Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries

CD20-TCB, a Novel T-Cell-Engaging Bispecific Antibody, Induces T-Cell-Mediated Killing in Relapsed or Refractory Non-Hodgkin Lymphoma: Biomarker Results From a Phase I Dose-Escalation Trial

BackgroundThe continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options.MethodsThe MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model.ResultsAll of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated using the anti-CD73 antibody, which bound other ‘triple negative’ breast cancer cell lines and produced significant tumour growth inhibitory activity in a MDA-MB-231 xenograft model.ConclusionsThis study has demonstrated that multiple methods are required to successfully analyse the membranome of a desired cell type. It has also successfully demonstrated that phenotypic antibody screening provides a mechanism for rapidly discovering and evaluating antibody tractable targets, which can significantly accelerate the therapeutic discovery process.

BackgroundInhibition of T cell activation and effector function via engagement of the co-inhibitory receptor PD-1 is a critical mechanism enabling tumors to evade host immunity. The two ligands for PD-1, PD-L1 and PD-L2, can be expressed by a variety of immunosuppressive stromal cells, particularly of the myeloid lineage, endothelial cells, and by tumors themselves. In addition to PD-1, PD-L1 engages B7-1 in an additional co-inhibitory interaction. Blocking only PD-1 or only PD-L1 thus does not relieve all inhibitory components of this pathway. We hypothesized that bispecific antibodies blocking both PD-L1 and PD-L2 could more fully restore tumor-specific T cell activation and potentiate anti-cancer immunotherapy. Furthermore, we speculated that enhancing the cytotoxic effector function of these antibodies might further enhance their efficacy through the depletion of tumor cells and supportive stroma.MethodsWe investigated the capacity of monoclonal antibodies capable of bivalent binding to both PD-L1 and PD-L2 to restore the function of PD-1-suppressed T cells in vitro. To assess the in vivo therapeutic efficiency of bispecific PD-Ligand antibodies with ADCC capacities, mouse IgG2a and modified human IgG1 versions were generated. We assessed their ADCC activity in vitro using a bioluminescent reporter assay, and their therapeutic efficiency in vivo in syngeneic or human-cell derived tumors.ResultsThe bispecific antibodies we generated restore the function of PD-1-suppressed T cells in vitro with equivalent efficiency to the FDA approved PD-1 antibody Pembrolizumab. Moreover, our modified human bispecific antibodies lead to significantly higher FcγRIIa activation than FDA-approved clinical human IgG1 PD-L1 antibodies in vitro. In vivo, ADCC-capable PD-Ligand bispecific antibodies suppress the growth of U2940 lymphoma in immunodeficient mice more efficiently than Rituximab, and in a syngeneic model of PD-L1/PD-L2 double positive colon carcinoma, these antibodies demonstrate superiority to PD-1 blocking antibodies to limit tumor growth and increase survival. Furthermore, treatment with our bispecific antibodies increases T cell proliferation and cytotoxicity and reduces density of immunosuppressive myeloid stroma in vivo.ConclusionsADCC-capable PD-Ligand bispecific antibodies display higher therapeutic potential than existing anti-PD-1 antibodies and represent a new class of PD-1 pathway therapeutics with significant potential for the treatment of a variety of human cancers.AcknowledgementsI would like to thank Anupallavi Srinivasamani, PhD student in Michael Curran’s lab, who performed a considerable amount of the work on this project before I joined.

CD20-TCB, a Novel T-Cell-Engaging Bispecific Antibody, Can be Safely Combined with the Anti-PD-L1 Antibody Atezolizumab in Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

T cell bispecific antibodies (TCBs) are potent molecules that upon simultaneous binding to tumor cells and T cells trigger strong T cell activation resulting in the killing of tumor cells. CEA TCB (RG7813) is a novel bispecific antibody targeting carcinoembryonic antigen (CEA), often overexpressed on solid tumors (e.g. colorectal, gastric, pancreatic, lung carcinoma etc.), and the CD3 epsilon chain present on T cells. CEA TCB bears several innovative technological features that distinguish it from other bispecific antibodies currently in (pre-)clinical development: (a) bivalency for tumor antigen translating into higher avidity, superior potency and better differentiation between high and low antigen-expressing cells; (b) head-to-tail fusion geometry for anti-tumor and CD3-binding domains, resulting in higher potency compared to conventional IgG-based TCBs; (c) extended half-life compared to non-Fc-based TCBs; (d) fully silent Fc ensuring lower risk of FcgR-mediated infusion reactions; and (e) robust production using standard manufacturing processes (enabled by “CrossMAb” and knob-into-hole bispecific antibody technologies). In vitro, CEA TCB mediates potent target-dependent T cell cytotoxicity, T cell activation, proliferation, and cytokine release in killing assays, exclusively in the presence of CEA-expressing target-cells. CEA TCB activity correlates with CEA expression level, showing higher potency against tumor cells with high expression of CEA. In vivo, CEA TCB induces dose- and time-dependent regression of CEA-expressing tumors with variable amounts of immune cell infiltrate. In fully humanized NOG mice, CEA TCB is efficacious in poorly-infiltrated tumors and converts non-inflamed into highly-inflamed tumors. Histological and FACS analyses revealed that CEA TCB recruits new T cells into tumors and/or expands pre-existing ones and is able to induce T cell re-localization from the tumor periphery into the tumor bed. Surprisingly, CEA TCB treatment also qualitatively alters the composition of intratumoral T cells resulting in an increased frequency of activated (CD69, CD25), proliferating (Ki67) and differentiated T cells (having effector memory phenotype) that are ready to kill (express high levels of Granzyme B). Taken together, these preclinical data show that CEA TCB is a novel tumor-targeted T cell bispecific antibody with promising anti-tumor activity and the novel ability to modify the tumor microenvironment. Phase 1 clinical trials with CEA TCB are currently ongoing. Future studies will focus on identification of combination partners that inhibit T cell suppression and unleash the full potential of T cell activity. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Johannes Sam, Valeria Nicolini, Nathalie Steinhoff, Oliver Ast, Peter Bruenker, Ralf Hosse, Thomas Hofer, Ekkehard Moessner, Christiane Jaeger, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2481. doi:10.1158/1538-7445.AM2015-2481

Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs that recognize the invariant CD3 signaling complex is a controlled polyclonal activation of T cells that, ideally, is exquisitely dependent on the presence of target cells. Otherwise, overt production of inflammatory cytokines and secondary reactions may occur as side effects, as can be observed with constitutively T-cell activating monoclonal antibodies to CD3 or CD28, and with bispecific antibodies bearing Fc gamma portions. Here we analyzed 2 distinct bispecific single-chain antibody constructs of the BiTE class, called MT110 and MT103 (or MEDI-538), for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of the BiTE molecules were sufficient to stimulate a high percentage of peripheral human T cells to express cytokines and surface activation markers, enter into cell cycle, and induce redirected lysis of target cells. However, in the absence of target cells, the 2 BiTE molecules even at high concentrations did not detectably activate T cells. Our data show that T cell activation by monomeric forms of MT110 and MT103 is highly conditional in that it is strictly dependent on the presence of cells expressing the proper target antigen. BiTE molecules therefore qualify for a highly controlled polyclonal T-cell therapy of cancer.

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

Combination checkpoint blockade promotes productive anti-tumor clinical responses often associated with an increase in immune-related adverse events. We developed optimized bispecific antibody candidates that simultaneously engage PD1 and CTLA4 (XmAb20717), CTLA4 and LAG3 (XmAb22841), and PD1 and ICOS (XmAb23104), that preferentially bind to cells co-expressing the targeted receptors. Because tumor infiltrating lymphocytes (TILs) typically express multiple immune checkpoints and costimulatory receptors, we hypothesized that these bispecific antibodies will enable selective targeting of tumor-reactive TILs, leading to safer and more cost-effective combination checkpoint blockade. The PD1 x CTLA4 bispecific antibody XmAb20717 and the PD1 x ICOS bispecific antibody XmAb23104 promoted superior in vitro T cell activation when compared to an anti-PD1 bivalent antibody. The CTLA4 x LAG3 bispecific antibody XmAb22841 combines productively with an anti-PD1 bivalent antibody achieving additive T cell activation through triple checkpoint blockade. Treatment with bispecific antibodies caused superior engraftment and activation of human T cells in NSG mice compared to an anti-PD1 bivalent antibody. Bispecific antibodies also potentiate allogeneic anti-tumor activity against human cancer cells in vivo. Each bispecific antibody promoted a unique RNA gene signature suggesting distinct mechanisms of action. These bispecific antibodies demonstrate compelling immune modulatory activity suggesting clinical development is warranted for the treatment of human malignancies. Citation Format: Michael Hedvat, Christine Bonzon, Matthew J. Bernett, Gregory L. Moore, Kendra Avery, Rumana Rashid, Alex Nisthal, Suzanne Schubert, Rajat Varma, Sung-Hyung Lee, Liz Bogaert, Irene W.L. Leung, Seung Chu, Umesh Muchhal, John Desjarlais. Simultaneous checkpoint-checkpoint or checkpoint-costimulatory receptor targeting with bispecific antibodies promotes enhanced human T cell activation [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 2784.

Population Pharmacokinetics and Novel Exposure-Response Analyses to Inform Optimal Biologic Dose Selection for CD20-TCB, a T-Cell-Engaging Bispecific Antibody, in Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Acute lymphocytic leukemia (ALL) is the most common form of leukemia in childhood and adolescence. Outcomes of relapsed/refractory adult B-ALL remain dismal with long-term survival of less than 10%. Although targeted therapies such as blinatumomab, inotuzumab ozogamicin, and CD19 CAR T cell therapy are a significant treatment advance for patients with B-ALL, there is still an unmet medical need for novel therapies for B-ALL. CRLF2-rearranged B-ALL, a subtype of Philadelphia chromosome-like B-ALL, is a high-risk disease subset with poor long-term outcomes. In an effort to identify new therapeutic targets for B-ALL, we validated by flow cytometry that patients with CRLF2-rearranged Ph-like ALL overexpress thymic stromal lymphopoietin receptor (TSLPR) at levels comparable with CD19. We subsequently generated antibodies against TSLPR from immunized H2L2 Harbor mice through single B cell cloning technology. The lead antibody was further engineered to a CD3-redirecting bispecific antibody (BsAb) using Xencor’s CD3e bispecific antibody technology to create 1B7/CD3 BsAb with an Fc-silent region. 1B7/CD3 BsAb exhibits high affinity binding to both human and cynomolgus monkey CD3e and TSLPR by BLI. Additionally, UHPLC-SEC analysis of 1B7/CD3 shows that this BsAb is stable without any aggregation or degradation present after five weeks incubation at 37 °C. In cell-based analyses,1B7/CD3 BsAb demonstrate potent antigen-specific T cell activation and tumor lysis activity against TSLPR+-REH and MHH-CALL4 cell lines as well as a primary B-ALL patient sample, but none against human peripheral blood mononuclear cells (PBMC) collected from healthy donors. Animal tumor models suggest that 1B7/CD3 triggers a dose-dependent tumor regression or growth inhibition in PBMC humanized TSLPR-REH CDX and BOS-1 PDX models across donors. Correspondingly, T cell activation and expansion were examined by CD69 and CD3+ T cells with the best effect observed at the 1mg/kg dose. 1B7/CD3 exhibits durable PK with T1/2 in NSG mice of up to ten days. Finally, 1B7/CD3 BsAb demonstrated a tolerable safety margin in a two-dose exploratory toxicity study in cynomolgus monkeys with MTD equal to or less than 1mg/kg. Consistent with clinical observation, we observed a transient increase of six major cytokines (IFNγ, IL-6, IL-8, IL-10, MCP-1 and TNFα) 4hr post the initial dosing, but no notable induction of cytokine levels after the second dosing (except for IL-8). Thus, our preclinical data provide the framework for the clinical evaluation of 1B7/CD3 BsAb in patients with CRLF2-rearranged B-ALL. Citation Format: Ze Tian, chunhua shi, Amin AI-Shami, GuoJun Yong, Jason K Allen, Jill Wardell Olson, Melinda G Smith, Qing Chang, Qing Shi, Junping You, Michelle A Gonzalez, Timothy E Lofton, Jasbir Kaur, Qi Zhang, DongXing Zha, Nitin Jain, Marina Y Konopleva, Timothy Heffernan, Jeffrey J Molldrem. preclinical development of a novel anti-tslpr bispecific antibody 1b7/cd3 targeting crlf2-rearranged ph-like b-all. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5655.

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

Coronary artery disease (CAD) is the leading cause of excess deaths in rheumatoid arthritis (RA). However, identification of features denoting patients with a risk of developing CAD is lacking. The composition of circulating peripheral blood mononuclear cell (PBMC) subsets in RA patients differs markedly from that in healthy controls with regard to the extent of T cell activation, with clonal expansion and differentiation to effector memory status, and presence of inflammatory monocytes. In this study, we sought to evaluate whether elevations in these PBMC subpopulations in RA patients could denote those with an increased risk of subclinical CAD, as determined by the presence of coronary artery calcification (CAC). The study cohort comprised 72 patients with RA who underwent cardiac computed tomography to assess CAC. PBMC subsets were determined by multiparameter flow cytometry. Multivariable logistic regression was used to determine the associations between PBMC subpopulations and the presence of CAC. Among the 72 patients with RA, 33% had CAC and exhibited significant increases in the levels of circulating CD4 T cell subsets denoting activation and differentiation to the effector memory phenotypes. Analogous increases in the levels of CD8 T cell subsets, as well as in the CD14(high)CD16+ intermediate monocyte subset, were also present in these patients, as compared to those without CAC. The increases in the CD4 and CD8 T cell subsets were highly intercorrelated, whereas the increases in CD14(high)CD16+ monocytes were independent of elevations in the CD4 T cell subsets. After adjustments for relevant confounders, the levels of CD4+CD56+CD57+ T cells and CD14(high)CD16+ monocytes remained associated with the presence of CAC. These findings indicate that PBMC subsets are markers for the presence of CAC and suggest that mechanisms of atherogenesis in RA may operate in part through the elevations in these subsets, raising further questions about the mechanisms underlying the presence of such alterations in cell composition in patients with RA and the potential for shared etiologic pathways between RA and cardiovascular disease.

A bispecific antibody enhances cytokine-induced killer-mediated cytolysis of autologous acute myeloid leukemia cells

Bispecific T-Cell Engager Antibodies in Multiple Myeloma- a Systematic Review of Phase 1 Clinical Trials

Amid advancements in antibody-based cancer immunotherapy, T-cell-dependent bispecific antibodies (TDBs) hold great promise in cancer treatment. Peripheral blood mononuclear cell (PBMC) humanized mice serve as a platform for preclinical characterization of TDBs, although its induction of xenogeneic graft-versus-host disease (GvHD) limits the experimental time window and introduces confounding factors. NSG-MHC I/II DKO mouse is a highly immunodeficient mouse knocked out for mouse MHC class I and II molecules, which delay GvHD onset following human PBMC engraftment. Here, we describe the materials and methods for generating PBMC humanized mouse models with NSG-MHC I/II DKO mice. Using this model, we demonstrate the feasibility of testing the in vivo efficacy of TDBs in NSG-MHC I/II DKO mice engrafted with the colorectal cancer cell line HT29.