IP Library › Granted Patent US 12,258,594
Granted Patent B2
US 12,258,594 · App. 16/704,857 · Granted Mar 25, 2025

Cas proteins with reduced immunogenicity and methods of screening thereof

Inventor: Feng Zhang (Cambridge, MA)
Assignees: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
C12N9/22C12N15/907C12N2310/20
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,258,594
App. No.
16/704,857
Granted
Mar 25, 2025
Kind
B2
Abstract

The present disclosure generally relates to systems, methods and compositions related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. The present disclosure also relates to methods, systems, and compostions modified to reduce immunogenicity. Additionally, the present disclosure relates to methods for developing or designing CRISPR-Cas system based therapy or therapeutics.

Claims (23)

1. A method of reducing immunogenicity of a S. pyogenes CRISPR-associated (SpCas9) protein as compared to a naturally occurring SpCas9 protein by mutating one or more immunogenic T cell epitopes in a SpCas9 protein; said method comprising the steps of

determining immunogenicity of the one or more T cell epitopes by measuring affinity of a peptide containing the T cell epitope for one or more MHC molecules,

wherein the amino acid sequence of the T cell epitope is chosen from WRQLLNAKL (SEQ TD NO: 223), VITLKSKLV (SEQ ID NO: 224), LFDDKVMKQ (SEQ ID NO: 226), LIHIDDSLTF (SEQ TD NO: 227), LVSDFRKDF (SEQ ID NO: 228), FLAAKNLSD (SEQ ID NO: 230), IEKIL,TFRI (SEQ TD NO: 233), IYIJLRKKLV (SEQ ID NO: 236), LAHIMIKFRG (SEQ ID NO: 237), IL,SARLSKS (SEQ TD NO: 238), LKALVRQQL (SEQ ID NO: 239), YKFIKPIL,E (SEQ ID NO: 240), LTLLKALVR (SEQ ID NO: 242), FYKFIKPIL, (SEQ ID NO: 243), LFKTNRKVT (SEQ TD NO: 244), MKQLKRRRY (SEQ ID NO: 245), WGRLSRKLI (SEQ ID NO: 246), ARLSKSRRL (SEQ ID NO: 248), IYLALAHMI (SEQ ID NO: 250), MIKFRGHFL (SEQ ID NO: 251), or FLYLASHYE (SEQ ID NO: 252),

ordering the T cell epitopes of the SpCas9 protein based on immunogenicity;

mutating one or more of the most immunogenic T cell epitopes;

determining for the reduced immunogenicity SpCas9 protein, Cas-crRNA complex formation and binding to a PAM-containing target; and

if the reduced immunogenicity SpCas9 protein can bind to a PAM-containing target, performing phage-assisted continuous evolution (PACE) to restore or improve Cas-crRNA complex formation and binding to a PAM-containing target of the reduced immunogenicity SpCas9 protein.

2. The method of claim 1 , which comprises mutating the SpCas9 proteins containing one or more mutations at one or more amino acid positions and screening the mutant proteins for one or more Cas activities.

3. The method of claim 1 , wherein nuclease activity of the SpCas9 protein is preserved.

4. The method of claim 1 , wherein one or more catalytically active site residues of the SpCas9 protein are unchanged.

5. The method of claim 1 , wherein one or more residues that determine complex formation with a guide are unchanged.

6. The method of claim 1 , wherein target specificity of a CRISPR system comprising the SpCas9 protein is maintained or increased.

7. The method of claim 1 , which comprises deleting, inserting, or mutating one or more amino acids in the immunogenic T cell epitope.

8. The method of claim 1 , wherein identification of a T cell epitope comprises determining the sequence of one or more peptides from the SpCas9 protein that bind to an MHC receptor; or comparison of the CRISPR protein to a database of peptides that bind to an MHC receptor.

9. The method of claim 8 , wherein the comparison is in silico.

10. The method of claim 1 , wherein the MHC receptor is a class II MHC receptor.

11. The method of claim 1 , wherein the SpCas9 protein is associated with a functional domain.

12. The method of claim 11 , wherein the functional domain comprises:

a mutation that reduces immunogenicity;

an activator, a repressor, or a DNA methylase; or

a base editor.

13. The method of claim 1 , wherein in the PACE step the selection phage (SP) encodes a catalytically dead mutant of the reduced immunogenicity SpCas9 protein, wherein the SpCas9 protein is fused to the ω subunit of bacterial RNA polymerase, and wherein the accessory plasmid (AP) encodes an sgRNA and a PAM and protospacer upstream of the M13 phage gene III, whereby mutations in the reduced immunogenicity SpCas9 protein restore or improve Cas-crRNA complex formation and binding to the PAM-containing target causing increased gene III expression.

14. The method of claim 1 , wherein ten or more of the most immunogenic T cell epitopes are mutated.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 26, 2020
From: ZHANG, FENG
To: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 052231/0343 →
Continuity (7)
Provisional Application 62775839 · Dec 5, 2018
Provisional Application 62775832 · Dec 5, 2018
Provisional Application 62775860 · Dec 5, 2018
Provisional Application 62775855 · Dec 5, 2018
Provisional Application 62775877 · Dec 5, 2018
Provisional Application 62775812 · Dec 5, 2018
Related Publication 20200199555A1 · Jun 25, 2020
References Cited (88)
US 11208640B2 · Ewaisha · 2021 [cited by examiner]
US 11566052B2 · Payne · 2023 [cited by examiner]
US 20180319850A1 · Payne · 2018 [cited by examiner]
US 20200172931A1 · Liu · 2020 [cited by examiner]
WO 2014093622A2 · 2014 [cited by applicant]
WO 2019005884A1 · 2019 [cited by applicant]
WO 2019005886A1 · 2019 [cited by applicant]
WO 2019060746A1 · 2019 [cited by applicant]
WO 2019071048A1 · 2019 [cited by applicant]
WO 2019084013A1 · 2019 [cited by applicant]
WO 2019084062A1 · 2019 [cited by applicant]
Chew et al., A multi-functional AAV-CRISPR-Cas9 and its host response. Nature Methods, 2016, vol. 13(10): 868-874. (Year: 2016). [cited by examiner]
Chew WL., Immunity to CRISPR Cas9 and Cas12 therapeutics. WIREs Syst Biol Med., 2018, 10:e1408, pp. 1-23. (Year: 2018). [cited by examiner]
Moreno et al., Exploring protein orthogonality in immune space: a case study with AAV and Cas9 orthologs. bioRxiv, version posted Jan. 10, 2018, pp. 1-24). (Year: 2018). [cited by examiner]
De Groot et al., De-immunization of therapeutic proteins by T-cell epitope modification. Dev. Biol., (Basel), Basel, Krager, 2005, vol. 122: 171-194. (Year: 2005). [cited by examiner]
Fridman et al., An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform. OncoImmunology, 2012, vol. 1:8: 1258-1270. (Year: 2012). [cited by examiner]
Ogishi et al., The landscape of T cell epitope immunogenicity in sequence space. bioRxiv preprint this version posted Oct. 9, 2018, pp. 1-63. (Year: 2018). [cited by examiner]
Vaughan et al., Deciphering the MHC-associated peptidome: a review of naturally processed ligand data. Expert Review of Proteomics, 2017, vol. 14, (9): 729-736. (Year: 2017). [cited by examiner]
Hu et al., Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 2018, vol. 556: 57-63. (Year: 2018). [cited by examiner]
Ferdosi SR., Genome-Driven Targeted Cancer Therapy. Ph.D., Thesis, Arizona State University, Dec. 2017, pp. 1-118. (Year: 2017). [cited by examiner]
Abelin, et al., “Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction”, Immunity, vol. 46, Issue 2, Feb. 21, 2017, 315-326. [cited by applicant]
Atanasova, et al., “EpiDOCK: A Molecular Docking-Based Tool for MHC Class II Binding Prediction”, Protein Engineering, Design and Selection, vol. 26, No. 10, May 9, 2013, 631-634. [cited by applicant]
Barra, et al., “Footprints of Antigen Processing Boost MHC Class II Natural Ligand Predictions”, Genome Medicine, vol. 10, No. 84, 2018, 15 pages. [cited by applicant]
Bhasin, et al., “A Hybrid Approach for Predicting Promiscuous MHC Class I Restricted T Cell Epitopes”, Journal of Biosciences, vol. 31, No. 1, Jan. 2007, 31-42. [cited by applicant]
Bhasin, et al., “SVM Based Method for Predicting HLA-DRB1*0401 Binding Peptides in an Antigen Sequence”, Bioinformatics, vol. 20, No. 3, 2004, 421-423. [cited by applicant]
Canver, et al., “BCL11A Enhancer Dissection by Cas9-Mediated In Situ Saturating Mutagenesis”, Nature, vol. 527, Nov. 12, 2015, 192-197. [cited by applicant]
Chen, et al., “Genome-Wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis”, Cell, vol. 160, No. 6, Mar. 12, 2015, 1246-1260. [cited by applicant]
Choi, et al., “EpiSweep: Computationally Driven Reengineering of Therapeutic Proteins to Reduce Immunogenicity While Maintaining Function”, Methods in Molecular Biology, vol. 1529, 2017, 375-398. [cited by applicant]
Cong, et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems”, Science, vol. 339, No. 6121, Feb. 15, 2013, 819-823. [cited by applicant]
Dhanda, et al., “Development of a Strategy and Computational Application to Select Candidate Protein Analogues with Reduced HLA Binding and Immunogenicity”, Immunology, vol. 153, No. 1, 2018, 118-132. [cited by applicant]
Dhanda, et al., “Prediction of IL4 Inducing Peptides”, Clinical and Developmental Immunology, vol. 2013, Article 263952, 2013, 9 pages. [cited by applicant]
Dimitrov, et al., “EpiTOP—A Proteochemometric Tool for MHC Class II Binding Prediction”, Bioinformatics, vol. 26, No. 16, 2010, 2066-2068. [cited by applicant]
Doench, et al., “Rational Design of Highly Active SgRNAs for CRISPR-Cas9-Mediated Gene Inactivation”, Nature Biotechnology, vol. 32, No. 12, Dec. 2014, 1262-1267. [cited by applicant]
Donnes, et al., “Integrated Modeling of the Major Events in the MHC Class I Antigen Processing Pathway”, Protein Science, vol. 14, No. 8, 2005, 2132-2140. [cited by applicant]
Donnes, et al., “Prediction of MHC Class I Binding Peptides, using Svmhc”, BMC Bioinformatics, vol. 3, No. 25, Sep. 2002, 8 pages. [cited by applicant]
Doytchinova, et al., “EpiJen: A Server for Multistep T Cell Epitope Prediction”, BMC Bioinformatics, vol. 7, Article No. 131, Feb. 2006, 11 pages. [cited by applicant]
Gao, et al., “Engineered Cpf1 Enzymes with Altered PAM Specificities”, Nature Biotechnology, vol. 35, No. 8, Dec. 4, 2016, 1-17. [cited by applicant]
Gfeller, et al., “Predicting Antigen Presentation-What Could We Learn From a Million Peptides?”, Frontiers in Immunology, vol. 9, Article 1716, Jul. 2018, 17 pages. [cited by applicant]
Guan, et al., “MHCPred: A Server for Quantitative Prediction of Peptide-MHC Binding”, Nucleic Acids Research, vol. 31, No. 13, Aug. 2003, 3621-3624. [cited by applicant]
Hakenberg, et al., “MAPPP: MHC Class I Antigenic Peptide Processing Prediction”, Applied Bioinformatics, vol. 2, No. 3, 2003, 155-158. [cited by applicant]
He, et al., “Vaxign: The First Web-Based Vaccine Design Program for Reverse Vaccinology and Applications for Vaccine Development”, Journal of Biomedicine and Biotechnology, vol. 2010, Article 297505, 2010, 15 pages. [cited by applicant]
Hsu, et al., “Development and Applications of CRISPR-Cas9 for Genome Engineering”, Cell, vol. 157, No. 6, Jun. 5, 2014, 1262-1278. [cited by applicant]
Hsu, et al., “DNA Targeting Specificity of RNA-Guided Cas9 Nucleases”, Nature Biotechnology, vol. 31, No. 9, Sep. 2013, 827-832. [cited by applicant]
Hu, et al., “Evolved Cas9 Variants with Broad PAM Compatibility and High DNA Specificity”, Nature, vol. 556, No. 7699, Apr. 5, 2018, 57-63. [cited by applicant]
Jensen, et al., “Improved Methods for Predicting Peptide Binding Affinity to MHC Class II Molecules”, Immunology, vol. 154, No. 3, 2018, 394-406. [cited by applicant]
Jiang, et al., “RNA-Guided Editing of Bacterial Genomes Using CRISPR-Cas Systems”, Nature Biotechnology, vol. 31, No. 3, Mar. 2013, 233-239. [cited by applicant]
Jurtz, et al., “NetMHCpan 4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data”, Journal of Immunology, vol. 199, No. 9, Nov. 2017, 3360-3368. [cited by applicant]
Konermann, et al., “Genome-Scale Transcriptional Activation by an Engineered CRISPR-Cas9 Complex”, Nature, vol. 517, No. 7536, Jan. 29, 2015, 583-588. [cited by applicant]
Konermann, et al., “Optical Control of Mammalian Endogenous Transcription and Epigenetic States”, Nature, vol. 500, No. 7463, Aug. 22, 2013, 472-476. [cited by applicant]
Larsen, et al., “An Integrative Approach to CTL Epitope Prediction: A Combined Algorithm Integrating MHC Class I Binding, TAP Transport Efficiency, and Proteasomal Cleavage Predictions”, European Journal of Immunology, … [cited by applicant]
Liu, et al., “Quantitative Prediction of Mouse Class I MHC Peptide Binding Affinity Using Support Vector Machine Regression (SVR) Models”, BMC Bioinformatics, vol. 7, Article No. 182, 2006, 13 pages. [cited by applicant]
Molero-Abraham, et al., “Selection of Conserved Epitopes from Hepatitis C Virus for Pan-Populational Stimulation of T-Cell Responses”, Clinical and Developmental Immunology, vol. 2013, Article ID 601943, 2013, 10 pages. [cited by applicant]
Nielsen, et al., “NetMHCpan, A Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence”, PLoS One, vol. 2, Issue 8, e796, Aug. 2007, 10 pages. [cited by applicant]
Nielsen, et al., “Prediction of MHC Class II Binding Affinity Using SMM-Align, A Novel Stabilization Matrix Alignment Method”, BMC Bioinformatics, vol. 8, Article No. 238, 2007, 12 pages. [cited by applicant]
Nielsen, et al., “Quantitative Predictions of Peptide Binding to Any HLA-DR Molecule of Known Sequence: NetMHCIIpan”, PLoS Computational Biology, vol. 4, Issue 7, Jul. 2008, 10 pages. [cited by applicant]
Nielsen, et al., “Reliable Prediction of T-Cell Epitopes Using Neural Networks with Novel Sequence Representations”, Protein Science, vol. 12, No. 5, 2003, 1007-1017. [cited by applicant]
Nishimasu, et al., “Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA”, Cell, vol. 156, No. 5, Feb. 27, 2014, 935-949. [cited by applicant]
Nishimasu, et al., “Crystal Structure of [cited by applicant]
Oyarzun, et al., “PREDIVAC: CD4+ T-Cell Epitope Prediction for Vaccine Design that Covers 95% of HLA Class II DR Protein Diversity”, BMC Bioinformatics, vol. 14, Article No. 52, 2013, 11 pages. [cited by applicant]
Parker, et al., “Scheme for Ranking Potential HLA-A2 Binding Peptides Based on Independent Binding of Individual Peptide Side-Chains”, The Journal of Immunology, vol. 152, No. 1, Jan. 1, 1994, 163-175. [cited by applicant]
Parker, et al., “Structure-Guided Deimmunization of Therapeutic Proteins”, Journal of Computational Biology, vol. 20, No. 2, Feb. 2013, 152-165. [cited by applicant]
Parnas, et al., “A Genome-Wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks”, Cell, vol. 162, No. 3, Jul. 30, 2015, 675-686. [cited by applicant]
Platt, et al., “CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling”, Cell, vol. 159, No. 2, Oct. 9, 2014, 440-455. [cited by applicant]
Ramanan, et al., “CRISPR/Cas9 Cleavage of Viral DNA Efficiently Suppresses Hepatitis B Virus”, Scientific Reports, vol. 5, No. 10833, Jun. 2, 2015, 9 pages. [cited by applicant]
Rammensee, et al., “SYFPEITHI: Database for MHC Ligands and Peptide Motifs”, Immunogenetics, vol. 50, 1999, 213-219. [cited by applicant]
Ran, et al., “Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity”, Cell, vol. 154, No. 6, Sep. 12, 2013, 1380-1389. [cited by applicant]
Ran, et al., “Genome Engineering Using the CRISPR-Cas9 System”, Nature Protocols, vol. 8, No. 11, Nov. 2013, 2281-2308. [cited by applicant]
Ran, et al., “In Vivo Genome Editing Using [cited by applicant]
Reche, et al., “Enhancement to the RANKPEP Resource for the Prediction of Peptide Binding to MHC Molecules using Profiles”, Immunogenetics, vol. 56, No. 6, Oct. 2004, 405-419. [cited by applicant]
Reche, et al., “PEPVAC: A Web Server for Multi-Epitope Vaccine Development Based on the Prediction of Supertypic MHC Ligands”, Nucleic Acids Research, vol. 33, 2005, W138-W142. [cited by applicant]
Zhang, et al., “MULTIPRED2: A Computational System for Large-Scale Identification of Peptides Predicted to Bind to HLA Supertypes and Alleles”, Journal of Immunological Methods, vol. 374, No. 1-2, Nov. 30, 2011, 53-61. [cited by applicant]
Sanchez-Trincado, et al., “Fundamentals and Methods for T- and B-Cell Epitope Prediction”, Journal of Immunology, vol. 2017, Article 2680160, Dec. 28, 2017, 14 pages. [cited by applicant]
Shalem, et al., “Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells”, Science, vol. 343, No. 6166, Jan. 3, 2014, 84-87. [cited by applicant]
Shalem, et al., “High-Throughput Functional Genomics Using CRISPR-Cas9”, Nature Reviews Genetics, vol. 16, No. 5, May 2015, 299-311. [cited by applicant]
Shmakov, et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, vol. 60, No. 3, Nov. 5, 2015, 385-397. [cited by applicant]
Singh, et al., “Propred: Prediction of HLA-DR Binding Sites”, Bioinformatics, vol. 17, No. 12, Dec. 2001, 1236-1237. [cited by applicant]
Singh, et al., “Propred1: Prediction of Promiscuous MHC Class-I Binding Sites”, Bioinformatics, vol. 19, No. 8, Jun. 2003, 1009-1014. [cited by applicant]
Slaymaker, et al., “Rationally Engineered Cas9 Nucleases with Improved Specificity”, Science, vol. 351, No. 6268, Jan. 1, 2016, 84-88. [cited by applicant]
Sturniolo, et al., “Generation of Tissue-Specific and Promiscuous HLA Ligand Databases Using DNA Microarrays and Virtual HLA Class II Matrices”, Nature Biotechnology, vol. 17, Jun. 1999, 555-561. [cited by applicant]
Swiech, et al., “In Vivo Interrogation of Gene Function in the Mammalian Brain Using CRISPR-Cas9”, Nature Biotechnology, vol. 33, No. 1, Jan. 2015, 102-106. [cited by applicant]
Wang, et al., “A Systematic Assessment of MHC Class II Peptide Binding Predictions and Evaluation of a Consensus Approach”, PLOS Computational Biology, vol. 4, No. 4, e1000048, 2008, 10 pages. [cited by applicant]
Wang, et al., “Genetic Screens in Human Cells Using the CRISPR/Cas9 System”, Science, vol. 343, No. 6166, Jan. 3, 2014, 80-84. [cited by applicant]
Wang, et al., “One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering”, Cell, vol. 153, No. 4, May 9, 2013, 910-918. [cited by applicant]
Wu, et al., “Genome-Wide Binding of the CRISPR Endonuclease Cas9 in Mammalian Cells”, Nature Biotechnology, vol. 32, No. 7, Jul. 2014, 670-676. [cited by applicant]
Xu, et al., “Sequence Determinants of Improved CRISPR SgRNA Design”, Genome Research, vol. 25, No. 8, Aug. 2015, 1147-1157. [cited by applicant]
Zetsche, et al., “A Split-Cas9 Architecture for Inducible Genome Editing and Transcription Modulation”, Nature Biotechnology, vol. 33, No. 2, Feb. 2015, 139-142. [cited by applicant]
Zetsche, et al., “Cpf1 is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System”, Cell, vol. 163, No. 3, Oct. 22, 2015, 759-771. [cited by applicant]
Zhang, et al., “Immune Epitope Database Analysis Resource (IEDB-AR)”, Nucleic Acids Research, vol. 36, Aug. 2008, W513-W518. [cited by applicant]