[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

CN116635407A - Nucleic acids encoding anchor modified antibodies and uses thereof - Google Patents

Nucleic acids encoding anchor modified antibodies and uses thereof Download PDF

Info

Publication number
CN116635407A
CN116635407A CN202180085965.3A CN202180085965A CN116635407A CN 116635407 A CN116635407 A CN 116635407A CN 202180085965 A CN202180085965 A CN 202180085965A CN 116635407 A CN116635407 A CN 116635407A
Authority
CN
China
Prior art keywords
human
segment
nucleic acid
light chain
locus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202180085965.3A
Other languages
Chinese (zh)
Inventor
J·马斯泰蒂斯
A·J·莫菲
J·麦克沃克
V·沃罗宁那
J·科洛马达
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regeneron Pharmaceuticals Inc
Original Assignee
Regeneron Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Priority to CN202311142098.XA priority Critical patent/CN117186227A/en
Priority claimed from PCT/US2021/064263 external-priority patent/WO2022140219A1/en
Publication of CN116635407A publication Critical patent/CN116635407A/en
Pending legal-status Critical Current

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Described herein are anchor modified immunoglobulin polypeptides, wherein an anchor immobilizes the immunoglobulin polypeptide to a receptor of interest. The anchor modified immunoglobulin polypeptides are typically characterized at the N-terminus by an anchor, such as a receptor binding portion of a ligand that binds to the receptor. The anchor modified immunoglobulin polypeptide can be prepared from a non-human animal genetically modified with a recombinant immunoglobulin segment encoding the anchor modified immunoglobulin polypeptide. Also provided are such non-human animals, as well as methods and compositions for making and using the same. Methods for producing anchor modified immunoglobulins from non-human animals are also provided, as are anchor modified immunoglobulins produced thereby.

Description

Nucleic acids encoding anchor modified antibodies and uses thereof
Cross reference to related applications
The present application claims the benefit of U.S. provisional application Ser. No. 63/129,893 filed on 12/23/2020 and U.S. provisional application Ser. No. 63/219,402 filed on 8/2021, 35/2020, to 35USC 119 (e), each of which is hereby incorporated by reference.
Sequence listing
The sequence table written in file 10507WO01_ST25.Txt is 47 kilobytes, created at 2021, 12, 17, and hereby incorporated by reference in its entirety.
Background
Monoclonal antibody products have completely altered the biopharmaceutical industry and have made significant progress in the treatment of several diseases. Despite these advances, and the knowledge gained by using monoclonal antibodies for therapeutic use, diseases associated with targets that are difficult to bind and/or obtain with monoclonal antibodies still exist, highlighting the need for different methods of developing effective therapeutic methods.
Disclosure of Invention
Disclosed herein are recognition that it is desirable to engineer non-human animals into improved in vivo systems for the identification and development of new antibody-based therapies, and in some embodiments, antibodies (e.g., monoclonal antibodies and/or fragments thereof), which can be used to treat a variety of diseases. The nucleic acids, non-human animals, methods and polypeptides disclosed herein relate to anchor modified immunoglobulins. Anchors as described herein generally include a receptor binding portion of a non-immunoglobulin polypeptide that binds to a cognate receptor. Anchors attached to immunoglobulins help to increase the affinity of the immunoglobulin for cognate receptors of the anchors, thereby improving the binding properties of the immunoglobulin. Nucleic acid molecules encoding anchor modified immunoglobulins and/or useful for modifying a non-human animal such that the non-human animal can prepare the anchor modified immunoglobulin de novo are described herein.
The anchor modified immunoglobulins described herein may be encoded at least in part by variable region (V) segments, such as immunoglobulin (Ig) heavy chain variable regions (V H ) Segment or Ig light chain variable region (V L ) Segments, modified to encode and operably linked to anchors between Ig leader sequences and Framework (FR) and Complementarity Determining Region (CDR) sequences of germline V segments.
Thus, nucleic acid molecules comprising targeting vectors and non-human animal genomes, including unrearranged or rearranged modified Ig V segments, are also described. Also described are non-human animal genomes, non-human animal cells, and non-human animals comprising the nucleic acid molecules described herein.
The recombinant nucleic acid molecules described herein may include a modified immunoglobulin (Ig) variable (V) segment encoding an anchor modified Ig polypeptide, wherein the modified Ig V segment includes a nucleic acid sequence encoding the anchor located between a nucleic acid sequence encoding an Ig signal peptide and a nucleic acid sequence encoding a Framework Region (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3, and CDR3 of a germline Ig V segment or variant thereof, wherein the anchor modified Ig polypeptide comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, and (iii) the FR1, CDR1, FR2, CDR2, FR3, and CDR3 of the germline Ig V segment or variant thereof, wherein the anchor comprises a receptor binding portion of a non-immunoglobulin polypeptide of interest that binds to a cognate receptor, and optionally wherein the recombinant nucleic acid molecule lacks any other V segment. The recombinant nucleic acid molecules described herein may also include one or more (not) rearranged Ig-diversity (D) segments, one or more (not) rearranged Ig-splicing (J) segments, and/or one or more Ig-constant region (C) genes.
In some embodiments, the Ig signal peptide is the Ig signal peptide of the germline Ig V segment or variant thereof. In some embodiments, the Ig signal peptide includes sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7). In some embodiments, the anchor comprises a linker that connects the receptor binding portion of the non-immunoglobulin polypeptide of interest to the FR1, CDR1, FR2, CDR2, FR3, and CDR3 of the germline Ig V segment or variant thereof. In some linker embodiments, the linker comprises the sequence GGGGS (SEQ ID NO: 5)
In some embodiments, the germline Ig V segment or variant thereof is a germline Ig heavy chain variable (V H ) Segments or variants thereof, e.g. human (h) germline Ig V segments or variants thereof, e.g. germline human (h) V H 1-2 segment, germ line hV H 1-3 segment, germ line hV H 1-8 segment, germ line hV H Segment 1, germ line hV H 1-24 segment, germ line hV H 1-45 segment, germ line hV H 1-46 segment, germ line hV H 1-58 segment, germ line hV H Segment 1-69, germ line hV H 2-5 segment, germ line hV H 2-26 segment, germ line hV H 2-70 segment, germ line hV H 3-7 segment, germ line hV H 3-9 segment, germ line hV H 3-11 segment, germ line hV H 313 segment, germ line hV H 3-15 segment, germ line hV H 3-16 segment, germ line hV H 3-20 segment, germ line hV H 3-21 segment, germ line hV H 3-23 segment, germ line hV H 3-30 segment, germ line hV H 3-30-3 segment, germ line hV H 3-30-5 segment, germ line hV H 3-33 segment, germ line hV H 3-35 segment, germ line hV H 3-38 segment, germ line hV H 3-43 segment, germ line hV H 3-48 segment, germ line hV H 3-49 segments, germ line hV H 3-53 segment, germ line hV H 3-64 segment, germ line hV H 3-66 segment, germ line hV H 3-72 segment, germ line hV H 3-73 segment, germ line hV H 3-74 segment, germ line hV H 4-4 segment, germ line hV H 4-28 segment, germ line hV H 4-30-1 segment, germ line hV H 4.30-2 segment, germ line hV H 4-30-4 segment, germ line hV H 4-31 segment, germ line hV H 4-34 segment, germ line hV H 4-39 segment, germ line hV H 4-59 segment, germ line hV H 4-61 segment, germ line hV H 5-51 segment, germ line hV H 6-1 segment, germ line hV H 7-4-1 segment, germ line hV H 7-81 segments or variants thereof. In some embodiments, the germline Ig V segment or variant thereof is germline hV H 1-69 or a variant thereof. In some embodiments, the variant is an allelic variant.
In some embodiments, the recombinant nucleic acid molecule may include a heavy chain variable region locus, e.g., may include the following operably linked and from 5 'to 3': (I) The modified Ig V H Segment (II) one or more Ig heavy chain diversity (D) H ) Segments, and (III) one or more Ig heavy chain linkages (J H ) A section. In some embodiments, the one or more Ig D of (II) H Segments comprising one, more or all human Ig D H Segments, and/or (III) the one or more Ig J H Segments comprising one, more or all human Ig J H A section. In some embodiments, the one or more Ig D of (II) H Segment and the one or more Ig J of (III) H Gene segments are recombined and form rearranged Ig D H /J H A sequence such that the recombinant nucleic acid molecule comprises, operably linked and from 5 'to 3': the modified Ig V H Gene segments and the rearranged Ig D H /J H Sequence.
In some embodiments, the modified Ig V H Gene segments and the rearranged Ig D H /J H The sequences are recombined and form rearranged Ig V encoding an anchor-modified Ig heavy chain variable domain H /D H /J H A sequence, wherein the anchor modified Ig heavy chain variable domain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, and (iii) the IgV resulting from the rearrangement H /D H /J H The FR1, complementarity Determining Regions (CDRs) 1, FR2, CDR2, FR3, CDR3 and FR4 are encoded by the sequences.
In some embodiments, the modified Ig V H The segments are modified Ig Vs that are not rearranged H A gene segment.
In some embodiments, the recombinant nucleic acids disclosed herein further comprise a nucleic acid encoding an Ig heavy chain constant region (C H ) Wherein the nucleic acid sequence encodes an IgC H In (I) said modified IgV H Segments, (II) the one or more Ig D H Segments and (III) the one or more Ig J H Downstream of and operatively connected to the section. In some embodiments, the IgC is encoded H Including Igmu gene encoding IgM isotype, igdelta gene encoding IgD isotype, iggamma gene encoding IgG isotype, igalpha gene encoding IgA isotype and/or Igepsilon gene encoding IgE isotype. In some embodiments, the recombinant nucleic acid molecules described herein comprise a nucleic acid sequence encoding an anchor modified Ig heavy chain, wherein the anchor modified Ig heavy chain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, (iii) the IgV comprising a rearrangement of H /D H /J H Sequence-encoded Ig heavy chain variable domains of said FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and (iv) Ig C H . In some embodiments, the IgC H Non-human Ig C H For example rodent IgC H For example, rat IgC H Or mouse Ig C H 。
In some embodiments, the germline Ig V segment or variant thereof is a germline Ig light chain variable (V L ) Segments or variants thereof. In some embodiments, the recombinant nucleic acid molecule may include a light chain variable region locus, e.g., may include the following operably linked and from 5 'to 3': (I) The modified V L Segments, and (II) one or more Ig light chain engagements (J L ) A section.
In some embodiments, the modified Ig V L Segments and the one or more Ig J L The segments are recombined and form rearranged Ig V encoding an anchor modified Ig light chain variable domain L /J L A sequence wherein the anchor modified Ig light chainThe variable domain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, and (iii) the IgV resulting from the rearrangement L /J L The FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are sequence encoded.
In some embodiments, the recombinant nucleic acid molecule may include a light chain variable region locus and a light chain constant region encoding Ig (C L ) Wherein the nucleic acid sequence encodes an IgC L Is operably linked thereto downstream of: (I) Modified Ig V L Segments and (II) the one or more Ig light chain engagements (J L ) A section. In some embodiments, the anchor-modified Ig light chain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, (iii) the IgV comprising a rearrangement of L /J L Sequence-encoded Ig light chain variable domains of said FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and (iv) Ig C L . In some embodiments, the IgC L Non-human Ig C L For example rodent IgC L For example, rat IgC L Or mouse Ig C L 。
In some light chain variable region locus embodiments, the germline Ig V L The segment or variant thereof is a germline Ig light chain variable kappa (vk) segment or variant thereof. Thus, in some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modified Ig vκ segment and (II) one or more Ig light chain engagement κ (jκ) segments. In some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modified Ig V kappa segment, (II) one or more Ig light chain engagement kappa (Jkappa) and (III) a nucleic acid sequence encoding an Ig light chain constant kappa region (Ckappa).
In some light chain variable region locus embodiments, the germline Ig V L The segment or variant thereof is a germline Ig light chain variable lambda (vλ) segment or variant thereof. Thus, in some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modified V lambda segment and (II) one or moreIg light chains bind lambda (Jlambda) segments. In some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modified Ig V lambda segment, (II) one or more Ig light chain engagement lambda (J lambda) segments, and a nucleic acid sequence encoding an Ig light chain constant lambda region (C lambda).
In some embodiments, the recombinant nucleic acid molecules described herein include sequences shown as SEQ ID NO. 8 or degenerate variants thereof, or SEQ ID NO. 10 or degenerate variants thereof.
Targeting vectors, non-human animal cells (e.g., host cells, embryonic stem cells, etc.), and non-human animals including nucleic acid molecules are also described.
Targeting vectors comprising embodiments of the recombinant nucleic acid molecules disclosed herein are also described. In some targeting vector embodiments, the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig heavy chain locus such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a non-human Ig C at the non-human Ig heavy chain locus H A recombinant nucleic acid molecule upstream of and operably linked to, optionally wherein said non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or wherein said non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, an endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces the non-human V at the non-human Ig heavy chain locus H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces a non-human Ig heavy chain locus at one but oneAll non-persons V H Segment or all non-human V H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus. In some embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig heavy chain locus such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent heavy chain locus in a rodent or rodent cell (e.g., a rodent embryonic stem cell), and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V H 、D H And/or J H Deletion of gene segments or combinations thereof, and optionally wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Gene segment, all non-human J H Gene segment and one or more non-human C H And (3) a gene. In some embodiments, the 5 'homology arm comprises the sequence shown as SEQ ID NO. 11 and/or the 3' homology arm comprises the sequence shown as SEQ ID NO. 12.
In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a recombinant nucleic acid molecule upstream of and operably linked to a non-human Ig CL at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain locus is endogenous rodentAnimal Ig light chain loci and/or wherein said non-human Ig light chain loci comprise human or humanized immunoglobulin light chain variable regions, endogenous Ig V L And/or J L Deletion of a gene segment or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces the non-human V at the non-human Ig light chain locus L A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain loci, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig light chain loci L Segment and all non-human J L A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces all non-human V at the non-human Ig light chain locus L Segment and all non-human J H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus. In some embodiments, the targeting vectors described herein comprise a nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain locus is an endogenous rodent light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, an endogenous Ig V L And/or J L Deletion of gene segments or combinations thereof, and optionally wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L Segment, all non-human J L A gene segment and the non-human CL gene.
In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain kappa locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule located upstream of and operably linked to a non-human Ig ck at the non-human Ig light chain kappa locus, optionally wherein the non-human Ig light chain kappa locus is an endogenous rodent light chain kappa locus and/or wherein the non-human Ig light chain kappa locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vk and/or jk gene segments, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces a non-human vk segment at the non-human Ig light chain kappa locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces one or more non-human vk segments and all non-human jk segments at the non-human Ig light chain kappa locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces all non-human vk segments and all non-human jk segments at the non-human Ig light chain kappa locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus. In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms targeting a non-human Ig light chain kappa locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus, optionally wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces a non-human vk segment at the non-human Ig light chain kappa locus, all non-human jk gene segments, and the non-human ck gene.
In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule upstream of and operably linked to a non-human Ig C lambda at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain lambda locus is an endogenous rodent light chain lambda locus and/or wherein the non-human Ig light chain lambda locus comprises a deletion of human or humanized immunoglobulin light chain variable regions, endogenous Ig V lambda and/or J lambda gene segments, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces a non-human vlambda segment at the non-human Ig light chain lambda locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces one or more non-human vlambda segments and all non-human jlambda segments at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces all non-human V lambda segments and all non-human J lambda segments at the non-human Ig light chain lambda locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus. In some embodiments, a targeting vector comprises a recombinant nucleic acid molecule as described herein and 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces the non-human va segment, all non-human jlambda gene segments, and the non-human clambda gene at the non-human Ig light chain lambda locus.
Also described herein is a non-human animal genome comprising a recombinant nucleic acid molecule and/or targeting vector as described herein. In some non-human animal genome embodiments, the non-human animal genome comprises a recombinant nucleic acid molecule as described herein at an endogenous Ig locus of the non-human animal genome, e.g., the non-human animal genome comprises a targeting vector as described herein, wherein the targeting vector comprises 5 'and 3' homology arms that target the endogenous Ig locus. In some embodiments, the non-human animal genome is a rodent genome. In some embodiments, the non-human animal genome is a rat genome. In some embodiments, the non-human animal genome is a mouse genome.
Also described herein are non-human animal or non-human animal cells comprising recombinant nucleic acid molecules, targeting vectors, and/or non-human animal genomes as described herein. In some non-human animal embodiments, a non-human animal as described herein comprises in its germline, e.g., in a germ cell, a recombinant nucleic acid molecule, targeting vector, or non-human animal genome as described herein, e.g., capable of delivering the recombinant nucleic acid molecule, targeting vector, and/or non-human animal genome as described herein to its offspring.
Methods of preparing non-human cells, non-human embryos, and/or non-human animals in vitro using the recombinant nucleic acid molecules described herein, e.g., targeting vectors, are also described. In some embodiments, an in vitro method of modifying an isolated cell comprises introducing a recombinant nucleic acid molecule as described herein into an isolated cell, for example, by contacting the cell with a targeting vector as described herein. In some method embodiments, the cell is a host cell. In some method embodiments, the cell is an Embryonic Stem (ES) cell. In some embodiments, the cell as described herein or prepared according to the methods described herein is a rodent cell, e.g., wherein the rodent cell is a rat cell or a mouse cell.
Also described are methods of making anchor modified antigen binding proteins using the nucleic acid molecules, the non-human cells, and/or the non-human animals as described herein. Also described are non-human animal embryos and non-human animals, which may include and/or develop (e.g., arise from) embryonic stem cells as described herein. Such embryos or non-human animals may be developed by a method comprising implanting ES cells as described herein into an embryo, and/or implanting an embryo comprising the ES cells into a suitable host, and maintaining the host under suitable conditions during the development of the ES cells or embryo into viable offspring.
In some non-human animal embodiments as described herein (e.g., embodiments in which the non-human animal comprises a recombinant nucleic acid molecule, targeting vector, and/or genome as described herein and/or is produced according to a method as described herein), the non-human animal comprises, as compared to a control non-human animal: (a) a significant number of mature B cells in the spleen, (B) a significant number of kappa positive B cells in the spleen, (c) a significant number of lambda positive B cells in the spleen, (d) a significant level of serum IgG and/or (e) a significant level of serum IgM. In some embodiments, a non-human animal as described herein is capable of generating an immune response comparable to a control non-human animal. In some embodiments, a non-human animal as described herein comprises a plurality of antigen binding proteins, each comprising an anchor and/or derived from a recombinant nucleic acid molecule, a targeting vector, and/or a non-human animal as described herein. In some embodiments, the non-human animal as described herein further comprises a cognate receptor for the non-immunoglobulin polypeptide of interest, wherein the receptor binding portion of the non-immunoglobulin polypeptide of interest serves as an anchor. In some non-human animal embodiments, the non-human animal as described herein comprises a plurality of antigen binding proteins that each specifically bind to the cognate receptor of the non-immunoglobulin polypeptide of interest, wherein the receptor-binding portion of the non-immunoglobulin polypeptide of interest serves as an anchor.
As described herein, the non-immunoglobulin polypeptide of interest (wherein the receptor binding portion of the non-immunoglobulin polypeptide of interest serves as an anchor) may comprise Atrial Natriuretic Peptide (ANP). In some embodiments, the c-terminal tail of ANP (e.g., NSFRY (SEQ ID NO: 3)) may be used as an anchor for cognate receptors. In some embodiments, the cognate receptor comprises a Natriuretic Peptide Receptor (NPR), such as NPR3 or a portion thereof.
In some non-human animal embodiments, wherein a non-human animal as described herein is immunized with the cognate receptor of the non-immunoglobulin polypeptide of interest (e.g., ANP), optionally wherein the cognate receptor comprises a Natriuretic Peptide Receptor (NPR), e.g., NPR3, the receptor binding portion thereof (e.g., nsfray (SEQ ID NO: 3)) is used as an anchor as described herein, the non-human animal as described herein further comprises a plurality of antigen binding proteins that bind to the cognate receptor, each of the antigen binding proteins comprising less than 1 x 10 9 And/or t1/2 over 30 minutes. In some embodiments, at least 15% of the plurality of antigen binding proteins are capable of blocking and/or blocking binding of the cognate receptor to the non-immunoglobulin polypeptide of interest. In some embodiments, greater than 50% of the plurality of antigen binding proteins bind to the cognate receptor expressed on the surface of the cell.
In some non-human animal embodiments described herein, the non-human animal is a rodent. In some non-human animal embodiments described herein, the non-human animal is a rat. In some non-human embodiments, the non-human animal is a mouse.
Also described are anchor modified antigen binding proteins encoded by the nucleic acid molecules described herein or prepared by the non-human animals described herein.
Described herein are methods of producing an antigen binding protein or obtaining a nucleic acid encoding the antigen binding protein, the method comprising (i) immunizing a non-human animal described herein or prepared according to the methods described herein (e.g., a non-human animal comprising a modified immunoglobulin (Ig) variable (V) segment encoding an anchor modified Ig polypeptide) with an antigen (e.g., the cognate receptor of a non-immunoglobulin polypeptide of interest, wherein the receptor-binding portion of the non-immunoglobulin polypeptide of interest serves as an anchor), and (ii) allowing the non-human animal to produce an immune response to the antigen, comprising an antibody that binds to the antigen or a nucleic acid encoding the antibody. Some embodiments further comprise recovering the antigen binding protein or nucleic acid encoding the antigen binding protein from the non-human animal or non-human animal cell, e.g., a B cell, and optionally fusing the B cell with a myeloma cell to form a hybridoma. Some embodiments further comprise cloning the recovered nucleic acid into an expression construct, and optionally expressing the expression construct in a host cell. In some cloning embodiments, the method further comprises cloning the recovered nucleic acid, wherein the recovered nucleic acid encodes an Ig variable domain in frame with a human Ig constant region coding sequence. Also described herein are the B cells, hybridomas fused to the B cells, or the host cells expressing nucleic acids recovered from the B cells. In some embodiments, the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide. In some embodiments, the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. In some embodiments, the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide, and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
Also described herein are anchor modified Ig polypeptides that are (a) encoded by a recombinant nucleic acid molecule described herein, a targeting vector described herein, or a non-human animal genome described herein, (b) expressed by a non-human animal or non-human animal cell described herein, (c) expressed by the non-human animal or non-human animal cell prepared according to a method described herein, and/or (d) produced by any method described herein.
Other features, objects, and advantages of the non-human animals, cells, nucleic acids, and compositions disclosed herein will be apparent from the detailed description of certain embodiments that follows. However, it is to be understood that the detailed description, while indicating certain embodiments, is given by way of illustration and not limitation.
Drawings
The drawings contained herein are comprised of the following figures for illustrative purposes only and are not limiting.
FIG. 1 shows ANP modified V useful as a Cas9/GA modified donor DNA H The illustrations of exemplary, non-limiting embodiments of gene segments are not drawn to scale. Restriction recognition sites (XhoI, mrel, ecoRI, avrII, mrel) and a Spectinomycin (Spectinomycin) resistance gene (SPEC) are also shown. In this non-limiting example, human V is modified with a sequence encoding the C-terminal tail of Atrial Natriuretic Protein (ANP) (NSFRY; SEQ ID NO: 3) H 1-69 gene segment to form V comprising ANP modification H 1-69 gene segment. In general, the unfilled shapes represent human sequences, the filled shapes represent murine sequences, and the dashed shapes represent non-human and non-murine sequences.
FIG. 2 shows the modification of V in BAC clone VI504 H 1-69 (shown as SEQ ID NO: 10). The following features are shown:
-a sense DNA sequence comprising
(a) From germ line human V H The last 7 codons of the bipartite signal peptide encoded by the 1-69 segment (see split arrow labeled "signal peptide"); the nucleotide sequence encoding the entire signal polypeptide is shown as SEQ ID NO. 6; and the amino acid sequence of the entire signal polypeptide is shown as SEQ ID NO. 7,
(b) Germline Ig V H 1-69 comprising exon 1, intron 1 and exon 2 (see split arrow labeled "VH" directly above the DNA sequence)
(c) Germline Ig V H Intron 1 of the 1-69 segment ("intron"),
(d) The nucleotide sequence encodes the C-terminal tail of Atrial Natriuretic Peptide (ANP) (SEQ ID NO: 3) and a G4S linker (SEQ ID NO: 5),
(e) Germline V encoding FR1, CDR1 (patterning frame), FR2, CDR2 (patterning frame), FR3 and CDR3 (patterning frame) H 1-69 sectionsAnd the amino acid sequence thereof (see resolution arrow labeled IgHV 1-69), and
(e) 23-mer recombination signal sequence (RS-23 split),
-ANP modified V H 1-69, comprising a leader sequence (labeled ANP-G4S-VH 1-69),
the crRNA binding site for cleavage of VI504 with Cas9 in vitro (labeled 5' vh1-69Cas9 and PAM),
5 'and 3' overlap for donor and VI504 Gibson assembly (black lines labeled as arrows of "5'VH1-69 overlap" and 3' VH1-69 overlap ", respectively), and
restriction enzyme sites indicated by diagonal striped boxes under the sequence: for ligating the spectinomycin-resistant cassette to the EcoRI and AvrII sites in the donor, for removing the XhoI site of the donor from the pUC vector backbone, and for removing the MreI site of the Spec cassette from the modified BAC prior to seamless repair by adaptor oligonucleotide-mediated Gibson assembly.
FIG. 3 shows modification of ANP according to example 1 H The segments 1-69 are inserted into BAC clone VI504 to create a representation of a non-limiting exemplary embodiment of targeting vector VI748, not drawn to scale. Typically, the unfilled shapes represent a sequence of people (e.g., the unfilled triangles represent people V H Segment 1-69, person D H Segment and person J H Segments), filled shapes represent murine sequences (e.g., filled triangles represent endogenous murine V H Segments (not labeled), filled arrows represent murine Adam6a "a" and Adam6b "b" genes, filled ovals representing Ig enhancers and filled arrows representing murine Ig μ gene "IgM"), and dashed shapes represent non-human and non-murine sequences (e.g., chloramphenicol (chloromycetin) resistance gene "CM", site-specific recombinase recognition site "Frt site", hygromycin resistance gene "HYG" and spectinomycin resistance gene "SPEC").
FIG. 4 shows a schematic representation of a non-limiting exemplary embodiment of insertion of a targeting vector into the genome of a mouse ES cell by electroporation, not to scale. After electroporation, this is notThe exemplary embodiment retains endogenous V H Segments, depicted as filled triangles upstream of Adam6a ("a") gene. Typically, the unfilled shapes represent a sequence of people (e.g., the unfilled triangles represent people V H Segment 1-69, person D H Segment and person J H Segments), filled shapes represent murine sequences (e.g., filled triangles represent endogenous murine V H Segments (not labeled), filled arrows represent murine Adam6a "a" and Adam6b "b" genes, filled ovals representing Ig enhancers and filled arrows representing murine Ig μ gene "IgM"), and dashed shapes represent non-human and non-murine sequences (e.g., chloramphenicol resistance gene "CM", site-specific recombinase recognition site "Frt site", hygromycin resistance gene "HYG" and spectinomycin resistance gene "SPEC")
FIGS. 5A-5F show results relating to non-limiting examples of the invention comparing mice from ANP-VH1-69 modification with controlsA population of spleen cells of the mice. FIGS. 5A-B provide V modified with ANP H 1-69 segment (ANP) modified mice or controls comprising humanized Ig loci +.>Animal (control) (A) total number of cells per spleen (y-axis), CD19 + Number of cells/spleen (y-axis) or (B) lymphocytes per spleen (y-axis) (CD 19) + Cells). FIGS. 5C-5D provide V modified with ANP H 1-69 segment (ANP) modified mice or controls comprising humanized Ig loci +.>Animal (control) (C) mature B cells of each spleen (CD 19) + IgD hi IgM int ) And transitional B cells (CD 19) + IgD int IgM hi ) Total number (y-axis) or (D) mature B cells per spleen (CD 19) + IgD hi IgM int ) And transitional B cells (CD 19) + IgD int IgM hi ) Is a percentage of (c). FIGS. 5E-F provide ANP modified V H 1-69 segment (ANP) modified mice or controls comprising humanized Ig lociAnimal (control) (E) CD19 of each spleen + κ + Cell and CD19 + λ + Total number of cells (y-axis) or (F) CD19 per spleen + κ + Cell and CD19 + λ + Percentage of cells (y-axis).
FIGS. 6A-6F show results relating to non-limiting embodiments of the invention, comparing the results from ANP-V H Femur isolated population bone marrow cells of 1-69 modified mice and controlsAnd (3) a mouse. FIGS. 6A-B provide V modified with ANP H 1-69 segment (ANP) modified mice or controls comprising humanized Ig lociAnimal (control) (A) total number of cells per femur (y-axis) and CD19 per femur + B cell number (y axis) or (B) lymphocyte per femur (CD 19) + Cells). FIGS. 6C-D provide ANP modified V H 1-69 segment (ANP) modified mice or controls comprising humanized Ig loci +.>Animal (control) (C) CD43 of each femur + ckit + Total number of primordial B cells (y-axis) and CD43 per femur - ckit - Number of anterior B cells (y-axis) or (D) CD43 per femur + ckit + Pronor-B cells or CD43 - ckit - Percentage of pre-B cells. FIGS. 6E-F provide (E) V modified with ANP H 1-69 segment (ANP) modified mice or controls comprising humanized Ig loci Animal (control) (E) total number of immature B cells and mature B cells per femur (y axis) or (F) percentage of immature B cells and mature B cells per femur (y axis).
FIG. 7 shows the results associated with a non-limiting example of the present invention, analyzed by Western blot analysis from ANP-V H 1-69 modified animals and controlsSerum IgG levels of control animals.
FIGS. 8A-B show results related to non-limiting examples of the invention, (A) serum mice (mIgG) or (B) ANP-V H 1-69 modified Animals (ANP) or controls comprising a humanized Ig locusConcentration of serum mIgM isolated from animals (control) (μg/mL; y-axis).
FIG. 9 shows results related to a non-limiting embodiment of the invention, comparing ANP-V H 1-69 modified mice (ANP) or controls comprising a humanized Ig locus on a protein antigenImmune responses (antibody titer; y-axis) of animals (control). Of the serum tested, 3 serum samples were from 3 rd enhanced bleeding, 1 from ANP-V H 1-69 modified mice after 5 th boost. For->Control mice, 2 serum samples were from bleeding after the 6 th boost, and 1 from after the 9 th boost.
FIG. 10 shows results related to a non-limiting embodiment of the present invention, comparing ANP-V H 1-69 modified mice (ANP) or controls comprising a humanized Ig locus on a protein antigenImmune responses (antibody titer; y-axis) of animals (control). Of the serum tested, 3 serum samples were from 3 rd enhanced bleeding, 1 from ANP-V H 1-69 modified mice after 5 th boost. For->Control mice, 2 serum samples were from bleeding after the 6 th boost, and 1 from after the 9 th boost.
FIG. 11 shows the results associated with a non-limiting example of the present invention, comparing the K binding of hNPR3-MMH to NPR3 monoclonal antibody (mAb) using a plot of box plot D Value (y-axis) of the monoclonal antibody from ANP-V H 1-69 modified mice (ANP) or controls comprising a humanized Ig locus on a protein antigen at 25 DEG CAnimals (control) were isolated.
Fig. 12: shows the results associated with a non-limiting example of the present invention, comparing t1/2 values (y-axis) of binding of hNPR3-MMH to NPR3 monoclonal antibodies (mAbs) from ANP-V using a plot of box plot H 1-69 modified mice (ANP) or controls comprising a humanized Ig locus on a protein antigen at 25 DEG CAnimals (control) were isolated.
Definition of the definition
The scope of the invention is defined by the claims appended hereto and is not limited by the particular embodiments described herein; those skilled in the art will recognize many modifications that are equivalent to those described embodiments or which are within the scope of the claims after reading this disclosure. Generally, unless explicitly indicated otherwise, terms shall be in accordance with their understood meaning in the art. Explicit definitions of certain terms are provided herein and below; the meaning of these and other terms in a particular instance throughout the specification will be apparent to those skilled in the art depending on the context. Additional definitions of the following terms and other terms are set forth throughout the specification. References cited within this specification, or related portions thereof, are incorporated herein by reference in their entirety.
Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.
The articles "a" and "an" in the specification and claims should be understood to include plural referents unless the contrary is explicitly stated. Claims or descriptions that contain "or" between one or more members of a group should be construed as satisfying situations where one, more than one, or all of the group members are present, applied to, or otherwise associated with a given product or method, unless indicated to the contrary or otherwise clearly differentiated by context. The present invention encompasses embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The invention also encompasses embodiments in which more than one or all of the members of the group are present in, used in, or otherwise associated with a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the listed claims are introduced into another claim (or any other claim concerned) that is dependent on the same base claim unless otherwise indicated or unless it would be apparent to one of ordinary skill in the art that contradiction or inconsistency would arise. When elements are presented in a list (e.g., in a markush group or the like), it should be understood that each subset of elements is also disclosed, and any element may be removed from the group. It should be understood that, in general, where the invention or aspects of the invention are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist of, or consist essentially of, such elements, features, etc. In the interest of brevity, these embodiments are not described in detail with the words of description in each case. It should also be understood that any embodiment or aspect of the present invention may be explicitly excluded from the claims, whether or not such specific exclusion is described in the specification.
As used in this patent application, the terms "about" and "approximately" are used interchangeably. Any numerical value, with or without about/approximately, used in the present application is intended to encompass any normal fluctuation, such as +/-5%, as would be appreciated by one of ordinary skill in the relevant art.
And (3) application: refers to administering the composition to a subject or system (e.g., to a cell, organ, tissue, organism, or related component or group of components thereof). Those of skill in the art will appreciate that the route of administration may vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, the purpose of administration, and the like.
For example, in some embodiments, administration to an animal subject (e.g., to a human or rodent) can be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, intratracheal (including by intratracheal instillation), transdermal, intravaginal, and/or intravitreal. In some embodiments, administration may involve intermittent administration. In some embodiments, administration may involve continuous administration (e.g., infusion) for at least a selected period of time. In some embodiments, antibodies produced by the non-human animals disclosed herein can be administered to a subject (e.g., a human subject or rodent). In some embodiments, the pharmaceutical composition comprises an antibody produced by a non-human animal disclosed herein. In some embodiments, the pharmaceutical composition may comprise a buffer, a diluent, an excipient, or any combination thereof. In some embodiments, a pharmaceutical composition comprising an antibody produced by a non-human animal disclosed herein can be contained in a container for storage or administration, such as a vial, syringe (e.g., IV syringe), or bag (e.g., IV bag).
Affinity: refers to the strength of interaction between an antigen binding protein and its binding partner, e.g., between an antibody and a particular epitope. Antibodies that specifically bind to an epitope relative to K of its target epitope D Typically about 10 -9 M or less (e.g., about 1X 10) -9 M、1×10 -10 M、1×10 -11 M or about 1X 10 -12 M)。K D By surface plasmon resonance (e.g. BIACORE TM ) The method comprises the steps of carrying out a first treatment on the surface of the An enzyme-linked immunoassay (ELISA) or other well known method.
Antibody: refers to immunoglobulin antigen binding proteins. Tetrameric antibodies include four polypeptide immunoglobulin (Ig) chains, e.g., two Ig heavy (H) chains and two Ig light (L) chains, interconnected by disulfide bonds. Each heavy chain comprises an Ig heavy chain variable domain and an Ig heavy chain constant region or domain (C H ). The heavy chain constant region comprises three domains C H 1、C H 2 and C H 3. Each Ig light chain includes an Ig light chain variable domain and an Ig light chain constant region (C L )。
The Ig heavy chain variable domain and the Ig light chain variable domain can each be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions, termed Framework Regions (FR). Each of the Ig heavy chain variable domain and the light chain variable domain comprises three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR 3).
Antigen binding proteins refer to immunoglobulins, antibodies, binding proteins, and the like, such as monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, fab fragments, F (ab') fragments, disulfide-linked Fv (sdFv), intracellular antibodies, minibodies, diabodies, and anti-idiotype (anti-Id) antibodies, as well as epitope-binding fragments of any of the foregoing. The terms "antibody" and "antibodies" also refer to covalent bifunctional antibodies, such as U.S. patent application publication 20070004909, which is incorporated by reference in its entirety, and Ig DARTS, such as those disclosed in U.S. patent application publication 20090060910, which is incorporated by reference in its entirety.
Biological activity: refers to any agent that is active in a biological system, in vitro, or in vivo (e.g., in vivo). For example, an agent that has a biological effect in an organism when present in that organism is considered to have biological activity.
In particular embodiments wherein the protein or polypeptide has biological activity, a portion of the protein or polypeptide that confers at least one biological activity on the protein or polypeptide is commonly referred to as a "bioactive" portion.
Homology: refers to two biomolecules (e.g., receptors and their ligands) that typically interact.
The method is equivalent to that of: refers to two or more agents, entities, conditions, sets of conditions, etc., that may be different from each other but sufficiently similar to allow comparison therebetween such that a conclusion may be reasonably drawn based on the observed differences or similarities. Those of ordinary skill in the art will understand how, in this context, for two or more such agents, entities, situations, sets of conditions, etc., a degree of identity that is deemed quite desirable in any given case.
Conservation: refers to conservative amino acid substitutions, i.e., the substitution of one amino acid residue with another amino acid residue having a side chain R group of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not significantly alter the functional properties of the protein, e.g., the ability of the non-immunoglobulin polypeptide of interest to bind to its cognate receptor. Examples of groups of amino acids whose side chains have similar chemical properties include: aliphatic side chains such as glycine, alanine, valine, leucine and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and sulfur-containing side chains such as cysteine and methionine. Conservative amino acid substitutions groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamic acid/aspartic acid, and asparagine/glutamine.
In some embodiments, conservative amino acid substitutions may be substitution of any native residue in the protein with alanine, as used, for example, in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have positive values in the PAM250 log likelihood matrix disclosed in Gonnet, G.H. et al, 1992, science (Science) 256:1443-1445, which is hereby incorporated by reference in its entirety. In some embodiments, if a substitution has a non-negative value in the PAM250 log likelihood matrix, the substitution is a moderately conservative substitution.
Control: refers to the meaning of "control" as understood in the art, i.e., a criterion for comparison of results. Typically, controls are used to improve experimental integrity by separating variables to draw conclusions regarding such variables. In some embodiments, the control is a reaction or assay that is performed concurrently with the test reaction or assay to provide a comparison. "control" may refer to a "control animal". The "control animal" may have a modification as described herein, a different modification than the modification described herein, or no modification (i.e., wild-type animal). In one experiment, a "test" (i.e., a variable tested) was applied. In the second experiment, the "control", no variable tested was applied. The control may be a positive control or a negative control.
In some embodiments, the control is a historical control (i.e., a test or assay previously performed, or a previously known amount or result). In some embodiments, the control is or includes a record printed or otherwise saved.
Degenerate variants of a reference nucleic acid molecule encode a polypeptide that has an amino acid sequence that is identical to the amino acid sequence encoded by the reference nucleic acid and that has a nucleic acid sequence that is substantially identical to the reference nucleic acid molecule, but differs due to the degeneracy of the genetic code.
Breaking: refers to the result of homologous recombination events with a DNA molecule (e.g., with an endogenous homologous sequence such as a gene or locus).
In some embodiments, the cleavage may effect or represent an insertion, a deletion, a substitution, a missense mutation, or a DNA sequence frameshift, or any combination thereof. Insertion may comprise insertion of the entire gene, a fragment of the gene, such as an exon (which may have an origin other than an endogenous sequence (e.g., a heterologous sequence)), or a coding sequence derived or isolated from a particular gene of interest. In some embodiments, the disruption may enhance expression and/or activity of a gene or gene product (e.g., a protein encoded by a gene). In some embodiments, the disruption may reduce expression and/or activity of the gene or gene product. In some embodiments, the disruption may alter the sequence of the gene or encoded gene product (e.g., encoded protein). In some embodiments, the disruption may alter the sequence of the chromosome or the chromosomal location in the genome. In some embodiments, the disruption may truncate or split the gene or encoded gene product (e.g., encoded protein). In some embodiments, the disruption may extend the gene or encoded gene product. In some such embodiments, cleavage may allow for assembly of the fusion protein. In some embodiments, the disruption may affect the level of the gene or gene product but not its activity. In some embodiments, the disruption may affect the activity of the gene or gene product but not its level. In some embodiments, the disruption may have no significant effect on the level of the gene or gene product. In some embodiments, the disruption may have no significant effect on the activity of the gene or gene product. In some embodiments, the disruption may have no significant effect on the level or activity of the gene or gene product. In some embodiments, the significant effect may be measured by, for example, but not limited to, the Stoneon's T test (Student's s T-test).
Endogenous locus or endogenous gene: refers to a genetic locus found in a parent or reference organism (or cell) prior to the introduction of a change, break, deletion, insertion, modification, substitution, or substitution described herein.
In some embodiments, the endogenous locus comprises all or part of the sequence found in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-bred organism (whether wild-type or engineered).
Endogenous promoters: refers to a promoter naturally associated with an endogenous gene or genetic locus, for example in a wild-type organism.
Engineering: generally referred to as manually manipulated aspects. As is commonly practiced and understood by those of skill in the art, even though the actual operation is an engineered polynucleotide or cell progeny of a previous entity, is still generally referred to as "engineered". Furthermore, those skilled in the art will recognize that the "engineering" described herein may be accomplished in a variety of available ways. A polynucleotide may be considered "engineered" when two or more sequences that are not sequentially joined together in nature are manually manipulated to join directly to each other in an engineered polynucleotide. In some embodiments, an engineered polynucleotide may include a regulatory sequence that is operably linked to a first coding sequence in nature but is not operably linked to a second coding sequence, the regulatory sequence being operably linked to the second coding sequence by a manual linkage. Alternatively or additionally, in some embodiments, a first nucleic acid sequence and a second nucleic acid sequence each encoding a polypeptide element or domain that are not linked to each other in nature may be linked to each other in a single engineered polynucleotide. In contrast, in some embodiments, a cell or organism may be considered "engineered" if the cell or organism has been manipulated such that its genetic information has been altered (e.g., new genetic material that was not previously present has been introduced, or genetic material that was previously present has been altered or removed).
In some embodiments, "engineering" may involve selection or design (e.g., nucleic acid sequences, polypeptide sequences, cells, tissues, and/or organisms) through the use of a computer system programmed to perform analysis or comparison, or otherwise analyze, recommend and/or select sequences, changes, or the like. Alternatively or additionally, in some embodiments, "engineering" may involve the use of in vitro chemical synthesis methods and/or recombinant nucleic acid techniques, such as nucleic acid amplification (e.g., via polymerase chain reaction), hybridization, mutation, transformation, transfection, etc., and/or any of a variety of controlled mating methods. As will be appreciated by those of skill in the art, various established such techniques (e.g., for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection, etc.) are well known in the art and are described in various general and more specific references cited and/or discussed in this specification see, e.g., sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual), 2 nd edition, cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y.), 1989; incorporated herein by reference in its entirety.
Gene: refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and/or a polypeptide product). For clarity, the term "gene" generally refers to a portion of a nucleic acid encoding a polypeptide; the term may optionally encompass regulatory sequences, as will be clear to one of ordinary skill in the art in this context. This definition is not intended to exclude the use of the term "gene" for non-protein encoding expression units, but rather to clarify that, in most cases, the term used in this document refers to a nucleic acid encoding a polypeptide.
In some embodiments, the gene comprises a coding sequence (i.e., a sequence encoding a particular product). In some embodiments, the gene comprises a non-coding sequence. In some embodiments, a gene may comprise both coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene may comprise one or more regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences that may control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression, etc.), for example.
The variable domains of immunoglobulin antigen receptors, such as antibodies, are encoded in a set of gene segments, also referred to herein as "segments," which are positioned along the chromosomal sequence and undergo somatic recombination to form the complete variable domain exons. The configuration of a genetic human gene segment, e.g., the germline configuration of a human gene segment, e.g., the order of the human gene segment in the human germline genome (e.g., the genome that passes to the next generation) can be found in: lefranc, M. -P., "Experimental and clinical immunogenetics (exp. Clin. Immunogenet.), 18,100-116 (2001), incorporated herein by reference in its entirety, also shows functional gene segments and pseudogenes found within germline-configured human immunoglobulin heavy chain loci. The gene segments are classified as variable (V) gene segments (each of which may also be referred to as V segments alone), diversity (D) gene segments (each of which may also be referred to as D segments alone), or junction (J) gene segments (each of which may also be referred to as J segments alone). Multiple copies of each type of gene segment are present in germline DNA, but only one copy of each type of receptor chain is expressed in receptor-bearing lymphocytes
A series of recombination events involving several genetic components acts to assemble immunoglobulins from an ordered arrangement of gene segments (e.g., V, D and J). Such assembly of gene segments is known to be imprecise, and thus immunoglobulin diversity is achieved by both combining different gene segments and forming unique linkages via imprecise joining. Further diversity is generated by a process known as somatic hypermutation, in which the variable region sequences of the immunoglobulins are altered to increase affinity and specificity for antigens. Ig gene segments, e.g., ig V segments, referred to herein include variants of germline Ig V segments. Variants of germline Ig segments, e.g., germline Ig V segments, include polymorphic (e.g., allelic) variants thereof, somatic hypermutated variants thereof, recombinant variants thereof, and degenerate variants thereof.
Sequence polymorphisms of the coding region of an Ig segment, such as the sequence of allelic variants of a germline Ig segment, are described below: palaros, N.et al, (1998) experimental and clinical immunogenetics, 15,8-18; barbie, V. and Lefranc, M. -P. (1998) experimental and clinical immunogenetics, 15,171-183; martinez, C.and Lefranc, M. -P. (1998) experimental and clinical immunogenetics, 15,184-193; palaros, N.et al, (1999) experimental and clinical immunogenetics, 16,36-60 (1999), and Ruiz, M.et al, (1999) experimental and clinical immunogenetics, 16,173-184, each of which is incorporated herein by reference in its entirety. The expression of the allelic germline Ig segments can also be found on the world wide web (www) in two formats, imgt.org/IMGTrepertoire/Proteins/#b, which shows an alignment of all known sequences assigned to different alleles by comparison with allele x 01, and imgt.org/IMGTrepertoire/Proteins/#c, which provides a description of nucleotide mutations and corresponding amino acid changes of different alleles by comparison with allele x 01. See also EP patent No. 3 128009, which is incorporated herein by reference in its entirety.
An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it comprises one or more of the following regions: FR1, FR2 and/or FR3 have at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% nucleic acid sequence homology to the germline segment, or encode an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence homology to the amino acid sequence encoded by the germline Ig V segment.
In some embodiments, an Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it comprises one or more of the following regions: FR1, FR2 and/or FR3 have a nucleic acid sequence homology of between 80% and 99% with the germline segment. In some embodiments, an Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it comprises one or more of the following regions: FR1, FR2 and/or FR3 have a nucleic acid sequence homology of between 85% and 99% with the germline segment. In some embodiments, an Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it comprises one or more of the following regions: FR1, FR2 and/or FR3 have a nucleic acid sequence homology of between 90% and 99% with the germline segment. In some embodiments, an Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it comprises one or more of the following regions: FR1, FR2 and/or FR3 have a nucleic acid sequence homology of between 95% and 99% with the germline segment.
An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, an amino acid having between 80% and 99% amino acid sequence homology with the amino acid sequence encoded by the germline Ig V segment. An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, an amino acid having between 85% and 99% amino acid sequence homology with the amino acid sequence encoded by the germline Ig V segment. An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, an amino acid having between 90% and 99% amino acid sequence homology with the amino acid sequence encoded by the germline Ig V segment. An Ig V segment can be considered a recombinant or somatically hypermutated variant of a germline Ig V segment if it encodes one or more of the following regions: FR1, FR2 and/or FR3, an amino acid having between 95% and 99% amino acid sequence homology with the amino acid sequence encoded by the germline Ig V segment.
An immunoglobulin molecule is a Y-shaped polypeptide consisting of two identical heavy chains and two identical light chains, each chain having two structural components: a variable domain and a constant domain. It is the variable domain of the heavy and light chains assembled from gene segments, while the constant domain is fused to the variable domain by RNA splicing. Although the mechanism of assembling (or ligating) gene segments is similar for heavy and light chains, the light chain requires only one ligation event (i.e., V and J), while the heavy chain requires two ligation events (i.e., D and J and V and DJ).
Typically, an immunoglobulin heavy chain variable domain is encoded by a variable domain exon that is variable by an immunoglobulin heavy chain (V H ) Gene segments (also called V H Segment) and immunoglobulin heavy chain diversity (D H ) Gene segment (also called D H Segment) and immunoglobulin heavy chain junction J H Gene segments (also called J H Segments) are recombined. Immunoglobulin light chain variable domains are typically encoded by variable domain exons that are variable by immunoglobulin light chains (V L ) Gene segments (also called V L Segment) to immunoglobulin light chain (J L ) Gene segments (also called J L Segment) is formed by somatic recombination.
The assembly of the gene segments of the heavy and light chain variable regions, referred to as VDJ recombination and VJ recombination, respectively, is guided by conserved non-coding DNA sequences flanking each gene segment, referred to as Recombination Signal Sequences (RSS), which ensure precise positional rearrangement of the DNA relative to V, D and J coding sequences (see, e.g., ramsden, d.a. et al, 1994, nucleic acids research (nuc.acids res.)) 22 (10): 1785-96; incorporated herein by reference in its entirety). Each RSS consists of a conserved block of seven nucleotides (heptamer) that is contiguous with the coding sequence (e.g., V, D or J segment), followed by a spacer (12 bp or 23 bp) and a second conserved block of nine nucleotides (nonamer). Although it is tolerable that there are considerable sequence differences between individuals for the 12Bp or 23Bp spacers, the length of these sequences will generally not vary. Recombination between immunoglobulin gene segments follows a rule commonly referred to as the 12/23 rule, wherein gene segments flanking an RSS with a 12bp spacer (or 12 mer) are generally flanked by gene segments flanking a 23bp spacer (or 23 mer; see, e.g., hiom, K. And M.Gellert,1998, molecular cells (mol. Cell.) "1 (7): 1011-9; incorporated herein by reference in its entirety).
Unless otherwise indicated, or unless a contradiction or inconsistency would be apparent to one of ordinary skill in the art, an unrearranged gene segment to which no reference is made is considered to include two RSS to which the gene segment naturally relates, e.g., flanking, operably linked, etc. In some embodiments, a gene segment not rearranged herein may include a gene segment in its germline (e.g., wild-type) configuration, e.g., germline V H Gene segment and germ line J H The gene segment is flanked on both sides by 23-mer RSS. In contrast, germline D H Gene segments, e.g. unrearranged D H The gene segment is flanked on each side by 12-mer RSS.
Thus, an unrearranged gene segment may also refer to a gene segment in its germline configuration, including any RSS associated with such germline configuration. Furthermore, a plurality of gene segments in their germline configuration generally refers not only to each individual gene segment being in its germline (e.g., unrearranged) configuration, but also to the order and/or position of functional gene segments. See, e.g., lefranc, m. -p., "experimental and clinical immunogenetics", 18,100-116 (2001); incorporated herein by reference in its entirety for human V, D and germline configuration of the J gene segments.
Each V segment comprises a nucleic acid sequence encoding an Ig signal or leader (L) peptide operably linked to a nucleic acid sequence encoding a portion of FR1, CDR1, FR2, CDR2, FR3, and CDR3 of an immunoglobulin variable domain. Each D gene segment contributes to CDR3 of the immunoglobulin heavy chain variable domain. Each J gene segment also contributes to CDR3 and FR4 of the immunoglobulin variable domain. The amino acid positions of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are based on unique numbering described in the following: lefranc et al, (2003) development and comparison immunology (Dev. Comp. Immunol.) 27:55-77, and can also be seen on www.imgt.org.
Heterologous: refers to agents or entities from different sources. For example, when used in reference to a polypeptide, gene, or gene product that is present in a particular cell or organism, the term clarifies the relevant polypeptide or fragment thereof, gene or fragment thereof, or gene product or fragment thereof: (1) is artificially engineered; (2) Introduction of the cell or organism (or precursor thereof) by manual (e.g., by genetic engineering); and/or (3) are not naturally produced by or present in the relevant cell or organism (e.g., the relevant cell type or organism type). Another example comprises a polypeptide or fragment thereof, a gene or fragment thereof, or a gene product or fragment thereof, which is typically present in a particular native cell or organism, but which has been modified, e.g., by mutation or placed under the control of a non-naturally associated and in some embodiments non-endogenous regulatory element (e.g., a promoter).
Host cell: refers to cells into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. Those skilled in the art will appreciate upon reading this disclosure that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".
In some embodiments, the host cell is or comprises a prokaryotic cell or a eukaryotic cell. In embodiments, the host cell is or includes a mammalian cell. Generally, a host cell is any cell suitable for accepting and/or producing a heterologous nucleic acid or protein, regardless of the life world to which the cell is assigned. Exemplary cells include those of prokaryotes and eukaryotes (single or multiple cells), bacterial cells (e.g., escherichia coli), bacillus (Bacillus spp.), streptomyces (Streptomyces spp.), etc.), mycobacterial cells, fungal cells, yeast cells (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (Schizosaccharomyces pombe), pichia pastoris (Pichia pastoris), pichia methanolica (Pichia methanolica), etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus infected insect cells, trichoplusia ni (Trichoplusia ni), etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas.
In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the following: CHO (e.g., CHO K1, DXB-11CHO, veggie-CHO), COS (e.g., COS-7), retinal cells, vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, haK, BHK), heLa, hepG2, WI38, MRC 5, colo205, HB 8065, HL-60 (e.g., BHK 21), jurkat, daudi, A431 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2/0, NS-0, MMT 060562, sertoli cells (seltoli cells), BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing. In some embodiments, the cells include one or more viral genes, such as retinal cells expressing viral genes (e.g.Cells). In some embodiments, the host cell is or comprises an isolated cell. In some embodiments, the host cell is part of a tissue. In some embodiments, the host cell is part of an organism.
Humanization: refers to a molecule of non-human origin (e.g., nucleic acid, protein, etc.) a portion of which has been replaced with a corresponding portion of a corresponding human molecule, thereby allowing the modified (e.g., humanized) molecule to retain its biological function and/or maintain a structure that performs the retained biological function. In contrast, "human" and the like encompass molecules derived from humans only, such as human nucleotides or proteins comprising only human nucleotide and amino acid sequences, respectively. The term "human (humanized)" is used to reflect that a human (humanized) molecule may be (a) a human molecule or (b) a humanized molecule.
Identity: by comparison of sequences, it is meant the identity determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity.
In some embodiments, clustalW v.1.83 (slow) alignment (with an open gap penalty of 10.0, an extended gap penalty of 0.1) and Gonnet similarity Matrix (MACVECTOR) are used TM 10.0.2,MacVector Inc., 2008) to determine identity as described herein.
Immunoglobulins refer to a class of polypeptides and nucleic acids encoding the polypeptides that are present in serum or expressed on B cells of the immune system, which function as antibodies, e.g., antigen binding proteins.
A non-immunoglobulin polypeptide refers to a ligand, such as a polypeptide, that binds to a cognate receptor. Exemplary and well known non-immunoglobulin polypeptides homologous receptor pairs include, but are not limited to, those non-immunoglobulin polypeptides that bind to, for example, homologous G protein-coupled receptors (GPCRs). Exemplary GPCRs include, but are not limited to, chemokine receptors, glucagon receptors (e.g., GLP1: GLP 1R), calcitonin receptors, melanocortin receptors. These and other cognate GPCRs, including non-immunoglobulin polypeptides conjugated thereto, are well known in the art. See, for example, wu et al, (2017) journal of molecular biology (J.mol. Biol.) 429:2726-45, which is incorporated herein by reference in its entirety. Additional non-limiting and exemplary non-immunoglobulin polypeptides (ligands) the cognate receptor pairs comprise
a. Ligands that bind to cognate receptor tyrosine kinases, such as, but not limited to, the following: epidermal Growth Factor (EGF), insulin, platelet-derived growth factor (PDGF), vascular Endothelial Growth Factor (VEGF), fibroblast Growth Factor (FGF), etc.
Dll: a Notch receptor pair,
b7 CD28/CLTA4/PD1 receptor pair,
d. arm plate protein, plexin receptor pair,
pcsk9/LDLR pairs,
HLA: LILR pair,
HLA: KIR pair,
h.RGD ligand, integrin pair,
i. amylin CALCR/RAMP, e.g. RAMP1/2/3 pair
j. Natriuretic peptide (e.g., ANP, BNP, CNP, etc.) and natriuretic peptide receptors (NPR, NPR3, etc.).
Ligand-receptor pairs may comprise proteases and inhibitors.
In vitro: refers to events that occur in an artificial environment, such as in a tube or reaction vessel, in a cell culture, etc., rather than within a multicellular organism.
In vivo: refers to events occurring in multicellular organisms such as humans and/or non-human animals. In the context of a cell-based system, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).
Separating: means that the substance and/or entity has been (1) isolated from at least some of the components with which it was associated at the time of initial production (whether in nature and/or in an experimental environment), and/or (2) is designed, produced, prepared and/or manufactured by hand. The isolated substance and/or entity may be separated from about 10 or more other components with which it was originally associated. In some embodiments, the isolated agent is at least about 80% or more pure. A substance is "pure" if it is substantially free of other components. In some embodiments, the material may still be considered "isolated" or even "pure" after being combined with certain other components (e.g., one or more carriers or excipients (e.g., buffers, solvents, water, etc.)) as will be appreciated by those skilled in the art; in such embodiments, the percent separation or purity of a material that does not comprise such carriers or excipients is calculated.
By way of example only, in some embodiments, a biopolymer, such as a polypeptide or polynucleotide, that occurs in nature is considered "isolated" in the following cases: (a) Due to its origin or derived source, is not associated with part or all of the components that accompany it in its natural state; (b) Which is substantially free of other polypeptides or nucleic acids from the same species as the species from which it is produced in nature; or (c) expressed by or otherwise associated with a component from a cell or other expression system that is not of a species that produces the biopolymer in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system that differs from the cellular system in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, the polypeptide that has undergone one or more purification techniques is associated with a) it in nature; and/or b) the extent to which other components associated therewith are separated when initially produced may be considered an "isolated" polypeptide.
Leader sequence or signal peptide: refers to immunoglobulin signaling or leader (L) peptides that direct an immunoglobulin heavy or light chain through the endoplasmic reticulum and are subsequently cleaved from the heavy or light chain prior to assembly of the final antibody. It may also refer to a nucleic acid sequence encoding a signal or leader peptide. Each V gene segment includes a leader sequence encoded by exon 1 and exon 2 of the segment (see, e.g., fig. 2), immediately upstream of the exon 2 sequences encoding FR1, CDR1, FR2, CDR2, FR3, and CDR3 of the germline Ig V segment. Ig signals or leader sequences are well known in the art. See, for example, lefranc et al, (2003) development and comparison immunology 27:55-77, incorporated herein by reference in its entirety, and also viewable on the world Wide Web (www) addressed as imgt. See also Lefranc and Lefranc (2020) & biomedical (Biomedicines) 8 (9): 1-117.
Non-human animals: refers to any vertebrate that is not a human. The non-human animal may be a round mouth animal, teleost fish, cartilaginous fish (e.g., shark or ray), amphibian, reptile, mammal, and bird. In some embodiments, the non-human mammal may be a primate, goat, sheep, pig, dog, cow, or rodent. In some embodiments, the non-human animal may be a rat or a mouse.
Nucleic acid in its broadest sense refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain and that is generally interchangeable with nucleic acid molecules, nucleic acid sequences, nucleotide molecules, and the terms are also interchangeable with one another.
In some embodiments, a "nucleic acid" is a compound and/or substance that is or can be incorporated into an oligonucleotide chain through a phosphodiester linkage. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide strand comprising a single nucleic acid residue. In some embodiments, a "nucleic acid" is or includes RNA; in some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, a "nucleic acid" is, includes, or consists of one or more natural nucleic acid residues. In some embodiments, a "nucleic acid" is, includes, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a "nucleic acid" in that the nucleic acid analog does not utilize a phosphodiester backbone. For example, in some embodiments, a "nucleic acid" is, includes, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds in the backbone rather than phosphodiester bonds, which are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the "nucleic acid" has one or more phosphorothioate and/or 5' -N-phosphoramidite linkages instead of phosphodiester linkages. In some embodiments, a "nucleic acid" is, includes, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a "nucleic acid" is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxo-adenosine, 8-oxo-guanosine, O (6) -methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof), includes or consists of one or more nucleoside analogs. In some embodiments, a "nucleic acid" includes one or more modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose) as compared to the sugars in a natural nucleic acid. In some embodiments, a "nucleic acid" has a nucleotide sequence encoding a functional gene product, such as RNA or a protein. In some embodiments, a "nucleic acid" has a nucleotide sequence encoding a polypeptide fragment (e.g., a peptide). In some embodiments, a "nucleic acid" comprises one or more introns. In some embodiments, a "nucleic acid" comprises one or more exons. In some embodiments, a "nucleic acid" comprises one or more coding sequences. In some embodiments, a "nucleic acid" is prepared by one or more of the following means: isolated from natural sources, enzymatic synthesis by complementary template-based polymerization (in vivo or in vitro), replication in recombinant cells or systems, and chemical synthesis. In some embodiments, a "nucleic acid" is at least 3 or more residues long. In some embodiments, a "nucleic acid" is single stranded; in some embodiments, a "nucleic acid" is double-stranded. In some embodiments, a "nucleic acid" has a nucleotide sequence that includes at least one element that encodes a polypeptide or fragment thereof, or is the complement of a sequence encoding a polypeptide or fragment thereof. In some embodiments, a "nucleic acid" has enzymatic activity.
Operatively connected to: refers to juxtaposition wherein the components so described are in a relationship permitting the components to function in their intended manner.
In other embodiments, the operative connection need not abut. For example, unrearranged variable region gene segments "operably linked" to each other can be rearranged to form rearranged variable region genes, which may not necessarily be contiguous with each other. Unrearranged variable region gene segments operably linked to each other and to a contiguous constant region gene can be rearranged to form a rearranged variable region gene that is expressed as a polypeptide chain of an antigen binding protein with the constant region gene. The control sequences "operably linked" to the coding sequences are linked in such a way that expression of the coding sequences is achieved under conditions compatible with the control sequences. "operably linked" sequences include expression control sequences that are contiguous with the gene of interest and expression control sequences that function in trans or remotely to control the gene of interest.
The term "expression control sequence" refers to a polynucleotide sequence that is necessary to affect the expression and processing of the coding sequence to which it is linked. "expression control sequences" include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and, when desired, sequences that enhance protein apocrine. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence, while in eukaryotes, typically, such control sequences include a promoter and a transcription termination sequence. The term "control sequences" is intended to encompass the presence of components critical to expression and processing, and may also encompass additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.
Physiological conditions: having the meaning as understood in the art under conditions of living and/or propagation of a cell or organism. In some embodiments, the term refers to conditions of the external or internal environment in which an organism or cellular system may occur in nature. In some embodiments, physiological conditions are those present in a human or non-human animal body, particularly those present at a target portion of interest and/or within a surgical site. Physiological conditions typically comprise, for example, a temperature range of 20-40 ℃, atmospheric pressure 1, pH 6-8, glucose concentration of 1-20mM, oxygen concentration at atmospheric level and gravity as it encounters on earth. In some embodiments, the conditions in the laboratory are manipulated and/or maintained under physiological conditions. In some embodiments, the physiological condition is encountered in an organism (e.g., a non-human animal).
Polypeptide: refers to any polymeric chain of amino acids.
In some embodiments, the polypeptide has an amino acid sequence that occurs in nature. In some embodiments, the polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, the polypeptide has an amino acid sequence of: which contain parts that exist in nature independently of each other (i.e., from two or more different organisms, such as human and non-human parts). In some embodiments, the polypeptide has an amino acid sequence engineered as a result of design and/or production by artificial action. In some embodiments, the polypeptide may comprise or consist of a plurality of fragments, each of which are found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., fragments directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in the polypeptide of interest in a different order than in the parent), such that the polypeptide of interest is a derivative of its parent polypeptide.
Recombination: refers to nucleic acids and/or polypeptides designed, engineered, prepared, expressed, produced or isolated by recombinant means, such as polypeptides expressed using recombinant expression vectors transfected into host cells (Hoogenboom H.R.,1997, trends in biotechnology (TIB Tech.)) 15:62-70; hoogenboom H.and Chames P.,2000, immunology Today, 21:371-378; azzazy H.and Highsmith W.E.,2002, clinical biochemistry (Clin. Biochem.) 35:425-445;Gavilondo J.V and Larrick J.W.,2002, biotechnology (biochoniques) 29:128-145), antibodies isolated from animals (e.g., mice) that are transgenic human immunoglobulin genes (see, for example, taylor, L.D.1992, 19920:95, and Sci.35:35, 35.35:95, 35.g., 35.35.95, 35.35.35, 35.95, and 35.35.35.95, 35.35.35.35.95, and 35.35.35.35.35.35.95, 35.35.35.35.35, and/or 4, 35.35.35.35.35.35.35, 35.35.35.35, and/or 35.35.35.35, respectively; each of which is incorporated herein by reference in its entirety) or by any other means that involves splicing selected sequence elements to one another.
In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed by computer simulation (in silico). In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements (e.g., from natural or synthetic sources). For example, in some embodiments, the recombinant polypeptide includes sequences found in the genome (or polypeptide) of the source organism of interest (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide includes sequences that exist separate from each other in nature (i.e., from two or more different organisms, such as human and non-human portions) in two different organisms (e.g., human and non-human organisms). In some embodiments, the recombinant polypeptide has an amino acid sequence derived from mutagenesis (e.g., in vitro or in vivo, e.g., in a non-human animal) such that the amino acid sequence of the recombinant polypeptide is a sequence that, while derived from and associated with the polypeptide sequence, may not naturally occur within the genome of the non-human animal.
Reference is made to: refers to a standard or control agent, animal, cohort, individual, population, sample, sequence, or value that is compared to the agent, animal, cohort, individual, population, sample, sequence, or value of interest. "reference" or "control" may refer to a "reference animal" or "control animal". The "reference animal" may have a modification as described herein, different from the modification described herein or no modification (i.e., wild-type animal). Typically, as will be appreciated by those of skill in the art, a reference agent, animal, group, individual, population, sample, sequence, or value is determined or characterized under conditions commensurate with the conditions used to determine or characterize the agent, animal (e.g., mammal), group, individual, population, sample, sequence, or value of interest.
In some embodiments of the present invention, in some embodiments,the testing and/or assaying of a reference agent, animal, group, individual, population, sample, sequence, or value is performed substantially simultaneously with the testing or assaying of the agent, animal, group, individual, population, sample, sequence, or value. In some embodiments, a reference agent, animal, cohort, individual, population, sample, sequence, or value is a historical reference, optionally embodied in a tangible medium. In some embodiments, the reference may refer to a control. " Control ", etc., e.g." control "as reference animal>"or" control "means ++including humanized heavy and kappa variable region loci>Mice, wherein the mice are capable of breeding. These->Control mice are generally described in the following: macdonald et al, (2014) Proc.Natl.Acad.Sci.USA 111:5147-52, which is incorporated herein by reference in its entirety, and supplemental information.
Somatic cell recombination: refers to V H 、D H And J H Recombination of gene segments at immunoglobulin heavy chain loci, or V L And J L Recombination of gene segments at immunoglobulin light chain loci. Somatic recombination occurs prior to antigen exposure to ad during B cell development in bone marrow. At the heavy chain locus, a D H And one J H Random recombination, removing all intermediate DNA in a process called D-J ligation. Next, random V will be H Segment reorganization to rearranged D H J H A section. Recombination at the immunoglobulin light chain locus occurs in a similar manner. V (V) L Gene segment and J L The gene segments combine and recombine in a process called V-J joining, removing themAll DNA in between.
Basically: refers to a qualitative condition that exhibits all or nearly all of the degree or characteristic of interest. Those of ordinary skill in the biological arts will appreciate that biological and chemical phenomena are rarely, if ever, accomplished and/or proceed to completion or to achieve or avoid absolute results. The term "substantially" is thus used herein to capture the potential complete absence inherent in many biological and chemical phenomena.
Substantial homology: refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered "substantially homologous" if they contain identical residues at corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues having suitably similar structural and/or functional characteristics. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and/or have "polar" or "nonpolar" side chains, as is well known to those of ordinary skill in the art. Substitutions of one amino acid for another amino acid of the same type may be generally considered "homologous" substitutions. Typical amino acid classifications are summarized below:
any of a variety of algorithms may be used to compare amino acid or nucleic acid sequences, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences, as is well known in the art. An exemplary such procedure is described below: altschul, S.F. et al, 1990, journal of molecular biology, 215 (3): 403-410; altschul, S.F. et al, 1996, methods of enzymology 266:460-80; altschul, S.F. et al, 1997, nucleic acids research 25:3389-402; baxevenis, a.d. and b.f. f. euellette (edit) & biology informatics: practical guidelines for gene and protein analysis (Bioinformatics: APractical Guide to the Analysis of Genes and Proteins), wiley,1998; and Misener et al, (editions) bioinformatics methods and protocols (Bioinformatics Methods and Protocols) (methods of molecular biology (Methods in Molecular Biology), volume 132), humana Press (Humana Press), 1998, the above-mentioned procedures generally provide an indication of the degree of homology in addition to identifying homologous sequences.
In some embodiments, two sequences are considered substantially homologous if at least 95% or more of the corresponding residues of the two sequences are homologous over the relevant sequence stretch of residues. In some embodiments, the relevant sequence segments are complete sequences. In some embodiments, the relevant sequence segment is at least 9 residues or more. In some embodiments, the relevant sequence segments comprise contiguous residues along the complete sequence. In some embodiments, the relevant sequence segments comprise non-contiguous residues along the complete sequence, e.g., non-contiguous residues that are brought together by the folded conformation of the polypeptide or a portion thereof. In some embodiments, the relevant sequence segment is at least 10 residues or more.
Substantial identity: refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered "substantially identical" if they contain identical residues at corresponding positions. Any of a variety of algorithms may be used to compare amino acid or nucleic acid sequences, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences, as is well known in the art. An exemplary such procedure is described below: altschul, S.F. et al, 1990, journal of molecular biology, 215 (3): 403-410; altschul, S.F. et al, 1996, methods of enzymology 266:460-80; altschul, S.F. et al, 1997, nucleic acids research 25:3389-3402; baxevenis, a.d. and b.f. f. euellette (edit) & biology informatics: practical guidelines for gene and protein analysis, wiley,1998; and Misener et al, (editions) bioinformatics methods and protocols (methods of molecular biology, volume 132), humana Press, 1998. In addition to identifying identical sequences, the above procedure generally provides an indication of the degree of identity.
In some embodiments, two sequences are considered to be substantially identical if at least 95% or more of the corresponding residues of the two sequences are identical over the relevant sequence segment of residues. In some embodiments, the relevant sequence segments are complete sequences. In some embodiments, the relevant sequence segment is at least 10 residues or more.
Targeting vector or targeting construct: refers to polynucleotide molecules comprising a targeting region. The targeting region comprises the sequence: the sequence is identical or substantially identical to a sequence in a target cell, tissue or animal and provides integration of the targeting construct to a location within the genome of the cell, tissue or animal via homologous recombination. Also included are targeting regions targeted using site-specific recombinase recognition sites (e.g., loxP or Frt sites).
In some embodiments, the targeting construct as described herein further comprises a specific nucleic acid sequence or gene of interest, a selectable marker, control and/or regulatory sequences, and other nucleic acid sequences that allow for recombination mediated by exogenously added proteins that help or facilitate recombination involving such sequences. In some embodiments, the targeting construct as described herein further comprises all or part of a gene of interest, wherein the gene of interest is a heterologous gene encoding all or part of a polypeptide having a similar function as the protein encoded by the endogenous sequence. In some embodiments, the targeting construct as described herein further comprises all or part of a humanized gene of interest, wherein the humanized gene of interest encodes all or part of a polypeptide having a similar function as the polypeptide encoded by the endogenous sequence. In some embodiments, the targeting construct (or targeting vector) may include a nucleic acid sequence that is manipulated by man. For example, in some embodiments, a targeting construct (or targeting vector) can be constructed to contain an engineered or recombinant polynucleotide comprising two or more sequences that are not linked together in order in nature, but are instead manually manipulated to be directly linked to each other in the engineered or recombinant polynucleotide.
Transgenic or transgenic construct: refers to a nucleic acid sequence (encoding, for example, a polypeptide of interest, in whole or in part) that is introduced into a cell by artificial, as described herein. The transgene may be partially or completely heterologous, i.e., exogenous, to the transgenic animal or cell into which it is introduced. A transgene may comprise one or more transcriptional regulatory sequences and any other nucleic acid, such as introns or promoters, which may be necessary for expression of a selected nucleic acid sequence. The transgene may comprise one or more selectable markers that allow subsequent selection of progeny (e.g., cells) that have received the transgene.
Transgenic animals, transgenic non-human animals or Tg + : are used interchangeably herein and refer to any non-naturally occurring non-human animal in which one or more cells of the non-human animal contain, in whole or in part, a heterologous nucleic acid and/or a gene encoding a polypeptide of interest.
In some embodiments, the heterologous nucleic acid sequence and/or gene is introduced into the cell directly or indirectly by introduction into the precursor cell, by intentional gene manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not encompass classical breeding techniques, but rather involves the introduction of recombinant DNA molecules. The molecule may integrate into the chromosome or it may be extrachromosomally replicated DNA. The term "Tg + "animals comprising heterozygous or homozygous for a heterologous nucleic acid and/or gene, and/or animals having single or multiple copies of a heterologous nucleic acid and/or gene.
Targeting vector: refers to a nucleic acid molecule capable of transporting a nucleic acid of interest associated therewith, particularly targeted insertion of the nucleic acid of interest into another nucleic acid molecule, such as a donor plasmid, non-human animal genome, or the like.
In some embodiments, the vectors are capable of extrachromosomal replication and/or expression of the nucleic acids to which they are attached in a host cell (such as a eukaryotic and/or prokaryotic cell). Vectors capable of directing expression of operably linked genes are referred to herein as "expression vectors" or "constructs".
Wild type: the meaning understood in the art refers to an entity that has a structure and/or activity found in nature in a "normal" (as opposed to mutant, diseased, engineered, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles).
Other features, objects, and advantages of the present invention will be apparent from the following detailed description of some embodiments. It is to be understood, however, that the detailed description, while indicating some embodiments of the invention, is given by way of illustration and not of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.
Detailed Description
Although antibody-based therapies have significant promise in the treatment of several diseases, the development of particularly effective antibody agents that bind to refractory targets remains a challenge. Described herein are anchor modified immunoglobulins (igs) and modified Ig V segments encoding the same, wherein the anchors comprise at least a receptor binding portion of a ligand (e.g., a non-immunoglobulin polypeptide) that binds to a cognate receptor. Animals comprising modified Ig V segments can produce a variety of anchor modified immunoglobulins in response to immunization of recipients homologous to the anchors.
Without being bound by theory, it is believed that the effect of the anchor is to increase the affinity of antibodies produced de novo in animals in response to antigen challenge by cognate receptors, e.g., by binding to cognate receptors simultaneously with antibodies that specifically bind cognate receptors and/or allowing affinity maturation of antibodies that would not normally be amplified in the absence of the anchor in response to antigen challenge by cognate receptors. Thus, the various immunoglobulins produced by the non-human animals disclosed herein may include anchor modified immunoglobulins that bind to cognate receptors with high affinity, thereby increasing the population of immunoglobulins from which primary candidates for previously refractory targets may be found.
Nucleic acid molecules, including targeting vectors and animal genomes, and the like.
The anchor modified immunoglobulins described herein may be encoded at least in part by variable region (V) segments, such as immunoglobulin (Ig) heavy chain variable regions (V H ) Segment or Ig light chain variable region (V L ) Segments modified to encode and operably linked to anchors between Ig leader sequences and Framework (FR) and Complementarity Determining Region (CDR) sequences of germline Ig V segments. Nucleic acid sequences encoding immunoglobulin (Ig) signal peptides and germline V segments (e.g., human germline V segments) are well known in the art, as are the amino acid sequences so encoded. See, e.g., lefranc, m. -p., "experimental and clinical immunogenetics", 18,100-116 (2001), incorporated herein by reference in its entirety, and web sites found in the world wide web (www) at an address imgt.
The nucleic acid molecules, including targeting vectors and animal genomes, as described herein may include nucleic acid sequences encoding any Ig signal peptides of germline V segments. In some embodiments, the modified Ig V segments comprise a nucleic acid sequence encoding a signal peptide of a first germline Ig V segment, a nucleic acid sequence encoding an anchor, and a nucleic acid encoding a Framework Region (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3, and CDR3 of a second germline Ig V segment, wherein the first germline Ig V segment and the second germline Ig V segment are different germline Ig V segments. In some embodiments, the modified Ig V segments comprise a nucleic acid sequence encoding a signal peptide of a first germline Ig V segment, a nucleic acid sequence encoding an anchor, and a nucleic acid encoding a Framework Region (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3, and CDR3 of a second germline Ig V segment, wherein the first germline Ig V segment and the second germline Ig V segment are the same germline Ig V segment. In some embodiments, the Ig signal peptide includes sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7).
Human (h) germline V segments (e.g., human germline variable heavy chains (hV H Or hIGVH), human germline variable kappa (hVkappa or hIGKV) and human germline variable lambda (hVlambda or hIGLV) segments, and murine (m) germline V segments (e.g., mouse germline variable heavy chains(mV H Or mIGVH), mouse germline variable kappa (mV kappa or mIGKV) and mouse germline variable lambda (mV lambda or mIGLV) segments may be found at world wide web (www) addresses imgt.org/IMGTrepertoire/Proteins/sequences logo/human/and imgt.org/IMGTrepertoire/Proteins/sequences logo/mouse/found, each of which is incorporated herein by reference in its entirety.
In some embodiments, the germline Ig V segment or variant thereof (e.g., a segment encoding FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a modified Ig V segment as described herein) is a human (h) germline Ig V segment or variant thereof, e.g., germline human (h) V H 1-2 segment, germ line hV H 1-3 segment, germ line hV H 1-8 segment, germ line hV H Segment 1, germ line hV H 1-24 segment, germ line hV H 1-45 segment, germ line hV H 1-46 segment, germ line hV H 1-58 segment, germ line hV H Segment 1-69, germ line hV H 2-5 segment, germ line hV H 2-26 segment, germ line hV H 2 segment 70, germ line hV H 3-7 segment, germ line hV H 3-9 segment, germ line hV H 3 11 segment, germ line hV H 3 segment 13, germ line hV H 3-15 segment, germ line hV H 3-16 segment, germ line hV H 3-20 segment, germ line hV H 3-21 segment, germ line hV H 3-23 segment, germ line hV H 3-30 segment, germ line hV H 3-30-3 segment, germ line hV H 3-30-5 segment, germ line hV H 3-33 segment, germ line hV H 3-35 segment, germ line hV H 3-38 segment, germ line hV H 3-43 segment, germ line hV H 3-48 segment, germ line hV H 3-49 segments, germ line hV H 3-53 segment, germ line hV H 3-64 segment, germ line hV H 3-66 segment, germ line hV H 3-72 segment, germ line hV H 3-73 segment, germ line hV H 3-74 segment, germ line hV H 4-4 segment, germ line hV H 4-28 segment, germ line hV H 4-30-1 segment, germ line hV H 4.30-2 segment, germ line hV H 4-30-4 segment, germ line hV H 4-31 segment, germ line hV H 4-34 segment, germ line hV H 4-39 segment, germ line hV H 4-59 segment, germ line hV H 4-61 segment, germ line hV H 5-51 segment, germ line hV H 6-1 segment, germ line hV H 7-4-1 segment, germ line hV H 7-81 segments or variants thereof. In some embodiments, the germline Ig V segment or variant thereof is germline hV H 1-69 or a variant thereof.
In some embodiments, the nucleic acid molecules described herein include only modified Ig hvs H Segments, e.g. not including any further hV H Segments or variants thereof.
In some embodiments, except for modified Ig hV H Outside the segment, the nucleic acid molecule as described herein further comprises an additional hV H A segment, for example, one, more than one, or each of: v (V) H 1-2、hV H 1-3、hV H 1-8、hV H 1 18、hV H 1-24、hV H 1-45、hV H 1-46、hV H 1-58、hV H 1-69、hV H 2-5、hV H 2-26、hV H 2 70、hV H 3-7、hV H 3-9、hV H 3 11、hV H 3 13、hV H 3-15、hV H 3-16、hV H 3-20、hV H 3-21、hV H 3-23、hV H 3-30、hV H 3-30-3、hV H 3-30-5、hV H 3-33、hV H 3-35、hV H 3-38、hV H 3-43、hV H 3-48、hV H 3-49、hV H 3-53、hV H 3-64、hV H 3-66、hV H 3-72、hV H 3-73、hV H 3-74、hV H 4-4、hV H 4-28、hV H 4-30-1、hV H 4 30-2、hV H 4-30-4、hV H 4-31、hV H 4-34、hV H 4-39、hV H 4-59、hV H 4-61、hV H 5-51、hV H 6-1、hV H 7-4-1、hV H 7-81, and variants thereof. In some embodiments including more than one or each of the following: v (V) H 1-2、hV H 1-3、hV H 1-8、hV H 1 18、hV H 1-24、hV H 1-45、hV H 1-46、hV H 1-58、hV H 1-69、hV H 2-5、hV H 2-26、hV H 270、hV H 3-7、hV H 3-9、hV H 3 11、hV H 3 13、hV H 3-15、hV H 3-16、hV H 3-20、hV H 3-21、hV H 3-23、hV H 3-30、hV H 3-30-3、hV H 3-30-5、hV H 3-33、hV H 3-35、hV H 3-38、hV H 3-43、hV H 3-48、hV H 3-49、hV H 3-53、hV H 3-64、hV H 3-66、hV H 3-72、hV H 3-73、hV H 3-74、hV H 4-4、hV H 4-28、hV H 4-30-1、hV H 4 30-2、hV H 4-30-4、hV H 4-31、hV H 4-34、hV H 4-39、hV H 4-59、hV H 4-61、hV H 5-51、hV H 6-1、hV H 7-4-1 and hV H 7-81 segment, said hV H The segments are in a germ line configuration.
In some embodiments, a nucleic acid molecule as described herein (e.g., targeting vector, non-human animal genome, etc.) may include an Ig heavy chain variable region, e.g., may include additional (not) rearranged V in addition to the anchor modified V segment H 、D H And/or J H Gene segments, and in some embodiments, additional (not) rearranged hvs H 、hD H And/or hJ H A gene segment. In some embodiments, a nucleic acid molecule as described herein (e.g., targeting vector, non-human animal genome, etc.) comprises only one (un) rearranged hV H Segment, one or more (un) rearranged hD H Segments and one or more (un) rearranged hJ H A segment in which the only one (un) rearranged hV H Segments are modified hvs comprising a nucleic acid sequence encoding an anchor as described herein H A section.
In some embodiments, a recombinant nucleic acid molecule as described herein (e.g., targeting vector, non-human animal genome, etc.) comprises one or more human D H A segment, for example, one of the following,More than one or each: hD H 1-1、hD H 1-7、hD H 1-14、hD H 1-20、hD H 1-26、hD H 2-2、hD H 2-8、hD H 2-15、hD H 2-21、hD H 3-3、hD H 3-9、hD H 3-10、hD H 3-16、hD H 3-22、hD H 4-4、hD H 4-11、hD H 4-17、hD H 4-23、hD H 5-5、hD H 5-12、hD H 5-18、hD H 5-24、hD H 6-6、hD H 6-13、hD H 6-19、hD H 6-25、hD H 7-27, and variants thereof. In some embodiments including more than one or each of the following: hD H 1-1、hD H 1-7、hD H 1-14、hD H 1-20、hD H 1-26、hD H 2-2、hD H 2-8、hD H 2-15、hD H 2-21、hD H 3-3、hD H 3-9、hD H 3-10、hD H 3-16、hD H 3-22、hD H 4-4、hD H 4-11、hD H 4-17、hD H 4-23、hD H 5-5、hD H 5-12、hD H 5-18、hD H 5-24、hD H 6-6、hD H 6-13、hD H 6-19、hD H 6-25 and hD H 7-27, said hD H The segments are in a germ line configuration.
In some embodiments, a recombinant nucleic acid (e.g., targeting vector, non-human animal genome, etc.) as described herein comprises one or more human J H A segment, for example one, more than one or each of the following: hJ H 1、hJ H 2、hJ H 3、hJ H 4、hJ H 5、hJ H 6, and variants thereof. In some embodiments including more than one or each of the following: hJ H 1、hJ H 2、hJ H 3、hJ H 4、hJ H 5 and hJ H 6 segment, said hJ H The segments are in a germ line configuration.
In some embodiments, a recombinant nucleic acid molecule as described herein (e.g., targeting vector, non-human animal genome, etc.) can include a heavy chain variable region locusFor example, the following operably connected and from 5 'to 3' may be included: (I) The modified Ig V H Segment (II) one or more Ig heavy chain diversity (D) H ) Segments, and (III) one or more of all Ig heavy chain linkages (J) H ) A section. In some embodiments, the one or more Ig D of (II) H Segments comprising one, more or all human Ig D H Segments, and/or (III) the one or more Ig J H Segments comprising one, more or all human Ig J H A section. In some embodiments, the one or more Ig D of (II) H Segment and the one or more Ig J of (III) H Gene segments are recombined and form rearranged Ig D H /J H A sequence such that the recombinant nucleic acid molecule comprises, operably linked and from 5 'to 3': the modified Ig V H Gene segments and the rearranged Ig D H /J H Sequence.
In some embodiments, the IgV H Gene segments and the rearranged Ig D H /J H The sequences are recombined and form rearranged Ig V encoding an anchor-modified Ig heavy chain variable domain H /D H /J H A sequence, wherein the anchor modified Ig heavy chain variable domain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, and (iii) the IgV resulting from the rearrangement H /D H /J H The FR1, complementarity Determining Regions (CDRs) 1, FR2, CDR2, FR3, CDR3 and FR4 are encoded by the sequences.
In some embodiments, the modified Ig V H The segments are modified Ig Vs that are not rearranged H A gene segment.
In some embodiments, the recombinant nucleic acids (e.g., targeting vectors, non-human animal genomes, etc.) as disclosed herein further comprise a nucleic acid encoding an Ig heavy chain constant region (C H ) Wherein the nucleic acid sequence encodes an IgC H In (I) said modified IgV H Segments, (II) the one or more Ig D H Segments and (III) the one or more Ig J H Downstream of and operatively connected to the section. In some casesIn embodiments, igC is encoded H Including Igmu gene encoding IgM isotype, igdelta gene encoding IgD isotype, iggamma gene encoding IgG isotype, igalpha gene encoding IgA isotype and/or Igepsilon gene encoding IgE isotype. In some embodiments, the recombinant nucleic acid molecules described herein comprise a nucleic acid sequence encoding an anchor modified Ig heavy chain, wherein the anchor modified Ig heavy chain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, (iii) the IgV comprising a rearrangement of H /D H /J H Sequence-encoded Ig heavy chain variable domains of said FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and (iv) Ig C H . In some embodiments, the IgC H Non-human Ig C H For example rodent IgC H For example, rat IgC H Or mouse Ig C H 。
In some embodiments, the germline Ig V segment or variant thereof (e.g., the segment or variant thereof encoding FR1, CDR1, FR2, CDR2, FR3, and CDR3 of a modified Ig V segment as described herein) is a germline Ig light chain variable (V L ) Segments or variants thereof. In some embodiments, the recombinant nucleic acid molecule may include a light chain variable region locus, e.g., may include the following operably linked and from 5 'to 3': (I) The modified V L Segments, and (II) one or more Ig light chain engagements (J L ) A section. In some embodiments, the modified Ig V L Segments and the one or more Ig J L The segments are recombined and form rearranged Ig V encoding an anchor modified Ig light chain variable domain L /J L A sequence, wherein the anchor modified Ig light chain variable domain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, and (iii) the IgV resulting from the rearrangement L /J L The FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are sequence encoded. In some embodiments, the recombinant nucleic acid molecule may include a light chain variable region locus and a light chain constant region encoding Ig (C L ) Wherein the nucleic acid sequence encodes an IgC L Is operably linked thereto downstream of: (I) Modified Ig V L Segments and (II) the one or more Ig light chain engagements (J L ) A section. In some embodiments, the anchor-modified Ig light chain comprises operably linked: (i) the Ig signal peptide, (ii) the anchor, (iii) the IgV comprising a rearrangement of L /J L Sequence-encoded Ig light chain variable domains of said FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and (iv) Ig C L . In some embodiments, the IgC L Non-human Ig C L For example rodent IgC L For example, rat IgC L Or mouse Ig C L 。
In some embodiments, the germline Ig V segment or variant thereof (e.g., the segment encoding FR1, CDR1, FR2, CDR2, FR3 and CDR3 of a modified Ig V segment as described herein or variant thereof) is a germline Ig light chain variable kappa (V kappa) segment or variant thereof, e.g., a human V kappa segment, for example, the hVκ1-5, hVκ1-6, hVκ1-8, hVκ1D-8, hVκ1-9, hVκ1-12, hVκ1D-12, hVκ1-13, hVκ1D-13, hVκ1-16, hVκ1D-16, hVκ1-17, hVκ1D-17, hVκ1-27, hVκ1-33, hVκ1D-33, hVκ1-37, hVκ1D-37, hVκ1-39, hVκ1D-39, hVκ1-NL1 a hVκ1D-42 segment, a hVκ1D-43 segment, a hVκ2-4 segment, a hVκ2-18 segment, a hVκ2D-18 segment, a hVκ2-24 segment, a hVκ2D-24 segment, a hVκ2-28 segment, a hVκ2D-28 segment, a hVκ2-29 segment, a hVκ2D-29 segment, a hVκ2-30 segment, a hVκ2D-30 segment, a hVκ2-40 segment, a hVκ2D-26 segment, a hVκ3-7 segment, a hVκ3D-7 segment, a hVκ3-11 segment, a hVκ3D-11 segment, a hVκ3-15 segment, hVκ 3D-15, hVκ 3-20, hVκ 3D-20, hVκ4-1, hVκ5-2, hVκ6-21, hVκ6D-41, hVκ7-3 and variants thereof. In some embodiments, in addition to the modified Ig hvκ segment, a nucleic acid molecule as described herein further comprises additional hvκ segments, e.g., one, more than one, or each of: the hVκ1D-8, hVκ1-9, hVκ1-12, hVκ1D-12, hVκ1-13, hVκ1D-13, hVκ1-16, hVκ1D-16, hVκ1-17, hVκ1D-17, hVκ1-27, hVκ1-33, hVκ1D-33, hVκ1-37, hVκ1D-37, hVκ1-39, hVκ1D-39, ahVκ1-NL1, hVκ1D-42, hVκ1D-43, hVκ2-4, hVκ2-18, hVκ2D-18, hVκ1D-33 a hVκ2-24 segment, a hVκ2D-24 segment, a hVκ2-28 segment, a hVκ2D-28 segment, a hVκ2-29 segment, a hVκ2D-29 segment, a hVκ2-30 segment, a hVκ2-40 segment, a hVκ2D-26 segment, a hVκ3-7 segment, a hVκ3D-7 segment, a hVκ3-11 segment, a hVκ3D-11 segment, a hVκ3-15 segment, a hVκ3D-15 segment, a hVκ3-20 segment, a hVκ3D-20 segment, a hVκ4-1 segment, a hVκ5-2 segment, a hVκ6-21 segment, and a hVκ6D-21 segment. In some embodiments including more than one or each of the following: a hVκ1D-8, a hVκ1-9, a hVκ1-12, a hVκ1D-12, a hVκ1-13, a hVκ1D-13, a hVκ1-16, a hVκ1D-16, a hVκ1-17, a hVκ1D-17, a hVκ1-27, a hVκ1-33, a hVκ1D-17, a hVκ1D-16, a hVκ1D-17, a hVκ1D-33, a hVκ1D-13, a hVκ1D-16, a hVκ1D-33, a hVκ1-33, a hKKKK1-12, a hVκ1-12, a hKK1-12, a hKKKK1-12, a hKK1-12, a hK1-33, a hVK1-33, a hK1-33, a h a hVκ1D-33 segment, a hVκ1-37 segment, a hVκ1D-39, a hVκ1NL1 segment, a hVκ1D-42 segment, a hVκ1D-43 segment, a hVκ2-4 segment, a hVκ2-18 segment, a hVκ2D-18 segment, a hVκ1D-NL1 segment, a hVκ1D-42 segment, a hVκ1D-43 segment, a hVκ2-4 segment, a hVκ2D-18 segment, a hVκ1D-39 segment, a hVκ1D-L-a hVκ2-24 region, a hVκ2D-24 region, a hVκ2-28 region, a hVκ2D-28 region, a hVκ2-29 region, a hVκ2D-29 region, a hVκ2-30 region, a hVκ2-40 region, a hVκ2D-26 region, a hVκ3-7 region, a hVκ3D-7 region, a hVκ3-11 region, a hVκ3D-11 region, a hVκ3-15 region, a hVκ3D-15 region, a hVκ3-20 region, a hVκ3D-20 region, a hVκ4-1 region, a hVκ5-2 region, a hVκ6-21 region, and a hVκ6D-21 region, the hvκ segment is in germline configuration.
In some embodiments, a nucleic acid molecule as described herein (e.g., targeting vector, non-human animal genome, etc.) may include an Ig light chain kappa variable region, e.g., may include additional (not) rearranged jκ segments in addition to the anchor modified hvκ segments, and in some embodiments, additional (not) rearranged hjκ gene segments. Thus, in some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modifiedAn Ig V kappa segment and (II) one or more Ig light chain binding kappa J kappa segments. In some embodiments, a recombinant nucleic acid (e.g., targeting vector, non-human animal genome, etc.) as described herein comprises one or more human J κ A segment, for example one, more than one or each of the following: hjκ1, hjκ2, hjκ3, hjκ4, hjκ5, and variants thereof. In some embodiments including more than one or each of the following: hjκ1, hjκ2, hjκ3, hjκ4, hjκ5 segments in germline configuration.
In addition, in some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modified Ig V kappa segment and (II) one or more Ig light chain engagement kappa (Jkappa) and a nucleic acid sequence encoding an Ig light chain constant kappa region (Ckappa).
In some embodiments, the germline Ig V segment or variant thereof (e.g., the segment encoding FR1, CDR1, FR2, CDR2, FR3 and CDR3 of a modified Ig V segment as described herein or variant thereof) is a germline Ig light chain variable lambda (V lambda) segment or variant thereof, e.g., a human V lambda segment, for example, a hV.lamda.1-36, a hV.lamda.1-40, a hV.lamda.1-41, a hV.lamda.1-44, a hV.lamda.1-47, a hV.lamda.1-50, a hV.lamda.1-51, a hV.lamda.1-62, a hV.lamda.2-5, a hV.lamda.2-8, a hV.lamda.2-11, a hV.lamda.2-14, a hV.lamda.2-18, a hV.lamda.2-23, a hV.lamda.2-33, a hV.lamda.2-34, a hV.lamda.3-1, a hV.lamda.3-9, a hV.lamda.3-10, a hV.lamda.3-12, a hV.lamda.3-13 hV 3-16, hV 3-19, hV 3-21, hV 3-22, hV 3-25, hV 3-27, hV 3-31, hV 3-32, hV 4-3, hV 4-60, hV 4-69, hV 5-37, hV 5-39, hV 5-45, hV 5-48, hV 5-52, hV 6-57, hV 7-43, hV 7-46, hV 8-61, hvλ9-49, hvλ10-54, hvλ11-55 and variants thereof. In some embodiments, in addition to the modified Ig hvλ segment, the nucleic acid molecule as described herein further comprises an additional hvλ segment, for example one, more than one, or each of: a hvλ1-36 segment, a hvλ1-40 segment, a hvλ1-41 segment, a hvλ1-44 segment, a hvλ1-47 segment, a hvλ1-50 segment, a hvλ1-51 segment, a hvλ1-62 segment, a hvλ2-5 segment, a hvλ2-8 segment, a hvλ2-11 segment, a hvλ2-14 segment, a hvλ2-18 segment, a hvλ2-23 segment, a hvλ2-33 segment, a hvλ2-34 segment, a hvλ3-1 segment, a hvλ3-9 segment, a hvλ3-10 segment, a hvλ3-12 segment, a hvλ3-13 segment, a hvλ3-16 segment the composition comprises a portion of hV.lamda.3-19, a portion of hV.lamda.3-21, a portion of hV.lamda.3-22, a portion of hV.lamda.3-25, a portion of hV.lamda.3-27, a portion of hV.lamda.3-31, a portion of hV.lamda.3-32, a portion of hV.lamda.4-3, a portion of hV.lamda.4-60, a portion of hV.lamda.4-69, a portion of hV.lamda.5-37, a portion of hV.lamda.5-39, a portion of hV.lamda.5-45, a portion of hV.lamda.5-48, a portion of hV.lamda.5-52, a portion of hV.lamda.6-57, a portion of hV.lamda.7-43, a portion of hV.lamda.7-46, a portion of hV.lamda.8-61, a portion of hV.lamda.lamda.9-49, a portion of hV.lamda.10-54, and a portion of hV.lamda.lamda.11-55. In some embodiments including more than one or each of the following: the hV 1-36, hV 1-40, hV 1-41, hV 1-44, hV 1-47, hV 1-50, hV 1-51, hV 1-62, hV 2-5, hV 2-8, hV 2-11, hV 2-14, hV 2-18, hV 2-23, hV 2-33, hV 2-34, hV 3-1, hV 3-9, hV 3-10, hV 3-12, hV 3-13, hV 3-16, hV 3-19, hV 3-21, hV 3-22, hV 3-25, hV 3-27, hV 3-31, hV 3-32, hV 4-4, V4-9, hV 4-5, hV 3-9, hV 3-7, hV 3-5, hV 3-22, the hvλ segment is in germline configuration.
In some embodiments, a nucleic acid molecule as described herein (e.g., targeting vector, non-human animal genome, etc.) may include an Ig light chain lambda variable region, e.g., may include additional (not) rearranged jlambda segments in addition to the anchor modified hvlambda segment, and in some embodiments, additional (not) rearranged hjlambda gene segments. Thus, in some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modified Ig V lambda segment and (II) one or more Ig light chain binding kappa J lambda segment. In some embodiments, a recombinant nucleic acid as described herein (e.g., targeting vector, non-human animal genome, etc.) comprises one or more human jλ segments, e.g., one, more than one, or each of: hJλ1, hJλ2, hJλ3, hJλ4, hJλ5, hJλ6, hJ λ7 segments, and variants thereof. In some embodiments including more than one or each of the following: hJ λ1, hJ λ2, hJ λ3, hJ λ4, hJ λ5, hJ λ6, hJ λ7, said hJ λsegment being in a germline configuration. In some embodiments, the recombinant nucleic acid molecules described herein include the following operably linked and from 5 'to 3': (I) The modified Ig V lambda segment, (II) one or more Ig light chain engagement lambda (J lambda) segments, and a nucleic acid sequence encoding an Ig light chain constant lambda region (C lambda).
Anchor
As described herein, an anchor includes a ligand (or portion thereof) that binds to a cognate receptor. In some embodiments, the ligand may be a non-immunoglobulin polypeptide. Thus, as described herein, an anchor may include a non-immunoglobulin polypeptide, such as a receptor binding portion of a non-immunoglobulin polypeptide. The anchor modifications described herein can be used to increase the affinity of an antigen binding protein (e.g., an antibody) for a refractory receptor.
Exemplary and well known non-immunoglobulin polypeptides homologous receptor pairs include, but are not limited to, those non-immunoglobulin polypeptides that bind to, for example, homologous G protein-coupled receptors (GPCRs). Exemplary GPCRs include, but are not limited to, chemokine receptors, glucagon receptors (e.g., GLP1: GLP 1R), calcitonin receptors, melanocortin receptors. These and other cognate GPCRs, including non-immunoglobulin polypeptides conjugated thereto, are well known in the art. See, for example, wu et al, (2017) journal of molecular biology 429:2726-45, which is incorporated herein by reference in its entirety. Additional non-limiting and exemplary non-immunoglobulin polypeptides (ligands) the cognate receptor pairs comprise
a. Ligands that bind to cognate receptor tyrosine kinases, such as, but not limited to, the following: epidermal Growth Factor (EGF), insulin, platelet-derived growth factor (PDGF), vascular Endothelial Growth Factor (VEGF), fibroblast Growth Factor (FGF), etc.
Dll: a Notch receptor pair,
b7 CD28/CLTA4/PD1 receptor pair,
d. arm plate protein, plexin receptor pair,
pcsk9/LDLR pairs,
HLA: LILR pair,
HLA: KIR pair,
h.RGD ligand, integrin pair,
i. natriuretic peptide (e.g., ANP, BNP, CNP, etc.) and natriuretic peptide receptors (NPR, NPR3, etc.).
Ligand-receptor pairs may comprise proteases and inhibitors.
In some embodiments, the anchor comprises a Natriuretic Peptide (NP), such as a receptor binding portion of NP. NPs include at least eight structurally related amino acid peptides stored as three different prohormones: atrial Natriuretic Peptide (ANP) prohormone, type B Natriuretic Peptide (BNP) prohormone, and type C Natriuretic Peptide (CNP) prohormone. The dendroaspin natriuretic peptide is a recently discovered form D Natriuretic Peptide (DNP) whose role in humans is still unclear.
ANP prohormone (proANP) is a 126 amino acid polypeptide that is expressed primarily by cardiomyocytes and produces several peptides with hypotensive, natriuretic, diuretic and/or kallikrein properties. These ANP prohormone-derived peptides were identified by their amino acid sequence starting at the N-terminus of ANP prohormone: for example, proANP 1-30 includes long-acting NPs, proANP 31-67 includes vasodilator factors, proANP 79-98 includes potassium-benefiting peptides and amino acids 99-126 (also referred to as ANP), and the like. Within the kidney, proANP is treated differently, resulting in the addition of four additional amino acids to the N-terminus, such as proANP 95-126 (also known as urodilatin).
proBNP prohormone (proBNP) is a 108 amino acid polypeptide that is also expressed primarily by cardiomyocytes. The proBNP hormone is processed in the human heart to form BNP (e.g., amino acids 77-108 of its 108 amino acid prohormone) and NT-proBNP (e.g., amino acids 1-76), both of which circulate in the human body. BNP is a reliable biomarker of ventricular dilation. Pandit et al, (2011) journal of indian endocrine and metabolism (ind.j. Endocrinol. Metab.) 15 (4) S345-53, which is incorporated herein by reference in its entirety.
Unlike ANP and BNP, CNP is expressed primarily by endothelial cells and renal epithelial cells. Two CNP molecules have been identified in the circulation. Furthermore, although CNP appears to lack natriuretic function, CNP may act as a regulator of vascular tone and growth in a paracrine or autocrine manner, and there are some indications that CNP may play a role in bone growth.
NPs exert their biological functions by binding specifically to cell surface receptors. Three specific receptors have been identified in mammalian tissues: two guanylate cyclase-coupled receptors (GC-A and GC-B, also known as NP receptors (NPR) -A and NPR-B, respectively). NPR-A and NPR-B act by activating a cGMP-dependent signaling cascade. In contrast, the third type C receptor (also known as NPR-C) is not coupled to guanylate cyclase and appears to be primarily involved in NP clearance. All three receptors bind with different affinities to ANP, BNP and CNP. The ligand selectivity of GC-A is ranked in order of ANP > BNP > CNP, the ligand selectivity of GC-B is ranked in order of CNP > ANP > BNP, and the ligand selectivity of NPR-C is ranked in order of ANP > CNP > BNP. Jaubert et al, (1999) Proc. Natl. Acad. Sci. USA (PNAS) 96 (18) 10278-283, incorporated herein by reference in its entirety.
NP receptors may be useful targets for the treatment of hypertension and cardiovascular diseases. However, although ANP and BNP have important diuretic, natriuretic and antihypertensive properties, CNP may play a role in bone growth. Thus, any antibody-based NP receptor targeting must take care of the different binding affinities of the receptor for NP.
In some embodiments, the anchor comprises the sequence ANP or a portion thereof. The nucleic acid sequences encoding human ANP are shown herein as NCBI accession No. NM-006172.4 and SEQ ID NO. 1. The amino acid sequences of human ANP are shown herein as NCBI accession number NP-006163 and SEQ ID NO. 2. In some embodiments, anchors described herein include a receptor binding portion of ANP, e.g., the C-terminal tail of ANP. In some embodiments, the receptor binding portion of ANP comprises the amino acid sequence NSFRY (SEQ ID NO: 3).
Joint
In some embodiments, the anchors comprise linkers connecting the receptor binding portion of the non-immunoglobulin polypeptide of interest to FR1, CDR1, FR2, CDR2, FR3 and CDR3 of the germline Ig V segment or variant thereof. In some embodiments, the length of the linker may be one amino acid. In some embodiments, the linker may be two amino acids in length. In some embodiments, the linker may be three amino acids in length. In some embodiments, the linker may be four amino acids in length, e.g., the linker may include the sequence GLSG (SEQ ID NO: 13). In some embodiments, the linker may be five amino acids in length, e.g., may include the sequence GGGGS (SEQ ID NO: 5). In some embodiments, the linker may be six amino acids in length, and may include the sequence GLSGSG (SEQ ID NO: 14), for example. In some embodiments, the linker may be seven amino acids in length. In some embodiments, the linker may include the sequence GLSGLSGS (SEQ ID NO: 15). In some embodiments, the linker may be nine amino acids in length. In some embodiments, the linker may be ten amino acids in length, and may include, for example, the sequence GLSGLSGLSG (SEQ ID NO: 16) or GLSGGSGLSG (SEQ ID NO: 17). In some embodiments, the first and second linkers are the same length and are each more than ten amino acids in length.
In some embodiments, the recombinant nucleic acid molecules described herein include sequences shown as SEQ ID NO. 8 or degenerate variants thereof, or SEQ ID NO. 10 or degenerate variants thereof.
Targeting vectors
Further provided are targeting vectors for use in the methods of making the genetically modified non-human animals, cells, tissues or embryos provided herein.
In one embodiment, a targeting vector is provided that includes an insert nucleic acid, e.g., a recombinant nucleic acid molecule comprising a modified Ig V segment as described herein, flanked by 5 'and 3' homology arms that can be homologously recombined with a locus of interest (e.g., an Ig heavy chain or light chain variable region locus). Examples of targeting vectors and components of targeting vectors (i.e., insert nucleic acids, polynucleotides of interest, expression cassettes, etc.) are described in detail herein below.
A homology arm and a target site (i.e., homologous genomic region) are "complement" or "complementary" to each other when they share a sufficient level of sequence identity with each other to serve as a substrate for homologous recombination reactions. "homology" means a DNA sequence that is identical to or shares sequence identity with a corresponding or "complementary" sequence. The sequence identity between a given target site and the corresponding homology arm found on the targeting vector may be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity shared by the homology arms of the targeting vector (or fragment thereof) and the target site (or fragment thereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity such that the sequence undergoes homologous recombination. Furthermore, the region of homology complementarity between the homology arm and the complementary target site may be of any length sufficient to promote homologous recombination at the recognition site of cleavage. For example, a given homology arm and/or complementary target site may include a homology complementing region of at least 5kb to 10kb, 5kb to 15kb, 10kb to 20kb, 20kb to 30kb, 30kb to 40kb, 40kb to 50kb, 50kb to 60kb, 60kb to 70kb, 70kb to 80kb, 80kb to 90kb, 90kb to 100kb, 100kb to 110kb, 110kb to 120kb, 120kb to 130kb, 130kb to 140kb, 140kb to 150kb, 150kb to 160kb to 170kb, 170kb to 180kb, 180kb to 190kb, 190kb to 200kb to 300kb or longer (as described in the vectors described elsewhere herein) such that the homology arm has sufficient homology to undergo homologous recombination with the corresponding target site within the genome of the cell. For ease of reference, homology arms are referred to herein as 5 'and 3' homology arms. This term relates to the relative position of the homology arms and the inserted nucleic acid within the targeting vector.
Thus, the homology arms of the targeting vector are designed to be complementary to the target site with the targeted locus. Thus, the homology arm may be complementary to a locus inherent to the cell, or alternatively, it may be complementary to a region of a heterologous or exogenous segment of DNA integrated into the genome of the cell, including but not limited to a transgene, an expression cassette, or a heterologous or exogenous region of genomic DNA. Alternatively, the homology arm of the targeting vector may be complementary to a region of an artificial chromosome or any other engineered genomic region contained in an appropriate host cell. Still further, the homology arm of the targeting vector may be complementary to or derived from a region of a BAC library, cosmid library, or P1 phage library. Thus, in particular embodiments, the homology arm of the targeting vector is complementary to a natural, heterologous, or exogenous eukaryotic, non-human, mammalian, non-human mammalian, human, rodent, mouse, or rat genomic locus of a given cell. In one embodiment, the homology arms are derived from synthetic DNA.
In some targeting vector embodiments, the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig heavy chain locus such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a non-human Ig C at the non-human Ig heavy chain locus H Recombinant nucleic acid molecules upstream of and operably linked to (e.g., comprising a modified IgV H Segments and optionally Ig D H And/or Ig J H A recombinant nucleic acid molecule of a segment), optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces the non-human V at the non-human Ig heavy chain locus H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces all but one non-human V at the non-human Ig heavy chain locus H Segment or all non-human V H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus.
In some embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig heavy chain locus such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus (e.g., comprising a modified Ig V H Segments and optionally Ig D H Segment, ig J H Segment and/or IgC H Recombinant nucleic acid molecule of a gene), optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus in a rodent or rodent cell (e.g., a rodent embryonic stem cell), and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, an endogenous Ig V H 、D H And/or J H Deletion of gene segments or combinations thereof, and optionally wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Gene segment, all non-human J H Gene segment and one or more non-human C H And (3) a gene.
In some embodiments, the 5 'homology arm comprises the sequence shown as SEQ ID NO. 12 and/or the 3' homology arm comprises the sequence shown as SEQ ID NO. 13.
In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a non-human Ig C at the non-human Ig light chain locus L Recombinant nucleic acid molecules upstream of and operably linked to (e.g., comprising a modified Ig V as described herein L Segments and optionally Ig J L A recombinant nucleic acid molecule of a segment), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, endogenous Ig V L And/or J L Deletion of a gene segment or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces the non-human V at the non-human Ig light chain locus L A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain loci, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig light chain loci L Segment and all non-human J L A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces all non-human V at the non-human Ig light chain locus L Segment and all non-human J H A section. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus.
In some embodiments, a targeting vector described herein comprises a nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a sequence that is homologous to the non-human Ig light chain locus Recombinant nucleic acid molecules in which non-human Ig light chain regulatory sequences are operably linked (e.g., comprising a modified IgV as described herein L Segments and optionally Ig J L Segment and/or IgC L Recombinant nucleic acid molecule of a gene), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, an endogenous Ig V L And/or J L Deletion of gene segments or combinations thereof, and optionally wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L Segment, all non-human J L Gene segment and the non-human C L And (3) a gene.
In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain kappa locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vk segment and optionally an Ig jk segment as described herein) located upstream of and operably linked to the non-human Ig ck at the non-human Ig light chain kappa locus, optionally wherein the non-human Ig light chain kappa locus is an endogenous rodent light chain kappa locus and/or wherein the non-human Ig light chain kappa locus comprises a deletion of human or humanized immunoglobulin light chain variable regions, endogenous Ig vk and/or J gene segments or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces a non-human vk segment at the non-human Ig light chain kappa locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces one or more non-human vk segments and all non-human jk segments at the non-human Ig light chain kappa locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces all non-human vk segments and all non-human jk segments at the non-human Ig light chain kappa locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus.
In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain kappa locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus (e.g., a recombinant nucleic acid molecule comprising a modified Ig vk segment and optionally an Ig jk segment and/or an Ig ck gene as described herein), optionally wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces the non-human V k segment, all non-human jk gene segments and the non-human ck gene at the non-human Ig light chain kappa locus.
In some targeting vector embodiments, a targeting vector comprises a recombinant nucleic acid molecule described herein and 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig lambda segment and optionally an Ig lambda segment) located upstream of and operably linked to the non-human Ig lambda at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain lambda locus is an endogenous rodent light chain lambda locus and/or wherein the non-human Ig light chain lambda locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig lambda and/or J lambda gene segments, or a combination thereof. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces a non-human vlambda segment at the non-human Ig light chain lambda locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces one or more non-human vlambda segments and all non-human jlambda segments at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces all non-human V lambda segments and all non-human J lambda segments at the non-human Ig light chain lambda locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus.
In some embodiments, a targeting vector comprises a recombinant nucleic acid molecule as described herein and 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus (e.g., a recombinant nucleic acid molecule comprising a modified Ig vlambda segment and optionally an Ig jlambda segment and/or an Ig clambda gene as described herein). In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces the non-human va segment, all non-human jlambda gene segments, and the non-human clambda gene at the non-human Ig light chain lambda locus.
In some embodiments, a targeting vector as described herein may further comprise a nucleotide sequence flanked by site-specific recombination target sequences. It has been recognized that any region or individual polynucleotide of interest within a targeting vector may also be flanked by such sites. The site-specific recombinase can be introduced into the cell by any means, including by introducing the recombinase polypeptide into the cell or by introducing a polynucleotide encoding the site-specific recombinase into the host cell. The polynucleotide encoding the site-specific recombinase may be located within the targeting vector or within a separate polynucleotide. The site-specific recombinase may be operably linked to a promoter active in the cell, including, for example, an inducible promoter, a promoter endogenous to the cell, a promoter heterologous to the cell, a cell-specific promoter, a tissue-specific promoter, or a developmental stage-specific promoter. The site-specific recombination target sequences that can flank the nucleotide sequence of interest or any polynucleotide in the targeting vector can include, but are not limited to, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT11, FRT71, attp, att, FRT, rox, or a combination thereof.
In some embodiments, the site-specific recombination site flanks the polynucleotide encoding the selectable marker within the targeting vector. In such cases, the nucleotide sequence between the site-specific recombination sites can be removed after integration of the targeting vector at the targeted locus.
In some embodiments, a targeting vector as described herein includes a selectable marker, which may be contained in a selection cassette. Such selectable markers include, but are not limited to, neomycin phosphotransferase (neo r ) Hygromycin B phosphotransferase (hyg) r ) puromycin-N-acetyltransferase (puro) r ) Pyricularia oryzae-killing bacteria-S deaminase (bsr) r ) Xanthine/guanine phosphoribosyl transferase (gpt) or herpes simplex virus thymidine kinase (HSV-k), or a combination thereof. In one embodiment, the polynucleotide encoding the selectable marker is operably linked to a promoter active in the cell. In one embodiment, the polynucleotide encoding the selectable marker is flanked by site-specific recombination target sequences.
Non-human animal genome
Also described herein is a non-human animal genome comprising a recombinant nucleic acid molecule and/or targeting vector as described herein. In some non-human animal genome embodiments, the non-human animal genome comprises a recombinant nucleic acid molecule as described herein at an endogenous Ig locus of the non-human animal genome, e.g., the non-human animal genome comprises a targeting vector as described herein, wherein the targeting vector comprises 5 'and 3' homology arms that target the endogenous Ig locus. In some embodiments, the non-human animal genome is a rodent genome. In some embodiments, the non-human animal genome is a rat genome. In some embodiments, the non-human animal genome is a mouse genome.
In a non-human animal genome embodiment, the genome comprises a non-human Ig heavy chain locus comprising a non-human Ig C at the non-human Ig heavy chain locus H Recombinant nucleic acid molecules upstream of and operably linked to (e.g., comprising a modified IgV H Segments and optionally Ig D H And/or Ig J H A recombinant nucleic acid molecule of a segment), optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig heavy chain locus H A section. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, the recombinant nucleic acid molecule replaces all but one of the non-human V at the non-human Ig heavy chain locus H Segment or all non-human V H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus.
In some embodiments, the non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus (e.g., comprising a modified Ig V H Segments and optionally Ig D H Segment, ig J H Segment and/or IgC H Recombinant nucleic acid molecules of genes), optionally wherein the non-human Ig heavy chain locus is a rodentEndogenous rodent Ig heavy chain loci in an animal or rodent cell (e.g., rodent embryonic stem cell), and/or wherein the non-human Ig heavy chain loci comprise human or humanized immunoglobulin heavy chain variable regions, endogenous Ig V H 、D H And/or J H Deletion of gene segments or combinations thereof, and optionally wherein the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Gene segment, all non-human J H Gene segment and one or more non-human C H And (3) a gene.
In a non-human animal genome embodiment, the genome comprises a non-human Ig light chain locus comprising a non-human Ig C located at the non-human Ig light chain locus L Recombinant nucleic acid molecules upstream of and operably linked to (e.g., comprising a modified Ig V as described herein L Segments and optionally Ig J L A recombinant nucleic acid molecule of a segment), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, endogenous Ig V L And/or J L Deletion of a gene segment or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L A section. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig light chain locus L Segment and all non-human J L A section. In some embodiments, the recombinant nucleic acid molecule replaces all non-human V at the non-human Ig light chain locus L Segment and all non-human J H A section. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus.
In non-human animal genomic embodiments, the Ig light chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus (e.g., comprising a modified Ig as described herein V L Segments and optionally Ig J L Segment and/or IgC L Recombinant nucleic acid molecule of a gene), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, an endogenous Ig V L And/or J L Deletion of gene segments or combinations thereof, and optionally wherein the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L Segment, all non-human J L Gene segment and the non-human C L And (3) a gene.
In non-human animal genome embodiments, the genome comprises a non-human Ig light chain kappa locus comprising a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vk segment and optionally an Ig jk segment as described herein) upstream of and operably linked to a non-human Ig ck at the non-human Ig light chain kappa locus, optionally wherein the non-human Ig light chain kappa locus is an endogenous rodent Ig light chain kappa locus and/or wherein the non-human Ig light chain kappa locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vk and/or jk gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human vk segment at the non-human Ig light chain k locus. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human vk segments and all non-human jk segments at the non-human Ig light chain k locus. In some embodiments, the recombinant nucleic acid molecule replaces all non-human vk segments and all non-human jk segments at the non-human Ig light chain k locus. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus.
In some embodiments, the recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vk segment and optionally an Ig jk segment and/or an Ig ck gene as described herein) is operably linked to a non-human Ig light chain k regulatory sequence at the Ig light chain k locus, optionally wherein the recombinant nucleic acid molecule replaces the non-human vk segment, all non-human jk gene segments, and the non-human ck gene at the non-human Ig light chain k locus.
In non-human animal genome embodiments, the genome comprises a non-human Ig light chain lambda locus comprising a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vlambda segment and optionally an Ig jlambda segment) upstream of and operably linked to a non-human Ig clambda at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain lambda locus is an endogenous rodent light chain lambda locus and/or wherein the non-human Ig light chain lambda locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vlambda and/or jlambda gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human vλ segment at the non-human Ig light chain λ locus. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human vλ segments and all non-human jλ segments at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces all non-human V lambda segments and all non-human J lambda segments at the non-human Ig light chain lambda locus. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus.
In some non-human animal genomic embodiments, the non-human Ig light chain lambda locus comprises the recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vlambda segment and optionally an Ig jlambda segment and/or an Ig clambda gene as described herein) operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus. In some embodiments, the recombinant nucleic acid molecule replaces a non-human vλ segment at the non-human Ig light chain λ locus, all non-human jλ gene segments, and the non-human cλ gene.
Non-human animal cell, non-human animal and preparation method thereof
Non-human animal and non-human animal cell
ProvidesNon-human animals and non-human animal cells expressing anchor modified immunoglobulins, e.g., from Ig loci modified to include recombinant nucleic acid molecules as described herein, including modified Ig V segments, e.g., ig heavy chain variable regions (V H ) Segment or Ig light chain variable region (V L ) Segments, modified to encode and operably linked to anchors between Ig leader sequences and Framework (FR) and Complementarity Determining Region (CDR) sequences of germline V segments. Thus provided are non-human animals, embryos, cells comprising recombinant nucleic acid molecules, targeting constructs, and/or animal genomes described herein.
In some non-human animal or non-human animal cell embodiments, the non-human animal or cell comprises a recombinant nucleic acid molecule as described herein (including a modified Ig V segment, e.g., an Ig heavy chain variable region (V H ) Segment or Ig light chain variable region (V L ) Segments, modified to encode and operably linked to anchors between Ig leader sequences and Framework (FR) and Complementarity Determining Region (CDR) sequences of germline V segments randomly located within the genome of the animal. In some embodiments, the non-human animal or cell comprises a recombinant nucleic acid molecule as described herein (including a modified Ig V segment, e.g., an Ig heavy chain variable region (V H ) Segment or Ig light chain variable region (V L ) A segment modified to encode an anchor between and operably linked to a Framework (FR) and Complementarity Determining Region (CDR) sequences of an Ig leader sequence and a germline V segment located at an endogenous Ig locus of the non-human animal, e.g., the non-human animal comprises a targeting vector as described herein, wherein the targeting vector comprises 5 'and 3' homology arms targeting the endogenous Ig locus. In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal cell is a rodent cell. In some embodiments, the non-human animal is a rat. In some embodiments, the non-human animal cell is a rat cell. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal cell is a mouse cell.
In an embodiment, the non-human animal or non-human animalThe somatic cell includes a non-human Ig heavy chain locus including a non-human Ig C located at the non-human Ig heavy chain locus H Recombinant nucleic acid molecules upstream of and operably linked to (e.g., comprising a modified IgV H Segments and optionally Ig D H And/or Ig J H A recombinant nucleic acid molecule of a segment), optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig heavy chain locus H A section. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, the recombinant nucleic acid molecule replaces all but one of the non-human V at the non-human Ig heavy chain locus H Segment or all non-human V H Segment, all non-human D H Segment and all non-human J H A section. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus.
In some embodiments, the non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus (e.g., comprising a modified Ig V H Segments and optionally Ig D H Segment, ig J H Segment and/or IgC H Recombinant nucleic acid molecule of a gene), optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus in a rodent or rodent cell (e.g., a rodent embryonic stem cell), and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, an endogenous Ig V H 、D H And/or J H Deletion of gene segments or combinations thereof, and optionally wherein said recombinationReplacement of one or more non-human V at the non-human Ig heavy chain locus by a nucleic acid molecule H Segment, all non-human D H Gene segment, all non-human J H Gene segment and one or more non-human C H And (3) a gene.
In some embodiments, the non-human animal or non-human animal cell comprises a non-human Ig light chain locus comprising a non-human Ig C located at the non-human Ig light chain locus L Recombinant nucleic acid molecules upstream of and operably linked to (e.g., comprising a modified Ig V as described herein L Segments and optionally Ig J L A recombinant nucleic acid molecule of a segment), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, endogenous Ig V L And/or J L Deletion of a gene segment or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L A section. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig light chain locus L Segment and all non-human J L A section. In some embodiments, the recombinant nucleic acid molecule replaces all non-human V at the non-human Ig light chain locus L Segment and all non-human J H A section. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus.
In some embodiments, the Ig light chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus (e.g., comprising a modified igv as described herein L Segments and optionally Ig J L Segment and/or IgC L Recombinant nucleic acid molecule of a gene), optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, an endogenous Ig V L And/or J L GeneDeletion of segments or combinations thereof, and optionally wherein the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L Segment, all non-human J L Gene segment and the non-human C L And (3) a gene.
In some embodiments, the non-human animal or non-human animal cell comprises a non-human Ig light chain kappa locus comprising a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vk segment and optionally an Ig jk segment as described herein) upstream of and operably linked to a non-human Ig ck at the non-human Ig light chain kappa locus, optionally wherein the non-human Ig light chain kappa locus is an endogenous rodent light chain kappa locus and/or wherein the non-human Ig light chain kappa locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vk and/or jk gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human vk segment at the non-human Ig light chain k locus. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human vk segments and all non-human jk segments at the non-human Ig light chain k locus. In some embodiments, the recombinant nucleic acid molecule replaces all non-human vk segments and all non-human jk segments at the non-human Ig light chain k locus. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus.
In some embodiments, the recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vk segment and optionally an Ig jk segment and/or an Ig ck gene as described herein) is operably linked to a non-human Ig light chain k regulatory sequence at the Ig light chain k locus, optionally wherein the recombinant nucleic acid molecule replaces the non-human vk segment, all non-human jk gene segments, and the non-human ck gene at the non-human Ig light chain k locus.
In some embodiments, the non-human animal or non-human animal cell comprises a non-human Ig light chain lambda locus comprising a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vlambda segment and optionally an Ig jlambda segment) upstream of and operably linked to a non-human Ig clambda at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain lambda locus is an endogenous rodent light chain lambda locus and/or wherein the non-human Ig light chain lambda locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vlambda and/or jlambda gene segments, or a combination thereof. In some embodiments, the recombinant nucleic acid molecule replaces a non-human vλ segment at the non-human Ig light chain λ locus. In some embodiments, the recombinant nucleic acid molecule replaces one or more non-human vλ segments and all non-human jλ segments at the non-human Ig light chain locus. In some embodiments, upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces all non-human V lambda segments and all non-human J lambda segments at the non-human Ig light chain lambda locus. In some embodiments, the recombinant nucleic acid molecule is operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus.
In some embodiments, the non-human Ig light chain lambda locus comprises the recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule comprising a modified Ig vlambda segment and optionally an Ig jlambda segment and/or an Ig clambda gene as described herein) operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus. In some embodiments, the recombinant nucleic acid molecule replaces a non-human vλ segment at the non-human Ig light chain λ locus, all non-human jλ gene segments, and the non-human cλ gene.
Method for preparing non-human animal or non-human animal cell
Methods of preparing non-human cells, non-human embryos, and/or non-human animals in vitro using the recombinant nucleic acid molecules described herein, e.g., targeting vectors, are also described. In some embodiments, an in vitro method of modifying an isolated cell comprises introducing a recombinant nucleic acid molecule as described herein into an isolated cell, for example, by contacting the cell with a targeting vector as described herein. In some method embodiments, the cell is a host cell. In some method embodiments, the cell is an Embryonic Stem (ES) cell. In some embodiments, the cell as described herein or prepared according to the methods described herein is a rodent cell, e.g., wherein the rodent cell is a rat cell or a mouse cell.
Also described are methods of making anchor modified antigen binding proteins using the nucleic acid molecules, the non-human cells, and/or the non-human animals as described herein. Also described are non-human animal embryos and non-human animals, which may include and/or develop (e.g., arise from) embryonic stem cells as described herein. Such embryos or non-human animals may be developed by a method comprising implanting ES cells as described herein into an embryo, and/or implanting an embryo comprising the ES cells into a suitable host, and maintaining the host under suitable conditions during the development of the ES cells or embryo into viable offspring.
As described herein, targeting vectors can be used to target Ig loci with human or humanized immunoglobulin variable regions. Immunoglobulin loci comprising human variable region gene segments are known in the art and can be found, for example, in U.S. patent No. 5,633,425; 5,770,429; no. 5,814,318; 6,075,181; 6,114,598; 6,150,584; 6,998,514; 7,795,494; 7,910,798; 8,232,449; 8,502,018; 8,697,940; 8,703,485; 8,754,287; 8,791,323; 8,809,051; 8,907,157; 9,035,128; 9,145,588;9,206,263; 9,447,177; 9,551,124; 9,580,491 and 9,475,559; each of which is incorporated by reference herein in its entirety; and U.S. patent publications 20100146647, 20110195454, 20130167256, 20130219535, 20130326647, 20130096287, and 2015/0110268, each of which is incorporated by reference herein in its entirety; and PCT publications No. WO 2007117410, no. WO 2008151081, no. WO 2009157771, no. WO 2010039900, no. WO 2011004192, no. WO 2011123708 and No. WO 2014093908, each of which disclosures is incorporated herein by reference in its entirety.
In some embodiments, the non-human animals as disclosed herein include exogenous fully human immunoglobulin transgenes comprising modified Ig V segments as described herein that are capable of rearranging in precursor B cells of mice (Alt et al, 1985, immunoglobulin genes in transgenic mice (Immunoglobulin genes in transgenic mice), "Trends Genet)," 1:231-236; incorporated herein by reference in its entirety). In these examples, a fully human immunoglobulin transgene comprising a modified Ig V segment as described herein may be (randomly) inserted and endogenous immunoglobulin genes may also be knocked out (Green et al, 1994, antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs (Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs), nature genetics (Nat Genet) 7:13-21; lonberg et al, 1994, antigen-specific human antibodies from mice comprising four different genetic modifications (Antigen-specific human antibodies from mice comprising four distinct genetic modifications), nature 368:856-859; jakobovits et al, 2007, from NOMouse technology to panitumumab, first human antibody products from transgenic mice (From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice), nature biotechnology (Nature Biotechn) 25:1134-3; each of said genes is incorporated by way of, for example, into the human locus by way of random gene insertion, or by way of a deletion of the endogenous gene, for example, into the human heavy chain, the human locus, tomika gene locus, and the like, and then, as part of the endogenous gene sequence is deleted (Tomizu-2000), double trans chromosome mice: maintaining expression of two separate human chromosome fragments containing Ig heavy and kappa loci and a fully human antibody (Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies), "Proc. Natl. Acad. Sci. USA (PNAS USA)," 97:722-727; incorporated by reference in its entirety).
In some embodiments, human or humanized immunoglobulin heavy and light chain loci comprising modified Ig V segments as described herein are located at endogenous immunoglobulin light and heavy chain loci, respectively. It has been shown that even human V H Gene segment replacement of single endogenous V H A gene segment may also result in an immune response that includes a humanized immunoglobulin variable domain. See, for example, tien et al, (2016) Cell 166:1471-84; incorporated herein by reference in its entirety.
A method of large scale in situ gene replacement of a mouse germline immunoglobulin variable locus with a human germline immunoglobulin variable locus while maintaining the ability of the mouse to produce offspring has been previously described. See, for example, U.S. Pat. nos. 6,596,541 and 8,697,940, each of which is incorporated by reference in its entirety. In particular, it is described that six megabases of mouse heavy and kappa light chain immunoglobulin variable gene loci are precisely replaced with their human counterparts while leaving the mouse constant regions intact. Thus, mice have been established in which the entire germline immunoglobulin variable profile is precisely replaced by equivalent human germline immunoglobulin variable sequences, while maintaining the mouse constant regions. The human variable region is linked to a mouse constant region to form a chimeric human-mouse immunoglobulin locus that rearranges and expresses at a physiologically appropriate level. The expressed antibodies are "reverse chimeras", i.e., they include human variable region sequences and mouse constant region sequences.
In some embodiments, mice having humanized immunoglobulin variable regions that express antibodies with human or humanized variable regions and mouse constant regions are referred to asAnd (3) a mouse.Humanized mice exhibit a substantially incompetent region with wild-type miceA divided fully functional humoral immune system. They display normal cell populations at all stages of B cell development. They exhibit normal lymphoid organ morphology.The antibody sequences of the mice exhibit normal V (D) J rearrangements and normal somatic hypermutation frequencies. The population of antibodies in these mice reflects the isotype profile resulting from normal class switching (e.g., normal isotype cis switching). For->Immunization of mice produces a robust humoral immune response that produces a large diverse antibody repertoire with human immunoglobulin variable domains suitable for use as therapeutic candidates. This platform provides a rich source of naturally affinity matured human immunoglobulin variable region sequences for the manufacture of pharmaceutically acceptable antibodies and other antigen binding proteins. It is precisely the exact replacement of the mouse immunoglobulin variable sequence with the human immunoglobulin variable sequence that is operably linked in a reverse chimeric fashion to the endogenous non-human constant region gene sequence, allowing for the manufacture +. >And (3) a mouse.
Mice modified in a reverse chimeric manner include, but are not limited to, mice modified to include a human (humanized) variable region (e.g., comprising (D), J, and one or more human V gene segments) operably linked to an endogenous constant region at an endogenous immunoglobulin locus, e.g., such as
(a) At the endogenous heavy chain locus:
(i) An unrearranged human (humanized) immunoglobulin heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the unrearranged human (humanized) immunoglobulin heavy chain variable region comprises a plurality of unrearranged human heavy chain variable regions V H Gene segments (e.g., human V with all functional humans unrearranged H Gene segment), one or more non-polynucleotidesRearranged immunoglobulin heavy chain D H Gene segments and one or more unrearranged immunoglobulin heavy chains J H A gene segment, wherein the gene segment,
optionally wherein the one or more unrearranged immunoglobulin heavy chains D H Gene segments and one or more unrearranged immunoglobulin heavy chains J H The gene segments are one or more unrearranged human immunoglobulin heavy chains D H Gene segments (e.g., all functional human D H Gene segment) and/or one or more unrearranged human immunoglobulin heavy chains J H Gene segments (e.g., all functional human J H A gene segment);
(ii) A restricted unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the restricted unrearranged human (humanized) heavy chain variable region consists essentially of an immunoglobulin heavy chain D that is unrearranged with one or more heavy chains H Gene segments and one or more unrearranged immunoglobulin heavy chains J H Single unrearranged human heavy chain variable region V with operably linked gene segments H Gene segment composition, optionally wherein the one or more unrearranged immunoglobulin heavy chains D H Gene segments and one or more unrearranged immunoglobulin heavy chains J H The gene segments are respectively one or more unrearranged human immunoglobulin heavy chains D H Gene segments and/or one or more unrearranged human immunoglobulin heavy chains J H A gene segment;
(iii) A histidine-modified unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the histidine-modified unrearranged human (humanized) heavy chain variable region comprises an unrearranged immunoglobulin heavy chain variable gene sequence comprising at least one non-histidine codon substituted with or inserted into a complementarity determining region 3 (CDR 3) coding sequence; or (b)
(iv) A heavy chain-only immunoglobulin coding sequence comprising an unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region, wherein the endogenous heavy chain constant region comprises (1) a complete endogenous IgM gene encoding an IgM isotype associated with a light chain and (2) a non-IgM gene, e.g., an IgG gene, lacking a sequence encoding a functional CH1 domain, wherein the non-IgM gene encodes a non-IgM isotype lacking a CH1 domain capable of covalently binding to the light chain constant domain;
and/or
(b) At the endogenous light chain locus:
(i) An unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region, wherein the unrearranged human (humanized) immunoglobulin light chain variable region comprises a plurality of unrearranged human light chain variable regions V L Gene segments (e.g.human V with all functional humans unrearranged L Gene segment) and one or more unrearranged immunoglobulin light chains J L A gene segment, wherein the gene segment,
optionally wherein the one or more unrearranged immunoglobulin light chains J L The gene segments are one or more unrearranged human immunoglobulin light chains J L Gene segments (e.g. all functional human J H L gene segment),
Optionally wherein the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain kappa (kappa) locus and the unrearranged human (humanized) immunoglobulin light chain variable region comprises a human variable kappa (V κ ) And bond kappa (J) κ ) A gene segment, and wherein the endogenous light chain constant region is an endogenous kappa chain constant region sequence and/or wherein the endogenous immunoglobulin light chain locus is an endogenous immunoglobulin light chain lambda (lambda), the unrearranged human (humanized) immunoglobulin light chain variable region comprises a human variable lambda (V λ ) And joint lambda (J) λ ) A gene segment, and the endogenous light chain constant region is an endogenous lambda chain constant region sequence, optionally wherein the endogenous immunoglobulin light chain lambda locus comprises (a) one or more human V' s λ A gene segment, (b) one or more human J λ A gene segment, and (C) one or more human C λ Gene segments, wherein (a) and (b) are in engagement with (c) anddentifrices immunoglobulin light chain constant C λ ) The gene segments are operably linked, and wherein the endogenous immunoglobulin lambda light chain locus further comprises: one or more rodent immunoglobulin lambda light chain enhancers (eλ), and one or more human immunoglobulin lambda light chain enhancers (eλ), optionally comprising three human eλ;
(ii) A common light chain coding sequence comprising a rearranged human (humanized) light chain variable region sequence operably linked to an endogenous light chain constant region, wherein the rearranged human (humanized) light chain variable region sequence comprises a sequence that is complementary to an immunoglobulin light chain J L Human light chain variable region V with gene segments rearranged together L A gene segment;
(iii) A constrained, unrearranged, human (humanized) light chain variable region operably linked to an endogenous light chain constant region, wherein the constrained, unrearranged, human (humanized) light chain variable region comprises a human immunoglobulin light chain linkage J operably linked to one or more unrearranged L ) No more than two unrearranged human immunoglobulin light chain variants of a gene segment (V L ) A gene segment;
(iv) A histidine-modified unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region, wherein the histidine-modified unrearranged human (humanized) light chain variable region comprises an unrearranged human (humanized) immunoglobulin light chain variable gene sequence comprising at least one non-histidine codon substituted with a histidine codon or inserted with at least one histidine codon in a complementarity determining region 3 (CDR 3) coding sequence; or (b)
(v) A histidine-modified rearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region, wherein the histidine-modified rearranged human (humanized) light chain variable region comprises a rearranged human (humanized) immunoglobulin light chain variable gene sequence comprising at least one non-histidine codon substituted with a histidine codon or inserted into at least one histidine codon in a complementarity determining region 3 (CDR 3) encoding sequence,
optionally, wherein the mouse further comprises
(i) A human (humanized) immunoglobulin heavy chain locus comprising a functional ADAM6 gene such that the mouse exhibits wild-type fertility in a non-human animal; and/or
(ii) An exogenous terminal deoxynucleotidyl transferase (TdT) gene for increasing antigen receptor diversity, optionally such that at least 10% of the rearranged variable region gene comprises a non-template addition,
the mice have been described previously. See, for example, U.S. patent No. 8,697,940; 8,754,287; 9,204,624; 9,334,334; 9,801,362; 9,332,742; and 9,516,868; U.S. patent publications 20110195454, 20120021409, 20120192300, 20130045492;20150289489;20180125043;20180244804; PCT publications WO2019/113065, WO 2017210586 and WO 2011163314; lee et al, (2014) Nature Biotechnology 32:356, each of which is incorporated herein by reference in its entirety.
One of skill in the art will readily recognize that any mouse or mouse cell (e.g., a mouse ES cell modified in a reverse chimeric fashion) may be modified to include a modified Ig V segment as described herein. In some embodiments, described herein are genetically modified non-human animals whose genomes, e.g., germline genomes, comprise:
an endogenous immunoglobulin locus comprising an immunoglobulin heavy chain variable region comprising a modified hV of the invention H Gene segment, human D H Gene segment and human J H A gene segment wherein the immunoglobulin heavy chain variable region is operably linked to a constant region, and/or
An endogenous chain locus comprising an immunoglobulin light chain variable region comprising a modified V of the invention L Segment and person J L A gene segment, wherein the immunoglobulin light chain variable region is operably linked to a constant region.
In some embodiments, the non-human animal, e.g., rodent, e.g., rat or mouse, includes in its genome one or more human V H 、D H And J H Segment replacement of one or more endogenous V at an endogenous immunoglobulin heavy chain locus H 、D H And J H A zone, and wherein the one or more people V H 、D H And J H Segments include modified human V as described herein H A gene segment and operably linked to an endogenous immunoglobulin heavy chain gene; and optionally non-rearranged or rearranged human V L And person J L Segments that are constant with a non-human, e.g., rodent, e.g., mouse or rat or human immunoglobulin light chain (C L ) The region genes (e.g., at endogenous non-human light chain loci) are operably linked.
In certain embodiments, a genetically modified non-human animal comprises in its genome, e.g., germline genome, an immunoglobulin locus (exogenous or endogenous) comprising an immunoglobulin variable region comprising one or more unrearranged human immunoglobulin variable region gene segments comprising a modified Ig V gene segment as described herein and an immunoglobulin constant region comprising an immunoglobulin constant region gene, and wherein the one or more unrearranged human immunoglobulin variable region gene segments are operably linked to the immunoglobulin constant region gene.
Generally, a genetically modified immunoglobulin locus comprises an immunoglobulin variable region (including immunoglobulin variable region gene segments) operably linked to an immunoglobulin constant region. In some embodiments, the genetically modified immunoglobulin loci comprise one or more human unrearranged immunoglobulin heavy chain variable region gene segments comprising a modified Ig V as described herein H A gene segment operably linked to a heavy chain constant region gene. In some embodiments, the genetically modified immunoglobulin locus comprises human unrearranged immunoglobulin variable regionsA kappa gene segment comprising a modified Ig vkappa gene segment as described herein operably linked to a kappa chain constant region gene. In some embodiments, the genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region lambda gene segment comprising a modified Ig vlambda gene segment as described herein operably linked to a kappa chain constant region gene. In some embodiments, the genetically modified immunoglobulin locus comprises a human unrearranged immunoglobulin variable region lambda gene segment comprising a modified Ig va gene segment as described herein operably linked to a lambda chain constant region gene.
In certain embodiments, the non-human animal comprises an unrearranged human (humanized) immunoglobulin heavy chain variable region at an endogenous heavy chain locus comprising a modified Ig V operably linked to an endogenous heavy chain constant region as described herein H A gene segment, wherein the immunoglobulin variable region comprises one or more unrearranged human Ig heavy chain variable region gene segments. In some embodiments, the one or more unrearranged human Ig variable region gene segments comprise a modified Ig V as described herein H Gene segment, one or more immunoglobulin heavy chain diversity (D H ) Segment and one or more immunoglobulin heavy chain adaptors (J H ) Segments (optionally one or more unrearranged human J H A section). In some embodiments, a modified igv as described herein H The gene segment is the only Ig V present in the heavy chain variable region H A section. In some embodiments, the unrearranged human Ig gene segments comprise all functional human D H A gene segment. In some embodiments, the unrearranged human Ig gene segments comprise all functional human J H A gene segment. Exemplary variable regions comprising Ig heavy chain gene segments are provided, for example, in the following: macdonald et al, proc.Natl.Acad.Sci.USA 111:5147-52, which is incorporated herein by reference in its entirety, and supplemental information.
In some embodiments, the non-human animals provided herein are at an endogenous heavy chain locusComprising a restricted unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region comprising at least a non-human IgM gene, wherein the restricted unrearranged human (humanized) heavy chain variable region is characterized by a single human V H Gene segments (e.g., single modified Ig V as described herein H Gene segment), a plurality of D H Gene segments (e.g., human D H Gene segment) and a plurality of J H Gene segments (e.g. human J H Gene segments), wherein the restricted immunoglobulin heavy chain locus is capable of rearranging and forming a plurality of different rearrangements, wherein each rearrangement is derived from the single human V H Gene segment, one of said D H Segment and one of said J H Segments, and wherein each rearrangement encodes a different heavy chain variable domain (e.g., as described in U.S. patent publication No. 20130096287, which is incorporated herein by reference in its entirety). In some embodiments, the single modified human V H The gene segment is V H 1-2 or V H 1-69。
In certain embodiments, the non-human animal comprises an unrearranged human (humanized) immunoglobulin light chain variable region at an endogenous light chain locus comprising a modified Ig V as described herein L A segment operably linked to an endogenous light chain constant region. In some embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region contains unrearranged human igκ variable region gene segments. In some embodiments, the unrearranged human (humanized) immunoglobulin variable region comprises one or more unrearranged human vκ segments, which may comprise a modified Ig vκ segment and one or more unrearranged human J as described herein K A section. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all human jκ segments. In some embodiments, the immunoglobulin variable region gene segments comprise four functional vk segments and all human jk segments. In some embodiments, the immunoglobulin variable region gene segments comprise 16 functional vk segments and all human jk segments (e.g., all functional human vk segments and jk segments). In some implementationsIn embodiments, the unrearranged human immunoglobulin variable region gene segments include all human vk segments and all human jk segments. Exemplary variable regions comprising Ig kappa gene segments are provided, for example, in the following: macdonald et al, proc.Natl.Acad.Sci.USA 111:5147-52, which is incorporated herein by reference in its entirety, and supplemental information.
In some embodiments, the restriction unrearranged human (humanized) light chain variable region operably linked to an endogenous light chain constant region is characterized in that the unrearranged human (humanized) light chain variable region comprises no more than two human V L Gene segments and a plurality of J L Gene segments (e.g., double light chain mice or DLC, as described in U.S. patent No. 9,796,788, incorporated herein by reference in its entirety), no more than two of human V L The gene segment may be a modified Ig V as described herein L A section. In some embodiments, V L The gene segment is a vk gene segment. In some embodiments, V L The gene segment is a V lambda gene segment. In some embodiments, the V kappa gene segments are IGKV3-20 and IGKV1-39. In some embodiments, the endogenous kappa light chain locus of the non-human animal at the mouse comprises exactly two unrearranged human vk gene segments and five unrearranged human vk gene segments operably linked to the mouse light chain constant region, optionally wherein the exactly two unrearranged human vk gene segments are a human vk 1-39 gene segment and a human vk 3-20 gene segment, wherein the five unrearranged human vk gene segments are a human jk 1 gene segment, a human jk 2 gene segment, a human jk 3 gene segment, a human jk 4 gene segment, and a human jk 5 gene segment, wherein the unrearranged human jk light chain gene segment is capable of rearranging and encoding a human variable domain of an antibody, and optionally further wherein the non-human animal does not comprise an endogenous vk gene segment capable of rearranging to form an immunoglobulin light chain variable region.
In certain embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region contains an unrearranged human igλ variable region gene segment comprising a modified igλ segment as described herein. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise a plurality of human vλ segments and one or more human jλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise one or more human va segments, one or more human jλ segments, and one or more human cλ constant region sequences. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all human vλ segments. In some embodiments, the unrearranged human immunoglobulin variable region gene segments comprise all human jλ segments. Exemplary variable regions comprising igλ gene segments are provided, for example, in the following: U.S. patent nos. 9,035,128 and 6,998,514, each of which is incorporated by reference herein in its entirety. In some embodiments, the unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region comprises (a) one or more human va gene segments, (b) one or more human jλ gene segments, and (C) one or more human cλ gene segments, wherein (a) and (b) are operably linked to (C) and endogenous (e.g., rodent) cλ gene segments, and wherein the endogenous immunoglobulin lambda light chain locus further comprises: one or more rodent immunoglobulin lambda light chain enhancers (eλ) and one or more human immunoglobulin lambda light chain enhancers (eλ), optionally comprising three human eλ.
In certain embodiments, an unrearranged human (humanized) immunoglobulin light chain variable region operably linked to an endogenous light chain constant region comprises an unrearranged human igλ variable region gene segment, e.g., a modified igλ segment as described herein, operably linked to an endogenous (e.g., rodent, e.g., rat or mouse) ck gene such that the non-human animal expresses an immunoglobulin light chain comprising a human variable domain sequence having λ derived from a vλ and jλ gene segment fused to an endogenous kappa constant domain, see, e.g., U.S. patent No. 9,226,484, which is incorporated herein by reference in its entirety.
In some embodiments, a humanized immunoglobulin kappa light chain locus, e.g., a humanized immunoglobulin endogenous kappa locus, comprises one or more human vλ gene segments (e.g., modified human vλ segments as described herein) upstream of (e.g., operably linked to) a cλ gene and one or more human jλ gene segments, e.g., that can replace the endogenous cκ gene. In some embodiments, the cλ gene is a rodent (e.g., rat or mouse) cλ gene. In some embodiments, the cλgene is a mouse cλ1 gene. In some embodiments, the cλ gene comprises one or more human cλ genes. In some embodiments, one or more human jλ gene segments and one or more cλ genes of such humanized immunoglobulin kappa light chain loci are present in the jλ -cλ cluster. In some embodiments, the genetically modified rodent (e.g., rat or mouse) is homozygous for such humanized immunoglobulin kappa light chain locus. In some embodiments, the genetically modified rodent (e.g., rat or mouse) is heterozygous for such a humanized immunoglobulin kappa light chain locus. In some embodiments, genetically modified rodents (e.g., rats or mice) that include such humanized immunoglobulin kappa light chain loci produce antibodies, e.g., in response to antigenic stimulation, that include, inter alia, lambda light chains, wherein each lambda light chain comprises a human lambda light chain variable domain operably linked to a human lambda light chain constant domain.
In some embodiments, the immunoglobulin variable region comprising unrearranged human immunoglobulin variable region gene segments further comprises a human immunoglobulin variable region intergenic sequence. In some embodiments, the immunoglobulin variable region comprises a non-human (e.g., rodent, rat, mouse) Ig variable region intergenic sequence. In some embodiments, the intergenic sequence is of endogenous species origin.
In some embodiments, the immunoglobulin variable region is a rearranged light chain variable region (universal light chain variable region). In some embodiments, the rearranged Ig light chain variable region gene is a human rearranged Ig light chain variable region gene. Exemplary rearranged Ig light chain variable regions are provided in the following: such as U.S. patent nos. 9,969,814, 10,130,181 and 10,143,186 and U.S. patent publication nos. 20120021409, 20120192300, 20130045492, 20130185821, 20130302836 and 20150313193, each of which is incorporated herein by reference in its entirety. In some embodiments, a non-human organism that includes a universal light chain variable region ("universal light chain" organism) is used to produce the bispecific antibody. In some embodiments, a common light chain coding sequence comprises a single rearranged human immunoglobulin light chain vk/jk sequence operably linked to an endogenous light chain constant region. In some embodiments, a single rearranged human immunoglobulin light chain vk/jk sequence is modified such that the single rearranged human immunoglobulin light chain sequence encodes an anchor operably linked to a universal light chain as described herein. In some embodiments, the single rearranged human immunoglobulin light chain vk/jk sequence is (i) a human vk 1-39/jk 5 sequence comprising a human vk 1-39 gene segment fused to a human jk 5 gene segment, or (ii) a human vk 3-20/jk 1 sequence comprising a human vk 3-20 gene segment fused to a human jk 1 gene segment.
In some embodiments, the immunoglobulin variable region is a light chain and/or heavy chain immunoglobulin variable region comprising an insertion and/or substitution of a histidine codon designed to introduce pH-dependent binding properties into antibodies produced in such non-human organisms. In some such embodiments, histidine codons are inserted and/or substituted in the nucleic acid sequence encoding CDR 3. Various such light and/or heavy chain immunoglobulin loci are provided in U.S. patent nos. 9,301,510, 9,334,334 and 9,801,362 and U.S. patent application publication No. 20140013456, each of which is incorporated herein by reference in its entirety. In some embodiments, the histidine-modified rearranged human (humanized) light chain variable region operably linked to the endogenous light chain constant region comprises a single rearranged human immunoglobulin light chain variable region gene sequence comprising human vk and jk segment sequences, optionally wherein the vk segment sequences are derived from human vk 1-39 or vk 3-20 gene segments, and wherein the single rearranged human immunoglobulin light chain variable region gene sequence comprises at least one non-histidine codon of the vk segment sequences replaced with a histidine codon expressed at a position selected from the group consisting of : 105. 106, 107, 108, 109, 111 and combinations thereof (according to IMGT numbering). In some embodiments, the histidine-modified unrearranged human (humanized) heavy chain variable region operably linked to an endogenous heavy chain constant region comprises an unrearranged human (humanized) immunoglobulin heavy chain variable gene sequence that is operably linked to an endogenous heavy chain constant region in a complementarity determining region 3 (CDR 3) coding sequence (e.g., in a modified (human) V as described herein H In the gene segment) includes substitution or insertion of at least one histidine codon with at least one non-histidine codon. In some embodiments, the unrearranged human (humanized) immunoglobulin heavy chain variable gene sequence comprises unrearranged human V H (e.g., modified (human) V as described herein H Segment), non-rearranged person D H Or synthesizing D H Human J without rearrangement H A gene segment, optionally wherein the unrearranged human V H Segments (e.g., modified (human) V as described herein H Segment) includes substitution or insertion of at least one non-histidine codon with a histidine codon. In some embodiments, the histidine-modified unrearranged human (humanized) light chain variable region operably linked to an endogenous heavy chain constant region comprises unrearranged V L And unrearranged J L A gene segment. In some embodiments, the histidine-modified unrearranged human (humanized) light chain variable region comprises no more than two unrearranged human V L (e.g., no more than two V.kappa.gene segments) and one or more unrearranged human J L (e.g., jκ) gene segments, no more than two of which are V L Each of the gene segments includes at least one non-histidine codon substituted with or inserted into a histidine codon in the CDR3 encoding sequence. In some embodiments, the no more than two unrearranged human vk gene segments are human vk 1-39 and vk 3-20 gene segments each comprising a non-histidine codon substituted one or more times with a histidine codon, and wherein the human vk and jk gene segments are capable of rearranging and the human vk and jk gene segments encode a human light chain comprising one or more histidines at a position selected from the group consisting ofVariable domain: 105. 106, 107, 108, 109, 111 (numbered according to IGMT) and combinations thereof, wherein one or more histidines are derived from one or more substitutions.
In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is a human heavy chain constant region gene. In some embodiments, the heavy chain constant region gene is of endogenous species origin. In some embodiments, the heavy chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the constant region gene is a mixture of human and non-human sequences. For example, in some embodiments, the constant region gene encodes a human CH1 region and a non-human (e.g., endogenous species source, mouse, rat) CH2 and/or CH3 region. In some embodiments, the heavy chain constant region gene is a cμ, cδ, cγ (cγl, cγ2, cγ3, cγ4), cα, or cε constant region gene. In some embodiments, the constant region gene is an endogenous constant region gene. In some embodiments, the constant region gene encodes a mutated CH1 region such that the non-human animal expresses heavy chain-only antibodies (see, e.g., U.S. patent No. 8,754,287, U.S. patent application publication No. 2015/0289489, each incorporated herein by reference in its entirety). In some embodiments, for example, where the goal is to generate heavy chains to make bispecific antibodies (e.g., in a universal or double light chain organism), the Fc domain of the heavy chain includes modifications that promote heavy chain heterodimer formation and/or inhibit heavy chain homodimer formation. Such modifications are provided, for example, in the following: U.S. Pat. nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228 and 8,679,785, and U.S. patent publication No. 2013/0195849, each of which are incorporated herein by reference in their entirety.
In some embodiments, the immunoglobulin constant region comprises a light chain constant region gene. In some embodiments, the light chain constant region gene is a kappa constant region gene. In some embodiments, the light chain constant region gene is a lambda constant region gene. In some embodiments, the light chain constant region gene is of endogenous species origin. In some embodiments, the light chain constant region gene is a mouse constant region gene or a rat constant region gene. In some embodiments, the light chain constant region gene is a mixture of human and non-human sequences.
In some embodiments, the immunoglobulin variable region comprising the human variable region gene segment and the immunoglobulin constant region gene to which the variable region gene segment is operably linked are located at an endogenous immunoglobulin locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous heavy chain locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous kappa locus. In some embodiments, the endogenous immunoglobulin locus is an endogenous lambda locus. In some embodiments, the constant region gene to which the human variable region gene segment is operably linked is an endogenous constant region gene.
In some embodiments, one or more endogenous immunoglobulin loci or a portion of one or more endogenous loci (e.g., variable and/or constant regions) in the genome of a non-human animal provided herein are inactivated. The endogenous immunoglobulin variable region gene locus and portions thereof may be inactivated using any method known in the art, including but not limited to deletion of the locus or a portion thereof from the genome of an organism, replacement of the locus or a portion thereof with a different nucleic acid sequence, inversion of a portion of the locus and/or movement of a portion of the locus to another location in the genome of a non-human organism. In some embodiments, inactivation of the locus is only partial. In some embodiments, the variable region of the locus is inactivated, but the constant region remains functional (e.g., because it is operably linked to a non-endogenous variable region gene segment).
In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin heavy chain locus. In some embodiments, the endogenous immunoglobulin heavy chain locus, or a portion thereof, is inactivated by deletion, substitution, translocation and/or inversion of at least a portion of the endogenous variable region of the endogenous heavy chain locus. In some embodiments, at least a portion of the variable region of the endogenous heavy chain locus that is deleted, substituted, shifted, and/or inverted comprises a J segment of the variable region. In some embodiments, the endogenous immunoglobulin heavy chain locus or portion thereof is inactivated by deletion, substitution, translocation and/or inversion of at least a portion of an endogenous constant region of the endogenous heavy chain locus. In some embodiments, at least a portion of the endogenous heavy chain locus of the deleted, substituted, shifted, and/or inverted constant region comprises the o μ gene of the endogenous constant region.
In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin kappa chain locus. In some embodiments, the endogenous immunoglobulin kappa chain locus or a portion thereof is inactivated by deletion, substitution, translocation and/or inversion of at least a portion of an endogenous variable region of the endogenous kappa chain locus. In some embodiments, at least a portion of the variable region of the endogenous kappa chain locus deleted, substituted, shifted and/or inverted comprises a J segment of the variable region. In some embodiments, the endogenous immunoglobulin kappa chain locus or a portion thereof is inactivated by deletion, substitution, translocation and/or inversion of at least a portion of an endogenous constant region of the endogenous kappa chain locus. In some embodiments, at least a portion of the constant region of the endogenous kappa chain locus deleted, substituted, shifted and/or inverted comprises a ck gene of the endogenous constant region.
In some embodiments, the genetically modified non-human animal comprises an inactivated endogenous immunoglobulin lambda chain locus. In some embodiments, the endogenous immunoglobulin lambda chain locus or a portion thereof is inactivated by deletion, substitution, translocation and/or inversion of at least a portion of the endogenous variable region of the endogenous lambda chain locus. In some embodiments, at least a portion of at least one V-J-C gene cluster in the endogenous lambda chain locus is deleted, substituted, shifted, and/or inverted. In some embodiments, the endogenous immunoglobulin lambda locus or a portion thereof is inactivated by deletion, substitution, translocation and/or inversion of at least a portion of the endogenous constant region of the endogenous lambda chain locus. In some embodiments, at least a portion of the constant region of the endogenous lambda chain locus deleted, substituted, shifted and/or inverted comprises the C gene of the endogenous constant region.
In various embodiments, the immunoglobulin locus modification does not affect fertility in a non-human animal. In some embodiments, the heavy chain locus comprises functionality, such as an endogenous ADAM6a gene, an ADAM6b gene, or both, and the genetic modification does not affect expression and/or function of the endogenous ADAM6a gene, ADAM6b gene, or both. In some embodiments, the genome of the genetically modified non-human animal further comprises functionality located ectopic, such as an endogenous ADAM6a gene, ADAM6b gene, or both. Exemplary non-human animals expressing exogenous ADAM6a and/or ADAM6b are described below: U.S. patent nos. 8,642,835 and 8,697,940, each of which is incorporated by reference herein in its entirety.
In some embodiments, the genetically modified non-human animal further comprises an exogenous terminal deoxynucleotidyl transferase (TdT) to increase antigen receptor diversity. An exemplary non-human animal expressing exogenous TdT is described in PCT publication WO 2017210586, which is hereby incorporated by reference in its entirety.
In some embodiments, the transcriptional control element comprises a RAG1 transcriptional control element, a RAG2 transcriptional control element, an immunoglobulin heavy chain transcriptional control element, an immunoglobulin k light chain transcriptional control element, an immunoglobulin l light chain transcriptional control element, or any combination thereof.
In some embodiments, the genome of the provided non-human animals further comprises one or more human immunoglobulin heavy and/or light chain genes (see, e.g., U.S. patent No. 8,502,018; U.S. patent No. 8,642,835; U.S. patent No. 8,697,940; U.S. patent No. 8,791,323; and U.S. patent application publication nos. 2013/0096287A1 and 2018/0125043 A1; and PCT publication No. WO2019/113065, each of which is incorporated herein by reference in its entirety). Alternatively, recombinant nucleic acid molecules comprising a modified Ig V segment as described herein can be introduced into a different modified strain, e.g.In embryonic stem cells of the strain (see, e.g., U.S. Pat. No. 8,502,018 or U.S. Pat. No. 8,642,835; general)Incorporated by reference in its entirety). In some embodiments, a non-human animal as described herein may be prepared by introducing a targeting vector as described herein into cells from a modified strain. As just one example, as described in U.S. patent nos. 8,642,835 and 8,697,940 (incorporated herein by reference in their entirety), a targeting vector as described herein can be introduced into a non-human animal that expresses an antibody having fully human variable regions and mouse constant regions. In some embodiments, the non-human animal as described herein is prepared to further include human immunoglobulin genes (variable and/or constant region genes). In some embodiments, a non-human animal as described herein comprises a modified Ig V segment as described herein and genetic material from a heterologous species (e.g., human), wherein the genetic material encodes, in whole or in part, one or more human heavy and/or light chain variable regions.
The non-human animals described herein may be prepared as described above or using methods known in the art to include additional human or humanized genes, generally depending on the intended use of the non-human animal. The genetic material of such additional human or humanized genes may be introduced by further altering the genome of cells (e.g., embryonic stem cells) having the above-described genetic modifications, or by other genetic modification lines as desired by culture techniques known in the art.
For example, as described herein, a non-human animal comprising a modified Ig V segment as described herein may further comprise (e.g., by cross breeding or multiple gene targeting strategies) one or more modifications, as described in the following: U.S. patent application publication nos. 2011-0195454A1, 2012-0021409A1, 2012-0192300A1, 2013-0045492A1, 2013-0185821A1, 2013-0198880A1, 2013-0302836A1, 2015-0059009 A1; international patent application publications WO 2011/097603, WO 2012/148873, WO 2013/134263, WO 2013/184761, WO 2014/160179, WO 2014/1601202; all of which are hereby incorporated by reference in their entirety.
Transgenic founder non-human animals can be identified based on the presence of modified Ig V segments in their genomes and/or the expression of anchor modified antibodies comprising amino acids corresponding to the receptor binding portion of the non-immunoglobulin polypeptide that binds to the cognate receptor. The transgenic founder non-human animal can then be used to breed additional non-human animals carrying the modified Ig V segments, thereby producing a series of non-human animals carrying one or more copies of the modified Ig V segments. In addition, transgenic non-human animals carrying modified Ig V segments can be further bred as desired with other transgenic non-human animals carrying other transgenes (e.g., human immunoglobulin genes).
Transgenic non-human animals can also be produced that contain a selected system that is capable of regulating or directing expression of the transgene. Exemplary systems include the Cre/loxP recombinase system of phage P1 (see, e.g., lakso, M. Et al, 1992, proc. Natl. Acad. Sci. USA 89:6232-6236, which is incorporated by reference in its entirety) and the FLP/Frt recombinase system of Saccharomyces cerevisiae (S. Cerevisiae) (O' Gorman, S. Et al, 1991, science 251:1351-1355, each of which is incorporated by reference in its entirety). Such animals can be provided by constructing a "double" transgenic animal, for example, by mating two transgenic animals, one containing a transgene comprising a selected modification (e.g., a modified Ig V segment), and the other containing a transgene encoding a recombinase (e.g., cre recombinase).
Although broadly discussed herein in mice (i.e., with modified human V H Section, D H And J H Mice of gene segments, all of which are operably linked to one or more murine heavy chain constant region genes), but other non-human animals including modified Ig v segments are also provided. Such non-human animals include any animal, including, for example, mammals, such as mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffalo), deer, sheep, goats, chickens, cats, and the like, that can be genetically modified to express the anchor-modified immunoglobulins described hereinDogs, ferrets, primates (e.g., marmosets, rhesus), and the like. For example, for those non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are employed to prepare non-human animals including genetic modifications. Such methods comprise, for example, modifying a non-ES cell genome (e.g., a fibroblast or induced pluripotent cell) and transferring the genetically modified genome to a suitable cell, such as an enucleated oocyte, using Somatic Cell Nuclear Transfer (SCNT), and seeding the modified cell (e.g., modified oocyte) in a non-human animal under conditions suitable for embryo formation.
Methods for modifying a non-human animal genome (e.g., porcine, bovine, rodent, chicken, etc.) include, for example, modifying the genome to include a modified Ig V segment as described herein using a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a Cas protein (i.e., CRISPR/Cas system). Guidance for methods for modifying the germline genome of a non-human animal can be found, for example, in the following: U.S. patent application publication Nos. 2015-0376628A1, 2016-0145646A1 and 2016-0177339 A1; incorporated herein by reference in its entirety.
In some embodiments, the non-human animals described herein are mammals. In some embodiments, the non-human animal as described herein is a small mammal, such as a small mammal of the general murine family (Dipodoidea) or the general murine family (muroiidea). In some embodiments, the genetically modified animal as described herein is a rodent. In some embodiments, the rodent as described herein is selected from a mouse, a rat, and a hamster. In some embodiments, the rodent as described herein is selected from the murine superfamily. In some embodiments, the genetically modified animal as described herein is from a family selected from the group consisting of: the species hamster (Calomyscidae) (e.g., hamster (mouse-like hamster)), the species hamster (Cricetidae) (e.g., hamster, new world rats and mice, voles), the species murine (Muridae) (true and rats, gerbil, spiny mice, coronaries), the species masomidae (Nesomyidae) (climbing, rock mice, caudal rats, motor gas rats and mice), the species spinosaidae (plaatacanthomidae) (e.g., spiny rod sleeping mice) and the species mole (spaxacidae) (e.g., mole, bamboo mice and zokors). In some embodiments, the genetically modified rodent as described herein is selected from a eukaryotic mouse or rat (murine), gerbil, acanthus, and coronal mouse. In some embodiments, the genetically modified mouse as described herein is a member from the murine family. In some embodiments, the non-human animal as described herein is a rodent. In some embodiments, the rodent as described herein is selected from a mouse and a rat. In some embodiments, the non-human animal as described herein is a mouse.
In some embodiments, the non-human animal as described herein is a rodent that is a mouse of a C57BL strain selected from the group consisting of: c57BL/A, C BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6J, C BL/6ByJ, C57BL/6NJ, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola. In some embodiments, the mice of the invention are 129 strains selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S 1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., festing et al, 1999, mammalian Genome (Mammalian Genome) 10:836; auerbach, W. Et al, 2000, biotechnology (Biotechnology) 29 (5): 1024-1028,1030,1032; incorporated herein by reference in its entirety). In some embodiments, the genetically modified mice as described herein are a mixed strain of the 129 strain described above and the C57BL/6 strain described above. In some embodiments, the mice as described herein are a mix of the 129 strain described above, or a mix of the BL/6 strain described above. In some embodiments, the 129 line of the hybrid line as described herein is a 129S6 (129/SvEvTac) line. In some embodiments, the mice as described herein are BALB strains, e.g., BALB/c strains. In some embodiments, the mouse as described herein is a mixed strain of a BALB strain and another of the foregoing strains.
In some embodiments, the non-human animal as described herein is a rat. In some embodiments, the rat as described herein is selected from the group consisting of a wista rat (Wistar rat), a LEA line, sildenafil Lei Pinji (Sprague Dawley strain), a Fischer line (Fischer strain), F344, F6, and black chinchilla (Dark Agouti). In some embodiments, the rat strain as described herein is a mixture of two or more strains selected from the group consisting of: wista, LEA, strengendole, fischer, F344, F6 and black mice.
Method for producing anchor modified immunoglobulins and anchor modified immunoglobulins
Several in vitro and in vivo techniques have been developed to produce antibody-based therapeutics. In particular, in vivo techniques are characterized by the production of transgenic animals (i.e., rodents) containing human immunoglobulin genes either randomly incorporated into the animal genome (see, e.g., U.S. Pat. No. 5,569,825, incorporated by reference in its entirety) or precisely placed at an endogenous immunoglobulin locus operably linked to an endogenous immunoglobulin constant region of the animal (see, e.g., U.S. Pat. Nos. 8,502,018; 8,642,835; 8,697,940; and 8,791,323, each of which is incorporated by reference in its entirety). Both of these methods have a success in producing promising antibody therapeutic candidates for use in humans. Further, both methods have advantages over in vitro methods in that the antibody candidate is selected from an in vivo generated antibody repertoire comprising affinity and specificity selections for the antigen within the internal environment of the host's immune system. In this way, antibodies bind to naturally occurring antigens (within the relevant biological epitopes and surfaces), rather than artificial environments or computer chip predictions that can accompany in vitro techniques. Although robust antibody libraries have been generated by in vivo techniques, antibodies to complex (e.g., viruses, channel polypeptides) or cytoplasmic antigens remain difficult. Further, the generation of antibodies to polypeptides sharing a high degree of sequence identity between species (e.g., human and mouse) remains a challenge due to immune tolerance.
The invention is therefore based on the recognition, inter alia, that in vivo systems are characterized by the generation of antibodies with anchors to help immobilize immunoglobulins to an antigen of interest (e.g., an anchor's cognate receptor) and to increase their affinity for the antigen, to promote the affinity of the molecule by its own affinity for its cognate receptor and/or to allow for somatic hypermutation of a larger immunoglobulin pool capable of recognizing the cognate receptor.
The provided non-human animals can be used to prepare human antibodies, wherein the human antibodies comprise variable domains derived from one or more variable region nucleic acid sequences encoded by genetic material of cells of the non-human animals as described herein. For example, a provided non-human animal is immunized with an antigen of interest (e.g., a receptor homologous to an anchor) under conditions and for a time sufficient for the non-human animal to mount an immune response to the antigen of interest. Antibodies are isolated from a non-human animal (or one or more cells, e.g., one or more B cells) and characterized using various assays that measure, for example, affinity, specificity, epitope mapping, ability to block ligand-receptor interactions, inhibitory receptor activation, and the like. In some embodiments, antibodies produced by a provided non-human animal include one or more human variable domains derived from one or more human variable region nucleotide sequences isolated from the non-human animal.
The non-human animals as described herein provide an improved in vivo system and source of biological material (e.g., cells) for the production of human antibodies that can be used in a variety of assays. In some embodiments, the provided non-human animals are used to develop a therapeutic agent that targets one or more receptors of a ligand receptor pair, as described herein. In some embodiments, provided non-human animals are used to identify, screen, and/or develop candidate therapeutic agents (e.g., antibodies, etc.) that bind to one or more G protein-coupled receptor (GPCR) polypeptides. In some embodiments, provided non-human animals are used to screen and develop candidate therapeutic agents (e.g., antibodies, etc.) that block the activity of one or more receptor tyrosine kinases, one or more human GPCR polypeptides, one or more Notch receptors, one or more of CD28, CTLA4, and PD1, plexin receptors, LDLR, LILR, KIR, and integrins or NPRs (e.g., NPR 3). In some embodiments, provided non-human animals are used to determine the binding profile of one or more human GPCR polypeptides, one or more Notch receptors, one or more of CD28, CTLA4, and PD1, plexin receptor, LDLR, LILR, KIR, and antagonists and/or agonists of integrin or NPR (e.g., NPR 3). In some embodiments, provided are non-human animals for determining one or more epitopes of one or more candidate therapeutic antibodies with one or more human GPCR polypeptides, one or more Notch receptors, CD28, CTLA4, and PD1, plexin receptors, LDLR, LILR, KIR, and integrins or NPRs (e.g., NPR 3).
In some embodiments, a non-human animal is provided for determining a pharmacokinetic profile of an antibody. In some embodiments, the one or more provided non-human animals and the one or more control or reference non-human animals are each exposed to a different dose (e.g., 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 7.5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 40mg/kg, or 50mg/kg or more) of one or more candidate therapeutic antibodies. Candidate therapeutic antibodies can be administered via a variety of desired routes of administration, including parenteral and non-parenteral routes of administration. Parenteral routes include, for example, intravenous, intra-arterial, portal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intraventricular, intracranial, intrapleural, or other injection routes. Parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, ocular. Administration may also be by continuous infusion, topical administration, sustained release from implants (gels, films, etc.), and/or intravenous injection, for example using intravenous fluid bags. Blood was isolated from non-human animals (humanized and controls) at multiple time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more). A variety of assays may be performed using samples obtained from non-human animals as described herein to determine pharmacokinetic properties of the administered candidate therapeutic antibodies, including but not limited to total IgG, anti-therapeutic antibody responses, agglutination, and the like.
In some embodiments, the provided non-human animals express antibodies, and thus cells, cell lines, and cell cultures can be produced for use as a source of antibodies for use in binding and functional assays, e.g., assaying for binding or function of an antagonist or agonist, particularly when the antagonist or agonist is specific for a human polypeptide sequence or epitope, or alternatively for a human polypeptide sequence or epitope that is functional in ligand-receptor interactions (binding). In some embodiments, the epitope bound by the candidate therapeutic antibody or siRNA can be determined using cells isolated from the provided non-human animals.
In some embodiments, cells from the provided non-human animals may be isolated and used on a specific basis, or may be maintained in culture for a number of generations. In some embodiments, cells from the provided non-human animals are immortalized (e.g., by use of viruses) and maintained in culture indefinitely (e.g., in continuous culture).
In some embodiments, a non-human animal as described herein provides an in vivo system for producing antibody variants that bind to a human target antigen. Such variants comprise antibodies having desirable functionality, specificity, low cross-reactivity to a common epitope shared by two or more human target antigens. In some embodiments, the provided non-human animals are used to generate an antibody stack to generate a range of antibody variants that are screened for desired or improved function.
In some embodiments, a non-human animal as described herein provides an in vivo system for generating an antibody library. Such libraries provide sources of heavy and light chain variable region sequences that can be grafted onto different Fc regions based on desired effector functions and/or used as a source of affinity maturation of variable region sequences using techniques known in the art (e.g., site-directed mutagenesis, error-prone PCR, etc.).
Kit for detecting a substance in a sample
The invention further provides a package or kit comprising one or more containers containing at least one non-human animal, non-human cell, DNA fragment and/or targeting vector described herein. The kit may be used in any suitable method (e.g., research methods). Optionally associated with one or more such containers may be a notice in the form prescribed by a government agency that prescribes manufacture, use or sale of a pharmaceutical or biological product, the notice reflecting (a) agency approval for manufacture, use or sale for human administration, (b) instructions for use or both, or a contract to manage transfer of materials and/or biological products (e.g., non-human animals or non-human cells as described herein) between two or more entities.
Non-limiting examples are described below:
example 1A recombinant nucleic acid molecule comprising a modified immunoglobulin (Ig) variable (V) segment encoding an anchor modified Ig polypeptide,
wherein the modified Ig V segment comprises a nucleic acid sequence encoding an anchor between the nucleic acid sequence encoding the Ig signal peptide and the nucleic acid sequences encoding the Framework Regions (FRs) 1, complementarity Determining Regions (CDRs) 1, FR2, CDR2, FR3 and CDR3 of the germline Ig V segment or variant thereof,
wherein the anchor modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) Said FR1, CDR1, FR2, CDR2, FR3 and CDR3 of said germline Ig V segment or variant thereof,
wherein the anchor comprises a receptor binding portion of a non-immunoglobulin polypeptide of interest that binds to a cognate receptor, and
optionally wherein the nucleic acid molecule lacks any other V segment.
Example 2. The recombinant nucleic acid molecule of example 1, wherein the Ig signal peptide is an Ig signal peptide of the germline Ig V segment or variant thereof.
Example 3 the recombinant nucleic acid molecule according to example 1 or example 2, wherein the germline Ig V segment or variant thereof is a germline Ig heavy chain variable (V H ) Segments or variants thereof such that
The modified Ig V segment is a modified Ig V comprising the nucleic acid sequence encoding an anchor H A segment comprising said anchor in said nucleic acid sequence encoding an Ig signal peptide and encoding said germline IgV H Between the nucleic acid sequences of the Framework Regions (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3 and CDR3 of the segments or variants thereof, and
the anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) The germline Ig V H The FR1, CDR1, FR2, CDR2, FR3 and CDR3 of the stretch or variant thereof.
Example 4 the recombinant nucleic acid molecule of any one of examples 1 to 3, wherein the germline Ig V segment or variant thereof is germline human (h) V H 1-2 segment, germ line hV H 1-3 segment, germ line hV H 1-8 segment, germ line hV H 1-18 segment, germ line hV H 1-24 segment, germ line hV H 1-45 segment, germ line hV H 1-46 segment, germ line hV H 1-58 segment, germ line hV H Segment 1-69, germ line hV H 2-5 segment, germ line hV H 2-26 segment, germ line hV H 2-70 segment, germ line hV H 3-7 segment, germ line hV H 3-9 segment, germ line hV H 3-11 segment, germ line hV H 3-13 segment, germ line hV H 3-15 segment, germ line hV H 3-16 segment, germ line hV H 3-20 segment, germ line hV H 3-21 segment, germ line hV H 3-23 segment, germ line hV H 3-30 segment, germ line hV H 3-30-3 segment, germ line hV H 3-30-5 segment, germ line hV H 3-33 segment, germ line hV H 3-35 segment, germ line hV H 3-38 segment, germ line hV H 3-43 segment, germ line hV H 3-48 segment, germ line hV H 3-49 segments, germ line hV H 3-53 segment, germ line hV H 3-64 segment, germ line hV H 3-66 segment, germ line hV H 3-72 segment, germ line hV H 3-73 segment, germ line hV H 3-74 segments, speciesIs hV line H 4-4 segment, germ line hV H 4-28 segment, germ line hV H 4-30-1 segment, germ line hV H 4-30-2 segment, germ line hV H 4-30-4 segment, germ line hV H 4-31 segment, germ line hV H 4-34 segment, germ line hV H 4-39 segment, germ line hV H 4-59 segment, germ line hV H 4-61 segment, germ line hV H 5-51 segment, germ line hV H 6-1 segment, germ line hV H 7-4-1 segment, germ line hV H 7-81 segments or variants thereof.
Example 5 the recombinant nucleic acid molecule of any one of examples 1 to 4, wherein the germline Ig V segment or variant thereof is germline hV H 1-69 or a variant thereof, optionally wherein said Ig signal peptide comprises sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7).
Embodiment 6. The recombinant nucleic acid molecule of any one of embodiments 3 to 5, comprising, operably linked and from 5 'to 3':
(I) The modified Ig V H A section;
(II) one or more Ig heavy chain diversity (D H ) A section; and
(III) one or more Ig heavy chain conjugation (J H ) A section.
Example 7. The recombinant nucleic acid molecule of example 6, wherein
The one or more Ig D of (II) H Segments comprising one, more or all human Ig D H Segments, and/or
The one or more Ig J's of (III) H Segments comprising one, more or all human Ig J H A section.
Example 8 the recombinant nucleic acid molecule according to example 6 or example 7, wherein the one or more Ig D of (II) H Segment and one or more Ig J of (III) H Gene segments are recombined and form rearranged Ig D H /J H A sequence such that the recombinant nucleic acid molecule comprises, operably linked and from 5 'to 3':
modified Ig V H A gene segment; and
the rearranged Ig D H /J H Sequence.
Example 9 the recombinant nucleic acid molecule of example 8, wherein the modified IgV H Gene segments and the rearranged Ig D H /J H The sequences are recombined and form rearranged Ig V encoding an anchor modified Ig heavy chain variable domain H /D H /J H The sequence of the sequences is set up,
wherein the anchor modified Ig heavy chain variable domain comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) IgV rearranged by the rearrangement H /D H /J H Sequence-encoded FR1, complementarity Determining Regions (CDRs) 1, FR2, CDR2, FR3, CDR3 and FR4.
Example 10 the recombinant nucleic acid molecule of any one of examples 3 to 8, wherein the modified IgV H The segments are modified Ig Vs that are not rearranged H A gene segment.
Example 11 the recombinant nucleic acid molecule of any one of examples 6 to 10, further comprising a nucleic acid encoding an Ig heavy chain constant region (C H ) Is a nucleic acid sequence of (a),
wherein said IgC is encoded H Downstream of and operably linked to the nucleic acid sequence of:
(I) The modified Ig V H A section;
(II) the one or more Ig D H A section; and
(III) the one or more Ig J H A section.
Example 12 the recombinant nucleic acid molecule according to example 11, wherein said Ig C-encoding H Including Igmu gene encoding IgM isotype, igdelta gene encoding IgD isotype, iggamma gene encoding IgG isotype, igalpha gene encoding IgA isotype and/or Igepsilon gene encoding IgE isotype.
Embodiment 13. The recombinant nucleic acid molecule of any one of embodiments 3 to 12, comprising a nucleic acid sequence encoding an anchor modified Ig heavy chain, wherein the anchor modified Ig heavy chain comprises operably linked:
(i) The Ig signal peptide;
(ii) The anchor;
(iii) Comprising Ig V rearranged H /D H /J H Ig heavy chain variable domains of said FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 encoded by sequences; and
(iv)Ig C H 。
embodiment 14. The recombinant nucleic acid molecule of any one of embodiments 11 to 13, wherein the Ig C H Non-human Ig C H 。
Example 15 the recombinant nucleic acid molecule of example 14, wherein the non-human Ig C H Is rodent Ig C H 。
Example 16 the recombinant nucleic acid molecule of example 15, wherein the non-human Ig C H Is rat Ig C H 。
Example 17 the recombinant nucleic acid molecule of example 15, wherein the non-human Ig C H Is mouse Ig C H 。
Example 18 the recombinant nucleic acid molecule according to example 1 or example 2, wherein the germline Ig V gene segment or variant thereof is a germline Ig light chain variable (V L ) Segments or variants thereof such that
The modified Ig V segment is a modified Ig V comprising the nucleic acid sequence encoding an anchor L A segment comprising said anchor in said nucleic acid sequence encoding an Ig signal peptide and encoding said germline IgV L Between the nucleic acid sequences of the Framework Regions (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3 and CDR3 of the segments or variants thereof, and
The anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) The germline Ig V L Said segment or variant thereofFR1, CDR1, FR2, CDR2, FR3 and CDR3.
Embodiment 19 the recombinant nucleic acid molecule of embodiment 18 comprising, operably linked and from 5 'to 3':
(I) The modified Ig V L A section; and
(II) one or more Ig light chain engagements (J L ) A section.
Example 20 the recombinant nucleic acid molecule of example 19, wherein the modified IgV L Segments and the one or more Ig J L The segments are recombined and form rearranged Ig V encoding an anchor modified Ig light chain variable domain L /J L The sequence of the sequences is set up,
wherein the anchor modified Ig light chain variable domain comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) IgV rearranged by the rearrangement L /J L Sequence-encoded FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
Example 21 the recombinant nucleic acid molecule of example 19 or example 20 further comprising a nucleic acid sequence encoding an Ig light chain constant region (C L ) Is a nucleic acid sequence of (a),
wherein said IgC is encoded L Downstream of and operably linked to the nucleic acid sequence of:
(I) The modified Ig V L A section; and
(II) the one or more Ig light chain engagements (J L ) A section.
Embodiment 22. The recombinant nucleic acid molecule of any one of embodiments 18 to 21, comprising a nucleic acid sequence encoding an anchor modified Ig light chain, wherein the anchor modified Ig light chain comprises operably linked:
(i) The Ig signal peptide;
(ii) The anchor;
(iii) Comprising Ig V rearranged L /J L Sequence-encoded said FR1, CDR1,Ig light chain variable domains of FR2, CDR2, FR3, CDR3, and FR 4; and
(iv)Ig C L 。
example 23 the recombinant nucleic acid molecule according to example 21 or example 22, wherein the IgC L Non-human Ig C L 。
Example 24 the recombinant nucleic acid molecule of example 23, wherein the non-human Ig C L Is rodent Ig C L 。
Example 25 the recombinant nucleic acid molecule of example 23, wherein the non-human Ig C L Is rat Ig C L 。
Example 26 the recombinant nucleic acid molecule of example 23, wherein the non-human Ig C L Is mouse Ig C L 。
Embodiment 27. The recombinant nucleic acid molecule of any one of embodiments 18 to 26, wherein the germline Ig V L The segment or variant thereof is a germline Ig light chain variable kappa (V kappa) segment or variant thereof such that
The modified Ig V segment is a modified Ig V kappa segment comprising the nucleic acid sequence encoding an anchor between the nucleic acid sequence encoding an Ig signal peptide and a nucleic acid sequence encoding a Framework Region (FR) 1, a Complementarity Determining Region (CDR) 1, FR2, CDR2, FR3 and CDR3 of the germline Ig V kappa segment or variant thereof, and
the anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) Said FR1, CDR1, FR2, CDR2, FR3 and CDR3 of said germline Ig vκ segment or variant thereof.
Embodiment 28. The recombinant nucleic acid molecule of embodiment 27, comprising the following operably linked and from 5 'to 3':
(I) The modified Ig vκ segment; and
(II) one or more Ig light chain engagement kappa (jk) segments.
Example 29 the recombinant nucleic acid molecule of example 28, further comprising a nucleic acid sequence encoding an Ig light chain constant kappa region (Ckappa),
wherein the nucleic acid sequence encoding Ig ck is located downstream of and operably linked to:
(I) The modified Ig vκ segment; and
(II) the one or more Ig jκ segments.
Embodiment 30. The recombinant nucleic acid molecule of any one of embodiments 18 to 26, wherein the germline Ig V L The segment or variant thereof is a germline Ig light chain variable lambda (V lambda) segment or variant thereof such that
The modified Ig V segment is a modified Ig V lambda segment comprising the nucleic acid sequence encoding an anchor between the nucleic acid sequence encoding an Ig signal peptide and a nucleic acid sequence encoding a Framework Region (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3 and CDR3 of the germline Ig V lambda segment or variant thereof, and
the anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) Said FR1, CDR1, FR2, CDR2, FR3 and CDR3 of said germline Ig V lambda segment or variant thereof.
Embodiment 31. The recombinant nucleic acid molecule of embodiment 30, comprising the following operably linked and from 5 'to 3':
(I) The modified Ig vλ segment; and
(II) one or more Ig light chains engages a lambda (jlambda) segment.
Example 32 the recombinant nucleic acid molecule of example 31, further comprising a nucleic acid sequence encoding an Ig light chain constant lambda region (C lambda),
wherein the nucleic acid sequence encoding Ig C lambda is located downstream of and operably linked to:
(I) The modified Ig vλ segment; and
(II) the one or more Ig jλ segments.
Embodiment 33. The recombinant nucleic acid molecule of any one of embodiments 1 to 32, wherein the anchor comprises a linker that connects the receptor binding portion of the non-immunoglobulin polypeptide of interest to the FR1, CDR1, FR2, CDR2, FR3, and CDR3 of the germline Ig V segment or variant thereof.
Embodiment 34. The recombinant acid molecule of embodiment 33, wherein the linker comprises the sequence GGGGS (SEQ ID NO: 5).
Embodiment 35. The recombinant nucleic acid molecule of any one of embodiments 1 to 34, wherein the anchor comprises a Natriuretic Peptide Receptor (NPR) binding portion of a Natriuretic Peptide (NP).
Embodiment 36. The recombinant nucleic acid molecule of embodiment 35, wherein the NPR-binding portion of the NP comprises a C-terminal tail of the NP.
Example 37. The recombinant nucleic acid molecule of example 35 or example 36, wherein the NP is Atrial Natriuretic Peptide (ANP).
Embodiment 38. The recombinant nucleic acid molecule of any one of embodiments 1 to 37, wherein the anchor comprises the sequence NSFRY (SEQ ID NO: 3).
Embodiment 39. The recombinant nucleic acid molecule of any one of embodiments 1 to 38, comprising a sequence selected from the group consisting of: a sequence shown as SEQ ID NO. 8 or a degenerate variant thereof, a sequence shown as SEQ ID NO. 10 or a degenerate variant thereof, SEQ ID NO. 11 of a degenerate variant thereof, and SEQ ID NO. 12 or a degenerate variant thereof.
Embodiment 40. A targeting vector comprising the recombinant nucleic acid molecule of any one of embodiments 1 to 10 and 33 to 39, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig heavy chain locus such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a non-human Ig C located at the non-human Ig heavy chain locus H A recombinant nucleic acid molecule upstream of and operably linked to, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus, and/or wherein the non-human Ig heavy chain locus comprises a human or humanizedImmunoglobulin heavy chain variable region, endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof.
Embodiment 41. The targeting vector of embodiment 40, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces the non-human V at the non-human Ig heavy chain locus H A section.
Embodiment 42. The targeting vector of embodiment 40 or embodiment 41, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain loci, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain loci H Segment, all non-human D H Segment and all non-human J H A section.
Embodiment 43. The targeting vector according to any of the embodiments 40 to 42, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain loci, the recombinant nucleic acid molecule replaces all non-human V at the non-human Ig heavy chain loci except one H Segment or all non-human V H Segment, all non-human D H Segment and all non-human J H A section.
Embodiment 44. The targeting vector of any one of embodiments 40 to 43, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus.
Embodiment 45. The targeting vector according to any of the embodiments 40 to 44, wherein the 5 'homology arm comprises the sequence shown as SEQ ID NO. 11 and/or the 3' homology arm comprises the sequence shown as SEQ ID NO. 12.
Embodiment 46. A targeting vector comprising the recombinant nucleic acid molecule according to any one of embodiments 1 to 17 and 33 to 39, wherein the targeting vector further comprises 5 'and 3' homology arms targeting a non-human Ig heavy chain locus such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, The targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus, and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, an endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof.
Embodiment 47. The targeting vector of embodiment 46, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain loci, the recombinant nucleic acid molecules displace one or more non-human V at the non-human Ig heavy chain loci H Segment, all non-human D H Gene segment, all non-human J H Gene segment and one or more non-human C H And (3) a gene.
Embodiment 48. A targeting vector comprising the recombinant nucleic acid molecule according to any one of embodiments 1 to 2, 18 to 20, 27 to 28, 30 to 31 and 33 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms targeting a non-human Ig light chain locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a non-human Ig C located at the non-human Ig light chain locus L A recombinant nucleic acid molecule upstream of and operably linked to, optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, an endogenous Ig V L And/or J L Deletion of a gene segment or a combination thereof.
Example 49 the targeting vector of example 48, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L A section.
Embodiment 50. The targeting vector of embodiment 48 or embodiment 49, wherein the homology between the targeting vector and the non-human Ig light chain locus is heavyIn group, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig light chain locus L Segment and all non-human J L A section.
Embodiment 51 the targeting vector of any one of embodiments 48 to 50, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces all non-human V at the non-human Ig light chain locus L Segment and all non-human J H A section.
Embodiment 52. The targeting vector of any one of embodiments 48 to 51, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus.
Example 53A targeting vector comprising the recombinant nucleic acid molecule according to any one of examples 1 to 2, 18 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms targeting a non-human Ig light chain locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, an endogenous IgV L And/or J L Deletion of a gene segment or a combination thereof.
Embodiment 54 the targeting vector of embodiment 53, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces a non-human V at the non-human Ig light chain locus L Segment, all non-human J L Gene segment and non-human C L And (3) a gene.
Embodiment 55. A targeting vector comprising the recombinant nucleic acid molecule according to any one of embodiments 1 to 2, 18 to 20, 27 to 28 and 33 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain kappa locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule upstream of and operably linked to a non-human Ig ck at the non-human Ig light chain kappa locus, optionally wherein the non-human Ig light chain kappa locus is an endogenous rodent light chain kappa locus, and/or wherein the non-human Ig light chain kappa locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vkappa and/or J kappa gene segments, or a combination thereof.
Embodiment 56. The targeting vector of embodiment 55, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces a non-human V kappa segment at the non-human Ig light chain kappa locus.
Embodiment 57. The targeting vector of embodiment 55 or embodiment 56, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces one or more non-human V kappa segments and all non-human J kappa segments at the non-human Ig light chain kappa locus.
Embodiment 58 the targeting vector of any one of embodiments 55 to 57, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces all non-human vk segments and all non-human jk segments at the non-human Ig light chain kappa locus.
Embodiment 59. The targeting vector of any one of embodiments 55 to 58, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus.
Embodiment 60. A targeting vector comprising the recombinant nucleic acid molecule according to any one of embodiments 1 to 2 and 18 to 29, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain kappa locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus.
Embodiment 61. The targeting vector of embodiment 60, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces the non-human V kappa segment, all non-human J kappa gene segments and non-human C kappa genes at the non-human Ig light chain kappa locus.
Embodiment 62. A targeting vector comprising the recombinant nucleic acid molecule according to any one of embodiments 1 to 2, 18 to 20, 30 to 31 and 33 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule upstream of and operably linked to a non-human Ig clambda at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain lambda locus is an endogenous rodent light chain lambda locus, and/or wherein the non-human Ig light chain lambda locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vlambda and/or J gene segments, or a combination thereof.
Embodiment 63. The targeting vector of embodiment 62, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces a non-human V lambda segment at the non-human Ig light chain lambda locus.
Embodiment 64. The targeting vector of embodiment 62 or embodiment 63, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces one or more non-human V lambda segments and all non-human J lambda segments at the non-human Ig light chain locus.
Embodiment 65. The targeting vector of any one of embodiments 62 to 64, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces all non-human V lambda segments and all non-human J lambda segments at the non-human Ig light chain lambda locus.
Embodiment 66. The targeting vector of any one of embodiments 62 to 65, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus.
Embodiment 67. A targeting vector comprising the recombinant nucleic acid molecule according to any one of embodiments 1 to 2, 18 to 26 and 30 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus.
Embodiment 68. The targeting vector of embodiment 67, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces the non-human V lambda segment, all non-human J lambda gene segments, and non-human C lambda genes at the non-human Ig light chain lambda locus.
Embodiment 69. A non-human animal genome comprising the recombinant nucleic acid molecule of any one of embodiments 1-39 or the targeting vector of any one of embodiments 40-68, optionally wherein the non-human animal is a rodent, optionally wherein the rodent is a rat or mouse.
Embodiment 70. The non-human animal genome of embodiment 69, wherein the recombinant nucleic acid is located at an endogenous Ig locus of the non-human animal genome.
Embodiment 71. A non-human animal or non-human animal cell comprising the recombinant nucleic acid molecule of any one of embodiments 1 to 39, the targeting vector of any one of embodiments 40 to 68, or the non-human animal genome of embodiment 69 or embodiment 70.
Embodiment 72. The non-human animal or non-human animal cell of embodiment 71 wherein the recombinant nucleic acid molecule, the targeting vector, or the non-human animal genome is in the germline of the non-human animal or non-human animal cell.
Example 73 an in vitro method of modifying an isolated cell, the method comprising introducing the recombinant nucleic acid molecule of any one of examples 1 to 39 into the isolated cell.
Embodiment 74. The in vitro method according to embodiment 73, wherein introducing comprises contacting the cells with the targeting vector according to any one of embodiments 40 to 68.
Embodiment 75. The in vitro method of embodiment 73 or embodiment 74 wherein the cell is a host cell.
Embodiment 76. The in vitro method of embodiment 73 or embodiment 74 wherein the cells are Embryonic Stem (ES) cells.
Embodiment 77 the in vitro method of any one of embodiments 73 to 76, wherein the cell is a rodent cell, optionally wherein the rodent cell is a rat cell or a mouse cell.
Example 78. A non-human animal embryo produced from the embryonic stem cell of example 76.
Example 79. A non-human animal produced from the embryonic stem cell of example 76.
Example 80. A method of making a non-human animal comprising implanting the ES cell of example 76 or an embryo comprising the ES cell into a suitable host and maintaining the host under suitable conditions during development of the ES cell or embryo into viable offspring.
Embodiment 81 the non-human animal of any one of embodiments 71-72 and 79 or the non-human animal prepared according to the method of embodiment 80, wherein the non-human animal comprises, as compared to a control non-human animal:
(a) A significant number of mature B cells in the spleen;
(b) A significant number of kappa positive B cells in the spleen;
(c) A significant number of lambda positive B-cells in the spleen;
(d) Serum IgG at comparable levels; and/or
(e) Serum IgM at comparable levels.
Embodiment 82. The non-human animal of any one of embodiments 71-72, 79 and 81 or the non-human animal prepared according to the method of embodiment 80, wherein the non-human animal is capable of producing an immune response comparable to a control non-human animal.
Embodiment 83 the non-human animal of any one of embodiments 71-72, 79 and 81-82 or the non-human animal prepared according to the method of embodiment 80, comprising a plurality of antigen binding proteins each comprising the anchor modified Ig polypeptide, optionally:
wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide,
wherein the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
Embodiment 84. The non-human animal of any one of embodiments 71-72, 79 and 81-83 or the non-human animal prepared according to the method of embodiment 80, wherein the non-human animal further comprises the cognate receptor for the non-immunoglobulin polypeptide of interest.
Example 85 the non-human animal of any one of examples 71-72, 79 and 81-84 or the non-human animal prepared according to the method of example 80, comprising a plurality of antigen binding proteins each comprising the anchor modified Ig polypeptide and specifically binding to the cognate receptor of the non-immunoglobulin polypeptide of interest, optionally:
wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide,
wherein the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
Embodiment 86 the non-human animal of any one of embodiments 84-85, wherein the cognate receptor is a Natriuretic Peptide Receptor (NPR).
Embodiment 87. The non-human animal of any of embodiments 84-86, wherein each of the plurality of antigen binding proteins comprises less than 1 x 10 9 And/or t1/2 over 30 minutes.
Embodiment 88 the non-human animal of any one of embodiments 84-87, wherein at least 15% of the plurality of antigen binding proteins block binding of the cognate receptor to the non-immunoglobulin polypeptide of interest.
Embodiment 89 the non-human animal of any of embodiments 84-88, wherein greater than 50% of the plurality of antigen binding proteins bind to the cognate receptor expressed on the cell surface.
Embodiment 90 the non-human animal of any one of embodiments 71-72, 79 and 81-89 or the non-human animal prepared according to the method of embodiment 80, wherein the non-human animal is a rodent, optionally wherein the rodent is a rat or mouse.
Example 91A method of producing an antigen binding protein or obtaining a nucleic acid encoding the antigen binding protein, the method comprising:
immunizing a non-human animal according to any one of examples 71-72, 79 and 81-90 or a non-human animal prepared according to the method of example 80 with an antigen;
Allowing the non-human animal to produce an antigen binding protein comprising the anchor modified Ig polypeptide or a nucleic acid encoding the antigen binding protein, which antigen binding protein binds to the antigen, optionally:
wherein the mass of said antigen binding protein confirms the presence of said anchor modified Ig polypeptide,
wherein the mass of the antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of the antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of the antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
Embodiment 92. The method of embodiment 91 further comprising recovering the antigen binding protein or nucleic acid encoding the antigen binding protein from the non-human animal or non-human animal cell.
Embodiment 93. The method of embodiment 92 wherein the non-human animal cell is a B cell or a hybridoma.
Example 94. A non-human animal cell recovered according to the method of example 91.
Embodiment 95. The non-human animal of embodiment 94, wherein the non-human animal cell is a B cell.
Embodiment 96. The non-human animal cell of embodiment 94 or 95, wherein the B cell is a mouse B cell.
Embodiment 97. A hybridoma cell comprising the non-human animal cell of any one of embodiments 94-95 fused to a myeloma cell.
Embodiment 98. An anchor modified Ig polypeptide expressed from the recombinant nucleic acid molecule of any one of embodiments 1 to 39, the targeting vector of any one of embodiments 40 to 68, the non-human animal genome of any one of embodiments 69 to 70, from the non-human animal or non-human animal cell of any one of embodiments 71 to 72 and 81 to 90, from the non-human animal or non-human animal cell prepared according to the method of any one of embodiments 73 to 76 and 80, or from the method of any one of embodiments 91 to 93, optionally:
wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide,
wherein the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
Example 99 the anchor modified Ig polypeptide of example 98, comprising the amino acid sequence depicted as SEQ ID NO. 3 at its N-terminus.
Other features of the embodiments will become apparent from the course of the following description of exemplary embodiments, which are given for illustration and are not intended to be limiting thereof.
Examples
The following examples are provided to describe how to make and use the methods and compositions disclosed herein to those of ordinary skill in the art and are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, temperatures are stated in degrees celsius and pressures are at or near atmospheric.
EXAMPLE 1 construction of immunoglobulin variable region comprising ANP modified immunoglobulin variable region Gene segments
This non-limiting example illustrates the construction of targeting vectors for integration of anchor modified immunoglobulin (Ig) variable region (V) gene segments into immunoglobulin variable regions of immunoglobulin loci. As described below, the coding sequence of the ANP C-terminal tail is operably linked to an Ig V gene segment, which may be operably linked to an Ig-joining (J) gene segment and an appropriate Ig-diversity (D) gene segment, such that upon V (D) J recombination, an antibody comprising the ANP C-terminal tail at the N-terminus of the immunoglobulin polypeptide chain is expressed.
V containing ANP modifications for insertion of immunoglobulin heavy chain variable regions were created using the following technique H Targeting vector for gene segments:techniques (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al, 2003, nature Biotechnology 21 (6): 652-659; incorporated herein by reference) and molecular biology techniques known in the art. Non-limiting exemplary strategies for constructing targeting vectors using sequences encoding the C-terminal tail of ANP are depicted in fig. 1-2.
Preparation of germline V by de novo DNA synthesis involving insertion of the germline V by a sequence encoding a peptide linker H 1-69 gene segment (Blue Heron Biotech, botull, WA) of the donor DNA fragment at the C-terminal tail of ANP (blue aigrette biotechnology company, bosell, washington). FIG. 1 shows "p466090" ANP-V H 1-69 Cas9 donor fragment. The spectinomycin resistance cassette "SPEC" was ligated to the EcoRI and avril sites of donor plasmid p46609 to prepare plasmid p46685 (fig. 1). Shown in FIG. 2 is a more detailed illustration of the resulting donor plasmid p 46609.
The donor fragment was used to modify BAC clone VI504 (MAID 6211). See fig. 2.BAC clone VI504 comprises from 5' to 3' a 5' mouse homology arm of about 20kb, an I-CeuI-AscI fragment of about 15kb containing mouse Adam6a gene "a", the Frt-Ub-Hyg-Frt cassette and mouse Adam6b gene "b", containing germline human V H About 9kb AscI-AsiSI fragment of 1-69 gene containing human D H And person J H An about 60kb fragment of the gene, and an about 8kb 3' mouse homology arm containing the mouse IgH intron enhancer (filled in oval), igM switch region and part of the mouse IgM gene (FIG. 3).
In step 1, human germline V is used and cut H The Cas9 complexed with a mixture of two grnas 5 'and 3' of the 1-69 gene digested VI504 in vitro. The resulting 3 'and 5' ends overlap with the p46685 end by 60 bp. The XhoI fragment of p46685 was then assembled with VI504 (MAID 6211) by Gibson assembly to prepare VI738. In step 2, VI738 was digested with MreI to remove the spectinomycin-resistant cassette. BAC was then repaired by conjugate oligonucleotide mediated Gibson assembly, leaving a seamless join (Δmrei) to make the final LTVEC VI748 (MAID 6833). LTVEC is identical to VI504 except that the ANP-G4S codon is inserted in VI 748.
Throughout the construction of the targeting vector using the primers described in table 1, correct assembly of the described donor fragments and V modified with NPs as described herein was confirmed by sequencing and polymerase chain reaction H 1-69 Gene segment targeting substitution BAC clone VI504 Germline (GL) V H 1-69 gene segments.
TABLE 1
Example 2V comprising NP modification H Generation of rodents of gene segments
This example demonstrates the production of a non-human animal (e.g., rodent) whose genome comprises an immunoglobulin heavy chain variable region comprising an NP-modified V H Gene segments, e.g. V comprising the C-terminal tail of ANP H A gene segment.
Linearizing and electroporating a VI748 targeting vector into mouse embryonic stem cells having a homozygous genome for the endogenous Ig heavy chain variable region locus, including except for the majority of 5' v H All endogenous V outside the 1-86 segment (1115 KO) H 、D H And J H Deletion of segments, and endogenous Ig light chain variable region kappa loci, which include replacement of all endogenous vk and jk segments with complete libraries of human vk and jk segments. The Ig heavy chain loci (with 50% Balb, 25% C57BL/6, 25% 129 background) of the ES cells used for electroporation of each targeting vector are depicted in fig. 4. Following electroporation, the electroporated cells are cultured in selection medium. Drug resistant colonies were picked 10 days after electroporation and passed through TAQMAN TM And karyotyping to obtain the correct targeting as described previously (Valenzuela et al, supra; frendewey, D. Et al, 2010, methods of enzymology 476:295-307, which is incorporated by reference in its entirety) Incorporated herein by reference), using detection anchors to modify V H 1-69 gene segments.
Forward primer: TGTGTCCTGTCCACAGGTG (SEQ ID NO: 34)
And (3) probe: CCAGTCCAACAGTTCCGGTACG (SEQ ID NO: 35)
Reverse primer: CAGCTGGACCTGGCTACC (SEQ ID NO: 36)
UsingMethods (DeChiara, T.M. et al, 2010, methods of enzymology 476:285-294; deChiara, T.M.,2009, methods of molecular biology 530:311-324; poueymiou et al, 2007, nature Biotechnology 25:91-99; incorporated herein by reference in its entirety), wherein targeted ES cells are injected into uncompacted 8-cell stage Swiss Webster embryos to produce healthy, fully ES cell-derived F0 generation mice modified for Anchor (ANP) V H The segments hybridize and express Anchor (ANP) modified antibodies. Such modified mice are referred to herein as ANP-V H 1-69 modified mice.
The drug selection cassette can optionally be removed by subsequent addition of a recombinase (e.g., by Cre treatment) or by breeding to a Cre delete mouse strain (see, e.g., international patent application publication No. WO 2009/114400, incorporated herein by reference in its entirety) to remove any loxed selection cassette introduced by the unremoved targeting construct, e.g., during ES cell phase or in an embryo. Optionally, the selection cassette remains in the mouse.
EXAMPLE 3 immunophenotyping of rodents by flow cytometry
To determine ANP-V H 1-69, bone marrow and spleen B cells were analyzed by flow cytometry. For this study, two were sacrificedControl mice (see, e.g., U.S. Pat. No. 8,502,018; 8,642,835; 8,697,940, each of which is incorporated herein by reference)One incorporated herein by reference in its entirety) and the three ANP-V described in example 2 H 1-69, and spleen and bone marrow thereof were collected. Bone marrow was collected from the femur by centrifugation at 8,000rpm for 2 minutes. The spleen was dissociated to obtain a single cell suspension. Erythrocytes from spleen and bone marrow preparations were lysed with ACK lysis buffer and then washed with DPBS containing 2% FBS. The isolated cells (total 1X 10 6 Individual) were incubated with anti-mouse CD16/CD32 (clone 2.4g2, bd) for 10 minutes on ice and then labeled on ice for 30 minutes with the antibody set described in table 2.
Table 2: ab stack for flow cytometry
After staining, cells were washed and fixed in 2% formaldehyde. Data acquisition was performed on a BD LSRFortessa flow cytometer and analyzed with FlowJo. Cell subsets were identified using the following strategy. Bone marrow maturation: immature B cells (B220 int IgM+), mature B cells (B220 high IgM+), primitive B cells (IgM-B220 int, c-KitintCD43 high), pre-B cells (IgM-B220 int, c-Kit-CD43 int). Spleen and bone marrow kappa/lambda: b cells (cd19+cd3-), T cells (cd3+cd19-), igk+b cells (cd19+igk+igl-), igl+b cells (cd19+igk-igl+). Spleen maturation: mature B cells (cd19+, b220+cd93-), follicular B cells (cd19+, b220+cd93-, cd21/35int igmit/+), marginal zone B cells (cd19+, b220+cd93-, cd21/35+igm+), transitional B cells (cd19+, b220+cd93+), T1B cells (cd19+, b220+cd93+, igm+cd23-), T2B cells (cd19+, b220+cd93+, igm+cd23+), and T3B cells (cd19+, b220+cd93+, igmit cd23+).
As shown in FIGS. 5A-5F, in the spleen, andANP-V compared to control mice H 1-69 modified mice were similar in B cell levels. ANP-V H The frequency of mature B cells of 1-69 modified mice appears to be the same as that ofThe frequencies observed in the control mice are similar, however similar to +.>ANP-V compared to control mice H The frequency of immature cells of 1-69 modified mice was slightly decreased. In the spleen, withANP-V compared to control mice H The frequency of lambda and kappa positive B cells was similar in 1-69 modified mice.
As shown in FIGS. 6A-6F, in bone marrow, andANP-V compared to control mice H 1-69 modified mice were similar in B cell levels. In bone marrow, and->ANP-V compared to control mice H 1-69 modified mice had more primitive B cells and fewer pre-B cells. And->ANP-V compared to control mice H 1-69 there were fewer mature and more immature B cells in the bone marrow of the modified mice.
To ANP-V H 1-69 the modified mice were subjected to further immunophenotyping analysis, and the levels of mouse IgG were analyzed by Western blotting. For the assay, the sample was isolated from ANP-V H 1-69 modified miceBlood was drawn from a subpopulation of control mice. Serum is collected in a serum separation tubeIn (BD company), incubate for 30 minutes and separate from blood by centrifugation at 9000rcf for five minutes at 4 ℃. Mouse serum was diluted 1:25 in PBS and then run on 4% to 20% Novex Tris-Glycine gel under non-reducing conditions. Gels were transferred onto polyvinylidene fluoride (PVDF) membranes according to manufacturer's specifications (BioRad Trans-Blot transfer system). The blots were then blocked overnight with 10% skim milk in Tris buffered saline with 0.05% tween 20 (TBST, sigma). PVDF membranes were incubated with anti-mouse IgG-HRP (sammer (Thermo)/Pierce (Pierce), catalog No. 31432) diluted 1:20,000 in 4% skim milk in TBST for one hour at room temperature. The blots were then washed five times for five minutes each and then developed for one minute with the Amersham ECL western blot detection reagent (general electric medical group (GE Healthcare Life Sciences)) according to manufacturer's specifications. The blots were then imaged using a GE Healthcare ImageQuant LAS-4000 cooled CCD camera gel recording system. Images are captured at 15 second intervals until 20 images are captured or the images are fully exposed.
As shown in FIG. 7, ANP-V as measured by Western blotting H 1-69 modified miceSerum IgG levels of control mice were comparable. The result shows that andANP mice Designer V mice had normal Ab levels compared to control mice.
To ANP-V H 1-69 the modified mice were subjected to further immunophenotyping assays and levels of total serum IgM and IgG were measured by ELISA. For ELISA, plates were coated with 1. Mu.g/mL of anti-mouse IgM+IgG+IgA (clone Ab102445, ai Bokang company (Abcam)) overnight at 4 ℃. Plates were then washed in DPBS with 0.1% tween 20 and blocked in 1% BSA in DPBS for 1 hour at room temperature. ANP-V H 1-69 modified mice orSerum from control mice and mouse IgM standard (bai biosystems, cat# 401604) or mouse IgG standard (sigma, cat# I8765) were serially diluted in 1% BSA in DPBS and incubated for 1 hour at room temperature. After incubation, plates were washed in DPBS with 0.1% Tween 20 and IgM or IgG was detected using anti-mouse IgM-HRP (Southern Biotech, catalog number 1021-05) or anti-mouse IgG-HRP (Southern Biotech, catalog number 1030-05). Further washing was performed, and then TMB substrate (BD company) was added. After allowing color development, the reaction was stopped with 1N sulfuric acid and the absorbance at 450nm was read on a SpectraMax plate reader. Standard plots were generated in GraphPad Prism using non-linear regression (curve fitting, four parameters) of IgM or IgG standards, and serum IgM and IgG levels were quantified.
As shown in FIGS. 8A-8B, serum IgG levels measured by ELISA were measured at ANP-V H 1-69 modified miceThe control mice were comparable. Serum IgM levels measured by ELISA at ANP-V H 1-69 modified mice and +.>The control mice were also comparable. Both results indicate that, withANP mice had normal Ab levels compared to control mice.
The retention of the antibody to the anchor was verified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Immunoglobulins isolated from mouse serum using protein a magnetic beads (zemer technologies (Thermo Scientific)) were run under reducing conditions by SDS-PAGE gel to isolate immunoglobulin heavy and light chains. Immunoglobulin heavy chains were excised and digested overnight with Lys-C enzyme (Promega). Salts were removed from digests with C18 zipper (Millipore) according to manufacturer's protocol. Peptides were eluted from each ziptip in 2.5ul of 70% ACN/0.1% TFA containing 10mg/ml of a-cyano-4-hydroxycinnamic acid (CHCA) and applied directly onto Bruker Anchorchip targets (Bruker Daltonics) mixed with a-cyano-4-hydroxycinnamic acid, which were dissolved in 70% acetonitrile +0.1% trifluoroacetic acid (TFA). After drying, each target was analyzed in a reflectance tube positive mode by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on Bruker Ultraflextreme MALDI MS (Bruker Daltonics).
MALDI-TOF MS showed that ANP modified immunoglobulins were found in ANP-V H- 1-69 modified mice remained intact in serum (data not shown).
Example 4 production of antibodies containing engineered immunoglobulin variable region gene segments in rodents
This example demonstrates the production of antibodies in rodents whose genome comprises an immunoglobulin heavy chain variable region comprising an engineered immunoglobulin variable region gene segment as described herein. The methods described in this example and/or immunization methods well known in the art can be used to immunize rodents containing engineered immunoglobulin variable region gene segments as described herein with a polypeptide or fragment thereof (e.g., a peptide derived from a desired epitope) or a combination of polypeptides or fragments thereof, as desired.
Briefly, a mouse cohort comprising an engineered immunoglobulin variable region gene segment as described herein is challenged with an antigen of interest, e.g., a receptor of interest that binds to a cognate receptor, using immunization methods known in the art. The antibody immune response was monitored by ELISA immunoassay (i.e., serum titer).
Immunization
Control (n=3) and ANP-V H 1-69 modified (n=4) mice were immunized with protein immunogens using standard immunization protocols, whichProtein immunogens include the extracellular domain of NPR3 fused to a C-terminal mFc tag (known as human NPR3 extracellular-mFc). Mice were bled prior to the onset of immunization and after the immunogen boost. The last blood draw prior to euthanasia of mice used for antibody isolation was subjected to titer analysis of human NPR3 protein (including the extracellular domain of NPR3 fused to the C-terminus of myc-myc-hexahistidine tag; referred to as human NPR3 extracellular-MMH) and engineered human NPR3 expressing cells (HEK 293 cells engineered to overexpress full-length human NPR 3; referred to as 293/hNPR3 cells). />
Antisera titer determination
The anti-NPR 3 antibody titer in serum was determined using ELISA. Ninety-six well microtiter plates (Pierce) were coated with 2 μg/mL human NPR3 extracellular-MMH antigen in phosphate buffered saline (PBS, irvine Scientific) overnight at 4 ℃. Plates were washed with PBS containing 0.05% Tween 20 (PBS-T, sigma-Aldrich) and blocked with 250. Mu.L of 0.5% bovine serum albumin (BSA, sigma-Aldrich) in PBS for 1 hour at room temperature. The plate was washed with PBS-T. Pre-immune and immune antisera were serially diluted three times in 0.5% BSA-PBS and added to the plate for 1 hour at room temperature. The plates were washed and goat anti-mouse IgG-Fc horseradish peroxidase (HRP) -conjugated secondary antibody (jackson immunoresearch laboratory limited (Jackson ImmunoResearch)) was added to the plates at a 1:5000 dilution and incubated for 1 hour at room temperature. The plates were washed and developed by incubation for 15 to 20 minutes using TMB/H2O2 as substrate. The reaction was stopped with acid and the plates were read on a spectrophotometer at 450nm (Victor, perkin-Elmer). Antibody titers were calculated using Graphpad PRISM software. Titer was defined as the interpolated serum dilution factor, whose binding signal was 2 times that of the background.
Antibody titer of cells
Using mesoscale discovery company (Meso Scale Discovery, MSD)Techniques determine anti-NPR 3 antibody titers on engineered cells. Ninety-six (sixteen) partsThe plate of the carbon wells (from MSD) was coated with 40,000 cells/well of 293/hNPR3 cells or HEK293 cells in PBS for 1 hour at 37 ℃. The cell coating solution was decanted and the plate was blocked by incubation with 150 μl of 2% bovine serum albumin (BSA, sigma-aldrich) in PBS for 1 hour at Room Temperature (RT), followed by washing with PBS. Pre-immune and immune antisera were serially diluted three times in 1% BSA-PBS and added to the plate for 1 hour at room temperature, followed by washing. Goat anti-mouse IgG-Fc ruthenium conjugated secondary antibodies (jackson immunoresearch laboratory limited and internal ruthenium label) were then added to the plates at 1 μg/mL and incubated for 1 hour at room temperature. 4 XRead Buffer T of MSD was diluted to 1 Xwithout surfactant and 150. Mu.L was added to each well and the MSD was followedAnd reading on an imager. Antibody titers were calculated using Graphpad PRISM software. Titer was defined as the interpolated serum dilution factor, whose binding signal was 2 times that of the background.
Results
Control and ANP-V H 1-69 modified mice were identified using recombinant hNPR3 protein and engineered human NPR3 expressing cells (HEK 293/hNPR 3) after immunization with NPR3 extracellular protein immunogens. ANP-V H Antibodies from 1-69 modified mice (n=4) showed a range of high antibody titers against hnpr3.Mmh protein, average titers of 465,625, and +.>Average titers 394,032 of control lines (n=3) were comparable (fig. 9). ANP-V H 1-69 modified mice showed average antibody titers of 268,763 and 3,948 for HEK293/hNPR3 expressing cells and parental HEK293 cells, respectively, indicating that the antisera are specific for NPR3.Similar results were obtained for control mice with average antibody titers of 72,826 and 5,219 for HEK293/hNPR3 expressing cells and parental HEK293 cells, respectively (fig. 10). These results indicate that ANP-V H 1-69 modified mice are capable of producingThe control mice strain had a comparable immune response.
Example 4 isolation of cells expressing and/or encoding rodent-produced nucleic acids containing engineered immunoglobulin variable region gene segments
When the desired immune response is achieved, spleen cells (and/or other lymphoid tissue) are harvested and fused with mouse myeloma cells to maintain their viability and form immortalized hybridoma cell lines. Hybridoma cell lines are screened (e.g., by ELISA assay) and selected to identify hybridoma cell lines that produce antigen-specific antibodies. The relative binding affinities and isoforms of hybridomas can also be characterized as desired. Using this technique, several antigen-specific chimeric antibodies (i.e., antibodies with human variable domains and rodent constant domains) were obtained.
DNA encoding the heavy and light chain variable regions can be isolated and linked to desired isoforms (constant regions) of the heavy and light chains for the preparation of fully human antibodies. Such antibody proteins may be produced in cells such as CHO cells. The relative binding affinity and/or neutralizing activity of the fully human antibodies to the antigen of interest is then characterized.
DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes. Initially, high affinity chimeric antibodies having human variable and rodent constant regions were isolated and characterized and selected for desired characteristics (including affinity, selectivity, epitopes, etc.). The rodent constant regions are replaced with the desired human constant regions to produce fully human antibodies. While the constant region selected may vary depending on the particular application, high affinity antigen binding and target-specific features are present in the variable region. Antigen-specific antibodies are also isolated directly from antigen-positive B cells (from immunized mice) without fusion to myeloma cells, as described, for example, in U.S. patent No. 7,582,298, which is expressly incorporated herein by reference in its entirety. Using this approach, several fully human antigen-specific antibodies (i.e., antibodies with human variable domains and human constant domains) were prepared.
Specifically, from NPR3 immunizationMice or ANP-V H Spleen cells of 1-69 modified mice were stained with a C-terminal human Fc tag (hNPR 3 extracellular-hFc) and a FITC anti-mFc expressed human NPR3 extracellular domain, as described in U.S. patent No. 7,582,298, which is incorporated herein by reference in its entirety. Single IgG positive and antigen positive B cells were isolated into separate wells of 384 well plates by Fluorescence Activated Cell Sorting (FACS). RT-PCR of antibody genes from these B cells was performed according to the method described by Wang and Stollar (J.Immunol. Methods (Journal ofImmunology Methods) 2000; 244:217-225), which is incorporated herein by reference in its entirety. Briefly, by Reverse Transcriptase (RT) reaction (SUPERCISPOT TM III, invitrogen) synthesized cDNA for each B cell. Each resulting RT product was then isolated and transferred to two corresponding wells on an isolated 384-well plate for amplification of the heavy and light chain sequences. A set of resulting RT products were first amplified by PCR using 5 'degenerate primers specific for the human IgG heavy chain variable region leader sequence and 3' primers specific for the mouse heavy chain constant region to form amplicons. The second round of PCR was performed on the amplicons using a 5 'degenerate primer set specific for framework 1 of the human IgG heavy chain variable region sequence or a specific primer for the ANP peptide (ANP mouse only) and a 3' degenerate primer set specific for framework 4 of the human IgG heavy chain variable region sequence. Another set of resulting RT products was first PCR amplified using 5 'degenerate primers specific for the human kappa light chain variable region leader sequence and 3' primers specific for the mouse kappa light chain constant region to form amplicons. Use of 5' degenerate primers specific for framework 1 of human kappa light chain variable region sequences The amplicon was subjected to a second round of PCR with the set of primers 3' degenerate to the framework 4 of the human kappa light chain variable region sequence. The heavy and light chain PCR products were cloned into antibody vectors containing human IgG1 heavy chain constant regions and kappa light chain constant regions, respectively. Recombinant hIgG1 antibodies were generated by transient transfection of CHO K1 cells for further screening.
Example 5: primary screening of supernatants isolated from CHO K1 cells
Characterized as containing cells transfected from CHO K1 (146 from ANP-V) H 1-69 modified mice and 65 fromControl) the binding properties of the pooled supernatant anti-NPR 3 monoclonal antibodies to hNPR3-mmH and kinetic binding parameters and equilibrium binding constants were determined using SPR techniques.
Determination of equilibrium dissociation constants (K) for NPR3 binding to different NPR3 monoclonal antibodies (mAbs) using a Biacore 4000 instrument using a real-time surface plasmon resonance biosensor D ) And dissociation rate at pH 6.0. All binding studies were performed in 10mM sodium phosphate, 137mM NaCl, 2.7mM KCl and 0.05% v/v surfactant Tween 20 (PBS-T) and prepared in pH7.4 (PBS-T_pH7.4) buffer at 25 ℃. The Biacore CM5 sensor chip surface was first derivatized by amine coupling with an anti-human Fc specific mouse mAb (REGN 2567) to capture different NPR3 mabs. A single 100nM concentration of human NPR3 extracellular domain expressed with a C-terminal myc-myc-6XHis tag (hNPR 3-MMH) prepared in PBS-T_pH7.4 running buffer was injected at a flow rate of 30 microliters/min for 90 seconds onto the NPR3mAb captured surface and its dissociation in PBS-T_pH7.4 running buffer was monitored for 90 seconds. After the dissociation phase, PBS-T buffer prepared at pH6.0 (PBS-T_pH6.0) was injected for 2 minutes. At the end of each cycle, the NPR3mAb captured surface was regenerated using a 12 second injection of 20mM phosphate.
Rate of association (k) a ) Dissociation rate (k) d ) Determined by fitting a real-time binding sensor map to a 1:1 binding model with mass transfer limitations using Biacore 4000 evaluation software, from PBS-t_pDissociation of hNPR3-MMH of NPR3mAb in H6.0 was determined by fitting the dissociation curve using a virus 2.0c curve fitting software. Calculating the binding dissociation equilibrium constant (K) from the kinetic rate D ) The dissociation half-life (t 1/2) is:
and->
The slave is also calculated and provided in Table 3Control and ANP-V H K of NPR3mAb isolated from 1-69 modified mice D And median and average values of t1/2 values.
From the slaveControl and ANP-V H K of NPR3mAb isolated from 1-69 modified mice D And t1/2 values were compared using the box and box plots, respectively, as shown in FIGS. 11-12.
Table 3 shows the results from ANP-V H 1-69 modified sumK of control mAb D And t 1/2 Average and median of (c), and K D Value sum t 1/2 A comparison of the values is shown in fig. 11 and 12, respectively. ANP-V H 1-69 modified sum->Both control mice were able to produce antibodies with a broad affinity range, derived from ANP-V H Average/median K of abs of 1-69 modified mice D Value ratio->Control mice are small, and t 1/2 Longer.These results indicate that ANP-V H 1-69 modified mice may be in the form of a specific +. >High affinity antibodies were produced in the higher population of control mice.
Table 3: median and mean K of hNPR3-MMH and NPR3 monoclonal antibodies (mAbs) D And t1/2 value, said monoclonal antibody being derived from at 25 DEG CControl and ANP-V H 1-69 modified mouse isolation
Luminex
A Luminex binding assay was performed to determine the binding of antibodies isolated from mice immunized with NPR3 to the NPR3 antigen stack. For this assay, the antigen is an amine coupled to Luminex microspheres. Microspheres for amine-coupled proteins were prepared as follows: about 1000 ten thousand MicroPlex microspheres (MicroPLex Microspheres, luminex, catalog No. LC 10000-00) were resuspended by vortexing in 500 μl of 0.1m nap 4 (pH 6.2) (activation buffer), and then centrifuged to remove the supernatant. The microspheres were resuspended in 160. Mu.L of activation buffer and the carboxylate groups (-COOH) were prepared by adding 15. Mu.L of 50mg/mL N-hydroxysuccinimide (NHS, semer technologies, cat. No. 24525) followed by 15. Mu.L of 50mg/mL 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide (EDC, semer technologies, cat. No. 22980) (25 ℃). After 10 minutes, the pH of the reaction was reduced to 5.0 by adding 500. Mu.L of 50mM MES at pH 5.0, and the microspheres were vortexed and subsequently centrifuged to remove the supernatant. The activated microspheres were immediately mixed with 500. Mu.L of 25. Mu.g/mL protein antigen [ human NPR3 extracellular domain expressed with C-terminal human Fc tag (hNPR 3 extracellular-hFc) and human NPR3 extracellular domain expressed with C-terminal mouse Fc tag complexed with human ANP (hNPR 3-extracellular-mFc-hANP) ] in 50mM MES pH 5.0. Each conjugated protein/antibody uses a unique bead region. The microsphere-protein mixture was incubated at 25℃for two hours. The coupling reaction was quenched by addition of 50 μl of 1M Tris-HCl at pH 8.0 and the microspheres were vortexed, centrifuged and washed three times with 800uL of PBS containing 0.05% tween 20 to remove unconjugated proteins and other reaction components. The microspheres were resuspended at 1000 ten thousand microspheres/mL in PBS with 2% BSA and 0.05% sodium azide.
Microspheres with amine-conjugated protein were mixed at 2700 beads/mL, then 75. Mu.L of microspheres were plated per well on a 96-well filter plate flat bottom plate (Milibo Corp., catalog number: MSBVN 1250) and mixed with 25. Mu.L of supernatant containing a single anti-NPR 3 antibody. The samples and microspheres were incubated at 25 ℃ for two hours and then washed twice with 200 μl of PBS containing 0.05% tween 20. To detect the bound antibody level of individual microspheres, 100. Mu.L of 2.5. Mu.g/mL R-phycoerythrin conjugated goat F (ab') 2 anti-human kappa (southern Biotechnology Co., catalog No. 2063-09) (in PBS containing 2% BSA and 0.05% sodium azide) was added and then incubated at 25℃for 30 minutes. The samples were washed twice with 200 μl of PBS containing 0.05% tween 20 and resuspended in 150 μl of PBS containing 0.05% tween 20. The plates were read on a Luminex FlexMap3D instrument with Luminex xPonent software version 4.2.
Antibodies that bind to hNPR 3-extracellular-hFc and hNPR 3-extracellular-mFc-hANP with binding signals greater than 1000 but less than 5000 and greater than 5000 are shown in table 4. A total of 439 samples were tested: from ANP-V H 236 samples from 1-69 modified mice and203 samples of control mice. Binding signals were measured as Median Fluorescence Intensity (MFI). As shown in Table 4, and from- >Antibodies isolated from ANP-V compared to control mice H 1-69 modified mouse antibodies to hNPR3 extracellular-hFc and hNPR3 extracellular-mFc-hANP in Luminex assay>5000, a higher percentage of complex binding.
Table 4: total number (percent) of anti-NRP 3 antibodies bound to amine-conjugated hNPR 3-extracellular-hffc and hNPR 3-extracellular-mFc-hnp in Luminex binding assay.
Blocking ability
An ELISA-based blocking assay was developed to determine the ability of anti-NPR 3 antibodies to block human NPR3 binding to human ANP (hANP). In this assay, recombinant human NPR3 protein, including a portion of the human NPR3 extracellular domain (amino acids Gly27-Ser 482) (hNPR 3-mFc, seq ID or accession number) fused at the C-terminus to the Fc portion of mouse IgG2a, was passively absorbed overnight at a PBS concentration of 2 μg/mL on a 96-well microtiter plate at 4 ℃. The non-specific binding sites were then blocked for 1 hour at room temperature using a 0.5% (w/v) solution of BSA in PBS. After washing the plates with PBS+Tween, either 1:10 dilution of anti-NPR 3 antibody supernatant or 1 μg/mL of non-binding human IgG1 isotype control antibody was added. Plates were incubated for 1 hour at room temperature, then recombinant biotinylated hANP (biotin-hANP; phoenix pharmaceutical Co., berlingame, calif. (Phoenix Pharmaceuticals INC, burlingame, calif.) was added to a final concentration of 200pM and incubated for 1 hour. The concentration of biotin-hANP is within the dynamic range of dose-dependent binding of biotin-hANP to plate-coated hNPR3 around the EC50 value. Plates were washed and visualized with 100ng/mL biotin-hANP conjugated to horseradish peroxidase (HRP) conjugated streptavidin assay plates, and using TMB substrate solution (BD Biosciences, san Jose, calif.) according to manufacturer's recommendations. In Victor TM Multi-label reader (Perkinelmer) TM ) Absorbance at 450nm was measured.
The percent blocking of the tested anti-NPR 3 antibodies was calculated using the following formula:
antibodies that block greater than or equal to 50% of biotin-hANP binding to hNPR3 are classified as blocking agents.
Evaluation of the samples from ELISA-based blocking assaysControl and ANP-V H 1-69 modified mouse anti-NPR 3 antibody supernatant blocked the ability of human ANP to bind to human NPR 3. As seen in table 5, 338 parts of anti-NPR 3 antibody supernatant was used to test for blocking binding of human ANP to human NPR3, 125 parts of which were fromControl mice, and 213 parts from ANP-V H 1-69 modified mice. One hundred thirty four parts of anti-NPR 3 antibody supernatant blocked binding of hNPR3-mFc to biotin-hANP with a blocking percentage of greater than or equal to 50%. Of the 134 blockers 17 are from +.>Isolated from control mice and 117 derived from ANP-V H 1-69, said blockers comprising +.>Control mice and ANP-V H 1-69 modified mice tested 14% and 55% of the number of antibodies. Of the first 50 blockers, 7 are from +.>Control mice, and 43 were derived from ANP-V H 1-69 modified mice, which account for 6% and 20% of the total mAb tested in the two strains of mice, respectively.
In this experiment, the human IgG1 isotype control antibody showed a percentage of blocking of 32% and was classified as a non-blocking agent.
In general, withANP-V compared to control mice H 1-69 repairDecorated mice produced more antibodies that bound to NPR3 with high specificity and blocked the binding of recombinant NPR3 protein to human ANP.
Table 5. Summary of anti-NPR 3 antibody supernatants blocking binding of human ANP to human NPR 3-mFc.
Determination of ANP-V from immunization with NPR3 recombinant proteins using Electrochemiluminescence (ECL) based assays H 1-69 modified sumAbility of isolated anti-human NPR3 monoclonal antibodies to bind to cells expressing human NPR3 (hNPR 3) in control mice.
Briefly, HEK293 was engineered to express human NPR3 by transfecting cells with a neomycin resistant pLVXN.NPR3 expression plasmid (amino acids M1-A541, uniProtKB-P17342) encoding human NPR3. Non-transfected HEK293 cells were included in the experiments as background binding controls that did not show detectable NPR3 expression by Fluorescence Activated Cell Sorting (FACS) with commercial Atrial Natriuretic Peptide (ANP). An anti-allergen human IgG1 antibody and an anti-idiotype mouse IgG2 antibody were included as unrelated antibody controls for binding.
Experiments were performed according to the following procedure. The NPR3 expressing cells and the parent cells cultured in the flask were washed once in ca2+/mg2+ -free 1xPBS buffer and incubated with the enzyme-free cell dissociation solution for 10 min at 37 ℃ to isolate the cells. Cell pellets were washed once with ca2+/mg2+ containing 1xPBS and counted with a CellometerTM Auto T cell counter (fine-resistant biotechnology company (Nexcelom Bioscience, lawrence, MA) of lorens, MA). Approximately 2.0X104 HEK293/hNPR3 or HEK293 cells per well were individually seeded onto 96-well carbon electrode plates (Meso Scale Discovery, rockville, md.) and incubated at 37℃for 1 hour to allow cell adhesion. The non-specific binding sites were blocked by incubation with 2% BSA (w/v) in 1xPBS containing Ca2+/Mg2+ for 1 hour at room temperature. anti-NPR 3 antibody supernatants, diluted 1:10 or 0.2 μg/mL control antibody, were added to plate-bound cells and then incubated for 1 hour at room temperature. Plates were then washed using an AquaMax2000 plate washer (MDS analytical techniques company of sanyvale, CA, MDS Analytical Technologies) with a cell wash head to remove unbound antibodies. Plate-bound human IgG was detected with a sulfa-tag conjugated goat polyclonal anti-human IgG antibody specific for heavy and light chains (Jackson Immunoresearch Labs, west Grove, PA) and plate-bound mouse IgG (commercial NPR3 antibody and mIgG isotype control antibody) was detected with a sulfa-tag conjugated goat polyclonal anti-mouse IgG antibody specific for fcγ fragment (jackson immune research laboratory, scove, PA) at room temperature for 1 hour. After washing, the plates were developed with a read buffer (mesoscale discovery company of rocyverer, maryland) and luminescence signals were recorded with a SECTOR Imager 600 (mesoscale discovery company of rocyverer, maryland) according to the manufacturer's recommendations. The ratio of binding signal on HEK293/hNPR3 to the parental HEK293 cells was calculated and reported as an indication of the binding specificity of the anti-NPR 3 antibodies. Antibodies with a binding signal greater than or equal to 200RLU and a binding ratio greater than or equal to 3 on HEK293/hNPR3 cells were classified as specific binders and antibodies with a binding signal less than 200RLU or a binding ratio less than 3 were classified as non-specific binders or non-binders.
Results:
the slave was evaluated in a electrochemiluminescence based binding assayControl and ANP-V H 1-69 ability of isolated anti-NPR 3 antibodies in modified mice to specifically bind to HEK293 cells expressing NPR 3.
The experimental results are summarized in table 6. A total of 338 anti-NPR 3 antibodies in the crude supernatant were tested for binding to HEK293/hNPR3 cells,of which 125 antibodies were derived fromControl mice were isolated and 213 antibodies were isolated from ANP-V H 1-69 in mice modified. There are a total of 250 anti-NPR 3 antibodies, 62 of which are fromControls, and 188 from ANP-V H 1-69, which shows a binding signal of greater than or equal to 200RLU on HEK293/hNPR3 cells and a binding ratio to parental HEK293 of greater than or equal to 3 on HEK293/hNPR3, indicates that these antibodies are able to specifically bind to NPR 3. From->Fifty percent of control mice and in contrast, from ANP-V H 88% of the anti-NPR 3 antibodies from 1-69 modified mice were specific NPR3 binders. Of the first 50 specific binders, 6 antibodies were from +.>Controls, and 44 from ANP-V H 1-69 modified mice, which indicated 5% from +.>anti-NPR 3 antibody supernatant and 21% from ANP-V in control mice H The anti-NPR 3 antibody supernatants of 1-69 modified mice were top binders.
In the experiments, commercial anti-hNPR 3-mIgG2 antibodies (contained as positive controls that specifically bind to HEK293/hNPR3 cells) and both human IgG1 and mouse IgG2 isotype controls did not bind to HEK293/hNPR3 cells as expected.
Overall, binding using HEK293/hNPR3 cells showed ANP-V H 1-69 modified mice are capable of producing antibodies that specifically bind to hNPR3 expressing cells and are derived from ANP-V H Abundance of high-potency conjugates of isolated antibodies in 1-69 modified miceMay be higher than from conventionalIsolated antibody in control mice.
Table 6: summary of anti-NPR 3 antibody supernatants bound to cells engineered to express human NPR 3.
Purified antibodies
To evaluate the origin of ANP-V H The ability of 1-69 modified mouse anti-NPR 3 antibodies to bind NPR3 an engineered cell line was established in HEK293 cells (human embryonic kidney 293, atcc) transfected to stably express full-length human NPR3 and subsequently subjected to high expression sorting. The resulting cell line was designated HEK293/hNPR3 high-sorting (ACL #9752, regeneration pharmaceutical Co., ltd.).
For flow cytometry binding evaluation, HEK293/hNPR3 high-sorting cells were lifted with enzyme-free cell dissociation buffer (Miibbo Co., catalog No. S-004-C), washed once and resuspended in staining buffer (1 XPBS, no calcium or magnesium (Euro. Technologies Co., irvine Scientific, catalog No. 9240)) with 2% filtered fetal bovine serum (Seradaigm Co., ltd., seradaigm, catalog No. 1500-500), and stained with fixable green-viability dye (Life technologies Co., ltd. (Life Technologies), catalog No. L23101), then washed again and plated in a V-bottom staining plate (Aisi in technologies Co., ltd. (Axygen Scientific), catalog No. P-96-450-V-C-S).
Will be from ANP-V H 1-69 modified miceControl mice isolated purified antibodies, commercial antibodies (GeneTex, GTX 84015) or isotype control antibodies were serially diluted from 100nM to 24.4pM in staining buffer 1:3 (with additional wells for staining buffer alone, without test molecules) and added to cells in the stained plates.
After the additional washing step, the washing step was carried out with CytoFix (BD biosciences, cat. No. 554655) before immobilization with the immobilization buffer647 labeled secondary detection antibody [ anti-human (Jackson immunoresearch laboratory Co., ltd., catalog No. 109-607-003) and anti-mouse (Jackson immunoresearch laboratory Co., ltd., catalog No. 115-607-003)]The samples were stained. All samples were filtered through 96-well filter plates (Pall, catalog No. 8027) into a U-bottom read plate (Corning, catalog No. 3799) before passing through an iQue PLUS cytometer, and the Mean Fluorescence Intensity (MFI) of each sample was measured. In iQue->Gating of data in software to calculate the geometric mean of each sample in the RL1-A channel and use +.>The results were further analyzed by a nonlinear regression (4-parameter logic) model in 8 software to obtain EC50 values. The maximum binding fold was calculated using the following equation:
In this equation, "MFI Geometric mean RL1-A "means the maximum Mean Fluorescence Intensity (MFI) value in the RL1-A channel of cells stained with purified antibodies. "MFI Geometric mean RL1-A, secondary antibody only control "means the MFI value in the RL1-A channel from cells stained with Alexa 647-labeled secondary detection antibody alone.
As shown in Table 7, from ANP-V expressed with the ANP tag ("+ANP) H 1-69 modified mice isolated from 12 purified anti-NPR 3 antibodies, ANP-V expressed from non-ANP markers ("-ANP") H 1-69 modified mice isolated from 12 purified anti-NPR 3 antibodies, and fromThe 8 purified anti-NPR 3 antibodies isolated in control mice were tested for specific binding to HEK293/hNPR3 highly sorted cells by flow cytometry. From ANP-V expressed with ANP tag H Eleven of the 12 purified anti-NPR 3 antibodies isolated from 1-69 modified mice showed specific binding to HEK293/hNPR3 cells with fold values ranging from 21 to 1012 and EC50 values ranging from 7.7nM to 1012 compared to the secondary detection antibody alone control>10nM. Never expressed with ANP-V H Of the 12 purified anti-NPR 3 antibodies isolated from 1-69 modified mice, 10 showed specific binding to HEK293/hNPR3 cells with fold values ranging from 132 to 819 and EC50 values ranging from 6.3nM to 819 compared to secondary detection antibody only control >10nM. From the slaveAll 8 purified anti-NPR 3 antibodies isolated in control mice showed specific binding to HEK293/hNPR3 cells with fold values ranging from 215 to 789 and EC50 values ranging from 2.4nM to 789 compared to secondary detection antibody only control>10nM. Isotype control antibodies showed no binding to HEK293/hNPR3 cells and commercial anti-NPR 3 antibodies bound to HEK293/hNPR3, 22 fold more binding than secondary detection antibody control only and EC50 was 7.6nM.
TABLE 7
Equivalent forms
Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only and the invention is described in detail by the following claims.
Those skilled in the art will appreciate typical criteria attributable to deviations or errors in the values obtained in the analysis or other processes described herein.
Publications, web sites, and other references cited herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference.
Sequence listing
<110> Ruizhen pharmaceutical Co (Regeneron Pharmaceuticals, inc.)
J Ma Sitai Di Si
A.J.Mofei
J microphone Woods
V.Voronana
J-Kelol motor
<120> nucleic acid encoding anchor-modified antibody and use thereof
<130> 009108.507WO1/10507WO01
<150> 63/129,893
<151> 2020-12-23
<150> 63/219,402
<151> 2021-07-08
<160> 32
<170> patent In version 3.5
<210> 1
<211> 855
<212> DNA
<213> Homo sapiens (Homo sapiens)
<400> 1
gagacaggga cagacgtagg ccaagagagg ggaaccagag aggaaccaga ggggagagac 60
agagcagcaa gcagtggatt gctccttgac gacgccagca tgagctcctt ctccaccacc 120
accgtgagct tcctcctttt actggcattc cagctcctag gtcagaccag agctaatccc 180
atgtacaatg ccgtgtccaa cgcagacctg atggatttca agaatttgct ggaccatttg 240
gaagaaaaga tgcctttaga agatgaggtc gtgcccccac aagtgctcag tgagccgaat 300
gaagaagcgg gggctgctct cagccccctc cctgaggtgc ctccctggac cggggaagtc 360
agcccagccc agagagatgg aggtgccctc gggcggggcc cctgggactc ctctgatcga 420
tctgccctcc taaaaagcaa gctgagggcg ctgctcactg cccctcggag cctgcggaga 480
tccagctgct tcgggggcag gatggacagg attggagccc agagcggact gggctgtaac 540
agcttccggt actgaagata acagccaggg aggacaagca gggctgggcc tagggacaga 600
ctgcaagagg ctcctgtccc ctggggtctc tgctgcattt gtgtcatctt gttgccatgg 660
agttgtgatc atcccatcta agctgcagct tcctgtcaac acttctcaca tcttatgcta 720
actgtagata aagtggtttg atggtgactt cctcgcctct cccaccccat gcattaaatt 780
ttaaggtaga acctcacctg ttactgaaag tggtttgaaa gtgaataaac ttcagcacca 840
tggacagaag acaaa 855
<210> 2
<211> 151
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 2
Met Ser Ser Phe Ser Thr Thr Thr Val Ser Phe Leu Leu Leu Leu Ala
1 5 10 15
Phe Gln Leu Leu Gly Gln Thr Arg Ala Asn Pro Met Tyr Asn Ala Val
20 25 30
Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu
35 40 45
Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser
50 55 60
Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val
65 70 75 80
Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala
85 90 95
Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys
100 105 110
Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser
115 120 125
Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu
130 135 140
Gly Cys Asn Ser Phe Arg Tyr
145 150
<210> 3
<211> 5
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 3
Asn Ser Phe Arg Tyr
1 5
<210> 4
<211> 15
<212> DNA
<213> artificial sequence
<220>
<223> G4S DNA sequence
<220>
<221> CDS
<222> (1)..(15)
<400> 4
ggt ggc ggc ggt agc 15
Gly Gly Gly Gly Ser
1 5
<210> 5
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 5
Gly Gly Gly Gly Ser
1 5
<210> 6
<211> 107
<212> DNA
<213> Homo sapiens (Homo sapiens)
<400> 6
gcagctacag gtaaggggct tcctagtcct aaggctgagg aagggatcct ggtttagtta 60
aagaggattt tattcacccc tgtgtcctgt ccacaggtgt ccagtcc 107
<210> 7
<211> 19
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 7
Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly
1 5 10 15
Val Gln Ser
<210> 8
<211> 327
<212> DNA
<213> artificial sequence
<220>
<223> ANP-VH1-69 without leader sequence
<220>
<221> CDS
<222> (1)..(327)
<400> 8
aac agc ttc cgg tac ggt ggc ggc ggt agc cag gtc cag ctg gtg cag 48
Asn Ser Phe Arg Tyr Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
1 5 10 15
tct ggg gct gag gtg aag aag cct ggg tcc tcg gtg aag gtc tcc tgc 96
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys
20 25 30
aag gct tct gga ggc acc ttc agc agc tat gct atc agc tgg gtg cga 144
Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg
35 40 45
cag gcc cct gga caa ggg ctt gag tgg atg gga ggg atc atc cct atc 192
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile
50 55 60
ttt ggt aca gca aac tac gca cag aag ttc cag ggc aga gtc acg att 240
Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile
65 70 75 80
acc acg gac gaa tcc acg agc aca gcc tac atg gag ctg agc agc ctg 288
Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
85 90 95
aga tct gag gac acg gcc gtg tat tac tgt gcg aga gac 327
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp
100 105
<210> 9
<211> 109
<212> PRT
<213> artificial sequence
<220>
<223> synthetic construct
<400> 9
Asn Ser Phe Arg Tyr Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln
1 5 10 15
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys
20 25 30
Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg
35 40 45
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile
50 55 60
Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile
65 70 75 80
Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
85 90 95
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp
100 105
<210> 10
<211> 583
<212> DNA
<213> artificial sequence
<220>
<223> ANP-VH1-69 Cas9/BHR donor
<400> 10
ctcgagcagc tacaggtaag gggcttccta gtcctaaggc tgaggaaggg atcctggttt 60
agttaaagag gattttattc acccctgtgt cctgtccaca ggtgtccagt ccaacagctt 120
ccggtacggt ggcggcggta gccaggtcca gctggtgcag tctggggctg aggtgaagaa 180
gcctgggtcc tcggtgaagg tctcctgcaa ggcttctgga ggcaccttca gcagctatgc 240
tatcagctgg gtgcgacagg cccctggaca agggcttgag tggatgggag ggatcatccc 300
tatctttggt acagcaaact acgcacagaa gttccagggc agagtcacga ttaccacgga 360
cgaatccacg agcacagcct acatggagct gagcagcctg agatctgagg acacggccgt 420
gtattactgt gcgagagaca cagtgtgaaa acccacatcc tgagagtgac aaaaaccctg 480
agggcgccgg cggaattcaa gcttcctagg cgccggcgag aaggcagctg tgccgggctg 540
aggagatgac aggggttatt aggtttaagg ctgtttactc gag 583
<210> 11
<211> 19726
<212> DNA
<213> mice (Mus musculus)
<400> 11
aagcttatct ctctgttgct cagactcatc taggaatttc agaaatttct gttctagcat 60
ctcttccagc ttttgtctcc aaccctcatt ctcttctttc tttttttttt taaattatat 120
gttctctgtc tttttaaaaa actttttaaa attaggtatt tatgtcattt acatttccaa 180
tgctatccca aaagtcccac ccacgctccc caacccacta tcccacccac ccactcccac 240
ttcttggccc tggcattcac agtgtactga gacatataaa gtttgcacaa ccaatgggcc 300
tctctttcca ctgatggccg actaggccat cttctgatac atatgcagct agagacacga 360
gattctgggg gtactggtta gttcatattg ttgttccacc tatagggttg cagatccttt 420
tagctccttg ggtactttct ctagctcctc cattgggggc cctgtgatcc atccaatagc 480
tgactgtgag catccacttc tgtgtttgct aggccccaga tagtctcaca agagacagct 540
atatctgggt cctttcagca aaatcttgct agtgtatgca acggtgtcag agtttggaag 600
ctgattatgg gatggatccc cggatatggc attctctagt tggttcatcc ttttgtctca 660
gctccaaact ttgtctctgt aactccttcc atgggtgttt tgttcccagt tctaaggagg 720
ggcaaagtat ccacactttg gtcttcattc ttcttgagtt tcatgtgttt tgcaaattgt 780
atcttatatc ttgggtattc taagtttctg ggctaatatc cacttatcag tgagtacaca 840
ttgtgtgagt tcttttgtga ttgggttacc tcactcagta tgatgccctc caggtccatc 900
catttgccta ggaatttcat aaattcattc tttttaatag ctcagtagta ctccattgtg 960
tagatgtacc acattttctg tattcattcc tctgttgagg ggcatctggg ttctttccag 1020
cttctggcta ttataaataa ggctgctatg aacatagtgg agcatgtgac cttcttaccg 1080
gttgggacat cttctggata tatgcccagg agaggtattg ctggatcttc cggtagtact 1140
atgtccaatt ttctgaggaa ctgacaaact gatttccaga gtggttagta ccagcttgca 1200
atcccaccaa caatgagagg agtgttcgtc tttctccaca tcctcaccag catgctgctg 1260
tcacctgaat ttttgatgct tagccattct gactggtgtg aggtggaatc tcagggttgt 1320
tttgatttgt atttccctga tgattaagga tgctgaacat tttctcaggt gcttctcagc 1380
cattcagtat tctttaggtg agaattcttt gtttagctct aagccccatt tttttaatgg 1440
ggttatttga ttttctggag tccaccttct tgagtttttt tttccatttt ttattacata 1500
atttcctcaa ttacatttcc aatgctatcc caaaagtccc ccataccctc ccccccccaa 1560
ttccctaccc accccttccc atttttttgg ccctggcgtt cccctgtact ggggcatata 1620
aagtttgtgt gtccaatggg cttctctttc cagtgatggc tgactaggcc atcttttgat 1680
acatatgcag ctagagtcaa gagctcccgg gtactggtta gttcataatg ttgttccacc 1740
tatagggttg cagatccctt tagcttcttg ggtactttct ctagctcctc cattgggagc 1800
cctgtgatcc atccaatagc tgactgtgag catccacttc tgtgtttgct aggccccggc 1860
atagtctcac aagagacagc tacatctggg tccttttgat aaaatcttgc tagtgtatgc 1920
aagggtgtca gcatttggaa gctgattatg gggtggatcc ctggatatgg cagtctctac 1980
atggtccatc cttttgtctc agctccaaac tttgtctctg taacttcttc catgagtgtt 2040
ttgttcccaa ttctaaggag gggcatagtg tccacacttc attcttcatt cttcttgagt 2100
ttcatgtgtt tagcaaattg tatcttatat cttgggtatc ctaggttttg ggctaatatc 2160
cacttatcag tgagtacata ttgtgtgagt tcctttgtaa atgtgttacc tcactcagga 2220
tgacgccctc caggtccatc catttggcta ggaatttcat aaattcattc tttttaatag 2280
ctgagtagta ctccattgtg taaatgtacc acattttctg tactcattcc tctgttgagg 2340
ggcatctggg ttctttatag gttctggcta ttataaataa ggttgctatg aacatagtgg 2400
agcatgtgtc cttcttaccg gttgagacat cttctggata tatgcccagg cgaggtattg 2460
ctggatcctc cggtagtact atgtccaatt ttctgaggaa ctgccagact gatttccaga 2520
gtggttgtac aagcctgcac tctcaccaac aatggaggag tgttcctctt tctccacatc 2580
cacgccagca tctgctgtca cctgaatttt tgatcttagc cattctgact ggtgtgaggt 2640
ggaatctcag ggttgttttg atttgcattt ccctgatgat taaggatgtt gaacattttt 2700
ttcaggtgct tctctgccat tcggtattcc tcaggtgaga attctttgtt cagttctgag 2760
ccccattttt taatggggtt atttgatttt ctgaagtcca ccttcttgag ttctttatat 2820
atgttggata ttagtcccct atctgattta cgataggtaa agatcctttc ccaatctgtt 2880
ggtggtcttt ttctcttatt gacggtgtct tttgccttgc agaaactttg gagtgagttc 2940
tttatatata ttggatatta gtcccctatc tgatttagga taggtaaaga tcctttccca 3000
atctgttggt gacctttttg tcttattgac ggtgtctttt gccttgcaga atctttgcaa 3060
ttttatgagg tcgcatttgt caattctcga tcttacagca caagtcattg ctgttctgtt 3120
caggaatttt tcctctgtgc ccatatcttc gaggctttta cctgctttct cctctatatg 3180
tttgagtgtc tctggtttaa tgtggagttc cttaatccac ttagatttga ccttagtaca 3240
aggagatagg aatggatcaa ttcgcattct tctacatgat aaccgctagt tgtgccagca 3300
ccatttgttg ataatgctgt cttttttcca ctggatggtt tttgctccct tgtctaagat 3360
caagtgacca taggtgtgtg ggttcatttc tgggtcttca attctatttc attggtctac 3420
ttgtctgttg ttataccagt accatgcaga ttttatcaca attgctctgt agtagagttt 3480
taggtcaggc atggtgatta caccagaggt tttttttatc cttgagcaga gtttttgcta 3540
tcctaggttt tgtgttattt cagatgaatt tgcagattgc cctttccagt tcgttgaaga 3600
attgagttgg aattttgatg gggattgcat tgaatctgta gattgctttg gcaatatagc 3660
catttttact atattgatcc tgccaatcca tgagcatggg agatctttcc atcttctcaa 3720
atcttcttta atttctttct tcagagactt gaagttcttg tcatacagat ctttcacttc 3780
cttagttaga gtcacgctaa ggtattttat attatttgtg actattgaga agggtgttgt 3840
ttccctaatt tctttctcag cctgtttatc ctttgtgtac agaaaagcca ttgacttgtg 3900
ttagttaatc tcatatccag ctacttcact gaagcggttt atcaggttta ggagttctct 3960
ggtgtaattt ttagggtcac tcatatatac tatcatatca tctgcaaaaa gtgacatttt 4020
gacttcttcc tttccaattt gtatcccctt gatctccttt tgttgtcgaa ttgctctggc 4080
aaggacatca agtactatat tgaataggta gggagaaaat cggcaccctt gtctagtccc 4140
tgattttagt aggattgctt caagtttctc accatttact ttgatgttgg ctactggttt 4200
gctgttgaat gctttttatc atgtttaggt atgggccttg aattcctgat ctttccaaga 4260
cttttatcat gaaagggtgt tggattttgt caaatgcttt ctccagcctt tcattctgag 4320
gttgtgtctg tctttttccc tgagatgggt ttcctgtaag cagcaaaatg ttgggtcctg 4380
tttgtgtagc ccgtctgtta ttctatgtct ttttattggg gagttgagtc cattgatatt 4440
aagatatatt aaggaaaagt aattgttgct tcctattatt tttgttttta aagttggcat 4500
tctgttcttg tggctgtctt cttttaggtt tgttgaagga ttcctttctt gctttttcta 4560
ggtcgtggtt tccatccttg tattcatttt ttttctgtta ttatcctttg aaggactgga 4620
ttcatggata gataatgtgt gaatttggtt ttgtcttgga atacttttgt ttctccatct 4680
acggtaattg agagtttggc tgggtatagt agcctgggct ggcaattgtg ttgtcttagt 4740
gtctatataa tgtctgtcca ggatcttctg gctttcatag tctgtggtga aaaatctggt 4800
gtaattctga taggcttgcc tttatatgtt acttgaattt ttcacttact gcttttaata 4860
ttctttcttt atttagtgca tttgttgttc tgattattat gtgtcgggag gaatttcttt 4920
tctggtccag tctatttgga gttctgtagg cttcttgtat gttcacgggc atctctttct 4980
ttaggtttgg gaagttttct tctataattt tgttgaagat atttgctggc ccttcaagtt 5040
gaaaatgttc attctcatct actcctatta ttcgtatggt tggtcttctc attgtgtcct 5100
ggatttcctg gatgttttga gttaggatct ttttgcattt tccattttct ttgattgttg 5160
tgcagatgtt ctctatggaa tcttctgcac ctgatattct ctcttccatc tcttgtagtc 5220
tgttgctgat gctcgcatct atggttccag atttctttcc tagggtttct atctccagtg 5280
ttgccccact ttgggttttc tgtatagtgt ctacttccct ttttagatct agtatggttt 5340
tgttcatttc catcacctgt ttgggtgtgt tttcctgttt ttctttaaag acttgcaact 5400
ctttagcaga gttctcctgt atttaagtga gttattaaag tccttcttga tgtccagtac 5460
cataattgtg agatatgcct ttaaatccaa gtctaggttt ttgggtgtgt tggggtgccc 5520
tggactggct gagttgggag tgctgcattc tgatgatggt gagtggtctt ggtttctgct 5580
agtaagattc ttacatctgc ctttcgccat ctggtaatct ctggagtcag ttgttaaagt 5640
tgtctctggt taaagcttgt tcctctcgtg attctgttat tctcttccag cagacctggg 5700
agactagctc tttcctgagt ttcagtggtc agagcactct ctgcaggcag gatttcctct 5760
ttcagggaag gtgcacagat atctggtgtt cagatttgcc tcctggcaga agatgatggc 5820
ctgaaacagg acctgtccca gaagctgtta gcttctgtag tcaacactgt cacctgtgca 5880
gactagtctc ggtggagtcc gggaaccaag atgtctcctg cagatgctct ggcattccct 5940
tctgggccgg gtgatcacct ctcctctggc agggaaggtg ccctggtgtc tggaacccga 6000
aaagggggct gcctcagaag ctctgtggct actgcctgtc ccagaagctg ttagcttctg 6060
tagtccacac tctcacctgt gcagactagt cttggtggag tctgggaacc aagatgtctc 6120
ccgcagatgc tccagccatt ctcctctttc tgttgcttat tttgacctat gaaatcctgg 6180
acatatagtt ctagtgttgc ttgtaatctc ttttctaagc caaggaattt tttttatcta 6240
gggcacaatc ttttgagaag acatattaaa tcaagagaat aaatattgca agaccaataa 6300
atgataaggt atctattttc tttaaatcca tcgctgtcaa accattcaaa atatcctcac 6360
ataaagccaa aaagatattt attgtgtttc ccatcttagt tgagttcaag tcaatatttt 6420
ggtgccattt tgttgcagta aatctctaac acaaatatgc ctgggcaatg aaaacacaac 6480
tcagttaata tgaatacaga ttgttcagat ctaccactac actaccatct tcttcatcta 6540
agagacccct tagaacttgc agtttctcca ggccttgtgc ttctgcgctg cttttcttct 6600
tcttcctctt ctacattgct tctctcataa acctacttct ttttttccct ccttctgttc 6660
catcttccct tttatctgcc caatcattag ctctccttta ttttacaaat taaggtgtga 6720
agccggtttc taggaaatca cctgagtgct gacttgttcc ttgttcagag ccacgcacag 6780
gagaacagaa ttaacatcaa atataattat ccccagggct atccacaaca cgtgcatcct 6840
ataagatcac cacggactaa tgctggtctt caattacaac ataaacaaca aaaaccccac 6900
atatatgtgg aaacaaatcg aactatacaa agaatcaatg aaaccaggag cttgttcttt 6960
gagaaaaatc aacaagatag ataaaccctt agccagacta accagagggc acagagacag 7020
tatccaaatt aataaagtca gaaatgaaag gaagacataa caatgaaata tatcttaaaa 7080
taattaatct gtttgtagac tattagcagt tgaaaatatt aaaatcatgt tctacaaacg 7140
tggaattatt attgataatt ttctcactgt gcttgaaatt agcattttct taatgtttaa 7200
cttcaaagag tttttgctat tttgaaatat taaacatata cttactgata aaataatttc 7260
cctcctaaca acactgataa tcttttttta agtaaactga ttattagaca atgtacacag 7320
atatataatg tgttttaaat actctcccac tgtcaggtgg tatcatatag ggcctttgaa 7380
tatattttta aatgtattat ttgtaatatt ttatggtctc tcctatgctt atttctgaaa 7440
gaatattttg tatgttttga aacaatttag tatttaacat tagatatagg atcctcagtt 7500
atggatagta ttaaatattc attaatgata tttttaaggt ataaaaggat atgaatataa 7560
aagtttaaca aattttatgt attatttgat tctaaaaata ctcaatatta ttaatatgtt 7620
tgatgtttaa aatgcattta aataataaaa acatttaaaa aaataaaatc aagaaatgag 7680
gttctaagca gaggtcaagg aaaatgagga atagaaaaat agtaaaaatc aatatgtcca 7740
tttattcaag gaaagctcct acatagacat tgcaccagat tagcaaatat tatggtcctc 7800
atattagttt aagttaggag actatgctta tgttatctat ttacattcta aggagcctag 7860
acatttgtga atggattaca ttataagagg aggatgtcta cttaagtagg catgaacgcc 7920
tgtgcattgc accctatgag ttccatcagc attccatgat tggagtatga agaacagcat 7980
tatagacatt acccagaacc ttagtggttc tagaatgcca agataaaaca atctaacctt 8040
ctggatagta gggataaatg ttcctatatc atcagaattc actggtgccc tgaggatgtt 8100
accctgctaa ctgacaattc acaggacatc acatggattc tgataagttg cagaaaagag 8160
gagatgcatt caattggtcc tcctccttct aagctgcaat attaggtgca tccaatttgt 8220
gaacttcaat ttagattaca atagacatga ataatctgaa ttcatgtagt acatattttt 8280
gttttaatat gagttaccat tgttcagaaa attaaataca catgatcaca tattcctaca 8340
tagtgctgtt agtttttcac atctctggga caatattcca aatatctcct tcattagtga 8400
aaatatcaac tactgtaaag cttagctaac atgcctttgc aggaataaga acatcctgga 8460
ttgaaagcta cacagggaga tgtaaaactt tctaagcaca cacattctcc atccattagg 8520
atcatggtcc atgagatttt tctctctctc ttcttcccat taaatgcatg tacatgcagg 8580
ttgggaaaca gattgtgttg cagaatacat ttgcttgatt tccacttcct tctcaatgca 8640
aatatttttg aagtgttaat tttgctgtga gtaccacagt ggttcttgct ctttctgttg 8700
actcctgtct gtgaatgttc caggaattca cacatggaca cacgtggggc tgcatctgag 8760
ctccagactc actgttgtcc ttctgtcctc agctgctctg gcccaggcac agcctcgtga 8820
attcaacaaa gaccctgatc tctcttgttt acacctcatt acaaatggga actgttagag 8880
gtggacccaa ctgcatttcc atgaggaaag cacatgagtt tgagagggtc gttgatgata 8940
aggtagaaac aactttaatt cataggctga gatatcagtc atcacctcca gataaacaag 9000
agccatttct tcctgcatct gagccctgta agcacactag ctttaggaat atgttactgc 9060
tgaagtcaga ttgggcaact tcatagtata caatagaaaa tctacctgca gatgagttca 9120
gaaccagcag ggggcacaat ggggccaaga atccctagca gagagatgtg gtgtgtgtgc 9180
aggggactct gcatcctctg tggtttcctt tcttaactta catgtacctg tagtgattga 9240
catgtaacgt ttccacgctc aaacactgtg aagatacttt gctaaacact tcaaagattt 9300
atgttttctt gatgtgtgca tgtgtgtatt cttttttgtt tttagacaca gggtttctct 9360
gtgtagtcct ggctgccctg gaactcactc tgtagaccag gctggcctcg aactcagaaa 9420
tctgcctgct tctgcctccc aagtgctgaa gttaaagaca tgtgccacca ttgcctggcc 9480
atgtgtgtat tcttgatgca ctcttctgtt gacagataca cagtttattt ccataattta 9540
tttattgtga tggtgctgca ataatcactt atgtacaaat gtttctgaag tatatttagt 9600
tttggtcatt tgggtgatta tttttttctt tctagtatat agcattttgg aaaggtagat 9660
attaattgta tgtatgggaa ggaggctgta aattctaata acttagctgc ttttgaaatt 9720
tgtcctcaat tctatcatcc ttgtaaccac cttaaatcca tctattagcc ttgtcacaag 9780
tgagccactg tctcaggctg caaatctttt tatagattag gtcgtgatgt tacatccaca 9840
gcctctgcac aatgctcagg ggtgggatat gggatgaatt ccctcagaca gcattaggac 9900
ttggatctca gcagactgat tcttgaccca aatgtctctt cttctctagc aggagtaagt 9960
ccttatctaa gatgtactct gctcatgaat atgcaaatca attgagtcta tggtggtaaa 10020
tatagggatg tctacacccc tcaaaaactt aagatcactg tcgtcttcac agtcacagga 10080
gtacacagga catcaccatg tgttggagct gtatcatcct cttcctgtta gcaacagctg 10140
cacgtaaggg gcttacagta gcaggcttga ggtctggcca tacactcatg tgacaatgac 10200
atccactctg tccttccctt cacaggtgtg cactcccagg tccagctgca gcagtctggg 10260
gctgagctgg tgaggcctgg ggcctcagtg aagatttcct gcaaggcttt tggctacacc 10320
ttcacaaacc atcatataaa ctgggtgaag cagaggcctg gacagggcct ggactggatt 10380
ggatatatta atccttataa tgattatact agctacagaa ccagaagttc aagggcaagg 10440
ccacattgac tgtagacaaa tcctccagca cagcctatat ggagcttagc agcctgacat 10500
ctgaggactc tgcagtctat tactgtgcaa gacacagtgc tacaaacaca tcctgagtgt 10560
gtcagaaacc ctggaggaga agcaagcaga gctggaatgg agatgacaga aagattatca 10620
tttagacttg ctcagaaaga gaaattttga atgcccattt attgcctctt ccttacagta 10680
ctatagtgcc tgtttttgtt gacattttca aactaatttc caaagtcact accacaattt 10740
acaatcacat aaaaagcaag caaggataac attattttct gtgcttactt gccatttata 10800
ttcttgctta ttctcatctc actgaggtca tattgggaca ttaaatttct ggggttactt 10860
tttattaaaa atttttcatt attcattcac tttacatcct tctagtcttc ctctcacaca 10920
tgccctatcc ctttctcctc tgagaggatg gagccctccc taccctcgta tccccttacc 10980
caggcacatc aagtgtctgc agtactagga atattctctg tcaatgctgc cagacaaggc 11040
agacaagtta ggggatcagg attcacagga aggcaacagc ttgagggaca gcccccactg 11100
aagttattgg tggattcaca tgaagactga gttgcacatc tgctacatat attcaggggt 11160
cctatttaca gctcaagtag actcttgttg gtggtttagt ctcttagaac cccaagtgtc 11220
caggttagtt gactctgtgg gtcttccttt ggagttccta tcccctccag atccctcagt 11280
tcttctccca actcttccat aagacacccg taggtccatc caatgtttgg ttttgggttt 11340
ttctgcatct gcttcagtca gctgctgggt ggagcatctc tgaggataat tatgagaagc 11400
tcttatgtgc aagcataaca ggatatcatt attagtgtca gggactggtg cttggccatg 11460
ggatgggtct caagtttggt cagttatttg gccattccca cagtctctga taatctttgt 11520
ccctgcattt cttgtagaca ggaaaaatat tgggttgaaa gttttgtggg tgggttggcg 11580
tctctattgc tccactgggc ttctttctgg atataggagt ttgcctcttc aggttccata 11640
ttcccaaagt agtgtgtcac actaaggtca ctcccataca gagggacact cattctcttg 11700
ccacgtctct gtccaccttc attggacctg aggttcctga atcatacaga actgcatgtg 11760
tgcaaccaca cagaacaagg ctatctatca gaggcctacc ataccaggac catcaaggtt 11820
caccttactc ccaatactga ctacaaaaag aacatcaagg accaatgcag tctatatgga 11880
taaacacact tgaaagaaca caaacaagat tgagggcaac atgacacctc caaagcatac 11940
ctaaccgagt acagcatgcc ctggatatcc taacacaatc aaaacacaag aaagttacct 12000
taaatccagt cttataaagg tgatgaaggc ctttaaatag gaaatgaatt aatccttagg 12060
ataatacagg acaatacatt cgaacagata gaggtcttta ggaggaaaga aataaatccc 12120
tcaaagacat acatgaaaat acaattaaac aggtgaaagt aataactaca atggtgtaag 12180
acctaaaaat ggaaatagaa gcaataaagt aacacaaact tagaatcttg aaggtggaaa 12240
acctagagaa caggaatact agatgcaagg atgatatctt ctaggtccat ccatttgctt 12300
gcacaattta tcatgtcctt gcttttaata gttgaacagt atttcattgt ttaaatgaac 12360
cacatgttct gtctccattc tctggatgag ggggtgagca agtttttcca cattctggct 12420
attacaaata gagctgctat gaacctagta gaaaacatat cctgtgtatg gtggagagtt 12480
ttggagtata tcaccaagag tgttatagct gggtcttcat gtagaactat tcctaatttt 12540
ctgagaaatc ccaagtcaga tttctagaat ggttgttcaa gtgttcactc caaccatcaa 12600
tggaggactg ttttccttgc cagcatgtgc tgtattttga gtttttgatc ctagccagtt 12660
ttatcctgca tttcacactt agatatggac tatggtacag gacagagaga aaccaacctt 12720
ctactcacca ggatattcta cctgctacca atttatttat ttatttattt atttatttat 12780
ttatttattt atttatttat attagagaac aacaccatgc agtttagaag aagtactaag 12840
acgtcagtga tgttatactg tgcctaacct tgcattgtac aatctcagct ttcaggtaag 12900
acagtgcatg actcttatgc agtgccaact gttttctgat tgtatttatg gtctattgcc 12960
taggaatgac ctcctctcaa ataaacatgg tcaaaagccc atggcctgag atgacagagc 13020
ccctagtaga ccctagttgt atttctgaag tttagatatc ataatgactt ataaatactt 13080
atgtttatac aatagattag agctgctctc agccatgacc aaggagcttc tgtgttcaat 13140
gaataatgat tgatgcagac attcgtgagt ggtcaaagtg gtgagaatga ttagagagtc 13200
ctcagccaca caagcgttaa tgatatgaac tttccaatat attaactgta ttaatgaata 13260
aatgcagaca tcatatgaga tctcattagt agttcttagg tattgcattt ttatatacaa 13320
ttatgcatat cagtacatta tagtgtataa aggaaattgt ctagcataat agagaaaaat 13380
aggacagtca agaaacaaaa gagtagaaat tatgggtgaa atatgcagtg tgaaatattt 13440
acatgaaaat tttaaccata tgtaaaattg ttatttttgt ttttcagaat gagtttgctc 13500
attctttgac atttttattc ctgtgtgaaa tatatcagga tcatatgtat cccattctga 13560
tggtctgact tccactggga atttccaata tatctcttcc aactaactga ccagtttctt 13620
tttttcttat tttctctctt tctcgttttg ttttgctttg ttttgttttt caagacaggg 13680
tttctctgtg tagctctggc tgtcctggaa ctcactttgt agatcaggct ggcttcgagc 13740
tcataaatcc acttgcctct gcctcctgag tgctgggatt aaaggagtgg ctaccacgcc 13800
cggctagttt ttttttttct tataagaaca acatttactg gatggtcact tacatattca 13860
gaggttcagt caattattat caaggcagaa gcatggcagt ggtccagtag tcatggcact 13920
ggggaaggag ctgagagatc tacatcttgc tccaaaggga aagaggaata gtctgacttc 13980
catgtgtttc agaggagggt ttcatttccc acccccacag tgacacactt cctccaacac 14040
ggccacacct cctaatattg ccactcttgg atcaagcata ttcacaccac aaaggaaagt 14100
ttagagataa acattaagaa aattaatgaa gtcattttat cttatatgct caacatgact 14160
agtacttaaa accataattt tacatgtaca atatttcatg gcataacata ttttttatat 14220
ttttattaga tattttcttt atttatattt caaatgtgat accctttccc aattcccctc 14280
caaaaatccc ctatgccttc ccctcatagc cagctcccaa acccacccac tcctgctttc 14340
tggtcctggc attcccctat actggggcat aaaaccttca caggaccaag tgcctcttct 14400
ccattgatgg ccaattaggc catcctctgc tacatatgca gctagagcca tgagttccac 14460
catgtgtttt ctttgattgg tggtttagtt ccagggagct ctgggggtat tggttagttc 14520
atattgttcc tcctatgggg ctgcaaaccc tttcagcccc ttgggtattt tttctagctc 14580
cttcattggg gaccctgtgc tccatccaat ggatgagtga gcctccactt ttgtatttgt 14640
caggaactgg cagagtctct caggagacaa ttatatcagg ctcctgtcag caaaatctcg 14700
ttggcatctg caatagtgtc tgggtttggt ggttgtttat gggatggatt tctgggtggg 14760
gcagtctctg gattgtcatt cctttagtct ctgcttccac ctttgtcttt gtaactccat 14820
ccatgggtat tttgttcccc cttcaaagaa ggatcaaaat atccacactt tagtcttcct 14880
tcttcttgag tctcatgtgt ttttcaaatt gtatcttggg tattctgagc ttctaggcta 14940
atatccactt atcagtgagt gattatcatg tctgttcttt tgtgattgag ttacctcact 15000
tagcatgata tcctccaggt ctatccattt gtctaagaat ttcataaagt cattgtcttt 15060
aatagctgca tcgtactcaa ttgtgtaaat gcaccacatt ttctttatcc attcctctgt 15120
tgagggacac ttggtttttc ccagcttctg gttattataa ataaggctgc tatgaacata 15180
gtggaacatg tgtccttagt acatgttgga acatcttctg ggtatatgcc caggagtggt 15240
attgctggat cttctggtgg tactatgtcc aaatttttgg ggaaccatca aactgatttc 15300
ctgagtggtt gtacaagctt gcaatcccac accagcaata gtggaatgtt catctttgtc 15360
caagtccttg ccagcatctg ctgtcacctg agtttttgat cttagccatt cttactggtg 15420
tgaggtggaa tcttggggtt gttttgattt gcatttccct gatgtttaag ggttttgaac 15480
atttttaggt gcttattaga catttggtat tcctcagttt agaaatcttt gtttagctct 15540
gtaccacatt tttgaatagg gttatttggt tttctggagt ctaacttctt gagttctttg 15600
tacatattgg atattagccc tctatcagat ttagaattag taaggatctt tccccaaact 15660
gttggtggtt cttttgtctt attgacagtg tactttgcct tagagaagct ttgcaatttt 15720
atgaggtccc atttgtcaat tcttgatctt atagtacaag ccattggtct tttgttcagg 15780
aatttttccc atgtgtccat atgttcaagg catttcccca ctttctccac tacaagtttt 15840
agtgtctctg gttttatgtg gaggtccttg atccacttag atttgagctt tgtacaagga 15900
gataagaatg gatagattca cattcttcta catgctctct gccagttgag ctagcaccat 15960
ttgttgaaaa tgctgtcttt tttttccccc actggatggt ttttagctct tttggccaag 16020
atcaagtgac cattggtgtg tgggttcatt tcttggtctt caattctagt tcactgactt 16080
acctgtttgt cactgtacaa ggaccatgca gcttttttca caattgctct gtagtacagc 16140
ttgaggtctg ggatggtgat tctaccagag agattctttt actgttgtga ataatttttg 16200
ctatcatagg atattttttt atttcagatg aatttacaaa ttgctctttc taactctgtg 16260
aacaattgag ttggaatttt gattgtgatt gctttgaata ctcaagatat aatttacaaa 16320
acacatgaaa cttaacaagg actactaaag tgcagatact tcgatccttc ttagaagggg 16380
gaacaaaata cccatagatg gagttacaga gacaaagttc ggagcagaga ctataggaac 16440
gaccatccag aggtccacct ggggatccat catgtaaaca accacccaaa acagacacta 16500
ttgtggatgc caagaagaac ttgctgacag gagtctgata tagctgtctc ttgagaggct 16560
ctgccagggc ctcagaaagt ggaggctcac agccatccat tggatggagc acagggtccc 16620
caatgaagga gctagagaaa gtactcaagg agctgaaggg gtttgcagcc ccataggagg 16680
aacaacaata tgaactaacc agtaccccca gagctccctg ggactaaacc accaatcaaa 16740
gaaaacacat ggagggactt gaagctcttg ctgcatttat agcagaggat ggcctagatg 16800
gtcatcaatg ggaggagagg tcaatggtcc tgggaaggtt ccatgcccca gtatagggga 16860
atgccagggc caggaagcag gagtgggtgg gctggggatc agggaggggg agatgatagg 16920
gcattttcag tggggaaact aggaaagagg ataacattta aaatataaat aaagaaaata 16980
tctaattaaa aaggattacc tatgtgcatg ggagctcatg agcagcaggg gtcactctaa 17040
ggccaataat ccacatagag cgatgagctg tgtgtgaaca ggactctgta tcctctgtgg 17100
tttcctttct taagtgtatt aactgatctg tccagctgtg attgacatgt gatgtctcca 17160
tgctcaagcc cagtaaagat tctctgttaa ataccttaca gacttatgtt tacttgtttt 17220
tatttgcttt tcatattttt ttaaaaagtc atacaatgta ttctaataac tcattctccc 17280
atctccaatt tattctaagt ttttcttaac tcatccaacc acacactttt taattctgat 17340
aaagcacccc cccccccaaa aaaaaaccca accaaccaaa aaaaaaaaaa gccaaggaat 17400
ttaaaagggg attgaaagca aataaaaact aaacaaaaaa gtaaaaacta cacacacaca 17460
cacacacaca cacacacaca cacacacaca cacacactca cacacacaca cacacaccac 17520
acacacacac acccatgcac gaacacacac acacacacac acacacacac acacacacac 17580
acacacacac acacatggaa tccagtaaaa ccacaactct ttacccatga tacacaggaa 17640
aatataagtc aaacaaacag aatggaagaa ggtggtatta taaaaatgtc tgcacaaata 17700
ccattaagtt cattttcttg ttggctacca actgctaagc ctgtctccct tgattaattg 17760
tgcttatcat cccctatgaa ctccattgga ggacactaat ttttccttct gtctccagga 17820
attgaagtgt tgcagaactc tcagtagctt tatttacctg cacaatacag cctctaatcc 17880
aaccagtgaa aattaccaca tgagagactt ccaaatgaaa gaacaggtaa agttgtctac 17940
tggcaagctt agtaatatca tgtaaatgcc ttagaattta atgacatatg tcatcctctg 18000
aggttaataa atccattttg gtgcatatat accctgaact caccactaac ataatacaac 18060
aattaaaaaa ttccaacatg gatgcagagg aatccctgag ggacatttgt tgatttgtga 18120
gcacaatata attatttttt gggggggaaa tgtctgaatg ttaactcttt accagtgata 18180
atctattcta ttaatgtgta cataggtagc actaattaaa atcactgtgt tatcaggtaa 18240
tgaaacagag gaagtaggat gctgggaaac agacttttgg aaggtcccaa gggaaaccac 18300
agggacctag tggtgataga ttatggtgag agtcctgaga gtggtcatag attatagcat 18360
atttcatatg caattgaaaa tttcaaagaa tgaaaatcct tatgaaatat agaaataaca 18420
actttactta tgtacatata cttcatagta caatttttac actgtgcata tttctcctgt 18480
aacatctggt tcctcctatt ttcctttatt ctcctagaca atttcactga tacaatctca 18540
tgtttttgta taaatagttg tatataacta ttaaatacat aagctgttaa tgagtcttca 18600
ttaatgtctg tgattttttt attgtcttaa ttaatactat tatctctaat tgcatccaca 18660
ttttcaaaag caatgtaaat ttcttactca tttctgttca aaaacttctg ttgttgtatc 18720
attaccatgc cttagtgata aaatcctttc ttgacacatc tatagctatt gctataattt 18780
agttattgat gatcctcctg caataatcat tgataggtaa atattttaag cacttttact 18840
tttagtcatt ttagtgagat ttgaagtagt atataacctg ttggaaaggc aaatattaat 18900
tccatatatg tgaaagaaga cgctaaaact aaaaacatta gccactttta gatatcttct 18960
ccttcttctt cttcttcttc ttcttcttct tcttcttctt cttcttcttc ttcttcttct 19020
tcttcttctt cttcttttct tcttcttctt ctccttctcc ttctccttct ccttctcctt 19080
ctcctcttcc tcctccttcc ttccttcctt ccttccttcc ttccttcctt ccttccttcc 19140
ttccttcctt ccttccttcc ttccttcctt ccttccttcc ttccttcctt ccttccttcc 19200
ttcctttctt tctttctttc tttctttctt tctttctttc tttctttctt tctttctttc 19260
tttctttctt tctttctttc tttctttctt ctcctcctcc ttctttttcc ttctccttcc 19320
ccttcacctt ccccttcctt cctctttccc ttccccttct ccttctcctc aatctacaat 19380
ctgttaacat attaacatgt cccagagtag agcaacagac tcaggtcaaa catctactga 19440
gaaatttgcc catgtagtta acatctacag catctgtcta ggggttacaa aaagtctatg 19500
ggatacaatt cctcagaaag gaataggatt tggacctgag catactgctg cctaacacat 19560
gaaatggcag ttcttctcca gctggactag gtccttaact aagaaatgca ctgctcatga 19620
atatgcaaat tacccaagtc tatggcagta aatacagaga tgtccacacc ctgaagacaa 19680
cctatgaaca atgttctctc cacagtccct gaagacactg attcta 19726
<210> 12
<211> 7928
<212> DNA
<213> mice (Mus musculus)
<400> 12
ctgagcattg cagactaatc ttggatattt gtccctgagg gagccggctg agagaagttg 60
ggaaataaac tgtctaggga tctcagagcc tttaggacag attatctcca catctttgaa 120
aaactaagaa tctgtgtgat ggtgttggtg gagtccctgg atgatgggat agggactttg 180
gaggctcatt tgagggagat gctaaaacaa tcctatggct ggagggatag ttggggctgt 240
agttggagat tttcagtttt tagaataaaa gtattagctg cggaatatac ttcaggacca 300
cctctgtgac agcatttata cagtatccga tgcataggga caaagagtgg agtggggcac 360
tttctttaga tttgtgagga atgttccaca ctagattgtt taaaacttca tttgttggaa 420
ggagagctgt cttagtgatt gagtcaaggg agaaaggcat ctagcctcgg tctcaaaagg 480
gtagttgctg tctagagagg tctggtggag cctgcaaaag tccagctttc aaaggaacac 540
agaagtatgt gtatggaata ttagaagatg ttgcttttac tcttaagttg gttcctagga 600
aaaatagtta aatactgtga ctttaaaatg tgagagggtt ttcaagtact cattttttta 660
aatgtccaaa attcttgtca atcagtttga ggtcttgttt gtgtagaact gatattactt 720
aaagtttaac cgaggaatgg gagtgaggct ctctcataac ctattcagaa ctgactttta 780
acaataataa attaagtttc aaatattttt aaatgaattg agcaatgttg agttggagtc 840
aagatggccg atcagaacca gaacacctgc agcagctggc aggaagcagg tcatgtggca 900
aggctatttg gggaagggaa aataaaacca ctaggtaaac ttgtagctgt ggtttgaaga 960
agtggttttg aaacactctg tccagcccca ccaaaccgaa agtccaggct gagcaaaaca 1020
ccacctgggt aatttgcatt tctaaaataa gttgaggatt cagccgaaac tggagaggtc 1080
ctcttttaac ttattgagtt caacctttta attttagctt gagtagttct agtttcccca 1140
aacttaagtt tatcgacttc taaaatgtat ttagaattca ttttcaaaat taggttatgt 1200
aagaaattga aggactttag tgtctttaat ttctaatata tttagaaaac ttcttaaaat 1260
tactctatta ttcttccctc tgattattgg tctccattca attcttttcc aatacccgaa 1320
gcatttacag tgactttgtt catgatcttt tttagttgtt tgttttgcct tactattaag 1380
actttgacat tctggtcaaa acggcttcac aaatcttttt caagaccact ttctgagtat 1440
tcattttagg agaaagactt tttttttaaa tgaatgcaat tatctagact tatttcagtt 1500
gaacatgctg gttggtggtt gagaggacac tcagtcagtc agtgacgtga agggcttcta 1560
agccagtcca catgctctgt gtgaactccc tctggccctg cttattgttg aatgggccaa 1620
aggtctgaga ccaggctgct gctgggtagg cctggacttt gggtctccca cccagacctg 1680
ggaatgtatg gttgtggctt ctgccaccca tccacctggc tgctcatgga ccagccagcc 1740
tcggtggctt tgaaggaaca attccacaca aagactctgg acctctccga aaccaggcac 1800
cgcaaatggt aagccagagg cagccacagc tgtggctgct gctcttaaag cttgtaaact 1860
gtttctgctt aagagggact gagtcttcag tcattgcttt agggggagaa agagacattt 1920
gtgtgtcttt tgagtaccgt tgtctgggtc actcacattt aactttcctt gaaaaactag 1980
taaaagaaaa atgttgcctg ttaaccaata atcatagagc tcatggtact ttgaggaaat 2040
cttagaaagc gtgtatacaa ttgtctggaa ttatttcagt taagtgtatt agttgaggta 2100
ctgatgctgt ctctacttca gttatacatg tgggtttgaa ttttgaatct attctggctc 2160
ttcttaagca gaaaatttag ataaaatgga tacctcagtg gtttttaatg gtgggtttaa 2220
tatagaagga atttaaattg gaagctaatt tagaatcagt aaggagggac ccaggctaag 2280
aaggcaatcc tgggattctg gaagaaaaga tgtttttagt ttttatagaa aacactacta 2340
cattcttgat ctacaactca atgtggttta atgaatttga agttgccagt aaatgtactt 2400
cctggttgtt aaagaatggt atcaaaggac agtgcttaga tccgaggtga gtgtgagagg 2460
acaggggctg gggtatggat acgcagaagg aaggccacag ctgtacagaa ttgagaaaga 2520
atagagacct gcagttgagg ccagcaggtc ggctggacta actctccagc cacagtaatg 2580
acccagacag agaaagccag actcataaag cttgctgagc aaaattaagg gaacaaggtt 2640
gagagcccta gtaagcgagg ctctaaaaag cacagctgag ctgagatggg tgggcttctc 2700
tgagtgcttc taaaatgcgc taaactgagg tgattactct gaggtaagca aagctgggct 2760
tgagccaaaa tgaagtagac tgtaatgaac tggaatgagc tgggccgcta agctaaacta 2820
ggctggctta accgagatga gccaaactgg aatgaacttc attaatctag gttgaataga 2880
gctaaactct actgcctaca ctggactgtt ctgagctgag atgagctggg gtgagctcag 2940
ctatgctacg ctgtgttggg gtgagctgat ctgaaatgag atactctgga gtagctgaga 3000
tggggtgaga tggggtgagc tgagctgggc tgagctagac tgagctgagc tagggtgagc 3060
tgagctgggt gagctgagct aagctggggt gagctgagct gagcttggct gagctagggt 3120
gagctgggct gagctggggt gagctgagct gagctggggt aagctgggat gagctggggt 3180
gagctgagct gagctggagt gagctgagct gggctgagct ggggtgagct gggctgagct 3240
gggctgagct gggctgagct ggggtgagct gagctggggt gagctgagct gagctggggt 3300
gagctgagct gagctggggt gagctggggt gagctgagct ggggtgagct gagctgagct 3360
ggggtgagct gagctggggt gagctgagct gagctggggt gagctgagct gagctgagct 3420
gagctgagct ggggtgagct gagctgagct gagctggggt gagctggggt gagctgagct 3480
gagctggagt gagctgagct gggctgagct ggggtgagct gggctgagct ggggtgagct 3540
gagctgagct gagctgagct ggggtgagct gagctgagct ggggtgagct gagctggggt 3600
gagctgggct gagctgagct gagctgagct gagctgagct gagctgagct gagctgagct 3660
gagctgagct gagctgagct gagctgagct gagctggggt gagctgagct gagctgggct 3720
gagctggggt gagctgggct gagctgggct gagctgggct gagctggggt gagctgagct 3780
ggggtgagct gagctgagct gggctgagct gagctgagct ggggtgagct gagctgagct 3840
ggggtgagct gagctgagct gagctggggt gagctgagct gagctgggct gagcagggct 3900
gagctggggt gagctgagct gagctggggt gagctgggct gagctgggct gagctgagct 3960
gagctgggct gagctgggct gagctgggct gagctgggct gagctgggct gagctggggt 4020
gagctgagct ggggtgagct ggggtgagct gagctggggt gagctgagct ggggtgagct 4080
gagctgagct ggggtgagct gagctggggt gagctgagct gagctggggt gagctgagct 4140
gagctggggt gagctgagct agggtgaact gggctgggtg agctggagtg agctgagctg 4200
aggtgaactg gggtgagccg ggatgttttg agttgagctg gggtaagatg agctgaactg 4260
gggtaaactg ggatgagctg tggtgagcgg agctggattg aactgagctg tgtgagctga 4320
gctggggtca gctgagcaag agtgagtaga gctggctggc cagaaccaga atcaattagg 4380
ctaagtgagc cagattgtgc tgggatcagc tgtactcaga tgagctggga tgaggtaggc 4440
tgggatgagc tgggctagct gacatggatt atgtgaggct gagctagcat gggctggcct 4500
agctgatgag ctaagcttga atgagcgggg ctgagctgga ctcagatgtg ctagactgag 4560
ctgtactgga tgatctggtg tagggtgatc tggactcaac tgggctggct gatgggatgc 4620
gccaggttga actaggctca gataagttag gctgagtagg gcctggttga gatggttcgg 4680
gatgagctgg gaaaagatgg actcggacca tgaactgggc tgagctgggt tgggagacca 4740
tgaattgagc tgaactgagt gcagctggga taaactgggt tgagctaaga atagactacc 4800
tgaattgtgc caaactcggc tgggatcaat tggaaattat caggatttag atgagccgga 4860
ctaaactatg ctgagctgga ctggttggat gtgttgaact ggcctgctgc tgggctggca 4920
tagctgagtt gaacttaaat gaggaaggct gagcaaggct agcctgcttg catagagctg 4980
aactttagcc tagcctgagc tggaccagcc tgagctgagt aggtctaaac tgagttaaaa 5040
atcaacaggg ataatttaac agctaattta acaagcctga ggtctgagat tgaatgagca 5100
gagctgggat gaactgaatg agtttcacca ggcctggacc agttaggcta ggacctcgtt 5160
ctatagaggc agactgtgtg ctacagtgga gtttcaagat gattccatga gtcctccccg 5220
cccccaacat aacccacctt cctcctaccc tacacgcctg tctggtgtgt aaatcccagc 5280
tttgtgtgct gatacagaag cctgagcccc tcccccacct ccacctacct attactttgg 5340
gatgagaata gttctcccag ccagtgtctc agagggaagc caagcaggac aggcccaagg 5400
ctacttgaga agccaggatc taggcctctc cctgagaacg ggtgttcatg cccctagagt 5460
tggctgaagg gccagatcca cctactctag aggcatctct ccctgtctgt gaaggcttcc 5520
aaagtcacgt tcctgtggct agaaggcagc tccatagccc tgctgcagtt tcgtcctgta 5580
taccaggttc acctactacc atatctagcc ctgcctgcct taagagtagc aacaaggaaa 5640
tagcagggtg tagagggatc tcctgtctga caggaggcaa gaagacagat tcttacccct 5700
ccatttctct tttatccctc tctggtcctc agagagtcag tccttcccaa atgtcttccc 5760
cctcgtctcc tgcgagagcc ccctgtctga taagaatctg gtggccatgg gctgcctggc 5820
ccgggacttc ctgcccagca ccatttcctt cacctggaac taccagaaca acactgaagt 5880
catccagggt atcagaacct tcccaacact gaggacaggg ggcaagtacc tagccacctc 5940
gcaggtgttg ctgtctccca agagcatcct tgaaggttca gatgaatacc tggtatgcaa 6000
aatccactac ggaggcaaaa acaaagatct gcatgtgccc attccaggta agaaccaaac 6060
cctcccagca ggggtgccca ggcccaggca tggcccagag ggagcagcgg ggtggggctt 6120
aggccaagct gagctcacac cttgaccttt cattccagct gtcgcagaga tgaaccccaa 6180
tgtaaatgtg ttcgtcccac cacgggatgg cttctctggc cctgcaccac gcaagtctaa 6240
actcatctgc gaggccacga acttcactcc aaaaccgatc acagtatcct ggctaaagga 6300
tgggaagctc gtggaatctg gcttcaccac agatccggtg accatcgaga acaaaggatc 6360
cacaccccaa acctacaagg tcataagcac acttaccatc tctgaaatcg actggctgaa 6420
cctgaatgtg tacacctgcc gtgtggatca caggggtctc accttcttga agaacgtgtc 6480
ctccacatgt gctgccagtg agtggcctgg gctaagccca atgcctagcc ctcccagatt 6540
agggaagtcc tcctacaatt atggccaatg ccacccagac atggtcattt gctccttgaa 6600
ctttggctcc ccagagtggc caaggacaag aatgagcaat aggcagtaga ggggtgagaa 6660
tcagctggaa ggaccagcat cttcccttaa gtaggtttgg gggatggaga ctaagctttt 6720
ttccaacttc acaactagat atgtcataac ctgacacagt gttctcttga ctgcaggtcc 6780
ctccacagac atcctaacct tcaccatccc cccctccttt gccgacatct tcctcagcaa 6840
gtccgctaac ctgacctgtc tggtctcaaa cctggcaacc tatgaaaccc tgaatatctc 6900
ctgggcttct caaagtggtg aaccactgga aaccaaaatt aaaatcatgg aaagccctcc 6960
caatggcacc ttcagtgcta agggtgtggc tagtgtttgt gtggaagact ggaataacag 7020
gaaggaattt gtgcgtactg tgactcacag ggatctgcct tcaccacaga agaaattcat 7080
ctcaaaaccc aatggtaggt atcccccctt cccttcccct ccaattgcag gacccttcct 7140
gtacctcata gggagggcag gtcctcttcc accctatcct cactactgtc ttcatttaca 7200
gaggtgcaca aacatccacc tgctgtgtac ctgctgccac cagctcgtga gcaactgaac 7260
ctgagggagt cagccacagt cacctgcctg gtgaagggct tctctcctgc agacatcagt 7320
gtgcagtggc ttcagagagg gcaactcttg ccccaagaga agtatgtgac cagtgccccg 7380
atgccagagc ctggggcccc aggcttctac tttacccaca gcatcctgac tgtgacagag 7440
gaggaatgga actccggaga gacctatacc tgtgttgtag gccacgaggc cctgccacac 7500
ctggtgaccg agaggaccgt ggacaagtcc actggtaaac ccacactgta caatgtctcc 7560
ctgatcatgt ctgacacagg cggcacctgc tattgaccat gctagcgctc aaccaggcag 7620
gccctgggtg tccagttgct ctgtgtatgc aaactaacca tgtcagagtg agatgttgca 7680
ttttataaaa attagaaata aaaaaaatcc attcaaacgt cactggtttt gattatacaa 7740
tgctcatgcc tgctgagaca gttgtgtttt gcttgctctg cacacaccct gcatacttgc 7800
ctccaccctg gcccttcctc taccttgcca gtttcctcct tgtgtgtgaa ctcagtcagg 7860
cttacaacag acagagtatg aacatgcgat tcctccagct acttctagat atatggctga 7920
aagcttgc 7928
<210> 13
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> GLSG
<400> 13
Gly Leu Ser Gly
1
<210> 14
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> linker-GGGGS
<400> 14
Gly Leu Ser Gly Ser Gly
1 5
<210> 15
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> linker-GLSGLSGS
<400> 15
Gly Leu Ser Gly Leu Ser Gly Ser
1 5
<210> 16
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> GLSGLSGLSG Joint
<400> 16
Gly Leu Ser Gly Leu Ser Gly Leu Ser Gly
1 5 10
<210> 17
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> GLSGGSGLSG
<400> 17
Gly Leu Ser Gly Gly Ser Gly Leu Ser Gly
1 5 10
<210> 18
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> 5' detection of VH1-69 FR3
<400> 18
acagaagttc cagggcagag 20
<210> 19
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> 3' detection of spec
<400> 19
tgtccactgg gttcgtgcct t 21
<210> 20
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> 5' lower detection (spec)
<400> 20
cagtatcagc ccgtcatact t 21
<210> 21
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> 3' VH1-69 overlap detection
<400> 21
taacccctgt catctcctc 19
<210> 22
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> 5' DNA target (PAM)
<400> 22
ggatcctggt ttagttaaag agg 23
<210> 23
<211> 42
<212> RNA
<213> artificial sequence
<220>
<223> 5' VH1-69 Cas9 crRNA
<400> 23
ggauccuggu uuaguuaaag guuuuagagc uaugcuguuu ug 42
<210> 24
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> 3' DNA target (PAM)
<400> 24
gacaaaaacc ctgagggaga agg 23
<210> 25
<211> 42
<212> RNA
<213> artificial sequence
<220>
<223> 3 VH1-69 Cas9 crRNA
<400> 25
gacaaaaacc cugagggaga guuuuagagc uaugcuguuu ug 42
<210> 26
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> 5' detection of VH1-69
<400> 26
ctgtgaaata ccctgcctc 19
<210> 27
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> 3' detection of spec
<400> 27
tgtccactgg gttcgtgcct t 21
<210> 28
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> 5' detection spec
<400> 28
cagtatcagc ccgtcatact t 21
<210> 29
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> 3' detection under M1116 (h 70)
<400> 29
ccccctcttg ctctctttct 20
<210> 30
<211> 80
<212> DNA
<213> artificial sequence
<220>
<223> VH1-69 MreI del conjugate oligonucleotides
<400> 30
gtgaaaaccc acatcctgag agtgacaaaa accctgaggg agaaggcagc tgtgccgggc 60
tgaggagatg acaggggtta 80
<210> 31
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> 5' detection of VH1-69 FR3
<400> 31
acagaagttc cagggcagag 20
<210> 32
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> 3' detection under M1116 (h 70)
<400> 32
ccccctcttg ctctctttct 20

Claims (99)

1. A recombinant nucleic acid molecule comprising a modified immunoglobulin (Ig) variable (V) segment encoding an anchor modified Ig polypeptide,
wherein the modified Ig V segment comprises a nucleic acid sequence encoding an anchor between the nucleic acid sequence encoding the Ig signal peptide and the nucleic acid sequences encoding the Framework Regions (FRs) 1, complementarity Determining Regions (CDRs) 1, FR2, CDR2, FR3 and CDR3 of the germline Ig V segment or variant thereof,
wherein the anchor modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) Said FR1, CDR1, FR2, CDR2, FR3 and CDR3 of said germline Ig V segment or variant thereof,
wherein the anchor comprises a receptor binding portion of a non-immunoglobulin polypeptide of interest that binds to a cognate receptor, and
optionally wherein the nucleic acid molecule lacks any other V segment.
2. The recombinant nucleic acid molecule of claim 1, wherein the Ig signal peptide is an Ig signal peptide of the germline Ig V segment or variant thereof.
3. The recombinant nucleic acid molecule of claim 1 or claim 2, wherein the germline Ig V segment or variant thereof is a germline Ig heavy chain variable (V H ) Segments or variants thereof such that
The modified Ig V segment is a modified Ig V comprising the nucleic acid sequence encoding an anchor H A segment comprising said anchor in said nucleic acid sequence encoding an Ig signal peptide and encoding said germline IgV H Between the nucleic acid sequences of the Framework Regions (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3 and CDR3 of the segments or variants thereof, and
the anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) The germline Ig V H The FR1, CDR1, FR2, CDR2, FR3 and CDR3 of the stretch or variant thereof.
4. The recombinant nucleic acid molecule of any one of claims 1-3, wherein the germline Ig V segment or variant thereof is germline human (h) V H 1-2 segment, germ line hV H 1-3 segment, germ line hV H 1-8 segment, germ line hV H 1-18 segment, germ line hV H 1-24 segment, germ line hV H 1-45 segment, germ line hV H 1-46 segment, germ line hV H 1-58 segment, germ line hV H Segment 1-69, germ line hV H 2-5 segment, germ line hV H 2-26 segment, germ line hV H 2-70 segment, germ line hV H 3-7 segment, germ line hV H 3-9 segment, germ line hV H 3-11 segment, germ line hV H 3-13 segment, germ line hV H 3-15 segment, germ line hV H 3-16 segment, germ line hV H 3-20 segment, germ line hV H 3-21 segment, germ line hV H 3-23 segment, germ line hV H 3-30 segment, germ line hV H 3-30-3 segment, germ line hV H 3-30-5 segment, germ line hV H 3-33 segment, germ line hV H 3-35 segment, germ line hV H 3-38 segment, germ line hV H 3-43 segment, germ line hV H 3-48 segment, germ line hV H 3-49 segments, germ line hV H 3-53 segment, germ line hV H 3-64 segment, germ line hV H 3-66 segment, germ line hV H 3-72 segment, germ line hV H 3-73 segment, germ line hV H 3-74 segment, germ line hV H 4-4 segment, germ line hV H 4-28 segment, germ line hV H 4-30-1 segment, germ line hV H 4-30-2 segment, germ line hV H 4-30-4 segment, germ line hV H 4-31 segment, germ line hV H 4-34 segment, germ line hV H 4-39 segment, germ line hV H 4-59 segment, germ line hV H 4-61 segment, germ line hV H 5-51 segment, germ line hV H 6-1 segment, germ line hV H 7-4-1 segment, germ line hV H 7-81 segments or variants thereof.
5. The recombinant nucleic acid molecule of any one of claims 1-4, wherein the germline Ig V segment or variant thereof is germline hV H 1-69 or a variant thereof, optionally wherein said Ig signal peptide comprises sequence MDWTWRFLFVVAAATGVQS (SEQ ID NO: 7).
6. The recombinant nucleic acid molecule of any one of claims 3 to 5, comprising the following operably linked and from 5 'to 3':
(I) The modified Ig V H A section;
(II) one or more Ig heavy chain diversity (D H ) A section; and
(III) one or more Ig heavy chain conjugation (J H ) A section.
7. The recombinant nucleic acid molecule of claim 6, wherein
The one or more Ig D of (II) H Segments comprising one, more or all human Ig D H Segments, and/or
The one or more Ig J's of (III) H Segments comprising one, more or all human Ig J H A section.
8. The recombinant nucleic acid molecule of claim 6 or claim 7, wherein the one or more Ig D of (II) H Segment and one or more Ig J of (III) H Gene segments are recombined and form rearranged Ig D H /J H A sequence such that the recombinant nucleic acid molecule comprises, operably linked and from 5 'to 3':
modified Ig V H A gene segment; and
the rearranged Ig D H /J H Sequence.
9. The recombinant nucleic acid molecule of claim 8, wherein the modified Ig V H Gene segments and the rearranged Ig D H /J H The sequences are recombined and form the coding anchorRearranged IgV of modified Ig heavy chain variable domains H /D H /J H The sequence of the sequences is set up,
wherein the anchor modified Ig heavy chain variable domain comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) IgV rearranged by the rearrangement H /D H /J H Sequence-encoded FR1, complementarity Determining Regions (CDRs) 1, FR2, CDR2, FR3, CDR3 and FR4.
10. The recombinant nucleic acid molecule of any one of claims 3-8, wherein the modified Ig V H The segments are modified Ig Vs that are not rearranged H A gene segment.
11. The recombinant nucleic acid molecule of any one of claims 6-10, further comprising a gene encoding an Ig heavy chain constant region (C H ) Is a nucleic acid sequence of (a),
wherein said IgC is encoded H Downstream of and operably linked to the nucleic acid sequence of:
(I) The modified Ig V H A section;
(II) the one or more Ig D H A section; and
(III) the one or more Ig J H A section.
12. The recombinant nucleic acid molecule of claim 11, wherein the encoding Ig C H Including Igmu gene encoding IgM isotype, igdelta gene encoding IgD isotype, iggamma gene encoding IgG isotype, igalpha gene encoding IgA isotype and/or Igepsilon gene encoding IgE isotype.
13. The recombinant nucleic acid molecule of any one of claims 3-12, comprising a nucleic acid sequence encoding an anchor modified Ig heavy chain, wherein the anchor modified Ig heavy chain comprises operably linked:
(i) The Ig signal peptide;
(ii) The anchor;
(iii) Comprising Ig V rearranged H /D H /J H Ig heavy chain variable domains of said FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 encoded by sequences; and
(iv)Ig C H 。
14. the recombinant nucleic acid molecule of any one of claims 11-13, wherein the Ig C H Non-human Ig C H 。
15. The recombinant nucleic acid molecule of claim 14, wherein the non-human Ig C H Is rodent Ig C H 。
16. The recombinant nucleic acid molecule of claim 15, wherein the non-human Ig C H Is rat Ig C H 。
17. The recombinant nucleic acid molecule of claim 15, wherein the non-human Ig C H Is mouse Ig C H 。
18. The recombinant nucleic acid molecule of claim 1 or claim 2, wherein the germline Ig V gene segment or variant thereof is germline Ig light chain variable (V L ) Segments or variants thereof such that
The modified Ig V segment is a modified Ig V comprising the nucleic acid sequence encoding an anchor L A segment comprising said anchor in said nucleic acid sequence encoding an Ig signal peptide and encoding said germline IgV L Between the nucleic acid sequences of the Framework Regions (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3 and CDR3 of the segments or variants thereof, and
the anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) By a means ofGermline Ig V L The FR1, CDR1, FR2, CDR2, FR3 and CDR3 of the stretch or variant thereof.
19. The recombinant nucleic acid molecule of claim 18, comprising the following operably linked and from 5 'to 3':
(I) The modified Ig V L A section; and
(II) one or more Ig light chain engagements (J L ) A section.
20. The recombinant nucleic acid molecule of claim 19, wherein the modified Ig V L A segment and the one or more J L The segments are recombined and form rearranged Ig V encoding an anchor modified Ig light chain variable domain L /J L The sequence of the sequences is set up,
wherein the anchor modified Ig light chain variable domain comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) IgV rearranged by the rearrangement L /J L Sequence-encoded FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
21. The recombinant nucleic acid molecule of claim 19 or claim 20, further comprising a gene encoding an Ig light chain constant region (C L ) Is a nucleic acid sequence of (a),
wherein said IgC is encoded L Downstream of and operably linked to the nucleic acid sequence of:
(I) The modified Ig V L A section; and
(II) the one or more Ig light chain engagements (J L ) A section.
22. The recombinant nucleic acid molecule of any one of claims 18-21, comprising a nucleic acid sequence encoding an anchor modified Ig light chain, wherein the anchor modified Ig light chain comprises operably linked:
(i) The Ig signal peptide;
(ii) The anchor;
(iii) Comprising Ig V rearranged L /J L Ig light chain variable domains of said FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 encoded by sequences; and
(iv)Ig C L 。
23. The recombinant nucleic acid molecule of claim 21 or claim 22, wherein the Ig C L Non-human Ig C L 。
24. The recombinant nucleic acid molecule of claim 23, wherein the non-human Ig C L Is rodent Ig C L 。
25. The recombinant nucleic acid molecule of claim 23, wherein the non-human Ig C L Is rat Ig C L 。
26. The recombinant nucleic acid molecule of claim 23, wherein the non-human Ig C L Is mouse Ig C L 。
27. The recombinant nucleic acid molecule of any one of claims 18-26, wherein the germline Ig V L The segment or variant thereof is a germline Ig light chain variable kappa (V kappa) segment or variant thereof such that
The modified Ig V segment is a modified Ig V kappa segment comprising the nucleic acid sequence encoding an anchor between the nucleic acid sequence encoding an Ig signal peptide and a nucleic acid sequence encoding a Framework Region (FR) 1, a Complementarity Determining Region (CDR) 1, FR2, CDR2, FR3 and CDR3 of the germline Ig V kappa segment or variant thereof, and
the anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) Said FR1, CDR1, FR2, CDR2, FR3 and CDR3 of said germline Ig vκ segment or variant thereof.
28. The recombinant nucleic acid molecule of claim 27, comprising the following operably linked and from 5 'to 3':
(I) The modified Ig vκ segment; and
(II) one or more Ig light chain engagement kappa (jk) segments.
29. The recombinant nucleic acid molecule of claim 28, further comprising a nucleic acid sequence encoding an Ig light chain constant kappa region (Ckappa),
wherein the nucleic acid sequence encoding Ig ck is located downstream of and operably linked to:
(I) The modified Ig vκ segment; and
(II) the one or more Ig jκ segments.
30. The recombinant nucleic acid molecule of any one of claims 18-26, wherein the germline Ig V L The segment or variant thereof is a germline Ig light chain variable lambda (V lambda) segment or variant thereof such that
The modified Ig V segment is a modified Ig V lambda segment comprising the nucleic acid sequence encoding an anchor between the nucleic acid sequence encoding an Ig signal peptide and a nucleic acid sequence encoding a Framework Region (FR) 1, complementarity Determining Regions (CDR) 1, FR2, CDR2, FR3 and CDR3 of the germline Ig V lambda segment or variant thereof, and
the anchor-modified Ig polypeptide comprises the following operably linked:
(i) The Ig signal peptide;
(ii) The anchor; and
(iii) Said FR1, CDR1, FR2, CDR2, FR3 and CDR3 of said germline Ig V lambda segment or variant thereof.
31. The recombinant nucleic acid molecule of claim 30, comprising the following operably linked and from 5 'to 3':
(I) The modified Ig vλ segment; and
(II) one or more Ig light chains engages a lambda (jlambda) segment.
32. The recombinant nucleic acid molecule of claim 31, further comprising a nucleic acid sequence encoding an Ig light chain constant lambda region (C lambda),
wherein the nucleic acid sequence encoding Ig C lambda is located downstream of and operably linked to:
(I) The modified Ig vλ segment; and
(II) the one or more Ig jλ segments.
33. The recombinant nucleic acid molecule of any one of claims 1-32, wherein the anchor comprises a linker that connects the receptor binding portion of a non-immunoglobulin polypeptide of interest to the FR1, CDR1, FR2, CDR2, FR3, and CDR3 of the germline Ig V segment or variant thereof.
34. The recombinant acid molecule of claim 33, wherein the linker comprises the sequence GGGGS (SEQ ID NO: 5).
35. The recombinant nucleic acid molecule of any one of claims 1-34, wherein the anchor comprises a Natriuretic Peptide Receptor (NPR) binding portion of a Natriuretic Peptide (NP).
36. The recombinant nucleic acid molecule of claim 35, wherein the NPR-binding portion of the NP comprises a C-terminal tail of the NP.
37. The recombinant nucleic acid molecule of claim 35 or claim 36, wherein the NP is Atrial Natriuretic Peptide (ANP).
38. The recombinant nucleic acid molecule of any one of claims 1-37, wherein the anchor comprises the sequence nsfray (SEQ ID NO: 3).
39. The recombinant nucleic acid molecule of any one of claims 1 to 38, comprising a sequence selected from the group consisting of: a sequence shown as SEQ ID NO. 8 or a degenerate variant thereof, a sequence shown as SEQ ID NO. 10 or a degenerate variant thereof, SEQ ID NO. 11 of a degenerate variant thereof, and SEQ ID NO. 12 or a degenerate variant thereof.
40. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 10 and 33 to 39, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig heavy chain locus, such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a non-human Ig C at the non-human Ig heavy chain locus H A recombinant nucleic acid molecule upstream of and operably linked to, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus, and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, an endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof.
41. The targeting vector according to claim 40, wherein upon homologous recombination between said targeting vector and said non-human Ig heavy chain locus, said recombinant nucleic acid molecule replaces a non-human V at said non-human Ig heavy chain locus H A section.
42. The targeting vector according to claim 40 or 41, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the recombinant nucleic acid molecule replaces one or more non-human V at the non-human Ig heavy chain locus H Segment, all non-human D H Segment and all non-human J H A section.
43. The targeting vector according to anyone of the claims 40 to 42, wherein upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeting vectorReplacement of all but one non-human V at the non-human Ig heavy chain locus by a recombinant nucleic acid molecule H Segment or all non-human V H Segment, all non-human D H Segment and all non-human J H A section.
44. The targeting vector according to anyone of the claims 40 to 43, wherein upon homologous recombination between said targeting vector and said non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at said non-human Ig heavy chain locus.
45. The targeting vector according to anyone of the claims 40 to 44, wherein said 5 'homology arm comprises the sequence shown as SEQ ID NO. 11 and/or said 3' homology arm comprises the sequence shown as SEQ ID NO. 12.
46. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 17 and 33 to 39, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig heavy chain locus, such that upon homologous recombination between the targeting vector and the non-human Ig heavy chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig heavy chain regulatory sequence at the non-human Ig heavy chain locus, optionally wherein the non-human Ig heavy chain locus is an endogenous rodent Ig heavy chain locus, and/or wherein the non-human Ig heavy chain locus comprises a human or humanized immunoglobulin heavy chain variable region, endogenous Ig V H 、D H And/or J H Deletion of a gene segment or a combination thereof.
47. The targeting vector according to claim 46, wherein upon homologous recombination between said targeting vector and said non-human Ig heavy chain locus, said recombinant nucleic acid molecule replaces one or more non-human V at said non-human Ig heavy chain locus H Segment, all non-human D H Gene segment, all non-human J H Gene segment and one or more non-human C H And (3) a gene.
48. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 20, 27 to 28, 30 to 31, and 33 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a non-human Ig C at the non-human Ig light chain locus L A recombinant nucleic acid molecule upstream of and operably linked to, optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, an endogenous Ig V L And/or J L Deletion of a gene segment or a combination thereof.
49. The targeting vector according to claim 48, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain locus, said recombinant nucleic acid molecule replaces a non-human V at said non-human Ig light chain locus L A section.
50. The targeting vector according to claim 48 or 49, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain locus, said recombinant nucleic acid molecule replaces one or more non-human V at said non-human Ig light chain locus L Segment and all non-human J L A section.
51. The targeting vector according to anyone of the claims 48 to 50, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the recombinant nucleic acid molecule replaces all non-human V at the non-human Ig light chain locus L Segment and all non-human J H A section.
52. The targeting vector according to anyone of the claims 48 to 51, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig heavy chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the Ig light chain locus.
53. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain locus, the targeted non-human Ig light chain locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain regulatory sequence at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain locus is an endogenous rodent Ig light chain locus, and/or wherein the non-human Ig light chain locus comprises a human or humanized immunoglobulin light chain variable region, endogenous Ig V L And/or J L Deletion of a gene segment or a combination thereof.
54. The targeting vector according to claim 53, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain locus, said recombinant nucleic acid molecule replaces a non-human V at said non-human Ig light chain locus L Segment, all non-human J L Gene segment and non-human C L And (3) a gene.
55. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 20, 27 to 28 and 33 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain kappa locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule upstream of and operably linked to a non-human Ig ck at the non-human Ig light chain kappa locus, optionally wherein the non-human Ig light chain kappa locus is an endogenous rodent light chain kappa locus, and/or wherein the non-human Ig light chain kappa locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig vk and/or jk gene segments, or a combination thereof.
56. The targeting vector of claim 55, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain kappa locus, said recombinant nucleic acid molecule replaces a non-human vkappa segment at said non-human Ig light chain kappa locus.
57. The targeting vector according to claim 55 or 56, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain kappa locus, said recombinant nucleic acid molecule replaces one or more non-human V kappa segments and all non-human J kappa segments at said non-human Ig light chain kappa locus.
58. The targeting vector of any one of the claims 55 to 57, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the recombinant nucleic acid molecule replaces all non-human vk segments and all non-human jk segments at the non-human Ig light chain kappa locus.
59. The targeting vector according to anyone of the claims 55 to 58, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at said Ig light chain kappa locus.
60. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1-2 and 18-29, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain kappa locus such that upon homologous recombination between the targeting vector and the non-human Ig light chain kappa locus, the targeted non-human Ig light chain kappa locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain kappa regulatory sequence at the Ig light chain kappa locus.
61. The targeting vector of claim 60, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain kappa locus, said recombinant nucleic acid molecule replaces a non-human vk segment, all non-human jk gene segments and a non-human ck gene at said non-human Ig light chain kappa locus.
62. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 20, 30 to 31 and 33 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule upstream of and operably linked to a non-human Ig C lambda at the non-human Ig light chain locus, optionally wherein the non-human Ig light chain rodent light chain lambda locus is an endogenous Ig light chain lambda locus, and/or wherein the non-human Ig light chain lambda locus comprises a human or humanized immunoglobulin light chain variable region, a deletion of endogenous Ig V lambda and/or J lambda gene segments, or a combination thereof.
63. The targeting vector of claim 62, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain lambda locus, said recombinant nucleic acid molecule replaces a non-human vlambda segment at said non-human Ig light chain lambda locus.
64. The targeting vector according to claim 62 or 63, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain lambda locus, said recombinant nucleic acid molecule replaces one or more non-human va segments and all non-human jlambda segments at said non-human Ig light chain locus.
65. The targeting vector according to anyone of the claims 62 to 64, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the recombinant nucleic acid molecule replaces all non-human V lambda segments and all non-human J lambda segments at the non-human Ig light chain lambda locus.
66. The targeting vector according to anyone of the claims 62 to 65, wherein upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus.
67. A targeting vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 2, 18 to 26 and 30 to 38, wherein the targeting vector further comprises 5 'and 3' homology arms that target a non-human Ig light chain lambda locus, such that upon homologous recombination between the targeting vector and the non-human Ig light chain lambda locus, the targeted non-human Ig light chain lambda locus comprises a recombinant nucleic acid molecule operably linked to a non-human Ig light chain lambda regulatory sequence at the Ig light chain lambda locus.
68. The targeting vector of claim 67, wherein upon homologous recombination between said targeting vector and said non-human Ig light chain lambda locus, said recombinant nucleic acid molecule replaces a non-human va segment, all non-human jlambda gene segments and a non-human cλ gene at said non-human Ig light chain lambda locus.
69. A non-human animal genome comprising the recombinant nucleic acid molecule of any one of claims 1 to 39 or the targeting vector of any one of claims 40 to 68, optionally wherein the non-human animal is a rodent, optionally wherein the rodent is a rat or mouse.
70. The non-human animal genome of claim 69, wherein a recombinant nucleic acid is located at an endogenous Ig locus of the non-human animal genome.
71. A non-human animal or non-human animal cell comprising the recombinant nucleic acid molecule of any one of claims 1 to 39, the targeting vector of any one of claims 40 to 68, or the non-human animal genome of claim 69 or claim 70.
72. The non-human animal or non-human animal cell of claim 71, wherein the recombinant nucleic acid molecule, the targeting vector, or the non-human animal genome is in the germline of the non-human animal or non-human animal cell.
73. An in vitro method of modifying an isolated cell, the method comprising introducing the recombinant nucleic acid molecule of any one of claims 1 to 39 into the isolated cell.
74. The in vitro method of claim 73, wherein introducing comprises contacting the cells with a targeting vector according to any one of claims 40 to 68.
75. The in vitro method of claim 73 or claim 74, wherein the cell is a host cell.
76. The in vitro method of claim 73 or claim 74, wherein the cells are Embryonic Stem (ES) cells.
77. The in vitro method of any one of claims 73 to 76, wherein the cell is a rodent cell, optionally wherein the rodent cell is a rat cell or a mouse cell.
78. A non-human animal embryo produced from the embryonic stem cell of claim 76.
79. A non-human animal produced from the embryonic stem cell of claim 76.
80. A method of making a non-human animal comprising implanting the ES cell of claim 76 or an embryo comprising the ES cell into a suitable host and maintaining the host under suitable conditions during the development of the ES cell or embryo into viable offspring.
81. The non-human animal of any one of claims 71-72 and 79 or the non-human animal prepared according to the method of claim 80, wherein the non-human animal comprises, as compared to a control non-human animal:
(a) A significant number of mature B cells in the spleen;
(b) A significant number of kappa positive B cells in the spleen;
(c) A significant number of lambda positive B-cells in the spleen;
(d) Serum IgG at comparable levels; and/or
(e) Serum IgM at comparable levels.
82. The non-human animal of any one of claims 71-72, 79 and 81 or the non-human animal prepared according to the method of claim 80, wherein the non-human animal is capable of producing an immune response comparable to a control non-human animal.
83. The non-human animal of any one of claims 71-72, 79 and 81-82 or the non-human animal prepared according to the method of claim 80, comprising a plurality of antigen binding proteins each comprising the anchor modified Ig polypeptide, optionally:
wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide,
wherein the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
84. The non-human animal of any one of claims 71-72, 79 and 81-83 or the non-human animal prepared according to the method of claim 80, wherein the non-human animal further comprises the cognate receptor for the non-immunoglobulin polypeptide of interest.
85. The non-human animal of any one of claims 71-72, 79 and 81-84 or the non-human animal prepared according to the method of claim 80, comprising a plurality of antigen binding proteins each comprising the anchor modified Ig polypeptide and specifically binding to the cognate receptor of the non-immunoglobulin polypeptide of interest, optionally:
wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide,
wherein the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
86. The non-human animal of any one of claims 84-85, wherein the cognate receptor is a Natriuretic Peptide Receptor (NPR).
87. The non-human animal of any one of claims 84-86, wherein each of the plurality of antigen binding proteins comprises less than 1 x 10 9 And/or t1/2 over 30 minutes.
88. The non-human animal of any one of claims 84-87, wherein at least 15% of the plurality of antigen binding proteins block binding of the cognate receptor to the non-immunoglobulin polypeptide of interest.
89. The non-human animal of any one of claims 84-88, wherein more than 50% of the plurality of antigen binding proteins bind to the cognate receptor expressed on the cell surface.
90. The non-human animal of any one of claims 71-72, 79 and 81-89 or the non-human animal prepared according to the method of claim 80, wherein the non-human animal is a rodent, optionally wherein the rodent is a rat or mouse.
91. A method of producing an antigen binding protein or obtaining a nucleic acid encoding the antigen binding protein, the method comprising:
immunizing a non-human animal according to any one of claims 71 to 72, 79 and 81 to 90 or a non-human animal prepared according to the method of claim 80 with an antigen;
Allowing the non-human animal to produce an antigen binding protein comprising the anchor modified Ig polypeptide or a nucleic acid encoding the antigen binding protein, which antigen binding protein binds to the antigen, optionally:
wherein the mass of said antigen binding protein confirms the presence of said anchor modified Ig polypeptide,
wherein the mass of the antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of the antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of the antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
92. The method of claim 91, further comprising recovering the antigen binding protein or nucleic acid encoding the antigen binding protein from the non-human animal or non-human animal cell.
93. The method of claim 92, wherein the non-human animal cell is a B cell or a hybridoma.
94. A non-human animal cell recovered according to the method of claim 91.
95. The non-human animal of claim 94, wherein the non-human animal cell is a B cell.
96. The non-human animal cell of claim 94 or 95, wherein the B cell is a mouse B cell.
97. A hybridoma cell comprising the non-human animal cell of any one of claims 94-95 fused to a myeloma cell.
98. An anchor modified Ig polypeptide expressed by the recombinant nucleic acid molecule of any one of claims 1 to 39, the targeting vector of any one of claims 40 to 68, the non-human animal genome of any one of claims 69 to 70, by the non-human animal or non-human animal cell of any one of claims 71 to 72 and 81 to 90, by the non-human animal or non-human animal cell prepared by the method of any one of claims 73 to 76 and 80, or by the method of any one of claims 91 to 93, optionally:
wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide,
wherein the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, or
Wherein the mass of each antigen binding protein confirms the presence of the anchor modified Ig polypeptide and the mass of each antigen binding protein is determined by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
99. The anchor-modified Ig polypeptide of claim 98, comprising an amino acid sequence at its N-terminus shown as SEQ ID No. 3.
CN202180085965.3A 2020-12-23 2021-12-20 Nucleic acids encoding anchor modified antibodies and uses thereof Pending CN116635407A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311142098.XA CN117186227A (en) 2020-12-23 2021-12-20 Nucleic acids encoding anchor modified antibodies and uses thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US63/129,893 2020-12-23
US202163219402P 2021-07-08 2021-07-08
US63/219,402 2021-07-08
PCT/US2021/064263 WO2022140219A1 (en) 2020-12-23 2021-12-20 Nucleic acids encoding anchor modified antibodies and uses thereof

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN202311142098.XA Division CN117186227A (en) 2020-12-23 2021-12-20 Nucleic acids encoding anchor modified antibodies and uses thereof

Publications (1)

Publication Number Publication Date
CN116635407A true CN116635407A (en) 2023-08-22

Family

ID=87642238

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202311142098.XA Pending CN117186227A (en) 2020-12-23 2021-12-20 Nucleic acids encoding anchor modified antibodies and uses thereof
CN202180085965.3A Pending CN116635407A (en) 2020-12-23 2021-12-20 Nucleic acids encoding anchor modified antibodies and uses thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202311142098.XA Pending CN117186227A (en) 2020-12-23 2021-12-20 Nucleic acids encoding anchor modified antibodies and uses thereof

Country Status (1)

Country Link
CN (2) CN117186227A (en)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5698679A (en) * 1994-09-19 1997-12-16 National Jewish Center For Immunology And Respiratory Medicine Product and process for targeting an immune response
CA2897963A1 (en) * 2013-02-20 2014-08-28 Regeneron Pharmaceuticals, Inc. Non-human animals with modified immunoglobulin heavy chain sequences
EP3064507A1 (en) * 2015-03-06 2016-09-07 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Fusion proteins comprising a binding protein and an interleukin-15 polypeptide having a reduced affinity for IL15ra and therapeutic uses thereof
SG11202008799PA (en) * 2018-03-24 2020-10-29 Regeneron Pharma Genetically modified non-human animals for generating therapeutic antibodies against peptide-mhc complexes, methods of making and uses thereof

Also Published As

Publication number Publication date
CN117186227A (en) 2023-12-08

Similar Documents

Publication Publication Date Title
EP2825036B1 (en) Histidine engineered light chain antibodies and genetically modified rodents for generating the same
CN106255410B (en) Non-human animals producing single domain binding proteins
KR102459666B1 (en) Non-human animals expressing ph-sensitive immunoglobulin sequences
CN108611369B (en) Mice expressing restricted immunoglobulin light chain repertoire
EP3046412B1 (en) Histidine engineered light chain antibodies and genetically modified non-human animals for generating the same
US20150250151A1 (en) Histidine Engineered Light Chain Antibodies and Genetically Modified Non-Human Animals for Generating the Same
JP2018508224A (en) Non-human animals that select light chain variable regions that bind antigen
US20220174920A1 (en) Non-human animals having an immunoglobulin heavy chain variable region that includes an engineered diversity cluster and uses thereof
KR20230018439A (en) Genetically Modified Non-Human Animals with Common Light Chain Immunoglobulin Loci
CN116635407A (en) Nucleic acids encoding anchor modified antibodies and uses thereof
US20220195014A1 (en) Nucleic acids encoding anchor modified antibodies and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination