Bispecific Formats Research Articles

Abstract B34: Quantitative systems pharmacology and immunotherapy: accelerating lead generation and optimization of a PD-1 x TIM-3 biotherapeutic in immuno-oncology

Abstract The goal of this collaboration was to provide early quantitative decision making guidance for the project team by developing and interrogating a quantitative systems pharmacology (QSP) model of the co-modulation inhibitory receptors PD-1 and TIM-3 in immuno-oncology. The QSP model was to: (1) provide predictions of the best-in-class profile for a PD-1 and TIM-3 dual antagonist biologic(s), (2) accelerate project timelines, (3) provide biological insights, and (4) reduce experimental costs. The QSP model was based on first principles as a system of elementary mass-action, mechanistic PKPD, ordinary differential equations. The model parameters and reactions were based on biophysics, and are interpretable. The model reactions include protein synthesis and elimination, ligand-receptor and drug-target formation and turnover, and drug administration and first order clearance. There were four versions of the model: PD-1 monospecific, TIM-3 monospecific, PD-1 x TIM-3 bispecific and fixed dose combination (FDC) targeting PD-1 and TIM-3. The monospecific models were then benchmarked against published data such that model parameter values were set to known values and unknown parameters were estimated. Once benchmarked, the FDC and bispecific models were analyzed by systematically investigating how tuning the model parameters (e.g., affinity, avidity, dose, half-life, target expression, etc.) impacted target inhibition, and to simulate patient variability. The model was in good agreement with published clinical data from nivolumab and pembrolizumab, and data from RMT3-23 in the TIM-3 driven mouse model. QSP model analysis predicted: (1) there would be diminishing returns on very tight binding biologics due to Target Mediated Drug Disposition (TMDD) that offsets potency, and (2) there is no advantage between FDC, 2-2 bispecific, and 2-1 bispecific formats, which are predicted to be roughly equivalent. As a result of these analyses, there was a significant reduction in the number of experiments, and acceleration of project timelines by (1) eliminating rounds of affinity maturation, as drug leads were in predicted optimal drug parameter ranges, and (2) eliminating the need to construct and evaluate bi-specific constructs and proceed with FDCs. Citation Format: Joshua F. Apgar, Jamie Wong, Ryan Phennicie, Mike Briskin, John M. Burke. Quantitative systems pharmacology and immunotherapy: accelerating lead generation and optimization of a PD-1 x TIM-3 biotherapeutic in immuno-oncology. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B34.

Mathematical PKPD and safety model of bispecific TfR/BACE1 antibodies for the optimization of antibody uptake in brain

Treatment of diseases of the central nervous system by monoclonal antibodies may be limited by the restricted uptake of antibodies across the blood–brain barrier (BBB). An antibody targeting transferrin receptor (TfR) has been shown to take advantage of the receptor-mediated transcytosis properties of TfR in order to cross the BBB in mice, with the uptake in the brain being dependent on the affinity to TfR. In the bispecific format with arms targeting both TfR and β-secretase 1 (BACE1), altering the affinity to TfR has been shown to impact systemic exposure and safety profiles. In this work, a mathematical model incorporating pharmacokinetic/pharmacodynamic (PKPD) and safety profiles is developed for bispecific TfR/BACE1 antibodies with a range of affinities to TfR in order to guide candidate selection. The model captures the dependence of both systemic and brain exposure on TfR affinity and the subsequent impact on brain Aβ40 lowering and circulating reticulocyte levels. Model simulations identify the optimal affinity for the TfR arm of the bispecific to maximize Aβ reduction while maintaining reticulocyte levels. The model serves as a useful tool to prioritize and optimize preclinical studies and has been used to support the selection of additional candidates for further development.

Abstract A151: Quantitative systems pharmacology approaches accelerate lead generation and optimization of a PD-1 x TIM-3 therapeutic in immuno-oncology

Abstract The goal of this collaboration was to provide early quantitative decision making guidance for the project team by developing and interrogating a quantitative systems pharmacology (QSP) model of the co-modulation inhibitory receptors PD-1 and TIM-3 in immuno-oncology. The QSP model was to: (1) provide predictions of the best-in-class profile for a PD-1 and TIM-3 dual antagonist, (2) accelerate project timelines, (3) provide biological insights, and (4) reduce experimental costs. The QSP model was based on first principles as a system of elementary mass-action, mechanistic PKPD, ordinary differential equations. The model parameters and reactions were based on biophysics, and are interpretable. The model reactions include protein synthesis and elimination, ligand-receptor and drug-target formation and turnover, and drug administration and first order clearance. There were four versions of the model: PD-1 monospecific, TIM-3 monospecific, PD-1 x TIM-3 bispecific and fixed dose combination (FDC) targeting PD-1 and TIM-3. The monospecific models were then benchmarked against published data such that model parameter values were set to known values and unknown parameters were estimated. Once benchmarked, the FDC and bispecific models were analyzed by systematically investigating how tuning the model parameters (e.g., affinity, avidity, dose, half-life, target expression, etc.) impacted target inhibition, and to simulate patient variability. The model was in good agreement with published clinical data from nivolumab and pembrolizumab, and data from RMT3-23 in the TIM-3 driven mouse model. QSP model analysis predicted: (1) there would be diminishing returns on very tight binding biologics due to Target Mediated Drug Disposition (TMDD) that offsets potency, and (2) there is no advantage between FDC, 2-2 bispecific, and 2-1 bispecific formats, which are predicted to be roughly equivalent. As a result of these analyses, there was a significant reduction in the number of experiments, and acceleration of project timelines by (1) eliminating rounds of affinity maturation, as drug leads were in predicted optimal drug parameter ranges, and (2) eliminating the need to construct and evaluate bi-specific constructs and proceed with FDCs. Citation Format: Joshua F. Apgar, Jamie Wong, Ryan Ryan Phennicie, Mike Briskin, John M. Burke. Quantitative systems pharmacology approaches accelerate lead generation and optimization of a PD-1 x TIM-3 therapeutic in immuno-oncology. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A151.

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

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.

Prosthetic Antigen Receptors.

Chimeric antigen receptors (CARs) have shown great promise for the immunological treatment of cancer. Nevertheless, the need to genetically engineer a patient's T-cells has presented significant production and safety challenges. To address these issues, we have demonstrated that chemically self-assembled nanorings (CSANs) displaying single chain antibodies can bind to both the CD3 ε subunit of the T-cell-receptor/CD3 complex and the CD22 antigen on malignant B cells such as B-leukemias or lymphomas. We demonstrate that the multivalent and bispecific format allows the antiCD3/antiCD22 CSANs to stably bind to T-cell surfaces for greater than 4 days, while being easily disassembled on the cell membrane by treatment with the nontoxic FDA approved drug, trimethoprim. In the presence of CD22+ Raji cells, T-cells modified with antiCD3/antiCD22 CSANs were shown to selectively up-regulate the production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) and to initiate cytotoxicity. Taken together, our results demonstrate that antiCD3/antiCD22 bispecific CSANs offer a potential alternative to CARs, as prosthetic antigen receptors.

Bispecific antibody targeting of CD47/CD19 to promote enhanced phagocytosis of patient B lymphoma cells.

e14016 Background: Many cancers up-regulate the expression of CD47 in order to evade immune surveillance and killing. This ‘don’t eat me’ signal is a molecular means for healthy cells to limit their phagocytic elimination by macrophages and other innate immune cells. This cell purging mechanism is mediated by the interaction of CD47 on the target cell and Signal Regulatory Protein Alpha (SIRPα) on myeloid cells. Development of anti-CD47 monoclonal antibodies may be hindered due to the ubiquitous expression of CD47, leading to rapid drug elimination kinetics through target-mediated drug disposition, an unfavorable pharmacokinetic profile and target related toxicity including chronic anemia. Methods: To overcome these clinical liabilities, we have developed an anti-CD47 antibody arm paired with a high affinity anti-CD19 arm in a fully human bispecific format (kl-body) for targeted blockade of the CD47-SIRPα interaction on B cells. Results: Using human B cell lymphoma lines the kl-body demonstrated selective binding, targeted blockade of the CD47-SIRPα interaction and cell killing through Fc region-dependent effector functions such as ADCP (antibody-mediated cellular phagocytosis) and ADCC (antibody-dependent cellular cytotoxicity). Studies using PBMCs derived from CLL patient samples demonstrated a correlation between poor prognostic markers and expression of CD47, and efficient phagocytosis in the presence of the kl-body. Moreover, the kl-body demonstrated enhanced phagocytosis of primary acute lymphoblastic leukemia (ALL) B cells and bone marrow aspirates both unresponsive to anti-CD20 mediated ADCP by rituximab. kl-bodies demonstrated favorable elimination kinetics and no effects on hematological parameters (e.g., red blood cell and platelet counts) in non-human primates. Conclusions: Our lead kl-body, NI-1701, is in preclinical enabling studies with an anticipated entry into clinical trials in 2016.

Advances in Bispecific Antibodies Engineering: Novel Concepts for Immunotherapies

Bispecific antibodies are emerging as novel approach for immunotherapies by combining two antigen-recognizing elements into a single construct that is able to simultaneously bind to two distinct targets. Bispecific antibodies can be applied to recruit immunological effector cells for killing of tumor cells or to simultaneously block two signaling pathways or cytokines. Subsequently, they have prompted significant interest for a number of therapeutic applications, both in cancer and in other indications. The growing interest in therapeutic antibodies along with the rapid progress in antibody engineering have yield a multitude of bispecific antibodies formats and derived molecules that differ in size, shape and function. Presently, two major classes of bispecific antibodies are most widely studied: immunoglobulin-like and small single chain Fv (scFv)-based bispecific antibodies. This review summarizes selected key methods used to generate bispecific antibodies, reports new concepts developed for immunotherapy and discusses their potential development and therapeutic benefits.

Immunotherapy with Long-Lived Anti-CD20 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human B Cell Lines and of Circulating and Lymphoid B Cells in Monkeys: A Potential Therapy for B Cell Lymphomas and Leukemias

CD20 is highly expressed on normal and malignant B cells, and is a well-established target of antibody therapeutics for B cell leukemias and lymphomas. However, a limitation of approved anti-CD20 antibodies such as rituximab, ofatumumab, and obinutuzumab is that they are unable to stimulate T cell-mediated killing of CD20+ B cells. To exploit the potent activity intrinsic to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed novel humanized bispecific antibodies that bind to both CD20+ B cells and CD3+ T cells. Such antibodies act via a mechanism known as "redirected T cell-cytotoxicity" (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).We first generated a series of affinity-optimized anti-CD20 × anti-CD3 bispecific antibodies and screened these using RTCC assays in which bispecifics stimulated killing of the CD20+ Ramos B cell line by purified human T cells. From this cell-based screen, we selected two candidates for further study in animal models. The bispecific antibodies XmAb13676 and XmAb13677 have identical T cell-engaging domains with 8 nM affinity for human CD3. XmAb13676 stimulated T cell killing of Ramos cells with an EC50 of ~53 ng/ml (~420 pM), while XmAb13677 (with higher affinity for CD20) had an EC50 of ~2 ng/ml (~16 pM).To assess in vivo half-life, we next dosed mice with 2 mg/kg of XmAb13676 or XmAb13677. In marked contrast to non-Fc domain-containing bispecific antibody formats, XmAb13676 and XmAb13677 had an extended serum half-life in mice of 6.7 and 6.6 days, respectively. Because these bispecifics were optimized for binding to human CD20 and CD3 targets and do not crossreact with mouse antigens, we evaluated efficacy in cynomolgus monkeys. We treated 3 monkeys per group with a single dose of XmAb13676 or XmAb13677 at 0.03, 0.3, or 3 mg/kg. Within 4 hr after dosing, T cells were strongly activated and stimulated depletion of over 97% of circulating CD40+ B cells, with the two 3 mg/kg groups showing greatest depletion. B cells continued to decrease for 24 to 48 hr after dosing, with the high-dose groups remaining at baseline levels for the duration of the study (29 days). CD4+ and CD8+ T cells in the circulation were activated immediately after treatment with XmAb13676 and XmAb13677, and this state was sustained for over 48 hr, as measured by greatly increased levels of the activation markers CD25 and CD69. Bispecific antibodies also induced rapid margination of CD4+ and CD8+ T cells from the circulation, with blood T cell populations returning to baseline from 2 to 7 days after dosing. Notably, CD40+ cells in lymph nodes and in bone marrow were depleted by over 90% at all doses, and at the higher dose levels, these B cell populations had not recovered by 29 days after treatment.Our results demonstrate that bispecific antibodies can recruit and activate T cells to efficiently kill CD20+ B cells not only in the circulation but also in the more resistant reservoir of lymphoid organs. These preclinical data in cynomolgus monkeys provide a rationale for clinical assessment of anti-CD20 × anti-CD3 bispecific antibodies in patients with CD20+ B cell leukemias and lymphomas. DisclosuresChu:Xencor: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Immunotherapy with Long-Lived Anti-CD123 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human AML Cell Lines and of CD123+ Cells in Monkeys: A Potential Therapy for Acute Myelogenous Leukemia

CD123 (IL-3 receptor alpha) is highly expressed on acute myeloid leukemia stem cells and blasts, and represents a promising target of antibody therapies for AML. Anti-CD123 antibodies such as CSL-362 and KHK2823 are currently in clinical development; however, a limitation of these molecules is that they are unable to stimulate T cell-mediated killing of CD123+ AML cells. To exploit the potent activity inherent to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed a novel bispecific antibody that recruits T cells to attack CD123+ AML stem and blast cells. Such antibodies act via a mechanism known as “redirected T cell-cytotoxicity” (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).We first generated a library of humanized and affinity-optimized anti-CD123 × anti-CD3 bispecific antibodies and assessed their potency using RTCC assays, in which bispecifics stimulated killing by human T cells of the CD123+ AML cell lines KG-1a and TF-1. From this cell-based screen, we selected the bispecific antibody XmAb14045 for testing in animal models. This antibody has 0.1 nM affinity for human CD123, and a T cell-engaging domain with 8 nM affinity for human CD3. XmAb14045 stimulated T cell-mediated killing of KG-1a and TF-1 cells with an EC50 < 1 ng/ml (8 pM). In contrast, XmAb14045 had no cytotoxic activity against the CD123− Raji B cell line, demonstrating target specificity of the T cells.XmAb14045 had a prolonged serum half-life in mice of 6.2 days, in marked contrast to non-Fc domain-containing bispecific formats. Because this antibody was optimized for human CD123 and CD3 targets and does not crossreact with mouse antigens, we evaluated efficacy in cynomolgus monkeys. We treated 3 monkeys per group with a single dose of XmAb14045 at 0.01, 0.1, or 1 mg/kg. We quantified CD123+ cell numbers, including basophils and plasmacytoid dendritic cells (pDC) as CD123+ surrogate populations for AML stem and blast cells. Within 4 hours of dosing, XmAb14045 strongly activated T cells and stimulated depletion of over 99% of circulating CD123+ cells within 1 hr, particularly at the 0.1 and 1 mg/kg doses. Basophil and pDC counts fell to baseline within 4 hr and remained low for several weeks. Circulating CD4+ and CD8+ T cells were activated immediately after dosing and this was sustained for 48 hr, as measured by markedly increased levels of the activation markers CD25 and CD69. Notably, XmAb14045 induced rapid margination of CD4+ and CD8+ T cells from the circulation, with blood T cell populations returning to baseline within several days. Bone marrow CD123+ cells were depleted by over 95% at all doses, and these cell populations had not recovered by 8 days after treatment.Our results demonstrate that bispecific antibodies can recruit and activate T cells to efficiently kill CD123+ cells not only in the circulation but also in the bone marrow. Results in monkeys also suggest that changes in basophil and/or plasmacytoid dendritic cell numbers are readily quantifiable in peripheral blood, and thus these populations may serve as biomarkers for clinical efficacy. Our preclinical data provide a rationale for clinical assessment of anti-CD123 × anti-CD3 bispecific antibodies in patients with acute myeloid leukemia. DisclosuresChu:Xencor: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

A Novel CD20xCD3 Bispecific Fully Human Antibody Induces Potent Anti-Tumor Effects Against B Cell Lymphoma in Mice

Bispecific antibodies that redirect effector T cells to kill tumor cells have shown considerable promise in both pre-clinical and clinical studies. However, these bispecific formats can have short half-lives necessitating constant infusion of the molecules into patients. We report here on a novel full-length human IgG CD20xCD3 bispecific antibody (REGN1979) that targets CD20 expressed on normal and malignant B cells and CD3 expressed on T cells in humans and cynomolgus monkeys. Our results demonstrate CD20-target cell-dependent activation and cytokine release by T cells, and efficient redirected T cell lysis of target tumor cells. Raji B cell lymphomas grown as tumors in NOD SCID IL2R gamma deficient (NSG) mice and co-implanted with human peripheral blood mononuclear (PBMC) cells were completely inhibited when treated at the time of implantation with a low dose (0.004 mg/kg; 2x/week) of REGN1979. As expected, T cells were required for this tumor inhibition, since treatment in the absence of human T cells was not effective. REGN1979 bispecific antibody also demonstrated potent activity against other tumor cells expressing CD20, as it significantly delayed CD20-transduced B16F10.9 tumor growth in immune-competent mice. Most importantly, REGN1979 induced dramatic tumor regression in large advanced (500-900 mm3) Raji tumors, associated with long-lasting tumor control. The tumor-infiltrating lymphocytes (TILs) in B cell lymphomas in these untreated NSG mice were found to express the inhibitory receptors Tim-3 and PD-1 and were the predominant fraction of T cells in the tumors and in the circulation. T cells in mice treated with REGN1979 showed decreased Tim-3 and PD-1 expression in the circulation accompanied by complete tumor regression. In further studies, REGN1979 (dosed at 0.4 mg/kg; 2x/week) was superior to rituximab therapy (dosed at 8 mg/kg; 5x/week) and comparable to the CD19xCD3 BiTE (dosed at 0.5 mg/kg; 5x/week) in suppressing established Raji tumors (200-400mm3). Pre-clinical studies in cynomolgus monkeys to assess activity of the bispecific antibody for depleting B cells in circulation and various lymphoid organs showed that a single injection of REGN1979 (0.1 mg/kg) was more potent at depleting CD20+ B cells in the mesenteric lymph nodes than a high dose of rituximab (30 mg/kg). In separate studies, REGN1979 was also found to have a long half-life (>14 days) in the circulation of monkeys following depletion of B cells. These studies show potent activity of a new class of fully human bispecific antibodies for treating tumors, and support clinical testing of REGN1979 in patients with CD20+ cancers. [Display omitted] DisclosuresVarghese:Regeneron Pharmaceuticals: Employment, Equity Ownership. Menon:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rodriguez:Regeneron Pharmaceuticals: Employment, Equity Ownership. Haber:Regeneron Pharmaceuticals: Employment, Equity Ownership. Olson:Regeneron Pharmaceuticals: Employment, Equity Ownership. Duramad:Regeneron Pharmaceuticals: Employment, Equity Ownership. Oyejide:Regeneron Pharmaceuticals: Employment, Equity Ownership. Smith:Regeneron Pharmaceuticals: Employment, Equity Ownership. Thurston:Regeneron Pharmaceuticals: Employment, Equity Ownership. Kirshner:Regeneron Pharmaceuticals: Employment, Equity Ownership.

Immunotherapy with Long-Lived Anti-CD38 × Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human Myeloma Cell Lines and CD38+ Cells in Monkeys: A Potential Therapy for Multiple Myeloma

CD38, being highly expressed on malignant plasma cells, is an attractive target of new therapies for multiple myeloma (MM). Several anti-CD38 antibodies including daratumumab are in clinical development; however, a limitation of such monospecific antibodies is their inability to stimulate cytotoxic T cell killing of myeloma cells. To exploit the potent mechanism of T cell immunotherapy yet preserve the favorable drug and dosing properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via a "redirected T cell-cytotoxicity" (RTCC) mechanism because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).We first generated a library of humanized and affinity-optimized anti-CD38 × anti-CD3 antibodies and measured their potency using RTCC assays in which antibodies stimulated killing of the human MM cell line RPMI8226 by human T cells. From this screen, we selected two candidates for further assessment. XmAb13243 and XmAb13551 have 21 and 0.2 nM affinities, respectively, for human CD38, and have identical T cell-engaging domains with 8 nM affinity for human CD3. XmAb13243 stimulated RTCC with an EC50 of 2.5 ng/ml (20 pM) after 24 hr, while XmAb13551 had an EC50 of ~100 pg/ml (~1 pM).In contrast to bispecific formats lacking an Fc domain, XmAb13243 and XmAb13551 had long half-lives in mice of ~7.6 and 8.3 days, respectively. Because these bispecifics were optimized for human CD38 and CD3 binding and do not crossreact with mouse antigens, we next evaluated efficacy in immunodeficient SCID mice engrafted with human PBMCs. In this model, engrafted human B cells differentiate into CD38+ plasma cells, which produce high levels of human Ig. Bispecific antibodies dosed at 0.2, 1, and 5 mg/kg, 7 and 15 days after engraftment, suppressed human IgG2, IgM, and IgE to below detectable levels by Day 14 (> 50-fold for IgG2, > 1,000-fold for IgM, and > 80-fold for IgE). Daratumumab at 5 mg/kg was markedly less potent than bispecifics, reducing IgG2 by 2-fold, IgM by 6-fold, and IgE by 3-fold. The control bispecific anti-RSV × anti-CD3 (which binds to T cells but not to CD38+ cells) had no effect on IgG2, IgM, or IgE levels. To investigate activity against an immune response requiring production of new human plasma cells, mice were vaccinated with tetanus toxoid 8 days after engraftment. Anti-CD38 × anti-CD3 bispecifics suppressed human anti-tetanus antibody titers to baseline (> 100-fold), while daratumumab suppressed titers by only 2-fold.We next assessed efficacy in cynomolgus monkeys. Unlike daratumumab, which does not crossreact with monkey CD38, XmAb13243 and XmAb13551 bind to both CD38 and CD3 in monkeys (23 and 0.3 nM, respectively, to CD38, and 6 nM to CD3 for both). We treated monkeys with a single dose of XmAb13243 or XmAb13551 at 2, 5, and 20 μg/kg. T cells were activated within 1 hr, as measured by dramatic increases in CD25 and CD69 activation markers. Within 8 hr, T cells depleted circulating CD38+ cells by > 95% at the 20 μg/kg dose.Our results demonstrate that XmAb13243 and XmAb13551 effectively recruit T cells to kill CD38+ cells in vivo. Our preclinical data in monkeys and humanized mice provide a rationale for clinical testing of anti-CD38 × anti-CD3 bispecific antibodies in patients with multiple myeloma and other CD38+ malignancies. DisclosuresChu:Xencor: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity.

Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.

Abstract 656: A bispecific HER2/CD3 targeting FynomAb with excellent tumor killing and favorable pharmacokinetic properties

Abstract There is increasing evidence that T cells are able to control tumor growth and increase survival of cancer patients. However, tumor-specific T cell responses are difficult to mount and sustain in cancer patients, and are limited by numerous immune escape mechanisms of tumor cells. A promising approach in the immunotherapy of cancer is to engage T cells to target tumor cells using bispecific therapeutics targeting a tumor-associated surface antigen and CD3e on T cells. Such therapeutics elicit T cell mediated lysis of tumor cells independent of T cell specificity. Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles. We have generated a novel bispecific FynomAb which can simultaneously bind HER2 on tumor cells and CD3 on T cells. The bispecific HER2/CD3 targeting FynomAb COVA420 potently redirected T cells to HER2 expressing tumor cells showing picomolar tumor cell lysis activity. We present for the first time that a tailored architecture of the FynomAb leads to optimal tumor cell killing properties. The activity of COVA420 was found to be highly specific, as no lysis of cells was observed in the absence of HER2 expression. In addition, COVA420 demonstrated an antibody-like pharmacokinetic profile in mice. We anticipate that the increased half-life of T cell recruiting FynomAbs compared to other bispecific formats translates into a significant benefit for patients, since it circumvents the need for continuous infusion and prolongs the intervals between successive treatments. In summary, bispecific T cell recruiting FynomAbs represent a novel platform technology to redirect T cells to tumor cells with optimal biophysical properties, long-half lives and tailored architectures. Citation Format: Ulrich Wuellner, Fabian Buller, Kristina Klupsch, Simon Brack, Irene Zbinden, Roger Santimaria, Isabella Attinger-Toller, Susann König-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2/CD3 targeting FynomAb with excellent tumor killing and favorable pharmacokinetic properties. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2014-656

A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

Production of stable bispecific IgG1 by controlled Fab-arm exchange

The manufacturing of bispecific antibodies can be challenging for a variety of reasons. For example, protein expression problems, stability issues, or the use of non-standard approaches for manufacturing can result in poor yield or poor facility fit. In this paper, we demonstrate the use of standard antibody platforms for large-scale manufacturing of bispecific IgG1 by controlled Fab-arm exchange. Two parental antibodies that each contain a single matched point mutation in the CH3 region were separately expressed in Chinese hamster ovary cells and manufactured at 1000 L scale using a platform fed-batch and purification process that was designed for standard antibody production. The bispecific antibody was generated by mixing the two parental molecules under controlled reducing conditions, resulting in efficient Fab-arm exchange of >95% at kg scale. The reductant was removed via diafiltration, resulting in spontaneous reoxidation of interchain disulfide bonds. Aside from the bispecific nature of the molecule, extensive characterization demonstrated that the IgG1 structural integrity was maintained, including function and stability. These results demonstrate the suitability of this bispecific IgG1 format for commercial-scale manufacturing using standard antibody manufacturing techniques.

Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Abstract 1243: Bispecific TandAbs: a safe and potent platform for T cell-mediated killing of CD19+ cells.

Abstract CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for therapeutic antibodies that treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The TandAb technology enables the development of bispecific antibody therapeutics for oncology indications that harness the cytotoxic nature of T cells. The CD3 RECRUIT-TandAb AFM11, a human bispecific tetravalent antibody with two binding sites for both CD3 and CD19, exhibits highly potent cytotoxic activity and possesses pharmacokinetic properties that are considerably improved relative to alternative bispecific formats. In vitro assays demonstrate high affinity binding of AFM11 to CD3+ and CD19+ cells leading to higher potency and efficacy of T cell recruitment and target cell lysis relative to alternative bispecific antibody formats(tandem scFv, diabody). Cytotoxicity assays show that AFM11 mediates target cell lysis by CD4+ and CD8+ T cells with EC50 values in the low to sub-picomolar range that is independent of the target cell CD19 density. AFM11 exhibits nearly identical cytotoxicity across a broad range of effector-to-target ratios, from 5:1 to 1:5, and facilitates serial target cell killing by T cells. Binding of antibodies to CD3 on T cells may elicit devastating cytokine release if it results in systemic rather than target-specific activation of T cells. Indeed, the anti-CD3 antibody OKT3 exhibits high toxicity in humans due to non-specific activation of T cells. Thus, in vitro assays were used to assess AFM11 off-target activity. AFM11 activates T cells inly in the presence of CD19+ cells, resulting in induction of CD69 and CD25 expression and the on-target release of IFN-γ, TNF-α, IL-2, and IL-6. However, depletion of CD19+ cells from PBMC cultures completely abrogates AFM11-mediated activation marker expression, T cell proliferation, and cytokine release, and supports strict CD19+ target-dependent T cell activation. Conversely, OKT3, produced cytokine release and T cell proliferation in all cultures except purified T cells. AFM11-mediated target cell lysis is specific for and restricted to CD19+ target cells since CD19− bystanders were not lysed in assays with mixed target populations. Binding of AFM11 to CD3 also did not induce anergy since its incubation with T cells for several days did not inhibit their potent cytotoxicity. AFM11 demonstrated a robust dose-dependent inhibition of growth of Raji tumor xenografts in NOD/scid mice reconstituted with human PBMC. Moreover, a single dose of AFM11 resulted in tumor growth inhibition similar to that produced by 5 daily injections. Altogether, these data suggest that AFM11 is a highly efficacious novel drug candidate for the treatment of hematological malignancies with advantages in both safety profile and anticipated dosing regimen. Citation Format: Eugene A. Zhukovsky, Uwe Reusch, Carmen Burkhardt, Stefan Knackmuss, Ivica Fucek, Markus Eser, Fionnuala McAleese, Kristina Ellwanger. Bispecific TandAbs: a safe and potent platform for T cell-mediated killing of CD19+ cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1243. doi:10.1158/1538-7445.AM2013-1243

Current strategies in antibody engineering: Fc engineering and pH‐dependent antigen binding, bispecific antibodies and antibody drug conjugates

Antibody engineering technologies are constantly advancing to improve the clinical effectiveness of monoclonal antibodies (mAbs). Effector functions may be modified by engineering the Fc region, for example to improve or reduce binding to Fc gamma receptors (FcγRs) or complement factors. Other examples for Fc engineering include modification of the half-life of immunoglobulin G (IgG); various studies have shown that half-life can be prolonged by increasing the affinity of Fc for the Fc neonatal receptor (FcRn). Furthermore, engineered pH-dependent antigen binding can be applied to enhance the recycling of IgG via FcRn, enabling binding to additional target molecules. Since bispecific approaches may elicit desired effects on disease targets, a variety of bispecific formats have been developed, including variants that structurally mimic IgG. Finally, antibody-drug conjugates (ADCs) create new opportunities for treatment of certain diseases. Advances in antibody generation, selection of highly cytotoxic molecules and production of stable linkers have paved the way to the development of many ADCs that can be tested in clinical trials. This review covers current antibody engineering strategies for the modification of therapeutic antibodies in the areas of Fc engineering and pH-dependent antigen binding, bispecific antibodies and ADCs.

Smarter Drugs

Antibodies capable of targeting more than one antigen are envisioned to expand therapeutic efficacy in complex disease settings. Several strategies have been developed to achieve multiple targeting, including antibody mixtures and bispecific formats. In recent years, several dual- and pan-specific antibodies have been described and represent an alternative approach. These antibodies bind to different targets using a single antigen-combining site while maintaining high affinity and specificity, thus challenging the 'one antibody, one antigen' dogma. Despite certain drawbacks, the simple IgG format of this drug class enables rapid progression into the clinic.

A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens

Bispecific antibodies based on full-length antibody structures are more optimal than fragment-based formats because they benefit from the favorable properties of the Fc region. However, the homodimeric nature of Fc effectively imposes bivalent binding on all current full-length bispecific antibodies, an attribute that can result in nonspecific activation of cross-linked receptors. We engineered a novel bispecific format, referred to as mAb-Fv, that utilizes a heterodimeric Fc region to enable monovalent co-engagement of a second target antigen in a full-length context. mAb-Fv constructs co-targeting CD16 and CD3 were expressed and purified as heterodimeric species, bound selectively to their co-target antigens, and mediated potent cytotoxic activity by NK cells and T cells, respectively. The capacity to co-engage distinct target antigens simultaneously with different valencies is an improved feature for bispecific antibodies with promising therapeutic implications.