Background Autologous chimeric antigen receptor (CAR) T cell therapies have revolutionized the treatment of some cancers and are now demonstrating effects in autoimmune disease. With several approved therapies on the market and cure rates approaching 50% in various hematologic malignancies, access to these life-saving therapies is paramount. However, challenges with cell manufacturing, scaling, and the need for inpatient treatment necessitate a truly off-the-shelf solution, especially for non-oncology indications. Utilizing the success of mRNA lipid nanoparticles (LNP) as COVID vaccines, Capstan Therapeutics has developed a novel targeted LNP (tLNP) platform that is purpose-built for specific delivery of therapeutic mRNAs to immune cells through functionalization with a targeting antibody. Results Capstan has developed rationally designed proprietary LNPs that exhibit significantly reduced delivery to liver compared to conventional LNPs and effectively deliver to T cells when functionalized with a T cell specific targeting antibody. These Capstan proprietary tLNPs were well tolerated following a single intravenous dose up through 6mg/kg in male Sprague Dawley rats, a highly sensitive species for evaluating LNP toxicity. In vitro screens for the optimization of payload mRNA components, including UTR sequences and codon usage, resulted in a several fold increase in CAR expression and promoted improved tumor cell killing. CD5 and CD8 antibody tLNPs delivered a reporter gene mRNA payload to human cells in vivo in a humanized mouse model at high efficiency and specificity. CD8 tLNPs specifically reprogrammed CD8 T cells effectively with minimal reporter expression in CD4 T cells, whereas CD5 tLNPs reprogrammed both CD8 and CD4 T cell populations. Delivery of an anti-CD19 mRNA CAR construct by both CD5 and CD8 tLNPs into a human PBL-engrafted Nalm6 tumor-bearing mouse model resulted in rapid clearance of the tumor. Repeat doses of tLNPs (BIWx5) at dose levels up through 30 µg/animal were well tolerated and the CAR was expressed on T cells in vivo after single and repeat dosing. Conclusions Capstan's tLNPs can specifically target and deliver a therapeutic CAR payload in vivo resulting in functional anti-tumor CAR T cells. This non-viral, redosable approach promises to improve access, efficacy, and safety, owing in part to lack of harsh lymphodepletion conditioning. Due to the versatility of this platform, treatments for various disease categories can be envisioned using different targeting binders to deliver a broad set of payloads to diverse cell populations. Ethics Approval This study complied with all relevant ethical regulations and all animal protocols were approved by the Explora BioLabs (AAALAC-accredited) Institutional Animal Care and Use Committee (IACUC).

Mechanisms of chimeric antigen receptor (CAR) T cell-mediated antitumor immunity and toxicity remain poorly characterized because few studies examine the intact tumor microenvironment (TME) following CAR T cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 CAR T cell therapy approved for patients with large B cell lymphoma. We devised multiplex immunostaining and ISH assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B cell lymphoma following axicabtagene ciloleucel infusion. We found that a majority of intratumoral CAR T cells expressed markers of T cell activation but, unexpectedly, constituted ≤5% of all T cells within the TME 5 days or more after therapy. Large numbers of T cells without CAR were also activated within the TME after axicabtagene ciloleucel infusion; these cells were positive for Ki-67, IFN-γ, granzyme B (GzmB), and/or PD-1 and were found at the highest levels in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and were found at their highest numbers in biopsies with CAR T cells. These data suggest that intratumoral CAR T cells are associated with non-CAR immune cell activation within the TME with both beneficial and pathological effects.

Introduction: Chimeric antigen receptor (CAR) T cell therapy is a transformative treatment modality in B cell malignancies. As next generation CARs are developed to improve durable response rates and expand into other indications of unmet need, the proper tools to identify, characterize, and modulate CAR activity will play a pivotal role in the optimization of this therapeutic option. This study developed and evaluated one approach, that of using monoclonal antibodies (mAbs) against common elements utilized in approved and investigational CARs.Methods: The mAbs KIP-1 and KIP-4 were raised in rabbits against the linkers of 2 single-chain variable fragments, the Whitlow linker (Whitlow, et al. Protein Eng. 1993) and the G4S linker (Huston JS, et al. PNAS. 1988), respectively, to generate universal detection reagents without adding an exogenous peptide recognition motif. Specificity and sensitivity were assessed by flow cytometric analysis of CAR T cells vs nontransduced T cells, immunohistochemistry (IHC) staining of embedded cell pellets with or without a CAR carrying the relevant linker, and epitope mapping by ELISA. Activation was assessed in a competitive stimulation assay in which nontransduced T cells and CAR T cells were mixed at defined ratios and stimulated broadly with OKT3 or specifically with KIP-1 or KIP-4.Results: Flow cytometric analysis demonstrated that CAR T cells with the Whitlow linker could be detected by phycoerythrin-conjugated KIP-1 at ≤ 10 ng per million cells with no detectable staining on nontransduced T cells above background levels. To further demonstrate specificity, IHC data revealed that KIP-1 bound to T cell pellets expressing CARs containing the Whitlow linker, but not the G4S linker. Epitope mapping revealed that the minimal KIP-1 epitope was contained within the amino acid sequence SGKPGSGE. Furthermore, KIP-1 was able to effectively identify CAR T cells in patient samples by both flow cytometry and IHC. Having established that KIP-1 was specific and sensitive, activation assays were performed to determine its CAR T cell-specific activating capacity. KIP-1 specifically activated CAR T cells, but not nontransduced T cells, as demonstrated by CAR T cell expansion and upregulation of cell surface activation markers, including CD69 and 4-1BB. KIP-4 was also assessed for specificity, sensitivity, and activation capacity with similar results.Conclusions: Taking advantage of the linear epitopes within the commonly-used Whitlow and G4S linkers, KIP-1 and KIP-4, respectively, can be used in a wide variety of phenotypic and functional assays with CARs of various specificities that share these linkers. These tools provide the means to realize the full potential of investigational CAR T cell products by supporting research, clinical, and manufacturing efforts.Citation Format: Stuart A. Sievers, Keith A. Kelley, Stephanie H. Astrow, Adrian Bot, Jed J. Wiltzius. Design and development of anti-linker antibodies for the detection and characterization of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1204.

Amyloid diseases are characterized by the deposition of proteins in the form of amyloid fibrils, in organs that eventually fail. The development of effective drug candidates follows from the understanding of the molecular processes that lead to protein aggregation. Here, we study amyloidogenic segments of transthyretin (TTR). TTR is a transporter of thyroxine and retinol in the blood and cerebrospinal fluid. When mutated and/or as a result of aging, TTR aggregates into amyloid fibrils that accumulate in organs such as the heart. Recently, we reported two amyloidogenic segments that drive amyloid aggregation. Here, we report the crystal structure of another six amyloidogenic segments of TTR. We found that the segments from the C‐terminal region of TTR form in‐register steric‐zippers with highly‐interdigitated, wet interfaces, whereas the β‐strand B from the N‐terminal region of TTR forms an out‐of‐register assembly, previously associated with oligomeric formation. Our results contribute fundamental information for understanding the mechanism of aggregation of TTR.

Anti-CD19 chimeric antigen receptor (CAR) T cells have caused remissions of B cell malignancies, but problems including cytokine-mediated toxicity and short persistence of CAR T cells in vivo might limit the effectiveness of anti-CD19 CART cells. Anti-CD19 CARs that have been tested clinically had single-chain variable fragments (scFvs) derived from murine antibodies. We have designed and constructed novel anti-CD19 CARs containing a scFv with fully human variable regions. T cells expressing these CARs specifically recognized CD19(+) target cells and carried out functions including degranulation, cytokine release, and proliferation. We compared CARs with CD28 costimulatory moieties along with hinge and transmembrane domains from either the human CD28 molecule or the human CD8 alpha molecule. Compared with T cells expressing CARs with CD28 hinge and transmembrane domains, T cells expressing CARs with CD8 alpha hinge and trans membrane domains produced lower levels of cytokines and exhibited lower levels of activation-induced cell death (AICD). Importantly, CARs with hinge and transmembrane regions from either CD8 alpha or CD28 had similar abilities to eliminate established tumors in mice. In anti-CD19 CARs with CD28 costimulatory moieties, lower levels of inflammatory cytokine production and AICD are potential clinical advantages of CD8 alpha hinge and transmembrane domains over CD28 hinge and transmembrane domains.

Background: Multiple myeloma (MM) is a usually fatal malignancy of plasma cells, with no current therapy considered curative. About 15% of patients diagnosed with MM are stratified as high risk with poor treatment outcomes and short (2-3 years) survival from diagnosis. Standard risk patients tend to live longer but undergo chronic and/or high intensity therapy and likely experience a relapsing and remitting disease pattern. Therefore, there is still a considerable unmet need for innovative therapies that improve outcomes in MM. One such approach is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against malignant cells. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. KITE-585 was developed as a CAR T cell immunotherapy product candidate directed against B cell maturation antigen (BCMA). BCMA is nearly ubiquitously expressed on MM cells, plasma cells and subsets of mature B cells, but with limited or absent expression on other tissues. Methods: We generated >50 fully human IgGs directed against BCMA using the BCMA protein as antigen and selection criteria including affinity, cross-reactivity and poly-specificity. Following assessment of the binding of the IgGs to a MM cell line known to express BCMA, >10 IgGs were identified that met the criteria for affinity and selectivity and had a >50-fold binding over background. The 8 IgGs that demonstrated the highest specific binding were then sequence-converted to single-chain variable fragments (scFvs) and incorporated into CARs. Results: In all but one case, human T cells engineered to express these CAR constructs exhibited specific cytolytic activity against MM cell lines (NCI-H929 and MM.1s). These CAR T cells demonstrated killing efficiencies of >95% at effector:target ratios of 1:1 over a 24-hour period. Similarly antigen-specific production of inflammatory cytokines was observed in response to target cell lines in vitro. Assessment of antigen-dependent proliferation over a 5 day period revealed >80% proliferation in the 7 constructs that showed cytolytic activity in vitro. Multiple different anti-BCMA CAR constructs representing distinct epitope binding bins of BCMA were then selected for in vivo evaluation. In two disseminated tumor models of luciferase labeled NCI-H929 or MM.1s cells injected intravenously (i.v.), a single i.v. injection of anti-BCMA CAR T-cells delayed the progression of disease and significantly increased survival when compared to control treatment. Conclusions: The results of these studies highlight the potential of targeting BCMA with adoptive transfer of engineered T cells for the treatment of MM. Given these positive findings, progress towards Phase 1 clinical studies in MM patients with KITE-585 is continuing. Citation Format: Gregor B. Adams, Jun Feng, Atefah Ghogha, Armen Mardiros, Jodi Murakami, Tammy Phung, Ruben Rodriguez, Stuart Sievers, Tassja J. Spindler, Jed Wiltzius, Clare Yarka, Sean C. Yoder, Tony Polverino. Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2017-4979

Some HIV-1-infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual's serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1(YU2)-infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection.

Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design.

The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.

Abstract HIV/AIDS remains one of the most serious threats to global public health. Although anti-HIV drugs have been effective among the wealthiest populations, new methods to prevent infections are needed to control HIV-1 infections globally. Strategies to combat HIV-1 benefit from structural knowledge of how antibodies recognize HIV envelope proteins and how the immune system eliminates viruses. Until recently, only a small number of broadly neutralizing antibodies against HIV-1 had been characterized, and the immunological basis for their breadth and potency remains poorly understood. However, it was recently demonstrated that antibodies could be engineered to greatly enhance their breadth and potency. Unfortunately, these and other engineering efforts can result in a decrease in antibody half-life in various animal models. This decrease in half-life correlates with polyreactivity, an increase in reactivity to a variety of antigens. In order to make better targets for passive delivery therapies, we are using a variety of computational and structure-based techniques. We have constructed several mutations in regions that have been predicted to have high aggregation propensities, and have shown that these novel reagents have reduced polyreactivity and longer in vivo half-lives, yet maintain potency in neutralization assays. Further characterization will help further our understanding of the relationship between antibody potency, polyreactivity, and half-life.

A New Glycan-Dependent CD4-Binding Site Neutralizing Exerts Pressure HIV-1 Abstract The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s of CD4bs is a central viral vulnerability site and isolation of new anti-HIV-1 CD4bs broadly neutralizing antibodies (bNAbs) provides information about viral escape mechanisms. Here we describe a new anti-HIV-1 bNAb that was isolated from an HIV-1 infected donor. The antibody, 179NC75, targets the CD4 binding site in a glycan-dependent man-ner. Although many CD4bs antibodies have been already described, a glycan-dependent mode of recognition is unusual for anti-HIV-1 CD4bs bNAbs. The glycan-dependent CD4bs antibodies have never been tested for their ability to neutralize HIV-1 in vivo . We infected humanized mice with HIV-1 YU2 and treated them with 179NC75 three weeks after infection. We observed a drop in viral load immediately after treatment followed by a viral rebound. The viral rebound was associated with specific escape mutations in the plasma virus envelope, resulting in a deletion of N276 glycan, and in some cases a glycan shift from position 276 to position 460. Similar signature mutations were found in the envelope of the autologous virus cloned from patient ’ s plasma. This defines the escape pathways from 179NC75, and shows that they are the same in humans and in HIV-1 YU2 infected humanized mice.

PURPOSE OF REVIEW:This review highlights recent developments in HIV-1 antibody engineering and discusses the effects of increased polyreactivity on serum half-lives of engineered antibodies.RECENT FINDINGS:Recent studies have uncovered a wealth of information about the relationship between the sequences and efficacies of anti-HIV-1 antibodies through a combination of bioinformatics, structural characterization and in vivo studies. This knowledge has stimulated efforts to enhance antibody breadth and potency for therapeutic use. Although some engineered antibodies have shown increased polyreactivity and short half-lives, promising efforts are circumventing these problems.SUMMARY:Antibodies are desirable as therapeutics due to their ability to recognize targets with both specificity and high affinity. Furthermore, the ability of antibodies to stimulate Fc-mediated effector functions can increase their utility. Thus, mAbs have become central to strategies for the treatment of various diseases. Using both targeted and library-based approaches, antibodies can be engineered to improve their therapeutic properties. This article will discuss recent antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. The polyreactivity of engineered HIV-1 bNAbs and the effect on serum half-life will be explored along with strategies to overcome problems introduced by engineering antibodies. Finally, advances in creating bispecific anti-HIV-1 reagents are discussed.

Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. At the morphological level, these fibers appear similar and are termed “amyloid.” From x‐ray and electron diffraction, we found that the adhesive segments of amyloid fibers are short protein sequences which form pairs of interdigitated, in‐register beta sheets. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that in at least some of the neurodegenerative diseases, smaller, often transient and polymorphic oligomers are the toxic entities. In attempts to determine structures for such oligomers, we have discovered segments of amyloid‐forming proteins that form toxic, antiparallel beta, out‐of‐register structures. In one case, the oligomer is a cylindrical barrel, formed from six anti‐parallel, out‐of‐register protein strands, which we term a cylindrin. In another case, the oligomer is an open, continuous cylindrin‐like structure that we term a corkscrew. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers, and are distinct in structure from amyloid fibrils. From the known structure of the spine of an amyloid fibril, we find it is possible to design an inhibitor of fibril formation.

AIDS Research and Human RetrovirusesVol. 30, No. S1 Glycans and Antibody Effector FunctionsEngineering Antibodies to Enhance Activity and Increase Half-lifeStuart A. Sievers, Sonal N. Patel, Kathleen Bennett, Florian Klein, Michel C. Nussenzweig, and Pamela J. BjorkmanStuart A. SieversCalifornia Institute of Technology, Biology and Biological Engineering, Pasadena, CA, United StatesSearch for more papers by this author, Sonal N. PatelCalifornia Institute of Technology, Biology and Biological Engineering, Pasadena, CA, United StatesSearch for more papers by this author, Kathleen BennettCalifornia Institute of Technology, Biology and Biological Engineering, Pasadena, CA, United StatesSearch for more papers by this author, Florian KleinRockefeller University, Laboratory of Molecular Immunology, New York, NY, United StatesSearch for more papers by this author, Michel C. NussenzweigRockefeller University, Laboratory of Molecular Immunology, New York, NY, United StatesSearch for more papers by this author, and Pamela J. BjorkmanCalifornia Institute of Technology, Biology and Biological Engineering, Pasadena, CA, United StatesSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5456.abstractAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Engineering Antibodies to Enhance Activity and Increase Half-life." AIDS Research and Human Retroviruses, 30(S1), p. A210FiguresReferencesRelatedDetailsCited byOvercoming low yields of plant-made antibodies by a protein engineering approach22 December 2015 | Biotechnology Journal, Vol. 11, No. 1Antibody engineering for increased potency, breadth and half-lifeCurrent Opinion in HIV and AIDS, Vol. 10, No. 3 Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Stuart A. Sievers, Sonal N. Patel, Kathleen Bennett, Florian Klein, Michel C. Nussenzweig, and Pamela J. Bjorkman.Engineering Antibodies to Enhance Activity and Increase Half-life.AIDS Research and Human Retroviruses.Oct 2014.A210-A210.http://doi.org/10.1089/aid.2014.5456.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

Amyloid fibers, once exclusively associated with disease, are acquiring utility as a class of biological nanomaterials. Here we introduce a method that utilizes the atomic structures of amyloid peptides, to design materials with versatile applications. As a model application, we designed amyloid fibers capable of capturing carbon dioxide from flue gas, to address the global problem of excess anthropogenic carbon dioxide. By measuring dynamic separation of carbon dioxide from nitrogen, we show that fibers with designed amino acid sequences double the carbon dioxide binding capacity of the previously reported fiber formed by VQIVYK from Tau protein. In a second application, we designed fibers that facilitate retroviral gene transfer. By measuring lentiviral transduction, we show that designed fibers exceed the efficiency of polybrene, a commonly used enhancer of transduction. The same procedures can be adapted to the design of countless other amyloid materials with a variety of properties and uses.

Amyloid fibers, once exclusively associated with disease, are acquiring utility as a class of biological nanomaterials. Here we introduce a method that utilizes the atomic structures of amyloid peptides, to design materials with versatile applications. As a model application, we designed amyloid fibers capable of capturing carbon dioxide from flue gas, to address the global problem of excess anthropogenic carbon dioxide. By measuring dynamic separation of carbon dioxide from nitrogen, we show that fibers with designed amino acid sequences double the carbon dioxide binding capacity of the previously reported fiber formed by VQIVYK from Tau protein. In a second application, we designed fibers that facilitate retroviral gene transfer. By measuring lentiviral transduction, we show that designed fibers exceed the efficiency of polybrene, a commonly used enhancer of transduction. The same procedures can be adapted to the design of countless other amyloid materials with a variety of properties and uses.

ALS is a terminal disease of motor neurons that is characterized by accumulation of proteinaceous deposits in affected cells. Pathological deposition of mutated Cu/Zn superoxide dismutase (SOD1) accounts for ∼20% of the familial ALS (fALS) cases. However, understanding the molecular link between mutation and disease has been difficult, given that more than 140 different SOD1 mutants have been observed in fALS patients. In addition, the molecular origin of sporadic ALS (sALS) is unclear. By dissecting the amino acid sequence of SOD1, we identified four short segments with a high propensity for amyloid fibril formation. We find that fALS mutations in these segments do not reduce their propensity to form fibrils. The atomic structures of two fibril-forming segments from the C terminus, (101)DSVISLS(107) and (147)GVIGIAQ(153), reveal tightly packed β-sheets with steric zipper interfaces characteristic of the amyloid state. Based on these structures, we conclude that both C-terminal segments are likely to form aggregates if available for interaction. Proline substitutions in (101)DSVISLS(107) and (147)GVIGIAQ(153) impaired nucleation and fibril growth of full-length protein, confirming that these segments participate in aggregate formation. Our hypothesis is that improper protein maturation and incompletely folded states that render these aggregation-prone segments available for interaction offer a common molecular pathway for sALS and fALS.

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases.