Accurately predicting whether a new molecule will be safe and effective in humans remains a fundamental challenge in biomedical research, requiring models that can reliably inform clinical outcomes. In oncology, drug attrition rates remain disproportionately high compared to other therapeutic areas, largely due to the poor translatability of conventional preclinical models that fail to capture the complexity of human immune-tumor-stroma interactions 1,2. Here, we describe human vascularized organoids (VascO), formed through self-assembly of an immune cell perfused capillary network and stromal compartment, interfacing with patient-derived colorectal cancer organoids that enable the combined safety and efficacy assessment of tumor-targeted immunotherapies. VascO generates functional and integrated vascular networks within tumor organoids that sustain organoid growth and recapitulate the morphology and functionality of tumor vasculature. Single‑cell transcriptomics reveals that the tumor organoids-educated VascO niche reprograms otherwise healthy fibroblasts into canonical cancer‑associated fibroblast states, adopting myofibroblastic or inflammatory signatures, and faithfully mirroring donor‑specific tumor cues. In parallel, VascO tumor vessels display an expanded tip‑cell program, aberrant morphology, and VEGF/ANG2‑driven permeability, recapitulating defining hallmarks of tumor vasculature. Immune perfusion with a tumor-targeted T cell bispecific antibody induces dynamic immune trafficking, infiltration and engagement of effector cells with the tumor tissue, leading to its effective killing. A protease-activated variant restricts T cell activation to the tumor microenvironment, retaining potent tumoricidal activity while sparing donor-matched healthy colon organoids, and thereby improving the therapeutic index. VascO thus models a patient-specific, tumor-educated microenvironment that is amenable to assessing the combined safety and efficacy of new drug candidates and provides a scalable platform to improve the preclinical assessment of innovative cancer immunotherapies. ### Competing Interest Statement All authors are current employees of Hoffmann-La Roche Ldt or were employed by the company while working on this study. The company provided support in the form of salaries for authors but did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Targeting various combinations of tumor antigens and immune cell receptors is of increasing importance in antibody-based cancer immunotherapy. Here, we present a novel modular P329G-engager platform that enables rapid combination of primary tumor-targeting and secondary immune effector antibodies. The platform utilizes two antibodies, each selected from: 1) a set of tumor-targeting adaptor antibodies, bearing P329G mutations in the Fc region, and 2) a set of P329G-targeting (bispecific) cell engagers, including innate and T cell engagers, costimulators and immunocytokines. Specifically, upon defining a tumor-associated cell surface target, a primary adaptor - tumor antigen-binding IgG1 antibody with Fc-silencing P329G L234A L235A mutations - is administered. Subsequently, a secondary antibody recognizing the P329G mutation is chosen from a panel of effector cell engagers with different modes of action - ADCC-competent P329G-innate cell engagers (P329G-ICE), P329G-T cell bispecifics (P329G-TCB), P329G-costimulators (P329G-CD28/4-1BBL), or P329G-immunocytokine (P329G-IL2v). In vitro assays showed that all P329G-targeting modalities induce anti-tumoral and/or immunomodulatory activity when both components were combined. In vivo, tumor shrinkage and T cell infiltration were confirmed in tumor-bearing humanized mice treated with P329G-mutated CEACAM5 adaptor IgG and P329G-TCB. Individually, neither the adaptor nor the P329G-TCB induced efficacy, validating the requirement for primary and secondary antibody assembly for T cell-engaging activity. These results provided evidence for the in vivo assembly and subsequent pharmacological activity, and provide preclinical proof-of-concept for the P329G-engager platform as an efficacious tool in drug discovery. Ultimately, this modular approach may enable mix-and-match drug assembly as a novel therapeutic principle in immunotherapy.

Background/Objectives: T cell bispecific antibodies (TCBs) result in the activation of T cell receptor signaling upon binding to tumor antigens providing signal 1 to T cells. To enhance and sustain their activity, a co-stimulatory signal 2 is required. Here CEACAM5-targeted 4-1BBL antibody fusion proteins for combination with CEA-TCB (cibisatamab, RG7802) are described in an investigation of the relationship between the CEACAM5 epitope and T cell activity. Methods: CEACAM5-targeted bispecific 4-1BBL antibody fusion proteins (CEA-4-1BBLs) were generated based on different CEACAM5 antibodies and characterized in vitro in Jurkat-4-1BB reporter and PBMC cell assays. The impact of shed CEA on in vitro activity and cynomolgus cross-reactivity was studied. In vivo efficacy was assessed in human stem cell humanized NSG mice xenograft models bearing MKN-45 and HPAFII tumors. Results: MFE23-4-1BBL and Sm9b-4-1BBL showed superior functional activity in Jurkat-4-1BB reporter and primary T cell assays when combined with the CD3 antibody V9, whereas T84.66-LCHA-4-1BBL and A5B7-4-1BBL performed better when combined with CEA-TCB. In humanized NSG mice MKN-45 and HPAFII xenograft models, T84.66-LCHA-4-1BBL mediated the best anti-tumor efficacy. Conclusions: For the assessment of the combination of CEA-TCB with CEA-4-1BBL, co-stimulatory antibody fusion protein in vitro assays are not sufficient to fully capture the complex relationships affecting efficacy. Thus, screening with different cell assays and in vivo efficacy studies in combination with CEA-TCB are essential to select the best candidate. Based on the totality of data on the T84.66-LCHA-4-1BBL antibody fusion protein comprising the CEACAM5 antibody, T84.66-LCHA was selected as the optimal combination partner for CEA-TCB.

CAR-T cell therapy is effective in many patients suffering from B cell malignancies, yet antigen escape is a major resistance mechanism by which efficacy can be diminished or lost. To counter this, we enhance anti-lymphoma CAR-T cells by generating conventional and adapter dual (ConvAD) CAR-T cells, which co-express a conventional antigen-specific CAR and the P329G adapter CAR that leverages Fc-mutated antibodies for redirection. ConvAD CAR-T cells display robust functionality against the primary lymphoma antigen of the conventional CAR while providing flexible redirection to additional targets via target-specific adapters in the event of antigen escape. We demonstrate the bimodal activity of ConvAD CAR-T cells acting through direct engagement of the conventional CAR as well as through binding of adapters, resulting in enhanced multispecific anti-lymphoma targeting. In vitro and in vivo, ConvAD CAR-T cells targeting combinations of ROR1, CD19, and CD20 prevent lymphoma outgrowth across models of stable antigen expression as well as single- or dual-antigen loss, and benchmark superior to both single-antigen specific and bispecific CAR-T cells. The ConvAD CAR platform thus addresses a medical need by offering an effective strategy for multi-antigen targeting, counteracting antigen escape.

Background/Objectives: T cell bispecific antibodies (TCBs) activate T cells for the killing of tumor cells upon binding to tumor antigens resulting in the activation of T cell receptor signaling providing the so-called signal 1 to T cells. In order to enhance and sustain the activity of T cells a co-stimulatory signal 2 is required. Here we describe CEACAM5-targeted 4-1BBL antibody fusion protein for combination with the CEA-TCB cibisatamab investigating the relationship of CEACAM5 epitope and activity. Methods: CEACAM5-targeted bispecific 4-1BBL antibody fusion proteins (CEA-4-1BBL) were generated using different CEACAM5 antibodies and characterized in vitro in Jurkat-4-1BB reporter and PBMC cell assays. The impact of shed CEA on in vitro activity was studied. In vivo efficacy was assessed in human stem cell humanized NSG mice xenograft models bearing MKN-45 and HPAFII tumors. Results: MFE23-4-1BBL and Sm9b-4-1BBL showed superior functional activity in Jurkat-4-1BB reporter and in primary T cell assays when combined with the CD3 antibody V9, whereas T84.66-LCHA-4-1BBL and A5B7-4-1BBL performed better when combined with CEA-TCB. In humanized NSG mice xenograft models T84.66-LCHA-4-1BBL showed overall the best anti-tumor efficacy. Concluions: In vitro assays and in vivo studies lead to different conclusions showing that in order to select the best candidate screening with different cell assays and in vivo efficacy studies together with the potential combination partner are essential. Based on the totality of data the T84.66-LCHA-4-1BBL antibody fusion protein based on the CEACAM5 antibody T84.66-LCHA was selected as optimal costimulatory antibody fusion protein for the combination with the CEA-TCB cibisatamab.

Abstract Novel antibody-engineering approaches have enabled the development of targeted innate cell engagers which redirect innate immune cells to eliminate cancer or suppressive immune cells. These antibodies function through binding to surface antigens on target cells and engaging activating receptors on innate immune cells such as natural killer cells and macrophages. Although early innate cell engagers have shown promise in controlling various cancer types, several major challenges remain in the clinics, including high tumor heterogeneity, low tumor immunogenicity, and reduced efficacy in solid tumors. Here, we present a universal innate cell engager as part of a novel adaptor-based antibody platform. Our universal innate cell engager utilizes a two-step approach combining the power of an adaptor and an effector antibody. First, an Fc-silenced adaptor antibody comprising P329G LALA mutations is used to target the desired antigen in the tumor microenvironment. Then, as an effector antibody, a universal innate cell engager directed against the P329G mutation on the adaptor antibody is used. Innate immune cells are recruited by the universal innate cell engager through its active Fc, leading to antibody-dependent cellular cytotoxicity against the target cells. To maximize innate cell activation, the Fc portion of the universal innate cell engager can be glycoengineered for enhanced FcgRIII affinity.In vitro and in vivo assays showed that our universal innate cell engager is inducing innate immune cell activation and innate immune cell-mediated target cell killing when combined with different antigen-targeted adaptor antibodies. Ultimately, this approach may enable off-the-shelf personalization via combination of universal effector cell engagers and selected adaptor antibodies specific to the patient’s tumor profile. Citation Format: Idil Hutter-Karakoc, Marlena Surowka, Diana Darowski, Christina Klaus, Claudia Ferrara Koller, Thomas Hofer, Anne Freimoser-Grundschober, Ekkehard Mössner, Stephane Leclair, Pablo Umana, Christian Munz, Maria Amann, Christian Klein. Recruiting innate immune cells universally: Development of a novel universal innate cell engager for cancer-immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A054.

Abstract Bispecific immune cell engagers are showing promise in cancer immunotherapy. To achieve optimal efficacy, there is a growing need for patient personalization, tackling intra-tumor heterogeneity, and simplified combination therapies. Here, we present a novel modular anti-P329G adaptor platform aiming to address these issues, which is based on the recognition of the P329G mutation in the Fc portion of a targeting adaptor antibody. This P329G mutation can serve to recruit different anti-P329G effector cell engagers, including innate cell engagers, T cell engagers, costimulators or immunocytokines. Specifically, upon identifying patient-specific tumor surface targets, the primary antigen-binding IgG1 antibodies bearing Fc-silencing P329G LALA mutations are applied. Subsequently, based on the tumor’s immune profile, the best-suited anti-tumoral effector cell type is chosen for recruitment and activation. Based on this information, the secondary antibody recognizing the P329G mutation is chosen from an array of effector cell engagers with different modes of action: P329G-T cell bispecifics (P329G-TCB), ADCC-competent P329G innate cell engagers (P329G-ICE), P329G costimulatory molecules (P329G-CD28/4-1BBL) or P329G immunocytokines (P329G-IL2v). In vitro assays showed all P329G modalities inducing anti-tumoral and/or immunomodulatory cell activity. Anti-tumoral efficacy of secondary antibodies was observed only in presence of tumor-targeted P329G adaptors, while no effect was observed in their absence. The readouts consisted of tumor killing quantification, CD4+ and CD8+ T cell activation, cytokine release and cytokine receptor signaling. As a proof of concept study in vivo, an experiment in MKN-45 (CEACAM5+)-bearing humanized mice was performed, using an anti-CEACAM5 P329G-IgG adaptor and a P329G-TCB. Both tumor volume shrinkage and T cell infiltration into the tumor confirmed anti-tumoral efficacy of the platform, as compared to a conventional CEACAM5-targeted TCB. Neither the individual P329G IgG nor the individual P329G-TCB induced anti-tumoral efficacy, validating the requirement for primary and secondary antibody binding for T cell engaging activity. These results provide in vitro and in vivo evidence that the universal P329G engager platform can be used as an efficacious cancer treatment. Ultimately, this modular approach may enable off-the-shelf personalization via combination of patient-specific antibodies and universal effector cell engagers based on the patient’s tumor target and immune profile. Citation Format: Marlena Surowka, Idil Hutter-Karakoc, Diana Darowski, Christina Claus, Claudia Ferrara Koller, Anne Freimoser-Grundschober, Thomas Hofer, Andrzej Sobieniecki, Denis Assisi, Stephane Leclair, Ekkehard Moessner, Pablo Umaña, Maria Amann, Christian Klein. P329G-Engager: A novel universal antibody-based adaptor platform for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6709.

FAP-drozitumab antibody-mediated apoptosis does not correlate with DR5.

FAP-drozitumab BsAb induces tumor growth inhibition in vivo.

Figure S2. Related to Figure 3. In vitro characterization of the FAP-DR5 BsAb RG7386; Table S4. Characterization of RG7386 binding avidities.

Figure S1. Related to Figure 1. Characterization of FAP-drozitumab BsAbs and antitumor efficacy in the presence of FAP-expressing fibroblasts in in vitro co-culture models; Table S1. BsAb binding to DR5 and FAP antigens; Table S2. Anti-tumor efficacy of FAP-drozitumab bispecific molecules is FAPdependent.

Characterization and optimization of RG7386 dosing in vivo.

Supplementary information on cell maintenance, and antibody selection and characterization

The clinical development of 4-1BB agonists for cancer immunotherapy has raised substantial interest during the past decade. The first generation of 4-1BB agonistic antibodies entering the clinic, urelumab (BMS-663513) and utomilumab (PF-05082566), failed due to (liver) toxicity or lack of efficacy, respectively. The two antibodies display differences in the affinity and the 4-1BB receptor epitope recognition, as well as the isotype, which determines the Fc-gamma-receptor (FcγR) crosslinking activity. Based on this experience a very diverse landscape of second-generation 4-1BB agonists addressing the liabilities of first-generation agonists has recently been developed, with many entering clinical Phase 1 and 2 studies. This review provides an overview focusing on differences and their scientific rationale, as well as challenges foreseen during the clinical development of these molecules.

Background The costimulatory receptor 4-1BB (CD137, TNFRSF9) plays an important role in sustaining effective T cell immune responses and is investigated as target for cancer therapy. Systemic 4-1BB directed therapies elicit toxicity or low efficacy, which significantly hampered advancement of 4-1BB-based immunotherapy. Therefore, targeted delivery of 4-1BB agonist to the tumor side is needed for eliciting antitumor efficacy while avoiding systemic toxicity. Methods We analyzed the immunostimulatory properties of a fibroblast activation protein (FAP)-targeted 4-1BB agonist (FAP-4-1BBL) by assessing tumor-infiltrating lymphocytes’ (TIL) activity from patients with non-small cell lung cancer and epithelial ovarian cancer. Results Combination treatment with FAP-4-1BBL and T cell receptor stimulation by either anti-CD3 or T cell bispecific antibodies significantly enhanced TIL activation and effector functions, including T cell proliferation, secretion of proinflammatory cytokines and cytotoxicity. Notably, costimulation with FAP-4-1BBL led to de novo secretion of interleukin (IL)−13. This was associated with cytokine-mediated tumor cell apoptosis, which was partially dependent on IL-13 alpha 1/2 receptors and STAT6 phosphorylation. Conclusions Our study provides mechanistic insights into T cell stimulation induced by FAP-4-1BBL in primary human tumors and supports the investigation of FAP-4-1BBL compound in early clinical trials.

Synthetic T cell redirecting therapies, using chimeric antigen receptor (CAR)-T cells or CD3-bispecific antibodies targeting B-cell surface antigens such as CD19 and CD20, currently in clinical development, are emerging as promising, potential therapeutic approaches for the treatment of non-Hodgkin lymphomas (NHL). CD3-bispecific antibodies and first generation CAR-T cells only provide T cell receptor stimulation, so-called "signal 1", to the redirected T cells, but lack costimulatory, so-called "signal 2", support of those T cells. Agonism of costimulatory receptors on T cells, such as CD28 and/or 4-1BB, can increase the strength and durability of a T cell-mediated response via multiple mechanisms. Co-stimulation can enhance T cell specific cytotoxicity, proliferation, secretion of Th1-polarizing cytokines, recruitment of additional T cells via increased chemokine secretion, T cell metabolic fitness, and resistance to T-cell exhaustion and to activation-induced T-cell death. Indeed, 2nd generation CAR-T cells that incorporate CD28 or 4-1BB co-stimulation have replaced 1st generation ones in clinical development. However, complex manufacturing logistics and the need of specialized clinical centers for the administration of CAR-T cells significantly limit their broad application. In order to provide an off-the-shelf, synthetic T cell redirection approach delivering both signals 1 and 2 to T cells, CD3-bispecific antibodies would need combination with systemically administered T-cell costimulatory agonists. Yet, clinical development of 1st generation costimulatory agonists has not been successful to date due to on-target, off-tumor immune-mediated toxicity, such as hepatotoxicity. To overcome this limitation, we have generated a novel 4-1BB costimulatory agonist, CD19-targeted 4-1BBL (CD19-4-1BBL, RG6076, RO7227166), and are developing it in combination with a potent CD20xCD3 T cell bispecific antibody, CD20-TCB (RG6026 or glofitamab). CD19-4-1BBL consists of a trimeric, human 4-1BBL fused to a monovalent CD19-targeting IgG1 antibody with an engineered Fc region devoid of FcgR binding. As effective agonism of 4-1BB receptor requires crosslinking of more than three receptor units on a T cell, CD19-4-1BBL is systemically inactive unless it binds to CD19 and clusters on the surface of targeted B-cells to hyper-crosslink multiple 4-1BB receptors on redirected T cells. In our off-the-shelf, combination approach, glofitamab binds to CD20 on B-cells and engages CD3 on redirected T cells, providing signal 1 and inducing the expression of 4-1BB on those T cells. CD19-4-1BBL can then target those activated T cells and provide them with signal 2. In preclinical experiments, we show that CD19-4-1BBL can boost glofitamab-mediated cytokine release by activated T cells in healthy donor as well as DLBCL patient-derived PBMCs. Using a human diffuse large B cell lymphoma (DLBCL) tumor-bearing (WSU-DLCL2) fully humanized mouse model, we observed a CD19-4-1BBL dose-dependent, synergistic combination effect with glofitamab, leading to strongly increased T cell accumulation in tumors, tumor growth inhibition and regression. Importantly, CD19-4-1BBL was also able to prevent tumor escape to glofitamab monotherapy at late treatment time points in a fully humanized mouse model bearing large OCI-Ly18 human DLBCL tumors. Glofitamab monotherapy has recently demonstrated encouraging activity in relapsed/refractory NHL patients with reported complete response rates in DLBCL in the same range as those of 2nd generation CAR-T cells that already incorporate both T cell signals 1 and 2. The preclinical data we report here provide a strong rationale for adding CD19-4-1BBL-mediated T cell signal 2 to glofitamab in the clinic to further boost treatment efficacy and deliver an off-the-shelf, enhanced T cell redirection approach alternative to CAR-T cell therapy. CD19-4-1BBL is currently in clinical trials (NCT04077723). Disclosures Herter: Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Sam:Roche Glycart AG:Current Employment.Ferrara Koller:Roche Glycart AG:Current Employment.Diggelmann:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company.Bommer:Roche Glycart AG:Current Employment.Schönle:Roche Glycart AG:Current Employment.Claus:Roche Glycart AG:Current Employment.Bacac:Roche Glycart AG:Current Employment, Patents & Royalties.Klein:Roche:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.Umana:Roche Glycart AG:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.

A persisting challenge in cancer immunotherapies (CIT) is the determination of patients responding early during therapy. As major effector cells, CD8 cytotoxic T cells are a primary target of many CIT approaches. ImaginAb is developing a human CD8 specific minibody 89Zr-Df-IAB22M2C to monitor CD8 tumor infiltrates non-invasively, longitudinally and throughout the whole body using positron emission tomography (PET). We evaluated pre-clinically the capability of this tracer to quantify CD8 tumor infiltrates upon treatment with T cell activating molecules using both, single agent FOLR1-T cell bispecific (TCB) antibody and the combination of CEA-TCB with a CEA-targeted 4-1BBL (CEA-4-1BBL). In the first study, HeLa-cervical cancer xenograft bearing CD34+ human stem cell engrafted NSG mice (HSC-NSG) were treated for 2 weeks with FOLR1-TCB (2 mg/kg, once weekly), an untargeted control DP47-TCB and vehicle followed by PET imaging with the CD8 specific 89Zr-Df-IAB22M2C tracer (90-95 µCi/10 µg). In the second study, MKN-45 gastric cancer bearing HSC-NSG mice were treated with CEA-TCB (2.5 mg/kg, 2x/week), CEA 4-1BBL (3.0 mg/kg, 1x/week) or the combination of both compounds for two weeks followed by PET imaging with the CD8 tracer (50-60 µCi/10 µg). Ex vivo γ-counting of tumors and organs of interest as well as CD8 IHC was performed for both studies. Treatment with FOLR1-TCB induced the highest tumor regression and strongest CD8+ T cell infiltrates. 89Zr-Df-IAB22M2C was able to detect tumor infiltrates by PET and γ-counting (13.6±2.7 %ID/g), whereas treatment with untargeted DP47-TCB induced a low T cell infiltrate (8.7±3.2 %ID/g) as compared to the vehicle group (5.6±1.2 %ID/g). In the second study, combined treatment with CEA-TCB and CEA-4-1BBL induced the highest tumor regression accompanied by the highest intratumoral CD8 T cell infiltrates, which were detectable by PET and γ-counting (8.95±2.47 %ID/g). Single agent CEA-TCB (6.91±1.61 %ID/g) and CEA-4-1BBL (6.24±1.11 %ID/g) showed higher signals compared to vehicle (4.87±1.18 %ID/g). The differences in CD8 infiltrate upon single and combination treatment was confirmed by IHC. The 89Zr-Df-IAB22M2 PET-tracer proved to be highly sensitive for the detection of intratumoral CD8 T cell infiltrates upon single and combination treatment with T cell activating compounds. These data correlated with CD8 IHC. These results provide further evidence that the CD8 imaging probe, which is currently in Phase II clinical testing (NCT03107663), may be a promising tool for the monitoring of CD8 T cells in patients treated with immunotherapies. Citation Format: Christoph M. Griessinger, Alessandro Mascioni, Fang Jia, Michael Torgov, Tapan Nayak, Preethi Latha Bhavani Mohan, Valeria G. Nicolini, Johannes Sam, Christina Claus, Claudia Ferrara-Koller, Marina Bacac, Pablo Umana, Ian Wilson, Christian Klein, Jean J. Tessier. Monitoring intratumoral CD8 T cell infiltrates in human stem cell engrafted mice during single agent and combination immunotherapy with T cell bispecific antibodies using the human PET-tracer 89Zr-Df-IAB22M2C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1129.

Abstract Co-stimulation through 4-1BB has shown promising anti-tumor activity in preclinical models, but the development of 4-1BB agonistic antibodies in the clinic has been hampered by Fc-mediated liver toxicity. Here, we describe a novel CD19-targeted 4-1BB ligand designed to deliver a safe but potent 4-1BB agonist to effector T and NK cells with the goal to improve treatment of B cell malignancies. The antibody fusion protein is composed of split trimeric 4-1BB ligands and a tumor antigen targeting moiety recognizing CD19 fused to a silent Fc part (CD19-4-1BBL). The construct is devoid of FcgR-mediated crosslinking responsible for Fc-mediated toxicity and reintroduces 4-1BB hyperclustering upon binding to CD19 on B cells exclusively. In mice and cynomolgus monkeys, CD19-4-1BBL shows IgG-like pharmacokinetic properties. When cross-linked via CD19 on Non-Hodgkin Lymphoma cell lines or normal B cells, CD19-4-1BBL is biologically active in co-stimulating T cells. As 4-1BB is an inducible protein on activated T cells and NK cells, we combined CD19-4-1BBL with a T cell bispecific Ab targeting CD20 (CD20-TCB) to provide initial T cell activation while engaging with CD19+CD20+ tumor cells. In vitro, pre-treatment with CD20-TCB mediates the clustering of CD19-4-1BBL molecules to the interaction synapse of T cells and tumor cells. Live imaging revealed that the addition of CD19-4-1BBL induces significantly prolonged T cell-tumor contact (>30 min), leading to fast and efficient killing of tumor cells. In vivo in WSU-DLCL2-bearing human stem cell engrafted NSG mice (HSC-NSG) mice, CD20-TCB treatment quickly up-regulates 4-1BB on activated T cells, resulting in tumor growth inhibition. The combination of CD19-4-1BBL and CD20-TCB (used at a suboptimal dose) synergizes to eradicate the tumor completely. The combination induces strong T cell infiltration into the tumor, accompanied by an elevated CD8/Treg ratio, as compared to the monotherapies. Similarly, the combination also induces complete remission in a “difficult-to-treat” Nalm6 tumor model associated with low and patchy CD20 expression, mimicking patterns seen in the ABC subtype of DLBCL. Finally, the combination of CD19-4-1BBL and the ADCC-enhanced Type II CD20 antibody obinutuzumab induces complete tumor remissions in WSU-DLCL2 tumor-bearing HSC-NSG mice, confirming effective 4-1BB co-stimulation on NK cells. Taken together, tumor-targeted cross-linking of 4-1BB mediated by CD19-4-1BBL provides safe and efficient co-stimulation of T cells that are pre-activated by a TCB, or of NK cells pre-activated by an ADCC mediating antibody. This novel and effective combination immunotherapy warrants clinical investigation and offers possible “chemo-free” treatment of B cell malignancies. Citation Format: Wei Xu, Johannes Sam, Mario Perro, John Challier, Christina Claus, Stanford Chen, Claudia Ferrara Koller, Michael Mølhøj, Stella Tournaviti, Marina Bacac, Tom Moore, Christian Klein, Pablo Umana. Design of CD19-4-1BBL, a novel CD19-targeted 4-1BB ligand for combination therapy with CD20 T-cell bispecific antibodies and CD20 antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 957.