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The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fcγ receptors (FcγR). Although an early goal has been enhanced FcγRIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as FcγRIIa. Here, we describe a set of engineered Fc variants with diverse FcγR affinities, including a novel substitution G236A that provides selectively enhanced binding to FcγRIIa relative to FcγRIIb. Variants containing this substitution have up to 70-fold greater FcγRIIa affinity and 15-fold improvement in FcγRIIa/FcγRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual FcγR to effector functions mediated by NK cells and macrophages. These experiments show that FcγRIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor FcγRIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers. [Mol Cancer Ther 2008;7(8):2517–27]

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Affinity measurements and antibody panels

Supplementary Fig. S1 from Optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor cells

Purpose:Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell–redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell–recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma.Experimental Design:Anti-CD38/CD3 XmAb T-cell–recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties.Results:AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow–invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented.Conclusions:These findings support the clinical development of AMG 424, an affinity-optimized T-cell–recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

Supplementary Figure 2 from Potent In vitro and In vivo Activity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia

Supplementary Figure 1 from Potent In vitro and In vivo Activity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia

Supplementary Figure Legends 1-3 from Potent In vitro and In vivo Activity of an Fc-Engineered Anti-CD19 Monoclonal Antibody against Lymphoma and Leukemia

T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve optimal activation. Solid tumor cells lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling at the T cell/tumor cell interface could enhance anti-tumor activity. We developed a CD28 bispecific platform that allows for the rapid generation of tumor-associated antigen (TAA) x CD28 bispecific antibodies that conditionally provide CD28 costimulation only in the presence of TAA and TCR engagement. Here, we present results for a set of bispecific antibodies with broad applicability across a range of solid tumors, including CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28. We developed optimized CD28 scFv and Fabs that cover a range of affinities and are only agonistic in the context of TCR engagement. A matrix of bispecifics pairing these CD28 binders with CEACAM5, Trop-2, STEAP1, and mesothelin antibodies of varying affinities and epitopes were generated using Xencor’s XmAb® bispecific antibody platform. Activities of these bispecifics were assessed in vitro by measuring T cell proliferation, cytokine production, and cytotoxicity in co-cultures of human cancer cell lines mixed with primary human T cells. CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28 bispecifics enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity only in concert with TCR engagement. IHC was used to compare TAA expression on cancer cell lines versus that found on cancer tissues, allowing categorization of cell lines as high density (tumor surrogate) or low density (normal tissue surrogate). CD28 bispecifics were identified with selective potency on high versus low expressing cell lines, suggesting a favorable therapeutic index. CD28 bispecific antibodies co-targeting CEACAM5, Trop-2, STEAP1, and mesothelin show promising activity and warrant further development across a range of solid tumors. Citation Format: Matthew A. Dragovich, Viralkumar Davra, Matthew S. Faber, Yoon Kyung Kim, Alex Nisthal, Veronica G. Zeng, Jonathan Jacinto, Juan E. Diaz, Thuy Truong, Jing Qi, Kendra N. Avery, Rumana Rashid, Sung-Hyung Lee, Seung Y. Chu, Christine Bonzon, Ruschelle Love, Matthew J. Bernett, James A. Ernst, Rena Bahjat, Norman J. Barlow, John R. Desjarlais, Michael Hedvat, Gregory L. Moore. Tumor-specific CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors [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 2983.

Bispecific antibodies that recruit and redirect T cells to attack tumor cells have tremendous potential for the treatment of various malignancies. In general, this class of therapeutics, known as CD3 bispecifics, promotes tumor cell killing by cross-linking a CD3 component of the T cell receptor complex with a tumor-associated antigen on the surface of the target cell. Importantly, this mechanism does not rely on a cognate interaction between the T cell receptor and a peptide:HLA complex, thereby circumventing HLA (human leukocyte antigen) restriction. Hence, CD3 bispecifics may find a key role in addressing tumors with low neoantigen content and/or low inflammation, and this class of therapeutics may productively combine with checkpoint blockade. A wide array of formats and optimization approaches has been developed, and a wave of CD3 bispecifics is proceeding into human clinical trials for a range of indications, with promising signs of therapeutic activity.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross–SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection. CA extern

The IL2Rβ/γc binding human cytokines IL2 and IL15 aid in the activation, proliferation, and survival of T and NK cells, and their therapeutic potential has been well established in animal models and human trials. However, therapeutic approaches utilizing these cytokines have suffered from low tolerability, fast clearance, and limited therapeutic window due to extensive activity on peripheral lymphocytes. Conversely, higher drug concentration and prolonged exposure are desirable to allow lymphocyte activation and proliferation at the tumor site, but this can be difficult to achieve due to dose-limiting toxicities associated with this axis. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, which is presented in trans to lymphocytes expressing IL2Rβ/γc. We hypothesized that we could selectively target tumor-reactive TILs by combining a reduced potency IL15/IL15Rα heterocomplex with an immune checkpoint(CP)-targeting arm to bias binding and activation to CP-positive TILs, potentially improving therapeutic index. Lymphocyte activation gene 3 (LAG3) was chosen as the CP targeting-arm due to its frequent co-expression with PD1, bias to CD8+ T cells, ability to easily combine with anti-PD1 agents, and recent promising results with anti-LAG3 agents in the clinic. First, potency-reduced IL15/IL15Rα were created by engineering amino acid substitutions in IL15 - at the IL2Rβ/γc interface - that reduced in vitro potency by at least 1000-fold. We then designed LAG3 x IL15 fusion proteins containing single-chain IL15/IL15Rα and LAG3-targeting arms attached to a heterodimeric-Fc region, relying on targeting avidity to recover potency on LAG3+ cells. In vitro proliferation of lymphocytes in human PBMCs, stimulated with sub-optimal concentrations of anti-CD3 to induce LAG3 expression, was monitored by CFSE dilution or by counting Ki67+ cells after incubation with LAG3 x IL15 for 4 days. In vivo activity was evaluated using humanized mouse models by measuring the extent of human leukocyte expansion. Lead LAG3 x IL15 were evaluated for pharmacodynamic activity, pharmacokinetics, and tolerability in non-human primates. LAG3 x IL15 showed >500-fold selectivity compared to a non-targeted IL15 in an in vitro proliferation assay of lymphocytes stimulated for induced LAG3 expression. In vitro potency was greatest on effector memory CD8 T cells. LAG3 x IL15 were 3-fold more potent on CD8 T cells compared to CD4 T cells and had very weak activity on NK cells, consistent with minimal LAG3 expression on this population. In mouse models, treatment with LAG3 x IL15 promoted significantly increased numbers of T cells. Moreover, LAG3 x IL15 combined productively with anti-PD1 to promote additional T cell expansion. These results demonstrate that LAG3 x IL15 show a promising profile of selective IL15 delivery to TIL with minimal peripheral activity. Citation Format: Matthew J. Bernett, Suzanne Schubbert, Michael Hackett, Lukasz J. Ochyl, Lizett E. Scott, Christine Bonzon, Rumana Rashid, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Connie Ardila, Umesh S. Muchhal, Norman J. Barlow, Rena Bahjat, John R. Desjarlais. LAG3-targeted IL15/IL15Rα-Fc (LAG3 x IL15) fusion proteins for preferential TIL expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2080.

Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. After processing of the inactive precursor within monocytes and macrophages, mature IL18 can promote the expansion, survival, and cytotoxicity of T and NK cells expressing its receptor subunits IL-18Rα (IL18R1) and IL18Rβ (IL18RAP). Preclinical studies with recombinant IL18 have demonstrated anti-tumor activity in animal models, including impressive synergy with both immune checkpoint inhibitors and CAR-T therapy. However, clinical development exhibited poor pharmacokinetics and an overall lack of efficacy despite heavy dosing. IL18 participates in a negative feedback loop with a very high affinity natural inhibitor, IL18 binding protein (IL18BP), whose expression is induced by IFNγ. As IL18BP upregulation was observed in early phase clinical trials, it likely limited the efficacy of recombinant IL18. Here, we have engineered next generation IL18 cytokines that aim to improve on the poor PK of WT and contend with IL18BP induction. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Following that principle, we generated monovalent IL18-Fc fusions upon our XmAb® heterodimeric Fc platform and introduced substitutions that could modulate IL18 stability, affinity toward the IL18 heterodimeric receptor, and affinity toward IL18BP. Initially, IL18-Fc was difficult to produce but yields improved to typical bispecific antibody levels after stabilization. An engineered disulfide bridge increased the WT thermal denaturation temperature from 45 °C to 65 °C, and demonstrated a significant improvement in serum clearance in mouse PK experiments. Variants at IL18 positions along the receptor and IL18BP interfaces were explored in vitro by measuring PD-L1 induction on KG-1 cells, with and without a high concentration of IL18BP. Recombining a select number of hits generated a potency series with variants exhibiting over a 2,000-fold reduction in PD-L1 induction potency as compared to WT IL18-Fc. Importantly, we identified variants that no longer detectably bound IL18BP, relieving natural inhibition of our engineered IL18-Fc. In vivo immune-mediated inflammation was explored in human PBMC engrafted mouse models of graft versus host disease (GvHD). We observed potency-dependent exacerbation of GvHD, with corresponding dramatic increases in the numbers and activation of T and NK cells as compared to a human PBMC only control. Analysis of serum from the study revealed up to 100-fold increases in IFNγ levels. Our engineered, reduced-potency IL18-Fc cytokines demonstrate robust inflammation activity in vivo, improved pharmacokinetics in mice, and insensitivity to IL18BP inhibition. Citation Format: Alex Nisthal, Sung-Hyung Lee, Christine Bonzon, Ruschelle Love, Kendra N. Avery, Rumana Rashid, Panida Lertkiatmongkol, Nicole Rodriguez, Hanh Nguyen, Araz Eivazi, Sher Karki, Norm Barlow, Seung Y. Chu, Greg L. Moore, John R. Desjarlais. Engineered IL18 heterodimeric Fc-fusions featuring improved stability, reduced potency, and insensitivity to IL18BP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3515.

Background TGFβ production by solid tumors and their microenvironment is a major mechanism used by tumors to avoid immunosurveillance. Blockade of TGFβ has been shown to promote an anti-tumor response; however, systemic blockade of TGFβ has also been associated with toxicity. We hypothesized that a PD1 x TGFβR2 bispecific antibody could selectively block the suppressive activity of TGFβ on tumor T cells and enhance their anti-tumor activity while avoiding the toxicity associated with systemic blockade. Methods We engineered bispecific antibodies that simultaneously engage PD1 and TGFβR2 using Xencor’s XmAb platform. The anti-TGFβR2 arm was tuned for optimal activity by introducing affinity-modulating amino acid substitutions. The activity of PD1 x TGFβR2 bispecifics was evaluated in vitro using a signaling assay to measure phosphorylated SMAD (pSMAD) by flow cytometry with exogenous TGFβ in unactivated and activated PBMC. In vivo activity was evaluated by monitoring the engraftment of human PBMC in NSG mice (huPBMC-NSG). Anti-tumor activity was assessed in huPBMC-NSG mice engrafted with established human cancer cell lines. Antibodies against other T cell targets were also incorporated into TGFβR2 bispecifics, and similarly evaluated in vitro and in vivo. Results PD1 x TGFβR2 bispecifics were confirmed to bind PD1 and block binding of TGFβ to TGFβR2. In vitro, we found that T cells from activated, serum-deprived PBMC exhibited robust induction of pSMAD in response to TGFβ, and PD1 x TGFβR2 bispecifics selectively inhibited pSMAD induction in PD1-positive T cells as demonstrated by over a 100-fold potency increase compared to an untargeted anti-TGFβR2 control. Additionally, we saw an enhancement of potency when evaluating blocking activity in activated (PD1-high) vs. unactivated (PD1-low) T cells. Similar selectivity was measured when comparing inhibition of pSMAD induction for activated T cells versus other PD1-negative, TGFβ-responsive immune cells. Intriguingly, TGFβR2 bispecifics incorporating antibodies against other T cell targets allowed for the targeting of a broader population of T cells while still conferring potent selectivity against target-negative cells. In vivo, treatment of huPBMC-NSG mice with TGFβR2 bispecifics promoted superior T cell engraftment and combined additively with PD1 blockade. Furthermore, TGFβR2 bispecific treatment of huPBMC-NSG mice containing established MDA-MB-231 triple-negative breast cancer tumors promoted an anti-tumor response that was also augmented with PD1 blockade. Conclusions Multiple PD1 x TGFβR2 bispecifics were engineered to selectively block TGFβR2 on PD1-positive T cells and evaluated in vitro and in vivo. Compelling activity, including additivity with PD1 blockade, suggests that clinical development is warranted for the treatment of human malignancies.

Background Metastatic castration-resistant prostate cancer (mCRPC) and Ewing sarcoma (EWS) are diseases for which immune therapies could potentially provide benefit. STEAP1 (Six Transmembrane Epithelial Antigen of the Prostate 1) is a cell surface protein with elevated expression in mCRPC 1 and EWS.2 Methods We designed AMG 509, a novel, half-life extended, STEAP1 x CD3 XmAb® 2+1 bispecific antibody to induce T cell-mediated cytotoxicity against STEAP1-expressing cancer cells. AMG 509 contains two identical anti-STEAP1 Fab domains, an anti-CD3 scFv domain, and an effectorless Fc domain that extends serum half-life. We characterized STEAP1 expression in normal and tumor tissues by immunohistochemistry, and we assessed the pharmacological properties of AMG 509 including binding, T cell-mediated redirected lysis, and in vivo antitumor activity. Results We detected high STEAP1 surface expression on 80% of primary prostate tumors (n=88), 89% of mCRPC lesions (n=114), including 84% of mCRPC bone metastases (n=31), and 63% of EWS samples (n=35). In contrast, in normal tissues (n=72), low STEAP1 expression was detected in only six other tissues, including the normal prostate. AMG 509 bound to recombinant human CD3e with a KD of 27.6 nM, and it bound specifically to 293T cells transfected with human STEAP1 with an EC50 of 3.8 nM. AMG 509 triggered potent T cell-redirected lysis of STEAP1-positive cancer cells, with a median EC50 of 37 pM across 19 cancer cell lines that endogenously express various levels of STEAP1. AMG 509-mediated cytotoxicity was specific, as it showed no activity against prostate cancer cells in which STEAP1 was knocked out. AMG 509 was 65-fold more potent in inducing the redirected lysis of prostate cancer cells in vitro than an XmAb® molecule with a single anti-STEAP1 Fab domain. AMG 509 had greater cytotoxic activity against high STEAP1-expressing cancer cells than against low STEAP1-expressing cancer cells, and it had minimal activity against normal cells. This preferential killing of high STEAP1-expressing cells is likely driven by the avidity conferred by the dual STEAP1-binding domains, a feature that may help reduce off-target effects in the clinic. In vivo, AMG 509 induced robust anti-tumor activity in prostate cancer and EWS mouse xenograft models, with concomitant CD8+ T-cell activation and expansion in tumors. Conclusions AMG 509 is a specific, first-in-class T cell-recruiting antibody with avidity-driven activity against STEAP1-positive malignancies. AMG 509 is currently being evaluated for safety, pharmacokinetics, and efficacy in a phase 1, first-in-human study in patients with mCRPC (NCT04221542). Acknowledgements The authors acknowledge Micah Robinson, PhD of Amgen Inc. for medical writing support. Trial Registration ClinicalTrials. gov Identifier: NCT04221542 Ethics Approval All animal experimental protocols were approved by an Institutional Animal Care and Use Committee (IACUC protocol number 2015-01243) and were conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) (Amgen) or the standards of the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (IACUC protocol number 15015x) in a facility certified with an Office of Laboratory Animal Welfare (OLAW) (UTHSA). References Grunewald TGP, Ranft A, Esposito I, Silva-Buttkus P da, Aichler M, Baumhoer D, Schaefer KL, Ottaviano L, Poremba C, Jundt G, Jurgens H, Dirksen U, Richter GHS, Burdach S. High STEAP1 expression is associated with improved outcome of Ewing’s sarcoma patients. Ann Oncol 2012; 23:2185–2190. Hubert RS, Vivanco I, Chen E, Rastegar S, Leong K, Mitchell SC, Madraswala R, Zhou Y, Kuo J, Raitano AB, Jakobvits A, Saffran SC, Afar DE. STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA 1999;96:14523–14528.

Background Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life. Methods IL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting MCF-7 cells into PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time. Results IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity in the huPBMC-MCF7 model was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Conclusions Combined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to small therapeutic index associated with recombinant native IL12 or IL12-Fc agents.

Background T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve complete activation. Solid tumors often lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling could be beneficial in solid tumors. We designed tumor-associated-antigen (TAA) x CD28 bispecific antibodies that conditionally costimulate CD28 only in the presence of TAA and TCR engagement. Clinical application of this class of antibodies has potential to enhance activity of either anti-PD(L)1 antibodies or TAA x CD3 T cell engagers. Methods We designed a stability and affinity optimized anti-CD28 antibody that can be paired with TAA of choice to engage CD28 monovalently using Xencor’s XmAb 2+1 and 1+1 platforms. In vitro T cell activation with these bispecifics was measured by T cell proliferation, cytokine production, and cytotoxicity, in co-cultures of human cancer cell lines mixed with primary human CD3-stimulated T cells. In vitro activity was validated in a CMV recall assay measuring CMV+ T cell proliferation of CMV+ PBMC co-cultured with cancer cell lines ectopically treated with pp65-derived NLV-peptide. In vivo anti-tumor and T cell proliferative activity of B7H3 x CD28 bispecific antibodies were determined in tumor-bearing huPBMC-NSG mice treated simultaneously with TAA x CD3 bispecific antibody. In vivo activity of PDL1 x CD28 antibodies was determined with hCD28 KI mice inoculated with MC38 tumors expressing hPDL1-antigen. Finally, safety and tolerability of B7H3 x CD28 and PDL1 x CD28 was determined in cynomolgus monkeys. Results B7H3 x CD28 and PDL1 x CD28 antibodies enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity in concert with CD3 stimulation only in the presence of target antigen. B7H3 x CD28, alone or in combination with anti-PD1 antibody, enhanced proliferation of CMV+ T cells recognizing cancer cells loaded with pp65-derived NLV peptide. PDL1 x CD28 also enhanced CMV+ cell expansion but did not synergize with anti-PD1 antibody treatment. B7H3 x CD28 significantly enhanced in vivo anti-tumor activity of TAA x CD3 antibodies while also promoting greater T cell expansion. In hCD28 mice inoculated with MC38 tumors expressing hPDL1, PDL1 x CD28 antibody inhibited tumor growth greater than an anti-PDL1 antibody alone. B7H3 x CD28 and PDL1 x CD28 were well tolerated in cynomolgus monkeys. Conclusions B7H3 x CD28 and PDL1 x CD28 bispecific antibodies show promising anti-tumor activity and warrant further development.

The spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics or vaccines, has paralyzed the globe While

BackgroundInterleukin-12 (IL12) is a proinflammatory cytokine that induces differentiation of Th1 cells and increased cytotoxicity of T and NK cells. Stimulation by IL12 leads to production of IFNγ and an inflammatory tumor microenvironment critical for anti-tumor responses. Studies in mice revealed IL12 can dramatically shrink syngeneic tumors, however human clinical studies resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior therapeutic index (TI) in non-human primates (NHP) by reducing receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve TI.MethodsIL12 is a heterodimer of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and IL12p40 to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc was assessed in huPBMC-NSG-DKO and huCD34+ MCF7 xenograft models. Surrogate mouse potency-reduced IL12-Fc were evaluated in syngeneic tumor models. Tolerability and pharmacodynamic activity were assessed in NHP.ResultsAn IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling and IFNγ production in an MLR assay compared to wild-type IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as single-agents and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Surrogate potency-reduced IL12-Fc had improved tolerability and greater selectivity of IFNγ production in tumors compared to spleen and less production of IL10 compared to wild-type IL12-Fc. In NHP, potency-reduced IL12-Fc had superior exposure with slower, more sustained accumulation of IFNγ and IP10, and a more gradual dose-dependent peak response, as well as more sustained margination of T and NK cells compared to wild-type IL12-Fc.ConclusionsPotency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow TI associated with wild-type IL12 or IL12-Fc agents.

BackgroundNatural Killer cell Engagers (NKEs) are multifunctional molecules that target activating receptors on the surface of NK cells, bind to tumor associated antigens and engage Fc gamma receptors expressed on effector cells of the immune system. NKEs promote tumor cell lysis by redirecting NK cells to their targets, and drive activation and proliferation of NK cells. Engagement of NK cells, an effector cell population of the innate immune system, provides an opportunity to target cancers with reduced expression of MHC molecules that are less responsive to therapies targeting the adaptive immune system. Therefore, NKEs have a potential to provide an additional treatment option to patients who respond poorly to T cell tailored immunotherapies.MethodsExpanding on Xencor's XmAb bispecific Fc platform, we developed NKE molecules targeting NKG2D, an activating receptor expressed on cytotoxic immune cells, B7H3, a pan tumor antigen, while simultaneously engaging Fc gamma receptors. Functional activity of NKEs was evaluated via assessing anti-tumor cytotoxicity and activation of NK and T cells in co-culture studies with human cancer cell lines.ResultsNKEs were engineered for synergistic effects on NK cells by the simultaneous engagement of NKG2D and Fc gamma receptors. Additionally, the NKG2D variable domains were selected for their ability to provide a co-stimulatory signal to T cells in the presence of TCR-mediated signaling. Developed NKEs showed cytotoxic activity and immune cell activation in co-culture studies of human cancer cell lines with either PBMCs, T cells or NK cells. Combination of NKEs with proinflammatory cytokines, such as IL15, showed enhancement of the cytotoxic activity against tumor cells and augmented NK cell activation.ConclusionsXmAb bispecific NKEs engineered to engage innate and adaptive immunity show encouraging tumor cell killing activity and synergistic cytotoxicity in combination with proinflammatory cytokines. These data have identified several promising candidate NKEs for future in vivo efficacy studies in mouse tumor models expressing B7H3.

BackgroundTGFbeta production by solid tumors and their microenvironment is a major mechanism used by tumors to avoid immunosurveillance. Blockade of TGFbeta has been shown to promote an anti-tumor response; however, systemic blockade of TGFbeta has also been associated with toxicity. We hypothesized that a T cell-targeted TGFbR2 bispecific antibody could selectively block the suppressive activity of TGFbeta on T cells and enhance their anti-tumor activity while avoiding toxicity associated with systemic blockade.MethodsWe engineered bispecific antibodies that simultaneously engage PD1 (activated) or CD5 (pan T) and block TGFbR2 using Xencor’s XmAb® platform. The anti-TGFbR2 arm was tuned for optimal activity by introducing affinity-modulating amino acid substitutions. The activity of TGFbR2 bispecifics was evaluated in vitro using a signaling assay to measure phosphorylated SMAD (pSMAD) by flow cytometry with exogenous TGFbeta in unactivated and activated PBMC. In vivo activity was evaluated by monitoring the engraftment of human PBMC in NSG mice (huPBMC-NSG). Anti-tumor activity was assessed in huPBMC-NSG mice engrafted with established human cancer cell lines.ResultsTGFbR2 bispecifics were confirmed to bind PD1 or CD5 and block binding of TGFbeta to TGFbR2. In vitro, we found that T cells from serum-deprived PBMC exhibited robust induction of pSMAD in response to TGFbeta, and TGFbR2 bispecifics selectively inhibited pSMAD induction in target-positive T cells as demonstrated by over a 100-fold potency increase compared to an untargeted anti-TGFbR2 control. Additionally, we saw an enhancement of potency when evaluating activity in target-high T cells versus target-low or -negative immune cells. Intriguingly, CD5-targeted TGFbR2 bispecifics allowed for the targeting of a broader population of T cells compared to PD1-targeting while still conferring potent selectivity against target-negative cells. In vivo, treatment of huPBMC-NSG mice with TGFbR2 bispecifics promoted superior T cell engraftment. Furthermore, TGFbR2 bispecific treatment of huPBMC-NSG mice containing established MDA-MB-231 triple-negative breast cancer tumors promoted an anti-tumor response that was augmented with PD1 blockade.ConclusionsPD1 x TGFbR2 and CD5 x TGFbR2 bispecific antibodies were engineered to selectively block TGFbR2 on target-positive T cells and evaluated in vitro and in vivo. These observations are compelling and suggest that development of these bispecifics is warranted for the treatment of human malignancies.

Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 may help to establish an inflammatory tumor microenvironment critical for anti-tumor responses. Preclinical studies in mice revealed that native IL12 can dramatically shrink syngeneic tumors, however clinical studies in humans resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and extend half-life. IL12 is a heterodimeric protein consisting of two subunits, so we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity of human IL12-Fc were assessed in a human PBMC engrafted mouse MCF7 tumor model. Surrogate mouse IL12-Fc were evaluated in additional murine tumor models. Tolerability and pharmacodynamic activity were assessed in non-human primates. IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to native IL12-Fc. Anti-tumor activity was achieved with potency-reduced IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that potency-reduced IL12-Fc retain strong anti-tumor activity, while potentially overcoming safety and tolerability issues related to narrow therapeutic index associated with recombinant native IL12 or IL12-Fc agents. Citation Format: Matthew J. Bernett, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Katrina Bykova, Michael Hackett, Norm Barlow, Irene Leung, Duc-Hanh Nguyen, Araz Eivazi, Seung Y. Chu, Rajat Varma, Umesh S. Muchhal, John R. Desjarlais. IL12 heterodimeric Fc-fusions engineered for reduced potency exhibit strong anti-tumor activity and improved therapeutic index compared to native IL12 agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1743.

There is a large unmet need for targeted therapies to treat ovarian cancer and other solid tumors. A promising strategy is the use of T cell engaging bispecific antibodies that recruit T cells to kill cancer cells by simultaneously binding to tumor-associated antigens (TAA) on cancer cells and CD3 on T cells. Effective and safe use of these therapeutics depends on selective targeting of cancer cells over normal tissue, feasible when a TAA is more highly expressed on cancer cells versus normal tissues. Selectivity can also be improved by using a mixed valency “2 + 1” bispecific format, coupled with target affinity tuning, to achieve avid binding to the TAA. Claudin-6 (CLDN6) is a tetraspan membrane protein, a member of the claudin family of tight junction proteins, and has been identified as a promising TAA for ovarian cancer due to its high expression on ovarian and other cancer tissues and low expression on normal tissue. However, a complicating factor is that many proteins in the claudin family have high sequence identity. Most similar to CLDN6 is CLDN9, and the two proteins differ at only 3/76 residues in their extracellular loops, creating a challenge for the development of highly selective CLDN6 antibodies. We analyzed CLDN6 and CLDN9 expression using a combination of normal tissue gene expression data (GTEx), cancer cell genomics data (TCGA), and immunohistochemistry (IHC) on a panel of cancer and normal tissues. Data confirmed the tumor-specific expression pattern of CLDN6 but indicated high CLDN9 expression in normal tissues, suggesting that strong antibody selectivity for CLDN6 versus CLDN9 is critical. We created CLDN6-selective T cell engaging bispecific antibodies by starting with a highly selective monoclonal antibody humanized from a mouse hybridoma. Selectivity was further improved by engineering the CDR regions. Variant antibodies were screened for binding to transiently transfected HEK293E cells that highly expressed either CLDN6, CLDN9, or other homologous claudin family members. Selective variants were converted into the XmAb® 2 + 1 bispecific antibody format and tested in T cell-dependent cellular cytotoxicity (TDCC) assays using cancer cell lines expressing CLDN6, or HEK293E cells transfected with claudin homologs. A set of lead CLDN6 x CD3 bispecifics displaying a range of potencies on CLDN6+ cancer cell lines and high selectivity for CLDN6 over CLDN9 and other homologous claudins was identified. Tolerability and pharmacokinetics of these molecules are currently being assessed in non-human primates, and activity will be evaluated in humanized mouse models of ovarian cancer. Citation Format: Matthew S. Faber, Sung-Hyung Lee, Yoon Kyung Kim, Jing Qi, Kendra N. Avery, Duc-Hanh T. Nguyen, Rumana Rashid, Araz Eivazi, Seung Y. Chu, Juan E. Diaz, Connie Ardila, Ruschelle Love, Alex Nisthal, Norman J. Barlow, Christine Bonzon, Umesh S. Muchhal, Matthew J. Bernett, John R. Desjarlais. Bispecific claudin-6 x CD3 antibodies in a 2 + 1 format demonstrate selectivity and activity on human ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1860.

T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve complete activation. Tumor cells lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling at the T cell/tumor cell interface could enhance anti-tumor activity. We designed PDL1 x CD28 bispecific antibodies that conditionally costimulate CD28 only in the presence of PDL1 and TCR engagement. As PD(L)1 signaling has been shown to directly inhibit CD28 costimulation, this novel bispecific modality has potential to promote CD28 costimulation while simultaneously preventing the suppression of the same signal. We designed a set of stability-optimized anti-CD28 antibodies that can be paired with anti-PDL1 antibodies to engage both PDL1 and CD28 monovalently using Xencor's XmAb® 1+1 bispecific antibody platform. In vitro T cell activation with these bispecifics was measured by T cell proliferation, cytokine production, and cytotoxicity, in co-cultures of human cancer cell lines mixed with primary human CD3-stimulated T cells. In vitro activity was validated in a CMV recall assay measuring CMV+ T cell proliferation of CMV+ PBMC co-cultured with cancer cell lines ectopically treated with pp65-derived NLV-peptide. In vivo activity was determined with hCD28 humanized mice inoculated with MC38 tumors stably expressing hPDL1-antigen. Finally, safety, tolerability, and pharmacodynamics of PDL1 x CD28 were determined in cynomolgus monkeys. PDL1 x CD28 bispecifics were generated by incorporating an anti-PDL1 mAb capable of blocking PDL1-PD1 interaction and anti-CD28 scFv covering a range of affinities. Multiple PDL1 x CD28 antibodies enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity in concert with CD3 stimulation only in the presence of PDL1. PDL1 x CD28 enhanced proliferation of CMV+ T cells recognizing cancer cells loaded with pp65-derived NLV peptide. In hCD28 mice inoculated with MC38 tumors expressing hPDL1, PDL1 x CD28 inhibited tumor growth significantly greater than an anti-PDL1 antibody alone. PDL1 x CD28 was well tolerated in cynomolgus monkeys. PDL1 x CD28 bispecific antibodies show promising anti-tumor activity and warrant further development. Citation Format: Gregory L. Moore, Veronica Zeng, Juan Diaz, Christine Bonzon, Kendra N. Avery, Ruschelle Love, Matthew Dragovich, Rumana Rashid, Michael Hackett, Irene W. Leung, Jing Qi, Charles G. Bakhit, Umesh S. Muchhal, Norman J. Barlow, John R. Desjarlais, Michael Hedvat. PDL1-targeted CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1880.

Bispecific T cell engagers simultaneously bind CD3 on T cells and tumor-associated antigens to promote T cell-mediated killing of tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. Glypican 3 (GPC3), a lipid-anchored cell surface protein, is over-expressed in the majority of hepatocellular carcinoma (HCC) and a smaller subset of lung squamous cell carcinoma. It is considered an ideal target because its expression is not detected in adult tissues, and its function can promote tumor growth through the Wnt/beta-catenin pathway. Although GPC3 demonstrates good differential expression, bispecific T cell engagers are powerful immunomodulatory agents and careful tuning can improve the therapeutic window on all targets. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for GPC3 and monovalent for CD3. Reducing the affinity of GPC3 encouraged avid binding and strong killing activity only on high GPC3 expressing cell lines while minimizing reactivity on low expressing cell lines. We found modulating the CD3 affinity, either directly through mutation or indirectly by the molecular format, contributes to the selectivity of the bispecific antibodies. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding GPC3 antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. Citation Format: Alex Nisthal, Nargess Hassanzadeh-Kiabi, Kendra N. Avery, Rumana Rashid, Juan E. Diaz, Umesh S. Muchhal, Seung Y. Chu, Gregory L. Moore, Katrina Bykova, John R. Desjarlais. Affinity tuned XmAb®2+1 GPC3 x CD3 bispecific antibodies demonstrate selective activity in liver cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1831.

Interleukin-12 (IL12) is a proinflammatory cytokine produced by activated antigen-presenting cells that induces differentiation of Th1 cells and increased proliferation and cytotoxicity of T and NK cells. Stimulation of these cells by IL12 leads to production of high levels of IFNγ. These immune-stimulating aspects of IL12 are promising for cancer treatment and may help to convert immunologically suppressed “cold” tumors into inflamed “hot” tumors. Preclinical studies in mice revealed that IL12 can have a dramatic effect on shrinking syngeneic tumors, however clinical studies in humans have resulted in severe toxicity and a small therapeutic window, limiting response rates. Prior work at Xencor demonstrated that reduced-potency IL15/IL15Rα-Fc fusion proteins exhibited superior pharmacokinetics, pharmacodynamics, and safety in non-human primates through reduction of receptor-mediated clearance. Applying similar principles to IL12, we created various IL12 heterodimeric Fc-fusions (IL12-Fc) with reduced potency in order to improve tolerability, slow receptor-mediated clearance, and prolong half-life. IL12 is a heterodimeric protein consisting of two subunits, and thus we engineered IL12-Fc fusions by fusing the IL12p35 subunit to one side of a heterodimeric (and inactive) Fc domain, and the IL12p40 subunit to the other side. These Fc-fusions were tuned for optimal activity by introducing amino acid substitutions at putative receptor-interface positions and screening for reductions of in vitro potency. In vitro activity was assessed on normal human PBMCs by measuring signaling in a STAT4 phosphorylation assay and IFNγ production in a mixed-lymphocyte reaction (MLR). In vivo anti-tumor activity was assessed by engrafting pp65-MCF-7 cells into human PBMC engrafted NSG MHC class I and II double-knockout mice and by measuring tumor volume, lymphocyte activation/proliferation, and IFNγ production over time. IL12-Fc were produced with good yield and purity. An IL12-Fc potency series was created, and weaker variants had up to a 10,000-fold reduction in STAT4 signaling potency and IFNγ production in an MLR assay compared to a wild-type IL12-Fc. In vivo anti-tumor activity in the huPBMC-pp65-MCF7 model was achieved with reduced-potency IL12-Fc as a single-agent and in combination with anti-PD1, with weaker variants maintaining anti-tumor activity at higher dose levels. Analysis of peripheral lymphocytes indicated increased numbers of T and NK cells as well as activation of CD8+ T cells, as evidenced by upregulation of CD25. Increased expression of immune checkpoints including PD1 was also observed. Analysis of serum cytokine indicated up to 200-fold increases in IFNγ levels. Combined, these data indicate that reduced-potency IL12-Fc retain strong anti-tumor activity, with potential for improvement of therapeutic index. Citation Format: Rajat Varma, Ke Liu, Christine Bonzon, Rumana Rashid, Nicole Rodriguez, Nargess Hassanzadeh-Kiabi, Connie Ardila, Seung Y. Chu, Umesh S. Muchhal, John R. Desjarlais, Matthew J. Bernett. Potency-reduced IL12 heterodimeric Fc-fusions exhibit strong anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5549.

Bispecific antibodies enable therapeutic modalities inaccessible to traditional mAbs, such as T cell engagers that bind both CD3 on T cells and a target antigen on tumor cells. These agents provide synthetic immunity by expanding, activating, and redirecting T cells against a target of interest. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. Prostate cancer (PC) is one of the most prevalent cancers in men, and end stage (castration-resistant prostate cancer) has no curative treatment option. Prostate specific membrane antigen (PSMA), a type II transmembrane protein with a large extracellular domain, has long generated interest as a therapeutic target. It is highly overexpressed in PC compared to normal tissue, and its expression has been shown to correlate with malignancy. Previous attempts to target PSMA include antibody-based radiotherapy and antibody drug conjugates, which have shown some success but can be hampered by the inherent toxicity of the modality. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for PSMA and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong activity on high PSMA expressing cell lines while minimizing reactivity on low expressing cell lines, matching PSMA's differential expression pattern. To ensure biologically valid antigen densities were being considered, IHC was conducted on paraffin embedded arrays of tumor tissue, normal tissue, and cell lines. Upon matching the staining intensity between the three sample types, we identified cell lines with corresponding PSMA antigen density that could serve as proxies of tumor and normal tissue. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. Citation Format: Alex Nisthal, Matthew Dragovich, Erik W. Pong, Veronica Zeng, Michael Hedvat, Christine Bonzon, Kendra Avery, Rumana Rashid, Connie Ardila, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. Affinity tuned XmAb®2+1 PSMA x CD3 bispecific antibodies demonstrate selective activity in prostate cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5663.

Mesothelin (MSLN) is a tumor-associated antigen highly expressed in ovarian tumors. Current treatment options for ovarian cancer have only modest efficacy and there remains a large unmet need for new targeted therapies. We generated targeted T cell engager bispecific antibodies that bind MSLN on tumor cells and CD3 on T cells. The humanized and affinity-optimized MSLN x CD3 bispecific antibodies are in an XmAb® 2+1 Fab2-scFv-Fc format that bind bivalently to MSLN and monovalently to CD3. The affinity of the MSLN-targeting arms was reduced to promote avid binding to MSLN expressing cancer cell lines and to minimize reactivity on normal surrogate cell lines. We correlated the expression of MSLN on cancer cell lines to 191 biopsy cores of serous and mucinous ovarian cancer tissues by IHC to identify surrogate cell lines to test for T cell-dependent cellular cytotoxicity (TDCC) activity. MSLN-transfected A549 cells were found to stain as intensely as ovarian cancer tumors expressing high amounts of MSLN. OVCAR-8 cancer cells were found to correlate with a medium MSLN level density on ovarian cancer tumors while ASPC-1 cells represented low MSLN-expressing tumors. Finally, we correlated expression of MSLN to non-cancer adjacent tissues and identified SKOV3, HT29, MCF7, and A549 (parental) cancer cells as appropriate surrogates of normal tissues. Reduction of MSLN affinity of MSLN x CD3 XmAb® 2+1 bispecific antibodies led to preferential killing of MSLN-high target cells, with up to 45-fold more potent TDCC activity on OVCAR8 and ASPC-1 cancer cells compared to the selected normal surrogate cell lines. Engineering of selective MSLN x CD3 bispecific antibodies allows for preferential killing of cancer cells, potentially minimizing on-target/off-tumor effects that may contribute to dose-limiting toxicities. Citation Format: Veronica G. Zeng, Matthew S. Faber, Matthew J. Bernett, Kendra N. Avery, John L. Zeytounian, Rumana Rashid, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais, Michael Hedvat. Affinity tuned Xmab®2+1 anti-mesothelin x anti-CD3 bispecific antibody induces selective T cell-dependent cellular cytotoxicity of human ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5654.

Bispecific T cell engagers simultaneously bind CD3 on T cells and tumor-associated antigens to promote T cell-mediated killing of tumor cells. While targeting lineage-restricted antigens such as CD19 or CD20 have found clinical success in hematopoietic cancers, targeting solid tumors requires high expressing tumor associated antigens (TAAs) with minimal normal tissue expression. One underexplored TAA is ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) for renal cell carcinoma (RCC). An activation marker for basophils, ENPP3 is a type II transmembrane protein that can promiscuously hydrolyze a large variety of nucleotide-containing molecules. Although its specific function in the tumor microenvironment is unknown, bulk RNAseq data from the TCGA describes broad overexpression of ENPP3 in clear cell and papillary RCC and less frequently in hepatocellular carcinoma (HCC). This pattern of expression has been confirmed by IHC and makes ENPP3 a potential target for a CD3 bispecific approach. Building upon the XmAb® heterodimeric Fc platform, we generated bispecific antibodies in an XmAb 2+1 Fab2-scFv-Fc format that are bivalent for ENPP3 and monovalent for CD3. Reducing the affinity of both specificities encouraged avid binding and strong potency on high ENPP3 expressing cell lines while minimizing reactivity on low expressing cell lines, matching ENPP3's differential expression pattern. In vitro T cell-dependent cellular cytotoxicity (TDCC) assays on the relevant cell lines confirmed selective potency on high versus low expressing cells. In vivo potency was evaluated by xenograft tumor models in human PBMC-engrafted NSG mice. In a KU812 tumor model, the lead candidate, XmAb30819, exhibited complete responses both as a single agent and in combination with checkpoint blockade. When tested against RXF393, an RCC cell line that overexpresses ENPP3 upon tumor engraftment, XmAb30819 demonstrated complete responses at all tested doses. Tolerability in non-human primates was evaluated in an escalating dose model and then confirmed in an expansion cohort. Dose-dependent T cell margination was observed, and doses that were active in the mouse tumor studies were well-tolerated. Preclinical studies have demonstrated the safety and efficacy of XmAb30819, an XmAb 2+1 ENPP3 x CD3 bispecific antibody for RCC. Citation Format: Alex Nisthal, Sung-Hyung Lee, Yoon Kyung Kim, Christine Bonzon, Rumana Rashid, Kendra N. Avery, Liz Bogaert, Connie Ardila, Irene W. Leung, Nicole Rodriguez, Umesh S. Muchhal, Gregory L. Moore, Seung Y. Chu, John R. Desjarlais. XmAb30819, an XmAb®2+1 ENPP3 x CD3 bispecific antibody for RCC, demonstrates safety and efficacy in in vivo preclinical studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2286.

e15001 Background: Interim safety and PD data from an ongoing first-in-human, multi-center, open-label dose escalation study of XmAb20717 (XmAb20717-01; NCT03517488) are reported here. The primary objectives were to determine safety, tolerability, and the MTD and/or recommended doses (RDs). Secondary objectives included preliminary anti-tumor activity and PK/PD. Methods: A 3+3 dose escalation design was used to establish an MTD/RD(s) for infusions on Days 1 and 15 of each 28-day cycle. DLT evaluation was based on Cycle 1 through Day 28. Patients with selected solid tumors (in indications both with and without approved checkpoint therapy) who have exhausted standard of care are eligible. Results: As of 05FEB 2020, 34 patients were treated in cohorts 1-6 at fixed doses of 0.15 to 10 mg/kg. Patients had a median age of 57 years (range 32-81), a median time since initial diagnosis of 42 months (range 3 -313), and a median of 4 prior systemic therapies (range 0-9). 68% of patients had a TNM stage of III/IV, and 68% had been exposed to checkpoint therapy. XmAb20717 treatment was generally well tolerated through the highest dose cohort tested. Overall rates of Gr3/4 immune-related AEs occurred in 8 (24%) patients including elevations of transaminases 3 (9%), rash 2 (6%), lipase and amylase 1 (3%, without clinical symptoms or radiographic evidence of pancreatitis), lipase (alone) 1 (3%), pruritus 1 (3%), hyperglycaemia 1 (3%), arthritis 1 (3%) and colitis 1 (3%), all reversible. Responses were evaluated based on RECIST 1.1 criteria, and there was 1 CR reported (melanoma, progressed on prior pembrolizumab) at 10 mg/kg (highest dose level). Dose-dependent pharmacodynamic activity consistent with dual PD-1/CTLA-4 blockade was noted, namely a proliferative burst of both CD8 and CD4 T cells and induction of IFN-inducible chemokines (Table). Conclusions: XmAb20717 is generally safe and has demonstrated PD activity in heavily pretreated patients with selected advanced solid tumors. Dose escalation continues. Clinical trial information: NCT03517488 . [Table: see text]

Purpose: Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell–redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell–recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma. Experimental Design: Anti-CD38/CD3 XmAb T-cell–recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties. Results: AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow–invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented. Conclusions: These findings support the clinical development of AMG 424, an affinity-optimized T-cell–recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

Bispecific monoclonal antibodies can bind two protein targets simultaneously and enable therapeutic modalities inaccessible by traditional mAbs. Bispecific formats containing a heterodimeric Fc region are of particular interest, as a heterodimeric Fc empowers both bispecificity and altered valencies while retaining the developability and druggability of a monoclonal antibody. We present a robust heterodimeric Fc platform, called the XmAb® bispecific platform, engineered for efficient development of bispecific antibodies and Fc fusions of multiple formats. First, we engineer a purification solution for proteins containing a heterodimeric Fc using engineered isoelectric point differences in the Fc region that enable straightforward purification of the heterodimeric species. Then, we combine this purification solution with a novel set of Fc substitutions capable of achieving heterodimer yields over 95% with little change in thermostability. Next, we illustrate the flexibility of our heterodimeric Fc with a case study in which a wide range of tumor-associated antigen × CD3 bispecifics are generated, differing in choice of tumor antigen, affinities for both tumor antigen and CD3, and tumor antigen valency. Finally, we present manufacturing data reinforcing the robustness of the heterodimeric Fc platform at scale.

Objective Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. The aim of this study was to characterize B cell immunosuppression mediated by the Fc-engineered antibody, XmAb5871, which coengages FcγRIIb with the B cell antigen receptor (BCR) complex and that is currently in clinical development for the treatment of rheumatoid arthritis (RA). Because rheumatoid factor (RF) might interfere with the binding of XmAb5871 to FcγRIIb, we correlated RF titers with the potency of XmAb5871. Methods We analyzed the expression of CD19, FcγRIIb, and CD86 on naive and memory B cells from 50 patients with RA and 66 healthy donors, quantified XmAb5871-induced promotion of FcγRIIb phosphorylation and suppression of calcium flux in activated B cells, measured CD86 inhibition in whole blood, and correlated RF and anti−citrullinated protein antibody (ACPA) levels with drug potency. We engrafted RA peripheral blood mononuclear cells (PBMCs) into SCID mice, treated them with XmAb5871, and quantified human total IgG, total IgM, and anti-tetanus IgG antibody levels in vivo. Results B cells from all donors expressed CD19 and FcγRIIb, and the expression of FcγRIIb was higher on naive, but not memory, B cells from donors with RA compared with healthy donors. BCR-mediated calcium flux was suppressed by XmAb5871 and was associated with FcγRIIb phosphorylation. XmAb5871 inhibited CD86 induction, and the levels of RF and ACPAs did not affect efficacy. XmAb5871 suppressed B cell activation regardless of disease severity. In SCID mice engrafted with PBMCs from a patient with RA, XmAb5871 suppressed humoral responses. Conclusion Coengagement of the BCR complex and FcγRIIb by XmAb5871 inhibits B cell activation and function. The similar potency in patients with RA and healthy donors and the absence of autoantibody interference suggest that XmAb5871 may represent a new therapeutic strategy to suppress autoreactive B cells in RA.

The CTLA4-Ig fusion proteins abatacept and belatacept are clinically proven immunosuppressants used for rheumatoid arthritis and renal transplant, respectively. Given that both biologics are typically administered chronically by infusion, a need exists for a next-generation CTLA4-Ig with more convenient dosing. We used structure-based protein engineering to optimize the affinity of existing CTLA4-Ig therapeutics for the ligands CD80 and CD86, and for the neonatal Fc receptor, FcRn. From a rationally designed library, we identified four substitutions that enhanced binding to human CD80 and CD86. Coupled with two IgG1 Fc substitutions that enhanced binding to human FcRn, these changes comprise the novel CTLA4-Ig fusion protein, XPro9523. Compared with abatacept, XPro9523 demonstrated 5.9-fold, 23-fold, and 12-fold increased binding to CD80, CD86, and FcRn, respectively; compared with belatacept, CD80, CD86, and FcRn binding increased 1.5-fold, 7.7-fold, and 11-fold, respectively. XPro9523 and belatacept sup...

Background Sequestration of IgE to prevent its binding to high-affinity IgE receptor FceRI on basophils and mast cells is an effective therapy for allergic asthma. IgE production requires differentiation of activated IgE + B cells into plasma cells upon allergen sensitization. B-cell receptor signaling is suppressed by the inhibitory IgG Fc receptor FcγRIIb; therefore, we reasoned that a therapeutic antibody that coengages FcγRIIb and IgE B-cell receptor would not only sequester IgE but also suppress its production by blocking IgE + B-cell activation and differentiation to IgE-secreting plasma cells. Objective To explore the effects of IgE sequestration versus IgE suppression by comparing omalizumab to FcγRIIb-optimized anti-IgE antibodies in humanized mouse models of immunoglobulin production. Methods By using a murine anti-IgE antibody as a template, we humanized, increased IgE binding, and modified its Fc domain to increase affinity for FcγRIIb. We next compared effects of this antibody (XmAb7195) versus omalizumab on the secretion of IgE and other isotypes in human PBMC cultures and in PBMC-engrafted severe combined immunodeficiency mice. Results Relative to omalizumab, XmAb7195 has a 5-fold higher affinity for human IgE and more than 400-fold higher affinity for FcγRIIb. In addition to sequestering soluble IgE, XmAb7195 inhibited plasma cell differentiation and consequent human IgE production through coengagement of IgE B-cell receptor with FcγRIIb. In PBMC-engrafted mice, XmAb7195 reduced total human IgE (but not IgG or IgM) levels by up to 40-fold relative to omalizumab. Conclusion XmAb7195 acts by IgE sequestration coupled with an FcγRIIb-mediated inhibitory mechanism to suppress the formation of IgE-secreting plasma cells and reduce both free and total IgE levels.

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B–dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

Regulatory T cells (Tregs) characterized as CD4 positive cells, expressing CD25 (IL-2Rα) and the forkhead transcription factor FoxP3 maintain immune tolerance in tissues by suppressing the function of both CD4 and CD8 effector T cells. It is well documented that there is a paucity and/or dysfunction of Tregs in most autoimmune diseases and therefore a promising therapeutic approach has been to restore their numbers and function. Treg homeostasis depends on IL-2 derived signals and Tregs are sensitive to IL-2 stimulation in part due to the high abundance of CD25. As a result, encouraging results have been seen in patients with autoimmune diseases who were treated with low doses of IL-2. Single or repeat low doses of IL-2 led to selective expansion of Tregs over effector cells and provided clinical benefit. Such IL-2 based therapies can be further improved by enhancing the selectivity of IL-2 for Tregs and improving the pharmacokinetics of the drug. Here we have developed an IL-2-Fc fusion protein that selectively acts on Tregs in vitro and in vivo. IL-2 mediated signal transduction occurs through the formation of a high affinity tetrameric complex consisting of IL-2, CD25, CD122(IL-2Rβ) and CD132(IL-2Rγ). IL-2-Fc fusion proteins were designed to be monomeric by fusing variant IL-2 molecules to one side of our heterodimeric Fc-region. To achieve Treg selectivity, we designed variants of IL-2 that have weakened interaction with CD122 and/or enhanced interaction with CD25 such that signaling is only observed in CD25 high cells. These IL-2-Fc variants were produced with good yield and purity. We identified novel variants that selectively led to phosphorylation of STAT5 transcription factor in Tregs from human peripheral blood mononuclear cells (PBMCs). These Treg-selective IL-2-Fc variants stimulated Tregs with varying potencies. Further, they selectively enhanced and sustained the proliferation of Tregs in vivo in non-human primates. These promising preclinical results suggest that clinical development of Treg selective IL-2-Fc fusion proteins for autoimmune diseases is warranted. Disclosures Varma: Xencor: Employment, Equity Ownership. Liu:Xencor: Employment, Equity Ownership. Avery:Xencor: Employment, Equity Ownership. Rashid:Xencor: Employment, Equity Ownership. Schubbert:Xencor: Employment, Equity Ownership. Leung:Xencor: Employment, Equity Ownership. Bonzon:Xencor: Employment, Equity Ownership. Bogaert:Xencor: Employment, Equity Ownership. Desjarlais:Xencor: Employment, Equity Ownership. Bernett:Xencor: Employment, Equity Ownership.

CD40 is highly expressed on various B-lineage malignancies and represents an attractive immunotherapy target for neoplastic disease. Previous work showed that engineering the Fc domain of an antibody for increased binding to Fcγ receptors (FcγRs) significantly enhanced Fc-mediated immune effector function and antitumor activity in vitro and in vivo. We developed a humanized anti-CD40 antibody similarly Fc-engineered for increased FcγR binding (XmAbCD40) and compared its efficacy with that of an anti-CD40 native IgG1 analog and the anti-CD20 antibody rituximab. XmAbCD40 increased antibody-dependent cell-mediated cytotoxicity (ADCC) up to 150-fold relative to anti-CD40 IgG1 against B-lymphoma, leukemia, and multiple myeloma cell lines, and significantly enhanced ADCC against primary tumors. XmAbCD40 was also superior to rituximab in enhancing ADCC (both in cell lines and primary tumors) and in augmenting antibody-dependent cellular phagocytosis. XmAbCD40 significantly inhibited lymphoma growth in disseminated and established mouse xenografts and was more effective than the IgG1 analog or rituximab. An anti-CD40 antibody constructed to abrogate FcγR binding showed no reduction of tumor growth, indicating that the in vivo antitumor activity of XmAbCD40 is primarily mediated via FcγR-dependent mechanisms. These data demonstrate that XmAbCD40 displays potent antitumor efficacy and merits further evaluation for the treatment of CD40+ malignancies.

IL15 and IL2 are two similar cytokines that stimulate the proliferation of lymphocytes, and their therapeutic potential has been well established in animal models and human trials. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain (γc) and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. Both cytokines share a similar biology, with the exception that IL2 has a greater preference for Tregs due to their high constitutive expression of IL2Rα. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, and this IL15/IL15Rα complex is presented in trans to lymphocytes expressing IL2Rβ and γc. It has been shown that recombinant IL15/IL15Rα heterodimer is stable and highly active. As potential drugs, both IL2 and IL15 are extremely potent and suffer from low tolerability and very fast clearance that limits therapeutic window. Seeking to engineer a more druggable version of IL15, we created various IL15/IL15Rα heterodimeric Fc-fusions (IL15/IL15Rα-Het-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and prolong half-life. We engineered IL15/IL15Rα-Het-Fc by fusing IL15 to one side of a heterodimeric Fc-region, and the sushi domain of IL15Rα to the other side, or by creating a single-chain IL15/IL15Rα that was attached to one side of a heterodimeric Fc-region. These Fc-fusions were tuned for optimal activity by engineering amino acid substitutions in IL15 - at the IL2Rβ or γc interface - that reduced overall in vitro potency. In vitro proliferation of lymphocytes in normal human PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days and by measuring signaling in a STAT5 phosphorylation assay for 15 minutes. In vivo activity was evaluated using a huPBMC-NSG mouse model by measuring the extent of human leukocyte engraftment by flow cytometry and IFNγ. Tolerability, immune stimulation, and pharmacokinetics were evaluated in non-human primates (NHP). IL15/IL15Rα-Het-Fc were produced with good yield and purity. The Fc-fusions enhanced proliferation of CD8+ T and NK cells in vitro, and potency of variants with substitutions at the IL2Rβ and/or γc interface was reduced up to ~700-fold compared to wild-type IL15/IL15Rα-Het-Fc. Treatment of huPBMC-NSG mice with IL15/IL15Rα-Het-Fc promoted enhanced T cell engraftment and elevated IFNγ in a dose dependent manner. NHP studies indicated half-lives of several days for IL15/IL15Rα-Het-Fc, which are significantly longer than the Citation Format: Matthew J. Bernett, Rajat Varma, Christine Bonzon, Rumana Rashid, Liz Bogaert, Ke Liu, Suzanne Schubbert, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. Potency-reduced IL15/IL15Rα heterodimeric Fc-fusions display enhanced in vivo activity through increased exposure [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 5565.

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.

Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V(H)/V(L) interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.

CD19 is a B cell–specific antigen expressed on chronic lymphocytic leukemia (CLL) cells but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified constant fragment (Fc)–domain designed to enhance binding of FcγRIIIa. Herein, we demonstrate that XmAb5574 mediates potent antibody-dependent cellular cytotoxicity (ADCC), modest direct cytotoxicity, and antibody-dependent cellular phagocytosis but not complement-mediated cytotoxicity against CLL cells. Interestingly, XmAb5574 mediates significantly higher ADCC compared with both the humanized anti-CD19 nonengineered antibody it is derived from and also rituximab, a therapeutic antibody widely used in the treatment of CLL. The XmAb5574-dependent ADCC is mediated by natural killer (NK) cells through a granzyme B–dependent mechanism. The NK cell–mediated cytolytic and secretory function with XmAb5574 compared with the nonengineered antibody is associated with enhanced NK-cell activation, interferon production, extracellular signal-regulated kinase phosphorylation downstream of Fcγ receptor, and no increased NK-cell apoptosis. Notably, enhanced NK cell–mediated ADCC with XmAb5574 was enhanced further by lenalidomide. These findings provide strong support for further clinical development of XmAb5574 as both a monotherapy and in combination with lenalidomide for the therapy of CLL and related CD19+ B-cell malignancies.

CD19 is a pan B-cell surface receptor expressed from pro–B-cell development until its down-regulation during terminal differentiation into plasma cells. CD19 represents an attractive immunotherapy target for cancers of lymphoid origin due to its high expression levels on the vast majority of non–Hodgkin's lymphomas and some leukemias. A humanized anti-CD19 antibody with an engineered Fc domain (XmAb5574) was generated to increase binding to Fcγ receptors on immune cells and thus increase Fc-mediated effector functions. In vitro, XmAb5574 enhanced antibody-dependent cell-mediated cytotoxicity 100-fold to 1,000-fold relative to an anti-CD19 IgG1 analogue against a broad range of B-lymphoma and leukemia cell lines. Furthermore, XmAb5574 conferred antibody-dependent cell-mediated cytotoxicity against patient-derived acute lymphoblastic leukemia and mantle cell lymphoma cells, whereas the IgG1 analogue was inactive. XmAb5574 also increased antibody-dependent cellular phagocytosis and apoptosis. In vivo, XmAb5574 significantly inhibited lymphoma growth in prophylactic and established mouse xenograft models, and showed more potent antitumor activity than its IgG1 analogue. Comparisons with a variant incapable of Fcγ receptor binding showed that engagement of these receptors is critical for optimal antitumor efficacy. These results suggest that XmAb5574 exhibits potent tumor cytotoxicity via direct and indirect effector functions and thus warrants clinical evaluation as an immunotherapeutic for CD19+ hematologic malignancies. [Cancer Res 2008;68(19):8049–57]

The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcgammaRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcgammaRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to approximately 430-fold greater affinity to FcgammaRIIb. Relative to native IgG1, the FcgammaRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcgammaRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcgammaRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcgammaRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.

We introduce a new method of humanization based on a novel and immunologically relevant metric of antibody humanness, termed human string content (HSC), that quantifies a sequence at the level of potential MHC/T-cell epitopes. Use of this quantity rather than global identity as an optimization goal enables the sampling of human diversity from distinct human germline sequences across the framework and CDR regions, and allows for the generation of multiple diverse candidate sequences. As a result engineering is carried out at finer sequence resolution relative to standard CDR grafting methods, providing for the optimization of antibody properties beyond immunogenicity such as antigen affinity and solution behavior. We have applied this method to the humanization of four antibodies with different antigen specificities. The resulting variable domains differ fundamentally from CDR-grafted antibodies in that they are immunologically more human and their humanness is derived from several discrete germline sequences. Furthermore, these antibodies bind their respective antigens better than or comparable to those of the parent antibodies without the need for affinity maturation.

Protein design is becoming an increasingly useful tool for optimizing protein drugs and creating novel biotherapeutics. Recent progress includes the engineering of monoclonal antibodies, cytokines, enzymes and viral fusion inhibitors.

Protein structure prediction by comparative modeling benefits greatly from the use of multiple sequence alignment information to improve the accuracy of structural template identification and the alignment of target sequences to structural templates. Unfortunately, this benefit is limited to those protein sequences for which at least several natural sequence homologues exist. We show here that the use of large diverse alignments of computationally designed protein sequences confers many of the same benefits as natural sequences in identifying structural templates for comparative modeling targets. A large-scale massively parallelized application of an all-atom protein design algorithm, including a simple model of peptide backbone flexibility, has allowed us to generate 500 diverse, non-native, high-quality sequences for each of 264 protein structures in our test set. PSI-BLAST searches using the sequence profiles generated from the designed sequences ("reverse" BLAST searches) give near-perfect accuracy in identifying true structural homologues of the parent structure, with 54% coverage. In 41 of 49 genomes scanned using reverse BLAST searches, at least one novel structural template (not found by the standard method of PSI-BLAST against PDB) is identified. Further improvements in coverage, through optimizing the scoring function used to design sequences and continued application to new protein structures beyond the test set, will allow this method to mature into a useful strategy for identifying distantly related structural templates.

An increasing number of engineered protein therapeutics are currently being developed, tested in clinical trials and marketed for use. Many of these proteins arose out of hit-and-miss efforts to discover specific mutations, fusion partners or chemical modifications that confer desired properties. Through these efforts, several useful strategies have emerged for rational optimization of therapeutic candidates. The controlled manipulation of the physical, chemical and biological properties of proteins enabled by structure-based simulation is now being used to refine established rational engineering approaches and to advance new strategies. These methods provide clear, hypothesis-driven routes to solve problems that plague many proteins and to create novel mechanisms of action. We anticipate that rational protein engineering will shape the field of protein therapeutics dramatically by improving existing products and enabling the development of novel therapeutic agents.