ITOC2 – 037. CEA TCB, A novel T-cell bispecific antibody with potent in vitro and in vivo antitumour activity against solid tumours

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

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786 Dose selection for DuoBody®-PD-L1×4-1BB (GEN1046) using a semimechanistic pharmacokinetics/pharmacodynamics model that leverages preclinical and clinical data

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.786 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A821-A821

Author(s):

Gaurav Bajaj ◽

Fereshteh Nazari ◽

Marc Presler ◽

Craig Thalhauser ◽

Ulf Forssmann ◽

...

Keyword(s):

T Cell ◽

Bispecific Antibody ◽

Receptor Occupancy ◽

Model Parameters ◽

Cell Trafficking ◽

Dynamic Binding ◽

Dose Selection ◽

Trimer Formation

BackgroundDuoBody-PD-L1×4-1BB (GEN1046) is a class-defining bispecific antibody, designed to elicit an anti-tumor immune response by simultaneous and complementary blockade of PD-L1 on tumor cells and conditional stimulation of 4-1BB on T-cells and NK cells. Optimizing target engagement for a bispecific antibody is challenging, as it involves binding with two targets, and predicting trimer levels in tumors based on affinity of individual arms and target expression. Here we describe a semimechanistic, physiologically based pharmacokinetic/pharmacodynamic (PK/PD) model that predicts a dosing regimen for DuoBody-PD-L1×4-1BB, which results in the formation of maximum levels of a therapeutically active 4-1BB-bispecific antibody-PD-L1 trimolecular complex (trimer), and optimal PD-L1 receptor occupancy (RO).MethodsAn integrated semimechanistic PK/PD model that describes the distribution of DuoBody-PD-L1×4-1BB into central and peripheral compartments and partitioning into tumor/lymph nodes was developed. The model used PK/PD data and physiological parameters from the literature for parameterizations of PD-L1 and 4-1BB expression levels and T-cell trafficking. The model incorporates dynamic binding of DuoBody-PD-L1×4-1BB to its targets to predict trimer formation and RO for PD-L1 in tumors. Model parameters were calibrated to match in vitro PD studies, such as analyses of T-cell proliferation and cytokine release, as well as clinical PK data. Sensitivity to model assumptions were assessed by varying PK/PD parameters, and assessing their impact on trimer formation and PD-L1 RO. The model was subsequently used to explore in vivo trimer levels and PD-L1 RO in tumors at various dosing regimens.ResultsThe model was able to adequately describe the PK of DuoBody-PD-L1×4-1BB in the central compartment. Simulations showed a bell-shaped response for average trimer levels in tumors that peaked at 100 mg every 3 weeks (Q3W), with doses >100 mg resulting in reduced trimer formation. Average PD-L1 receptor occupancy at the 100 mg dose was predicted to be approximately 70% over 21 days and increased at higher doses. Based on these model predictions, and available safety, anti-tumor activity, and PD data from the ongoing GCT1046-01 trial (NCT03917381), 100 mg Q3W was chosen as the expansion dose for further evaluation in Part 2 of the study.ConclusionsThis semimechanistic PK/PD model provides a novel approach for dose selection of bispecific antibodies such as DuoBody-PD-L1×4-1BB, by using preclinical and clinical PK/PD data to predict formation of optimal trimer levels and PD-L1 receptor occupancy.AcknowledgementsThe authors thank Friederike Gieseke and Zuzana Jirakova at BioNTech SE; Kalyanasundaram Subramanian at Applied Biomath LLC for their valuable contributions.Trial RegistrationWritten informed consent, in accordance with principles that originated in the Declaration of Helsinki 2013, current ICH guidelines including ICH-GCP E6(R2), applicable regulatory requirements, and sponsor policy, was provided by the patients.

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New emerging targets in cancer immunotherapy: the role of GITR

ESMO Open ◽

10.1136/esmoopen-2020-000738 ◽

2020 ◽

Vol 4 (Suppl 3) ◽

pp. e000738

Author(s):

Giulia Buzzatti ◽

Chiara Dellepiane ◽

Lucia Del Mastro

Keyword(s):

T Cell ◽

Cell Function ◽

Cytotoxic T Lymphocyte ◽

Antitumour Activity ◽

Cell Activity ◽

Phase I Clinical Trials ◽

Effector T Cell ◽

Number Of Patients

In the last decade, immunotherapies have revolutionised anticancer treatment. However, there is still a number of patients that do not respond or acquire resistance to these treatments. Despite several efforts to combine immunotherapy with other strategies like chemotherapy, or other immunotherapy, there is an ‘urgent’ need to better understand the immune landscape of the tumour microenvironment. New promising approaches, in addition to blocking co-inhibitory pathways, such those cytotoxic T-lymphocyte-associated protein 4 and programmed cell death protein 1 mediated, consist of activating co-stimulatory pathways to enhance antitumour immune responses. Among several new targets, glucocorticoid-induced TNFR-related gene (GITR) activation can promote effector T-cell function and inhibit regulatory T-cell (Treg) function. Preclinical data on GITR-agonist monoclonal antibodies (mAbs) demonstrated antitumour activity in vitro and in vivo enhancing CD8+ and CD4+ effector T-cell activity and depleting tumour-infiltrating Tregs. Phase I clinical trials reported a manageable safety profile of GITR mAbs. However, monotherapy seems not to be effective, whereas responses have been reported in combination therapy, in particular adding PD-1 blockade. Several clinical studies are ongoing and results are awaited to further develop GITR-stimulating treatments.

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Abstract 1886: Anti -GPRC5D/CD3 T cell-redirecting bispecific antibody with potent in vitro and in vivo antitumor efficacy against multiple myeloma

10.1158/1538-7445.am2021-1886 ◽

2021 ◽

Author(s):

Tatsushi Kodama ◽

Yu Kochi ◽

Waka Nakai ◽

Hideaki Mizuno ◽

Takeshi Baba ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cell ◽

Bispecific Antibody ◽

Antitumor Efficacy

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A Novel Bispecific Antibody Targeting CD3 and Lewis Y with Potent Therapeutic Efficacy against Gastric Cancer

Biomedicines ◽

10.3390/biomedicines9081059 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1059

Author(s):

Jie Chen ◽

Zhidi Pan ◽

Lei Han ◽

Yuexian Zhou ◽

Huifang Zong ◽

...

Keyword(s):

Gastric Cancer ◽

T Cell ◽

Therapeutic Efficacy ◽

Bispecific Antibody ◽

Target Cells ◽

Lewis Y ◽

Antibody Targeting ◽

Anti Tumor Activity

Lewis Y antigen, a glycan highly expressed on most epithelial cancers, was targeted for cancer treatment but lacked satisfactory results in some intractable and refractory cancers. Thus, it is highly desirable to develop an effective therapy against these cancers, hopefully based on this target. In this work, we constructed a novel T cell-engaging bispecific antibody targeting Lewis Y and CD3 (m3s193 BsAb) with the IgG-[L]-scfv format. In vitro activity of m3s193 BsAb was evaluated by affinity assay to target cells, cytotoxicity assay, cytokines releasing assay, and T cells proliferation and recruiting assays. Anti-tumor activity against gastric cancer was evaluated in vivo by subcutaneous huPBMCs/tumor cells co-grafting model and huPBMCs intravenous injecting model. In vitro, m3s193 BsAb appeared to have a high binding affinity to Lewis Y positive cells and Jurkat cells. The BsAb showed stronger activity than its parent mAb in T cell recruiting, activation, proliferation, cytokine release, and cytotoxicity. In vivo, m3s193 BsAb not only demonstrated higher therapeutic efficacy in the huPBMCs/tumor co-grafting gastric carcinoma model than the parent mAb but also eliminated tumors in the model of intravenous injection with huPBMCs. Strong anti-tumor activity of m3s193 BsAb revealed that Lewis Y could be targeted in T cell-engaging BsAb for gastric cancer therapy.

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CD20 Tcb (RG6026), a Novel "2:1" T Cell Bispecific Antibody for the Treatment of B Cell Malignancies

Blood ◽

10.1182/blood.v128.22.1836.1836 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1836-1836 ◽

Cited By ~ 13

Author(s):

Marina Bacac ◽

Pablo Umaña ◽

Sylvia Herter ◽

Sara Colombetti ◽

Johannes Sam ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Bispecific Antibody ◽

Ex Vivo ◽

Tumor Growth Inhibition ◽

Tumor Lysis

Abstract Despite the recent advancements in treatment options with the introduction of anti-CD20 monoclonal antibody therapy, approximately 50% of patients with non-Hodgkin's lymphomas (NHL) will not sustain a durable response to standard of care (SOC) treatment. Thus, there remains a continuous need for safer and more effective anti-cancer therapies in this indication. T-cell bispecific antibodies (TCBs) represent a new class of disease targeting agents shown to promote the activation of a patient's own T cells to attack and kill cancer cells. CD20 TCB is a new bispecific antibody with IgG-like pharmacokinetic properties whose unique "2:1" structure leads to increased tumor antigen avidity, T cell activation, and tumor cell killing, as compared to other T cell engaging bispecific antibody molecular formats. The molecule comprises two CD20 binding Fabs (derived from the Type II CD20 IgG1 obinutuzumab), one CD3e binding Fab (fused to one of the CD20 Fabs via a short flexible linker), and an engineered, heterodimeric Fc region with completely abolished binding to FcgRs and C1q. In vitro, CD20 TCB was shown to dose-dependently induce tumor lysis with EC50 values in the range of 0.05 - 3.1 pM. The "2:1"format of CD20 TCB was shown to confer superior potency (up to 10 - 1000x) when compared to CD20 TCBs having the conventional "1:1" IgG-based format (i.e., one binding domain for CD20 and one for CD3). CD20 TCB-mediated tumor lysis resulted in T-cell activation, proliferation and cytokine release with up-regulation of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis upon tumor lysis. CD20 TCB also demonstrated potent ex vivo activity in whole bone marrow aspirate samples of NHL and CLL patients (n=17). Such primary tumor samples preserve the native tumor microenvironment and bear low effector to target cell ratios ranging in this study from 0.02 to 0.8 (average value 0.3). CD20 TCB activity was consistently superior to that of the "1:1" CD20 TCB and demonstrated faster, more profound and more potent B cell depletion with EC50 values ranging from 0.002 to 2.7 nM. In vivo, CD20 TCB displayed potent anti-tumor activity in xenograft models in stem cell humanized mice and induced regression of large, aggressive WSU-DLCL2 lymphoma tumors (0.5 mg/kg, weekly administration). In addition to tumor regression, CD20 TCB treatment led to fast and complete elimination of peripheral blood B cells within 24 h after the first administration (0.05, 0.15 and 0.5 mg/kg, weekly administration) and to a complete elimination of B cells in spleen, bone marrow and lymph nodes after two administrations. B cell depletion was paralleled by transient decrease of T-cell counts in the peripheral blood and by the peak of cytokine release 24 h after the first administration, followed by rapid recovery and return to baseline levels at 72 h post treatment. Tumor growth inhibition mediated by CD20 TCB was accompanied by increase in intra-tumor T-cell infiltration, up-regulation of PD-1 receptor on T cells and PD-L1 in the tumor. Combination studies of CD20 TCB with PD-L1 blocking antibody led to more profound and faster tumor growth inhibition. Taken together, the preclinical data show that CD20 TCB is a novel differentiated CD20-targeting T cell bispecific antibody with promising anti-tumor activity and the ability to modify the tumor microenvironment. CD20 TCB consistently demonstrated superior potency compared to other CD20 TCBs with a conventional "1:1" IgG format. This translated into superior efficacy in vitro, ex-vivo and in vivo, which could not be matched by increasing doses of the "1:1" TCBs. The molecule is now scheduled to start clinical trial by December 2016. Disclosures Bacac: Roche: Employment, Equity Ownership, Patents & Royalties. Umaña:Roche: Employment, Equity Ownership, Patents & Royalties. Herter:Roche: Employment, Patents & Royalties. Colombetti:Roche: Employment. Sam:Roche: Employment. Le Clech:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Richard:Roche: Employment. Nicolini:Roche: Employment. Gerdes:Roche: Employment. Lariviere:Roche: Employment. Neumann:Roche: Employment. Klein:Roche: Employment, Patents & Royalties.

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Development of a fully human t-cell engaging bispecific antibody for the treatment of multiple myeloma.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.60 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 60-60

Author(s):

Ben Buelow ◽

Priya Choudhry ◽

Starlynn Clarke ◽

Kevin Dang ◽

Laura Davison ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cell ◽

T Cell Activation ◽

Cell Activation ◽

Bispecific Antibody ◽

Human Pbmcs ◽

Human T Cell

60 Background: T-cell engaging bispecific antibody (T-BsAb) therapies are highly efficacious and well suited for targets with low expression on tumor cells. Recently, T-BsAbs with high activation of CD3 have been shown to overstimulate T cells, leading to toxicity and decreased efficacy. Teneobio has developed a fully human BCMA-specific T-BsAb using a low-activating αCD3 that is highly effective in vitro and in vivo against MM but stimulates minimal cytokine release. Methods: UniRats were immunized with either CD3 or BCMA antigens and antigen-specific UniAbs were identified by Ab repertoire sequencing and high-throughput gene assembly, expression, and screening. Antigen-specific VH sequences with the desired target affinity were selected using recombinant proteins and cells. In vitro efficacy studies included T-cell activation by cytokine- and tumor cell kill by calcein-release assays. In vivo efficacy of the molecules was evaluated in NSG mice harboring myeloma cells and human PBMCs. Results: BCMA-specific UniAbs bound plasma cells with sub-nM affinity. Strong and weak T cell agonists were identified that bound human T cells with high and low affinities respectively. T-BsAbs with a strong and a weak αCD3 demonstrated T-cell activation and tumor-cell cytotoxicity in vitro; T-BsAbs with a weak αCD3 showed markedly reduced cytokine production even at doses that showed maximum tumor cell lysis. In vivo, BCMAxCD3 T-BsAbs reduced tumor load and increased survival when co-administered with human PBMCs as compared to controls. Conclusions: Our results suggest that T-BsAbs with low-activating αCD3 arms may have a favorable toxicity profile while maintaining efficacy in the treatment of MM.

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