Characterization of anti-drug antibody responses to the T-cell engaging bispecific antibody cibisatamab to understand the impact on exposure.

An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure.

Costimulation blockade in combination with IL-2 permits regulatory T cell sparing immunomodulation that inhibits autoimmunity.

Blockade of CD28 costimulation with CTLA-4-Ig/Abatacept is used to dampen effector T cell responses in autoimmune and transplantation settings. However, a significant drawback of this approach is impaired regulatory T cell homeostasis that requires CD28 signaling. Therefore, strategies that restrict the effects of costimulation blockade to effector T cells would be advantageous. Here we probe the relative roles of CD28 and IL-2 in maintaining Treg. We find provision of IL-2 counteracts the regulatory T cell loss induced by costimulation blockade while minimally affecting the conventional T cell compartment. These data suggest that combining costimulation blockade with IL-2 treatment may selectively impair effector T cell responses while maintaining regulatory T cells. Using a mouse model of autoimmune diabetes, we show combined therapy supports regulatory T cell homeostasis and protects from disease. These findings are recapitulated in humanised mice using clinically relevant reagents and provide an exemplar for rational use of a second immunotherapy to offset known limitations of the first.

Simlukafusp alfa (FAP-IL2v) immunocytokine is a versatile combination partner for cancer immunotherapy.

Simlukafusp alfa (FAP-IL2v, RO6874281/RG7461) is an immunocytokine comprising an antibody against fibroblast activation protein α (FAP) and an IL-2 variant with a retained affinity for IL-2Rßγ > IL-2 Rßγ and abolished binding to IL-2 Rα. Here, we investigated the immunostimulatory properties of FAP-IL2v and its combination with programmed cell death protein 1 (PD-1) checkpoint inhibition, CD40 agonism, T cell bispecific and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. The binding and immunostimulatory properties of FAP-IL2v were investigated in vitro and compared with FAP-IL2wt. Tumor targeting was investigated in tumor-bearing mice and in a rhesus monkey. The ability of FAP-IL2v to potentiate the efficacy of different immunotherapies was investigated in different xenograft and syngeneic murine tumor models. FAP-IL2v bound IL-2 Rßγ and FAP with high affinity in vitro, inducing dose-dependent proliferation of natural killer (NK) cells and CD4+/CD8+ T cells while being significantly less potent than FAP-IL2wt in activating immunosuppressive regulatory T cells (Tregs). T cells activated by FAP-IL2v were less sensitive to Fas-mediated apoptosis than those activated by FAP-IL2wt. Imaging studies demonstrated improved tumor targeting of FAP-IL2v compared to FAP-IL2wt. Furthermore, FAP-IL2v significantly enhanced the in vitro and in vivo activity of therapeutic antibodies that mediate antibody-dependent or T cell-dependent cellular cytotoxicity (TDCC) and of programmed death-ligand 1 (PD-L1) checkpoint inhibition. The triple combination of FAP-IL2v with an anti-PD-L1 antibody and an agonistic CD40 antibody was most efficacious. These data indicate that FAP-IL2v is a potent immunocytokine that potentiates the efficacy of different T- and NK-cell-based cancer immunotherapies.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease.

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.

Target Expression, Generation, Preclinical Activity, and Pharmacokinetics of the BCMA-T Cell Bispecific Antibody EM801 for Multiple Myeloma Treatment.

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis.

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR.

XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer.

The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the "knobs-into-holes" technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2-3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.

A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors.

PURPOSE: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. EXPERIMENTAL DESIGN: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. RESULTS: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA-expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1-positive tumors. CONCLUSIONS: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286-97. ©2016 AACR.

Immuno-PET and Immuno-SPECT of Rheumatoid Arthritis with Radiolabeled Anti-Fibroblast Activation Protein Antibody Correlates with Severity of Arthritis.

UNLABELLED: One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA. METHODS: SPECT and PET with (111)In- and (89)Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score. RESULTS: Both (111)In-28H1 and (89)Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of (111)In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis. CONCLUSION: SPECT/CT imaging with the anti-FAP antibody (111)In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. (89)Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of (89)Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on.

Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells.

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.

Kinetic screening of antibody-Im7 conjugates by capture on a colicin E7 DNase domain using optical biosensors.

Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark V(NAR) single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors.

Excystation of Eimeria tenella sporozoites impaired by antibody recognizing gametocyte/oocyst antigens GAM22 and GAM56.

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.

Eimeria tenella: genomic organization and expression of an 89kDa cyclophilin.

Though parasite cyclophilins are promising new drug targets, Eimeria tenella cyclophilins have not been characterized yet. Here, we describe an 89kDa cyclophilin, designated EtCYP89. It is expressed throughout the developmental cycle of E. tenella, both in the intracellular stages in chicken and in extracellular sporulated oocysts and sporozoites. The EtCYP89 protein contains two Ser-rich domains in its NH2-terminus separated by a His-rich stretch. WD40 repeats are localized in the central part of the protein followed by a cyclophilin domain at the COOH-terminus. Both protein and genomic organization of EtCyp89 are conserved in comparison with its ortholog TgCyp81.6 in Toxoplasma gondii, except for the absence of a Ser- and His-rich NH2-terminus in TgCYP81.6. In particular, those 13 residues are conserved which are responsible for binding the anti-coccidial drug cyclosporine A.

Selection of human anti-CD28 scFvs from a T-NHL related scFv library using ribosome display.

Engineered antibodies have become an invaluable source of biopharmaceuticals against a wide range of diseases. About 200 antibody-based biologicals have been tested in clinical trials. Single chain variable fragments of antibodies (scFvs) provide binding specificity and offer an increased ease of in vitro display selection. Here, we present the generation of a human scFv library from peripheral blood lymphocyte RNA of a patient with relapsed T-cell non-Hodgkin lymphoma (T-NHL) who experienced a rare case of "spontaneous" remission. Antibodies against human T-cell antigen CD28, a co-stimulatory protein that influences the immune response by amplification of the transcriptional effects of T-cell receptors, might have contributed to the patient's remission. The scFv library was panned against CD28 using ribosome display and further subjected to affinity maturation. Isolated scFv were assessed for binding specificity and affinity and may provide the basis for the development of novel immunotherapeutic strategies. This work demonstrates the selection of a fully human antibody fragment from a patient-derived gene pool by in vitro ribosome display technology.

In vitro display technologies reveal novel biopharmaceutics.

Display technologies are fundamental to the isolation of specific high-affinity binding proteins for diagnostic and therapeutic applications in cancer, neurodegenerative, and infectious diseases as well as autoimmune and inflammatory disorders. Applications extend into the broad field of antibody (Ab) engineering, synthetic enzymes, proteomics, and cell-free protein synthesis. Recently, in vitro display technologies have come to prominence due to the isolation of high-affinity human antibodies by phage display, the development of novel scaffolds for ribosome display, and the discovery of novel protein-protein interactions. In vitro display represents an emerging and innovative technology for the rapid isolation and evolution of high-affinity peptides and proteins. So far, only one clinical drug candidate produced by in vitro display technology has been approved by the FDA for use in humans, but several are in clinical or preclinical testing. This review highlights recent advances in various engineered biopharmaceutical products isolated by in vitro display with a focus on the commercial developments.

Ribosome display for improved biotherapeutic molecules.

Ribosome display presents an innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Displayed proteins are bound to and recovered from target molecules in multiple rounds of selection in order to enrich for specific binding proteins. No transformation step is necessary, which could lead to a loss of library diversity. A cycle of display and selection can be performed in one day, enabling the existing gene repertoire to be rapidly scanned. Proteins isolated from the panning rounds can be further modified through random or directed molecular evolution for affinity maturation, as well as selected for characteristics such as protein stability, folding and functional activity. Recently, the field of display technologies has become more prominent due to the generation of new scaffolds for ribosome display, isolation of high-affinity human antibodies by phage display, and their implementation in the discovery of novel protein-protein interactions. Applications for this technology extend into the broad field of antibody engineering, proteomics, and synthetic enzymes for diagnostics and therapeutics in cancer, autoimmune and infectious diseases, neurodegenerative diseases and inflammatory disorders. This review highlights the role of ribosome display in drug discovery, discusses advantages and disadvantages of the system, and attempts to predict the future impact of ribosome display technology on the development of novel engineered biopharmaceutical products for biological therapies.

A new generation of protein display scaffolds for molecular recognition.

Engineered antibodies and their fragments are invaluable tools for a vast range of biotechnological and pharmaceutical applications. However, they are facing increasing competition from a new generation of protein display scaffolds, specifically selected for binding virtually any target. Some of them have already entered clinical trials. Most of these nonimmunoglobulin proteins are involved in natural binding events and have amazingly diverse origins, frameworks, and functions, including even intrinsic enzyme activity. In many respects, they are superior over antibody-derived affinity molecules and offer an ever-extending arsenal of tools for, e.g., affinity purification, protein microarray technology, bioimaging, enzyme inhibition, and potential drug delivery. As excellent supporting frameworks for the presentation of polypeptide libraries, they can be subjected to powerful in vitro or in vivo selection and evolution strategies, enabling the isolation of high-affinity binding reagents. This article reviews the generation of these novel binding reagents, describing validated and advanced alternative scaffolds as well as the most recent nonimmunoglobulin libraries. Characteristics of these protein scaffolds in terms of structural stability, tolerance to multiple substitutions, ease of expression, and subsequent applications as specific targeting molecules are discussed. Furthermore, this review shows the close linkage between these novel protein tools and the constantly developing display, selection, and evolution strategies using phage display, ribosome display, mRNA display, cell surface display, or IVC (in vitro compartmentalization). Here, we predict the important role of these novel binding reagents as a toolkit for biotechnological and biomedical applications.