Matthew J. Bernett

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

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-Hodgkins lymphomas and some leukemias. A humanized anti-CD19 antibody with an engineered Fc domain (XmAb5574) was generated to increase binding to Fcgamma 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 Fcgamma 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.

Fc-engineered anti-CD40 antibody enhances multiple effector functions and exhibits potent in vitro and in vivo antitumor activity against hematologic malignancies

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.

Reduction of total IgE by targeted coengagement of IgE B-cell receptor and FcγRIIb with Fc-engineered antibody

BACKGROUND Sequestration of IgE to prevent its binding to high-affinity IgE receptor FcεRI 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.

Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function

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.

Engineering fully human monoclonal antibodies from murine variable regions.

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.

Immune suppression in cynomolgus monkeys by XPro9523: an improved CTLA4-Ig fusion with enhanced binding to CD80, CD86 and neonatal Fc receptor FcRn.

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 suppressed human T cell proliferation and IL-2 production more potently than abatacept. XPro9523 also suppressed inflammation in the mouse collagen-induced arthritis model. In cynomolgus monkeys, XPro9523 saturated CD80 and CD86 more effectively than abatacept and belatacept, potently inhibited IgM and IgG immunization responses, and demonstrated longer half-life. Pharmacokinetic modeling of its increased potency and persistence suggests that, in humans, XPro9523 may demonstrate superior efficacy and dosing convenience compared with abatacept and belatacept.

Abstract 3633: Anti-SSTR2 × anti-CD3 bispecific antibody induces potent killing of human tumor cellsin vitroand in mice, and stimulates target-dependent T cell activation in monkeys: A potential immunotherapy for neuroendocrine tumors

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors (NETs) and small cell lung cancer (SCLC). Treatment options for NETs include somatostatin analogs and radionuclides; however, such therapies suffer from short half-life, modest efficacy, and toxicities due to inhibition of other SSTRs. Reasoning that a targeted immunotherapy against SSTR2 would provide a new therapeutic modality for NETs, we designed XmAb18087, a humanized and affinity-optimized bispecific antibody that engages T cells to stimulate redirected T cell-mediated cytotoxicity (RTCC) of SSTR2+ tumor cells. XmAb18087 possesses an Fc domain that maintains long half-life, yet lacks binding to Fcγ receptors to reduce Fc-mediated effector functions. XmAb18087 stimulated robust RTCC of SSTR2+ cell lines including medullary thyroid carcinoma (TT), lung carcinoma (A549), and CHO cells overexpressing SSTR2, with EC50s of ~1 to 100 ng/ml. XmAb18087 also upregulated CD69 and CD25 activation markers on CD4 and CD8 T cells. T cell responses were target-specific, because SSTR2¯ cell lines were not depleted, and because a control bispecific (anti-RSV x CD3) was ineffective. In addition, XmAb18087 (3 mg/kg weekly) reduced tumor burden of an established A549 xenograft in NSG mice engrafted with 107 human PBMC. We next assessed XmAb18087 activity in cynomolgus monkeys. As SSTR2 is not expressed in peripheral blood, target cell depletion cannot be monitored in vivo. However, CD3 bispecifics induce effects such as lymphocyte extravasation, cytokine induction, and T cell activation, which can serve as pharmacologic markers for activity in target organs. XmAb18087 dosed once at 30 or 60 μg/kg rapidly activated peripheral T cells, as quantified by CD69 and CD25 induction (peaking at ~8-12 hr). T cells rapidly extravasated from blood, with a nadir by 8 hr. Cytokines IL6 and TNF were rapidly induced, peaking at ~1-8 hr and returning to baseline by 48 hr. To explore repeat dosing, XmAb18087 was dosed at 1 or 10 μg/kg on Days 0 and 7 in a second study. The first dose of both 1 and 10 μg/kg again stimulated peripheral T cell activation, extravasation, and cytokine induction. These responses decreased markedly after the second dose, suggesting that SSTR2+ target cells remained depleted for at least 7 days. In summary, these results on human cells, in mice, and in monkeys support clinical assessment of XmAb18087 in SSTR2+ cancers including NETs and SCLC. In monkeys, T cell activation, extravasation, and cytokine induction were readily measured in peripheral blood and are indicative of T cell-mediated depletion of SSTR2+ target cells. Importantly, these responses may also serve as useful surrogate markers of NET depletion in clinical trials of XmAb18087. Citation Format: Sung-Hyung Lee, Seung Y. Chu, Rumana Rashid, Sheryl Phung, Irene W. Leung, Umesh S. Muchhal, Gregory L. Moore, Matthew J. Bernett, Suzanne Schubbert, Connie Ardila, Christine Bonzon, Paul Foster, David E. Szymkowski, John R. Desjarlais. Anti-SSTR2 × anti-CD3 bispecific antibody induces potent killing of human tumor cells in vitro and in mice, and stimulates target-dependent T cell activation in monkeys: A potential immunotherapy for neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3633. doi:10.1158/1538-7445.AM2017-3633

A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats

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.

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

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

Abstract 1595: IL15/IL15Rα heterodimeric Fc-fusions with extended half-lives

IL15 and IL2 are similar cytokines that stimulate the proliferation and differentiation of B cells, T cells, and NK cells. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. IL2Rα is highly expressed on Tregs, and the therapeutic benefit of IL2 for cancer treatment has been limited for this reason. IL15 is produced by monocytes and dendritic cells and functions as a stabilized heterodimeric complex with membrane-bound IL15Rα present on the same cells. This IL15/IL15Rα complex is presented in trans to NK cells and CD8+ T cells expressing IL2Rβ and the common gamma chain. It has been shown that recombinant IL15/IL15Rα heterodimer is highly active. Currently there are no approved versions of recombinant IL15 although several clinical trials are ongoing. As potential drugs, cytokines suffer from a very fast clearance that hinders favorable dosing. Consequently, a more druggable version of IL15 would consist of the IL15/IL15Rα complex coupled with slower clearance. We created various IL15/IL15Rα heterodimeric Fc-fusions in an effort to facilitate production, promote FcRn-mediated recycling of the complex, and thus prolong half-life. We engineered IL15/IL15Rα heterodimeric Fc-fusions by either 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 the linker regions between IL15/IL15Rα and the Fc and/or by engineering substitutions on IL15 at the IL15/IL2Rβ or IL15/gamma chain interface. In vitro proliferation of T and NK cells in healthy PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and measuring the extent of T cell engraftment by flow cytometry as well as IFNγ. PK was evaluated in C57BL/6J mice. IL15/IL15Rα heterodimeric Fc-fusions were successfully produced with favorable yields. The Fc-fusions enhanced proliferation of T and NK cells in vitro. Treatment of huPBMC-NSG mice with weekly doses of IL15/IL15Rα heterodimeric Fc-fusions promoted enhanced T cell engraftment and elevated levels of IFNγ in a dose dependent manner. Severe graft versus host disease was observed in treated mice. Little activity was seen with a comparable weekly dose of recombinant IL15. PK in C57BL/6J mice indicated half-lives of several days for the IL15/IL15Rα heterodimeric Fc-fusions, which are significantly longer than the Together, these data indicate that IL15/IL15Rα heterodimeric Fc-fusions demonstrate the high activity of IL15 with a more favorable PK profile. Citation Format: Matthew J. Bernett, Christine Bonzon, Rumana Rashid, Rajat Varma, Kendra N. Avery, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. IL15/IL15Rα heterodimeric Fc-fusions with extended half-lives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2017-1595