Granted Patent
B2
US 12,291,798 · App. 17/817,959 · Granted May 6, 2025
Generation of binding molecules
Inventors:
Mark Throsby (Utrecht, NL); Ton Logtenberg (Driebergen, NL); John De Kruif (Bilthoven, NL)
Assignee:
Merus N.V.
C40B50/06C07K16/00C07K16/1018C07K16/2863C07K16/32C07K16/468C12Q1/6869C07K2317/21C07K2317/56C07K2317/565C40B30/04
View Patent ↗
Loading inventors, assignments & file history…
Abstract
Provided are methods for efficiently and comprehensively screening antibody repertoires from B cells to obtain and produce molecules with binding characteristics and functional activities for use in human therapy.
Claims (43)
1. A method for producing a population of antibodies which bind to an antigen, said method comprising:
a) obtaining B cells from lymph nodes, spleen, blood, and bone marrow from a transgenic non-human animal immunized with said antigen;
wherein said transgenic non-human animal expresses a rearranged human antibody light chain variable region (VL) from a VL nucleic acid,
wherein said transgenic non-human animal expresses a human antibody heavy chain variable region (VH) repertoire from a repertoire of VH nucleic acids,
said B cells expressing a repertoire of VHs and a limited immunoglobulin light chain variable (VL) region repertoire,
b) obtaining nucleic acids encoding antibody heavy chain variable (VH) regions from said B cells,
c) obtaining nucleotide sequences of the obtained nucleic acids of step b),
d) determining the frequency of nucleotide sequences from step c) which encode VH regions,
e) providing host cells, wherein each host cell comprises at least one vector comprising a nucleotide sequence encoding at least one VH region having a sequence from those determined in step d) wherein each said host cell expresses the rearranged human VL nucleic acid of step a) and said at least one VH region,
f) culturing said host cells from step e) and allowing for expression of said VL and VH regions, and
g) obtaining said population of antibodies from the cultured host cells of step f) which bind said antigen.
2. The method of claim 1 , wherein the method is without B cell immortalization.
3. A method for producing a population of antibodies which bind to an antigen, said method comprising:
a) inducing selective clonal expansion of B cells in a transgenic non-human animal,
wherein said transgenic non-human animal expresses a rearranged human antibody light chain variable region (VL) from a VL nucleic acid,
wherein said transgenic non-human animal expresses a human antibody heavy chain variable region (VH) repertoire from a repertoire of VH nucleic acids,
said B cells expressing a repertoire of VHs and a limited immunoglobulin light chain variable (VL) region repertoire,
b) obtaining nucleic acids encoding antibody heavy chain variable (VH) regions from said B cells,
c) obtaining nucleotide sequences of the obtained nucleic acids of step b),
d) determining the frequency of nucleotide sequences from step c) which encode VH regions,
e) providing host cells, wherein each host cell comprises at least one vector comprising a nucleotide sequence encoding at least one VH region having a sequence from those determined in step d) wherein each said host cell expresses the rearranged human VL nucleic acid of step a) and said at least one VH region,
f) culturing said host cells from step e) and allowing for expression of said VL and VH regions, and
g) obtaining said population of antibodies from the cultured host cells of step f) which bind said antigen.
4. The method of claim 3 , wherein the selective clonal expansion of B cells is induced by DNA tattoo vaccination.
5. A method for producing a population of antibodies which bind to an antigen, said method comprising:
a) providing a collection of B cell clones comprising a collection of VH regions that are enriched for VH regions encoding antibodies directed to said antigen,
wherein said collection of B cell clones is from a transgenic non-human animal,
wherein said transgenic non-human animal expresses a rearranged human antibody light chain variable region (VL) from a VL nucleic acid,
wherein said transgenic non-human animal expresses a human antibody heavy chain variable region (VH) repertoire from a repertoire of VH nucleic acids,
said B cell clones expressing a repertoire of VHs and a limited immunoglobulin light chain variable (VL) region repertoire,
b) obtaining nucleic acids encoding antibody heavy chain variable (VH) regions from said B cell clones,
c) obtaining nucleotide sequences of the obtained nucleic acids of step b),
d) determining the frequency of nucleotide sequences from step c) which encode VH regions,
e) providing host cells, wherein each host cell comprises at least one vector comprising a nucleotide sequence encoding at least one VH region having a sequence from those determined in step d) wherein each said host cell expresses the rearranged human VL nucleic acid of step a) and said at least one VH region,
f) culturing said host cells from step e) and allowing for expression of said VL and VH regions, and
g) obtaining said population of antibodies from the cultured host cells of step f) which bind said antigen.
6. The method of claim 5 , wherein said collection of B cell clones is obtained through an antigen-recognition process.
7. The method of claim 5 , wherein the 20, 50, 100, or 200 most abundant B cell clones are selected.
8. The method of claim 5 , wherein the method is without amplification of nucleic acids encoding antibody heavy chain variable regions obtained in step b).
9. The method of claim 1 , further comprising subjecting a sample of said cultured host cells of step g) to at least one functional assay to confirm binding to said antigen, and selecting at least one cell that produces an antibody which binds said antigen.
10. The method of claim 1 , wherein the transgenic non-human animal has been immunized with a nucleic acid encoding said antigen or with a protein form of said antigen.
11. The method of claim 1 , wherein the transgenic non-human animal is a rodent.
12. The method of claim 1 , wherein the transgenic non-human animal is a mouse.
Assignments (4)
NOTICE OF GRANT OF SECURITY INTEREST IN PATENTS
Recorded Jan 30, 2026
From: MERUS B.V.
To: WILMINGTON TRUST, NATIONAL ASSOCIATION
Reel/Frame 074562/0322 →
SECURITY INTEREST
Recorded Jan 29, 2026
From: MERUS B.V.
To: MORGAN STANLEY SENIOR FUNDING, INC.
Reel/Frame 074532/0434 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Aug 18, 2023
From: THROSBY, MARK; LOGTENBERG, TON; DE KRUIF, JOHN
To: MERUS BIOPHARMACEUTICALS B.V.
Reel/Frame 064650/0038 →
Continuity (6)
Provisional Application
61539116
· Sep 26, 2011
References Cited (325)
US 6596541B2
· Murphy
· 2003
[cited by applicant]
US 7105348B2
· Murphy
· 2006
[cited by applicant]
US 7262028B2
· Van Berkel
· 2007
[cited by applicant]
US 7329530B2
· Houtzager
· 2008
[cited by applicant]
US 7429486B2
· Van Berkel
· 2008
[cited by applicant]
US 7579446B2
· Bakker
· 2009
[cited by applicant]
US 7696330B2
· Meulen
· 2010
[cited by applicant]
US 7740852B2
· Bakker
· 2010
[cited by applicant]
US 7777010B2
· Logtenberg
· 2010
[cited by applicant]
US 7858086B2
· Geuijen
· 2010
[cited by applicant]
US 7901919B2
· Houtzager
· 2011
[cited by applicant]
US 7919257B2
· Hoogenboom
· 2011
[cited by applicant]
US 7927834B2
· Van Berkel
· 2011
[cited by applicant]
US 7932360B2
· Van Berkel
· 2011
[cited by applicant]
US 7960518B2
· Throsby
· 2011
[cited by applicant]
US 7968092B2
· Throsby
· 2011
[cited by applicant]
US 8052974B2
· Throsby
· 2011
[cited by applicant]
US 8106170B2
· Ter Meulen
· 2012
[cited by applicant]
US 8148497B2
· Bakker
· 2012
[cited by applicant]
US 8192927B2
· Van Den Brink
· 2012
[cited by applicant]
US 8211431B2
· Throsby
· 2012
[cited by applicant]
US 8241631B2
· Throsby
· 2012
[cited by applicant]
US 8268756B2
· Logtenberg
· 2012
[cited by applicant]
US 8470327B2
· Throsby et al.
· 2013
[cited by applicant]
US 8502018B2
· Murphy
· 2013
[cited by applicant]
US 8642513B2
· Throsby et al.
· 2014
[cited by applicant]
US 8911738B2
· Throsby et al.
· 2014
[cited by applicant]
US 9012371B2
· Logtenberg et al.
· 2015
[cited by applicant]
US 9145588B2
· Throsby et al.
· 2015
[cited by applicant]
US 9248182B2
· De Kruif
· 2016
[cited by applicant]
US 9738701B2
· Hogenboom et al.
· 2017
[cited by applicant]
US 9765133B2
· Logtenberg et al.
· 2017
[cited by applicant]
US 9908946B2
· Throsby et al.
· 2018
[cited by applicant]
US 9944695B2
· Logtenberg et al.
· 2018
[cited by applicant]
US 9951124B2
· Logtenberg et al.
· 2018
[cited by applicant]
US 20030096225A1
· Logtenberg
· 2003
[cited by applicant]
US 20060015949A1
· Lonberg
· 2006
[cited by applicant]
US 20060205077A1
· Schwenk
· 2006
[cited by applicant]
US 20080070799A1
· Bakker
· 2008
[cited by applicant]
US 20090130652A1
· Throsby
· 2009
[cited by applicant]
US 20100069614A1
· Houtzager
· 2010
[cited by applicant]
US 20100172917A1
· Ter Meulen
· 2010
[cited by applicant]
US 20120021409A1
· Mcwhirter
· 2012
[cited by applicant]
US 20120101000A1
· Zhou et al.
· 2012
[cited by applicant]
US 20120192300A1
· Babb
· 2012
[cited by applicant]
US 20140245468A1
· McWhirter et al.
· 2014
[cited by applicant]
US 20180002405A1
· Hoogenboom et al.
· 2018
[cited by applicant]
US 20180142003A1
· Logtenberg et al.
· 2018
[cited by applicant]
US 20180142005A1
· Logtenberg et al.
· 2018
[cited by applicant]
US 20180142006A1
· Logtenberg et al.
· 2018
[cited by applicant]
US 20200319181A1
· Logtenberg et al.
· 2020
[cited by applicant]
EP 0814159
· 1999
[cited by applicant]
EP 1439234
· 2004
[cited by applicant]
EP 2147594
· 2013
[cited by applicant]
JP 2004008218
· 2004
[cited by applicant]
JP 2006109711
· 2006
[cited by applicant]
WO WO1992003918
· 1992
[cited by applicant]
WO WO1993012227
· 1993
[cited by applicant]
WO WO1994002602
· 1994
[cited by applicant]
WO WO1998052976
· 1998
[cited by applicant]
WO WO1998050431
· 1999
[cited by applicant]
WO WO1999050657
· 1999
[cited by applicant]
WO WO1999045962
· 1999
[cited by applicant]
WO WO2002066630
· 2002
[cited by applicant]
WO WO2003052416
· 2003
[cited by applicant]
WO WO2004009618
· 2004
[cited by applicant]
WO WO2004106375
· 2004
[cited by applicant]
WO WO2005068622
· 2006
[cited by applicant]
WO WO2006117699
· 2007
[cited by applicant]
WO WO2008054606
· 2009
[cited by applicant]
WO WO2008076379
· 2009
[cited by applicant]
WO WO2009089004
· 2009
[cited by applicant]
WO WO2009100896
· 2009
[cited by applicant]
WO WO2009157771
· 2010
[cited by applicant]
WO WO2010136598
· 2010
[cited by applicant]
WO WO2011014469
· 2011
[cited by applicant]
WO WO2011097603
· 2011
[cited by applicant]
WO WO2011146514
· 2012
[cited by applicant]
“Murinae” Encyclopedia of Life 2018 http://eoLorg/pages/2847355/hierarchy_entries/57454206/overview.
[cited by applicant]
“Murine.” Merriam-Webster.com. Merriam-Webster, n.d. Web. Apr. 2, 2018.https://www.merriam-webster.com/dictionary/murine.
[cited by applicant]
Abidor et al., “Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions,” Biophysical Joural (1994) 67:427-435.
[cited by applicant]
Agca et al., “Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically,” BMC Neuroscience (2008)…
[cited by applicant]
Al-Lazikani et al., “Standard conformations for the Canonical structures of immunoglobulins,” J Mol Biol (1997) 273:927-948.
[cited by applicant]
Assignment by inventors, dated Oct. 1 and 2, 2012, 2 pages.
[cited by applicant]
Attaelmannan et al., “Understanding and Identifying Monoclonal Gammopathies,” Clinical Chemistry (2000) 46(8B):1230-1238.
[cited by applicant]
Aucouturier et al., “Monoclonal Lg L chain and L chain V domain fragment crystallization in myeloma-associated Fanconi's syndrome,” J Immunol (1993) 150(8) 3561-3568.
[cited by applicant]
Babcook et al., “A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities,” Proc Natl Acad Sci USA (1996) 93:7843-7848.
[cited by applicant]
Banchereau et al., “Long-term human B cell lines dependent on interleukin-4 and antibody to CD40,” Science (1991) 251(4989):70-72.
[cited by applicant]
Betz et al., “Elements regulating somatic hypermutation of an immunoglobulin kappa gene: critical role for the intron enhancer/matrix attachment region,” Cell (1994) 77(2):239-248.
[cited by applicant]
Bins et al., “A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression,” Nature Medicine (2005) 11(8):899-904.
[cited by applicant]
Boder et al., “Yeast surface display for screening combinatorial polypeptide libraries,” Nat Biotechnol (1997) 15(6):553-557.
[cited by applicant]
Bogen et al., “A rearranged lambda 2 light gene chain retards but does not exclude kappa and lambda 1 expression,” Eur. J. Immunol (1991) 21:2391-2395.
[cited by applicant]
Breiner et al., “Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse,” Gene. (1982) 18(2): 165-174.
[cited by applicant]
Bruggemann et al., “A repertoire of monoclonal antibodies with human heavy chains from transgenic mice,” Proc. Natl. Acad. Sci. USA (1989) 86:6709-6713.
[cited by applicant]
Buehr et al., “Capture of Authentic Embryonic Stem Cells from Rat Blastocysts,” Cell (2008) 135:1287-1298.
[cited by applicant]
Carter, “Bispecific human lgG by design,” Journal of Immunological Methods (2001) 248:7-15.
[cited by applicant]
Chao et al., “Isolating and engineering human antibodies using yeast surface display,” Nature Protocols (2006) 1(2):755-769.
[cited by applicant]
Clackson et al., “Making antibody fragments using phage display libraries,” Nature (1991) 352(6336):624-628.
[cited by applicant]
Clark et al. “A future for transgenic livestock,” (2003) Nat. Rev. Gen. 4:825-833.
[cited by applicant]
Clark, “Antibody humanization: a case of the ‘Emperor's new clothes’?” Immunology Today (2000) 21(8):397-402.
[cited by applicant]
Cobaugh et al., “Synthetic antibody libraries focused towards peptide ligands,” J Mol Biol (2008) 378(3):622-633.
[cited by applicant]
Cronkhite et al., “Male and female germline specific expression of an EGFP reporter gene in a unique strain of transgenic rats,” Developmental Biology (2005) 284:171-183.
[cited by applicant]
Crowe, “Recent advances in the study of human antibody responses to influenza virus using optimized human hybridoma approaches,” Vaccine (2009) 27S:G47-51.
[cited by applicant]
Dammers et al., “Organization of the Immunoglobulin Heavy- and Light-Chain Loci in the Rat,” In Azad K. Kaushik and Yfke Pasman (Eds.) 2014, Comparative Immunoglobulin Genetics (pp. 108-130). New Jersey, US: Apple Acade…
[cited by applicant]
Davies et al., “Creation of mice expressing human antibody light chains by introduction of a yeast artificial chromosome containing the core region of the human immunoglobulin K locus,” Biotechnology (1993) 11:911-914.
[cited by applicant]
De Kruif et al., “Human immunoglobulin repertoires against tetanus toxoid contain a large and diverse fraction of high-affinity promiscuous V(H) genes,” J Mol Biol (2009) 387(3):548-558.
[cited by applicant]
De Wildt et al., “Analysis of Heavy and Light Chain Pairings Indicates that Receptor Editing Shapes the Human Antibody Repertoire,” J Mol Biol (1999) 285:895-901.
[cited by applicant]
Dechiara et al., “VelociMouse: Fully ES Cell-Derived FO-Generation Mice Obtained from the Injection of ES Cells into Eight-Cell-Stage Embryos,” Gene Knockout Protocols: Second Edition, Ralf Kuhn (Ed.), Humana Press (200…
[cited by applicant]
Decision for Application 06121086.0, dated May 7, 2018, 23 pages.
[cited by applicant]
Esposito et al., “Phage display of a human antibody against
[cited by applicant]
Ettinger et al., “IL-21 induces differentiation of human naïve and memory B cells into antibody-secreting plasma cells,” The Journal of Immunology (2005) 176:7867-7879.
[cited by applicant]
European Search Report for Application No. 12186010.0, dated May 22, 2013, 11 pages.
[cited by applicant]
Fecteau et al., “A New Memory CD27 lgG+ B Cell Population in Peripheral Blood Expressing VH Genes with Low Frequency of Somatic Mutation,” The Journal of Immunology (2006) 177:3728-3736.
[cited by applicant]
Feldhaus et al., “Flow-cytometric isolation of human antibodies from a nonimmune
[cited by applicant]
Fischer, “Sequencing antibody repertoires: The next generation,” mAbs (2011) 3:17-20.
[cited by applicant]
Friedrich et al., “Promoter traps in embryonic stem cells: a genetic screen to identify, and mutate developmental genes in mice,” Genes & Development (1991) 5:1513-1523.
[cited by applicant]
Fuchs et al., “Targeting recombinant antibodies to the surface of
[cited by applicant]
Ge et al., “Rapid construction and characterization of synthetic antibody libraries without DNA amplification,” Biotechnology and Bioengineering (2010) 106(3):347-357.
[cited by applicant]
GenBank Accession No. DQ187586-1, Protein ID ABA26122.1, Rabquer, B.J. et al., “Differential variable gene usage between pneumococcal polysaccharide specific B cells isolated 5-10 days and 4-6 weeks post-vaccination,” 1…
[cited by applicant]
Genbank, M87478.1, “Human rearranged lgK mRNA VJC region,” Oct. 28, 1994, 1 page.
[cited by applicant]
Glanville et al., “Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire,” PNAS (2009) 106(48):20216-20221.
[cited by applicant]
Gonzalez-Fernandez et al., “Analysis of somatic hypermutation in mouse Peyer's patches using immunoglobulin κ light-chain transgenes,” Proc Natl Acad Sci USA (1993) 90:9862-9866.
[cited by applicant]
Good et al., “Kinetics of human B cell behavior and amplification of proliferative responses following stimulation with IL-21,” J Immunol (2006) 177:5236-5247.
[cited by applicant]
Goodnow et al., “Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice,” Nature (1988) 334:676-682.
[cited by applicant]
Gordon, “Transgenic technology and laboratory animal science,” ILAR Journal (1997) 38:32-41.
[cited by applicant]
Goyenechea et al., “Cells strongly expressing IgK transgenes show clonal recruitment of hypermutation: a role for both MAR and the enhancers,” The EMBO Journal (1997) 16(13):3987-3994.
[cited by applicant]
Goyenechea et al., “Modifying the sequence of an immunoglobulin V-gene alters the resulting pattern of hypermutation,” Proc Natl Acad Sci USA (1996) 93:13979-13984.
[cited by applicant]
Green et al., “Antigen-specific human monoclonal antibodies from mice engineered with human lg heavy and light chain YACs,” Nature Genetics (1994) 7:13-21.
[cited by applicant]
Gulli et al., “Epidermal Growth Factor-induced Apoptosis in A431 Cells Can Be Reversed by Reducing the Tyrosine Kinase Activity,” Cell Growth & Differentiation (1996) 7:173-178.
[cited by applicant]
Gunasekaran et al., “Enhancing antibody Fc heterodimer formation through electrostatic steering effects,” Journal of Biological Chemistry (2010) 285(25):19637-19646.
[cited by applicant]
Hamra et al.,m “Production of transgenic rats by lentiviral transduction of male germ-line stem cells,” PNAS USA (2002) 99:14931-14936.
[cited by applicant]
Harding et al., “The immunogenicity of humanize and fully human antibodies,” mAbs (2010) 2:3:256-265.
[cited by applicant]
Harvey et al., “Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from
[cited by applicant]
Hengstschlager et al., “A λ1 transgene under the control of a heavy chain promoter and enhancer does not undergo somatic hypermutation,” Eur J Immunol (1994) 24:1649-1656.
[cited by applicant]
Hochedlinger et al., “Monoclonal mice generated by nuclear transfer from mature B and T donor cells,” Nature (2002) 415:1035-1038.
[cited by applicant]
Homig-Holzel et al., “Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-kappaB pathway and promotes lymphomagenesis,” J. Exp. Med. (2008) 205(6):1317-1329.
[cited by applicant]
Hoogenboom, “Selecting and screening recombinant antibody libraries,” Nature Biotechnology (2005) 23(9):1105-1116.
[cited by applicant]
ImMunoGeneTics Information System, for analysed sequence CHEB VK, http://www.imgt.org/IMGT_vquest/vquest, at least as early as Apr. 25, 2012.
[cited by applicant]
Ishii et al., “TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines,” Nature (2008) 451:725-730.
[cited by applicant]
Jacob et al., “Activity of DNA vaccines encoding self or heterologous Her-2/neu in Her-2 or neu transgenic mice,” Cellular Immunology (2006) 240:96-106.
[cited by applicant]
Jacob et al., “Combining human and rat sequences in Her-2 DNA vaccines blunts immune tolerance and drives antitumor immunity,” Cancer Research (2010) Cancer Res 70(1):119-128.
[cited by applicant]
Jakobovits et al., “From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice,” Nature Biotechnology (2007) 25(10):1134-1143.
[cited by applicant]
Jechlinger, “Optimization and delivery of plasmid DNA for vaccination,” Expert Rev Vaccines (2006) 5(6):803-825.
[cited by applicant]
Jiang et al., “A novel strategy for generation of monoclonal antibodies from single B cells using RT-RCR technique and in vitro expression,” Biotechnol Prog (2006) 22(4):979-988.
[cited by applicant]
Jin et al., “A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood,” Nature Medicine (2009) 15(9):1088-1093.
[cited by applicant]
Jolly et al., “Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice,” Nucleic Acids Research (1997) 25(10):1913-1919.
[cited by applicant]
Kato et al., “Cell activation by CpG ODN leads to improved electrofusion in hybridoma production,” J Immunological Methods (2011) 373:102-110.
[cited by applicant]
Kim et al., “Subspecialization of CXCR5
[cited by applicant]
Kling et al. News in Brief , “Big Pharma vies for mice,” Nature Biotechnology (2007) 25(6):613-614.
[cited by applicant]
Klohn et al., “IBC's 23
[cited by applicant]
Klotz et al., “Somatic hypermutation of a λ
[cited by applicant]
Kohler et al., “Continuous cultures of fused cells secreting antibody of predefined specificity,” Nature (1975) 256:495-497.
[cited by applicant]
Kong et al., “A λ 3′ enhancer drives active and untemplated somatic hypermutation of a λ
[cited by applicant]
Kwakkenbos et al., “Generation of stable monoclonal antibody-producing BCR+ human memory B cells by genetic programming,” Nat. Med. (2010) 16(1):123-128.
[cited by applicant]
Kwaks et al., “Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells,” Treneds in Biotechnology (2006) 24(3):137-142.
[cited by applicant]
Larbouret et al., “In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas,” Clin. Cancer Res. (2007) 13(11):3356-3362.
[cited by applicant]
Larrick et al., “Rapid Cloning of Rearranged Immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction,” Biochemical and Biophysical Resarch Communication (1989) 160(3):1250-1…
[cited by applicant]
Lee et al., “Selection of human antibody fragments by phage display,” Nat Protoc (2007) 2:3001-3008.
[cited by applicant]
Li et al., “Characterization of PEG-mediated electrofusion of human erythrocytes,” Biophys J (1994) 67(6):2361-2366.
[cited by applicant]
Li et al., “Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts,” Cell (2008) 135(7):1299-1310.
[cited by applicant]
Lie et al., “Advances in quantitative PCR technology: 5′ nuclease assays,” Curr. Opin. Biotechnol (1998) 9 (1):43-48.
[cited by applicant]
Ling et al., “Modulation of the murine immune response to human lgG by complexing with monoclonal antibodies,” Immunology (1987) 62(1):1-6.
[cited by applicant]
Logtenberg, “Antibody cocktails: next-generation biopharmaceuticals with improved potency,” Trends in Biotechnology (2007) 25(9):390-394.
[cited by applicant]
Lonberg et al., “Antigen-specific human antibodies from mice comprising four distinct genetic modifications,” Nature (1994) 368:856-859.
[cited by applicant]
Lonberg, “Human antibodies from transgenic animals,” Nature Biotechnology (2005) 23(9):1117-1125.
[cited by applicant]
Love et al., “A microengraving method for rapid selection of single cells producing antigen-specific antibodies,” Nature Biotechnology (2006) 24(6):703-707.
[cited by applicant]
Manz et al., “Maintenance of serum antibody levels,” Annu Rev Immunol (2005) 23:367-286.
[cited by applicant]
Mao et al., “Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization,” Nature Biotechnology (2010) 28(11): 1195-1202.
[cited by applicant]
Mao et al., “Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain,” Blood (2001) 97(1):324-326.
[cited by applicant]
Mazor et al., “Isolation of engineered, full-length antibodies from libraries expressed in
[cited by applicant]
McCreath et al., “Production of gene-targeted sheep by nuclear transfer from cultured somatic cells,” Nature. (2000) 405(6790): 1066-1069.
[cited by applicant]
Meijer et al., “Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing,” J. Mol. Biol. (2006) 358(3):764-772.
[cited by applicant]
Mendez et al., “Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice,” Nature Genetics (1997) 15:146-156.
[cited by applicant]
Merchant et al., “An efficient route to human bispecific lgG,” Nat Biotechnol (1998) 16(7):677-681.
[cited by applicant]
Merus, “MeMo—the ingenious mouse, for improved antibody therapeutics,” www.merus.nl, 3 pages (2011).
[cited by applicant]
Meyer et al., “The importance of the 3′-enhancer region in immunoglobulin ϰ gene expression,” Nucleic Acids Research (1990) 18(19):5609-5615.
[cited by applicant]
Middendorp et al., “Impaired precursor cell differentiation in Bruton's tyrosine kinase-deficient mice,” The Journal of Immunology (2002) 168:2695-2703.
[cited by applicant]
Mohapatra et al., “Designer monoclonal antibodies as drugs: the state of the art,” Expert Rev Clin Immunol (2008) 4(3):305-307.
[cited by applicant]
Mullins et al., “Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene,” Nature (1990) 344:541-544.
[cited by applicant]
Murphy, Kenneth, The Development and Survival of Lymphocytes, Janeway's Immunobioloby, 8th Edition, Taylor & Francis, Chapter 8, pp. 275-290 (2011).
[cited by applicant]
Nagle, M. “Regeneron helps make Sanofi VelocImmune to its ‘weak’ pipeline,” (2007) Retrieved on http://www.outsourcing-pharma.com/Preclinical-Research/Regeneron-helps-make-Sanofi-VelocImmune-to-its-weak-pipeline. Retrie…
[cited by applicant]
Nemazee, “Receptor editing in lymphocyte development and central tolerance,” Nat Rev Immunol (2006) 6(10):728-740.
[cited by applicant]
Neuberger et al., “Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice,” Nature (1989) 338:350-352.
[cited by applicant]
Niemann et al., “Transgenic farm animals: present and future,” (2005) Rev. Sci, Tech. Off. Int. Spiz. 24:285-298.
[cited by applicant]
Nissim et al., “Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents,” EMBO J (1994) 13(3):692-698.
[cited by applicant]
O'Brien et al., “Somatic hypermutation of an immunoglobulin transgene in κ transgenic mice,” Nature (1987) 326:405-409.
[cited by applicant]
Odegard et al., “Targeting of somatic hypermutation,” Nat Rev Immunol (2006) 6(8):573-583.
[cited by applicant]
Ogunniyi et al., “Screening individual hybridomas by microengraving to discover monoclonal antibodies,” Nature Protocols (2009) 4(5):767-782.
[cited by applicant]
Pelanda et al., “A prematurely expressed lgκ transgene, but not a VκJκ gene segment targeted into the lgκ locus, can rescue B cell development in λ5-deficient mice,” Immunity (1996) 5:229-239.
[cited by applicant]
Peled et al., “The biochemistry of somatic hypermutation,” Annu Rev Immunol (2008) 26:481-511.
[cited by applicant]
Persson et al., “A Focused Antibody Library for Improved Hapten Recognition,” J. Mol. Biol. (2006) 357:607-620.
[cited by applicant]
Pokorna et al., “DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants,” Genetic Vaccines and Therapy (2008) 6(4):1-8.
[cited by applicant]
Ponsel et al., “High affinity, developability and functional size: the holy grail of combinatorial antibody library generation,” Molecules (2011) 16(5):3675-3700.
[cited by applicant]
Popov et al., “A human immunoglobulin λ locus is similarly well expressed in mice and humans,” J Exp Med (1999) 189(10):1611-1619.
[cited by applicant]
Poulsen et al., “Limits for antibody affinity maturation and repertoire diversification in hypervaccinated humans,” The Journal of Immunology (2011) 187:4229-4235.
[cited by applicant]
Prelle et al., “Pluripotent Stem Cells—Model of Embryonic Development, Tools for Gene Targeting, and Basis of Cell Therapy,” (2002) Anat. Histol. Embryol. 31:169-186.
[cited by applicant]
Presta et al., “Engineering of therapeutic antibodies to minimize immunogenicity and optimize function,” Advanced Drug Delivery Reviews (2006) 58:640-656.
[cited by applicant]
Quaak et al., “GMP production of pDERMATT IT for vaccination against melanoma in a phase I clinical trial,” Eur J Pharm Biopharm (2008) 70:429-438.
[cited by applicant]
Ravn et al., “By-passing in vitro screening-next generation sequencing technologies applied to antibody display and in silico candidate selection,” Necleic Acids Research (2010) 38(21):e193.
[cited by applicant]
Reddy et al., “Isolation of monoclonal antibodies without screening by mining the variable gene repertoire of plasma cells,” Nat Biotechnol (2010) 28(9):1-28.
[cited by applicant]
Reddy et al., “Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells,” Nature Biotechnology (2010) 28(9):965-971.
[cited by applicant]
Reddy et al., “Systems analysis of adaptive immunity by utilization of high-throughput technologies,” Current Opinion in Biotechnology (2011) 22:584-589.
[cited by applicant]
Retter et al., “Receptor Editing Occurs Frequently during Normal B Cell Development,” J. Exp. Med., vol. 188(7):1231-1238 (1998).
[cited by applicant]
Rickert et al., “B lymphocyte-specific, Cre-mediated mutagenesis in mice,” Nucleic Acids Research (1997) 25(6):1317-1318.
[cited by applicant]
Roitt, A. , Immunology, Mir, Moscow, pp. 134, 214 (2000).
[cited by applicant]
Ruuls et al., “Novel human antibody therapeutics: The age of the Umabs,” Biotechnol journal (2008) 3:1157-1171.
[cited by applicant]
Sasaki et al., “Canonical NF-kB Activity, Dispensable for B Cell Development, Replaces BAFF-Receptor Signals and Promotes B Cell Proliferation upon Activation,” Immunity (2006) 24:729-739.
[cited by applicant]
Schaffitzel et al., “Ribosome display: an in vitro method for selection and evolution of antibodies from libraries,” J Immunol Methods (1999) 231(1-2):119-135.
[cited by applicant]
Schmidlin et al., “New insights in the regulation of human B cell differentiation,” Trends Immunol (2009) 30(6):277-285.
[cited by applicant]
Scott, “Mice with a human touch,” Nature Biotechnology (2007) 25:1075-1077.
[cited by applicant]
Shapiro-Shelef et al., “Regulation of plasma-cell development,” Nature Review Immunol (2005) 5:230-242.
[cited by applicant]
Sharpe et al., “Somatic hypermutation of immunoglobulin kappa may depend on sequences 3′ of Cϰ and occurs on passenger transgenes,” The EMBO Journal (1991) 10(8):2139-2145.
[cited by applicant]
Sheppard et al., “Rat kappa-chain J-segment genes: two recent gene duplications separate rat and mouse,” Cell. (1982) 29(1):121-7.
[cited by applicant]
Singer et al., “Transcription: The Transfer of DNA Sequence Information to RNA,” Genes & Genomes, University Science Books, CA, (1991) Chapter 3.2, pp. 134-145.
[cited by applicant]
Sirac et al., “Role of the monoclonal κ chain V domain and reversibility of renal damage in a transgenic model of acquired Fanconi syndrome,” Blood (2006) 108:536-543.
[cited by applicant]
Smith et al., “Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen,” Nature Protocols (2009) 4(3):372-385.
[cited by applicant]
Smith-Gill et al., “Contributions of Immunoglobulin Heavy and Light Chains to Antibody Specificity for Lysozyme and Two Haptens,” The Journal of Immunology (1987) 139(12):4135-4144.
[cited by applicant]
Stevens, Sean, “Human Antibody Discovery, VelocImmune—A novel platform,” Pharma Focus Asia, Issue 8, pp. 72-74 (2008).
[cited by applicant]
Stevenson et al., “DNA vaccines to attack cancer,” PNAS (2004) 101 (Suppl. 2):14646-14652.
[cited by applicant]
Storb et al., “Transgenic mice with μ and κ genes encoding antiphosphorylcholine antibodies,” J Exp Med (1986) 164:627-641.
[cited by applicant]
Story et al., “Profiling antibody responses by multiparametric analysis of primary B cells,” PNAS (2008) 105(46):17902-17907.
[cited by applicant]
Sun et al., “Immunoglobulin genes and diversity: what we have learned from domestic animals,” J Animal Sci Biotechnol (2012) 3:18.
[cited by applicant]
Tajiri et al., “Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity,” Cytometry (2007) 71A:961-967.
[cited by applicant]
Taylor et al., “A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins,” Nucleic Acids Research (1992) 20(23):6287-6295.
[cited by applicant]
Taylor et al., “Human immunoglobulin transgenes undergo rearrangement, somatic mutation and class switching in mice that lack endogenous lgM,” Int Immunol (1994) 6(4):579-591.
[cited by applicant]
The Netherlands Manual for the Handling of Applications for Patents, Designs and Trade Marks Throughout the World, Arnold and Siedsma (ed.), Kluwer IP Law (2017) 3 pages.
[cited by applicant]
Third Party Observations against EP 12186010.0, dated Mar. 13, 2017, 8 pages.
[cited by applicant]
Third Party Observations pursuant to Article 115 EPC for EP 17174827.0, dated Jul. 5, 2019, 27 pages.
[cited by applicant]
Third party Opposition for EP 2604625, filed Mar. 23, 2018, 118 pages.
[cited by applicant]
Third party Opposition for EP 2604625, filed Mar. 27, 2018, 74 pages.
[cited by applicant]
Throbsy et al., “Isolation and characterization of human monoclonal antibodies from individuals infected with west nile virus §,” Journal of Virology (2006) 80(14):6982-6992.
[cited by applicant]
Tiller et al., “Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning,” J. Immunol. Methods (2008) 329(1-2):112-124.
[cited by applicant]
Tokimitsu et al., “Single Lymphocyte Analysis with a Microwell Array Chip,” Cytometry (2007) 71A:1003-1010.
[cited by applicant]
Torres et al., Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, Oxford, (1997) Chapters 10-11, pp. 42-53.
[cited by applicant]
Traggai et al., “An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus,” Nature Medicine (2004) 10(8):871-875.
[cited by applicant]
Ueda et al., “Establishment of Rat Embryonic Stem Cells and Making of Chimera Rats,” (2008) PLoS ONE 3(7):e2800.
[cited by applicant]
Van Den Berg, “Formulation and delivery of dermal DNA vaccines,” Gildeprint Drukkerijen B.V (2009) 160 pages.
[cited by applicant]
Van Doorn., Additional post-filing data and letter filed by the patentee, 1 page, dated Jun. 13, 2013.
[cited by applicant]
Weeratna et al., “CPG ODN allows lower dose of antigen against hepatitis B surface antigen in BALB/c mice,” Immunology and Cell Biology (2003) 81:59-62.
[cited by applicant]
Weiner., “Fully Human Therapeutic Monoclonal Antibodies,” J. Immunother (2006) 29(1):1-9.
[cited by applicant]
Wheeler et al., “Transgenic Technology and Applications in Swine,” (2001) Theriogenology, 56:1345-1369.
[cited by applicant]
Whittington et al., “DNA vaccination controls Her-2
[cited by applicant]
Winter et al., “Insertion of 2 KB of Bacteriophage DNA Between an Immunoglobulin Promoter and Leader Exon Stops Somatic Hypermutation in a kappa Transgene,” Molecular Immunology (1997) 34(5):359-366.
[cited by applicant]
Wrammert et al., “Rapid cloning of high-affinity human monoclonal antibodies against influenza virus,” Nature (2008) 453:667-672.
[cited by applicant]
Wu et al., “Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin,” Nature Biotechnology (2007) 25(11):1290-1297.
[cited by applicant]
Xiang et al., “The downstream transcriptional enhancer, Ed, positively regulates mouse lgκ gene expression and somatic hypermutation,” The Journal of Immunology (2008) 180:6725-6732.
[cited by applicant]
Yang et al., “Homologous recombination based modification in
[cited by applicant]
Yarilin, A.A., Osnovy Immunologii, [Fundamentals of Immunology], Meditsina, Moscow, p. 194 (1999).
[cited by applicant]
Yarilin, A.A., Osnovy Immunologii, [Fundamentals of Immunology], Meditsina, Moscow, p. 195 (1999).
[cited by applicant]
Yu et al., “An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies,” Journal of Immunoloaical Methods (2008) 336:142-151.
[cited by applicant]
Zubler et al., “Theoretical and practical aspects of B-cells activation: murine and human systems,” Immunological Reviews (1987) 99:281-299.
[cited by applicant]
Brinster et al., “Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice,” Nature 306:332-336 (1983).
[cited by applicant]
Heinrichs et al., “Universal cloning and direct sequencing of rearranged antibody V genes using C region primers, biotin-captured cDNA and one-side PCR,” Journal of Immunological Methods 178:241-251 (1995).
[cited by applicant]
Hieter et al., “Cloned Human and Mouse Kappa Immunoglobulin Constant and J Region Genes Conserve Homology in Functional Segments,” Cell, 22:197-207 (1980).
[cited by applicant]
Hieter et al., “Evolution of Human Immunoglobulin κ J Region Genes,” The Journal of Biological Chemistry 257(3):1516-1522 (1982).
[cited by applicant]
Larrick et al., “Polymerase Chain Reaction Using Mixed Primers: Cloning Of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells,” Biotechnology 7:934-938 (1989).
[cited by applicant]
Nakatani et al., “Functional Expression of Human Monoclonal Antibody Genes Directed Against Pseudomonal Exotoxin a In Mouse Myeloma Cells,” BioTechnology 7:805-810 (1989).
[cited by applicant]
Declaration of Prof. Olivier Devuyst, Nov. 15, 2023, 8 pgs.
[cited by applicant]
Declaration of Prof. Thomas Fehr, Jun. 1, 2022, 9 pgs.
[cited by applicant]
Hutchinson et al., “Free Ig Light Chains Interact with Sphingomyelin and Are Found on the Surface of Myeloma Plasma Cells in an Aggregated Form,” J Immunol. 185(7):4179-4188 (2010).
[cited by applicant]
Kuby—Immunology, sixth edition, chapter 5, pp. 105-136 (2006).
[cited by applicant]
Mullins et al., “Nuclear transfer in rodents,” J. Physiol. 554:4-12 (2003).
[cited by applicant]
Response to a USPTO Office Action filed by the Patentee on Oct. 17, 2013, in relation to the prosecution of related U.S. Appl. No. 12/589,181, 10 pgs.
[cited by applicant]
Zhou et al., “Generation of Fertile Cloned Rats by Regulating Oocyte Activation,” Science 302:1179 (2003) 2 pgs.
[cited by applicant]
Zhu et al., “Production of human monoclonal antibody in eggs of chimeric chickens,” Nature Biotechnology 23(9):1159-1169 (2005).
[cited by applicant]