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  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

• the germline modification further comprises a native nucleic acid sequence comprising a switch tandem repeat element (Sp) and Im promoter, wherein Ip drives constitutive expression of IgGl truncated for CHI domain (IgGlACHl).
• Sp switch tandem repeat element
• Im promoter IgGl truncated for CHI domain
• the IgH locus comprises a native nucleic acid sequence comprising an endogenous enhancer.
• the enhancer comprises Em, 3’RR, 3’gIE, 5’hsRl, or a combination thereof.
• the non-human animal is a mammal. In some embodiments, the mammal is a mouse.
• the mouse does not express a wild-type IgM protein, a wild- type IgD protein, a wild-type IgE protein, a wild-type IgG3 protein, or a combination thereof. In some embodiments, the mouse does not express a wild-type IgA protein, a wild-type IgG2b protein, a wild-type IgG2c protein, or a combination thereof.
• the exogenous nucleic acid sequence comprises 65 human V H gene segments, 27 human D H gene segments, and 6 J H gene segments.
• the stem cell is an embryonic stem cell.
• the method further comprises preserving a native nucleic acid sequence comprising a switch tandem repeat element (Sp) and Im promoter, wherein Ip drives constitutive expression of IgGl truncated for CHI domain (IgGlACHl).
• Sp switch tandem repeat element
• Im promoter IgGl truncated for CHI domain
• deleting an endogenous nucleic acid sequence comprising one or more heavy-chain C-region genes comprises CRISPR/Cas9 genome editing.
• the IgH allele (or the genome) can lack endogenous nucleic acid encoding each of the IgG2a constant domains, endogenous nucleic acid encoding each of the IgG2b constant domains, endogenous nucleic acid encoding each of the IgG2c constant domains, endogenous nucleic acid encoding each of the IgG3 constant domains, or endogenous nucleic acid encoding each of the IgG4 constant domains.
• the genetically modified non-human IgH allele can lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains comprising a CHI constant domain of an IgG subclass.
• the IgG subclass can comprise an IgGl, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclass.
• the IgG subclass can be the IgGl subclass.
• the genetically modified non-human IgH allele can comprise a nucleic acid sequence (Cyl-ACHl) encoding a CHI -truncated IgGl constant domain (IgGlACHl).
• the genetically modified non-human IgH allele can comprise one or more shark gene segments.
• the one or more shark gene segments can comprise VNAR- L38968, VNAR-L38967, or both.
• the shark gene segments can comprise a nucleic acid sequence selected from SEQ ID NOs:55-56.
• the DNA can comprise one or more human VH gene segments.
• the DNA can comprise one or more human JH gene segments.
• the genetically modified non-human IgH allele can encode an IgG heavy chain antibody, and the IgG heavy chain antibody can comprise a kappa light chain variable domain, a lambda light chain variable domain, or both.
• the genetically modified non-human IgH allele can comprise one or more exogenous human lambda light chain (LV) gene segments.
• LV human lambda light chain
• the genetically modified non-human IgH allele can lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains comprising a CHI constant domain of an IgG subclass.
• the IgG subclass can comprise an IgGl, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclass.
• the IgG subclass can be the IgGl subclass.
• the genetically modified non-human IgH allele can comprise a nucleic acid sequence (Cyl-ACHl) encoding a CHI -truncated IgGl constant domain (IgGlACHl).
• the genetically modified non-human IgH allele can comprise an Im promoter, an Im exon, or both.
• the genetically modified non-human IgH allele can comprise a switch tandem repeat element (Sp).
• IgGl expression can be driven by the Em, Im promoter, Sp, or any combination thereof.
• the genetically modified non-human IgH allele can lack one or more endogenous switch regions comprising Sy3, Syl, Sy2b, Sy2c, Ss, Sa, or any combination thereof.
• the genetically modified non-human IgH allele can comprise the following components (from 5’ to 3’): Em, Im promoter, Im exon, Sp, Cyl-ACHl, 3’gIE, 5’hsRl, and 3’RR.
• the docking cassette can comprise a nucleic acid sequence encoding a selection marker.
• the selection marker can comprise geneticin, hydromycin, puromycin, or any combination thereof.
• the genetically modified non-human IgH allele can encode an IgG heavy chain antibody.
• the genetically modified non-human IgH allele can comprise exogenous V gene segments, exogenous D gene segments, exogenous J gene segments, or any combination thereof.
• the exogenous gene segments can be selected from the group consisting of human, mouse, rat, bovine, alpaca, and shark gene segments.
• the exogenous gene segments can comprise human gene segments.
• the genetically modified non-human IgH allele can comprise one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments.
• the exogenous gene segments can be selected from the group consisting of human, mouse, rat, bovine, alpaca, and shark gene segments.
• the exogenous gene segments can comprise human gene segments.
• the genetically modified non-human IgH allele can comprise one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments.
• the genetically modified non-human IgH allele can comprise at least 10, 20, 30, 40, 50, 60, 80, 100, 120, or 126 human VH gene segments.
• the genetically modified non-human IgH allele can comprise at least 10, 15, 20, 25, or 27 human DH gene segments.
• the genetically modified non-human IgH allele can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments.
• this document features a recombinant vector system comprising at least one nucleic acid construct encoding a CRISPR/Cas genome editing system comprising a Cas protein and at least one guide RNA (gRNA), wherein the Cas protein and at least one gRNA form a complex that deletes one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the deleted one or more nucleic acid sequences encode at least a portion of one or more endogenous constant domains comprising a CHI constant domain of an IgG subclass, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof.
• gRNA guide RNA
• Figures 8A-8D are schematics of a Singularity Sapiens allelic series (SSV4-5) showing sequential integration of human IGH-BAC4 and IGH-BAC5 via RMCE into a clone containing human IGH-BAC1, human IGH-BAC2, and human IGH-BAC3, followed by removal of the selection marker cassette with via expression of ⁇ FC31 recombinase.
• SSV4-5 Singularity Sapiens allelic series
• Figure 10 illustrates an example of BAC recombineering.
• Source BACs are modified by bacterial homologous recombination (recombineering) to incorporate the appropriate selectable markers and recombination sites at the desired positions. Shown is an engineering process ofhlgH-BACl.
• FIG. 14A is a schematic showing deletion of the entire mouse IG Lambda locus via CRISPR mediated NHEJ.
• FIG. 14B is a PCR result confirming the generation of the IGL KO allele in ES cells.
• Singularity Sapiens allele containing all human JH segments can be used as a platform to integrate via sequential RCME a series of engineered hIGKV-BACs containing human VK segments from the human IG Kappa locus of chromosome 2.
• the resulting Singularity Sapiens VK-JH-containing allele can produce antibodies that contain a human variable Kappa segment contiguous with a human JH segment followed by a mouse constant region (e.g., a mouse IgGlACHl region).
• a truncated IgGl of about 40 kDa was detected in Singularity Musculus mice (as compared to the full length IgGl of about 50 kDa in the wild type mice), whereas IgM and IgG2b were not detectable ( Figure 15D).
• a truncated IgGl of about 40 kDa corresponding to the human VDJ-mouse IgGl -ACHl, was detected in immunized Singularity Sapiens mice (SSV1), which did not express IgM or full length IgGl as seen in the wild type mice ( Figure 17).
• the products of template switch reverse transcription were then amplified in the first round of PCR reaction using the 5’RACE adapter forward primer (5’-CTACACTC- TTTCCCTACACGACGCTCTTCCGATCT-3’; SEQ ID NO:38) and a reverse primer specific to the IgGl CH2 domain (5 ’-GGTGGTTGTGCAGGCCCTCATG-3 ’ ; SEQ ID NO:39).
• SEC size exclusion chromatography
• IgGl heavy chain antibody is a IgGlACHl protein.
• Embodiment 25 A The engineered non-human animal of any one of Embodiments 22 A- 24A, wherein the IgG heavy chain antibody lacks a light chain.
• Embodiment 33 A The engineered non-human animal of Embodiment 32 A, wherein the exogenous nucleic acid sequence comprises a bar code.
• Embodiment 34A The engineered non-human animal of any one of Embodiments 15A- 33A, wherein the non-human animal is a mammal.
• Embodiment 40 A The method of Embodiment 39 A, wherein the deletion of the nucleic acid sequence encoding the CHI domain of the IgGl C-region gene comprises exon 1.
• Embodiment 47 A The method of Embodiment 46A, wherein the IgGl heavy chain antibody is a IgGlACHl protein.
• Embodiment 7B The genetically engineered mouse of Embodiment 6B, wherein the enhancer is Em, 3’RR, 3’gIE, 5’hsRI, or a combination thereof.
• Embodiment 27B The engineered non-human animal of any one of Embodiments 15B- 26B, wherein the non-human animal does not express wild-type IgM protein, a wild-type IgD protein, a wild-type IgE protein, a wild-type IgG3 protein, or a combination thereof.
• Embodiment 40B A method of producing a genetically modified non-human animal capable of producing a humanized heavy-chain antibody comprising (a) deleting an endogenous nucleic acid sequence comprising one or more heavy-chain C-region genes from an endogenous immunoglobulin heavy chain locus in a stem cell of a non-human animal; (b) implanting the stem cell into a blastocyst; (c) implanting the blastocyst into a pseudo pregnant mouse to obtain a chimeric mouse; (d) crossing the chimeric mouse to a wild-type mouse to produce offspring; (e) screening the offspring for heterozygosity; and (1) identifying a founder mouse carrying a deletion of one or more heavy-chain C-region genes; and wherein said non-human animal is capable of producing a humanized heavy chain antibody.
• Embodiment 62B A method of producing a soluble humanized heavy-chain antibody in the engineered non-human animal of any one of Embodiments 40B-61B comprising (a) administering to the non-human animal an antigen; (b) isolating one or more B cells from the non-human animal; (c) isolating mRNA from the one or more B cells; (d) sequencing the mRNA; (e) identifying clonal type based on the mRNA sequence; and (1) performing phylogenetic analysis of the clonal type; thereby producing a soluble humanized heavy-chain antibody.
• Embodiment 3C The DNA of any one of Embodiments 1C-2C, wherein the genetically modified non-human IgH allele lacks one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains comprising a CHI constant domain of an IgG subclass.
• Embodiment 11C The DNA of any one of Embodiments 1C- IOC, wherein the genetically modified non-human IgH allele comprises a switch tandem repeat element (Sp).
• Sp switch tandem repeat element
• Embodiment 12C The DNA of any one of Embodiments 1 C-l 1 C, wherein IgGl expression is driven by the Em, Im promoter, Sp, or any combination thereof.
• Embodiment 46C The DNA of any one of Embodiments 42C-45C, wherein the DNA comprises one or more human JH gene segments.
• Embodiment 47C The DNA of any one of Embodiments 1C-46C, wherein the genetically modified non-human IgH allele encodes an IgG heavy chain antibody, and wherein the IgG heavy chain antibody comprises a kappa light chain variable domain, a lambda light chain variable domain, or both.
• Embodiment 72C The genetically modified non-human animal of any one of Embodiments 68C-71 C, wherein the IgG heavy chain antibody comprises a hinge domain, CH2 domain, a CH3 domain, or any combination thereof.
• Embodiment 75 C The genetically modified non-human animal of any one of
• Embodiment 76C The genetically modified non-human animal of any one of Embodiments 68C-75C, wherein the IgG heavy chain antibody comprises a kappa light chain variable domain, a lambda light chain variable domain, or both.
• Embodiment 79C The method of any one of Embodiments 77C-78C, wherein the IgG constant domain comprises a constant domain of an IgG subclass.

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