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. 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.

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.

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.

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., neoepitope presentation) without costimulation may be ineffective or even induce T-cell anergy. We designed the bispecific antibody XmAb808 to co-engage the tumor-associated antigen B7-H3 with CD28 to promote T-cell costimulation within the tumor microenvironment. XmAb808 costimulation was measured by its ability to activate and expand T cells and enhance T cell-mediated cancer cell killing in cocultures of human peripheral blood mononuclear cells (PBMCs) and cancer cells, and in mice engrafted with human PBMCs and tumor xenografts. XmAb808 avidly bound cancer cells and stimulated interleukin (IL)2 and interferon (IFN)γ secretion from T cells cocultured with cancer cells engineered to deliver Signal 1 to T cells via a surface-expressed anti-CD3 antibody. XmAb808 enhanced expression of the anti-apoptotic factor Bcl-xL and CD25, promoting survival and IL2-dependent expansion of T cells coupled with increased T cell-mediated cytotoxicity in vitro. XmAb808 combined with a EpCAM×CD3 bispecific antibody to enhance target cell killing through IL2-dependent expansion of CD25+ T cells. This combination also suppressed pancreatic tumor xenograft growth in mice. Further, XmAb808 combined with an anti-PD1 antibody to suppress breast tumor xenograft growth in mice. XmAb808 as monotherapy and in combination with an anti-PD1 antibody is currently in clinical development in patients with advanced solid tumors. Our results suggest that XmAb808 may also combine with tumor antigen-targeted anti-CD3 (Signal 1) T-cell engagers.

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.

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. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials. Results: The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range ∼1.0-4.1 days) compared to wild-type IL-15 (half-life ∼1.1 h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1+ lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003 mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data. Conclusion: This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine.

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. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.

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.

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.

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. 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 Fcgamma 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.

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.

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. XmAb5574 infusion caused an immediate and dose-related B-cell depletion in the blood (to <10% of baseline levels) concomitant with a sustained reduction of natural killer (NK) cells. NK cells had fully recovered by day 15, whereas B-cell recovery was underway by day 57. B cells in secondary lymphoid tissues were depleted (to 34%-61% of vehicle), with involuted germinal centers apparent in the spleen. Anti-CD19 IgG1 had comparable serum exposure to XmAb5574 but demonstrated no B-cell depletion and no sustained NK-cell reduction. Thus, increasing FcgammaR binding affinity dramatically increased B-cell clearing. We propose that effector cell functions, possibly those involving NK cells, mediate XmAb5574 potency in cynomolgus monkeys, and that enhancing these mechanisms should advance the treatment of B-cell malignancies in humans.

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. 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.

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.

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. Variants containing this substitution have up to 70-fold greater FcgammaRIIa affinity and 15-fold improvement in FcgammaRIIa/FcgammaRIIb 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 FcgammaR to effector functions mediated by NK cells and macrophages. These experiments show that FcgammaRIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor FcgammaRIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.

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.

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. 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.

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. In this review we discuss what is known and what is not known about the interactions between therapeutic antibodies and the immune system, with the goal being progress toward clear target profiles for effector engineering efforts.

Designing proteins for therapeutic applications.

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.

A de novo redesign of the WW domain.

We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small beta-sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring L-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS-WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5 degrees C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.