improves the cell/background classification accuracy. MATERIALS AND METHODS: The paper statement was tested on an image database composed of three cell lines: adherent CHO, adherent L929, and Sf21 in suspension. Local phase and local energy images were generated using the low-pass monogenic signal framework with axial derivative images as input. Machine learning was then employed to investigate the discriminative power of the local phase. Three classifier models were utilized: random forest (RF), support vector machine (SVM) with a linear kernel, and SVM with a radial basis function (RBF) kernel. RESULTS: The improvement, averaged over cell lines, of classifying 5×5 sized patches extracted from the local phase image instead of the defocused image was 7.3% using the RF, 11.6% using the linear SVM, and 10.2% when a RBF kernel was employed instead of the linear one. Furthermore, the feature images can be sorted by increasing discriminative power as follows: at-focus signal, local energy, defocused signal, local phase. The only exception to this order was the superiority of local energy over defocused signal for suspended cells. CONCLUSIONS: Local phase computed using the low-pass monogenic signal framework considerably outperforms the defocused image for the purpose of pixel-patch cell/background classification in bright-field microscopy.

Granulocyte-macrophage colony-stimulating factor is a key mediator in inflammatory and arthritic pain.

OBJECTIVES: Better therapies are needed for inflammatory pain. In arthritis the relationship between joint pain, inflammation and damage is unclear. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important for the progression of a number of inflammatory/autoimmune conditions including arthritis; clinical trials targeting its action in rheumatoid arthritis are underway. However, its contribution to inflammatory and arthritic pain is unknown. The aims of this study were to determine whether GM-CSF controls inflammatory and/or arthritic pain. METHODS: A model of inflammatory pain (complete Freund's adjuvant footpad), as well as two inflammatory arthritis models, were induced in GM-CSF(-/-) mice and development of pain (assessment of weight distribution) and arthritic disease (histology) was assessed. Pain was further assessed in a GM-CSF-driven arthritis (methylated bovine serum albumin/GM-CSF) model and the cyclooxygenase-dependence determined using indomethacin. RESULTS: GM-CSF was absolutely required for pain development in both the inflammatory pain and arthritis models, including for IL-1-dependent arthritic pain. Pain in a GM-CSF-driven arthritis model, but not the disease itself, was abolished by the cyclooxygenase inhibitor, indomethacin, indicating separate pathways downstream of GM-CSF for pain and arthritis control. CONCLUSIONS: GM-CSF is key to the development of inflammatory and arthritic pain, suggesting that pain alleviation could result from trials evaluating its role in inflammatory/autoimmune conditions.

Granulocyte-macrophage colony-stimulating factor is a key mediator in experimental osteoarthritis pain and disease development.

INTRODUCTION: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be important in the development of inflammatory models of rheumatoid arthritis and there is encouraging data that its blockade may have clinical relevance in patients with rheumatoid arthritis. The aims of the current study were to determine whether GM-CSF may also be important for disease and pain development in a model of osteoarthritis. METHODS: The role of GM-CSF was investigated using the collagenase-induced instability model of osteoarthritis. We studied both GM-CSF-/- mice and wild-type (C57BL/6) mice treated prophylactically or therapeutically with a monoclonal antibody to GM-CSF. Disease development (both early and late) was evaluated by histology and knee pain development was measured by assessment of weight distribution. RESULTS: In the absence of GM-CSF, there was less synovitis and matrix metalloproteinase-mediated neoepitope expression at week 2 post disease induction, and less cartilage damage at week 6. GM-CSF was absolutely required for pain development. Therapeutic neutralization of GM-CSF not only abolished the pain within 3 days but also led to significantly reduced cartilage damage. CONCLUSIONS: GM-CSF is key to the development of experimental osteoarthritis and its associated pain. Importantly, GM-CSF neutralization by a therapeutic monoclonal antibody-based protocol rapidly and completely abolished existing arthritic pain and suppressed the degree of arthritis development. Our results suggest that it would be worth exploring the importance of GM-CSF for pain and disease in other osteoarthritis models and perhaps clinically for this form of arthritis.

In vitro affinity maturation of HuCAL antibodies: complementarity determining region exchange and RapMAT technology.

Monoclonal antibodies gain ever-increasing importance in the treatment of human diseases across a broad range of indications. Diverse technologies currently exist, which are used to generate recombinant therapeutic antibodies that are basically indistinguishable from naturally occurring human immunoglobulins. We describe how human combinatorial antibody libraries are used together with unique optimization techniques to produce such therapeutically relevant proteins, for instance in the areas of oncology and inflammation.

In vitro affinity maturation of human GM-CSF antibodies by targeted CDR-diversification.

The mammalian immune system applies somatic hypermutation to select for antibodies with improved dissociation rates in vivo up to an intrinsic limit, previously termed as affinity ceiling. However, for certain therapeutic applications it may be desirable to further improve antibody affinities beyond that limit. In this study the selection of antibodies specific for the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) from the HuCAL GOLD human antibody library is described. In order to increase affinity and also functional activity, in vitro affinity maturation of a pool of lead Fab candidates was carried out. CDR-L3 and parallel CDR-H2 diversification using trinucleotide consensus cassettes were followed by the combination of optimized CDR-L3 and CDR-H2 leading to a 5000-fold improved affinity finally reaching a K(D) of 400 fM. Cytokine neutralizing potential of MOR04357 was evaluated in a TF-1 proliferation assay. Along with affinity optimization a 2000-fold increase in potency was observed compared to the parental antibody. Due to species cross-reactivity MOR04357 also blocks rat GM-CSF induced proliferation of FDCP-1 cells. Receptor inhibition studies showed that MOR04357 prevents the interaction of GM-CSF with the GM-CSF receptor alpha chain. As a consequence this leads to a blockade in signal transduction as measured by abolished STAT5 phosphorylation in the presence of GM-CSF and antibody. Due to its pro-inflammatory role GM-CSF has been implicated in the pathophysiology of inflammatory diseases like rheumatoid arthritis or asthma. Based on the mode of action described herein MOR04357 shows favourable antibody features as a potential drug candidate.