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Whilst treatment of multiple myeloma (MM) with daratumumab significantly extend patient lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/rMM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T-cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38 including those with a lower sensitivity to daratumumab. In a killing assay, wherein multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity towards MM cells compared to daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in two therapeutic mouse models, unlike daratumumab. Lastly, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/rMM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently developed in a phase 1 clinical study.

Supplementary Figure 1 shows more details on the mode of action and design of the used antibodies. Supplementary Figure 2 displays additional APC activation data of the human and murine FAP-CD40 molecules. Supplementary Figure 3 shows additional data for FRC in vitro assays and the OVA vaccination study. Supplementary Figure 4 shows additional data on the in vivo study comparing FAP-huCD40 and SGN40 moIgG1. Supplementary Figure 5 displays additional data on the in vivo comparison of selicrelumab and FAP-huCD40. Supplementary Figure 6 shows the data of a FAP-huCD40 dose escalation study in cynomolgus monkeys.

Supplemental data details

Detection of tumor targeting by in vivo imaging (fluorescent intensity, FLI)of CEA TCB formats having bivalent or monovalent binding to CEA and directly labeled with Alexa647.

Supplemental Video 3. In vitro visualization of the dynamics of CD20-TCB (white) during interaction between CD8+ T-cells (green) and tumor cells (blue). The video focuses on an example of CD20-TCB-mediated CD8+ T-cell cytotoxicity of tumor cells. T-cells surround the tumor cells and create stable synapses where CD20-TCB is localized (white). Tumor cells are killed upon certain time of T-cell/tumor cell interaction as evidenced by the formation of tumor blebs.

Purpose:CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma.Experimental Design:FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models.Results:FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects.Conclusions:FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

Pharmacokinetic profiles of CEA TCB in mice, additional information on CD3 signaling, T cell activation, tumor cell lysis and cytokine release upon CEA TCB and untargeted control TCB treatment.

Supplemental Figure 1. Assessment of the binding characteristics and cynomolgus monkey cross-reactivity of CD20-TCB. Supplemental Figure 2. Tumor cell lysis mediated by different CD20-targeting TCB antibodies. Supplemental Figure 3. T-cell and B-cell counts along with E:T ratio upon CD20-TCB treatment of primary bone marrow aspirates from patients with lymphoma and leukemia. Supplemental Figure 4. Quantification of interactions between T-cells and tumor cells from live imaging experiments and kinetic analysis of T-cell activation and exhaustion marker expression. Supplemental Figure 5. Secretion of cytokines, cytotoxic granules and T-cell proliferation upon tumor lysis. Supplemental Figure 6. Assessment of the cytokine-mediated endothelial cell activation. Supplemental Figure 7. Characterization of stem cell humanized mice (HSC-NSG) generated in house.

Purpose: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB.Experimental Design: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells.Results: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA–expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1–positive tumors.Conclusions: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286–97. ©2016 AACR.

Representative videos of intravital two-photon (2P) microscopy of tumors treated with CEA TCB. Tumor cells red, T cells green.

List of antibodies used for flow cytometry

Supplemental Video 1. In vitro visualization of the dynamics of untargeted TCB (white) during the interaction between CD8+ T-cells (green) and tumor cells (blue).

Additional details on methods used for SPR assays, NanoString RNAseq and cynomolgus monkey studies

Purpose: Despite promising clinical activity, T-cell–engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell–binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785–97. ©2018 AACR.See related commentary by Prakash and Diefenbach, p. 4631

List of all genes analyzed in NanoString gene expression analysis comparing gene expression in tumors of selicrelumab and FAP-huCD40-treated mice

Representative videos of intravital two-photon (2P) microscopy of tumors treated with untargeted TCB. Tumor cells red, T cells green.

List, Source and CEA binding sites of Cell Lines used in the study

Additional data of cynomolgus monkey study showing body weight and plasma cytokine levels over the course of the study

Assessment of binding of CEA TCB to human and cynomolgus monkey CEA- and CD3-expressing cells, internalization and ADCC, CDC activity.

Supplemental Material and Methods describing CEA TCB production and purification process, determination of affinity and avidity, genetic characterization of cell lines, determination of CEA binding sites, assessment of T cell proliferation and activation, Jurkat NFAT assay, confocal microscopy, FRET, SDPK, in vivo efficacy and imaging studies. Supplemental Video Legends are also within the file.

Supplemental Video 2. In vitro visualization of the dynamics of CD20-TCB (white) during the interaction between CD8+ T-cells (green) and tumor cells (blue).

Supplemental materials and methods

Supplemental Table 1. Overview of CD20-targeting T-cell bispecific molecules evaluated in the current study. Supplemental Table 2. Tumor cell lines and EC50 values of tumor cell lysis. Supplemental Table 3. Assessment of the activity in ex vivo primary tumor samples derived from aggressive lymphoma and leukemia patients. Supplemental Table 4. Absolute values of cytokines measured by multiplex analysis of blood samples reported as heatmap in Figure 3e, 4b, 5d, and 6b. Supplemental Table 5. Average score of perivascular CD3 positive T-cells in the lung (assessed by blinded pathologist analysis) referring to T-cell staining of lungs from tumor-bearing, HSC NSG mice 7 days after the first treatment and 24 h after the second treatment (treatment groups are indicated on Figure 6D panels).

Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1− PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell – T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.

Expansion of antigen-specific effector CD8 T cells is central to the control of chronic viral infections and cancer. In particular, differentiation of antigen-experienced, PD-1+TCF-1+ stem -like CD8 T cells, termed here “resource T cells”, into effector cells is critical for the success of immunotherapies based on immune checkpoint PD-1 signaling blockade1-4. In the accompanying article, Hashimoto et al. show that in chronic infections, administration of the cytokine interleukin- 2 (IL-2) triggers an alternative differentiation path from resource T cells leading to the expansion of a distinct population of effector CD8 T cells, termed “better effectors”, which are similar to effector cells generated in an acute infection. IL-2 binding to the non-signaling component of its receptor, IL-2Rα-chain, also known as CD25, was essential to trigger this alternative differentiation path and to expand better effectors with distinct transcriptional and epigenetic profiles. However, as IL-2 binding to CD25 constitutively expressed on immunosuppressive regulatory T cells and on some endothelial cells contributes to unwanted systemic effects of IL-2 therapy that have hampered its broad clinical application, a new generation of engineered IL-2Rβγ-biased agonists with reduced/abolished CD25 binding is currently being developed5-9. Here we show that IL-2Rβγ-biased agonists devoid of CD25 binding are unable to preferentially expand better effectors in cancer models, and describe a novel antibody-cytokine fusion protein, PD1-IL2v, that overcomes the need for CD25 binding by using PD-1 binding in cis in order to generate this distinct effector cell population. PD1-IL2v comprises an engineered IL-2 variant devoid of CD25 binding, fused to a high affinity PD-1 blocking antibody. In cis binding of PD1-IL2v to PD-1 and IL-2Rβγ on the same cell surface recovers the ability to differentiate resource CD8 T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models, while providing superior treatment efficacy. In contrast, PD-1/-L1 blocking antibodies alone, or their combination with clinically relevant doses of IL-2-based molecules with abolished CD25 binding and not targeted to PD-1, could not expand this unique subset of better effector T cells, and rather led to the accumulation of terminally-differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Expansion and differentiation of antigen-experienced PD-1

ISB 1442 is a fully human bispecific antibody (BsAb) using BEAT (Bispecific Engagement by Antibodies based on the T-cell receptor) platform to target CD38 and CD47 as treatment for CD38+ malignancies, including multiple myeloma (MM). ISB 1442 is designed with a bi-paratopic anti-CD38 arm that strongly binds to CD38+ tumor cells and an anti-CD47 arm made of a single Fab designed to block interaction between CD47 and the signal-regulatory protein alpha (SIRPα) with low affinity. This approach is expected to induce minimal unintended effects on red blood cells (RBC) compared to anti-CD47 monoclonal antibody (mAb) magrolimab as it enables the CD47 binding only upon avidity induced CD38 crosslinking. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells, autologous macrophages, as well as complement from human serum. In the MMoAK assay, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab. In vivo, in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower RBC hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. On the contrary, due to the higher expression of CD38 in monkey RBC compared to human, ISB 1442 showed more pronounced binding on RBC than magrolimab, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Finally, by integrating in vitro pharmacology data along with available clinical information on benchmark antibodies, a quantitative systems pharmacology model was developed to simulate potential efficacious dose range in MM. In summary, we report a novel approach for the treatment for CD38+ cancers by co-targeting CD38 and CD47. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity by overcoming known primary and acquired tumor escape mechanisms of resistance relative to daratumumab. Citation Format: Stefano Sammicheli, Camille Grandclement, Elie Dheilly, Maria Panagopoulou, Evangelia Martini, Perrine Suere, Blandine Pouleau, Carole Estoppey, Julia Frei, Jeremy Loyau, Thierry Monney, Adam Drake, Alain Rubod, Marie Agnes Doucey, Vinu Menon, Venkatesha Udupa, Sunitha GN, Daniel Rasmussen, Jeppe Koch Olsen, Roberto Gionannini, Girish Gudi, Ankita Srivastava, Cyril Konto, Mario Perro. ISB 1442, a first-in-class CD38 and CD47 bispecific antibody innate cell modulator for the treatment of CD38+ malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2903.

Diffuse large B cell lymphomas (DLBCL) are a highly heterogeneous subtype of Non Hodgkin Lymphoma (NHL), accounting for about 25% of NHL [1]. Despite an increased progression-free survival upon therapy, 40-50% of patients develop relapse/refractory disease, therefore there remains an important medical need [2]. T cell recruiting therapies, such as the CD20xCD3 T cell bi-specific antibody CD20-TCB (RG6026 or glofitamab), represent a novel approach to target all stages of DLBCL, especially those that fail to respond to multiple lines of treatment [3, 4]. We aimed for a better understanding of the molecular features related to the mode of action (MoA) of CD20-TCB in inducing Target/T cell synapse formation and human T cell recruitment to the tumor. To directly evaluate the correlation between synapse, cytokine production and anti-tumor efficacy using CD20-TCB, we developed an innovative preclinical human DLBCL in vivo model that allowed tracking in vivo human T cell dynamics by multiphoton intravital microscopy (MP-IVM). By ex vivo and in vivo approaches, we revealed that CD20-TCB is inducing strong and stable synapses between human T cell and tumor cells, which are dependent on the dose of CD20-TCB and on LFA-1 activity but not on FAS-L. Moreover, despite CD20-TCB being a large molecule (194.342 kDa), we observed that intra-tumor CD20-TCB-mediated human T cell-tumor cell synapses occur within 1 hour upon CD20-TCB administration. These tight interactions, observed for at least 72 hours post TCB administration, result in tumor cell cytotoxicity, resident T cell proliferation and peripheral blood T cell recruitment into tumor. By blocking the IFNγ-CXCL10 axis, the recruitment of peripheral T cells was abrogated, partially affecting the efficacy of CD20-TCB treatment which rely only on resident T cell proliferation. Altogether these data reveal that CD20-TCB’s anti-tumor activity relies on a triple effect: i) fast formation of stable T cell-tumor cell synapses which induce tumor cytotoxicity and cytokine production, ii) resident T cell proliferation and iii) recruitment of fresh peripheral T cells to the tumor core to allow a positive enhancement of the anti-tumor effect.

Purpose: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. Experimental Design: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. Results: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. Conclusions: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

Introduction Cancer Immune Therapies have shown unprecedented results in improving tumor control.1–3 However, many patients are still refractory to treatment. A deeper understanding of the mode of action of the different CITs sub-classes may help improving therapeutic approaches to reach better anti-tumor response. For this reason, we developed a multi-photon intra-vital microscopy (MP-IVM) approach to study in vivo, at single cell level, the tumor microenvironment upon treatment with CD20-targeting T-cell bispecific antibodies (TCB) [4] in a preclinical model of diffuse large B cell lymphomas (DLBCL). Methods To selectively monitor clinical lead molecules in the context of human T cell responses, we developed a skinfold chamber model [5] in last generation humanized mice [6] that allows visualization, by MP-IVM, of labelled human T cells co-injected intra-dermally with WSU-DLCL2, a human DLBCL. We have used this model to investigate T cells recruitment to tumors upon CD20-TCB therapy: by intra-venously injecting labeled T cells in mice treated with selected blocking antibodies, we were able to identify dedicated pathways induced by CD20-TCB and regulating T cell influx into the tumor bed. Furthermore, we developed a user-independent quantification platform to assess changes in the dynamics of T cell motility and time of interaction with tumor cells. Results/Discussion We have developed an experimental preclinical model that aims to reduce xenoreaction (human T cell reaction against mouse tissue) by utilizing T cells derived from humanized mice, educated within murine thymus. We demonstrate that such model is optimal to quantify human T cell dynamics in vivo. We show that CD20-TCB localizes in the tumor and acts on tumor-resident T cell motility within 1 hour post i.v. injection (defined as functional PK), causing a sharp reduction in their speed (from 4 to 2 µm/min) and an increase in tumor/T cell interaction time; those changes last up to 72h post-treatment. In addition, we prove how the initial tumor/T cell interaction mediated by CD20-TCB lead to peripheral T cells recruitment into the tumor. This mechanism is dependent on the presence of tumor-resident T cells and on IFNg-CXCL10 pathway. Inhibiting any of these two parameters resulted in reduced T cells infiltration from the periphery and reduced anti-tumor efficacy. Conclusion We developed a reliable imaging and analysis pipeline to investigate in vivo T cell dynamics and recruitment and applied it to the study of CD20-TCB treatment of DLBCL model. Our approach has shed new lights into the MoA of this new class of immune-therapeutics, demonstrating that the IFNγ-CXCL10 pathway is involved in T cell recruitment upon CD20-TCB treatment. Disclosure Information F. Cremasco: A. Employment (full or part-time); Significant; Roche Glycart AG. E. Menietti: A. Employment (full or part-time); Significant; Roche Glycart AG. D. Speziale: A. Employment (full or part-time); Significant; Roche Glycart AG. J. Sam: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Sammicheli: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Chen: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Richard: A. Employment (full or part-time); Significant; Roche Glycart AG. C. Klein: A. Employment (full or part-time); Significant; Roche Glycart AG. P. Umana: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Bacac: A. Employment (full or part-time); Significant; Roche Glycart AG. S. Colombetti: A. Employment (full or part-time); Significant; Roche Glycart AG. M. Perro: A. Employment (full or part-time); Significant; Roche Glycart AG.

SUMMARY Recently developed approaches for highly multiplexed imaging have revealed complex patterns of cellular positioning and cell-cell interactions with important roles in both cellular- and tissue-level physiology. However, tools to quantitatively study cellular patterning and tissue architecture are currently lacking. Here, we develop a spatial analysis toolbox, the histo-cytometric multidimensional analysis pipeline (CytoMAP), which incorporates data clustering, positional correlation, dimensionality reduction, and 2D/3D region reconstruction to identify localized cellular networks and reveal features of tissue organization. We apply CytoMAP to study the microanatomy of innate immune subsets in murine lymph nodes (LNs) and reveal mutually exclusive segregation of migratory dendritic cells (DCs), regionalized compartmentalization of SIRPa dermal DCs, and preferential association of resident DCs with select LN vasculature. The findings provide insights into the organization of myeloid cells in LNs and demonstrate that CytoMAP is a comprehensive analytics toolbox for revealing features of tissue organization in imaging datasets., In Brief Stoltzfus et al. present CytoMAP, a spatial analytics platform that incorporates diverse statistical and visualization modules for analysis of cellular positioning, cell-cell interactions, global tissue structure, and heterogeneity of tissue microenvironments. Exploration of myeloid cell localization in lymph nodes reveals fundamental positional relationships between dendritic cell subsets and local vasculature., Graphical Abstract

OBJECTIVE Depleting pathogenic B cells could treat systemic lupus erythematosus (SLE). However, depleting B cells in an inflammatory setting such as lupus is difficult. This study was undertaken to investigate whether a type II anti-CD20 monoclonal antibody (mAb) with a different mechanism of action, obinutuzumab (GA101), is more effective than a type I anti-CD20 mAb, rituximab (RTX), in B cell depletion in lupus, and whether efficient B cell depletion results in amelioration of disease. METHODS We treated lupus-prone MRL/lpr mice expressing human CD20 on B cells (hCD20 MRL/lpr mice) with either RTX or GA101 and measured B cell depletion under various conditions, as well as multiple clinical end points. RESULTS A single dose of GA101 was markedly more effective than RTX in depleting B cells in diseased MRL/lpr mice (P < 0.05). RTX overcame resistance to B cell depletion in diseased MRL/lpr mice with continuous treatments. GA101 was more effective in treating hCD20 MRL/lpr mice with early disease, as GA101-treated mice had reduced glomerulonephritis (P < 0.05), lower anti-RNA autoantibody titers (P < 0.05), and fewer activated CD4+ T cells (P < 0.0001) compared to RTX-treated mice. GA101 also treated advanced disease, and continual treatment prolonged survival. Using variants of GA101, we also elucidated B cell depletion mechanisms in vivo in mice with lupus. CONCLUSION Albeit both anti-CD20 antibodies ameliorated early disease, GA101 was more effective than RTX in important parameters, such as glomerulonephritis score. GA101 proved beneficial in an advanced disease model, where it prolonged survival. These data support clinical testing of GA101 in SLE and lupus nephritis.

Leukocyte function-associated antigen 1 (LFA-1) is the most widely expressed member of the β2 integrin family of cell-cell adhesion molecules. Although LFA-1 is thought to regulate multiple aspects of T cell immunity, its role in the response of CD8+ T cells to viral infections remains unclear. Indeed, compelling clinical evidence shows that loss of LFA-1 function predisposes to infection in humans but animal models show limited to no susceptibility to infection. Here, we addressed this conundrum in a mouse model of infection with lymphocytic choriomeningitis virus (LCMV), where CD8+ T cells are necessary and sufficient to confer protection. To this end, we followed the fate and function of wild-type and LFA-1 deficient virus-specific CD8+ T cells and assessed the effect of blocking anti-LFA-1 monoclonal antibody in the outcome of infection. Our analysis of viral clearance and T cell responses using transcriptome profiling reveals a role for LFA-1 as a gatekeeper of effector T cell survival and dysfunction that when defective can predispose to LCMV infection.

Recently developed approaches for highly-multiplexed 2-dimensional (2D) and 3D imaging have revealed complex patterns of cellular positioning and cell-cell interactions with important roles in both cellular and tissue level physiology. However, robust and accessible tools to quantitatively study cellular patterning and tissue architecture are currently lacking. Here, we developed a spatial analysis toolbox, Histo-Cytometric Multidimensional Analysis Pipeline (CytoMAP), which incorporates neural network based data clustering, positional correlation, dimensionality reduction, and 2D/3D region reconstruction to identify localized cellular networks and reveal fundamental features of tissue organization. We apply CytoMAP to study the microanatomy of innate immune subsets in murine lymph nodes (LNs) and reveal mutually exclusive segregation of migratory dendritic cells (DCs), regionalized compartmentalization of SIRPa− dermal DCs, as well as preferential association of resident DCs with select LN vasculature. These studies provide new insights into the organization of myeloid cells in LNs, and demonstrate that CytoMAP is a comprehensive analytics toolbox for revealing fundamental features of tissue organization in quantitative imaging datasets.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti–4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor–mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP–4-1BBL (RG7826) and CD19–4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen–mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP–4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer–bearing rhesus monkey. Combination of FAP–4-1BBL with tumor antigen–targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP– or CD19–4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8(+) T cells. FAP– and CD19–4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

Cancer Immunotherapy with checkpoint inhibitors (CPI) has been a major breakthrough in cancer treatment and it has been proven effective in around 30% of inflamed tumors. However, why the majority of solid tumors do not respond to CPIs is not well understood, and knowledge of resistance mechanisms (primary or acquired) and associated biomarkers could provide great benefit for more successful application of CPIs and other immunotherapies. Recent advances in highly multiplexed imaging techniques is starting to reveal that the organization of immune cells within tumors can be an important prognostic indicator of disease outcome. However, the complexity and heterogeneity of cancer microenvironments, as well as the paucity of tools to study such 3-dimensional spatial data, still limits our understanding of immune cell organization and function within tumors. Here, we developed and implemented a workflow to image resected tumors in two or three dimensions, using up to 13 fluorescent parameters. To resolve local cellular networks and reveal fundamental features of tissue organization within tumors, we utilized a newly-developed spatial analysis toolbox (CytoMAP), that incorporates machine learning, clustering, positional correlations, dimensionality reduction and region reconstruction. We employed this multiplexed imaging approach to explore the organization of immune cells in several syngeneic murine cancer models, representing inflamed and not-inflamed subtypes. We identified hallmark features of baseline tumor architecture, such as interaction between immune cells and blood vessels, and baseline organization and localization of innate and adaptive immune subsets, even across datasets with extensive intra- and inter- tumor heterogeneity. We, additionally, applied CytoMAP to understand how immunotherapies change tumor microenvironment. Treatment of MC38 tumors with CPI lead to an increased area of the tumor mass occupied by regions containing high number of activated CD8+ T cell and MHC-II+ macrophages but low number of Tregs and scattered tumor cells, identifying areas of active tumor killing. The relative area occupied by these ‘immune-control' regions, compared to that found at baseline, might better define responder and non-responder tumors. In conclusion, the combination of highly multiplexed confocal imaging and quantitative image analysis with CytoMAP allows deconvolution of the heterogeneity of the tumors into functional regions, providing a predictive tool to assess the response to different immune-therapies. Citation Format: Elena Menietti, Caleb Stoltzfus, Brandy Olin, Dario Speziale, Leo Kunz, Thomas Poeshinger, Sara Colombetti, Michael Gerner, Mario Perro. Understanding the complexity of the tumor microenvironment by 3-dimensional multiplexed imaging and artificial intelligence [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 370.

Introduction: In the rapidly growing field of chimeric antigen receptor (CAR) engineered T cells new approaches aim to make CAR-T cell therapy safer and more effective. Recent, designs aim towards universal or modular CARs that do not directly recognize the target antigen itself, but instead facilitate contact to CAR-adaptor molecules, which in turn bind to the target antigen. Current CAR-adaptor molecules include human antibodies of the IgG1 isotype binding to CD16 or antibodies modified by peptide or hapten tags. We developed a modular CAR-T approach that recognizes the P329G mutation clinically used to silence the Fc effector function of therapeutic antibodies. These modular anti-P329G-CAR-T cells are only functional in the presence of antibodies that possess the P329G mutation. Methods: The anti-P329G interaction with P329G-containing Fc fragment was analyzed using surface plasmon resonance and co-crystallography. Lentivirus transfected anti-P329G CAR-T cells were characterized in vitro for their selectivity and potential to mediate antigen specific tumor cell lysis, cytokine secretion and proliferation. Immunological synapse formation was investigated using confocal microscopy. Results: Anti-P329G-CAR-T cells allow the precise recognition of the P329G mutation present in therapeutic IgG1 based adaptor-molecules. Crystal structure- and SPR-analysis revealed a 1:1 binding stoichiometry with low nanomolar affinity of the P329G-Fab fragment applied in the CAR for P329G-containing IgG1 antibodies. Potent tumor cell lysis was demonstrated for multiple tumor antigens e.g. CD20, HER2, FOLR1, EpCAM, FAP and others. For all tested antigens, a huIgG1 dose-dependent activation of anti-P329G-CAR-T cells as well as dose-dependent tumor cell lysis was observed. For selected antigens P329G-CAR-T activity was found comparable to the activity mediated by T cell bispecific antibodies recognizing the respective tumor antigen. Finally, the immunological synapse formed by P329-CAR-T cells was compared to the one formed by T cell bispecific antibodies in a 2+1 format. Conclusions: P329G-CAR-T cells mediate potent and specific tumor cell killing using various tumor targeted antibodies as adaptor molecules. Based on these data in vivo studies to investigate efficacy and safety of the approach are foreseen. Notably, this approach allows control of CAR-T activity and potential side effects by titrating the adaptor molecule, as well as the simultaneous targeting of more than one antigen at the same time with the goal to prevent tumor escape mechanisms. Combining the P329G-CAR with allogenic T-cells may provide a truly off-the-shelf P329G-CAR-T cell therapy approach. Citation Format: Diana Darowski, Christian Jost, Zeno Riester, Mohamed Benmebarek, Kay Stubenrauch, Anne Freimoser-Grundschober, Uwe Wessels, Jörg Benz, Ekkehard Mössner, Renier Myburgh, Floriana Cremasco, Mario Perro, Pablo Umana, Sebastian Kobold, Christian Klein. Anti-P329G-CAR-T cells as a novel universal CAR-T cell platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4229.

Based on the unprecedented clinical efficacy of PD-1/PD-L1 pathway checkpoint inhibitors (CPI), non-redundant immune checkpoints like TIM-3, LAG-3, TIGIT or BTLA are currently being targeted, by combinatorial approaches using monospecific or bispecific antibodies. Up-regulation of TIM-3 has been described as an adaptive CPI resistance mechanism, and internal prevalence data on archival samples of CPI-naïve and -experienced patients showed co-expression of PD-1 and TIM-3 in various tumor types, consistent with literature reports. Here, we describe RG7769 (PD1-TIM3), a novel avidity driven heterodimeric PD-1/TIM-3 1+1 bispecific CrossMabVH-VL intentionally designed as high affinity PD-1 (KD 250 pM, 37°C) and low affinity TIM-3 (KD 130 nM, 37°C) Fab-moieties to specifically target PD-1+ and PD-1+ TIM-3+ T cells through avidity gain, while bypassing PD-1- TIM3+ myeloid and NK cells. In contrast to IgG4-based PD-1 antibodies and conventional IgG1-based TIM-3 Fc-effector function competent antibodies, RG7769 harbors a PG LALA containing heterodimeric KiH IgG1 Fc-region rendering the BsAb refractory to drug shaving by FcgR-expressing macrophages in the TME, while retaining IgG-pharmacokinetics. RG7769 binds to PD-1 with higher affinity than pembrolizumab and nivolumab. X-ray crystallography demonstrated that the humanized PD-1 binding Fab recognizes a unique glycosylated epitope on PD-1, and potently blocks the PD-1/PD-L1 and PD-1/PD-L2 interactions in both biochemical and reporter cell line assays. The humanized TIM-3 binding arm was identified for maximal functional activity using mixed lymphocyte reaction (MLR) assays. Compared with bivalent TIM-3 antibodies, RG7769 shows reduced binding to TIM-3+ myeloid and NK cells, but binds preferentially to dysfunctional T cells expressing PD-1 or both PD-1 and TIM-3, like tumor infiltrating lymphocytes (TILs) in the tumor microenvironment. By virtue of its monovalency, RG7769 induced low antibody internalization on activated T cells when compared with bivalent TIM-3 antibodies, overcoming a major cellular sink for TIM-3 antibodies. In functional assays, RG7769 showed increased IFN-γ secretion by in vitro generated tumor-specific T-cells, increased ex vivo tumor-specific effector functions of T cells from PBMCs of melanoma patients, and enhanced the anti-tumor-activity of TILs from melanoma patients when compared to the monospecific parental PD-1 antibody. Finally, RG7769 showed superior efficacy in controlling s.c. MC38 tumor growth in huPD-1/huTIM-3 transgenic C57/BL6 mice compared to the parental PD-1 antibody. In summary, these preclinical data support the use of RG7769 as a monotherapy and as combination partner for the treatment of patients with solid/hematological tumors. A phase I study is currently ongoing in patients with advanced metastatic solid tumors (NCT03708328). Citation Format: Laura Laura Codarri Deak, Stefan Seeber, Mario Perro, Patrick Weber, Laura Lauener, Standford Chen, Sonja Offner, Stefan Dengl, Friederike Hesse, Adrian Zwick, Marco Boettger, Alexander Bujotzek, Jörg Benz, Guy Georges, Georg Fertig, Valeria Lifke, Jens Fischer, Stephane Leclair, Victor Levitsky, Marta Canamero, Juha Lindner, Sara Colombetti, Stefanie Bendels, Christophe Boetsch, Matthias Fueth, Merlind Muecke, Henry Kao, Pablo Umana, Christian Klein. RG7769 (PD1-TIM3), a novel heterodimeric avidity-driven T cell specific PD-1/TIM-3 bispecific antibody lacking Fc-mediated effector functions for dual checkpoint inhibition to reactivate dysfunctional T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2270.

Vascular inflammation is a major contributor to the severity of acute kidney injury. In the context of vasospasm-independent reperfusion injury we studied the potential anti-inflammatory role of the Gα-related RGS protein, RGS4. Transgenic RGS4 mice were resistant to 25 minute injury, although post-ischemic renal arteriolar diameter was equal to the wild type early after injury. A 10 minute unilateral injury was performed to study reperfusion without vasospasm. Eighteen hours after injury blood flow was decreased in the inner cortex of wild type mice with preservation of tubular architecture. Angiotensin II levels in the kidneys of wild type and transgenic mice were elevated in a sub-vasoconstrictive range 12 and 18 hours after injury. Angiotensin II stimulated pre-glomerular vascular smooth muscle cells (VSMC) to secrete the macrophage chemoattractant, RANTES; a process decreased by angiotensin II R2 (AT2) inhibition. However, RANTES increased when RGS4 expression was suppressed implicating Gα protein activation in an AT2-RGS4-dependent pathway. RGS4 function, specific to VSMC, was tested in a conditional VSMC-specific RGS4 knockout showing high macrophage density by T2 MRI compared to transgenic and non-transgenic mice after the 10 minute injury. Arteriolar diameter of this knockout was unchanged at successive time points after injury. Thus, RGS4 expression, specific to renal VSMC, inhibits angiotensin II-mediated cytokine signaling and macrophage recruitment during reperfusion, distinct from vasomotor regulation.

Purpose: Despite promising clinical activity, T-cell–engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell–binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785–97. ©2018 AACR.See related commentary by Prakash and Diefenbach, p. 4631

SummaryT cells are activated by antigen (Ag)-bearing dendritic cells (DCs) in lymph nodes in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8+ T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, whereas higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation but yielded different transcriptome signatures at 12 hr and 24 hr. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure, resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics.