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.

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

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. The avidity-driven activity of AMG 509 enables selectivity for tumor cells with high STEAP1 expression compared with normal cells. AMG 509 is the first STEAP1 TCE to advance to clinical testing, and we report a case study of a patient with mCRPC who achieved an objective response on AMG 509 treatment. SIGNIFICANCE: Immunotherapy in prostate cancer has met with limited success due to the immunosuppressive microenvironment and lack of tumor-specific targets. AMG 509 provides a targeted immunotherapy approach to engage a patient's T cells to kill STEAP1-expressing tumor cells and represents a new treatment option for mCRPC and potentially more broadly for prostate cancer. See related commentary by Hage Chehade et al., p. 20. See related article by Kelly et al., p. 76. This article is featured in Selected Articles from This Issue, p. 5.

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.

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. 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.

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. 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.

eCodonOpt: a systematic computational framework for optimizing codon usage in directed evolution experiments.

We present a systematic computational framework, eCodonOpt, for designing parental DNA sequences for directed evolution experiments through codon usage optimization. Given a set of homologous parental proteins to be recombined at the DNA level, the optimal DNA sequences encoding these proteins are sought for a given diversity objective. We find that the free energy of annealing between the recombining DNA sequences is a much better descriptor of the extent of crossover formation than sequence identity. Three different diversity targets are investigated for the DNA shuffling protocol to showcase the utility of the eCodonOpt framework: (i) maximizing the average number of crossovers per recombined sequence; (ii) minimizing bias in family DNA shuffling so that each of the parental sequence pair contributes a similar number of crossovers to the library; and (iii) maximizing the relative frequency of crossovers in specific structural regions. Each one of these design challenges is formulated as a constrained optimization problem that utilizes 0-1 binary variables as on/off switches to model the selection of different codon choices for each residue position. Computational results suggest that many-fold improvements in the crossover frequency, location and specificity are possible, providing valuable insights for the engineering of directed evolution protocols.

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.

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. To assess the therapeutic potential of directed coengagement of FcεRI and FcγRIIb in the absence of FcεRI crosslinking, we developed a fusion protein comprising the coupled Fc domains of murine IgE and human IgG1. As a key functional component of this tandem Fcε-Fcγ biologic, we engineered its IgG1 Fc domain to bind to human FcγRIIb with 100-fold enhanced affinity relative to native IgG1 Fc. Using mast cells from mice transgenic for human FcγRIIb, we show that this tandem Fc binds with high affinity to murine FcεRI and human FcγRIIb on mast cells, triggers phosphorylation of FcγRIIb, and inhibits FcεRI-dependent calcium mobilization. Control tandem Fc biologics containing a native IgG1 Fc domain or lacking binding to Fcγ receptors were markedly less active, demonstrating that the affinity-optimized tandem Fc can inhibit degranulation through stimulation of FcγRIIb signaling as well as through competition with allergen-IgE immune complex for FcεRI binding. We propose that in the context of a fully human tandem Fc biologic, high-affinity coengagement of FcεRI and FcγRIIb has potential as a novel therapy for allergy and other mast cell and basophil-mediated pathologies.

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

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

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions.

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complement-enhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.

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.

IPRO: an iterative computational protein library redesign and optimization procedure.

A number of computational approaches have been developed to reengineer promising chimeric proteins one at a time through targeted point mutations. In this article, we introduce the computational procedure IPRO (iterative protein redesign and optimization procedure) for the redesign of an entire combinatorial protein library in one step using energy-based scoring functions. IPRO relies on identifying mutations in the parental sequences, which when propagated downstream in the combinatorial library, improve the average quality of the library (e.g., stability, binding affinity, specific activity, etc.). Residue and rotamer design choices are driven by a globally convergent mixed-integer linear programming formulation. Unlike many of the available computational approaches, the procedure allows for backbone movement as well as redocking of the associated ligands after a prespecified number of design iterations. IPRO can also be used, as a limiting case, for the redesign of a single or handful of individual sequences. The application of IPRO is highlighted through the redesign of a 16-member library of Escherichia coli/Bacillus subtilis dihydrofolate reductase hybrids, both individually and through upstream parental sequence redesign, for improving the average binding energy. Computational results demonstrate that it is indeed feasible to improve the overall library quality as exemplified by binding energy scores through targeted mutations in the parental sequences.

Identifying residue-residue clashes in protein hybrids by using a second-order mean-field approach.

In this article, a second-order mean-field-based approach is introduced for characterizing the complete set of residue-residue couplings consistent with a given protein structure. This information is subsequently used to classify protein hybrids with respect to their potential to be functional based on the presenceabsence and severity of clashing residue-residue interactions. First, atomistic representations of both the native and denatured states are used to calculate rotamer-backbone, rotamer-intrinsic, and rotamer-rotamer conformational energies. Next, this complete conformational energy table is coupled with a second-order mean-field description to elucidate the probabilities of all possible rotamer-rotamer combinations in a minimum Helmholtz free-energy ensemble. Computational results for the dihydrofolate reductase family reveal correlation in substitution patterns between not only contacting but also distal second-order structural elements. Residue-residue clashes in hybrid proteins are quantified by contrasting the ensemble probabilities of protein hybrids against the ones of the original parental sequences. Good agreement with experimental data is demonstrated by superimposing these clashes against the functional crossover profiles of bidirectional incremental truncation libraries for Escherichia coli and human glycinamide ribonucleotide transformylases.

Predicting out-of-sequence reassembly in DNA shuffling.

We present an analysis for calculating the frequency of out-of-sequence reassembly in DNA shuffling experiments. Out-of-sequence annealing events are undesirable since they typically encode non-functional proteins with missing or repetitive regions. The approach builds on the e Shuffle framework for the prediction of crossover formation using equilibrium thermodynamics and complete sequence information to model the reassembly process. An in silico case study of a set of subtilases reveals that, as expected, the presence of significant sequence identity between distant portions of the parental sequences gives rise to out-of-sequence annealing events that upon reassembly generate sequences with missing or repetitive DNA segments. The frequency of these events increases as the fragment length decreases. Interestingly, out-of-sequence annealing events are at a minimum near the annealing temperature of 55 degrees C used in the original DNA shuffling protocol. Neither parental sequence identity nor number of shuffled parents significantly alter the extent of out-of-sequence reassembly

Using multiple sequence correlation analysis to characterize functionally important protein regions.

Protein co-evolution under structural and functional constraints necessitates the preservation of important interactions. Identifying functionally important regions poses many obstacles in protein engineering efforts. In this paper, we present a bioinformatics-inspired approach (residue correlation analysis, RCA) for predicting functionally important domains from protein family sequence data. RCA is comprised of two major steps: (i) identifying pairs of residue positions that mutate in a coordinated manner, and (ii) using these results to identify protein regions that interact with an uncommonly high number of other residues. We hypothesize that strongly correlated pairs result not only from contacting pairs, but also from residues that participate in conformational changes involved during catalysis or important interactions necessary for retaining functionality. The results show that highly mobile loops that assist in ligand association/dissociation tend to exhibit high correlation. RCA results exhibit good agreement with the findings of experimental and molecular dynamics studies for the three protein families that are analyzed: (i) DHFR (dihydrofolate reductase), (ii) cyclophilin, and (iii) formyl-transferase. Specifically, the specificity (percentage of correct predictions) in all three cases is substantially higher than those obtained by entropic measures or contacting residue pairs. In addition, we use our approach in a predictive fashion to identify important regions of a transmembrane amino acid transporter protein for which there is limited structural and functional information available.