methods of producing mice genetically modified with T cell receptors, producing T cell receptors and

专利摘要:
  METHODS OF PRODUCTION OF GENETICALLY MODIFIED MICE WITH T-CELL RECEPTORS, OF PRODUCTION OF T-CELL RECEPTORS AND OF T-CELL IDENTIFICATION, AS WELL AS NUCLEIC ACIDThe present invention relates to a method for producing a genetically modified non-human animal by modifying its genome in order to comprise a locus of the non-rearranged T cell receptor (TCR) gene (Alpha) comprising at least one V segment Human (Alpha) and at least one human J (Alpha) segment, operably linked to a non-human TCR (Alpha) constant gene sequence; and / or locus of the non-rearranged TCR (Beta) variable gene comprising at least one human V (Beta) segment, at least one human D (Beta) segment and at least one human J (Beta) segment, operably linked to a non-human TCR (Beta) constant gene sequence. The present invention further relates to methods for producing human T cell receptor for antigen of interest and for identifying T cell with specificity against antigen of interest, as well as nucleic acid.
公开号:BR112014009203A2
申请号:R112014009203-6
申请日:2012-10-26
公开日:2020-10-27
发明作者:Lynn MacDonald；Andrew J. Murphy；John McWhirter；Naxin Tu；Vera Veronina；Cagan Gurer；Karolina MEAGHER；Sean Stevens
申请人:Regeneron Pharmaceuticals, Inc.；
IPC主号:

专利说明:

[001] [001] This claim claims the priority benefit for the US Provisional Order. No. 61 / 552,582 deposited on October 28, 2011; US Provisional Order. No. 61 / 621,198 filed on April 6, 2012; and US Provisional Order. No. 61 / 700,908 filed on September 14, 2012, all of which are incorporated herein by reference. Field of the Invention
[002] [002] The present invention relates to a genetically modified non-human animal, for example, a rodent (for example, a mouse or a rat), which comprises in its genome the loci of the T cell receptor variable region gene ( Humanized or human (for example, the loci of the TORa and TCR variable region gene be / or the loci of the TORd and TORg variable region gene), and expresses human or humanized TOR polypeptides (for example, poly- TOCRa and TCRb peptides and / or TCRd and TOCRg polypeptides) from the loci of the human or humanized TOR variable region gene. A non-human animal with the loci of the human or humanized TOR variable region gene comprises segments of the non-rearranged human TCR variable region gene (for example, segments V, D and / or J) on the loci of the endogenous non-human TOR gene . The invention also relates to embryos, tissues, and cells (e.g., T cells) that comprise the loci of the human or humanized TOR variable region gene and express human or humanized TOR polypeptides. Methods are also provided to make the non-human animal genetically modified comprising the gene loci of the human or humanized TOR variable region; and method for using non-human animals, embryos, tissues and cells that comprise the gene loci of the variable region of human or humanized TOR and express human or humanized TOR polypeptides from these loci. Background of the Invention
[003] [003] In the adaptive immune response, foreign antigens are recognized by the receptor molecules in B lymphocytes (for example, immunoglobulins) and T lymphocytes (for example, T cell receptors or TCRs). While pathogens in the blood and extracellular space are recognized by antibodies in the course of the human immune response, the destruction of pathogens within cells is mediated in the course of the cellular immune response by T cells.
[004] [004] T cells recognize and attack the antigens presented to them in the context of a Major Histocompatibility Complex (MHC) on the cell surface. Antigen recognition is mediated by TOCRs expressed on the surface of T cells. The two main classes of T cells serve this function: cytotoxic T cells, which express a protein on the surface of CD8 cells, and helper T cells, which express a protein on the surface of CDA4 cells. Cytotoxic T cells activate signaling cascades that result in direct destruction of the cell presenting the antigen (in the context of MHC |), while helper T cells differ in various classes, and their activation (initiated by the recognition of antigen presented in the context of MHC II) results in the destruction of pathogens mediated by macrophages and the stimulation of antibody production by B cells.
[005] [005] Because of their specificity to the antigen, antibodies are currently widely studied as to their potential therapeutic agent against numerous human disorders. To generate antibodies capable of neutralizing human targets, while simultaneously preventing the activation of immune responses against such antibodies,
[006] [006] Similar to an antibody, a T cell receptor comprises a variable region, encoded by non-rearranged locus (loci a and b, or loci of gd) comprising segments of variable region V (D) J, and this variable region confers , by means of T cells, its specificity of binding to the antigen. Also similar to an antibody, the specificity of TOR to its antigen can be used for the development of new therapeutic agents. Thus, there is a need in the art for non-human animals (for example, rodents, for example, rats or mice) that comprise genetically variable segments of non-rearranged human T cells capable of rearranging to form genes that they encode the variable domains of the T cell receptor, including the domains that are congenital to each other, and including the domains that specifically bind to an antigen of interest. There is also a need for non-human animals that comprise loci of variable region of T cells that comprise conservative humanizations, including non-human animals that comprise non-human gene segments
[007] [007] Non-human animals, for example, rodents, comprising non-human cells that express humanized molecules that function in the cellular immune response are provided. Non-human animals that comprise the loci of the non-rearranged TCR variable region gene are also provided. In vivo and in vitro systems that are supplied comprise humanized rodent cells, where the rodent cells express one or more humanized immune system molecules. TCR loci of humanized, non-rearranged rodents encoding humanized TOR proteins are also provided.
[008] [008] In one aspect, a genetically modified non-human animal (for example, a rodent, for example, a mouse or rat) is provided that comprises in its genome (a) the gene locus of the variable TORa region not rearranged comprising at least one human Voa segment and at least one human Ja segment, functionally linked to a non-human TCRa constant region gene sequence (e.g., from a rodent, e.g., a mouse or rat), and / or (b) a non-rearranged TCRb variable region locus comprising at least one human Vb segment, at least one human Db segment, and at least one human Jb segment, functionally linked to a sequence of non-human TCRb constant region gene (for example, a rodent, for example, a mouse or rat).
[009] [009] In one embodiment, the non-rearranged TCRa variable region gene locus replaces the non-human (e.g., rodent) TCRa variable region gene locus in an endogenous TORa variable region gene locus. In one embodiment, the non-rearranged TCRb variable region gene locus replaces the endogenous non-human (for example, rodent) variable region TORb gene locus. In one embodiment, the non-human (eg rodent) segments Va and Ja are unable to rearrange themselves to form a rearranged Vo / Ja sequence. In one mode, the endogenous non-human Vb, Db and Jb segments (for example, rodent) are unable to rearrange themselves to form a rearranged Vb / Db / Jb sequence. In one embodiment, the non-human animal comprises a deletion such that the animal's genome does not comprise a functional Va segment and a functional Ja segment. In one embodiment, the non-human animal comprises a deletion such that the animal's genome does not comprise a functional endogenous Vb segment, a functional endogenous Db segment, and a functional endogenous Jb segment. In one embodiment, the animal comprises a deletion of all functional endogenous Va and Ja segments. In one embodiment, the rodent comprises a deletion of all functional endogenous Vb, Db and Jb segments. In some modalities, the human Va and Ja segments rearrange to form a rearranged Vo / Ja sequence. In some embodiments, the human Vb, Db and Jb segments rearrange to form a rearranged Vb / Db / Jb sequence. Thus, in various modalities, the non-human animal (eg, rodent) expresses a T cell receptor comprising a human variable region and a non-human constant region (eg, rodent) on a T cell surface.
[0010] [0010] In some respects, non-human animal T cells undergo development of T cells in the thymus to produce CD4 and CD8 single-positive T cells. In some respects, the non-human animal comprises a normal ratio of CD3 + splenic T cells to total splenocytes. In several modalities, the non-human animal generates a population of central and effective memory T cells in the periphery.
[0011] [0011] In one embodiment, the locus of the TCRa variable region gene rearranged in the non-human animal described here comprises 61 human Ja segments and 8 human Voa segments. In another embodiment, the locus of the non-rearranged TCRa variable region gene in the non-human animal comprises a complete repertoire of human Jo segments and a complete repertoire of human Vo segments.
[0012] [0012] In one embodiment, the locus of the non-rearranged TCRb variable region gene in the non-human animal described here comprises 14 human Jb segments, 2 human Db segments, and 14 human Vb segments. In another embodiment, the locus of the TORb variable region gene not rearranged in the non-human animal comprises a complete repertoire of human Jb segments, a complete repertoire of human Db segments, and a complete repertoire of human Vb segments.
[0013] [0013] In an additional embodiment, the non-human animal described here (for example, a rodent) further comprises nucleotide sequences of variable human TORd segments in a humanized TCR locus. In one embodiment, the non-human animal (for example, a rodent) further comprises at least one human Vd, Dd and Jd segment, for example, a complete repertoire of human Vd, DdedJdhuman segments in the locus of humanized TCORa.
[0014] [0014] In one embodiment, the non-human anima retains a locus of non-human TCRa and / or TOCORb, where the locus is a non-functional locus.
[0015] [0015] In one aspect, the invention provides a genetically modified mouse comprising in its genome (a) a non-rearranged TORa variable region locus comprising a repertoire of human Jo segments and a repertoire of human Vo segments, linked in a functional manner to a sequence of the non-human TORa constant region gene (for example, mouse mouse), and / or (b) a non-rearranged TORb variable region gene locus comprising a repertoire of human Jb segments, a repertoire human Db segments, and a repertoire of human Vb segments, functionally linked to a non-human TCORb constant region gene sequence (for example, from a mouse or rat). In one embodiment, the mouse comprises a complete repertoire of human Va segments. In one embodiment, the mouse comprises a complete repertoire of human Vb segments. In one embodiment, the mouse comprises a complete repertoire of human Vo segments and human Ja segments. In one modality, the mouse comprises a complete repertoire of human Voa segments and human Vb segments. In one embodiment, the mouse comprises a complete repertoire of human Va, Ja, Vb, Db, and Jb segments.
[0016] [0016] In one embodiment, the mouse comprises at least one mouse endogenous Va segment and at least one mouse endogenous Ja segment, where the endogenous segments are unable to rearrange to form a rearranged Vo / Ja sequence, and also comprises at least one mouse endogenous Vb segment, at least one mouse endogenous Db segment, and at least one mouse endogenous Jb segment,
[0017] [0017] In one embodiment, the locus of the non-rearranged TCRa variable region gene comprising human TCRa variable region segments replaces the mouse TORa variable region genes at the locus of the endogenous mouse TORa variable region gene, and the locus of the non-rearranged TORb variable region gene comprising human TORb variable region segments replaces mouse TORb variable region genes at the locus of the endogenous mouse TCRb variable region gene.
[0018] [0018] In one embodiment, the human Va and Ja segments rearrange to form a rearranged human Vo / Joaa sequence, and the human Vb, Db and Jb segments rearrange to form a rearranged human Vb / Db / Jb sequence. In one embodiment, the rearranged human Vo / Ja sequence is functionally linked to a mouse TCRa constant region sequence. In one embodiment, the rearranged human Vb / Db / Jb sequence is functionally linked to a mouse TCORb constant region sequence. Thus, in various modalities, the mouse expresses a T cell receptor on the surface of a T cell, where the T cell receptor comprises a human variable region and a constant mouse region.
[0019] [0019] In one embodiment, the mouse further comprises a repertoire of segments of variable region of human TORd (for example, human Vd, Jd and Dd segments) in a humanized TCRa locus. In one embodiment, the human TORd variable region segment repertoire is a complete repertoire of human TORd variable region segments. In one embodiment, the human TORd variable region segments are at the endogenous TORa locus. In one embodiment, the human TCRd variable region segments replace the endogenous mouse TORA variable region segments.
[0020] [0020] In one embodiment, the genetically modified mouse expresses a T cell receptor comprising a human variable region and a constant mouse region on a T cell surface. In one aspect, the mouse T cells pass by developing thymic T cells to produce CD4 and CD8 single-positive T cells. In one aspect, the mouse comprises a normal ratio of CD3 + splenic T cells to total splenocytes; in one respect, the mouse generates a population of central and effective memory T cells for an antigen of interest.
[0021] [0021] Methods are also provided to produce the genetically modified non-human animals (for example, rodents, for example, mice or rats) described here.
[0022] [0022] In one aspect, a method for producing a humanized rodent (for example, a mouse or rat) is provided, comprising replacing segments from variable regions of TORa and TCRb, but not genes from constant regions of rodents, with segments of variable region of TCORa and TCRb, in endogenous TOR loci of rodents. In one embodiment, the method comprises replacing the segments of the variable TORa region of rodents (Vo and / or Ja) with segments of the variable region of the human TORa (Va and / or Ja), where the segments of the variable region of TCRa are functionally linked to a non-human TOR constant region gene to form a humanized TCRa locus; and replace rodent TCRb variable region segments (Vb and / or Db and / or Jb) with human TORb variable region segments (Vb and / or Db and / or Jb), where the variable region segments of TOCRb are functionally linked to a non-human TOR constant region gene to form a humanized TCRb locus. In one embodiment, the humanized rodent is a mouse and the mouse germline comprises the human TORa variable region segments that are functionally linked to a constant sequence of endogenous mouse TORa in an endogenous TCRa locus; and the mouse germline comprises the segments of variable region of human TCRb that are functionally linked to a constant sequence of endogenous mouse TCORb in a locus of endogenous TCRb.
[0023] [0023] In one embodiment, a method is provided here to produce a genetically modified non-human animal (eg, rodent, eg mouse or rat) that expresses a T cell receptor comprising a human or human variable region a non-human constant region (eg, rodent) on a T cell surface comprising: replacing an endogenous non-human TORa variable gene locus in a first non-human animal with a human TCRa variable gene locus - not rearranged, comprising at least one human Va segment and at least one human Ja segment, where the locus of the humanized TORa variable region gene is functionally linked to the non-human endogenous TCRa constant region; replace a non-human endogenous TCRb variable region gene locus in a second non-human animal with a non-rearranged humanized TCRb variable region locus comprising at least one human Vb segment, at least one human Db segment, and at least one human Jb segment, where the locus of the humanized TCRb variable region gene is functionally linked to the endogenous TCORb constant region; and cross the first and second non-human animals to obtain a non-human animal that expresses a
[0024] [0024] In a method modality, the non-human endogenous Va and Ja segments (for example, rodent) are unable to rearrange themselves to form a rearranged Vo / Jo sequence and the non-human endogenous Vb, Db and Jb segments ( for example, rodents) are unable to rearrange themselves to form a rearranged Vb / Db / Jb sequence. In a method embodiment, the human Va and Jo segments rearrange to form a rearranged Vo / Ja sequence and the human Vb, Db and Jb segments rearrange to form a rearranged Vb / Db / Jb sequence. In one embodiment of the method, the locus of the humanized TORa variable region gene not rearranged comprises 61 human Ja segments and 8 human Vo segments, and the locus of the humanized non-rearranged TORb variable region gene comprises 14 human Vb segments, 2 human Db segments, and 14 human Jb segments. In another modality of the method, the locus of the humanized variable TORa variable region gene not rearranged comprises a complete repertoire of Jo segments. humans and a complete repertoire of human Va segments, and the locus of the variable humanized TORb variable region not rearranged comprises a complete repertoire of human Vb segments, a complete repertoire of human Db segments, and a complete repertoire of human Jb segments.
[0025] [0025] In one aspect of the method, T cells from the non-human animal (eg, rodent) undergo the development of thymic T cells to produce CD4 and CD8 single-positive T cells. In one aspect, the non-human animal (for example, the rodent) comprises a normal ratio of CD3 + splenic T cells to total splenocytes. In one aspect, the non-human animal (for example, rodent) generates a population of T cells of central memory and effect an antigen of interest.
[0026] [0026] In some embodiments, the replacement of the locus of the non-human endogenous TORa variable region gene described here is done in a single ES cell, and the single ES cell is introduced into a non-human embryo (eg, rodent , for example, a mouse or rat) to produce a genetically modified non-human animal (ie, the first non-human animal, for example, the first rodent); and the replacement of the locus of the endogenous non-human TCRb variable region gene described here is done in a single ES cell, and the single ES cell is introduced into a non-human embryo (for example, a rodent, for example, a mouse or rat ) to produce a genetically modified non-human animal (ie a second non-human animal, for example, the second rodent). In one embodiment, the first rodent and the second rodent are crossed to form a progeny, where the progeny comprises in its germline a locus of the humanized TORa variable region gene and a locus of the human TCORb variable region gene - used.
[0027] [0027] In a method modality, the non-human animal is a rodent, for example, a mouse. Thus, the present invention also provides a method for producing a genetically modified mouse.
[0028] [0028] Cells, for example, isolated T cells (for example, cytotoxic T cells, helper T cells, memory T cells, etc.), derived from non-human animals (for example, rodents, for example) are also provided here mice or rats) described here. The tissues and embryos derived from the non-human animals described here are also provided.
[0029] [0029] In one aspect, a method for producing a human TCR variable domain is provided, comprising modifying genetics,
[0030] [0030] In one aspect, a method for producing a nucleic acid sequence encoding a variable domain of the human TOR that binds to an epitope of interest is provided, comprising ex- by a non-human animal as described herein to an epitope of interest - interest, keep the non-human animal under sufficient conditions for the animal to present the epitope of interest to a humanized TCR of the animal, and identify a nucleic acid of the animal that encodes a polypeptide of variable domain of the human TOR that binds to the epithelium. po of interest.
[0031] [0031] In one aspect, the use of a non-human animal as described here is provided to produce a humanized TOR receptor. In one aspect, the use of a non-human animal as described here is provided to produce a variable domain of the human TOR. In one aspect, the use of a non-human animal as described herein is provided to produce a nucleic acid sequence encoding a variable domain of the human TOR.
[0032] [0032] In one aspect, the use of nucleic acid sequence encoding a variable domain of the human TOR or fragment thereof to produce an antigen-binding protein is provided. In one embodiment, the antigen-binding protein comprises a variable domain of the TOR comprising a variable domain of the T-CRa and / or human TORb that binds to an antigen of interest.
[0033] [0033] In one aspect, the use of a non-human animal as described here is provided to produce a non-human cell that expresses a humanized T cell receptor on its surface.
[0034] [0034] In one aspect, a humanized T cell receptor from a non-human animal as described here is provided.
[0035] [0035] In one aspect, a nucleic acid sequence encoding a variable domain of the human TOR or fragment thereof, produced in a non-human animal as described herein, is provided.
[0036] [0036] Any of the modalities and aspects described here can be used together, unless otherwise indicated or clear from the context. Other modalities will become clear to those skilled in the art from a review of the detailed description later. The following detailed description includes exemplified representations of various embodiments of the invention, which are not restrictive of the claimed invention. The attached figures form part of this specification and, together with the description, serve only to illustrate the modalities and not to limit the invention. Brief Description of Drawings
[0037] [0037] Figure 1 represents the interaction in a mouse between a TCR molecule and an MHC molecule: the left panel shows a mouse T cell (upper) from a mouse with humanized TCR comprising a cell receptor T with variable domains of the human TOR and constant domains of the mouse TCR, which recognizes an antigen (gray ball) presented through a class MHC | by a cell showing antigen (lower); the right panel shows the same for an MHC class Il. The MHCI and MHCII complexes are shown together with their respective coreceptors, CD8 and CDA4. The mouse regions are black and the human regions are white.
[0038] [0038] Figure 2 represents (not to scale) the general organization of a mouse (upper panel, first locus) and human (upper panel, second locus) TCRa locus. The bottom panel illustrates a strategy to replace segments of variable region of TORa in a mouse with segments of variable region of human TORa (open symbols) in the endogenous locus of mouse in chromosome 14; a humanized TCRa locus having human Va and Ja segments is shown with a mouse constant region and a mouse enhancer; in the modality shown, the locus of TCRd is deleted in the course of humanization.
[0039] [0039] Figure 3 represents (not in scale) a progressive strategy for the humanization of the mouse TCRa locus, where the TORa variable region gene segments are sequentially added upstream of an initial humanization of a deleted mouse locus (MAID1540). The mouse sequence is indicated by closed symbols; the human sequence is indicated by open symbols. MAID refers to the modified allele ID number. TRAV = Va segment of TCR, TRAJ = Ja segment of TCR (hTRAJ = human TRAJ), TRAC = Ca domain of TOR, TORD = TCRd.
[0040] [0040] Figure 4 is a detailed representation (not to scale) of the progressive humanization strategy at the locus of TCRa. Figure 4A represents the deletion of segments V and J from the mouse TORa. Figure 4B represents the strategy for insertion of 2 human segments V and 61 human segments J into the deleted TORa locus of mice. Figure 4C represents the strategy for the insertion of additional human V segments, resulting in a total of 8 V segments and 61 human J segments. Figure 4D represents the strategy for the insertion of additional human V segments, resulting in a total of 23 V segments and 61 human J segments. Figure 4E represents the strategy for the insertion of additional human V segments resulting in 35 V segments and 61 human J segments. Figure 4F represents the strategy for the insertion of additional human segments resulting in 48 segments V and 61 segments
[0041] [0041] Figure 5 represents (not to scale) a modality of the humanization strategy of the mouse TCRa locus, in which human TORd sequences (TCRd Vs, TCRd Ds, TCRd Js, TORd intensifier (intensifier) , and TORd constant (C)) are also located in the humanized TORa locus. The mouse sequence is indicated by closed symbols; the human sequence is indicated by open symbols. LTVEC refers to a large targeting vector; hNTRD = human TCRd.
[0042] [0042] Figure 6 represents (not to scale) the general locus of mouse TORb (upper panel, first locus; on chromosome 6 of mice) and human TORb (upper panel, second locus; on human chromosome 7). The bottom panel illustrates a strategy to replace segments of variable region of TCORb in the mouse (closed symbols) with segments of variable region of human TORb (open symbols) in the endogenous locus of the mouse in the mouse chromosome 6. The locus of humanized TCRb having human Vb, Db and Jb segments is shown with constant regions of mouse and a mouse enhancer; in the modality shown, the humanized locus retains mouse trypsogen genes (solid rectangles); and in the particular modality shown, a single mouse V segment is retained upstream of the mouse 5 'trypsinogen genes.
[0043] [0043] Figure 7 represents (not to scale) a progressive strategy for the humanization of the mouse TCRb locus, where the gene segments of the variable TORb region are sequentially added to a variable locus of TCRb deleted from cam-
[0044] [0044] Figure 8 is a detailed representation of the strategy of progressive humanization in the locus of TCRb. Figure 8A represents the strategy for deletion of V segments of mouse TCRb. Figure 8B represents the strategy for the insertion of 14 V segments in the deleted TCRb locus. Figure 8C represents the strategy for the insertion of 2 D segments and 14 J segments in the TCRb locus (i), followed by deletion of the loxP site (ii), resulting in 14 human V segments, 2 human D segments and 14 human segments J. Figure 8D represents the strategy for inserting additional human V segments resulting in 40 V segments, 2 D segments and 14 segments J. Figure 8E represents the strategy for inserting additional human V segments resulting in 66 segments V, 2 D segments and 14 J segments. Figure 8F represents the replacement of mouse V segment downstream of a mouse intensifier, resulting in 67 V segments, 2 D segments, and 14 J segments. In this particular embodiment, a mouse V segment is retained 5 'from the mouse trypsinogen genes.
[0045] [0045] Figure 9 represents FACS analysis histograms representative for percentage splenic cells (where the Y axis is the number of cells, the X axis is the average fluorescence intensity, and the port shows the frequency of T cells CD3 + within the single population of lymphocytes) colored with anti-CD3 antibody in a naturally occurring mouse (WT); a homozygous mouse for a deleted TOCRa locus (first upper panel; MAID 1540 in figure 3); a homozygous mouse for a deleted TCRa locus and comprising 8 human Va segments and 61 human Ja segments
[0046] [0046] Figure 10 is a FACS contour plot representing mouse thymus cells from a naturally occurring mouse (WT), humanized homozygous TORa (1767 HO; hTCRa); humanized homozygous TCRb mouse (1716 HO; hT-CRb); and humanized homozygous TOCRo / b mouse (1716 HO 1767 HO; hTCRo / b) stained with anti-CD4 (Y axis) and anti-CD8 (X axis) antibodies (top panel), and anti-CD44 (Y axis) antibodies and anti-CD25 (X axis) (bottom panel). The FACS graph on the top panel allows to distinguish double-negative (DN), double-positive (DP), single-positive CD4 (CD4 SP), and single-positive CD8 (SP CD8) cells. The FACS graph in the lower panel allows to distinguish several stages of double negative T cells during the development of T cells (DN1, DN2, DN3 and DN4). 1716 and 1767 refer to MAID numbers identified in Figures 3 and 7.
[0047] [0047] Figure 11 demonstrates either the frequency (upper panel) or absolute number (lower panel) of DN, DP, CD4 SP, and CD SP cells in the thymus or WT mice; hTCRa (1767 HO); hTCRb (1716 HO) or hTCRoyb (1716 HO 1767 HO) (n = 4).
[0048] [0048] Figure 12 is a representative FACS analysis of splenic cells from a WT mouse; hTCRa (1767 HO); hTCRb (1716 HO) or hTCRo / b (1716 HO 1767 HO): the left panel represents the staining analysis of singlet cells based on anti-CD19 antibody (Y axis; colored for B lymphocytes) or anti-CD3 antibody (X axis ; labeled for T lymphocytes); the middle panel represents the analysis of CD3 + cells based on anti-CD4 (Y axis) or anti-CD8 (X axis) antibody staining; and the right panel represents the analysis of CD4 + or CD8 + cells based on the staining of anti-CDA44 (Y axis) or anti-CD62L (X axis) antibodies, the stains allow to distinguish several types of T cells in the periphery (T cells viruses versus central memory T cells (Tcm) versus effector memory T cells (TeffiTem)).
[0049] [0049] Figure 13 shows the number of CD4 + T cells (left panel) or CD8 + (right panel) per spleen (Y axis) of WT, hTCRa (1767 HO) mice; hTCRb (1716 HO) or hTCRo / b (1716 HO 1767 HO) (n = 4).
[0050] [0050] Figure 14 shows the number of virgin T cells, Tom and Tefflem per spleen (Y axes) of CD4 + T cells (upper panel) or CD8 + (lower panel) of WT, hTCRa (1767 HO) mice; hT-CRb (1716HO) ouhTCRo / b (1716 HO 1767 HO) (n = 4).
[0051] [0051] Figure 15 illustrates tables summarizing the expression (determined by FACS analysis using variable segment specific antibodies) of various B segments of human TCRb in CD8 + splenic T cells (figure 15A) or CD4 + T cells (figure 15B ) from WT mice, hTCRa (1767 HO); hTCRb (1716 HO) or hTCRo / b (1716
[0052] [0052] Figure 16 represents the expression mRNA (Y-axes) of various V segments of human TCRb present in mice WT, hTCRa (1767 HO); hTCRb (1716 HO) or hTCRo / b (1716 HO 1767 HO) in thymic or splenic T cells. Figure 16A represents the analysis of the variable segment mMRNA expression of human TCORb (NTRBV) 18, 19, 20 and 24. Figure 16B represents the analysis of hTRBV mRNA expression 25, 27, 28 and 29.
[0053] [0053] Figure 17 represents FACS histograms representative of splenic cells (where the Y axis is the number of cells, the X axis is the average fluorescence intensity, and the port shows the frequency of CD3 + T cells within the single lymphocyte population. ) stained with anti-CD3 antibody in a WT mouse, a mouse homozygous for a deleted TCRa locus (TCRA AV), a mouse homozygous for a deleted TCRa locus with 2 human V segments and 61 human J segments ( TCRA 2 hV; MAID 1626 of figure 3), a mouse homozygous for the locus of TCORa deleted with 8 human V segments and 61 human J segments (TCRA 8 hV; MAID 1767 of figure 3), and a mouse homozygous for the locus of T- CRa deleted with 23 human V segments and 61 human J segments (TCRA 23 hV; MAID 1979 in figure 3).
[0054] [0054] Figure 18, in the upper left panel, is a representative FACS analysis of CD3 + T cells of the thymus obtained from either a WT mouse or a hTCRa homozygous mouse with 23 human V segments and 61 human J segments (1979 HO ) stained with either anti-CD4 antibody (Y axis) or anti-CD8 antibody (X axis); in the lower left panel, it is a FACS analysis of DN T cells or a WT mouse or a 1979 mouse stained with either anti-CD44 (Y axis) or anti-CD25 (X axis); on the right panel, are
[0055] [0055] Figure 19, in the left panel, is a representative FACS analysis of splenic lymphocytes from a WT or 1979 HO mouse colored or with anti-CD19 antibodies or anti-CD3 antibodies; on the right panel are graphs of the percentage of splenocytes (Y-axis) obtained from WT mice and 1979 HO mice (n = 4) that are CD3 +.
[0056] [0056] The present invention provides genetically modified non-human animals, for example, rodents, for example, mice or rats, which express humanized T cell receptors.
[0057] [0057] The term "conservative", when used to describe a conservative amino acid substitution, includes the replacement of an amino acid residue with another amino acid residue having a side chain group R with similar chemical properties (for example , charge or hydrophobicity). Conservative amino acid substitutions can be achieved by modifying a nucleotide sequence to introduce a nucleotide change that will encode conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest for a protein, for example, the ability of a T cell to recognize a peptide presented by an MHC molecule. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine and isoleucine; aliphatic hydroxyl side chains such as serine and threonine; side chains containing amide 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 substitution groups include, for example, valine / leucine / isoleucine, - phenylalanine / tyrosine, lysine / arginine, —alanine / valine, glutamate / aspartate, and asparagine / glutamine. In some embodiments, a conservative amino acid substitution may be a substitution of any native residue in a protein for alumina, as used, for example, in alumina scan mutagenesis. In some embodiments, a conservative substitution that is made has a positive value in the log PAM250 probability matrix described by Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256: 1443-45), incorporated herein by reference. In some modalities, the substitution is a moderately conservative substitution where the substitution has a non-negative value in the log PAM250 probability matrix.
[0058] Thus, the invention encompasses a genetically modified non-human animal expressing humanized TOR a and b polypeptides (and / or humanized TOR d and g polypeptides) comprising
[0059] [0059] One skilled in the art would understand that in addition to the nucleic acid residues encoding humanized TOR a and b polypeptides described here, due to the degeneration of the genetic code, other nucleic acids can encode the polypeptides of the invention. So, in addition to a genetically modified non-human animal that comprises nucleotide sequences encoding humanized TOR polypeptides in its genome described here, a non-human animal that comprises nucleotide sequences in its genome that differ from those described here due to the degeneration of the genetic code is also provided.
[0060] [0060] The term "identity", when used in conjunction with the sequence, includes identity as 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 modalities described here, identities are determined using a ClustalW v alignment. 1.83 (slow) employing an open gap penalty of 0.1, and using a Gonnet similarity matrix (MacVector'Y "10.0.2, MacVector Inc., 2008). The length of the strings compared to the sequence identity will depend on the particular sequences.In different ways, the identity is determined by comparing the sequence of a mature protein from its N-terminus to its C-terminus In several modalities, when comparing a human / non-human chimeric sequence with a human sequence, the human / non-human chimeric sequence (but not the non-human sequence) is used in making a comparison for the purpose of verifying a level of identity between a human sequence and a human portion of a human / non-human chimeric sequence (for example, comparing
[0061] [0061] The terms "homology" or "homologous" in relation to the sequences, for example, nucleotide or amino acid sequences, mean two sequences which, through optimal alignment and comparison, are identical in at least 75% of the nucleotides or amino acids, at least approximately 80% nucleotides or amino acids, at least approximately 90 to 95% nucleotides or amino acids, for example, more than 97% nucleotides or amino acids. One skilled in the art would understand that, for optimal gene targeting, the targeting construct should contain arms homologous to endogenous DNA sequences (ie, "homology arms"); thus, homologous recombination can occur between the targeting construct and the target endogenous sequence.
[0062] [0062] The term "functionally linked" refers to a juxtaposition where the components so described are in a relationship that allows them to function in their intended manner. As such, a nucleic acid sequence encoding a protein can be functionally linked to regulatory sequences (for example, promoter, enhancer, silencer, etc.) in order to retain the appropriate transcriptional regulation. In addition, various portions of the humanized protein of the invention can be functionally linked to retain appropriate folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless otherwise stated, several domains of the humanized protein of the invention are functionally linked together.
[0063] [0063] The term "substitution" in relation to gene replacement refers to locating exogenous genetic material in an endogenous genetic locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. In one case, an endogenous non-human gene or fragment thereof is replaced by a corresponding human gene or fragment thereof. A corresponding human gene or fragment thereof is a human gene or fragment thereof which is an orthogen, homologous or is substantially identical or the same in structure and / or function, to the endogenous non-human gene or fragment thereof . As shown in the Examples below, the locus nucleotide sequences of the endogenous non-human TOR a and b variable region gene have been replaced by nucleotide sequences corresponding to the loci of the human TOR a and b variable region gene.
[0064] [0064] "Functional", as used here, for example, in relation to a functional protein, refers to a protein that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, a substitution in an endogenous locus (for example, substitution in the endogenous TORa, TORb, TOCRd and / or endogenous non-human TOCRg gene loci) results in a locus that fails in express a functional indigenous protein.
[0065] [0065] The TCR locus or the TCR gene locus (e.g., TOCRa locus or TOCRb locus), as used herein, refers to genomic DNA comprising the TCR coding region, including the region of TCR coding, including non-rearranged V (D) J sequences, intensifier sequence, constant sequence, and any sequence upstream or downstream (RTU, regulatory regions, etc.), or intervening DNA sequence (introns, etc. .). The variable locus of TCR or locus of TOR variable region gene (for example, locus of TCRa variable region or locus of TCRb variable region), refers to DNA comprising the region that includes
[0066] [0066] In various embodiments, the invention generally provides genetically modified non-human animals where non-human animals comprise in the genome the loci of the humanized TCR variable region gene not rearranged.
[0067] [0067] T cells bind to epitopes in small antigenic determinants on the surface of cells presenting antigens that are associated with a major histocompatibility complex (MHC; in mice) or human leukocyte antigen complex (HLA, in humans). T cells bind to these epitopes through a T cell receptor complex (TCR) on the surface of the T cell. T cell receptors are heterodimeric structures composed of two types of chains: an a (alpha) and b (beta) chain , or a g (gamma) and d (delta) chain. The a chain is encoded by the nucleic acid sequence located within locus a (on human or mouse chromosome 14), which also encompasses the entire d locus, and the b chain is encoded by the nucleic acid sequence located within the locus b (on mouse chromosome 6 or human chromosome 7). Most T cells have an ab TCR; while the minority of T cells has a gd TCR. The interactions of TORs with MHC class molecules | (showing CD8 + T cells) and MHC class | (showing CD4 + T cells) are shown in figure 1 (closed symbols represent non-human sequences; open symbols represent human sequences, showing a
[0068] [0068] The T cell receptor polypeptides a and b (and similarly polypeptides g and d) are linked together via a disulfide bond. Each of the two polypeptides that make up the TCR contains an extracellular domain comprising constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and the cytoplasmic tail are also part of the constant region). The variable region of the TOR determines its specificity to the antigen, and similar to the immunoglobulins, comprises 3 complementarity determining regions (CDRs). Also similar to immunoglobulin genes, the loci of the T cell receptor variable region gene (for example, TORa locus and TOCRb) contain a number of un rearranged V (D) J segments (variable segments (V ), junction (J) and, in TCRb ed, diversity segments (D)). During the development of T cells in the thymus, the locus of the TCRa variable region gene undergoes rearrangement, such that the resulting TORoa chain is encoded by a specific combination of VJ segments (Vo / Ja sequence); and the locus of the TCRb variable region gene undergoes rearrangement, such that the resulting TOCRb chain is encoded by a specific combination of VDJ segments (Vb / Db / Jb sequence).
[0069] [0069] Interactions with thymic stroma cause thymocytes to pass through various stages of development, characterized by the expression of various cell surface markers. A summary of characteristic cell surface markers at various stages of development in the thymus is shown in Table 1. Does the rearrangement at the locus of the TORb variable region gene begin at the DN stage and ends during the DNA4 stage, while the rearrangement of the locus of the TOCRa variable region gene occurs in the DP stage. After completing the TCRb locus rearrangement, cells express the TCRb chain on the cell surface along with the substitute chain, pTa. To see,
[0070] [0070] The virgin CD4 + and CD8 + T cells leave the thymus and enter the peripheral lymphoid organs (eg spleen) where they are exposed to antigens and are activated to expand clonally and differ in a number of T cells effector cells (Teff), for example, cytotoxic T cells, Tree cells, TH17, Ty, T1W2, etc. Subsequent to infection, a number of T cells persist as memory T cells, and are classified either as central memory T cells (Tcm) or effector memory T cells (Tem). Sallusto et al., (1999) Two subsets of memory T Iymphocytes with distinct homing potentials and effector functions, Nature 401: 708-12 and Commentary by Mackay (1999) Dual personality of memory T cells, Nature 401: 659-60. Sallusto and colleagues proposed that, after initial infection, Tem cells represent a readily available group of memory T cells initiated by antigen in peripheral tissues with effector functions, while Tem cells represent T cells from antigen-initiated memory in peripheral lymphoid organs that, through secondary provocation, can become new effector T cells. While all memory T cells express CD4545 isoforms of CDA45 (virgin T cells express CD45RA isoforms), Tem are characterized by the expression of L selectin (also known as CD62L) and CCR7 +, which are important for organ binding and signaling peripheral lymphoids and lymph nodes. Thus, all T cells found in peripheral lymphoid organs (for example, virgin T cells, Tem cells, etc.) express CD62L. In addition to CD45RO, all memory T cells are known to express a number of different cell surface markers, for example,
[0071] [0071] While the variable domain of TCR works mainly in antigen recognition, the extracellular portion of the constant domain, as well as transmembrane, and cytoplasmic domains of the TCR also serve important functions. A complete TOR receptor complex requires more than polypeptides a and b or g and d; Additional molecules required include CD3g, CD3d, and CD3e, as well as the 6-chain homodimer (CC). Upon completing the TCRb rearrangement, when cells express TCRb / pTa, this pre-TCR complex exists along with CD3 on the cell surface. TORa (or pTa) on the cell surface has two basic residues in its trans-membrane domain, one of which recruits a CD3ge heterodimer, and another recruits 66 via its respective acid residues. TCRb has an additional basic residue in its transmembrane domain that is believed to replicate the CD3de heterodimer. See, for example, Kuhns et al., (2006) Deconstructing the Form and Function of the TCOR / CD3 Complex, Community 24: 133-39; Wucherpfennig et al. (2009) Structural Biology of the T-cell Receiver: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb. Perspect. Biol. 2: a005140. The assembled complex, comprising the heterodimer TCRab, CD3ge, CD3de, and 6, is expressed on the surface of the T cell. Polar residues in the transmembrane domain have been suggested to serve as quality control to exit the endoplasmic reticulum; it has been shown that in the absence of CD3 subunits, the TCR chains are retained in the ER and targeted for degradation. See, for example, Call and Wucherpfennig (2005) The T Cell Receptor: Critical Role of the Membrane Environment in Receptor Assembly and Functone, Annu. Rev. Immunol. 23: 101-25.
[0072] [0072] The assembled complex CD3 and 66 chains provide components for TOR signaling as the TCRab heterodimer (or TCRgd heterodimer) lacks signal transduction activity. The CD3 chains have an Activation Reason based on Tyrosine Immunoreceptor (ITAM) each, while the chain of 6 contains three tandem ITAMs. ITAMs contain tyrosine residues capable of being phosphorylated by associated kinases. Thus, the assembled TCR-CD3 complex contains 10 ITAM motifs. See, for example, Love and Ha-yes (2010) ITAM-Mediated Signaling by the T-Cell Antigen Receptor, Cold Spring Harb. Perspect. Biol. 2: 2002485. After TCR coupling, the! TAM motifs are phosphorylated by the Src family of tyrosine kinases, LcK and Fyn, which initiates a signaling cascade, resulting in Ras activation, calcium mobilization, actin cytoskeleton rearrangements, and activation of transcription factors, all leading to T cell differentiation, proliferation and effector actions. See also, Jane- way's Immunobiology, 7th Ed., Murphy et al., Eds., Garland Science, 2008; both of which are incorporated by reference.
[0073] [0073] Additionally, the transmembrane and cytoplasmic domains of TORb have a role in mitochondrial targeting and in the induction of apoptosis; in fact, naturally occurring N-terminally truncated TORb molecules exist in thymocytes. Shani et al. (2009) (2009) Incomplete T-cell receptor - b peptides target the myth-chondrion and induce apoptosis, Blood 113: 3530-41. Thus, several important functions are served by the constant region of TOR (which, in various modalities, comprises a portion of extracellular domain, as well as transmembrane and cytoplasmic domain); and in several modalities, the structure of this region should be taken into account when designing humanized TCRs or genetically modified non-human animals expressing the same.
[0074] [0074] Transgenic mice for rearranged T cell receptor sequences are known in the art. The present invention
[0075] [0075] In one embodiment, the invention provides non-human animals (for example, rodents, for example, rats, mice) that are genetically modified and include in their genome segments of variable region of human TOR not rearranged (segments V (D) J), where non-rearranged human TOR variable region segments replace, in a locus of the endogenous non-human (eg, rodent) TCR variable region gene (eg, variable region gene locus TORa, b, de / or g), endogenous non-human TOR variable region segments. In one embodiment, the locus of the variable non-rearranged human TOR gene replaces the locus of the variable non-human TOR gene.
[0076] [0076] In another embodiment, the invention provides non-human animals (for example, rodents, for example, rats, mice) genetically modified that comprise in their genome segments of variable region of human TOR not rearranged (segments V (D) J), where the non-rearranged human TOR variable region segments are functionally linked to a non-human TOR constant region gene sequence resulting in a humanized TOR locus, where the TOR locus humanized is at a site in the genome other than the endogenous non-human TOR locus.
[0077] [0077] In one aspect, the genetically modified non-human animals of the invention comprise segments of the variable region of human TOR in their genome, while retaining segment segments of the non-human TCR constant region (for example, rodent, for example , mouse, rat). In various modalities, the constant regions include the transmembrane domain and the cytoplasmic tail of the TCR. Thus, in various embodiments of the present invention, genetically modified non-human animals retain the transmembrane domain and cytoplasmic tail of the endogenous non-human TOR. In other embodiments, non-human animals comprise non-human endogenous TOR constant region gene sequences, for example, non-human endogenous TOR transmembrane domain and cytoplasmic tail. As indicated above, the TCR constant region participates in a signaling cascade initiated during antigen-initiated T-cell activation; thus, the endogenous TOR constant region interacts with a variety of anchor and non-human signaling proteins in the T cell. Thus, in one aspect, the genetically modified non-human animals of the invention express humanized T cell receptors that have the ability to recruit a variety of endogenous anchor or non-human signaling molecules, for example, CD3 molecules (for example, CD3g, CD3d, CD3e), chain 6, Lck, Fyn, ZAP-70, etc. A non-limiting list of molecules that are recruited to the TCR complex is described in Janeway's Immunobiology. In addition, similar to VELOCIMMUNEGO mice, which exhibit normal B cell development processes and normal clonal selection which are believed to be due at least in part to the location of variable regions in endogenous mouse loci and the maintenance of constant domains. In mice, in one aspect, the non-human animals of the present invention exhibit normal T cell development and differentiation processes.
[0078] [0078] In some embodiments, a non-human animal that is supplied comprises in its genome variable regions of human non-rearranged TCRa, where the non-rearranged human TCORa variable region segments are functionally linked in a sequence of a non-human TCRa constant region gene resulting in a humanized TCRa locus. In one mode, the humanized TORa locus is at a site in the genome other than the endogenous non-human TCORa locus. In another mode, the non-rearranged human TORa variable region segments replace the endogenous non-human TORa variable region segments, while retaining the endogenous non-human TORa constant region. In one embodiment, the locus of the non-rearranged human TCRa variable region gene replaces the endogenous non-human TORa variable region gene locus. In some modalities, the animal retains sequences of the constant region and variable region
[0079] [0079] In other modalities, a non-human animal that is supplied comprises in its genome variable regions of non-rearranged human TCRb, where the segments of non-rearranged human TCORb variable region are functionally linked to a non-human TORb constant region gene sequence resulting in a humanized TCORb locus. In one mode, humanized TCRb locus is at a site in the genome other than the endogenous non-human TCORb locus. In another modality, the non-rearranged human TOCORb variable region segments replace the endogenous non-human TCORb variable region segments, while retaining the endogenous non-human TORb constant region. In one embodiment, the locus of the non-rearranged human TCRb variable region gene replaces the locus of the endogenous non-human TCORb variable region gene. In some modalities, the animal retains endogenous non-human TCORa constant and variable region gene sequences. Thus, the animal expresses a TCR comprising a chimeric human / non-human (i.e., humanized) TCRb chain and a non-human TCRb chain.
[0080] [0080] In some specific embodiments, the invention provides a genetically modified non-human animal (eg, rodent, eg mouse or rat) that comprises in its genome (a) a locus of the receptor region variable gene T cell (TCR) a not rearranged comprising at least one human Vo segment and at least one Jo segment. human, functionally linked to a non-human TCRa constant region gene sequence (eg, rodent, mouse or rat, for example) and / or (b) a locus of the TORb variable region gene not rearranged comprising at least one human Vb segment, at least one human Db segment, and at least one human Jb segment, functionally linked to a non-human TCORb constant region gene sequence (eg, rodent, for example) example, mouse or rat) endogenous.
[0081] [0081] In various embodiments of the invention, the non-rearranged human or humanized TOR variable region locus (e.g., TORa variable region locus and / or TCRb) is comprised in the germ line of the non-human animal ( eg, rodent, eg mouse or rat). In various modes, the substitutions of VCR (D) J segments of TCR with VR (D) J segments of human TCR that are not rearranged (for example, Va and Ja segments, and / or Vb and Db and Jb) are in one locus (locus) of endogenous non-human TCR variable region, where non-rearranged human Ve Je / or VeDeyJ segments are functionally linked to non-human TOR constant region genes.
[0082] [0082] In some embodiments of the invention, the non-human animal comprises two copies of the locus of the non-rearranged human or humanized TCRa variable region gene and / or two copies of the locus of the non-human or humanized TORb variable region gene rearranged. Thus, the non-human animal is homozygous for one or both of the gene loci of variable region of TORa and human or humanized TCORb not rearranged. In some embodiments, the non-human animal comprises a copy of the locus of the human or humanized TCRa variable region gene not rearranged and / or a copy of the locus of the human or humanized TORb variable region gene not rearranged. Thus, the non-human animal is heterozygous for one or both loci of the human or humanized TORa and TCORb variable region gene not rearranged.
[0083] [0083] In one embodiment, the locus of the variable region gene of
[0084] [0084] Similarly, in one embodiment, the locus of the non-rearranged TORb variable region gene comprising human variable region segments (for example, human Vb, Db and Jb segments) is positioned in the non-human genome such that the human variable region segments replace the corresponding non-human variable region segments. In one embodiment, the non-rearranged TORb variable region gene locus comprising human variable region segments replaces the endogenous TORb variable region gene locus. In one aspect, the endogenous non-human Vb, Db and Jb segments are unable to rearrange themselves to form a rearranged Vb / Db / Jb sequence. Thus, in one aspect, the human Vb, Db and Jb segments at the locus of the non-rearranged TCRb variable region gene are able to rearrange themselves to form a rearranged Vb / Db / Jb sequence.
[0085] [0085] In yet another embodiment, both loci of the non-rearranged TCRa and b variable region gene comprising human variable region segments replace the respective endogenous TORa and b variable region loci. In one aspect, the Va and Ja segments are unable to rearrange themselves to form a rearranged Va / Ja sequence, and the endogenous non-human Vb, Db and Jb segments are unable to rearrange themselves to form a Vb sequence / Db / Jb rearranged. Thus, in one aspect, the Va and Ja segments at the locus of the non-rearranged TORa variable region gene are able to rearrange themselves to form a rearranged human Vo / Jo sequence and the human Vb, Db and Jb segments at the locus of the gene variable region of non-rearranged TORb are able to be rearranged to form a rearranged Vb / Db / Jb sequence.
[0086] [0086] In some aspects of the invention, the non-human animal comprising a locus of the humanized TCRa and / or TORb gene (comprising a locus of the variable region of TOCRa and / or non-rearranged TCRb) retains a locus of the region gene variable of endogenous non-human TCRa and / or TCRb. In one embodiment, the locus of the variable TORa and / or non-human TCRb region gene is a non-functional locus. In one embodiment, the non-functional locus is an inactivated locus, for example, an inverted locus (for example, nucleic acid sequence encoding the locus of the variable region gene is in inverted orientation with respect to the constant region sequence, so that no successful rearrangement is possible using variable region segments from the inverted locus). In one embodiment, the locus of the humanized TCRa and / or TOCORb variable region gene is positioned between the locus of the endogenous non-human TCRa and / or TCRb variable region and the locus of the TCRa and / or constant region gene Endogenous non-human TORb.
[0087] [0087] The number, nomenclature, position, as well as other aspects of the V and J and / or V, D and J segments of the human and mouse TOR loci can be verified using the IMGT database,
[0088] [0088] In one embodiment, the non-human animal comprises a humanized TCRoa locus comprising a DNA fragment comprising a contiguous human sequence from human Vo40 to Vo41 (segment Va is also called "TRAV" or "T-CRAV") and a DNA fragment comprising a contiguous human sequence of 61 human Ja segments (Ja segment is also called "TRAJ" or "TCRAJ"). In one embodiment, the non-human animal comprises a humanized TCRa locus comprising a DNA fragment comprising a contiguous human sequence from TRAV35 to human TRAV41 and a DNA fragment comprising a contiguous human sequence from 61 human TRAJsS. In one embodiment, the non-human animal comprises a humanized T-CRa locus comprising a DNA fragment comprising a human contiguous human TRAV22 to TRAV41 sequence and a DNA fragment comprising a human contiguous human 61 TRAJ sequence . In one embodiment, the non-human animal comprises a humanized TCORa locus comprising a DNA fragment comprising a contiguous human sequence from TRAV13-2 to TRAV41 and a DNA fragment comprising a contiguous human sequence from 61 human TRAJS. In one embodiment, the non-human animal comprises a humanized TCRa locus comprising a DNA fragment comprising a contiguous human sequence of human TRAV6 to TRAV41 and 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRa locus comprising a DNA fragment comprising a contiguous human sequence from TRAV1- 1 to human TRAV41 and 61 human TRAJs. In various embodiments, DNA fragments comprising contiguous human sequences of variable region segments of human TOCRa also comprise restriction enzyme sites, selection cassettes, endonuclease sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various modalities, these additional sites do not interfere with the proper functioning (for example, rearrangement, cutting, etc.) of various genes in the TORa locus.
[0089] [0089] In one embodiment, the locus of humanized TORa
[0090] [0090] The variable locus of mouse TCORb has approximately 0.6 megabases and comprises a total of 33 Vb segments, 2 Db segments and 14 Jb segments (figure 6). The variable locus of human TCRb is approximately 0.6 megabases and comprises a total of 67 Vb segments, 2 Db segments and 14 Jb segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., a rodent, e.g., mouse or rat) comprises at least one human Vb segment, at least one human Db segment, and at least one Jo segment. human. In one embodiment, the non-human animal comprises a humanized TOCRb locus comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, 55, 60, or up to 67 human Vb segments. In some embodiments, the humanized TORb locus comprises 8, 14, 40, 66 or 67 human Vb segments. Thus, in some modalities, the locus of humanized TCRb in the non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% human Vb; in some modalities, it may comprise approximately 20%, approximately 60%, approximately 15%, approximately 98%, or 100% human Vb.
[0091] [0091] In one embodiment, the non-human animal comprises a humanized TCRb locus comprising a DNA fragment comprising a contiguous human sequence from Vb18 to Vb29-1 (segment Vb is also called "TRBV" or "TCRBV"). In one embodiment, the non-human animal comprises a humanized TCRb locus comprising a DNA fragment comprising a contiguous human sequence from human TRBV1I18 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence from Db1- Jb1 (i.e., human Db1-Jb1-1- Jb1-6 segments), and a separate DNA fragment comprising a contiguous human sequence of human Db2-Jb2 (i.e., Db2-Jb2-1-Jb2-7 segments humans). In one embodiment, the non-human animal comprises a humanized TORb locus comprising a DNA fragment comprising a contiguous human sequence from human TRBV6-5 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence from Human Db1-Jb1 (i.e., Db1-Jb1-1-Jb1-6 segments), and a separate DNA fragment comprising a contiguous human sequence of human Db2-Jb2 (i.e., human Db2-Jb2-1-Jb2-7 ). In one embodiment, the non-human animal comprises a humanized TOCRb locus comprising a DNA fragment comprising a human contiguous human TRBV1 to TRBV29-1 sequence, a separate DNA fragment comprising a human contiguous human Db1-Jb1 sequence , and a separate DNA fragment comprising a contiguous human sequence of human Db2-Jb2. In one embodiment, the non-human animal comprises a humanized TOCRb locus comprising a DNA fragment comprising a contiguous human sequence of human TRBV1 to human TRBV29-1,
[0092] [0092] In one embodiment, the locus of humanized TORb comprises 14 human Jb segments, or 100% human Jb segments, and 2 human Db segments or 100% human Jb segments. In another embodiment, the humanized TORb locus comprises at least one human Vb segment, for example, 14 human Vb segments, and all of the mouse Db and Jb segments. In a particular embodiment, the humanized TORb locus comprises 14 human Vb segments, 2 human Db segments, and 14 human Jb segments. In another particular embodiment, the humanized TORb locus comprises a complete repertoire of human Vb, Db, and Jb segments, that is, all human variable b region gene segments encoded by locus b or 67 Vb segments, 2 segments Db, and 14 human Jb segments. In one embodiment, the non-human animal comprises a (e.g., 5 ') non-human Vb segment at the humanized TCORb locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vb, Db, and Jb segments at the TCRb locus.
[0093] [0093] In various modalities, when the non-human animal (for example, rodent) comprises a repertoire of human TORa and TCRb (and optionally TCRd and TORg) variable region segments (for example, a complete repertoire of variable region segments) - speed), the repertoire of several segments (for example, the complete repertoire of several segments) is used by the animal to generate a diverse repertoire of TCR molecules for various antigens.
[0094] [0094] In several respects, non-human animals comprise contiguous portions of the human genomic TOR variable loci comprising segments V, D and J or DeJ, ouVeuy, ouV arranged as in a non-rearranged human genomic variable locus, for example, comprising promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc., arranged as in a variable locus of human genomic TOR. In other aspects, the various segments are arranged as in a variable locus of non-rearranged non-human genomic TCR. In various modalities of the humanized TCR and / or b locus, the humanized locus may comprise two or more human genomic segments that do not appear in a juxtaposed human genome, for example, a fragment of V segments of the localized human V locus in a human genome proximal to the constant region, juxtaposed to a fragment of V segments of the human V locus located in a human genome at the upstream end of the human V locus.
[0095] [0095] In both mice and humans, the TCRd gene segments are located with the TCRa locus (see figures2eo5) The TCRd segments J and D are located between the segments Va and Ja, while the segments of TCRd V are interconnected through the TOCRa locus, with the majority located among the various Va segments. The number and locations of various TORd segments can be determined from the IMGT database. Due to the genomic arrangement of the TORA gene segments within the TCRa locus, successful rearrangement at the TORa locus generally deletes the TCRd gene segments.
[0096] [0096] In some embodiments of the invention, a non-human animal comprising a locus of the non-rearranged human TORa variable region gene also comprises at least one Vd segment, for example, up to the complete repertoire of human Vd segments. we. Thus, in some modalities, the replacement of the locus of the endogenous TORa variable region gene results in a replacement of at least one non-human Vd segment by a human Vd segment. In other embodiments, the non-human animal of the invention comprises a complete repertoire of human Vd, Dd, and Jd segments at the non-rearranged humanized TCRa locus; in still other modalities, the non-human animal comprises a locus of human non-rearranged TCRd completely in the locus of humanized TORoa not rearranged (that is, a locus of TCRd including segments of human variable region, as well as human intensifier and region constant). An exemplified modality for building a humanized TORa locus not rearranged comprising the complete TOCRd locus not rearranged is shown in figure 5.
[0097] [0097] In yet another embodiment, the non-human animal of the invention further comprises a locus of humanized TCRg not rearranged, for example, a locus of TORg comprising at least one human segment Vg and at least one human segment Jg ( for example, a complete repertoire of human Vg and Jg variable region segments). The locus of human TORg is on human chromosome 7, while the locus of mouse TORg is on mouse chromosome 13. See the IMGT database for more details on the TCRg locus.
[0098] [0098] In one aspect, the non-human animal (for example, rodent, for example, mouse or rat) comprising the loci of the
[0099] [0099] In various embodiments of the invention, the humanized T cell receptor polypeptides described herein comprise human leader sequences. In alternative embodiments, the humanized TOR receptor nucleic acid sequences are designed such that the humanized TOR polypeptides comprise non-human sequences.
[00100] [00100] The humanized TOR polypeptides described here can be expressed under the control of endogenous non-human regulatory elements (for example, rodent regulatory elements), for example, promoter, silencer, intensifier, etc. The humanized TCR polypeptides described here can alternatively be expressed under the control of human regulatory elements. In various modalities, the non-human animals described here further comprise regulatory sequences and other sequences normally found in situ in the human genome.
[00101] [00101] In various modalities, the human variable region of the humanized TOR protein is capable of interacting with several proteins on the surface of the same cell or another cell. In one embodiment, the human variable region of the humanized TCR interacts with MHC proteins (for example, MHC class | or Il proteins) presenting antigens on the surface of the second cell, for example, an antigen presenting cell (APC). In some modalities, the MHC | or | l is a non-human protein (for example, rodent, for example, mouse or rat). In other modalities, the MHC | or | l is a human protein. In one aspect, the second cell, aAPC, is an endogenous non-human cell expressing a human or humanized MHC molecule. In a different embodiment, the second cell is a human cell expressing a human MHC molecule.
[00102] [00102] In one aspect, the non-human animal expresses a humanized T cell receptor with a non-human constant region on the surface of a T cell, where the receptor is able to interact with non-human molecules, for example, anchor molecules or signaling expressed in the T cell (for example, CD3 molecules, the 6 chain, or other proteins anchored to the TCR through the CD3 or β chain molecules).
[00103] [00103] Thus, in one aspect, a cell complex is provided, comprising a non-human T cell that expresses a TCR that comprises a humanized TCRa chain, as described here, and humanized TCORb chain as described here, and an apprehended cell. - non-human antigen carrier comprising an antigen bound to an MHC | or MHC Il. In one embodiment, the non-human TCORa and TCRb chains are complexed with a non-human zeta (6) chain homodimer and CD3 heterodimers. In one embodiment, the cell complex is an in vivo cell complex. In one embodiment, the cell complex is a cell complex in vitro.
[00104] [00104] The genetically modified non-human animal can be selected from a group consisting of a mouse, rat, rabbit, pig, bovine (eg cow, ox, buffalo), gazelle, sheep, goat, chicken, cat , dog, ferret, primate (e.g.,
[00105] [00105] In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, for example, of the superfamily Dipodoidea or Muroidea. In a modality, the genetically modified animal is a rodent. In a modality, the rodent is selected from a mouse, a rat and a hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (for example, mouse-like hamsters), Cricetidae (for example, hamster, New World rats, mice, dormouse), Muridae (true mice and rats, desert rats, spiny mice, crested rats), Nesomyidae (tree rats, rock rats, white-tailed rats, Mada-gascar rats and mice), Platacanthomyidae (for example, field rat prickly), and Spalacidae (for example, mole rats, bamboo rats, and zoors). In a specific modality, the genetically modified rodent is selected from a real mouse or rat (family Muridae), a desert rat, a spiny mouse, and a crested rat. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one mode,
[00106] [00106] In a specific modality, the non-human animal is a rodent that is a mouse from a class C57BL selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J , C57BL / 6ByJ, C57BL / GNJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In another embodiment, the mouse is from a class 129 selected from the group consisting of a class that is 129P1, 129P2, 129P3, 129X1, 12981 (for example, 129S1 / SV, 129S1 / SvIlm), 12982, 12984, 12985 , 129S9 / SvEvH, 129S6 (129 / SvEvTac), 12987, 12988, 129T1, 129T2 (see, for example, Festing et al. (1999) Revised nomencliature for strain 129 mice, Mammalian Genome 10: 836, see also , Auerbach et al. (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines). In a specific embodiment, the genetically modified mouse is a mixture of a previously mentioned class 129 and a previously mentioned C57BL / 6 class. In another specific embodiment, the mouse is a mixture of the previously mentioned classes 129, or a mixture of the previously mentioned classes BL / 6. In a specific embodiment, the 129 class of the mixture is a 129S6 class (129 / SvEvTac). In another modality, the mouse is of a BALB class, for example, BALB / c class. In yet another modality, the mouse is a mixture of a BALB class and another class mentioned earlier.
[00107] [00107] In one embodiment, the non-human animal is a mouse. In one embodiment, the mouse is selected from a Wistar mouse, a LEA class, a Sprague Dawley class, a Fischer class, F344, F6, and Dark Agouti. In one embodiment, the mouse class is a mixture
[00108] [00108] Thus, in one embodiment, the invention provides a genetically modified mouse comprising in its genome a locus of the variable region of the human or humanized TOR not rearranged, for example, the locus of the region gene TCRa, TORb, TORA, and / or TORg variable. In some embodiments, the locus of the variable region of the human or humanized TOR not rearranged replaces the locus of the variable region gene of the endogenous mouse TCR. In other modalities, the locus of the human or humanized TOR variable region gene not rearranged is at a site in the genome other than the endocrine mouse TCR locus. In some embodiments, the locus of the variable region gene of the human or humanized TCR that is not rearranged is functionally linked to the mouse TOR constant region.
[00109] [00109] In one embodiment, a genetically modified mouse is provided, where the mouse comprises in its genome a locus of the T cell receptor variable region (T-CR) gene to not rearranged comprising at least one segment Human and at least one human Va segment, functionally linked to a mouse TCRa constant region gene sequence, and a non-rearranged TOCRb variable region locus comprising at least one segment Human Jb, at least one human Db segment, and at least one human Vb segment, functionally linked to a mouse TOCRb constant region gene sequence. In a specific modality, the mouse comprises in its genome a locus of the non-rearranged TOCRa variable region gene comprising a complete repertoire of human Ja segments and a complete repertoire of human Va segments, functionally linked to a gene sequence mouse TCRa constant region, and a locus of the non-rearranged TCRb variable region gene comprising a complete repertoire of human Jb segments, a complete repertoire of human Db segments, and a complete repertoire of human WVb segments, linked from functional way to a mouse TCRb constant region gene sequence.
[00110] [00110] In some embodiments, the non-rearranged TOCRa variable region locus comprising human TCORa variable region segments replaces the endogenous mouse TCRade variable region gene locus and the variable region gene locus of the non-rearranged TCRb comprising variable region segments of human TCORb replaces the locus of the endogenous mouse TCRb variable region gene. In some embodiments, the endogenous mouse segments Va and Ja are unable to rearrange themselves to form a rearranged Vo / Jo sequence, and the endogenous mouse segments Vb, Db and Jb are unable to rearrange themselves to form a Vb / Db / sequence Jb rearranged. In some embodiments, the human Va and Jo segments rearrange to form a rearranged human Vo / Ja sequence, and the human WVb, DbedJb segments rearrange to form a rearranged human Vb / Db / Jb sequence.
[00111] [00111] In various modalities, the non-human animals (for example, rodents, for example, mice or rats) described here produce T cells that are capable of undergoing thymic development, processing T cells from DN1 to DN2 to DN3 for DN4 for DP and for CD4 or CD8 SP T cells. Such T cells of the non-human animal of the invention express the cell surface molecules typically produced by a T cell during a particular stage of thymic development (for example, CD25, CD44, Kit, CD3, pTa, etc.). Thus, the non-human animals described here
[00112] [00112] In various modalities, the non-human animals described here produce T cells that are capable of undergoing normal T cell differentiation on the periphery. In some embodiments, the non-human animals described here are capable of producing a normal repertoire of effector T cells, for example, CTL (cytotoxic T lymphocytes), TW1, THW2, Treo, TH17, etc. Thus, in these modalities, the non-human animals described here generate effector T cells that fulfill the different functions typical of the particular type of T cell, for example, they recognize, bind, and respond to foreign antigens. In various embodiments, the non-human animals described here produce effector T cells displaying peptide fragments from cytosolic pathogens expressed in the context of MHC | molecules; they recognize peptides derived from antigens degraded in intracellular vesicles and presented by MHC Il molecules on the surface of macrophages and induce macrophages to kill microorganisms; they produce cytokines that trigger B cell differentiation; activate B cells to produce opsonizing antibodies; induce epithelial cells to produce chemokines that recruit neutrophils to infection sites, etc.
[00113] [00113] In additional modalities, the non-human animals described here comprise a normal number of CD3 + T cells in the periphery, for example, in the spleen. In some modalities, the percentage of peripheral CD3 + T cells in the non-human animals described here is comparable to that of naturally occurring animals (that is, animals comprising all segments of endogenous TOR variable regions). In one embodiment, the non-human animals described here comprise a normal ratio of splenic CD3 + T cells to total splenocytes.
[00114] [00114] In other respects, the non-human animals described here are capable of generating a population of memory T cells in response to an antigen of interest. For example, non-human animals generate both central memory T cells (Tcm) and effector memory T cells (Tem) for an antigen, for example, antigen of interest (for example, antigen being tested for development) vaccine, etc.).
[00115] [00115] DNI and DN2 cells that do not receive enough signals (for example, Notch signals) can develop into B cells, myeloid cells (for example, dendritic cells), mast cells and NK cells See, for example, Yashiro- Ohtani et al. (2010) Notch regulation of early thymocyte development, Seminars in Immunology 22: 261-69. In some embodiments, the non-human animals described here develop normal numbers of B cells, myeloid cells (eg, dendritic cells), mast cells and NK cells. In some modalities, the non-human animals described here develop a population of normal dendritic cells in the thymus.
[00116] [00116] The predominant type of T cell receptors expressed on the surface of T cells is TORo / b, with the minority of cells expressing TCRd / g. In some embodiments of the invention, T cells of non-human animals comprising the loci of TORa and / or humanized b exhibit normal use of the loci of TORo / b and TORd / g, for example, the use of the loci of TORo / b and TORd / g which is similar to the naturally occurring animal (for example, the T cells of the non-human animals described here express TCRo / b and TCRd / gem proteins in proportions comparable to that expressed by naturally occurring animals). Thus, in some embodiments, non-human animals comprising endogenous TCRa / b loci and endogenous non-human T-CRd / g locus exhibit normal use of all loci.
[00117] [00117] In addition to the genetically engineered non-human animals described here, a non-human embryo (for example, a rodent embryo, for example, a mouse or a rat embryo) is also provided, where the embryo comprises a donor ES cell that is derived from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein. In one aspect, the embryo comprises an ES donor cell that comprises a non-rearranged humanized TOR locus, and embryo host cells.
[00118] [00118] A tissue is also provided, where the tissue is derived from a non-human animal (for example, a mouse or rat) as described here, and expresses a humanized TOR polypeptide (for example, TORa polypeptide and / or TOCRb, or TCRd, and / or T-CRg).
[00119] [00119] In addition, a non-human cell isolated from a non-human animal as described here is provided. In one embodiment, the cell is an ES cell. In one embodiment, the cell is a T cell. In one embodiment, the T cell is a CD4 + T cell. In another fashion, the T cell is a CD8 + T cell.
[00120] [00120] Also provided is a non-human cell comprising a chromosome or fragment thereof from a non-human animal as described here. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described here. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as a result of a nuclear transfer.
[00121] [00121] A non-human cell expressing a TOR protein comprising a human variable region and a non-human constant region is also provided. The TOR protein can comprise TOCRa, TCRb, or a combination thereof. In one embodiment, the cell is a T cell, for example, a CD4 + or CD8r + T cell.
[00122] [00122] In one aspect, a non-human induced pluripotent cell comprising a non-rearranged humanized TOR locus encoding a humanized TOR polypeptide as described herein is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described here.
[00123] [00123] In one aspect, a hybridoma or quadroma is provided, derived from a non-human animal cell as described here. In one embodiment, the non-human animal is a rodent, for example, a mouse or a rat.
[00124] [00124] A method is also provided to produce a genetically modified non-human animal (eg, rodent, for example, mouse or rat) described here. The method for producing a genetically modified non-human animal results in the cujogenoma animal comprising a non-rearranged humanized TOR locus (for example, a non-rearranged TORa, TOCRb, TORd, and / or human TORg). In one embodiment, the method for producing a genetically modified non-human animal (eg, rodent, eg mouse or rat) that expresses a T cell receptor comprising a human variable region and a region
[00125] [00125] Thus, the nucleotide constructs used to generate genetically engineered non-human animals described here are also provided. In one aspect, the nucleotide construct comprises: 5 'and 3' homology arms, a human DNA fragment comprising human TOR variable region gene segment (s), and a selection cassette flanked by sites recombination. In one embodiment, the human DNA fragment is a fragment of a TCRa gene and comprises at least one segment of a variable region of human TCORa. In another embodiment, the human DNA fragment is a TCRb fragment and comprises at least one variable region gene segment of human TORb. In one respect, at least one homology arm is a non-human homology arm and is homologous to the non-human TOR locus (for example, non-human TCRaorTCRb locus).
[00126] [00126] A selection cassette is a nucleotide sequence inserted in a targeting construct to facilitate the selection of cells (for example, ES cells) that have integrated the construct of interest. A number of suitable selection cassettes are known in the art. Generally, a selection cassette enables positive selection in the presence of a particular antibiotic (eg, Neo, Hyg, Pur, CM, Spec, etc.). In addition, a selection cassette can be flanked by recombination sites, which allow deletion of the selection cassette by treatment with recombinase enzymes. The commonly used sites of recombination are loxP and Frt, recognized by enzymes Cre and Flp, respectively, but others are known in the art.
[00127] [00127] In one embodiment, the selection cassette is located at the 5 'end of the human DNA fragment. In another way, the selection cassette is located at the 3 'end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA fragment, for example, within the human intron. In another modality, the selection cassette is located at the junction of the human and mouse DNA fragment.
[00128] [00128] Several exemplified modalities of the targeting strategy to generate genetically engineered non-human animals, the constructs, and the targeting vectors used for it are shown in figures 3, 4, 5,7 and 8.
[00129] [00129] Upon completion of gene targeting, genetically modified ES cells or non-human animals are screened to confirm successful incorporation of an exogenous nucleotide sequence of interest or expression of exogenous polypeptide (for example, region segments human TOR variable). Technical numbers are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCT (eg, real time PCR using TAQMANO), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (eg, mice) endowed with the genetic modification of interest can be identified by trimming the loss of mouse alleles and / or the gain of human alleles using a allele modification described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Bio-
[00130] [00130] The description also provides a method for modifying a locus of the TOR variable region gene (e.g., TCRa gene locus, TOCRb, TORd, and / or TORg) of a non-human animal to express a TOR protein humanized described here. In one embodiment, the invention provides a method for modifying a locus of the TCR variable region gene to express a humanized TCR protein on a T cell surface where the method comprises replacing a locus of the region gene in a non-human animal variable of endogenous non-human TOR by a locus of the genus of variable region of humanized TOR. In a modality where the locus of the TOR variable region gene is a locus of the TORa variable region gene, the locus of the humanized TOR variable region gene comprises at least one human Va segment and at least one human Jo segment . In a modality where the locus of the variable region gene of TOR is a locus of the variable region gene of TCRb, the locus of the variable region of humanized TOR not rearranged comprises at least one human Vb segment, at least one segment Human Db, and at least one human Jb segment. In several respects, the locus of the humanized TOR variable region gene not rearranged is functionally linked to the endogenous non-human TCR constant region.
[00131] [00131] A humanized TOR protein produced by a non-human animal (eg, rodent, eg mouse or rat) as described here is also provided, where the humanized TOR protein comprises a human variable region and a region non-human constant. Thus, the humanized TOR protein comprises human complementary determining regions (ie, human CDR1, 2 and 3) in its variable domain and a constant non-human region.
[00132] [00132] Although the Examples that follow describe a genetically engineered non-human animal whose genome comprises the locus of the humanized TORa variable region gene and / or humanized TCRb, one skilled in the art would understand that a similar strategy can be used to produce animals genetically elaborated whose genome comprises the locus of the variable region gene of humanized TCRd and / or humanized TORg. A genetically engineered non-human animal with humanization of all four loci of the TCR variable region gene is also provided. Use of Genetically Modified TCR Animals
[00133] [00133] In various modalities, the genetically modified non-human animals of the invention produce T cells with humanized TCR molecules on their surface, and as a result, they would recognize the peptides presented to them by MHC complexes in a similar way to human. The genetically modified non-human animals described here can be used to study the development and function of human T cells and the immune tolerance process; to test human vaccine candidates; to generate TCRs with certain specificities for TCR gene therapy; to generate TCR libraries for disease-associated antigens (e.g., tumor-associated antigens (TAAs); etc.
[00134] [00134] There is a growing interest in T cell therapy in the art, as T cells (eg cytotoxic T cells) can be targeted for attack and lead to the destruction of the antigen of interest, eg viral antigen, antigen bacterial, tumor antigen, etc., or cells that present it. Initial studies in cancer T-cell therapy aim at isolating tumor lymphocyte infiltrates (TILs; lymphocyte populations in the tumor mass that presumably comprise T cells reactive to tumor antigens) from the tumor cell mass, expanding them in vitro using T cell growth factors, and transferring them back to the patient in a process called adoptive T cell transfer. See, for example, Restifo et al. (2012) Adoptive immuno-therapy for cancer: harnessing the T cell response, Nature Reviews 12: 269-81; Linnermann et al. (2011) T-Cell Receptor Gene The Rapy: Critical Parameters for Clinical Success, J. Invest. Dermatol. 131: 1806-16. However, the success of these therapies has been limited to melanoma and renal cell carcinoma; and the adoptive transfer of TIL is not specifically directed to tumor-associated antigens (TAAs). Linnermann et al.
[00135] [00135] Attempts have been made to initiate TOR gene therapy where T cells are either selected or programmed to target an antigen of interest, for example, a TAA. Current TCR gene therapy relies on the identification of sequences of TORs that target specific antigens, for example, antigens associated with the tumor. For example, Rosenberg and colleagues published several studies in which they transduced peripheral blood lymphocytes derived from a melanoma patient with genes encoding TOCRa and b chains specific to melanoma-associated MART-1 antigen epitopes, and used the resulting expanded lymphocytes for adoptive therapy of T. Johnson et al. (2009) Gene the rapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen, Blood 114: 535-46; Morgan et al. (2006) Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes, Science 314: 126-29. MART-1-specific TCRs were isolated from patients who experienced tumor regression after TIL therapy. However, the identification of such TOCRs, particularly TORs with high avidity (which are more likely to be therapeutically useful) is complicated by the fact that most tumor antigens are self-antigens, and TCRs targeting these antigens are often or deleted or have suboptimal affinity, mainly due to immunological tolerance.
[00136] [00136] In several embodiments, the present invention solves this problem by providing genetically engineered non-human animals comprising in its genome a locus of the variable region gene of non-rearranged human TCR. The non-human animal described here is capable of generating T cells with a diverse repertoire of humanized T cell receptors. Thus, the non-human animals described here can be a source of a diverse repertoire of humanized T cell receptors, for example, highly avid humanized T cell receptors for use in adoptive T cell transfer.
[00137] [00137] Thus, in one embodiment, the present invention provides a method for generating a T cell receptor to a human antigen comprising immunizing a non-human animal (for example, a rodent, for example, a mouse or a rat) described here with an antigen of interest, allow the animal to establish an immune response, isolate an activated T cell with specificity for the antigen of interest from the animal, and determine the nucleic acid sequence of the T cell receptor expressed by antigen-specific T cell.
[00138] [00138] In one embodiment, the invention provides a method for producing a human T cell receptor specific for an antigen of interest (for example, an antigen associated with the disease) comprising immunizing a non-human animal described here with the antigen of interest; allowing the anima to establish an immune response; isolating a T cell reactive to the antigen of interest from the animal; determine a nucleic acid sequence from a variable region of
[00139] [00139] In one embodiment, the nucleotide sequence encoding a specific T cell receptor for an antigen of interest is expressed in a cell. In one embodiment, the cell expressing the TCR is selected from a CHO, COS, 293, HeLa, PERC.67Y cell, etc.
[00140] [00140] The antigen of interest can be any antigen that is known to cause or be associated with a disease or condition, for example, an antigen associated with the tumor, an antigen of viral, bacterial or pathogenic origin, etc. Many antigens associated with the tumor are known in the art. A selection of tumor-associated antigens is presented in the Cancer Immunity (A Journal of the Cancer Research Institute) Peptide Database (archiv.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm). In some embodiments of the invention, the antigen of interest is a human antigen, for example, an antigen associated with a human tumor. In some embodiments, the antigen is a cell type-specific intracellular antigen, and a T cell receptor is used to kill a cell expressing the antigen.
[00141] [00141] In one embodiment, a method is provided to identify a T cell with specificity against an antigen of interest, for example, a tumor-associated antigen, comprising immunizing a non-human animal described here with the antigen of interest, allow the animal to establish an immune response, and isolate a T cell with antigen specificity from the non-human animal.
[00142] [00142] The present invention provides new methods for adoptive T-cell therapy. Thus, a method is provided here to treat or ameliorate a disease or condition (eg, cancer) in a subject (eg, a mammalian subject, for example) example, a human subject) comprising immunizing a non-human animal described here with an antigen associated with the disease or condition, allowing the animal to establish an immune response, isolating a population of antigen-specific T cells from the animal, and infusing blood - antigen-specific T cells in the subject. In one embodiment, the invention provides a method for treating or ameliorating a disease or condition in a human subject, comprising immunizing the non-human animal described here with an antigen of interest (for example, an antigen associated with the disease or condition. , for example, a tumor-associated antigen), allow the animal to establish an immune response, isolate a population of antigen-specific T cells from the animal, determine the nucleic acid sequence of a T cell receptor expressed by the specific T cells of antigen, clone the nucleic acid sequence of the T cell receptor into an expression vector (e.g., retroviral vector), introduce the vector into T cells derived from the subject such that T cells express the antigen-specific T cell receptor, and infuse T cells in the subject. In one embodiment, the T cell receptor nucleic acid sequence is still humanized prior to introduction into the subject-derived T cells, for example, the sequence encoding the non-human constant region is modified to further assemble a human TOR constant region (for example, the non-human constant region is replaced by a human constant region). In some embodiments, the disease or condition is cancer. In some modalities, a population of antigen-specific T cells is expanded before infusion into the subject. In some modalities, the subject's immune cell population is immunodepleted before the infusion of antigen-specific T cells. In some embodiments, the antigen-specific TCR is a highly avid TCR, for example, a highly avid TCR for a tumor-associated antigen. In some ways, the T cell is a cytotoxic T cell. In other embodiments, the disease or condition is caused by a virus or bacteria.
[00143] [00143] In another mode, a disease or condition is an autoimmune disease. Treg cells are a subpopulation of T cells that maintain tolerance to autoantigens and prevent pathological autoreactivity. Thus, methods are also provided to treat autoimmune disease that rely on the generation of antigen-specific Treg cells in the non-human animal of the invention described here.
[00144] [00144] A method for treating or ameliorating a disease or condition (for example, cancer) in a subject comprising introducing the cells affected by the subject's disease or condition (for example, cancer cells) is also provided here. a non-human animal, allow the animal to establish an immune response to cells, isolate a cell-reactive T cell population from the animal, determine the nucleic acid sequence of a T cell receptor expressed by T cells, clone the sequence T cell receptor into a vector, introduce the vector into T cells derived from the subject, and infuse the subject's T cells by infecting the T cell receptor in the subject.
[00145] [00145] The use of a non-human animal as described here to produce nucleic acid sequences encoding human TOR variable domains (for example, variable TORa and / or b domains) is also provided here. In one embodiment, a method is provided to produce a nucleic acid sequence encoding a variable domain of human TOR, comprising immunizing a non-human animal as described here with an antigen of interest, allowing the non-human animal to establish an immune response to the antigen of interest, and obtain from it a nucleic acid sequence encoding a variable domain of human TOR that binds to the antigen of interest. In one embodiment, the method further comprises producing a nucleic acid sequence encoding a human TOR variable domain that is functionally linked to a non-human TOR constant region, comprising isolating a T cell from a non-human animal described herein. and obtaining from it the nucleic acid sequence encoding the TCR variable domain linked to the TCR constant region.
[00146] [00146] Also provided herein is the use of a non-human animal as described herein to produce a human therapeutic agent, comprising immunizing the non-human animal with an antigen of interest (e.g., a tumor-associated antigen), allowing the animal establish a immune response, obtain T cells reactive to the antigen of interest from the animal, obtain a nucleic acid sequence from T cells encoding a humanized TCR protein that binds to the antigen of interest, and employ the nucleic acid sequence encoding a humanized TCR protein in a human therapeutic agent.
[00147] [00147] Thus, a method is also provided to produce a human therapeutic agent, comprising immunizing a non-human animal as described here with an antigen of interest, allowing the non-human animal to establish an immune response, obtaining T cells from the animal reactive to the antigen of interest, obtain from the T cells a nucleic acid sequence encoding a humanized T cell receptor that binds to the antigen of interest, and employ the humanized T cell receptor in a human therapeutic agent.
[00148] [00148] In one embodiment, the human therapeutic agent is a T cell (for example, a human T cell, for example, a T cell derived from a human subject) infecting a nucleic acid sequence of interest (for example, transfected or transduced or otherwise introduced with the nucleic acid of interest) such that the T cell expresses the humanized TOR protein with affinity for an antigen of interest. In one aspect, a subject in whom the therapeutic person is employed is in need of therapy for a particular disease or condition, and the antigen is associated with the disease or condition. In one aspect, the T cell is a cytotoxic T cell, the antigen is a tumor-associated antigen, and the disease or condition is cancer. In one aspect, the T cell is derived from the subject.
[00149] [00149] In another embodiment, the human therapeutic agent is a T cell receptor. In one embodiment, the therapeutic receptor is a soluble T cell receptor. Much effort has been expanded to generate soluble T cell receptors or variable regions of TOR to use therapeutic agents. The generation of soluble T cell receptors depends on obtaining variable TOR regions rearranged. One approach is to design single chain TORs comprising TCRa and T-CRb, and, similar to the scFv immunoglobulin format, fuse them via a linker (see, for example, International Application No. WO 2011/044186). The resulting scTv, if analogous to scFv, would provide a soluble and thermally stable form of T-CRo / b binding protein. Alternative approaches included designing a soluble TCR having constant TCRb domains (see, for example, Chung et al., (1994) Functional three-domain single-chain T-cell receptors, Proc. Natl. Acad. Sci. USA. 91 : 12654-58); as well as to elaborate a non-native disulfide bond at the interface between the constant domains of TCR (reviewed in Boulter and Jakobsen (2005) Stable, soluble, high-affinity, engineered T cell receptors: novel antibody-like proteins for specific targeting of peptide antigens, Clinical and Experimental Immunology 142: 454-60; see also, US Patent No. 7,569,664). Other formats of soluble T cell receptors have been described. The non-human animals described here can be used to determine a sequence of a T cell receptor that binds with high affinity to an antigen of interest, and subsequently design a soluble T cell receptor based on the sequence.
[00150] [00150] A soluble T cell receptor derived from the TOR receptor sequence expressed by the non-human animal can be used to block the function of a protein of interest, for example, a viral, bacterial or tumor-associated protein. Alternatively, a soluble T cell receptor can be fused to a portion that can kill an infected or cancerous cell, for example, cytotoxic molecules (for example, a chemotherapeutic), toxin, radionuclide, prodrug, antibody, etc. A soluble T cell receptor can also be fused to an immunomodulatory molecule, for example, a cytokine, chemokine, etc. A soluble T cell receptor can also be fused to an immune inhibitory molecule, for example, a molecule that inhibits a T cell from killing other cells by infecting an antigen recognized by the T cell. Such soluble T cell receptors fused to inhibitory molecules. immune cells can be used, for example, to block autoimmunity. Several exemplified immune inhibitory molecules that can be fused to a soluble T cell receptor are reviewed by Ravetch and Lanier (2000) Immune Inhibition
[00151] [00151] The present invention also provides methods for studying the immune response in the context of human TCR, including human TOR rearrangement, T cell development, T cell activation, immunological tolerance, etc.
[00152] [00152] Methods for testing vaccine candidates are also provided. In one embodiment, a method is provided here to determine whether a vaccine will activate an immune response (for example, T cell proliferation, cytokine release, etc.) and lead to the generation of effector T cells, as well as T cells from memory (for example, central and effector memory T cells). Examples
[00153] [00153] The invention will be further illustrated by the following non-limiting examples. These Examples are presented to assist in understanding the invention, but are not intended to limit its scope in any way, and should not be construed as limiting its scope. The Examples do not include detailed descriptions of conventional methods that would be well known to those skilled in the art (molecular cloning techniques, etc.). Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is indicated in Celsius, and pressure is atmospheric or close to it. Example 1. Generation of mice with loci from the humanized TOR variable region gene
[00154] [00154] Mice comprising a variable locus deletion of TCR (a or b) and replacement of endogenous Ve Jou V, DeJ segments are produced using VELOCIGENEPGO genetic engineering technology (see, for example, US Patent No. 6,586. 251 and Valenzuela, DM, et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.
[00155] [00155] Targeted ES cell clones are introduced into 8-cell (or early) mouse embryos by the VELOCIMOUSEG method (Poueymirou, WT et al. (2007). FO gene generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyzes (Nat. Biotech. 25: 91 to 99). SPEED (FO mice completely derived from the donor ES cell) endowed with humanized TOR locus are identified by screening for loss of endogenous TOR variable alleles and gain of human alleles using an allele modification assay (Valenzuela and others). FO puppies are genotyped and crossed for homozygosity. Homozygous mice for variable loci of humanized TCRa and / or TCRb (for example, comprising a subset of variable segments of human TORa and / or TORb) are produced and phenotyped as described herein.
[00156] [00156] All mice were housed and crossed at the specific pathogen-free facility in Regeneron Pharmaceuticals. All animal experiments were approved by IACUC and Regeneron Pharmaceuticals. Example2: Progressive humanization of a variable locus of TCRa
[00157] [00157] 1.5 megabases of DNA in a mouse TCRa locus corresponding to 110 mouse V segments and 60 mouse J segments have been replaced by 1 megabase of DNA corresponding to 54 V segments and 61 human TCRa segments u - using a progressive humanization strategy summarized in Figures 2 and 3. The junctional nucleic acid sequences of several targeting vectors used for the progressive humanization strategy of TCRa locus are summarized in Table 2, and included in the Lis - Sequence loading. Table2: Junctional nucleic acid sequences for various SEQID TORa locus targeting vectors Description
[00158] [00158] The human TORa variable region segments are numbered as in the IMGT database. At least 100 bp in each junction (approximately 50 bp from each end) are included in the Sequence Listing.
[00159] [00159] Specifically, as shown in Figure 4A, the DNA of the mouse BAC clone RP23-6A14 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1539) to replace the TORAJ1-TCRAJ28 region of locus of endogenous mouse TCRa by an Ub-hygromycin cassette followed by a loxP site. The DNA of the mouse BAC clone RP23- 117i19 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1535) to replace -15 kb of region surrounding (and including) TORAV1 of the endogenous mouse TCRa and locus by a PGK-neomycin cassette followed by a loxP site. ES cells equipped with a double-directed chromosome (ie, a single locus of endocrine mouse TCRa targeted with both targeting vectors) were confirmed by karyotyping and screening methods (for example, TAQ-
[00160] [00160] The first human targeting vector for TCRa. ti- —nha191,660 bp of human DNA from BAC clones CTD2216p1 and CTD2285m07 (Invitrogen) that contained the first two consecutive human TCORaV gene segments (TRAV40 & 41) and 61 TCRayJ gene segments (50 functional ). This BAC was modified by homologous recombination to contain a 403 bp Not1 site downstream (3) of the TCRaJ1 gene segment to link a 3 'mouse homology arm and an AsSiS | 5 'for binding to that human fragment: the 3' homology arm contained endogenous mouse TCRa sequences from the BAC clone RP23- 6A14 and the 5 'homology arm contained the endogenous 5S TCRaV TORa sequence from mouse to from mouse BAC clone RP23-117i19. This human-mouse chimeric BAC was used as a targeting vector (MAID 1626) to make an initial insertion of TCRa gene segments plus a cassette / oxp-ub-hygromycin-loxp into the mouse TCORa loci (figure 4B). The junctional nucleic acid sequences (SEQ ID Nos: 1-3) for the MAID targeting vector 1626 are described in Table 2.
[00161] [00161] Subsequently, a series of human targeting vectors that was produced used the same 5 'mouse arm that contained the mouse 5' TCORaV endogenous TORa sequence from the mouse BAC clone RP23-117i19 with alternating selection cassettes loxP-neomycin-loxP and JloxP-hygromycin-loxP (or frt-hygromycin-frt for MAID 1979).
[00162] [00162] To generate a human TCRa mini-locus containing a total of 8 human TCORaV gene segments (7 functional) and 61 human TOCRaJ gene segments (50 functional), the DNA of the human BAC clone RP11-349p11 ( Invitrogen) was modified by homologous combination and used as a targeting vector (MAID 1767) (figure 4C). That 104,846 bp added of human DNA containing the next 6 (5 functional) consecutive human TCRoaV gene segments (TRAV35 to TRAV39) and a 5 'loxP-ub-neomycin-loxP cassette. The resulting TCRa locus contained a 5 'loxp-ub-neomycin-loxP cast plus a total of 8 TCRaV gene segments (7 functional) and 61 human TCORaJ gene segments functionally linked to constant region genes of mouse TORa and intensifiers. The junction nucleic acid sequences (SEQ ID Nos: 4 and 5) for the MAID 1767 targeting vector are described in Table 2.
[00163] [00163] To generate a human TCRa mini-locus containing a total of 23 TOCRaV gene segments (17 functional) and 61 human TCRoaJ gene segments, the DNA of the 5 'mouse BAC clone to 3 ": an exclusive I | -Ceul site, a 20 kb 5 'mouse TORA arm of the mouse TORA locus to be used for homologous recombination in ES cells, and a loxP-Ub-Hyg-loxP cassette in orientation reverse, was modified by homologous bacterial recombination to contain from 5 'to 3': an exclusive site | - Ceul, a 20 kb 5 'mouse TORA arm from the mouse TORA locus, a frt-pgk-Hyg cassette -frt, and an exclusive AsiS | site The DNA of the human BAC clone RP11-622020 (Invitrogen), infecting human TCRaV22-V34 modified by homologous recombination to contain a Spec cassette flanked by exclusive sites | -Ceul and Asi-
[00164] [00164] To generate a human TORa mini-locus containing a total of 35 TCRaV gene segments (28 functional) and 61 human TCRaJ gene segments, the DNA of the human BAC clone CTD2501-k5 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1769) (figure 4E). 124,118 bp of human DNA was then added which contained the next 12 consecutive human TCRaV gene segments (11 functional) (TRAV13-2 to TRAV21) and a 5 '/ oxp-ub-neomycin-loxP cassette. The resulting TCRa locus contained a 5 'loxp-ub-neomycin-loxP cassette plus a total of 35 human TCRaV gene segments (28 functional) and 61 human TCORaJ gene segments functionally linked to constant region genes and mouse TCRa enhancer. Junctional nucleic acid sequences (SEQ ID Nos: 8 and 9) for the targeting vector MAID 1769 are described in Table 2.
[00165] [00165] To generate a human TCORa mini-locus containing a total of 48 TCRaV gene segments (39 functional) and 61 human TCRaJ gene segments, the DNA of the human BAC clone RP11-92F11 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1770) (figure 4F). 145,505 bp of human DNA was added which contained the next 13 consecutive human TOCRaJ gene segments (TRAV6 to TRAVB8.5) and a 5 '/ oxp-ub-hygromycin-loxP cassette. The resulting TORa locus contains a 5 'loxp-ub-hygromycin- / loxP cassette plus a total of 48 TCRaV gene segments (39 functional) and 61 human TCRaJ gene segments functionally linked to region genes constant and intensifying mouse TCORa. Junctional nucleic acid sequences (SEQ ID NOs: 10 and 11) for the MAID 1770 targeting vector are described in Table 2.
[00166] [00166] To generate a human TCRa mini-locus containing a total of 54 TOCRaV gene segments (45 functional) and 61 human TOCRaJ gene segments, the DNA of the human BAC clone RP11-780M 2 (Invitrogen ) was modified by homologous recombination and used as a targeting vector (MAID 1771) (figure 4G). 148,496 bp of human DNA was added which contained the next 6 consecutive human TCRaV gene segments (TRAV1I-1 to TRAVS5) (6 functional) and a 5 '/ oxp-ub-neomycin-loxP cassette. The resulting TCRa locus contains a 5 'loxp-ub-neomycin-loxP cassette plus a total of 54 TCRaV gene segments (45 functional) and 61 human TCRaJ gene segments functionally linked to region genes constant and intensifying mouse TCORa. Junctional nucleic acid sequences (SEQ ID NOs: 12 and 13) for the MAID 1771 targeting vector are described in Table 2.
[00167] [00167] In any of the steps above, the selection cassettes
[00168] [00168] 0, 6 megabases of DNA in mouse TCRb locus corresponding to 33 V segments, 2 D segments, and 14 mouse J segments were replaced by 0.56 megabases of DNA corresponding to 67 V segments, 2 D segments, and 14 J segments of human TCRb using a progressive humanization strategy summarized in figures 6 and 7. The junctional nucleic acid sequences of various targeting vectors used for the progressive humanization strategy of TCRb locus are summarized in Table 3, and included in the Sequence Listing. Table 3: Junctional nucleic acid sequences for various locus targeting vectors of TCORb MAID | SEQID Description NO. NO 14 Junctional nucleic acid sequence between the 3 'end of the mouse sequence upstream of the variable TOCRb locus (close to the upstream mouse trypsogen genes) and the 5' end of the frt-Ub-Neo cassette -frt. 15 Junctional nucleic acid sequence between the 3 'end of the frt-Ub-Neo-frt cassette and the 5' end of the human TORVb18-TCRVb29-1 insert. 16 Junctional nucleic acid sequence between the 3 'end of the human TCRVb18-TCRVb29-1 insert and the 5' end of the mouse sequence downstream of the mouse TCRVb segments (close to the mouse trypsinogen genes upstream ). 17 Junctional nucleic acid sequence between the 3 'end of the downstream mouse trypsinogen genes and the 5' end of the T-1715 human insert CRDb1-TCRJb1-1-TCRJb1-6, including the Iceul site. 18 Junctional nucleic acid sequence between the 3 'end of the human insert TOCRDb1-TCRJb1-1-TCRJb1-6 and the 5' end of the loxP-Ub-Hyg-loxP cassette.
[00169] [00169] The variable region segments of human TCRb are numbered as in the IMGT database. At least 100 bp in each junction (approximately 50 bp from each end) are included in the Sequence Listing.
[00170] [00170] Specifically, the DNA of the mouse BAC clone RP23-153p19 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1544) to replace the 17 kb region (including TCORBV30) upstream of the group of 3 'trypsinogen gene at the endogenous mouse TORb locus with a PGK-neo cassette followed by a loxP site (figure 8A). The mouse BAC clone DNA RP23-461h15 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1542) to replace the 8355 bp region (including TORBV2 and TCRBV3) downstream of the gene group 5 'trypsinogen in the endogenous mouse T-CRb locus with an Ub-hygromycin cassette followed by a loxP site. ES cells with a double-directed chromosome (ie, a single locus of endogenous mouse TCORb targeted with both targeting vectors) have been confirmed by karyotyping and screening methods (for example, TAQMANT'Y) known in technical. The modified ES cells were treated with CRE recombinase, mediating the deletion of the region between the 5 'and 3' loxP sites (consisting of the endogenous mouse TORb locus from TCORBV2 to TCRBV30) and leaving behind only a single loxP site, hygromycin and the TCRBDs, TCRBJs sequences, constant and intensifying. A mouse TCRVb was left upstream of the 5 'group of trypsinogen genes, and a mouse TCRBb was left downstream of the mouse Eb, as noted in Figure 8A.
[00171] [00171] The first human TCRb targeting vector had 125,781 bp of human DNA from the CTD2559j2 clone (Invitrogen) that contained the first 14 consecutive human TCORbV gene segments (TRBV18-TRBV29-1) . This BAC was modified by homologous recombination to contain a 5 'AsiS | and a 3 'Aspin site for the connection of 5' and 3 'mouse homology arms. Two different homology arms were used to attach to this human fragment: one set of homology arms contained the endogenous TCORb sequence surrounding the mouse trypsinogen genes downstream from the BAC clone RP23-153p19 and another set contained a sequence of Endogenous TCORb surrounding the mouse trypsinogen genes upstream of the mouse BAC clone “RP23-461h15. This chimeric human-mouse BAC was used as a targeting vector (MAID 1625) to make an initial insertion of TCRb gene segments plus a frt-ub-neomycin-frt cassette into the mouse TCORb locus, and resulted in a mini -locus of human TORb containing 14 human TCRbV (8 functional) (figure 8B). The junction nucleic acid sequences (SEQ ID Nos: 14-16) for the targeting vector MAID 1625 are described in Table 3.
[00172] [00172] In order to replace mouse TCRb D and J segments with human TORb D and J segments, the DNA of the mouse BAC clone RP23-302p18 (Invitrogen) and the human BAC clone RP11-701D14 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1715) in ES cells that contained the TCRbV mini-locus described above (i.e., MAID 1625). This modification replaced the -18540 bp region (from 100 bp downstream of the polyA of the 3 'trypsinogen genes to 100 bp downstream of the J segments in the D2 group that included mouse TORBD1-J1, constant region 1 of mouse and mouse TCRBD2-J2 at the endogenous mouse TORb locus for -25425 bp of sequence containing human TCRBD1-J1, cassette / oxP Ub- hygromycin-loxP, mouse constant region 1, human TCRBD2-J2 ( figure 8C (i)). ES cells equipped with a double-directed chromosome (ie, a single locus of endogenous mouse TORb targeted with both targeting vectors) were confirmed by karyotyping and screening methods (for example, TAQMANT'Y) known in the art. The modified ES cells were treated with CRE recombinase thus mediating the deletion of the hygromycin cassette leaving behind only a single loxP site downstream of human J segments in the D1J group (figure 8C (ii)). Junctional nucleic acid sequences (SEQ ID NOs: 17 to 21) for the targeting vector MAID 1715 are described in Table 3.
[00173] [00173] Subsequently, a series of human targeting vectors that was produced utilized the same 5 'mouse arm contained the endogenous TORb sequence surrounding the mouse trypsinogen genes upstream of the mouse BAC clone RP23 -461h15 with alternative selection cassette.
[00174] [00174] To generate a mini-locus of human TORb containing a total of 40 V segments of human TCRb (30 functional) and the D and J segments of human TOCRb, the DNA of human BAC clones RP11-134h14 and RP11- 785k24 (Invitrogen) was modified by homologous recombination and combined into a targeting vector (MAID 1791) using standard bacterial homologous recombination, digestion / binding restriction, and other cloning techniques. The introduction of the MAID 1791 targeting vector resulted in the addition of 198,172 bp of human DNA that contained the next 26 consecutive human TCRb V gene segments (22 functional) (TRBV6-5 to TRBV1I7) and a 5 'frt-ub- hygromycin-frt. The resulting TORb locus contained a 5 'frt-ub-hygromycin-frt cassette plus a total of 40 human TCRb degeneV segments (30 functional) and human TCORb D and J gene segments functionally linked to genes from mouse TCRb constant region and enhancers (figure 8D). Junctional nucleic acid sequences (SEQ ID NOs: 22 and 23) for the MAID 1791 targeting vector are described in Table 3.
[00175] [00175] To generate a mini-locus of human TORb containing a total of 66 V segments of TCRb (47 functional) and segments D and J of human TOCRb, the DNA from the human BAC clone RP11- 902B7 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1792). This resulted in the addition of 159,742 bp of human DNA that contained the next consecutive TCRb V gene segments (17 functional) (TRBV1 to TRBV12-2) and a 5 'frt-ub-neomycin-frt cassette. The resulting TOCRb locus contained a 5 'frt-ub-neomycin-frt cassette plus a total of 66 human TCRb V gene segments (47 functional) and human TOCORb gene D and J segments functional to mouse TCORb constant genes and enhancers (figure 8E). Junctional nucleic acid sequences (SEQ ID NOs: 24 and 25) for the targeting vector MAID 1792 are described in Table 3.
[00176] [00176] In any of the steps above, the selection cassettes are removed by deletion with Cre or Flp recombinase. For example, as shown in figure 7, MAID 1716 corresponds to MAID 1715 with the deletion of the hygromycin cassette.
[00177] [00177] Finally, a mini-locus of human TORb containing a total of 67 V segments of human TCRb (48 functional) and the D and J segments of human TCORb was generated. TORBV31 is located —9.4 kb 3 'of TORBC 2 (the second TCRB constant region sequence) and is in the opposite orientation to the other T-CRBV segments. The equivalent human segment V is TORBV30, which is located in a similar position in the locus of human TORB.
[00178] [00178] To humanize TORBV31, the mouse BAC clone containing mouse TCORBV31, was modified by homologous bacterial recombination to produce LTIVEC MAID 6192 (figure 8F). To the entire coding region, starting at the start codon in exon 1, O3 intron UTR, and recombination signal sequences (RSS)
[00179] [00179] Junctional nucleic acid sequences (SEQ ID NOs: 26 and 27) for the MAID 6192 targeting vector are described in Table 3. MAID 6192 DNA is electroporated in MAID ES cells
[00180] [00180] Similar engineering strategy is used to optionally delete the V segment of remaining mouse 5 'TCRb. Example4: Generation of TCRawWTCRb mice
[00181] [00181] At each stage of progressive humanization of loci of TCRa and TCRb, mice homozygous for the variable locus of humanized TCRa can be crossed with homozygous mice for variable loci of humanized TORb to form progeny comprising the variable loci of TORa and Humanized TOCRb. The progeny is crossed for homozygosity in relation to the loci of humanized T-CRae and TCRb.
[00182] [00182] In one embodiment, mice homozygous for the variable locus of humanized TORa comprising 8 human Va segments and 61 human Ja segments (MAID 1767; "1767 HO") were crossed with mice homozygous for the humanized variable TCRb locus comprising 14 human Vb segments, 2 human Db segments and 14 human Jb segments (MAID 1716; "1716 HO"). The progeny was crossed for homozygosity in relation to both the humanized loci. Example 5: The production of splenic T cells in homozygous mice for locus of TCRa and / or humanized TCRb.
[00183] [00183] The spleens of naturally occurring mice (WT); mice with the deleted mouse TCRa locus ("MAID 1540", see figure 3); mice homozygous for the human TORa locus ("MAID 1767", see figure 3); mice with deleted TCRb V segments with the exception of two remaining mouse V segments ("MAID 1545", see figure 7); mice homozygous for the human TORb locus, also comprising the remaining two mouse V segments ("MAID 1716", see figure 7); and homozygous mice for both human TORa and TCRb loci, such as the TCRb locus also comprising the remaining two mouse V segments ("MAID 1767 1716") were sprayed with Collagenase D (Roche Bioscience) and erythrocytes were cleared with ACH lysis buffer, followed by washing in RPMI medium.
[00184] [00184] Splenocytes from a single representative animal WT, MAID 1540, 1767, 1545, 1716, and 1716 1767 were evaluated by flow cytometry. In summary, cell suspensions were made using standard methods. 1 x 10 th cells were incubated with anti-mouse CD16 / CD32 (2.4 G2, BD) on ice for 10 minutes colored with the appropriate antibody cocktail for 30 minutes on ice. After staining, the cells were washed and then fixed in 2% formaldehyde. Data acquisition was performed in an LSRIIl / Cantoll / LSRFortessa flow cytometer and analyzed with FlowJo.
[00185] [00185] For splenocyte staining, antidFITO-CD3 from
[00186] [00186] To determine whether mice homozygous for the humanized TCRa and / or TOCORb locus exhibited normal T cell development in the thymus, splenocytes from four of each corresponding WT animal, 1767 HO, 1716 HO, and 1716 HO 1767 HO (7 to 10 weeks old) were used in flow cytometry to evaluate T cell production at various stages of development, as well as to evaluate the frequency and absolute number of each T cell DN, DP , CD4 SP, and CD8 SP.
[00187] [00187] Cell type determinations were made based on the presence of cell surface markers CD4, CD8, CD44, and CDZ25, as summarized in Table 1. The correlation between cell type designation and expression of cell markers cell surface in the thymus is as follows: double-negative cells (DN) (CD4- CD8-), double-positive cells (DP) (CD4 + CD8 +), single-positive CD4 cells (CD4 + CD8-), single CD8 cells -positives (CD4- CD8 +), cells 1 / DN1 —double negative (CD4- CD8-, CD25- CD44 +), cells 2 / DN2 double-negative (CD4- CD8-, CD25 + CD44 +), cells 3 / DN3 double- negative (CD4- CD8-, CD25 + CD44-), double negative 4 / DN4 cells (CD4- CDB8-, CD25- CD44-).
[00188] [00188] Thymocytes were evaluated by flow cytometry. In summary, cell suspensions were made using methods
[00189] [00189] As shown in figures 10 and 11, the homozygous mice for TCRa, TCRb, and both TCRa and TCRb were able to produce a T cell DN1, DN2, DN3, DNA4, DP, CD4 SP, and CD8 SP, indicating that T cells produced from humanized loci are able to undergo T cell development in the thymus.
[00190] [00190] To determine whether mice homozygous for the locus of TOCRa and / or humanized TCRb exhibited normal T cell differentiation in the periphery (eg, spleen), four of each WT animal, 1767 HO, 1716 HO, and 1716 HO 1767 HO of corresponding age (7-10 weeks old) were used in flow cytometry to evaluate the production of various types of T cells in the spleen (CD3 +, CD4 +, CD8 +, virgin T, Tem, and Tefflfem) , as well as assessing the absolute number of each type of T cell in the spleen.
[00191] [00191] Cell type determinations were made based on the presence of CD19 cell surface markers (B cell marker), CD3 (T cell marker), CD4, CD8, CD44, and CD62L (selectin L). The correlation between cell type designation and the expression of cell surface markers in the spleen is as follows: T cells (CD3 +), CD4 T cells (CD3 + CD4 + CD8-), CD8 T cells (CD3 + CD4- CD8 +) , effector / effector memory CD4 T cells (CD3 + CD4 + CD8- CD62L- CD44 +), central memory CD4 T cells (CD3 + CD4 + CD8- CD62L + CD44 +), virgin CD4 T cells (CD3 + CD4 + CD8- CD62L + CD44-), T cells Effector CD8 / effector memory (CD3 + CD4-CD8 + CD62L- CD44 +), CD8 central memory T cells
[00192] [00192] Splenocytes were evaluated by flow cytometry. In summary, cell suspensions were made using standard methods. Flow cytometry was conducted as described in Example 5.
[00193] [00193] As shown in figures 12 to 14, the T cells in the spleen of mice homozygous for TORa, TCRb, and both humanized TCRa and TCRb were able to undergo T cell differentiation, and both CD4 + and CD8 + T cells. were present. In addition, memory T cells were detected in the spleens of the mice tested.
[00194] [00194] V segment expression of human TCRb was assessed for protein and RNA level using flow cytometry and real-time PCR TAQMAN'Y, respectively, in mice homozygous for the humanized TCORb locus (1716 HO) and homozygous mice for both loci of TORa and TCRb (1716 HO 1767 HO).
[00195] [00195] For flow cytometry, splenic T cells were prepared and the analysis conducted as described in Example 5. By flow cytometry, the TORb repertoire kit (IOTESTE Beta Mark, Beckman Coulter) was used. The kit contains anti-human antibodies specific to a number of human TORBVs, for example, h- TRBV-18, -19, -20, -25, -27, -28, and -29.
[00196] [00196] The results are summarized in figure 15. The tables presented
[00197] [00197] For real-time PCR, the total RNA was purified from the spleen and thymus using MAGMAX'TY-96 for the Total Microarray RNA Isolation Kit (Ambion from Life Technologies) according to the manufacturer's specifications. Genomic DNA was removed using MAGMAX'YTURBO'YDNase and TURBO DNase buffer from the MAGMAWX ki listed above (Ambion from Life Technologies). MRNA (up to 2.5 µg) was reverse transcribed into cDNA using SUPERS- CRIPTO VILO "" Master Mix (Invitrogen from Life Technologies). cONA was diluted 2-5 ng / uL, and 10-25 ng cDNA was amplified with TAQMANÔO Gene Expression Master Mix (Applied Biosystems from Life Technologies) using the ABI 7900HT Sequence Detection System (Applied Biosystems), using the primers and Taqman MGB probes (Applied Biosystems) or BHQ1 / BHQ-Plus probes (Biosearch Technologies) shown in Table 4 according to the manufacturer's instructions. The relative expression of each gene was normalized for the control of murine TOR beta 1 constant region (TRBC1). Table 4: Primers and probes used to detect RCR expression of TCRb V segments and constant region in mice with humanized TCR by real-time PCR (TAQMANTY), the Sequence Sequence SED Sequence
[00198] [00198] “As shown in figures 16A-B, the homozygous mice for the humanized TCORb locus (1716 HO) and homozygous mice for both the humanized TORa locus and TCRb (1716 HO 1767 HO) exhibited the expression of RNA from various human TCRb segments in both the spleen and thymus. Mice also exhibited RNA expression from mouse TRBV-1 and TRBV-3 segments (data not shown), but no mouse TRBV-1 protein was detected by flow cytometry (data not shown).
[00199] [00199] The mouse TRBV-31 segment is replaced by the human TRBV-30 segment as shown in figure 8F, and the mice are generated from ES MAID 6192 cells as described here. The spleens and thymuses of the resulting homozygous animals are tested for the use of human Vb segments, including
[00200] [00200] The homozygous mice with humanized TCRoa characterized in the previous examples contained 8 human Va segments and 61 human Jo segments (1767 HO, see figure 3). Homozygous mice with humanized TCRa comprising 23 human Voa segments and 61 Jo segments. humans (1979 HO, see figure 3) were tested for their ability to generate splenic CD3 + T cells and exhibited T cell development in the thymus.
[00201] [00201] The experimental data were obtained using flow cytometry using appropriate antibodies as described in the previous examples. As shown in figure 17, a homozygous mouse for 23 human Vo segments and 61 human Ja segments produced a significant number of splenic CD3 + T cells, and the percentage of peripheral CD3 + T cells was comparable to that of the animals of occurrence natural (figure 19).
[00202] [00202] Furthermore, thymocytes in 1979 HO mice were able to undergo T cell development and contained T cells in the DN1, DN2, DN3, DNA4, DP, CD4 SP, and CD8 SP stages (figure 18).
[00203] [00203] “Homozygous mice for a complete repertoire of segments of variable region of human TCORa (that is, 54 Va segments and 61 Ja segments) and homozygous mice for a complete repertoire of segments of variable region of human TORb (67 human Vb segments, 2 human Db segments, and 14 segments
[00204] [00204] Flow cytometry is conducted to determine the presence of DN1, DN2, DN3, DN4, DP, CD4 SP and CD8 SP T cells in the thymus using anti-CD4, -CD8, -CD25, and -CD44 antibodies of mouse, as described above in Examples 5 and 6. Flow cytometry is also conducted to determine the number of CD3 + T cells in the periphery, as well as to evaluate the differentiation of T cells in the periphery (for example, presence of T cells effectors and memory in the periphery). The experiment is conducted using anti-CD3, -CD19, - CDA4, -CD8, -CD44, and -CD62L mouse antibodies, as described above in Examples 5 and 7.
[00205] [00205] Finally, flow cytometry and / or real-time PCR are conducted to determine whether T cells in 1771 HO 6192 HO mice use a complete repertoire of V segments of TCRB and TCRA. For protein expression using flow cytometry, the TORb repertoire kit (IOTESTE Beta Mark, Beckman Coulter), containing anti-human hNTCRBV specific antibodies, is used (see Example 8). For RNA expression using real-time PCR, spleen and thymus cDNAs are amplified using human TCR-V primers and Taqman probes, according to the manufacturers' instructions and as described above in Example 8. Equivalents
[00206] [00206] Those skilled in the art will recognize, or be able to verify the use of more than routine experimentation, many equivalents of the specific modalities of the invention described here. Such equivalents are covered by the following claims.
[00207] [00207] The entire content of all non-patent documents, patent applications and patents cited throughout this application is incorporated by reference here.

权利要求:
Claims (25)
[1]
1. Method for producing a genetically modified non-human animal, characterized by the fact that the method comprises the steps of modifying a non-human animal in its genome to understand: a locus of the variable T cell receptor (TCR) gene not rearranged comprising at least one human Vo segment and at least one Jo segment. human, operably linked to a non-human TORa constant gene sequence; and / or a non-rearranged TOR B variable gene locus comprising at least one human VB segment, at least one human DB segment and at least one human JB segment, operably linked to a constant TCRB gene sequence not human.
[2]
Method according to claim 1, characterized in that the locus of the variable gene of non-rearranged TORa replaces a locus of the variable gene of endogenous non-human TCORa and / or the locus of the variable gene of non-rearranged TCRB replaces a locus variable endogenous non-human TCORB gene.
[3]
3. Method according to claim 1 or 2, characterized by the fact that the endogenous non-human Va and Ja segments are unable to rearrange themselves to form a rearranged Vo / Jo sequence and / or the VB, DB and JB segments endogenous non-humans are unable to rearrange themselves to form a rearranged VB / DB / JB sequence.
[4]
Method according to any one of claims 1 to 3, characterized in that the animal (i) lacks a functional endogenous non-human TCRa locus and / or (ii) lacks a variable non-human TCRB locus functional endogenous.
[5]
5. Method according to claim 4, characterized by the fact that (i) the absence of the functional endogenous non-human TCORa locus comprises a deletion selected from the group consisting of (a) a deletion of all Va gene segments endogenous, (b) a deletion of all endogenous Ja gene segments, and (c) a combination of these and / or (ii) the absence of the variable locus of functional endogenous rodent TOCRB comprises a deletion selected from the group consisting of (a) a deletion of all endogenous VB gene segments, (b) a deletion of all endogenous DB gene segments, (c) a deletion of all endogenous JB gene segments and (d) a combination of them.
[6]
Method according to any one of claims 1 to 5, characterized in that the human Va and Ja segments rearrange to form a rearranged human Vo / Joa sequence and / or the VB, DB and JB segments rearrange to form a rearranged human VB / DB / JB sequence.
[7]
Method according to claim 6, characterized in that the animal expresses a T cell receptor comprising a variable region of human TORa on the surface of a T cell and / or a variable region of human TORB on the surface of a T cell.
[8]
Method according to any one of claims 1 to 7, characterized in that the animal's T cells undergo the development of thymic T cells to produce CD4 and CD8 single-positive T cells.
[9]
Method according to any one of claims 1 to 8, characterized by the fact that the animal comprises a normal ratio of CD3 + splenic T cells to total splenocytes.
[10]
10. Method according to any one of the claims
sections 1 to 9, characterized by the fact that the animal generates central memory and effector T cells for an antigen of interest.
[11]
11. Method according to any one of claims 1 to 10, characterized in that the locus of the variable gene of non-rearranged TCRa comprises a complete repertoire of human Voa segments and a complete repertoire of human Ja segments and / or the The non-rearranged TOCRB variable gene locus comprises a complete repertoire of human VB segments, a complete repertoire of human DB segments, and a complete repertoire of human JB segments.
[12]
12. Method according to any one of claims 1 to 11, characterized in that the animal retains a locus of the endogenous non-human TCRa variable gene and / or a locus of the endogenous non-human TCRB variable gene, and in that the locus is a non-functional locus.
[13]
13. Method according to any one of claims 1 to 12, characterized in that the animal expresses a T cell receptor comprising a human variable region and a non-human constant region on the surface of a T cell.
[14]
14. Method according to any one of claims 1 to 13, characterized by the fact that the animal still comprises nucleotide sequences of human Vo segments.
[15]
15. Method according to claim 14, characterized by the fact that the animal also comprises a complete repertoire of human Vo segments, a complete repertoire of human Dô segments, and a complete repertoire of human Jô segments.
[16]
16. Method according to any one of claims 1 to 15, characterized by the fact that the animal is a rodent.
[17]
17. Method according to claim 16, characterized by the fact that the rodent is a mouse.
[18]
18. Method according to claim 17, characterized by the fact that the modification is made in a single ES cell and the single ES cell is introduced into a mouse embryo to produce the mouse.
[19]
19. Method according to any one of claims 1 to 18, characterized by the fact that it comprises: modifying a first non-human animal genome in a first non-human animal to understand the variable humanized TORa non-rearranged gene locus comprising at least one human Va segment and at least one human Ja segment, in which the locus of the variable humanized TCRa gene not rearranged is operationally linked to a constant region of non-human TCRa; modifying a second non-human animal genome into a second non-human animal to understand the non-rearranged humanized TCRB variable gene locus comprising at least one human VB segment, at least one human DB segment, and at least one JB segment human to generate a locus of the humanized TCORB variable gene, wherein the locus of the humanized TCRB variable gene is operably linked to the endogenous non-human TCRB constant region; and crossing the first and second non-human animals to obtain a non-human animal that expresses a T cell receptor comprising a human variable region and a non-human constant region.
[20]
20. Method for producing a human T cell receptor for an antigen of interest, characterized by the fact that it comprises: immunizing a non-human animal produced according to the method, as defined in any of claims 1 to 19,
with the antigen of interest; allow the animal to generate an immune response; isolate a T cell reactive to the antigen of interest from the animal; determining a nucleic acid sequence of a variable region of human TOR expressed by the T cell; cloning the human TOR variable region into a nucleotide construct comprising a nucleic acid sequence from a human TOR constant region, wherein the human TCR variable region is operably linked to the human TCR constant region; and expressing a human T cell receptor in a cell.
[21]
21. Nucleic acid, characterized by the fact that it comprises a sequence that encodes a variable TCRoa gene locus comprising at least one human non-rearranged Va segment and at least one human non-rearranged Ja segment, operably linked to a non-human TCRa constant gene sequence.
[22]
22. Nucleic acid, characterized by the fact that it comprises a sequence that encodes a variable TCRB gene locus comprising at least one human non-rearranged VB segment, at least one human non-rearranged DB segment and at least one non-rearranged JB segment human, operationally linked to a non-human TCRB constant gene sequence
[23]
23. Method for identifying a T cell with specificity to an antigen of interest, characterized by the fact that it comprises the steps of immunizing a non-human animal produced according to the method, as defined in any of claims 1 to 19, as an antigen of interest, allow the animal to generate a response
immune and isolate a T cell with specificity to the antigen of interest from the non-human animal.
[24]
24. Method according to claim 23, characterized by the fact that the antigen of interest is an antigen associated with the tumor, a viral antigen or a bacterial antigen.
[25]
25. Invention, characterized in any form of its embodiments or in any applicable category of claim, for example, product or process or use encompassed by the material initially described, revealed or illustrated in the patent application.

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2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-05-11| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|

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2021-06-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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申请号 | 申请日 | 专利标题

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