Bispecific Antibody Formats Research Articles

Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing.

A novel bispecific antibody format was applied to generate T cell-engaging antibodies. The TriFab format is a trivalent IgG-shaped entity composed of two Fab arms that bind to antigens on the surface of tumor cells, which are linked via flexible peptides to a CD3 binding moiety that replaces the CH2 domains of conventional IgGs. The distinctive feature of these T cell recruiting bispecifics is that their CD3 variable regions are incorporated between domains, rather than N- or C-terminally fused to an Fc or antibody fragments. T cell recruiting TriFabs resemble in size and shape, are expressed and show biophysical properties similar to regular IgGs. Transmission electron microscopy (TEM) demonstrates high flexibility of the cell surface binding arms as well as target antigen accessibility of the interspersed CD3 binding domain. Functional co-culturing assays of peripheral blood mononuclear cells (PBMCs) and different tumor cell lines (MCF7 and A431) revealed a dose-dependent T cell-mediated cytotoxicity that was induced by the TriFabs targeting either LeY or EGFR cell surface antigens.

Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.

Bispecific Antibodies for the Treatment of Acute Myeloid Leukemia.

Bispecific antibodies combine antigen recognition sites from two or more antibodies into a single construct allowing simultaneous binding to multiple targets. Bispecific antibodies exist which can redirect immune effector cells against acute myeloid leukemia (AML) targets. This review will highlight the progress to date and the challenges in developing bispecific antibodies for the treatment of AML. Currently, a number of bispecific antibody formats including bispecific T cell engagers, dual affinity retargeting proteins, and tandem diabodies are in clinical development for AML. These antibodies target antigens present on AML blasts, including CD33, and the low affinity IL3 receptor, CD123. T cell redirecting bispecific antibodies in early phase clinical trials for AML include AG330, flotetuzumab, JNJ-63709178, and AMV564. Bispecific antibodies represent a promising immunotherapeutic approach for the treatment of cancer. The results of ongoing studies in AML will elucidate the potential for these agents in AML.

Characterization and analysis of scFv-IgG bispecific antibody size variants

ABSTRACTBispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

Relative Contribution of Framework and CDR Regions in Antibody Variable Domains to Multimerisation of Fv- and scFv-Containing Bispecific Antibodies.

Bispecific antibodies represent an emerging class of antibody drugs that are commonly generated by fusion of Fv or scFv antigen binding domains to IgG or Fab scaffolds. Fv- or scFv-mediated multimerisation of bispecific antibodies via promiscuous vH-vL pairing can result in sub-optimal monomer levels during expression, and hence, undesirable therapeutic protein yields. We investigate the contribution of disulphide stabilised Fv and scFv to Fab-Fv and Fab-scFv multimerisation. We show that monomer levels of isolated Fv/scFv cannot always be used to predict monomer levels of Fab-linked Fv/scFv, and that Fab-scFv monomer levels are greater than the equivalent Fab-Fv. Through grafting bispecifics with framework/CDR-‘swapped’ Fv and scFv, we show that monomer levels of disulphide stabilised Fab-Fv and Fab-scFv can be improved by Fv framework ‘swapping’. The Fab-Fv and Fab-scFv can be considered representative of the significant number of bispecific antibody formats containing appended Fv/scFv, as we also used Fv framework ‘swapping’ to increase the monomer level of an IgG-scFv bispecific antibody. This research may, therefore, be useful for maximising the monomeric yield of numerous pharmaceutically-relevant bispecific formats in pre-clinical development.

Expanding the Boundaries of Biotherapeutics with Bispecific Antibodies.

Bispecific antibodies have moved from being an academic curiosity with therapeutic promise to reality, with two molecules being currently commercialized (Hemlibra® and Blincyto®) and many more in clinical trials. The success of bispecific antibodies is mainly due to the continuously growing number of mechanisms of actions (MOA) they enable that are not accessible to monoclonal antibodies. One of the earliest MOA of bispecific antibodies and currently the one with the largest number of clinical trials is the redirecting of the cytotoxic activity of T-cells for oncology applications, now extending its use in infective diseases. The use of bispecific antibodies for crossing the blood–brain barrier is another important application because of its potential to advance the therapeutic options for neurological diseases. Another noteworthy application due to its growing trend is enabling a more tissue-specific delivery or activity of antibodies. The different molecular solutions to the initial hurdles that limited the development of bispecific antibodies have led to the current diverse set of bispecific or multispecific antibody formats that can be grouped into three main categories: IgG-like formats, antibody fragment-based formats, or appended IgG formats. The expanded applications of bispecific antibodies come at the price of additional challenges for clinical development. The rising complexity in their structure may increase the risk of immunogenicity and the multiple antigen specificity complicates the selection of relevant species for safety assessment.

Abstract 5768: MG1122, a whole IgG-like bispecific antibody targeting mesothelin and CD3, induces T cell-mediated killing of MSLN-expressing tumor cells

Abstract Mesothelin (MSLN) is an antigen overexpressed in several malignancies, including mesothelioma and ovarian and pancreatic adenocarcinoma. It has been studied as a marker for diagnosis and a target for immunotherapy. Here, we adopted a bispecific antibody format for recruiting cytotoxic T cells to kill tumor cells. MG1122 is a novel, whole IgG-like bispecific antibody which recognizes CD3 on T cells and MSLN on tumor cells. MG1122 induced effective killing of MSLN-expressing tumor cells. This response was associated with robust activation of T cells as shown by nuclear factor of activated T cells (NFAT) activation, CD25 and CD69 upregulation and increased cytokine release. In addition, using a live cell analysis system, we found that levels of activated caspase 3/7 were amplified in the tumor cells by MG1122 treatment. Although MSLN is a surface antigen, it also exists as a secreted isoform, which can lead to tumor evasion against MSLN targeting antibodies. However, despite the high concentration of soluble MSLN, MG1122 still effectively bound and showed strong killing effects against MSLN-expressing tumor cells. Taken together, our MSLN/CD3 bispecific antibody, MG1122 offers a promising opportunity to redirect T cells to kill MSLN-expressing cancer cells. Citation Format: Yun-Jung Lee, Okjae Lim, Munkyung Kim, Jeewon Lee, Kisu Kim, Junhong Jung, SuA Lee, Sung Keun Kim, Haenaem Kwon, Yangmi Lim, Yong Yea Park, Jonghwa Won. MG1122, a whole IgG-like bispecific antibody targeting mesothelin and CD3, induces T cell-mediated killing of MSLN-expressing tumor cells [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 5768.

Bispecific Antibodies: Formats and Areas of Application

Bispecific antibodies capable of simultaneously binding two targets have been studied for many years with a view to their implementation in clinical practice. Unique biological and pharmacological properties, as well as the diversity of their formats, make it possible to consider bispecific antibodies as promising agents for use in various procedures: from visualization of intracellular processes to targeted anticancer therapy. Bispecific antibodies help to determine more precisely the therapeutic target, thereby increasing the efficiency of therapy and reducing the probability of side effects. The present review describes the main formats of bispecific antibodies, methods for their generation, and possibilities for practical application.

A HER2 bispecific antibody can be efficiently expressed in Escherichia coli with potent cytotoxicity.

Bispecific antibodies have been actively studied for cancer therapy due to their potent cytotoxicity against tumor cells. A number of bispecific antibody formats have exhibited strong tumor cytotoxicity in vitro and in vivo. However, effective production of bispecific antibodies remains challenging for the majority of bispecific antibody formats. In the present study, a bispecific antibody was designed that links a conventional antigen-binding fragment (Fab) against cluster of differentiation 3 antigen (CD3) to a camel single domain antibody (VHH) against human epidermal growth factor receptor 2 (HER2). This bispecific antibody may be secreted and purified efficiently from Escherichia coli culture medium. The purified bispecific antibody is able to trigger T cell-mediated HER2-specific cytotoxicity in vitro and in vivo. The data gathered in the present study suggest that this bispecific format may be applied to other tumor antigens to produce bispecific antibodies more efficiently.

Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Förster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the CH3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reaction.

ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy

ABSTRACTThe redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific antibody formats. Here, we have engineered the ATTACK (Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing), a novel T cell-recruiting bispecific antibody which combines three EGFR-binding single-domain antibodies (VHH; clone EgA1) with a single CD3-binding single-chain variable fragment (scFv; clone OKT3) in an intermediate molecular weight package. The two specificities are oriented in opposite directions in order to simultaneously engage cancer cells and T cell effectors, and thereby promote immunological synapse formation. EgA1 ATTACK was expressed as a homogenous, non-aggregating, soluble protein by mammalian cells and demonstrated an enhanced binding to EGFR, but not CD3, when compared to the previously characterized tandem bispecific antibody which has one EgA1 VHH and one OKT3 scFv per molecule. EgA1 ATTACK induced synapse formation and early signaling pathways downstream of TCR engagement at lower concentrations than the tandem VHH-scFv bispecific antibody. Furthermore, it demonstrated extremely potent, dose-dependent cytotoxicity when retargeting human T cells towards EGFR-expressing cells, with an efficacy over 15-fold higher than that of the tandem VHH-scFv bispecific antibody. These results suggest that the ATTACK is an ideal format for the development of the next-generation of T cell-redirecting bispecific antibodies.

Tethered-variable CL bispecific IgG: an antibody platform for rapid bispecific antibody screening.

Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties. This technology is broadly applicable in the generation of BsIgG from a variety of antibody isotypes, including human BsIgG1, BsIgG2 and BsIgG4. The practicality of the tcBsIgG platform is demonstrated by screening BsIgGs generated from FGF21-mimetic anti-Klotho-β agonistic antibodies in a combinatorial manner. This screen identified multiple biepitopic combinations with enhanced agonistic activity relative to the parental monoclonal antibodies, thereby demonstrating that biepitopic antibodies can acquire enhanced functionality compared to monospecific parental antibodies. By design, the tcBsIgG format is amenable to high-throughput production of large panels of bispecific antibodies and thus can facilitate the identification of rare BsIgG combinations to enable the discovery of molecules with improved biological function.

Abstract 1639: Combination of PD1 blockade and T cell costimulation by bispecific antibodies promotes human T cell activation and proliferation

Abstract Tumor infiltrating lymphocytes (TILs) express multiple checkpoint receptors, in contrast to lymphocytes found in the periphery (Matsuzaki et al PNAS 2010, Fourcade et al Cancer Res 2012, Gros et al JCI 2014). Checkpoint blockade has demonstrated increased clinical response rates relative to other treatment options; however, many patients fail to achieve a response to checkpoint blockade. We sought to identify an additional therapeutic modality to stack with checkpoint blockade that could increase patient response rate. We hypothesized that engagement of T cell costimulatory receptors in combination with checkpoint blockade could further increase T cell activation and proliferation. The combination of checkpoint blockade with costimulation could be accomplished using a bispecific antibody format, with the potential benefits of reduced cost and more selective targeting of TILs to improve safety. Antibodies binding to immune checkpoint PD1 and a T cell costimulatory receptor were assembled in a bispecific antibody platform with substitutions in the Fc domain to suppress effector function. PD1 x costimulation (PD1 x costim) bispecific antibodies were evaluated in vitro by measuring antibody binding and de-repression of Staphylococcal enterotoxin B (SEB) stimulated PBMCs. IL2 and IFNγ production was measured by immunoassay. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and the extent of T cell engraftment is monitored by flow cytometry. We produced PD1 x costim bispecific antibodies that bound PD1 and a T cell costimulatory receptor monovalently. The bispecifics bound to SEB-stimulated T cells more tightly than monovalent controls, indicating that a single bispecific molecule was capable of avid simultaneous co-engagement of both PD1 and a costimulatory receptor. The bispecifics enhanced IL2 and IFNγ production in an in vitro SEB stimulation assay relative to control (p < 0.001, n = 19 donors). IL2 and IFNγ production was superior to anti-PD1 or anti-costim antibodies alone (p < 0.001), indicating productive and synergistic combination of checkpoint blockade plus costimulation. Treatment of huPBMC-NSG mice with checkpoint bispecifics promoted enhanced T cell engraftment relative to control. Engraftment levels promoted by bispecifics were superior to those found for anti-PD1 treatment alone. For example, in one run of the model, while anti-PD1 treatment alone promoted a 2-fold increase in human CD45+ cells, a PD1 x costim bispecific antibody induced a 16-fold increase. Combination of checkpoint blockade and costimulation with bispecific antibodies is feasible and promotes strong T cell activation in vitro and in vivo. Compelling activity suggests clinical development is warranted for the treatment of human malignancies. Citation Format: Gregory L. Moore, Michael Hedvat, Matthew J. Bernett, Rajat Varma, Suzanne Schubbert, Christine Bonzon, Kendra N. Avery, Rumana Rashid, Alex Nisthal, Liz Bogaert, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais. Combination of PD1 blockade and T cell costimulation by bispecific antibodies promotes human T cell activation and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2017-1639

Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence

Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected.

Strategies for Bispecific Single Chain Antibody in Cancer Immunotherapy.

Genetic engineering has resulted in more than 50 recombinant bispecific antibody formats over the past two decades. Bispecific scFv antibodies represent a successful and promising immunotherapy platform that retargets cytotoxic T cells to tumor cells, with one scFv directed to tumor-associated antigens and the other to T cells. Based on this antibody construct, strategies for both specific tumor targeting and T cell activation are reviewed here. Three distinct types of tumor antigens are considered to optimize specificity and safety in bispecific scFv based treatment: cancer-testis antigens, neo-antigens and virus-associated antigens. In terms of T cell activation, although CD3 has been widely applied in bispecific scFvs being developed, CD28 and CD137 among co-stimulatory signals are also ideal candidates to be evaluated. Besides, LIGHT and HIV-Tat101 have drawn much attention as their potential roles in modulating antitumor responses.

Bispecific Antibodies as a Development Platform for New Concepts and Treatment Strategies.

With the development of molecular cloning technology and the deep understanding of antibody engineering, there are diverse bispecific antibody formats from which to choose to pursue the optimal biological activity and clinical purpose. The single-chain-based bispecific antibodies usually bridge tumor cells with immune cells and form an immunological synapse because of their relatively small size. Bispecific antibodies in the IgG format include asymmetric bispecific antibodies and homodimerized bispecific antibodies, all of which have an extended blood half-life and their own crystalline fragment (Fc)-mediated functions. Besides retargeting effector cells to the site of cancer, new applications were established for bispecific antibodies. Bispecific antibodies that can simultaneously bind to cell surface antigens and payloads are a very ideal delivery system for therapeutic use. Bispecific antibodies that can inhibit two correlated signaling molecules at the same time can be developed to overcome inherent or acquired resistance and to be more efficient angiogenesis inhibitors. Bispecific antibodies can also be used to treat hemophilia A by mimicking the function of factor VIII. Bispecific antibodies also have broad application prospects in bone disorders and infections and diseases of the central nervous system. The latest developments of the formats and application of bispecific antibodies will be reviewed. Furthermore, the challenges and perspectives are summarized in this review.

Generation of Bispecific Antibodies by Fc Heterodimerization and their Application.

Bispecific antibodies with binding specificities for two different antigens have prompted a lot of interest into their development and application. Currently, more than ten bispecific antibodies have been clinically validated for the treatment of various diseases, including cancers and inflammatory diseases. Intensive studies in antibody engineering drive the generation of different bispecific antibody formats that differ in size and shape. However, the most prominent formats, such as IgG-single-chain (sc) Fv or dual-variable domain (DVD) IgG, deviating from the natural IgG structure, may lead to manufacturing difficulties or increase the potential risk of immunogenicity. Thus, the recent efforts focus on the development of bispecific antibodies by Fc heterodimerization that maintain the native structure of the antibody IgG molecule. This review summarizes the various techniques and methods to generate bispecific antibody molecules with Fc heterodimerization, and discusses perspectives of their application.

Abstract B088: Generation of immuno-modulatory receptor binding bispecific antibodies to modulate tumor immunity

Abstract Introduction: Monoclonal antibodies blocking CTLA-4 or PD-1 ligand interaction have been shown to induce durable clinical responses in a subset of melanoma and NSCLC patients. The clinical efficacy of these antibodies has been linked to their ability to restore robust anti-tumor immune responses. Based on clinical and preclinical data it is expected that combinations of antibodies blocking inhibitory and/or costimulatory molecules could benefit patients that do not respond to existing immunotherapies. However dual blockade of immuno-modulatory receptors (iMODs) has also been shown to increase immune-related toxicity as has been shown for a combination of CTLA-4 and PD-1 antibodies. Bispecific antibodies are a novel therapeutic modality capable of specifically binding two different targets. As such they may be ideally suited to address dual blockade of iMODs. Previous work on other cell surface targets has shown that bispecific antibodies can exert functional activities that cannot be reproduced by monoclonal antibody combinations. In addition, bispecific antibodies that more selectively target specific cell populations, based on the co-expression of the two targets, could have reduced safety liabilities in patients. We report here the generation and characterization of common light chain (cLC) Fab panels against PD-1, PD-L1, LAG-3, CD137, OX40 and TIM-3 for combination in a full length IgG1 bispecific antibody format and present the functional evaluation of some of these combinations in human in vitro functional assays. Methods: Humanized cLC mice (MeMo®) were immunized with recombinant protein or DNA, and materials harvested from these mice were used to generate Fab regions against these antigens using phage display. Furthermore, large and diverse synthetic cLC Fab-phagemid libraries were used to select iMOD specific Fabs. The resulting cLC Fabs were cloned into bispecific antibody expression constructs for further testing. Recombinant proteins (both wild type and domain swapped orthologs) were generated for each target and used for binding and competition in both ELISA and FACS. Reporter assays using NFAT or NFkB-luc in Jurkat cells were used for each target to measure functional activity of antibodies. Furthermore, primary human assays using CD3 or SEB activation with cytokine release were used as a read-out of T cell activity. Results: Two parallel strategies were applied to generate panels of cLC Fabs against PD-1, PD-L1, LAG-3, CD137, OX40 and TIM-3; mouse immunization and phage selection yielded >1350 specific Fab arms whereas synthetic library selections yielded >850 Fabs. Initial characterization and binning of representative clones were carried out in bispecific IgG format (iMOD specific Fabs combined with a Tetanus toxoid-specific control Fab arm) allowing for stringent ranking of the iMOD panels in a monovalent format for (domain) specificity, cynomolgus cross-reactivity, affinity, stability, and ligand blocking potency. The resulting characteristics of the identified bins were used to generate diverse bispecific antibody panels that combined different iMOD Fabs. These were first tested in reporter assays to select functional clones, that were then confirmed in a SEB driven T cell proliferation assay. Several bispecific candidates have been identified that show significantly increased cytokine production and other functional parameters compared to benchmark antibodies. Conclusion: Large diverse Fab panels binding six different iMODs were generated for use in bispecific antibody combinations. Characterization and screening of large panels of bispecific IgG has been carried out in relevant functional assays. Results from the screening of bispecific antibodies targeting iMOD combinations support the concept of empirical screening to identify potent immunomodulatory combinations. Citation Format: Rinse Klooster, Cecile Geuijen, Mark Throsby. Generation of immuno-modulatory receptor binding bispecific antibodies to modulate tumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B088.

Single domain antibody-based bispecific antibody induces potent specific anti-tumor activity

ABSTRACTBispecific antibodies have emerged as powerful therapeutic agents given their high specificity and ability to induce a potent immune response. Various bispecific antibody formats have been designed and studied regarding their applications in cancer therapy, though associated with issues of short half-life or manufacturing difficulties. Herein, a novel bispecific antibody, SS-Fc, was constructed by pairing 2 single-domain antibodies, anti-CD16 and anti-CEA, which were fused with CH3 “knobs into holes” mutations individually. SS-Fc was expressed and purified from E.coli. In vitro and in vivo experiments confirmed that SS-Fc can form a heterodimeric bispecific antibody when expressed and purified from E. coli. By engaging natural killer (NK) cells through an anti-CD16 single domain antibody, the SS-Fc bispecific antibody exhibited potent in vitro and in vivo cytotoxicity against cancer cells with carcinoembryonic antigen (CEA) expression. Thus, SS-Fc represents a novel bispecific antibody format that can be applied to a wide range of both discovery and clinical applications.

MOR209/ES414, a Novel Bispecific Antibody Targeting PSMA for the Treatment of Metastatic Castration-Resistant Prostate Cancer.

Treatment of metastatic, castration-resistant prostate cancer (mCRPC) remains a highly unmet medical need and current therapies ultimately result in disease progression. Immunotherapy is a rapidly growing approach for treatment of cancer but has shown limited success to date in the treatment of mCRPC. We have developed a novel humanized bispecific antibody, MOR209/ES414, built on the ADAPTIR (modular protein technology) platform, to redirect T-cell cytotoxicity toward prostate cancer cells by specifically targeting T cells through CD3ε to prostate cancer cells expressing PSMA (prostate-specific membrane antigen). In vitro cross-linking of T cells with PSMA-expressing tumor cells by MOR209/ES414 triggered potent target-dependent tumor lysis and induction of target-dependent T-cell activation and proliferation. This activity occurred at low picomolar concentrations of MOR209/ES414 and was effective at low T-effector to tumor target cell ratios. In addition, cytotoxic activity was equivalent over a wide range of PSMA expression on target cells, suggesting that as few as 3,700 PSMA receptors per cell are sufficient for tumor lysis. In addition to high sensitivity and in vitro activity, MOR209/ES414 induced limited production of cytokines compared with other bispecific antibody formats. Pharmacokinetic analysis of MOR209/ES414 demonstrated a serum elimination half-life in NOD/SCID γ (NSG) mice of 4 days. Administration of MOR209/ES414 in murine xenograft models of human prostate cancer significantly inhibited tumor growth, prolonged survival, and decreased serum prostate-specific antigen levels only in the presence of adoptively transferred human T cells. On the basis of these preclinical findings, MOR209/ES414 warrants further investigation as a potential therapeutic for the treatment of CRPC. Mol Cancer Ther; 15(9); 2155-65. ©2016 AACR.