A B7-H3-targeted CD28 bispecific antibody enhances the activity of anti-PD1 and CD3 T-cell engager immunotherapies.

T-cell activation is a multistep process requiring T-cell receptor engagement by peptide-major histocompatibility complexes (Signal 1) coupled with CD28-mediated costimulation (Signal 2). Tumors typically lack expression of CD28 ligands, so tumor-specific Signal 1 (e.g.

Translational PK/PD and the first-in-human dose selection of a PD1/IL15: an engineered recombinant targeted cytokine for cancer immunotherapy.

Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities.

Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15.

AMG 509 (Xaluritamig), an Anti-STEAP1 XmAb 2+1 T-cell Redirecting Immune Therapy with Avidity-Dependent Activity against Prostate Cancer.

Unlabelled: The tumor-associated antigen STEAP1 is a potential therapeutic target that is expressed in most prostate tumors and at increased levels in metastatic castration-resistant prostate cancer (mCRPC). We developed a STEAP1-targeted XmAb 2+1 T-cell engager (TCE) molecule, AMG 509 (also designated xaluritamig), that is designed to redirect T cells to kill prostate cancer cells that express STEAP1. AMG 509 mediates potent T cell-dependent cytotoxicity of prostate cancer cell lines in vitro and promotes tumor regression in xenograft and syngeneic mouse models of prostate cancer in vivo.

Dissecting strategies to tune the therapeutic potential of SARS-CoV-2-specific monoclonal antibody CR3022.

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.

Targeting Multiple Myeloma with AMG 424, a Novel Anti-CD38/CD3 Bispecific T-cell-recruiting Antibody Optimized for Cytotoxicity and Cytokine Release.

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.

Redirected T Cell Cytotoxicity in Cancer Therapy.

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.

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.

Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcγ receptor IIb inhibitory receptor.

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.

Increasing FcγRIIa affinity of an FcγRIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested as an essential mechanism for the in vivo activity of cetuximab, an epidermal growth factor receptor (EGFR)-targeting therapeutic antibody. Thus, enhancing the affinity of human IgG1 antibodies to natural killer (NK) cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc portion has been demonstrated to improve NK cell-mediated ADCC and to represent a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) effector cells express the highly homologous FcγRIIIb isoform, which is described to be ineffective in triggering ADCC.

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.

Suppression of mast cell degranulation through a dual-targeting tandem IgE-IgG Fc domain biologic engineered to bind with high affinity to FcγRIIb.

Mast cells and basophils play a central role in allergy, asthma, and anaphylaxis, as well as in non-allergic inflammatory, neurological and autoimmune diseases. Allergen-mediated cross-linking of IgE bound to FcεRI leads to cellular activation, and the low-affinity Fc receptor FcγRIIb is a key inhibitor of subsequent degranulation. FcγRIIb, when coengaged with FcεRI via allergen bound to IgE, stimulates ITIM domain-mediated inhibitory signaling that efficiently suppresses mast cell and basophil activation.

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.

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.

Modulation of antibody effector function.

Several novel technologies have evolved over the last decade for the modification of antibodies to enhance their inherent effector functions. All focus on the constant Fc domain and utilize either amino acid substitutions or glycoform perturbations to modulate their interaction with Fc receptors and the effector cells that bear them. We review these technologies with an emphasis on their validation with animal models and human clinical data.

Antibody-mediated coengagement of FcγRIIb and B cell receptor complex suppresses humoral immunity in systemic lupus erythematosus.

Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF.

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.

Enhanced antibody half-life improves in vivo activity.

Improved affinity for the neonatal Fc receptor (FcRn) is known to extend antibody half-life in vivo. However, this has never been linked with enhanced therapeutic efficacy. We tested whether antibodies with half-lives extended up to fivefold in human (h)FcRn transgenic mice and threefold in cynomolgus monkeys retain efficacy at longer dosing intervals.

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.

CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain-engineered monoclonal antibody.

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 FcgammaRIIIa. 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.

The impact of Fc engineering on an anti-CD19 antibody: increased Fcgamma receptor affinity enhances B-cell clearing in nonhuman primates.

CD19, a B cell-restricted receptor critical for B-cell development, is expressed in most B-cell malignancies. The Fc-engineered anti-CD19 antibody, XmAb5574, has enhanced Fcgamma receptor (FcgammaR) binding affinity, leading to improved FcgammaR-dependent effector cell functions and antitumor activity in murine xenografts compared with the non-Fc-engineered anti-CD19 IgG1 analog. Here, we use XmAb5574 and anti-CD19 IgG1 to further dissect effector cell functions in an immune system closely homologous to that of humans, the cynomolgus monkey.

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-Hodgkin's 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.

Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells.

The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fcgamma receptors (FcgammaR). Although an early goal has been enhanced FcgammaRIIIa 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 FcgammaRIIa. Here, we describe a set of engineered Fc variants with diverse FcgammaR affinities, including a novel substitution G236A that provides selectively enhanced binding to FcgammaRIIa relative to FcgammaRIIb.

Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies.

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.

Optimizing engagement of the immune system by anti-tumor antibodies: an engineer's perspective.

A unique property of monoclonal antibodies, and a principal reason for their success as cancer therapeutics, is their ability to engage the immune system. A growing set of data supporting the relevance of Fc-mediated effector functions to anti-tumor efficacy has motivated efforts to enhance the interactions between antibodies and Fc receptors expressed on immune cells. Although current approaches have considerable promise for improved clinical performance, the immunobiology of tumors, antibodies, and Fc receptors continues to evolve.

A molecular immunology approach to antibody humanization and functional optimization.

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.