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  1. Biologics Research Projects

Immunogenicity of Protein-based Therapeutics

Principal Investigator: Zuben E. Sauna, PhD
Office / Division / Lab: OTAT / DPPT / HB

General Overview

A major problem with protein-based therapeutics is their immunogenicity, that is, their tendency to trigger an unwanted immune response against themselves. One form of immune response is activation of B cells, which produce antibodies that bind to the proteins and reduce or eliminate their therapeutic effects. Such antibodies can also cause complications that can be life-threatening. Therefore, a critical part of determining the clinical safety and efficacy of protein-based therapeutic products is measuring their tendency to trigger antibody formation.

The immune response to protein-based therapeutics also involves T-cells, which help to activate B cells so they produce antibodies, including those that block protein therapeutics. This happens if the natural protein made by the body is defective in some way. In that case, the T-cells respond to a normal, artificial protein therapeutic as if it were foreign, since it is different from the defective, natural protein. A T-cell response mismatch like this sometimes occurs in the case of the protein FVIII, a protein that is critical to the body's ability to form blood clots to stop bleeding. People who do not have sufficient quantities of FVIII, or whose FVIII is defective in some way, suffer from hemophilia A, a disease in which blood clotting is defective and leads to excessive bleeding. The problem of defective FVIII is genetically based. Although there is no cure for hemophilia A, infusion of the FVIII therapeutic protein has been one of the most successful examples of management of a chronic disease. Unfortunately, the development of anti-drug antibodies against the infused FVIII is a significant impediment to this strategy. The treatment of patients that develop an immune response is more complex, less effective and exceedingly expensive. It now appears that individual variations in the tendency to develop of anti-drug antibodies may also be based on genetic differences. This is reflected in the clinical observation that persons with hemophilia A of Black African descent are twice as likely as patients of European Caucasian descent to produce antibodies against factor VIII proteins given as replacement therapy.

One strategy for preventing mismatches between natural FVIII and replacement FVIII is to design genetically engineered FVIII proteins so they do not trigger immune reactions. But there are so many differences among the immune systems of people that it is not likely that researchers will be able to design a FVIII protein that is safe for all of them. Therefore we propose to take a personalized approach to predicting--and avoiding--immune responses to FVIII proteins. Our long-term goal is to develop a gene-based approach to identifying individuals whose immune system is likely to react to specific versions of genetically engineered therapeutic proteins so these patients can be treated with versions of these proteins that are less likely to cause immune responses.

We are also addressing the problem of differences in the three-dimensional structures of protein-drugs and natural proteins that trigger B-cells to produce antibodies against therapeutic proteins. The current method for predicting whether certain parts of such proteins will trigger antibody formation is challenging and expensive. Therefore, we are using tiny pieces of DNA-like molecules called aptamers to probe the proteins and determine their exact shapes. Aptamers are made of strings of molecules called nucleic acids that fold up into specific shapes that depend on which nucleic acids are present and the order in which they occur in the aptamer. Therefore, by identifying which aptamer binds tightly to a specific part of a molecule, we can predict the shape of that part of the molecule, somewhat like predicting the shape of a lock by knowing the shape of the key that fits into it.

We are now using this technique to determine the shapes of both FVIII and the part of the anthrax toxin called protective antigen. If an aptamer loses its ability to bind to FVIII, for example, that would indicate that part of this blood clotting protein has changed shaped, increasing the likelihood that it will trigger an immune reaction that reduces its therapeutic activity. We are using this approach to determine if therapeutic proteins have shapes that will trigger antibody production. And we are collaborating with the Center for Drug Evaluation and Research to adapt this technology to analyze new protein products developed as copies of existing, approved protein drugs (biosimilars) to ensure they will be safe and effective.

Scientific Overview

1) Predicting interaction of T-cell epitopes with specific MHC Class II antigens.

Factor VIII (FVIII) is an essential component of the coagulation cascade and individuals deficient in coagulation factors exhibit lifelong bleeding disorders. The development of immunogenicity against therapeutic (infused) versions of FVIII is a significant impediment to the successful treatment of hemophiliacs.

About 50% of the cases of hemophilia A are caused by an inversion of the F gene's exons 1-22, which results in the production of a polypeptide representing those exons, but not 23-26. However, there is a nested gene within the F8 promoter that translates exons 23-26. Nevertheless, the 1-22 inversion means that the overlapping peptides generated from this protein do not include the junction between 1-22 and 23-26. While the peptides from the infused drug-protein FVIII that cover this junction would thus be foreign to the patient's immune system, they are not generally immunogenic. Rather, immunogenicity due to the differences between endogenous and infused FVIII is likely due to a variety of facts, especially single nucleotide polymorphisms (SNPs), but also missense mutations and deletions, and nonsense mutations, as well as inversions,

Therefore, the ideal (but not likely) solution to the immunogenicity problem of infused FVIII would be to design FVIII replacements that match each patient's haplotype and HLA type to avoid triggering an immune response. While designing such biologics to match every patient is not practical, it might be possible in cases where there are clear and significant differences between specific populations (e.g., between those of European Caucasian and Black African descent). In such cases it would be desirable to adapt the designs of endogenous FVIII to each group to ensure that one population does not get a disproportionate share of the benefits of a single version of FVIII while the other population bears a disproportionate share of the risks from the same infused FVIII.

Current technology permits the identification of haplotype backgrounds for FVIII as well as the development of at least a limited array of personalized Factor VIII drug-products. Therefore, our short term goal is to determine the 1) quantitative distribution of different haplotypes (SNPs) in individuals of European-Caucasian and Black-African descent; 2) distribution of MHC Class II antigens in these populations; 3) composition of FVIII used as drugs; and 4) disease-causing mutation, deletion or inversion in the F8 gene (FVIII) of individual patients. We will use this data to predict the immunogenicity of individual FVIII products in different populations and/or individual patients.

2) Development of aptamers as a tool for the investigation of protein-drug conformational epitopes.

Aptamers, nucleic acids capable of forming complex conformations, are potential tools for mapping protein conformation, identification, and prediction of immunogenic sites, and for circumventing immunogenicity. Our laboratory is developing single-stranded DNA aptamers to recombinant human Factor VIII.

We designed a naive DNA library to generate aptamers using defined 5’ and 3’ regions for PCR flanking a randomized 60-base region. The naïve DNA library was denatured and the ssDNA segments were allowed to fold into unique 3-dimensional shapes. (The 60 random bases would theoretically result in 460 unique conformers.) We incubated the pool of folded ssDNA with FVIII and through iterative SELEX (systematic evolution of ligands by exponential enrichment) cycles, we were able to select protein-binding aptamers.

Our laboratory selected a sampling of individual aptamers in cycles 3, 5 and 8 and cloned and sequenced them. We are using these clones to characterize the aptamers through analysis of the predicted 3-D structure, binding properties, and the effect on FVIII activity. In addition, we are doing in silico comparisons of these clones to follow the evolution of the aptamers.

3) Use of diverse analytical techniques to evaluate protein characteristics that may correlate with immunogenicity.

In collaboration with Drs. Mansoor Khan and Rakhi Shah (Division of Product Quality, CDER) we will analyze drug excipient interactions using thermal methods (differential scanning calorimetry, microcalorimetry, thermogravimetric analysis), spectroscopic techniques (Fourier transform infra-red, near infra-red, Raman), crystallography (X-ray diffraction), and nuclear magnetic resonance.

4) Characterization of conformation sensitive antibodies.

An alternative method to studying conformational epitopes of therapeutically important proteins is to develop and characterize antibodies that are sensitive to conformational changes. In collaboration with Dr. Chava Kimchi-Sarfaty (CBER) we characterized several antibodies that are sensitive to the conformation of the zinc metalloprotease ADAMTS13, a multi-domain protein that cleaves von Willebrand Factor and is implicated in thrombotic thrombocytopenic purpura. Our results suggest that these antibodies might be useful reagents for distinguishing functional and non-functional ADAMTS13, and for analyzing conformational transitions during the catalytic cycle.


J Med Genet 2017 May;54(5):338-45
Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.
Simhadri VL, Hamasaki-Katagiri N, Lin BC, Hunt R, Jha S, Tseng SC, Wu A, Bentley AA, Zichel R, Lu Q, Zhu L, Freedberg DI, Monroe DM, Sauna ZE, Peters R, Komar AA, Kimchi-Sarfaty C

J Thromb Haemost 2017 Apr;15(4):721-34
Modulating immunogenicity of Factor IX by fusion to an immunoglobulin Fc domain: a study using hemophilia B mouse model.
Levin D, Lagasse HA, Burch E, Strome S, Tan S, Jiang H, Sauna ZE, Golding B

F1000Res 2017 Feb 7;6:113
Recent advances in (therapeutic protein) drug development.
Lagasse HA, Alexaki A, Simhadri VL, Katagiri NH, Jankowski W, Sauna ZE, Kimchi-Sarfaty C

Sci Transl Med 2017 Jan 11;9(372):aag1286
Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools.
Lamberth K, Reedtz-Runge SL, Simon J, Klementyeva K, Pandey GS, Padkjær SB, Pascal V, León IR, Gudme CN, Buus S, Sauna ZE

Biomed Res Int 2015;2015:456348
Muscular dystrophy: disease mechanisms and therapies.
Pandey SN, Kesari A, Yokota T, Pandey GS

Pers Med 2015;12(4):403-15
Personalized approaches to the treatment of hemophilia A and B.
Simhadri VL, Banerjee AS, Simon J, Kimchi-Sarfaty C, Sauna ZE

PLoS One 2015 Jul 15;10(7):e0132433
Small ncRNA expression-profiling of blood from hemophilia A patients identifies miR-1246 as a potential regulator of Ffctor 8 gene.
Sarachana T, Dahiya N, Simhadri VL, Pandey GS, Saini S, Guelcher C, Guerrera MF, Kimchi-Sarfaty C, Sauna ZE, Atreya CD

Nucleic Acids Res 2015 Jul 13;43(12):5699-707
Large scale analysis of the mutational landscape in HT-SELEX improves aptamer discovery.
Hoinka J, Berezhnoy A, Dao P, Sauna ZE, Gilboa E, Przytycka TM

Blood 2015 Jan 8;125(2):223-8
The intron-22-inverted F8 locus permits intracellular factor VIII synthesis, explaining its low inhibitor risk and suggesting a role for pharmacogenomics.
Sauna ZE, Lozier JN, Kasper CK, Yanover C, Nichols T, Howard TE

Trends Biotechnol 2015 Jan;33(1):27-34
Fc fusion as a platform technology: potential for modulating immunogenicity.
Levin D, Golding B, Strome SE, Sauna ZE

J Interferon Cytokine Res 2014 Dec;34(12):931-7
Pharmacogenetics and the Immunogenicity of Protein Therapeutics.
Pandey GS, Sauna ZE

Res Comput Mol Biol 2014;8394:115-28
AptaCluster - A Method to Cluster HT-SELEX Aptamer Pools and Lessons from its Application.
Hoinka J, Berezhnoy A, Sauna ZE, Gilboa E, Przytycka TM

Trends In Genetics 2014 Jul;30(7):308-21
Exposing synonymous mutations.
Hunt RC, Simhadri VL, Iandoli M, Sauna ZE, Kimchi-Sarfaty C

Br J Haematol 2014 Apr;165(1):154-8
Single-nucleotide variations defining previously unreported ADAMTS13 haplotypes are associated with differential expression and activity of the VWF-cleaving protease in a Salvadoran congenital thrombotic thrombocytopenic purpura family.
Kim B, Hing ZA, Wu A, Schiller T, Struble EB, Liuwantara D, Kempert PH, Broxham EJ, Edwards NC, Marder VJ, Simhadri VL, Sauna ZE, Howard TE, Kimchi-Sarfaty C

Nat Med 2013 Oct;19(10):1318-24
Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A.
Pandey GS, Yanover C, Miller-Jenkins LM, Garfield S, Cole SA, Curran JE, Moses EK, Rydz N, Simhadri V, Kimchi-Sarfaty C, Lillicrap D, Viel KR, Przytycka TM, Pierce GF, Howard TE, Sauna ZE, the PATH (Personalized Alternative Therapies for Hemophilia) Study Investigators

Trends Pharmacol Sci 2013 Oct;34(10):534-48
Building better drugs: developing and regulating engineered therapeutic proteins.
Kimchi-Sarfaty C, Schiller T, Hamasaki-Katagiri N, Khan MA, Yanover C, Sauna ZE

PLoS Comput Biol 2013 May;9(5):e1003066
Polymorphisms in the F8 gene and MHC-II variants as risk factors for the development of inhibitory anti-factor VIII antibodies during the treatment of hemophilia a: a computational assessment.
Pandey GS, Yanover C, Howard TE, Sauna ZE

Biomed Res Int 2013;2013:793502
Detection of intracellular Factor VIII protein in peripheral blood mononuclear cells by flow cytometry.
Pandey GS, Tseng SC, Howard TE, Sauna ZE

J Biol Chem 2012 Dec 28;287(53):44361-71
Cyclosporin A impairs the secretion and activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat).
Hershko K, Simhadri V, Blaisdell A, Hunt RC, Newell J, Tseng SC, Hershko AY, Choi JW, Sauna ZE, Wu A, Bram RJ, Komar AA, Kimchi-Sarfaty C

Haemophilia 2012 Nov;18(6):933-40
Analysis of F9 point mutations and their correlation to severity of haemophilia B disease.
Hamasaki-Katagiri N, Salari R, Simhadri VL, Tseng SC, Needlman E, Edwards NC, Sauna ZE, Grigoryan V, Komar AA, Przytycka TM, Kimchi-Sarfaty C

J Thromb Haemost 2012 Sep;10(9):1961-5
Observations regarding the immunogenicity of BDD-rFVIII derived from a mechanistic personalized medicine perspective.
Sauna ZE, Ameri A, Kim B, Yanover C, Viel KR, Rajalingam R, Cole SA, Howard TE

PLoS One 2012;7(6):e38864
Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches.
Edwards NC, Hing ZA, Perry A, Blaisdell A, Kopelman DB, Fathke R, Plum W, Newell J, Allen CE, S G, Shapiro A, Okunji C, Kosti I, Shomron N, Grigoryan V, Przytycka TM, Sauna ZE, Salari R, Mandel-Gutfreund Y, Komar AA, Kimchi-Sarfaty C

Bioinformatics 2012 Jun 15;28(12):i215-i223
Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.
Hoinka J, Zotenko E, Friedman A, Sauna ZE, Przytycka TM

Biologicals 2012 May;40(3):191-5
Plasma derivatives: new products and new approaches.
Sauna ZE, Pandey GS, Jain N, Mahmood I, Kimchi-Sarfaty C, Golding B

PLoS One 2012;7(2):e31948
Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins.
Zichel R, Chearwae W, Pandey GS, Golding B, Sauna ZE

Nat Rev Genet 2011 Oct;12(10):683-91
Understanding the contribution of synonymous mutations to human disease.
Sauna ZE, Kimchi-Sarfaty C

Nat Biotechnol 2011 Oct 13;29(10):870-3
Pharmacogenetics and the immunogenicity of protein therapeutics.
Yanover C, Jain N, Pierce G, Howard TE, Sauna ZE

Biochemistry 2011 May 10;50(18):3724-35
Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5'-Fluorosulfonylbenzoyl 5'-Adenosine: Evidence for an ATP Analogue That Interacts with Both Drug-Substrate-and Nucleotide-Binding Sites.
Ohnuma S, Chufan E, Nandigama K, Jenkins LM, Durell SR, Appella E, Sauna ZE, Ambudkar SV

Biochemistry 2010 Jun 1;49(21):4440-9
The signaling interface of the yeast multidrug transporter Pdr5 adopts a cis conformation, and there are functional overlap and equivalence of the deviant and canonical Q-loop residues.
Ananthaswamy N, Rutledge R, Sauna ZE, Ambudkar SV, Dine E, Nelson E, Xia D, Golin J

PLoS One 2009 Aug 5;4(8):e6506
Characterization of conformation-sensitive antibodies to ADAMTS13, the von Willebrand cleavage protease.
Sauna ZE, Okunji C, Hunt RC, Gupta T, Allen CE, Plum E, Blaisdell A, Grigoryan V, Geetha S, Fathke R, Soejima K, Kimchi-Sarfaty C

Cytometry A 2009 Aug;75(8):675-81
Detection of intracellular ADAMTS13, a secreted zinc-metalloprotease, via flow cytometry.
Geetha S, Allen CE, Hunt RC, Plum E, Garfield S, Friedman SL, Soejima K, Sauna ZE, Kimchi-Sarfaty C

J Biol Chem 2008 Dec 12;283(50):35010-22
Mutations define cross-talk between the N-terminal nucleotide-binding domain and transmembrane helix-2 of the yeast multidrug transporter Pdr5: possible conservation of a signaling interface for coupling ATP hydrolysis to drug transport.
Sauna ZE, Bohn SS, Rutledge R, Dougherty MP, Cronin S, May L, Xia D, Ambudkar SV, Golin J