Therapeutic Antibodies – Larry Green, Hiroaki Shizuya, Ablexis LLC
Abstract for “Genetic engineering non-human animals to produce chimeric antibodies”
The invention provides nonhuman cells and mammals with a genome that encodes chimeric antibodies, and methods for producing transgenic cells. Some aspects of the invention include humanized antibodies, chimeric antibodies and pharmaceutical compositions. Some aspects of the invention relate to the diagnostic and treatment methods that use the antibodies of this invention.
Background for “Genetic engineering non-human animals to produce chimeric antibodies”
“Technical Field”
“The invention is directed to the general production of chimeric immunoglobulin chain, antibodies, and other non-human animals, cells and cells.
“Description of Related Art”
Monoclonal antibodies (mAbs), which are used in disease therapies, have revolutionized medicine. mAb-based drugs can now be used to treat cancer, autoimmunity and inflammation, macular degeneration, and other conditions. The technologies available for the generation and discovery mAbs to treat diseases and disorders have many limitations. They are inefficient, lack or loss in sufficient potency, loss or absence of specificity, and induction of an immune reaction against the therapeutic mAb. First attempts at using mAbs for therapeutic purposes were hampered by the immunogenicity and composition of the mAbs. The mouse amino acid sequence caused severe allergic reactions in humans and reduced the drug’s pharmacokinetics and potency.
“Chimerized mAbs (cmAbs), which are derived from recombinant DNA technology, combine a mouse-derived variable region with a human constant area. Humanizing mAbs in vitro is another method to generate antibodies. This reduces the amount of amino acids sequence from mice in therapeutic mAbs. Technology for displaying antibodies to create ‘fully-human’? In vitro antibodies have not yet been able to replicate the natural process of antibody maturation that takes place in an in vivo immune reaction (see pg. 1122-23, Lonberg, Nat. Biotech. (2005) 23:1117-1125.) These mAbs can trigger an immune response that could reduce efficacy or be life-threatening. The process is often time-consuming, expensive, and can result in a slow and expensive recovery. These molecular processes can also lead to loss of affinity or epitope shifting, which can reduce potency and cause undesirable changes in specificity.
Transgenic mice can be engineered to produce fully human antibodies. Human antibody transgenes have been introduced to functionally replace Ig loci in activated mouse immunoglobulin. Many of these transgenic mice models lack key components for the antibody development process. These include sufficient diversity in genes from which antibody variable region are generated and the ability to make IgD. Loset et al. Immunol. (2004) 172.2925-2934. What are the important cis regulatory elements necessary for class switch mutation (CSR) or a functional 3? locus control area (LCR) (e.g. U.S. Patent. No. 7,049,426; Pan et al. J. Immunol. (2000) 30:1019-1029). Transgenic mice may contain yeast artificialchromosomes and human miniloci integrated transgenes. Some transchromosomes are more meiotically unstable than others. These transgenic mice have suboptimal function due to decreased activity with trans-acting and endogenous components (Ig) compared to wild-type mice. Ig? Ig?
“Knockin mice” have been genetically engineered so that they can produce chimeric antibodies that contain human V domains added to mouse C domains. These domains remain intact with all the genomic DNA downstream from the J gene cluster. (See U.S. Pat. Nos. 5,770,429 & 6,596,541 respectively and U.S. Patent Application No. 2007/0061900). These mice have human V regions that can be used to add to human constant gene genes using molecular biological techniques. They can also be expressed using recombinant methods to make fully-human antibodies. These antibodies may show a reduction or loss in activity, potency, solubility, etc. When the human V region of an antibody is removed from its context in the mouse C domains where it was developed, and then added to a human region to create a fully human antibody. Due to the unique structure of the mouse immunoglobulin Lambda locus, and the fact that the human endogenous 3 is different from the one in the human locus, and the differing nature of the human immunoglobulin Lambda locus, The described knock-in method could result in a defective enhancer for the mouse lambda lous.
Methods for transgene DNA creation to introduce into eukaryotic species, especially metazoans, have used DNA from genomic libraries that were made from isolated natural DNA. The process of recombination is slow, cumbersome and error-prone. It takes cloned DNA from natural sources to create the desired transgene design. Sometimes it is possible to create a transgene using a specific strain, organism or haplotype of an organism. However, a genomic library may not be available for this species. These obstacles prevent the creation transgenes with complexly reconfigured sequences, and/or transgenes that contain chimeric DNA sequences from different species, strains, or haplotypes. This prevents the engineering of transgenes that are highly tailored for eukaryotes (especially metazoans).
Current methods for developing therapeutic mAbs can alter the functions of the antibody such as solubility and potency. These were not selected during initial stages of development. Current methods can also produce dangerous immune responses upon administration. Current chimeric and human mAb-producing mice lack the appropriate genetic content to function properly. This includes genetic diversity, transact regulatory elements, signaling domains and genetic stability. It would be useful to create methods and compositions that allow for enhanced discovery and enhancement of therapeutic antibodies. These compositions could also retain their potency and specificity throughout the process of antibody generation, discovery and development without eliciting an immuno response. Transgene compositions may contain DNA sequences that have been so complicatedly altered that it is impossible to construct these improvements or derive products from them. Although mice are the most popular species due to their economic value and proven utility, it is possible to find a solution that works across many species. This invention allows for the creation and introduction of transgenes. It also improves the genetic background in which transgenes could function in mice. In particular, it generates improved antibodies in transgenic animals.
“The invention is applicable to non-human animals, cells, transgenes and antibodies, methods, compositions including pharmaceutical compositions, and kits of various embodiments. The present invention is more specifically related to methods, compositions, and kits relating chimeric Ig chains, antibodies, and human antibodies, as well as the human antibodies and their fragments, engineered from variable domains of the chimeric antigens. The invention may also include mammals in certain embodiments.
“One embodiment of this invention is a method for producing a cell that contains a chimeric immuneglobulin-chain genome. This involves the following steps: (1) creating a DNA construct from scratch, which includes one or more nonendogenous V, J, and/or D gene segments, and one or two non-endogenous constant area gene segments. (2) introducing said construct into a cell’s genome. In some embodiments, the non endogenous variable domain can be human. Another embodiment of the chimeric constant area includes a segment of the mouse constant domain gene. One embodiment encodes the chimeric constant area using a non-endogenous sequence of polynucleotides derived from non-endogenous species, haplotypes, and/or alleles. Another embodiment encodes the non-endogenous variable region using a polynucleotide sequence that has been derived from multiple species, alleles, and/or haplotypes. The chimeric immunoglobulin chains are light in certain embodiments.
“In some other embodiments, chimeric immunoglobulin chains are a heavy chain. A related embodiment includes a non-endogenous CH1 region in the chimeric constant area. Another related embodiment of the method includes the steps of creating a second DNA structure in silico. This construct is non-endogenous and contains an immunoglobulin light chains. The second DNA construct is then produced. Finally, the second construct is introduced into the cell’s genome. One embodiment of the non-endogenous light chains includes one or more human V. gene segments. Another embodiment of the non-endogenous light chains includes one or more human J? C? gene segments. Another embodiment of the non-endogenous lighting chain includes 8 or more human V?? gene segments. A similar embodiment also includes 7 or more human J.-C? gene segment pairs.”
“One embodiment refers to a nonhuman cell that has a genome that contains a chimeric immuneglobulin-chain. The genome is composed of a nonendogenous variable domain, and a constant region. This cell is created by a process which includes (1) designing a DNA construct from scratch, and (2) creating the DNA construct and (3) inserting it into the cell’s genome. A non-human animal is another embodiment. A non-human animal can also produce a chimeric immunoglobulin heavier chain. Certain embodiments offer a non-human animal produced chimeric antibody.
“Another embodiment provides a chimeric immunoglobulin heavier chain that includes a nonendogenous variable region and a nonendogenous variable area. The non-endogenous variable region is derived from non-human animals. A related embodiment includes a non-endogenous CH1 region. One embodiment of chimeric immunoglobulin heavy chains includes a nonendogenous variable region and a region containing a chimeric constant. The non-endogenous polynucleotide sequence that is derived from non-endogenous alleles, haplotypes, and/or species encodes the chimeric constant area. A chimeric immunoglobulin chain with a non-endogenous domain and a constant region is another embodiment. The non-endogenous domain is encoded using a polynucleotide sequence that has been derived from at least two species, alleles, and/or haplotypes.
“Another embodiment refers to a polynucleotide that encodes the disclosed chimeric immuneglobulin heavy chains. Particular embodiments of the polynucleotide include both coding and uncoding sequences. The polynucleotide can be synthetic in certain embodiments. One embodiment refers to a construct that includes the polynucleotide, a polynucleotide encoded the disclosed chimeric immuneglobulin heavy chains.”
“Another embodiment provides a synthetic chimeric antibody or antigen-binding fragment of it. It comprises (1) a human immunoglobulin heavy (chimeric) chain. The chimeric light chain is composed of a non endogenous immunoglobulin (non-endogenous immunoglobulin) chain. The chimeric high chain constant area is made from non-endogenous alleles, haplotypes, and/or species. Another embodiment includes a “chimeric antibody” or an antigen binding fragment. It comprises (1) a Chimeric Immunoglobulin Heavy Chain, which has a nonendogenous heavy-chain variable domain and a constant region. (2) A non-endogenous immuneglobulin light Chain, in which said non-endogenous high-chain variable domain is derived either from one or more species, alleles, and/or haplotypes. One embodiment is a chimeric antibody, or an antigen binding fragment thereof. It comprises a chimeric immuneglobulin heavy chains, which has a nonendogenous variable region and a variable domain. The variable domain is derived not from any human animal. A related embodiment of the disclosed chimeric antigen, or antigen binding fragment thereof, includes a non-endogenous lighter chain.
“One embodiment of this invention provides a cell that is non-human and has a genome that contains a chimeric immuneglobulin heavy-chain comprising a variable domain with a non endogenous non-human origin and a constant region. A non-endogenous immuneglobulin light chain is also included in a similar embodiment. Particular embodiments include a non-endogenous Ig. light chain and a non endogenous Ig? light chain. The cell may also contain an inactivated locus of endogenous immunoglobulin. One embodiment produces a chimeric antibody from the cell.
Another embodiment of a nonhuman cell includes a genome with a chimeric immuneglobulin heavy-chain that has a nonendogenous variable domain, a constant region and a chimeric constant area. The constant region can be derived from non-endogenous alleles, species and/or haplotypes. A non-human cell is provided with a genome that contains a chimeric immunoglobulin heavier chain. This includes a nonendogenous variable region and a nonendogenous variable area. The non-endogenous variabledomain is derived from multiple species, alleles, and/or haplotypes. A non-human cell is also provided with a genetic transgene that encodes a synthetic chimeric antigen, or an antigen binding fragment thereof. This chimeric heavy chains (1) contains a non endogenous heavy-chain variable domain, and a constant region of chimeric high-chain. The genome of the disclosed cell also includes a non-endogenous immuneglobulin light chains in certain embodiments. One embodiment of the cell’s genome includes a non-endogenous Ig. light chain and a non endogenous Ig? light chain. Particular embodiments include an inactivated immunoglobulin locus. A different embodiment allows for the production of chimeric antibodies by the cell.
“Another embodiment is a nonhuman animal that contains a genome that includes a chimeric immuneglobulin heavy chains comprising a variable domain with a nonendogenous non-human origin and a constant region. A related embodiment of the invention includes a polynucleotide sequence that encodes a non-endogenous immuneglobulin light chain. In some embodiments, the genome contains a non-endogenous Ig. Light chain and non-endogenous Ig light chain. Another embodiment of the animal includes an inactivated exogenous immunoglobulin locus. The animal may be a mouse in certain embodiments. A different embodiment uses chimeric antibodies that are produced by non-human animals.
Another embodiment of the invention creates a nonhuman animal that has (1) a human-like chimeric immunoglobulin-heavy chain. The chimeric chain is composed of a nonendogenous heavy-chain variable domain and a constant region. (2) A non-endogenous immunoglobulin-light chain. In this case, the constant region of the chimeric high chain is derived from non-endogenous species, haplotypes, and/or alleles. A non-human animal is provided with a genome that includes (1) a Chimeric Immunoglobulin Heavy Chain, where the chimeric chain has a nonendogenous heavy-chain variable domain and a constant region. (2) A non-endogenous immuneglobulin Light Chain, where the non-endogenous high-chain variable domain is derived either from one or more species, alleles, and/or haplotypes. A non-human animal is provided with a genetic transgene that encodes a synthetic chimeric antigen, or an antigen binding fragment thereof. The genome comprises (1) a synthesized transgene that encodes a chimeric immuneglobulin heavy chains, which includes a nonendogenous heavy-chain variable domain and constant region. The genome may also include a non-endogenous immuneglobulin light chains, in particular embodiments. The genome of an animal may also include a non-endogenous Ig Light chain and non-endogenous Ig light chain. Particular embodiments include an inactivated endogenous immuneglobulin locus. A second embodiment uses a chimeric antigen produced by the animal.
“One embodiment of this invention provides a nonhuman animal with an inactivated Ig locus. The endogenous Ig lous is comprised of a deletion that prevents the formation of a functional variable region and a constant area capable of driving primary cell development. In some embodiments, the endogenous immuneglobulin locus can be a heavy-chain locus. Other embodiments of the endogenous immuneglobulin locus are light chain. A third embodiment includes a non-human cell with an inactivated Ig locus. In this case, the deletion prevents the formation of a functional variable region and a constant area that can drive primary B cell development.
“One embodiment presents a DNA construct that includes a first flanking sequence and a transgene. The second flanking sequence is composed of a polynucleotide sequence that can introduce a deletion in an Ig locus. This will prevent the formation of a functional variable region and support primary B cell development. The DNA construct is also provided in a kit. A second embodiment describes a method of inactivating an endogenous immuneglobulin locus. This involves impairing formation of a functional variabledomain and impairing formation of a constant area capable of driving primary cell development.
Another embodiment of the invention describes a method for producing an antibody library. This involves providing a nonhuman animal with a genome that contains a chimeric immuneglobulin-heavy chain. The chimeric heavy chains comprises a nonendogenous heavy-chain variable domain and a constant region. An antibody display library that includes immunoglobulin heavy chains variable regions is provided by one embodiment of the invention. This is a library consisting of non-human animals with a genome that contains a chimeric immunoglobulin chain. The chimeric heavychain comprises a non endogenous heavy-chain variable domain and a constant region. Variable regions are derived using chimeric antibodies.
“BRIEF DESCRIPTION ABOUT THE VIEWS FROM THE DRAWINGS”
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“Overview”
“The present invention comprises chimeric antibodies. Non-human animals can produce chimeric, humanized antibodies. Methods of producing such cells and animals are also included. Also, compositions and kits containing the antibodies are included. Specific embodiments of this invention include mammals as the non-human animal.
“Chimeric antibodies and antigen-binding fragments of them, as described herein, comprise a non-endogenous variable region and a constant region in the chimeric heavy chains. An IgH chain may include one or more of the following: a non-endogenous V and D gene segments, a CH1 domain and endogenous H2 and CH3 domains. An antibody or antigen-binding piece of an IgH chain, which includes the chimeric IgH chains, may also include an IgL chain with an amino acid sequence that is encoded by a non-endogenous sequence. An antibody or antigen-binding piece of an antibody comprising the chimeric IgH chains described herein also includes an IgL chain with an amino acid sequence encoded by both endogenous and not-endogenous nucleotide sequencings.
This prevents any alteration in the V-domain conformation due to the in vitro switch from an initial C region, especially a CH1 region and optionally a portion from a hinge region from one species (e.g. mouse), during the in vivo immune reaction to a second region, particularly the CH1 domain, and optionally a part of the hinge area from another species (e.g. human). The present invention does not result in the loss or reduction in activity or potency of antibodies made from antibodies from other chimeric animal producing animals. This could be due to altered conformation in the VH domain, particularly a CH1 domain, or by changes in antigen binding, possibly caused by a change in the length or flexibility in the upper hinge regions (the sequence of peptides from the end CH1 to the first cysteine in the hinge that forms an intra-heavy chain disulfide link and which is variable in length and composition The following references are found in Immunology (1997) 159.3372-3382. The cysteine residues, which form inter-heavy chains disulfide bonds, bounded the middle hinge region.
“Definitions”
“Before we describe certain embodiments in detail, please understand that the invention is not limited by particular biological systems or compositions. They can vary. The terminology used in this specification is intended only to describe particular illustrative embodiments and is not meant to be restrictive. The terms used herein generally refer to the ordinary meaning of the term in the art within the context and context in which it is used. Some terms are described below and elsewhere in this specification to help the practitioner understand the compositions and methods and how they can be made and used. It is important to understand the context in which a term is used. This will help you determine the meaning and scope of the term. The definitions provided herein are meant to be illustrative and not limitative. The singular forms?a? and?an? are used in the claims and disclosure. ?an,? ?an,? If the context requires otherwise, include plural forms.
“As used herein, ?antibody? “Antibody” and “immunoglobulin?” Ig is an acronym for immunoglobulin. It refers to proteins molecules that are produced by B cells and recognize and bind specific antigens. They can be membrane bound or secreted. Monoclonal antibodies are those that are produced by one clone (or more) of B cells. They recognize the same epitope, have the same amino acid sequence and nucleic acids, and polyclonal antibodies are those that are produced from multiple clones (or more). Polyclonal antibodies are those that are produced by multiple B cell clones and recognize different epitopes.
“Antibody molecules, also known as Ig molecules, are usually composed of two identical heavy chain and two identical light chains, linked by disulfide bond. IgL and Ig? There are two types of IgL, Ig? Both IgH and IgL contain a variable region (V) or domain, and a constant region (C) or domain. Multiple copies of variable (V), diversity(D), and joining (J] gene segments are contained in the IgH region that encodes the V region. Ig? Ig? and Ig? encode the V region. They contain multiple copies of J and V gene segments. To develop antigen specificity, the V region that encodes IgH or IgL loci is subject to gene rearrangement. The secreted IgH C region consists of three C domains: CH1, CH2, and CH3, with optionally CH4 (C). A hinge region and three C domains. The membrane-bound IgH C region has intra- and membrane domains. The antibody’s isotype is determined by the IgH constant area. IgM, IgD and IgG1 are the IgG2 IgG3, IgG3, IgG4, IgA, IgE in humans. It will be appreciated by non-human mammals that encode multiple Ig isotypes to be able switch isotype classes.
“A ?Fab? “A?Fab?” domain or fragment is the N-terminal IgH portion, which includes V region and CH1 domains of IgH and the entire IgL. A?F?2? Domain includes the Fab domain, a portion of hinge region, and the 2 IgH are linked via disulfide linking in the middle hinge area. F(ab)2 and Fab are both?antigen-binding fragments. ?antigen binding fragments? The?Fc is the C-terminal portion (CH2 and CH3 domains) of the IgH. domain. The Fc domain is the portion of the Ig recognized by cell receptors, such as the FcR, and to which the complement-activating protein, C1q, binds. The 5 encodes the lower hinge region. The CH2 exon’s lower hinge region is responsible for flexibility in the antibody’s ability to bind to FcR receptors.
“As used herein, chimeric antibody?” Refers to an antibody encoded using a polynucleotide sequencing that contains polynucleotide combinations derived from multiple species.
“A ?humanized? An antibody is a chimeric antigen that contains more human sequences than its parent molecule. When administered to humans, humanized antibodies are less immunogenic than non-humanized antibodies from other species. A humanized antibody could include the variable region of an appended chimeric antibody to a human constant area. The chimeric antibodies described in this article can be used to create a fully human antibody.
“As used herein ?chimeric Ig chain? An Ig heavy or light chain encoded with a polynucleotide sequencing containing sequences derived of two or more species. A chimeric Ig heavy chains may include human VH and DH gene segments, JH gene segments, and mouse CH2 or CH3 gene segments.
“?Polypeptide,? ?peptide? or ?protein? These terms are interchangeable to refer to a chain of amino acid that is linked together by chemical bond. An IgH, IgL or V domain protein can be a polypeptide.
“?Polynucleotide? A chain of nucleic acid that is linked by chemical bonds. The polynucleotides can include DNA, cDNA and RNA as well as gene sequences or segments.
“Polynucleotides can be extracted from living sources such as prokaryotic cells, eukaryotic cells, viruses, or in vitro manipulative techniques using standard molecular biology techniques or DNA synthesis or a combination of several techniques.
“?Locus? “?Locus?” refers to a location that contains one or more genes, exons or genes. locus, the cis regulatory element and the binding areas to which trans-acting factor bind. As used herein, ?gene? or ?gene segment? Refers to the sequence of polynucleotide polynucleotides that encodes a specific polypeptide, or a portion thereof. The term?gene segment is used herein. ?gene segment? and?exon? are interchangeable terms. These terms can be interchanged and may refer to a specific polynucleotide that encodes a peptide or a portion thereof. A gene or segment of a gene may also contain one or more transcriptional control elements (e.g. promoters, enhancers or other non-coding areas (e.g. cis regulatory element, e.g. 5). and/or 3? ”
“Inactivated Ig locus” is the term used herein. An Ig locus that doesn’t encode a functional Ig Chain. Functional variable region? Produce from an Ig locus is a polynucleotide sequence that can undergo V-(D-J) recombination. It is transcribed into a variable-region polypeptide and then translated into a protein that can be expressed on a cell’s surface. What is a?functional heavy-chain constant region? A constant region that can be operationally connected to a variable area and drives primary B cell development. Primary B cell development is the process of developing B cells in primary lymphoid organs (e.g. bone marrow). It includes the stages of pro-B and late pro-B cells (i.e. IgH DJ rearranging), pro-B and large pre-B cells (i.e. IgL VJ rearranging), as well as the development of small pre-B cells (i.e. IgL VJ rearranging). What is meant by “driving?” By?driving?, it means that the functional heavy-chain constant region can be, e.g. anchoring to cell membrane, signal transduction and/or binding to an Fc receptor. What is a?functional lightchain constant region? A constant region that can be operationally connected to a variable area and bound to heavy chain to accelerate B cell development beyond the pre-B cell stage.
“?Impair? “?Impairment” refers to the introduction or modification of a gene that causes a mutation or deletion that results in, for example, a variable or constant region that is not functional. Homozygous deletions of C could be an example. An IgH that is responsible for primary B cell development in certain mammals and strains of mice is affected.”
“?Mutation? A mutation is a modification in the sequence of a polynucleotide (or polypeptide) that naturally occurs. Mutations can cause functional changes. Mutations can include the addition or deletion of nucleotides. ?Deletion? ?Deletion? refers to the deletion of one or more nucleotides in the naturally occurring endogenous sequence polynucleotide. Frameshift mutations can be introduced by additions and deletions. Deletions can also result in the deletion of entire genes, gene segments, or modules. Sometimes, the deletion of a part of an naturally occurring endogenous sequence can occur in conjunction with the addition or removal of a non-endogenous one. A homologous recombination may result in the deletion of a part of an endogenous polynucleotide sequencing sequence. Another aspect is that an endogenous sequence of polynucleotides may be deleted after two non-endogenous recognition sequences for a site specific recombinase are introduced, e.g. a loxP site. Then, exposure to the CRE recombinase.
“Endogenous” is a term that refers to a polynucleotide sequence that is naturally occurring in a cell or animal. A polynucleotide sequence that occurs naturally in a cell or animal. ?Orthologous? Refers to a sequence of polynucleotides that encodes the same polypeptide in another species. The term “syngeneic” is used. The term?syngeneic? refers to a sequence of polynucleotides that can be introduced into an animal from the same species as it is. Introduced into a mouse gene segment. Note that polynucleotide sequences from individuals of different species may contain regions of significant variation.
“Homologous” as used herein means: or a?homologous sequence. A polynucleotide sequence with a high percentage of identity or a highly similar sequence (e.g. A polynucleotide sequence that is highly similar or high in identity (e.g. 30%, 40%. 50%. 60%. 70%. 80%. 90%) to another polynucleotide sequencing or segment thereof. A DNA construct may include a sequence homologous with a part of an endogenous sequence, for example, to facilitate recombination at a specific location. In both prokaryotic or eukaryotic cells, homologous recombination can occur.
“As used herein, ?flanking sequence? Or?flanking sequence? A DNA sequence that is adjacent to a non endogenous sequence in a DNA construction that is homologous with an endogenous sequence or a previously combined non-endogenous sequence, either a whole or part thereof. The invention may include one or more flanking sequences. For example, the flanking sequence on the 3? 5, and 6? End of the non-endogenous sequencing or a flanking sequence on 3? End of the non-endogenous sequence or a flanking sequence on the 3? oder 5? End of the non-endogenous sequencing. The flanking sequence could be homologous with an endogenous, within-a-endogenous sequence, or it may be homologous with an endogenous, adjacent, (or outside) of, an endogenous, sequence.
“The phrase ?homologous recombination-competent cell? A cell capable of homologously recombining DNA fragments with regions of overlapping homology. Examples of homologous recombination-competent cells include, but are not limited to, induced pluripotent stem cells, hematopoietic stem cells, bacteria, yeast, various cell lines and embryonic stem (ES) cells.”
“A ?non-human animal? Any animal that is not a human, such as reptiles, birds, and mammals, is considered a?non-human animal? ?Non-human mammal? A non-human mammal is an animal that is not related to humans and falls under the Mammalia class. Non-human mammals can include rodents and non-human primates like camelids, rodents and bovines as well as ovines and equines. The preferred non-human mammals are those that rely on somatic hypermutation or gene conversion to generate antibodies diversity. Mice are a favorite non-human mammal.
“Transgenic” is a term that refers to a cell or animal that has a non-endogenous polynucleotide sequence. “Transgenic” refers to an animal or cell that contains a non-endogenous sequence of polynucleotides, such as a transgene from another species. A transgenic mouse is one that has a human VH segment embedded in its genome, but not the endogenous IgH locus. Conversely, a transgenic mice is one that has a human VH segment embedded directly into its genome, replacing the endogenous VH locus. mouse. Transgenic cells and nonhuman mammals may express the non-endogenous mononucleotide sequence with or without the endogenous genes.
“As used herein, ?replace? It can be used to refer to either direct or functional replacement. What is meant by ‘direct replacement? It refers to the replacement of an endogenous DNA sequence with an engineered sequence that contains a non-endogenous gene sequence, such as homologous recombination. Homologous recombination is used to remove the endogenous DNA sequence. Or, it can be deleted if the endogenous sequence remains between two non-endogenous sequences. What is meant by “functional replacement?” It refers to the fact that an endogenous sequence of DNA does not perform the function, such as the polypeptide made from engineered DNA sequences. A transgene, which encodes a chimeric IgH-chain and is insert into the genome without the IgH locus, can replace an endogenous IgH gene.
“A ?humanized? “A?humanized?” animal is a nonhuman animal (e.g., a mouse) that has a genetic structure that includes gene sequences from the non-human animals, and one or more gene segments or regulatory sequences that have been replaced by analogous human sequences.
“Vector” is the term used herein. A nucleic acids molecule into which another fragment of nucleic acids can be integrated without affecting the vector’s ability for replication. A virus, plasmid, or a cell from a higher organism can all be vectors. Vectors can be used to introduce recombinant or foreign DNA into host cells, where it is then replicated.
A vector can contain a polynucleotide agent, which allows for manipulation, such as the introduction of the polynucleotide to a target cell. A vector can either be a cloning or expression vector. This allows for the manipulation of the polynucleotide. An expression vector may contain the necessary expression elements to enable sustained transcription of the polynucleotide encoding. The regulatory elements can also be operatively linked with the polynucleotide before it is cloned into a vector.
An expression vector (or polynucleotide), generally contains or encodes an expression sequence. This sequence can provide constitutive, tissue-specific, or developmental stage-specific expression of the encoding protein. It can also include a polynucleotide recognition sequence and a ribosome recognition or internal ribosome entrance site. Other regulatory elements, such as enhancers, can also be included. The vector can also contain elements that are required to replicate in either a prokaryotic and eukaryotic host, or both, depending on the situation. These vectors include plasmid, retroviruses, lentiviruses, bacteriophage and adenovirus vectors. They can also be used to replicate in a prokaryotic or eukaryotic host system. Or can be made by someone skilled in the art (see Meth. Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb 25:37-42, 1993; Kirshenbaum et al., J. Clin. Invest 92, 381-387, 1993 (each of which is incorporated by reference).
A DNA vector used in the invention may contain both positive and negative selection marks. Negative and positive markers are genes that confer drug resistance on cells expressing them. E.coli has several suitable selection markers. These include Km (Kanamycin-resistant gene), TetA (tetracycline-resistant gene) and beta lactamase (ampicillin-resistant gene). Mammalian cells can be cultured with suitable selection markers, including hyg (hygromycin resistant gene), puro [puromycin resistance genes] and G418 (?neomycin resistant gene). You can also use selection markers to identify metabolic genes that convert a substance into toxic substances. The gene thymidinekinase, when expressed, converts drug gancyclovir to a toxic substance. Treatment of cells with gancylcovir may result in a negative selection for genes that don’t express thymidine kinase.
“In a similar aspect, the selection marker can be?screenable? markers. Such as yellow fluorescent protein, red fluorescent protein and red fluorescent protein (RFP), GFP like proteins, and Luciferase.
There are many types of vectors that are available in the art. These include viral, bacterial and yeast vectors. Any suitable DNA vector may include a cosmid or bacteriophage as well as a plasmid and cosmid. The DNA vector may be a BAC in certain cases. Each DNA vector is chosen according to the amount of DNA that will be inserted into the construct. One embodiment of the DNA constructs is bacterial artificial chromosomes (or fragments thereof).
“The term “bacterial artificial chromosome” “BAC” or?bacterial artificial chromosome? The term BAC, as it is used herein, refers to a bacterial genome vector. BACs such as those derived form E. coli may be used to insert, delete, or replace DNA sequences in non-human mammalian cell or animal cells via homologous recombination. Complex genomic DNA can be maintained by E.coli in the form BACs. (See Shizuya, Kouros-Mehr and Keio J Med. 2001, 50(1).26-30. BACs have a greater DNA stability than cosmids and yeast artificial chromosomes. BAC libraries of human genomic DNA DNA are more accurate than libraries in cosmids and yeast artificial chromosomes. U.S. Application Ser. Nos. Nos.
Before humanization of the locus, DNA fragments comprising an Ig gene locus or a portion thereof are taken from the same non-human mammal species. Multiple BACs that contain overlapping fragments can be humanized. The overlapping fragments are recombined to create a continuous IgH/IgL locus. The resulting chimeric Ig locus contains the human gene segments that are operably linked with the non-human mammal Ig genes segments. This allows for the creation of a functional Ig locus. It is capable of gene rearrangement, thereby generating a diverse repertoire of chimeric antibody.
“These are processes for combining BACs or of engineering a fragment of a Chimeric Ig Locus or BAC. A bacterial cell such as E.coli must be transformed with a host Ig locus-containing BAC or a portion thereof. The BAC containing Bacillus is transformed with a vector that contains the desired human Ig genes segment and a flanking homology sequence. The homology of the shared sequence mediates homologous cross-over between human Ig genes on the vector and non-human mammal Ig genes on the BAC. Selectable and/or screenable markers may be used to detect homologously recombined BABs. Humanized BACs can easily be isolated from bacteria and used to produce knock-in human cells. There are methods of combining BACs, engineering insertions or deletions within DNA on BACs, and methods for creating genetically modified mice from them. See, e.g., U.S. Pat. No. 5,770,429; Fishwild, D. et al. (1996) Nat. Biotechnol. 14:845-851; Valenzuela et al. Nature Biotech. (2003) 21:652-659; Testa et al. Nature Biotech. (2003) 21.443-447. Nature Biotech. (2003) 21:447-451.”
The first step of recombination may be performed in an E.coli strain that is lacking in sbcB or sbcC, recB activity, recC, recC, or recD activity, and that has a temperature sensitive mutation, recA. The recombination step results in the isolation of a recombined DNA structure, which can have the different sequences and orientations described.
The length of the regions that are used for BAC recombineering must be long enough to allow for homologous recombination. The flanking regions can be anywhere from 0.1 to 19 km, with a typical length of about 1 kb – 15 kb or 2 kb – 10 kb.
To recombinate BACs into larger or more tailored BACs that contain portions of the Igloci, it is necessary for E.coli to be transformed with a BAB carrying either a portion of the Ig locus or another target sequence. The BAC containing E. coli can then be transformed with a recombination virus (e.g. plasmid, BAC) that contains the desired Ig gene segment. This could be one or more of the human VH, DH, or JH gene segments, which will be joined to a portion of the mouse IgH locus. Both vectors share a region of sequence similarity. The presence of functional recA within the E. coli creates a shared identity region that mediates cross-over between Ig gene segments on the recombination and non-human mammal Ig genes on the BAC. Selectable and/or screenable markers may be used to select homologously recombined BABs. Humanized and chimeric BACs are easily purified from E. coli. They can then be used to produce transgenic and knockin non-human cells or animals.
“Alternatively, DNA fragments containing the Ig locus that can be incorporated into non-human animals are obtained from DNA synthesized intracellularly. Many organisms’ genomes have been fully sequenced (e.g. human, chimpanzees, rhesus monkeys, mouse, rat and dog), and are freely available with annotation. Information on the sequences and transcriptomes of many other organisms is also publicly available. The location and activity of non-coding regulatory elements and gene segments has been mapped and studied at the mouse and human immunoglobulin loci.
“In silico” is a term that refers to the use of a computer or algorithm to model a naturally occurring or in vitro process. “In silico” is a term that refers to the use or algorithm of a computer to model an in vitro or naturally occurring process. It also includes the creation of a nucleotide/polypeptide sequence or the production of such sequences using a cell-free system (e.g. using automated chemical synthesizing). You can manipulate the sequences of Ig loci and then recombine them in silico with commonly used software for nucleic acids sequence analysis. Recombination can be performed in silico within the same locus, between loci of the same species or between loci of different species. In silico recombination can be used to create either a functional or non-functional, activated sequence. Precise nucleotide-by-nucleotide engineering allows for precise manipulation of sequence composition that can be applied to precisely engineer the function of the transgene and after transcription and translation, result in precisely engineered composition and function of the polypeptide product of the locus.”
“Sequences from an Ig locus can also be recombined with sequences from a non immuneglobulin locus in silico, from the same species or from another. These sequences can include genes for positive or negative drug selection markers like G418, puro, hyg and tk, site specific recombinase sequences such as lox P sites and their variants, and frt sites. They also contain precisely delineated sequences that drive homologous replication. Once the desired sequence has been assembled in silico it can be synthesized and assembled with no errors (Kodumal, Proc. Natl. Acad. Sci. (2004) 101:15573-15578). Contractual services are available for the synthesis, assembly, and sequencing large DNAs (e.g. DNA 2.0, Menlo Park; Blue Heron Biotechnology Bothell, Wash.; or Eurogentec San Diego, Calif.). These synthetic DNA sequences can be carried in vectors like plasmids or BACs, and can be transferred to other vectors like YACs.
“Construct” is a term that refers to a sequence of DNA artificially constructed by genetic engineering. “Construct” is a sequence or combination of DNA that has been artificially created by genetic engineering, recombineering, or synthesis. Examples of constructs are, for instance, transgenes or vectors (e.g. BACs. P1s. Lambda bacteriophage. Cosmids. plasmids. YACs. MACs). One embodiment of the DNA constructs is linearized before being introduced into a cell. Another embodiment does not allow for the linearization of DNA constructs prior to their introduction into a cell.
“As used herein, ?loxP? “As used herein,?loxP? and?CRE?” The site-specific recombination systems derived from P1 bacteriaiophage are called?CRE? IoxP sites have 34 nucleotides. If DNA is flanked by a loxP-site and exposed to CRE-mediated recombination (CRE-mediated recombination), the DNA on the other side is removed and the two loxP websites become one. It is well-documented that the CRE/lox system (including variant-sequence CRE sites and variants CRE) can be used for genetic engineering in many species including mice.
“A similar system can be used to achieve similar results, using frt sites and the flp-recombinase of S. cerevisiae. The flp/frt can be used to facilitate any CRE/loxP-mediated deletional events in mammalian cells cultured.
“As used herein, the terms ‘immunize? ?immunization,? ?Immunization? and?immunizing? The act of exposing an animal’s adaptive immune system to an antigen is called?immunizing. You can inject, inhale, or ingestion the antigen. The adaptive immune response (i.e.,.) to the antigen is increased upon second exposure. The T and B cell responses are enhanced upon second exposure to the same antigen.
“?Antigen? “Antigen” refers to any peptide, lipid or nucleic acid, as well as any hapten, that are recognized by the adaptive immuno system. Antigens can include pollen, bacterial cell walls components, and the rh factor. ?Target antigen? A target antigen is an antigen, protein, lipid, saccharide or amino acid that is recognized by adaptive immune system. It is a substance, peptide or lipid that is used to generate an immune response against an infectious agent, endogenous or exogenous cells, or products thereof. Target antigens can include, but not be limited to, bacterial or viral components, tumor-specific antgens, cell surface molecules, and any antigens against whom antibodies or other binding protein have been produced by in vivo and in vitro methods.
“The term “pharmaceutical” is used interchangeably with the word “pharmaceutical.” “The term?pharmaceutical? or?pharmaceutical drugs? Any pharmacological, therapeutic, or active biological agent that can be administered to a patient or subject is referred to herein. In some embodiments, the subject is an animal. Preferably, a mammal. Most preferably, a human.
“Pharmaceutically acceptable carrier” is a general term that refers to any material that may be used in conjunction with the pharmaceutical drug. It refers to all material that can be used in conjunction with the drug, and which is not likely to cause adverse reactions.
“Administering” is a term that refers to administering a drug or other agent. “Administering” as it is used herein refers to any method of delivering, introducing, transporting, or transferring a pharmaceutical drug or another agent to a subject. These modes include oral, topical, intraperitoneal and intramuscular administrations, as well as subcutaneous administrations.
“Non-Human Mammals, Cells Encoding Chimeric Ig heavy Chains”
“Non-human animals or cells are comprised of one or more altered Ig loci, e.g. IgH, Ig?? and/or Ig?” It may include non-endogenous Ig genes segments that can replace endogenous segments. The altered loci can be used to replace endogenous gene segments in certain embodiments. Other embodiments replace the endogenous genes segments with the altered loci.
The non-endogenous gene segments can be derived from any species and may also include syngeneic genes. Non-endogenous sequences can be derived from humans, mice, nonhuman primates, camelids and rodents. The non-human cell/animal may, as mentioned above, be any nonhuman animal. The transgenic cells and animals described in this article may contain DNA sequences from any combination of species. Chimeric mice cells, as well as mice containing human and camelid Ig polynucleotide sequencings, are possible. The transgenic animal or cell may also contain non-endogenous DNA of more than one species. A transgenic mouse genome could include both human and camelid DNA sequences.
“The transgenic animals and cells described herein contain one or more non-endogenous segments of the V gene. Specific embodiments use mammal as the preferred non-human animal. In some embodiments, the animal or cell may also include one or more non-endogenous segments of J gene. A cell or animal that has a chimeric IgH-chain optionally includes one or more non endogenous D gene segments.
“In one embodiment, the cell/animal comprises a genome that encodes a chimeric IgH and transgenic light chains. A transgenic light chain could be an IgG? The transgenic light chain may be an Ig? light chain. The transgenic light chains may also be chimeric or contain only non-endogenous sequences of amino acids. Particular embodiments include a cell or animal with a genome that encodes non-endogenous IgH and Ig? In particular embodiments, the cell or animal contains a genome encoding non-endogenous IgH, Ig? gene segments. A non-endogenous CH1domain is used to replace a CH1domain in an endogenous CH gene. In some embodiments, the non endogenous CH1 domain can be orthologous with the endogenous CH1 region. In some other embodiments, non-endogenous CH1 domain is not orthologous with the endogenous CH1 region. Another embodiment replaces more than one CH1 domain with a non-endogenous domain. A related embodiment replaces all endogenous CH1domains with a non-endogenous one. An orthologous human CH1 could replace each endogenous C. gene (e.g., human C1 CH1 replaces mice C?1CH1 and human C2 CH1 replaces mice C?2CH1 etc.). Another embodiment replaces each endogenous C1 domain with a CH1 domain. A single human IgG type is one that is more commonly used in therapeutic mAbs. It is typically C?1, C??2 or C?4. This is to facilitate in vivo maturation and clinical relevance of a human V domain within a CH1 domain.
“Optionally the upper hinge sequences endogenous C genes can be replaced by orthologous non-endogenous Chine sequences. Alternately, the middle and upper hinge sequences of endogenous C gene genes can be replaced by the orthologous non?endogenous C hinges. In order to drive inter-heavy chains dimerization via disulfide bonding, the human C4 middle hinge sequence could be modified to include a proline at position 229 instead of a serine. The endogenous C?2 CH2 domain includes the lower hinge region. To ensure optimal binding to the endogenous Fc.R, this gene cannot be replaced. These options include Fab domain plus higher hinge or F(ab?) which provide non-endogenous heavy chains Fab domains, Fab domain plus lower hinge, and Fc?R. )2, respectively. The human upper hinge regions can be replaced by the upper. This will increase the likelihood that the variable region of the resulting antibody will retain its optimal characteristics when converted to human IgG.
“Another embodiment incorporates fully non-endogenous, e.g., human, Ig including the C regions comprising CH1-hinge-CH2-CH3(?CH4) and the cognate syngeneic, e.g., mouse, membrane and intracellular domains so as to provide native intracellular signal transduction and to enable association of the IgH in the B-cell receptor with Ig? Ig? IgG containing B-cell receptor and allow endogenous signaling from IgG, IgG? IgG containing B cell receptor. Another embodiment of the invention is that the membrane and intracellular domains of the constant heavy chain heavy chain are identical or non-cognate heavy chain isotypes. This can be done easily using the methods described below.
“In another embodiment, transgenic animals and cells comprising a Chimeric IgH Chain described herein include a constant region encoded using a non-endogenous sequence of polynucleotide derived from more than one species. A transgenic mouse with a genome that encodes a chimeric IgH chains constant region is one example. It would include a human CH1 and upper hinge regions as well as rat CH2 or CH3 domains. It is preferable that animals with a xenogeneic consistent region can interact (e.g. binding) an endogenous FCR.
“In another embodiment, transgenic animals and cells comprising a Chimeric IgH Chain described herein include a constant region encoded with a non-endogenous and an endogenous sequence of polynucleotide. A transgenic mouse with a genome that encodes a chimeric IgH chains constant region includes a human CH1 domain and human upper hinge regions. The Balb/c mouse CH2 coding sequences are embedded in C57BL/6 (??B6?). It is possible to create genomic DNA that contains all the genetic information of B6 except for the Balb/c sequence exons for CH2 or CH3.
“In one embodiment, the composite IgH sequencing comprises at least 3kb upstream from the VH6 promoter through D gene cluster through 3. JH6 and is in germline configuration. Another embodiment of the composite IgH sequence includes at least 3 kb downstream from the VH6 promoter via the D gene cluster through 3. The JH6 is human-specific and germline, with the exception that the D gene cluster may be replaced by a part or all of a xenogeneic specie. Another aspect of the invention is that additional human VH genes are upstream from human VH6. Another aspect of the invention is that the additional VH genes exist in germline form. Alternatively, additional VH genes have smaller sizes than those in the human genome. These units include upstream regulatory elements like cis-regulatory element binding sites and binding sites to trans-acting factor binding sites and introns. One aspect states that the unit size must be less than 10 kb. The unit size in another aspect is 5 kb. Another aspect is that the VH genes are chosen from the subset common VH genes within the human haplotypes. Another aspect is that VH genes, DH gene and JH genes are selected to represent a particular allele, such as the most common in human populations. Another aspect is that the VH gene’s individual codons are codon-optimized to allow efficient expression in non-human mammals. Another aspect is that the individual codons can be used as a template to facilitate somatic hypermutation.
“In an alternative embodiment, the composite IgH sequencing comprises a sequence of mouse DNA beginning at least 3 kb downstream from the promoter for VH genes in the closest cluster D, e.g. VH5-2 through 3? JH4 in germline form and into which the coding sequencings have been replaced all or part by human coding. E.g., coding for mouse VH5-2 is replaced with coding for human VH6-1. The mouse DH coding series has been replaced by human DH coding and the mouse JH coding sections have been replaced by human JH coding. The additional JH genes can be added to the genome by various methods, such as inserting human JH sequences with their cis regulatory element, such recombination signals sequences downstream from the JH4, or completely omitting them.
“In some other embodiments, the entire mouse VH coding sequences can be replaced by VL coding sequences. In some embodiments, the whole DH gene cluster can be of mouse sequence. In other embodiments, the whole DH gene cluster can be of xenogeneic species. Another aspect of the invention is that there are additional VH gene sequences upstream from VH6 coding sequences. This means that all sequences are mouse-specific, except for the coding sequences functional VH gene sequences.
“Another aspect is that the additional VH genes exist in germline configuration. Alternative aspects include additional VH genes with smaller sizes than those in the mouse genome. These units contain upstream regulatory elements, such as binding sites for trans-acting factor binding sites and cis-regulatory element bindings. One aspect states that the unit size is less than 10 kb. Another aspect states that the unit size should be 5 kb or smaller.
“In another aspect, VH genes are chosen from a subset that is functional. The replacing human VH coding sequence is from a functional human VH genetic sequence and the replacement mouse VH coding sequence from a functional mouse VH genetic sequence. Another aspect is that the VH coding sequences for human are chosen from the subset common VH genes within the human haplotypes. Another aspect is that the VH coding sequences for replacing VH coding, DH coding and JH coding sequences reflect a particular allele, such as the most common in human populations. Another aspect is that some or all the VH coding, DH coding, and JH coding sequences being replaced are not from humans. Another aspect is that the individual codons in the VH gene have been codon-optimized to allow efficient expression in non-human mammals. Another aspect is that the individual codons can be used as a template to facilitate somatic hypermutation.”
“In another embodiment, the composite IgH series further comprises 3? The most 3? JH The mouse sequence immediately below mouse JH4 through mouse E? via C? Through C? via C? All the C?3 promoters for mice in germline configuration, with the exception of replacing the CH1 domains for mouse C? C? C? by their human counterparts. Sometimes, the upper mouse hinge regions can be replaced with their human counterparts. The mouse C? The genes are set up in germline configuration, with the exception that their CH1 domains are replaced by human CH1 domains.”
“In certain cases, the mouse’s upper hinge regions can be replaced by human ones. In certain embodiments, the mouse C.3 coding sequences can be replaced by human and mouse CH1 and CH2, respectively. Another embodiment uses the entire germline-configured mouse C3 sequence starting at the promoter and continuing through the intracellular domains to the 3? The untranslated sequence, poly(A), and the promoter upstream of the switch region are replaced with the complete sequences from C1 in germline configuration. Human CH1 replaces mouse CH1 from C1 to effectively replace C?3 by chimeric C1 Some embodiments replace a mouse constant coding sequence with human CH1 or mouse CH2, and intercellular and membrane domains of different mouse constant region isotypes. Other embodiments further modify the sequence of CH2 and CH3 domains to increase binding to Fc receptors.
“In another embodiment, the cell/non-human animal includes a locus encoded with a human Ig lightchain comprising a human Ig?” Variable region. A related embodiment of the Ig light chain locus also includes a human Ig. constant region. The composite Ig? is one embodiment. The sequence includes mouse DNA sequence starting at least 3 kb downstream of the V? promoter. V?3-1 through 3? The mouse J?5 gene is located most proximal to mouse J?1 (V?3-1) through 3? Replaced by human J? Code sequences. Another embodiment uses the sequence J?5 through C? The sequence from J?5 through C? is in mouse and germline configuration. “The coding sequences have been completely or partially replaced by human coding.”
“In another aspect, there is a 3??LCR region downstream of the C? gene. The 3? LCR and RS elements can be used in mouse or germline configurations. Another aspect of the invention is that there are additional V? There are additional V? genes upstream from the coding sequences to human V?4-1. This means that the entire sequence is mouse, except for the coding sequences of functional V?? Replaced with the human V? genes.”
“In another aspect, the additional V?” Genes are in germline configuration. The additional V is another alternative. The genes have a smaller size than the one in the mouse genome. They contain upstream regulatory elements like cis-regulatory element and binding sites for transacting factors. One aspect of the unit size is less than 10 kb. The unit size in another aspect is 5 kb. Another aspect is the V? The V???? genes are chosen from the subset that is most commonly shared among human haplotypes. genes that are common among human haplotypes. V? V? genes and J? Genes are selected to reflect a particular allele, such as the dominant one in human populations. The individual codons for the V? are another aspect. The individual codons of the V? gene have been codon-optimized to allow efficient expression in a non-human mammal. Another aspect is that the individual codons can be used as a template to facilitate somatic hypermutation.”
“In another embodiment, the human Ig Light Chain locus includes all or part of a human Ig?” light chain locus, and an Ig? 3?LCR or a functional fragment thereof. The human Ig? is one embodiment. The entire human Ig’s light chain locus is included in one embodiment. locus. Another embodiment of the human Ig is light chain locus comprises human V? coding sequences and 1-7 J?-C? coding sequence pair, in which the human C? is replaced by syngeneic? C? The human Ig? is another example. light chain locus comprises human V? coding sequences, 1-7 human J? Code sequences and one human C? coding sequence in which the human coding sequences look like a human Ig? locus configuration.”
“In particular embodiments the Ig? 3? LCR or a functional fraction thereof is from a mammal chosen from the group consisting human, non-human primate and rat. One embodiment of the Ig? 3? LCR or a functional segment thereof is human. Ig? is a particular embodiment. 3? LCR or a functional fragment thereof binds NF??b. The Ig? 3? LCR or a functional portion thereof is from mice and has been mutagenized to restore binding of the NF?b. The 3? is used in other embodiments. LCR or a functional segment thereof in the human Ig. locus is an IgG? 3? LCR or functional fragment thereof.”
Summary for “Genetic engineering non-human animals to produce chimeric antibodies”
“Technical Field”
“The invention is directed to the general production of chimeric immunoglobulin chain, antibodies, and other non-human animals, cells and cells.
“Description of Related Art”
Monoclonal antibodies (mAbs), which are used in disease therapies, have revolutionized medicine. mAb-based drugs can now be used to treat cancer, autoimmunity and inflammation, macular degeneration, and other conditions. The technologies available for the generation and discovery mAbs to treat diseases and disorders have many limitations. They are inefficient, lack or loss in sufficient potency, loss or absence of specificity, and induction of an immune reaction against the therapeutic mAb. First attempts at using mAbs for therapeutic purposes were hampered by the immunogenicity and composition of the mAbs. The mouse amino acid sequence caused severe allergic reactions in humans and reduced the drug’s pharmacokinetics and potency.
“Chimerized mAbs (cmAbs), which are derived from recombinant DNA technology, combine a mouse-derived variable region with a human constant area. Humanizing mAbs in vitro is another method to generate antibodies. This reduces the amount of amino acids sequence from mice in therapeutic mAbs. Technology for displaying antibodies to create ‘fully-human’? In vitro antibodies have not yet been able to replicate the natural process of antibody maturation that takes place in an in vivo immune reaction (see pg. 1122-23, Lonberg, Nat. Biotech. (2005) 23:1117-1125.) These mAbs can trigger an immune response that could reduce efficacy or be life-threatening. The process is often time-consuming, expensive, and can result in a slow and expensive recovery. These molecular processes can also lead to loss of affinity or epitope shifting, which can reduce potency and cause undesirable changes in specificity.
Transgenic mice can be engineered to produce fully human antibodies. Human antibody transgenes have been introduced to functionally replace Ig loci in activated mouse immunoglobulin. Many of these transgenic mice models lack key components for the antibody development process. These include sufficient diversity in genes from which antibody variable region are generated and the ability to make IgD. Loset et al. Immunol. (2004) 172.2925-2934. What are the important cis regulatory elements necessary for class switch mutation (CSR) or a functional 3? locus control area (LCR) (e.g. U.S. Patent. No. 7,049,426; Pan et al. J. Immunol. (2000) 30:1019-1029). Transgenic mice may contain yeast artificialchromosomes and human miniloci integrated transgenes. Some transchromosomes are more meiotically unstable than others. These transgenic mice have suboptimal function due to decreased activity with trans-acting and endogenous components (Ig) compared to wild-type mice. Ig? Ig?
“Knockin mice” have been genetically engineered so that they can produce chimeric antibodies that contain human V domains added to mouse C domains. These domains remain intact with all the genomic DNA downstream from the J gene cluster. (See U.S. Pat. Nos. 5,770,429 & 6,596,541 respectively and U.S. Patent Application No. 2007/0061900). These mice have human V regions that can be used to add to human constant gene genes using molecular biological techniques. They can also be expressed using recombinant methods to make fully-human antibodies. These antibodies may show a reduction or loss in activity, potency, solubility, etc. When the human V region of an antibody is removed from its context in the mouse C domains where it was developed, and then added to a human region to create a fully human antibody. Due to the unique structure of the mouse immunoglobulin Lambda locus, and the fact that the human endogenous 3 is different from the one in the human locus, and the differing nature of the human immunoglobulin Lambda locus, The described knock-in method could result in a defective enhancer for the mouse lambda lous.
Methods for transgene DNA creation to introduce into eukaryotic species, especially metazoans, have used DNA from genomic libraries that were made from isolated natural DNA. The process of recombination is slow, cumbersome and error-prone. It takes cloned DNA from natural sources to create the desired transgene design. Sometimes it is possible to create a transgene using a specific strain, organism or haplotype of an organism. However, a genomic library may not be available for this species. These obstacles prevent the creation transgenes with complexly reconfigured sequences, and/or transgenes that contain chimeric DNA sequences from different species, strains, or haplotypes. This prevents the engineering of transgenes that are highly tailored for eukaryotes (especially metazoans).
Current methods for developing therapeutic mAbs can alter the functions of the antibody such as solubility and potency. These were not selected during initial stages of development. Current methods can also produce dangerous immune responses upon administration. Current chimeric and human mAb-producing mice lack the appropriate genetic content to function properly. This includes genetic diversity, transact regulatory elements, signaling domains and genetic stability. It would be useful to create methods and compositions that allow for enhanced discovery and enhancement of therapeutic antibodies. These compositions could also retain their potency and specificity throughout the process of antibody generation, discovery and development without eliciting an immuno response. Transgene compositions may contain DNA sequences that have been so complicatedly altered that it is impossible to construct these improvements or derive products from them. Although mice are the most popular species due to their economic value and proven utility, it is possible to find a solution that works across many species. This invention allows for the creation and introduction of transgenes. It also improves the genetic background in which transgenes could function in mice. In particular, it generates improved antibodies in transgenic animals.
“The invention is applicable to non-human animals, cells, transgenes and antibodies, methods, compositions including pharmaceutical compositions, and kits of various embodiments. The present invention is more specifically related to methods, compositions, and kits relating chimeric Ig chains, antibodies, and human antibodies, as well as the human antibodies and their fragments, engineered from variable domains of the chimeric antigens. The invention may also include mammals in certain embodiments.
“One embodiment of this invention is a method for producing a cell that contains a chimeric immuneglobulin-chain genome. This involves the following steps: (1) creating a DNA construct from scratch, which includes one or more nonendogenous V, J, and/or D gene segments, and one or two non-endogenous constant area gene segments. (2) introducing said construct into a cell’s genome. In some embodiments, the non endogenous variable domain can be human. Another embodiment of the chimeric constant area includes a segment of the mouse constant domain gene. One embodiment encodes the chimeric constant area using a non-endogenous sequence of polynucleotides derived from non-endogenous species, haplotypes, and/or alleles. Another embodiment encodes the non-endogenous variable region using a polynucleotide sequence that has been derived from multiple species, alleles, and/or haplotypes. The chimeric immunoglobulin chains are light in certain embodiments.
“In some other embodiments, chimeric immunoglobulin chains are a heavy chain. A related embodiment includes a non-endogenous CH1 region in the chimeric constant area. Another related embodiment of the method includes the steps of creating a second DNA structure in silico. This construct is non-endogenous and contains an immunoglobulin light chains. The second DNA construct is then produced. Finally, the second construct is introduced into the cell’s genome. One embodiment of the non-endogenous light chains includes one or more human V. gene segments. Another embodiment of the non-endogenous light chains includes one or more human J? C? gene segments. Another embodiment of the non-endogenous lighting chain includes 8 or more human V?? gene segments. A similar embodiment also includes 7 or more human J.-C? gene segment pairs.”
“One embodiment refers to a nonhuman cell that has a genome that contains a chimeric immuneglobulin-chain. The genome is composed of a nonendogenous variable domain, and a constant region. This cell is created by a process which includes (1) designing a DNA construct from scratch, and (2) creating the DNA construct and (3) inserting it into the cell’s genome. A non-human animal is another embodiment. A non-human animal can also produce a chimeric immunoglobulin heavier chain. Certain embodiments offer a non-human animal produced chimeric antibody.
“Another embodiment provides a chimeric immunoglobulin heavier chain that includes a nonendogenous variable region and a nonendogenous variable area. The non-endogenous variable region is derived from non-human animals. A related embodiment includes a non-endogenous CH1 region. One embodiment of chimeric immunoglobulin heavy chains includes a nonendogenous variable region and a region containing a chimeric constant. The non-endogenous polynucleotide sequence that is derived from non-endogenous alleles, haplotypes, and/or species encodes the chimeric constant area. A chimeric immunoglobulin chain with a non-endogenous domain and a constant region is another embodiment. The non-endogenous domain is encoded using a polynucleotide sequence that has been derived from at least two species, alleles, and/or haplotypes.
“Another embodiment refers to a polynucleotide that encodes the disclosed chimeric immuneglobulin heavy chains. Particular embodiments of the polynucleotide include both coding and uncoding sequences. The polynucleotide can be synthetic in certain embodiments. One embodiment refers to a construct that includes the polynucleotide, a polynucleotide encoded the disclosed chimeric immuneglobulin heavy chains.”
“Another embodiment provides a synthetic chimeric antibody or antigen-binding fragment of it. It comprises (1) a human immunoglobulin heavy (chimeric) chain. The chimeric light chain is composed of a non endogenous immunoglobulin (non-endogenous immunoglobulin) chain. The chimeric high chain constant area is made from non-endogenous alleles, haplotypes, and/or species. Another embodiment includes a “chimeric antibody” or an antigen binding fragment. It comprises (1) a Chimeric Immunoglobulin Heavy Chain, which has a nonendogenous heavy-chain variable domain and a constant region. (2) A non-endogenous immuneglobulin light Chain, in which said non-endogenous high-chain variable domain is derived either from one or more species, alleles, and/or haplotypes. One embodiment is a chimeric antibody, or an antigen binding fragment thereof. It comprises a chimeric immuneglobulin heavy chains, which has a nonendogenous variable region and a variable domain. The variable domain is derived not from any human animal. A related embodiment of the disclosed chimeric antigen, or antigen binding fragment thereof, includes a non-endogenous lighter chain.
“One embodiment of this invention provides a cell that is non-human and has a genome that contains a chimeric immuneglobulin heavy-chain comprising a variable domain with a non endogenous non-human origin and a constant region. A non-endogenous immuneglobulin light chain is also included in a similar embodiment. Particular embodiments include a non-endogenous Ig. light chain and a non endogenous Ig? light chain. The cell may also contain an inactivated locus of endogenous immunoglobulin. One embodiment produces a chimeric antibody from the cell.
Another embodiment of a nonhuman cell includes a genome with a chimeric immuneglobulin heavy-chain that has a nonendogenous variable domain, a constant region and a chimeric constant area. The constant region can be derived from non-endogenous alleles, species and/or haplotypes. A non-human cell is provided with a genome that contains a chimeric immunoglobulin heavier chain. This includes a nonendogenous variable region and a nonendogenous variable area. The non-endogenous variabledomain is derived from multiple species, alleles, and/or haplotypes. A non-human cell is also provided with a genetic transgene that encodes a synthetic chimeric antigen, or an antigen binding fragment thereof. This chimeric heavy chains (1) contains a non endogenous heavy-chain variable domain, and a constant region of chimeric high-chain. The genome of the disclosed cell also includes a non-endogenous immuneglobulin light chains in certain embodiments. One embodiment of the cell’s genome includes a non-endogenous Ig. light chain and a non endogenous Ig? light chain. Particular embodiments include an inactivated immunoglobulin locus. A different embodiment allows for the production of chimeric antibodies by the cell.
“Another embodiment is a nonhuman animal that contains a genome that includes a chimeric immuneglobulin heavy chains comprising a variable domain with a nonendogenous non-human origin and a constant region. A related embodiment of the invention includes a polynucleotide sequence that encodes a non-endogenous immuneglobulin light chain. In some embodiments, the genome contains a non-endogenous Ig. Light chain and non-endogenous Ig light chain. Another embodiment of the animal includes an inactivated exogenous immunoglobulin locus. The animal may be a mouse in certain embodiments. A different embodiment uses chimeric antibodies that are produced by non-human animals.
Another embodiment of the invention creates a nonhuman animal that has (1) a human-like chimeric immunoglobulin-heavy chain. The chimeric chain is composed of a nonendogenous heavy-chain variable domain and a constant region. (2) A non-endogenous immunoglobulin-light chain. In this case, the constant region of the chimeric high chain is derived from non-endogenous species, haplotypes, and/or alleles. A non-human animal is provided with a genome that includes (1) a Chimeric Immunoglobulin Heavy Chain, where the chimeric chain has a nonendogenous heavy-chain variable domain and a constant region. (2) A non-endogenous immuneglobulin Light Chain, where the non-endogenous high-chain variable domain is derived either from one or more species, alleles, and/or haplotypes. A non-human animal is provided with a genetic transgene that encodes a synthetic chimeric antigen, or an antigen binding fragment thereof. The genome comprises (1) a synthesized transgene that encodes a chimeric immuneglobulin heavy chains, which includes a nonendogenous heavy-chain variable domain and constant region. The genome may also include a non-endogenous immuneglobulin light chains, in particular embodiments. The genome of an animal may also include a non-endogenous Ig Light chain and non-endogenous Ig light chain. Particular embodiments include an inactivated endogenous immuneglobulin locus. A second embodiment uses a chimeric antigen produced by the animal.
“One embodiment of this invention provides a nonhuman animal with an inactivated Ig locus. The endogenous Ig lous is comprised of a deletion that prevents the formation of a functional variable region and a constant area capable of driving primary cell development. In some embodiments, the endogenous immuneglobulin locus can be a heavy-chain locus. Other embodiments of the endogenous immuneglobulin locus are light chain. A third embodiment includes a non-human cell with an inactivated Ig locus. In this case, the deletion prevents the formation of a functional variable region and a constant area that can drive primary B cell development.
“One embodiment presents a DNA construct that includes a first flanking sequence and a transgene. The second flanking sequence is composed of a polynucleotide sequence that can introduce a deletion in an Ig locus. This will prevent the formation of a functional variable region and support primary B cell development. The DNA construct is also provided in a kit. A second embodiment describes a method of inactivating an endogenous immuneglobulin locus. This involves impairing formation of a functional variabledomain and impairing formation of a constant area capable of driving primary cell development.
Another embodiment of the invention describes a method for producing an antibody library. This involves providing a nonhuman animal with a genome that contains a chimeric immuneglobulin-heavy chain. The chimeric heavy chains comprises a nonendogenous heavy-chain variable domain and a constant region. An antibody display library that includes immunoglobulin heavy chains variable regions is provided by one embodiment of the invention. This is a library consisting of non-human animals with a genome that contains a chimeric immunoglobulin chain. The chimeric heavychain comprises a non endogenous heavy-chain variable domain and a constant region. Variable regions are derived using chimeric antibodies.
“BRIEF DESCRIPTION ABOUT THE VIEWS FROM THE DRAWINGS”
“FIG. “FIG. coli.”
“FIG. “FIG.
“FIG. “FIG.
“FIG. “FIG. coli.”
“FIG. “FIG.
“Overview”
“The present invention comprises chimeric antibodies. Non-human animals can produce chimeric, humanized antibodies. Methods of producing such cells and animals are also included. Also, compositions and kits containing the antibodies are included. Specific embodiments of this invention include mammals as the non-human animal.
“Chimeric antibodies and antigen-binding fragments of them, as described herein, comprise a non-endogenous variable region and a constant region in the chimeric heavy chains. An IgH chain may include one or more of the following: a non-endogenous V and D gene segments, a CH1 domain and endogenous H2 and CH3 domains. An antibody or antigen-binding piece of an IgH chain, which includes the chimeric IgH chains, may also include an IgL chain with an amino acid sequence that is encoded by a non-endogenous sequence. An antibody or antigen-binding piece of an antibody comprising the chimeric IgH chains described herein also includes an IgL chain with an amino acid sequence encoded by both endogenous and not-endogenous nucleotide sequencings.
This prevents any alteration in the V-domain conformation due to the in vitro switch from an initial C region, especially a CH1 region and optionally a portion from a hinge region from one species (e.g. mouse), during the in vivo immune reaction to a second region, particularly the CH1 domain, and optionally a part of the hinge area from another species (e.g. human). The present invention does not result in the loss or reduction in activity or potency of antibodies made from antibodies from other chimeric animal producing animals. This could be due to altered conformation in the VH domain, particularly a CH1 domain, or by changes in antigen binding, possibly caused by a change in the length or flexibility in the upper hinge regions (the sequence of peptides from the end CH1 to the first cysteine in the hinge that forms an intra-heavy chain disulfide link and which is variable in length and composition The following references are found in Immunology (1997) 159.3372-3382. The cysteine residues, which form inter-heavy chains disulfide bonds, bounded the middle hinge region.
“Definitions”
“Before we describe certain embodiments in detail, please understand that the invention is not limited by particular biological systems or compositions. They can vary. The terminology used in this specification is intended only to describe particular illustrative embodiments and is not meant to be restrictive. The terms used herein generally refer to the ordinary meaning of the term in the art within the context and context in which it is used. Some terms are described below and elsewhere in this specification to help the practitioner understand the compositions and methods and how they can be made and used. It is important to understand the context in which a term is used. This will help you determine the meaning and scope of the term. The definitions provided herein are meant to be illustrative and not limitative. The singular forms?a? and?an? are used in the claims and disclosure. ?an,? ?an,? If the context requires otherwise, include plural forms.
“As used herein, ?antibody? “Antibody” and “immunoglobulin?” Ig is an acronym for immunoglobulin. It refers to proteins molecules that are produced by B cells and recognize and bind specific antigens. They can be membrane bound or secreted. Monoclonal antibodies are those that are produced by one clone (or more) of B cells. They recognize the same epitope, have the same amino acid sequence and nucleic acids, and polyclonal antibodies are those that are produced from multiple clones (or more). Polyclonal antibodies are those that are produced by multiple B cell clones and recognize different epitopes.
“Antibody molecules, also known as Ig molecules, are usually composed of two identical heavy chain and two identical light chains, linked by disulfide bond. IgL and Ig? There are two types of IgL, Ig? Both IgH and IgL contain a variable region (V) or domain, and a constant region (C) or domain. Multiple copies of variable (V), diversity(D), and joining (J] gene segments are contained in the IgH region that encodes the V region. Ig? Ig? and Ig? encode the V region. They contain multiple copies of J and V gene segments. To develop antigen specificity, the V region that encodes IgH or IgL loci is subject to gene rearrangement. The secreted IgH C region consists of three C domains: CH1, CH2, and CH3, with optionally CH4 (C). A hinge region and three C domains. The membrane-bound IgH C region has intra- and membrane domains. The antibody’s isotype is determined by the IgH constant area. IgM, IgD and IgG1 are the IgG2 IgG3, IgG3, IgG4, IgA, IgE in humans. It will be appreciated by non-human mammals that encode multiple Ig isotypes to be able switch isotype classes.
“A ?Fab? “A?Fab?” domain or fragment is the N-terminal IgH portion, which includes V region and CH1 domains of IgH and the entire IgL. A?F?2? Domain includes the Fab domain, a portion of hinge region, and the 2 IgH are linked via disulfide linking in the middle hinge area. F(ab)2 and Fab are both?antigen-binding fragments. ?antigen binding fragments? The?Fc is the C-terminal portion (CH2 and CH3 domains) of the IgH. domain. The Fc domain is the portion of the Ig recognized by cell receptors, such as the FcR, and to which the complement-activating protein, C1q, binds. The 5 encodes the lower hinge region. The CH2 exon’s lower hinge region is responsible for flexibility in the antibody’s ability to bind to FcR receptors.
“As used herein, chimeric antibody?” Refers to an antibody encoded using a polynucleotide sequencing that contains polynucleotide combinations derived from multiple species.
“A ?humanized? An antibody is a chimeric antigen that contains more human sequences than its parent molecule. When administered to humans, humanized antibodies are less immunogenic than non-humanized antibodies from other species. A humanized antibody could include the variable region of an appended chimeric antibody to a human constant area. The chimeric antibodies described in this article can be used to create a fully human antibody.
“As used herein ?chimeric Ig chain? An Ig heavy or light chain encoded with a polynucleotide sequencing containing sequences derived of two or more species. A chimeric Ig heavy chains may include human VH and DH gene segments, JH gene segments, and mouse CH2 or CH3 gene segments.
“?Polypeptide,? ?peptide? or ?protein? These terms are interchangeable to refer to a chain of amino acid that is linked together by chemical bond. An IgH, IgL or V domain protein can be a polypeptide.
“?Polynucleotide? A chain of nucleic acid that is linked by chemical bonds. The polynucleotides can include DNA, cDNA and RNA as well as gene sequences or segments.
“Polynucleotides can be extracted from living sources such as prokaryotic cells, eukaryotic cells, viruses, or in vitro manipulative techniques using standard molecular biology techniques or DNA synthesis or a combination of several techniques.
“?Locus? “?Locus?” refers to a location that contains one or more genes, exons or genes. locus, the cis regulatory element and the binding areas to which trans-acting factor bind. As used herein, ?gene? or ?gene segment? Refers to the sequence of polynucleotide polynucleotides that encodes a specific polypeptide, or a portion thereof. The term?gene segment is used herein. ?gene segment? and?exon? are interchangeable terms. These terms can be interchanged and may refer to a specific polynucleotide that encodes a peptide or a portion thereof. A gene or segment of a gene may also contain one or more transcriptional control elements (e.g. promoters, enhancers or other non-coding areas (e.g. cis regulatory element, e.g. 5). and/or 3? ”
“Inactivated Ig locus” is the term used herein. An Ig locus that doesn’t encode a functional Ig Chain. Functional variable region? Produce from an Ig locus is a polynucleotide sequence that can undergo V-(D-J) recombination. It is transcribed into a variable-region polypeptide and then translated into a protein that can be expressed on a cell’s surface. What is a?functional heavy-chain constant region? A constant region that can be operationally connected to a variable area and drives primary B cell development. Primary B cell development is the process of developing B cells in primary lymphoid organs (e.g. bone marrow). It includes the stages of pro-B and late pro-B cells (i.e. IgH DJ rearranging), pro-B and large pre-B cells (i.e. IgL VJ rearranging), as well as the development of small pre-B cells (i.e. IgL VJ rearranging). What is meant by “driving?” By?driving?, it means that the functional heavy-chain constant region can be, e.g. anchoring to cell membrane, signal transduction and/or binding to an Fc receptor. What is a?functional lightchain constant region? A constant region that can be operationally connected to a variable area and bound to heavy chain to accelerate B cell development beyond the pre-B cell stage.
“?Impair? “?Impairment” refers to the introduction or modification of a gene that causes a mutation or deletion that results in, for example, a variable or constant region that is not functional. Homozygous deletions of C could be an example. An IgH that is responsible for primary B cell development in certain mammals and strains of mice is affected.”
“?Mutation? A mutation is a modification in the sequence of a polynucleotide (or polypeptide) that naturally occurs. Mutations can cause functional changes. Mutations can include the addition or deletion of nucleotides. ?Deletion? ?Deletion? refers to the deletion of one or more nucleotides in the naturally occurring endogenous sequence polynucleotide. Frameshift mutations can be introduced by additions and deletions. Deletions can also result in the deletion of entire genes, gene segments, or modules. Sometimes, the deletion of a part of an naturally occurring endogenous sequence can occur in conjunction with the addition or removal of a non-endogenous one. A homologous recombination may result in the deletion of a part of an endogenous polynucleotide sequencing sequence. Another aspect is that an endogenous sequence of polynucleotides may be deleted after two non-endogenous recognition sequences for a site specific recombinase are introduced, e.g. a loxP site. Then, exposure to the CRE recombinase.
“Endogenous” is a term that refers to a polynucleotide sequence that is naturally occurring in a cell or animal. A polynucleotide sequence that occurs naturally in a cell or animal. ?Orthologous? Refers to a sequence of polynucleotides that encodes the same polypeptide in another species. The term “syngeneic” is used. The term?syngeneic? refers to a sequence of polynucleotides that can be introduced into an animal from the same species as it is. Introduced into a mouse gene segment. Note that polynucleotide sequences from individuals of different species may contain regions of significant variation.
“Homologous” as used herein means: or a?homologous sequence. A polynucleotide sequence with a high percentage of identity or a highly similar sequence (e.g. A polynucleotide sequence that is highly similar or high in identity (e.g. 30%, 40%. 50%. 60%. 70%. 80%. 90%) to another polynucleotide sequencing or segment thereof. A DNA construct may include a sequence homologous with a part of an endogenous sequence, for example, to facilitate recombination at a specific location. In both prokaryotic or eukaryotic cells, homologous recombination can occur.
“As used herein, ?flanking sequence? Or?flanking sequence? A DNA sequence that is adjacent to a non endogenous sequence in a DNA construction that is homologous with an endogenous sequence or a previously combined non-endogenous sequence, either a whole or part thereof. The invention may include one or more flanking sequences. For example, the flanking sequence on the 3? 5, and 6? End of the non-endogenous sequencing or a flanking sequence on 3? End of the non-endogenous sequence or a flanking sequence on the 3? oder 5? End of the non-endogenous sequencing. The flanking sequence could be homologous with an endogenous, within-a-endogenous sequence, or it may be homologous with an endogenous, adjacent, (or outside) of, an endogenous, sequence.
“The phrase ?homologous recombination-competent cell? A cell capable of homologously recombining DNA fragments with regions of overlapping homology. Examples of homologous recombination-competent cells include, but are not limited to, induced pluripotent stem cells, hematopoietic stem cells, bacteria, yeast, various cell lines and embryonic stem (ES) cells.”
“A ?non-human animal? Any animal that is not a human, such as reptiles, birds, and mammals, is considered a?non-human animal? ?Non-human mammal? A non-human mammal is an animal that is not related to humans and falls under the Mammalia class. Non-human mammals can include rodents and non-human primates like camelids, rodents and bovines as well as ovines and equines. The preferred non-human mammals are those that rely on somatic hypermutation or gene conversion to generate antibodies diversity. Mice are a favorite non-human mammal.
“Transgenic” is a term that refers to a cell or animal that has a non-endogenous polynucleotide sequence. “Transgenic” refers to an animal or cell that contains a non-endogenous sequence of polynucleotides, such as a transgene from another species. A transgenic mouse is one that has a human VH segment embedded in its genome, but not the endogenous IgH locus. Conversely, a transgenic mice is one that has a human VH segment embedded directly into its genome, replacing the endogenous VH locus. mouse. Transgenic cells and nonhuman mammals may express the non-endogenous mononucleotide sequence with or without the endogenous genes.
“As used herein, ?replace? It can be used to refer to either direct or functional replacement. What is meant by ‘direct replacement? It refers to the replacement of an endogenous DNA sequence with an engineered sequence that contains a non-endogenous gene sequence, such as homologous recombination. Homologous recombination is used to remove the endogenous DNA sequence. Or, it can be deleted if the endogenous sequence remains between two non-endogenous sequences. What is meant by “functional replacement?” It refers to the fact that an endogenous sequence of DNA does not perform the function, such as the polypeptide made from engineered DNA sequences. A transgene, which encodes a chimeric IgH-chain and is insert into the genome without the IgH locus, can replace an endogenous IgH gene.
“A ?humanized? “A?humanized?” animal is a nonhuman animal (e.g., a mouse) that has a genetic structure that includes gene sequences from the non-human animals, and one or more gene segments or regulatory sequences that have been replaced by analogous human sequences.
“Vector” is the term used herein. A nucleic acids molecule into which another fragment of nucleic acids can be integrated without affecting the vector’s ability for replication. A virus, plasmid, or a cell from a higher organism can all be vectors. Vectors can be used to introduce recombinant or foreign DNA into host cells, where it is then replicated.
A vector can contain a polynucleotide agent, which allows for manipulation, such as the introduction of the polynucleotide to a target cell. A vector can either be a cloning or expression vector. This allows for the manipulation of the polynucleotide. An expression vector may contain the necessary expression elements to enable sustained transcription of the polynucleotide encoding. The regulatory elements can also be operatively linked with the polynucleotide before it is cloned into a vector.
An expression vector (or polynucleotide), generally contains or encodes an expression sequence. This sequence can provide constitutive, tissue-specific, or developmental stage-specific expression of the encoding protein. It can also include a polynucleotide recognition sequence and a ribosome recognition or internal ribosome entrance site. Other regulatory elements, such as enhancers, can also be included. The vector can also contain elements that are required to replicate in either a prokaryotic and eukaryotic host, or both, depending on the situation. These vectors include plasmid, retroviruses, lentiviruses, bacteriophage and adenovirus vectors. They can also be used to replicate in a prokaryotic or eukaryotic host system. Or can be made by someone skilled in the art (see Meth. Enzymol., Vol. 185, Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994; Flotte, J. Bioenerg. Biomemb 25:37-42, 1993; Kirshenbaum et al., J. Clin. Invest 92, 381-387, 1993 (each of which is incorporated by reference).
A DNA vector used in the invention may contain both positive and negative selection marks. Negative and positive markers are genes that confer drug resistance on cells expressing them. E.coli has several suitable selection markers. These include Km (Kanamycin-resistant gene), TetA (tetracycline-resistant gene) and beta lactamase (ampicillin-resistant gene). Mammalian cells can be cultured with suitable selection markers, including hyg (hygromycin resistant gene), puro [puromycin resistance genes] and G418 (?neomycin resistant gene). You can also use selection markers to identify metabolic genes that convert a substance into toxic substances. The gene thymidinekinase, when expressed, converts drug gancyclovir to a toxic substance. Treatment of cells with gancylcovir may result in a negative selection for genes that don’t express thymidine kinase.
“In a similar aspect, the selection marker can be?screenable? markers. Such as yellow fluorescent protein, red fluorescent protein and red fluorescent protein (RFP), GFP like proteins, and Luciferase.
There are many types of vectors that are available in the art. These include viral, bacterial and yeast vectors. Any suitable DNA vector may include a cosmid or bacteriophage as well as a plasmid and cosmid. The DNA vector may be a BAC in certain cases. Each DNA vector is chosen according to the amount of DNA that will be inserted into the construct. One embodiment of the DNA constructs is bacterial artificial chromosomes (or fragments thereof).
“The term “bacterial artificial chromosome” “BAC” or?bacterial artificial chromosome? The term BAC, as it is used herein, refers to a bacterial genome vector. BACs such as those derived form E. coli may be used to insert, delete, or replace DNA sequences in non-human mammalian cell or animal cells via homologous recombination. Complex genomic DNA can be maintained by E.coli in the form BACs. (See Shizuya, Kouros-Mehr and Keio J Med. 2001, 50(1).26-30. BACs have a greater DNA stability than cosmids and yeast artificial chromosomes. BAC libraries of human genomic DNA DNA are more accurate than libraries in cosmids and yeast artificial chromosomes. U.S. Application Ser. Nos. Nos.
Before humanization of the locus, DNA fragments comprising an Ig gene locus or a portion thereof are taken from the same non-human mammal species. Multiple BACs that contain overlapping fragments can be humanized. The overlapping fragments are recombined to create a continuous IgH/IgL locus. The resulting chimeric Ig locus contains the human gene segments that are operably linked with the non-human mammal Ig genes segments. This allows for the creation of a functional Ig locus. It is capable of gene rearrangement, thereby generating a diverse repertoire of chimeric antibody.
“These are processes for combining BACs or of engineering a fragment of a Chimeric Ig Locus or BAC. A bacterial cell such as E.coli must be transformed with a host Ig locus-containing BAC or a portion thereof. The BAC containing Bacillus is transformed with a vector that contains the desired human Ig genes segment and a flanking homology sequence. The homology of the shared sequence mediates homologous cross-over between human Ig genes on the vector and non-human mammal Ig genes on the BAC. Selectable and/or screenable markers may be used to detect homologously recombined BABs. Humanized BACs can easily be isolated from bacteria and used to produce knock-in human cells. There are methods of combining BACs, engineering insertions or deletions within DNA on BACs, and methods for creating genetically modified mice from them. See, e.g., U.S. Pat. No. 5,770,429; Fishwild, D. et al. (1996) Nat. Biotechnol. 14:845-851; Valenzuela et al. Nature Biotech. (2003) 21:652-659; Testa et al. Nature Biotech. (2003) 21.443-447. Nature Biotech. (2003) 21:447-451.”
The first step of recombination may be performed in an E.coli strain that is lacking in sbcB or sbcC, recB activity, recC, recC, or recD activity, and that has a temperature sensitive mutation, recA. The recombination step results in the isolation of a recombined DNA structure, which can have the different sequences and orientations described.
The length of the regions that are used for BAC recombineering must be long enough to allow for homologous recombination. The flanking regions can be anywhere from 0.1 to 19 km, with a typical length of about 1 kb – 15 kb or 2 kb – 10 kb.
To recombinate BACs into larger or more tailored BACs that contain portions of the Igloci, it is necessary for E.coli to be transformed with a BAB carrying either a portion of the Ig locus or another target sequence. The BAC containing E. coli can then be transformed with a recombination virus (e.g. plasmid, BAC) that contains the desired Ig gene segment. This could be one or more of the human VH, DH, or JH gene segments, which will be joined to a portion of the mouse IgH locus. Both vectors share a region of sequence similarity. The presence of functional recA within the E. coli creates a shared identity region that mediates cross-over between Ig gene segments on the recombination and non-human mammal Ig genes on the BAC. Selectable and/or screenable markers may be used to select homologously recombined BABs. Humanized and chimeric BACs are easily purified from E. coli. They can then be used to produce transgenic and knockin non-human cells or animals.
“Alternatively, DNA fragments containing the Ig locus that can be incorporated into non-human animals are obtained from DNA synthesized intracellularly. Many organisms’ genomes have been fully sequenced (e.g. human, chimpanzees, rhesus monkeys, mouse, rat and dog), and are freely available with annotation. Information on the sequences and transcriptomes of many other organisms is also publicly available. The location and activity of non-coding regulatory elements and gene segments has been mapped and studied at the mouse and human immunoglobulin loci.
“In silico” is a term that refers to the use of a computer or algorithm to model a naturally occurring or in vitro process. “In silico” is a term that refers to the use or algorithm of a computer to model an in vitro or naturally occurring process. It also includes the creation of a nucleotide/polypeptide sequence or the production of such sequences using a cell-free system (e.g. using automated chemical synthesizing). You can manipulate the sequences of Ig loci and then recombine them in silico with commonly used software for nucleic acids sequence analysis. Recombination can be performed in silico within the same locus, between loci of the same species or between loci of different species. In silico recombination can be used to create either a functional or non-functional, activated sequence. Precise nucleotide-by-nucleotide engineering allows for precise manipulation of sequence composition that can be applied to precisely engineer the function of the transgene and after transcription and translation, result in precisely engineered composition and function of the polypeptide product of the locus.”
“Sequences from an Ig locus can also be recombined with sequences from a non immuneglobulin locus in silico, from the same species or from another. These sequences can include genes for positive or negative drug selection markers like G418, puro, hyg and tk, site specific recombinase sequences such as lox P sites and their variants, and frt sites. They also contain precisely delineated sequences that drive homologous replication. Once the desired sequence has been assembled in silico it can be synthesized and assembled with no errors (Kodumal, Proc. Natl. Acad. Sci. (2004) 101:15573-15578). Contractual services are available for the synthesis, assembly, and sequencing large DNAs (e.g. DNA 2.0, Menlo Park; Blue Heron Biotechnology Bothell, Wash.; or Eurogentec San Diego, Calif.). These synthetic DNA sequences can be carried in vectors like plasmids or BACs, and can be transferred to other vectors like YACs.
“Construct” is a term that refers to a sequence of DNA artificially constructed by genetic engineering. “Construct” is a sequence or combination of DNA that has been artificially created by genetic engineering, recombineering, or synthesis. Examples of constructs are, for instance, transgenes or vectors (e.g. BACs. P1s. Lambda bacteriophage. Cosmids. plasmids. YACs. MACs). One embodiment of the DNA constructs is linearized before being introduced into a cell. Another embodiment does not allow for the linearization of DNA constructs prior to their introduction into a cell.
“As used herein, ?loxP? “As used herein,?loxP? and?CRE?” The site-specific recombination systems derived from P1 bacteriaiophage are called?CRE? IoxP sites have 34 nucleotides. If DNA is flanked by a loxP-site and exposed to CRE-mediated recombination (CRE-mediated recombination), the DNA on the other side is removed and the two loxP websites become one. It is well-documented that the CRE/lox system (including variant-sequence CRE sites and variants CRE) can be used for genetic engineering in many species including mice.
“A similar system can be used to achieve similar results, using frt sites and the flp-recombinase of S. cerevisiae. The flp/frt can be used to facilitate any CRE/loxP-mediated deletional events in mammalian cells cultured.
“As used herein, the terms ‘immunize? ?immunization,? ?Immunization? and?immunizing? The act of exposing an animal’s adaptive immune system to an antigen is called?immunizing. You can inject, inhale, or ingestion the antigen. The adaptive immune response (i.e.,.) to the antigen is increased upon second exposure. The T and B cell responses are enhanced upon second exposure to the same antigen.
“?Antigen? “Antigen” refers to any peptide, lipid or nucleic acid, as well as any hapten, that are recognized by the adaptive immuno system. Antigens can include pollen, bacterial cell walls components, and the rh factor. ?Target antigen? A target antigen is an antigen, protein, lipid, saccharide or amino acid that is recognized by adaptive immune system. It is a substance, peptide or lipid that is used to generate an immune response against an infectious agent, endogenous or exogenous cells, or products thereof. Target antigens can include, but not be limited to, bacterial or viral components, tumor-specific antgens, cell surface molecules, and any antigens against whom antibodies or other binding protein have been produced by in vivo and in vitro methods.
“The term “pharmaceutical” is used interchangeably with the word “pharmaceutical.” “The term?pharmaceutical? or?pharmaceutical drugs? Any pharmacological, therapeutic, or active biological agent that can be administered to a patient or subject is referred to herein. In some embodiments, the subject is an animal. Preferably, a mammal. Most preferably, a human.
“Pharmaceutically acceptable carrier” is a general term that refers to any material that may be used in conjunction with the pharmaceutical drug. It refers to all material that can be used in conjunction with the drug, and which is not likely to cause adverse reactions.
“Administering” is a term that refers to administering a drug or other agent. “Administering” as it is used herein refers to any method of delivering, introducing, transporting, or transferring a pharmaceutical drug or another agent to a subject. These modes include oral, topical, intraperitoneal and intramuscular administrations, as well as subcutaneous administrations.
“Non-Human Mammals, Cells Encoding Chimeric Ig heavy Chains”
“Non-human animals or cells are comprised of one or more altered Ig loci, e.g. IgH, Ig?? and/or Ig?” It may include non-endogenous Ig genes segments that can replace endogenous segments. The altered loci can be used to replace endogenous gene segments in certain embodiments. Other embodiments replace the endogenous genes segments with the altered loci.
The non-endogenous gene segments can be derived from any species and may also include syngeneic genes. Non-endogenous sequences can be derived from humans, mice, nonhuman primates, camelids and rodents. The non-human cell/animal may, as mentioned above, be any nonhuman animal. The transgenic cells and animals described in this article may contain DNA sequences from any combination of species. Chimeric mice cells, as well as mice containing human and camelid Ig polynucleotide sequencings, are possible. The transgenic animal or cell may also contain non-endogenous DNA of more than one species. A transgenic mouse genome could include both human and camelid DNA sequences.
“The transgenic animals and cells described herein contain one or more non-endogenous segments of the V gene. Specific embodiments use mammal as the preferred non-human animal. In some embodiments, the animal or cell may also include one or more non-endogenous segments of J gene. A cell or animal that has a chimeric IgH-chain optionally includes one or more non endogenous D gene segments.
“In one embodiment, the cell/animal comprises a genome that encodes a chimeric IgH and transgenic light chains. A transgenic light chain could be an IgG? The transgenic light chain may be an Ig? light chain. The transgenic light chains may also be chimeric or contain only non-endogenous sequences of amino acids. Particular embodiments include a cell or animal with a genome that encodes non-endogenous IgH and Ig? In particular embodiments, the cell or animal contains a genome encoding non-endogenous IgH, Ig? gene segments. A non-endogenous CH1domain is used to replace a CH1domain in an endogenous CH gene. In some embodiments, the non endogenous CH1 domain can be orthologous with the endogenous CH1 region. In some other embodiments, non-endogenous CH1 domain is not orthologous with the endogenous CH1 region. Another embodiment replaces more than one CH1 domain with a non-endogenous domain. A related embodiment replaces all endogenous CH1domains with a non-endogenous one. An orthologous human CH1 could replace each endogenous C. gene (e.g., human C1 CH1 replaces mice C?1CH1 and human C2 CH1 replaces mice C?2CH1 etc.). Another embodiment replaces each endogenous C1 domain with a CH1 domain. A single human IgG type is one that is more commonly used in therapeutic mAbs. It is typically C?1, C??2 or C?4. This is to facilitate in vivo maturation and clinical relevance of a human V domain within a CH1 domain.
“Optionally the upper hinge sequences endogenous C genes can be replaced by orthologous non-endogenous Chine sequences. Alternately, the middle and upper hinge sequences of endogenous C gene genes can be replaced by the orthologous non?endogenous C hinges. In order to drive inter-heavy chains dimerization via disulfide bonding, the human C4 middle hinge sequence could be modified to include a proline at position 229 instead of a serine. The endogenous C?2 CH2 domain includes the lower hinge region. To ensure optimal binding to the endogenous Fc.R, this gene cannot be replaced. These options include Fab domain plus higher hinge or F(ab?) which provide non-endogenous heavy chains Fab domains, Fab domain plus lower hinge, and Fc?R. )2, respectively. The human upper hinge regions can be replaced by the upper. This will increase the likelihood that the variable region of the resulting antibody will retain its optimal characteristics when converted to human IgG.
“Another embodiment incorporates fully non-endogenous, e.g., human, Ig including the C regions comprising CH1-hinge-CH2-CH3(?CH4) and the cognate syngeneic, e.g., mouse, membrane and intracellular domains so as to provide native intracellular signal transduction and to enable association of the IgH in the B-cell receptor with Ig? Ig? IgG containing B-cell receptor and allow endogenous signaling from IgG, IgG? IgG containing B cell receptor. Another embodiment of the invention is that the membrane and intracellular domains of the constant heavy chain heavy chain are identical or non-cognate heavy chain isotypes. This can be done easily using the methods described below.
“In another embodiment, transgenic animals and cells comprising a Chimeric IgH Chain described herein include a constant region encoded using a non-endogenous sequence of polynucleotide derived from more than one species. A transgenic mouse with a genome that encodes a chimeric IgH chains constant region is one example. It would include a human CH1 and upper hinge regions as well as rat CH2 or CH3 domains. It is preferable that animals with a xenogeneic consistent region can interact (e.g. binding) an endogenous FCR.
“In another embodiment, transgenic animals and cells comprising a Chimeric IgH Chain described herein include a constant region encoded with a non-endogenous and an endogenous sequence of polynucleotide. A transgenic mouse with a genome that encodes a chimeric IgH chains constant region includes a human CH1 domain and human upper hinge regions. The Balb/c mouse CH2 coding sequences are embedded in C57BL/6 (??B6?). It is possible to create genomic DNA that contains all the genetic information of B6 except for the Balb/c sequence exons for CH2 or CH3.
“In one embodiment, the composite IgH sequencing comprises at least 3kb upstream from the VH6 promoter through D gene cluster through 3. JH6 and is in germline configuration. Another embodiment of the composite IgH sequence includes at least 3 kb downstream from the VH6 promoter via the D gene cluster through 3. The JH6 is human-specific and germline, with the exception that the D gene cluster may be replaced by a part or all of a xenogeneic specie. Another aspect of the invention is that additional human VH genes are upstream from human VH6. Another aspect of the invention is that the additional VH genes exist in germline form. Alternatively, additional VH genes have smaller sizes than those in the human genome. These units include upstream regulatory elements like cis-regulatory element binding sites and binding sites to trans-acting factor binding sites and introns. One aspect states that the unit size must be less than 10 kb. The unit size in another aspect is 5 kb. Another aspect is that the VH genes are chosen from the subset common VH genes within the human haplotypes. Another aspect is that VH genes, DH gene and JH genes are selected to represent a particular allele, such as the most common in human populations. Another aspect is that the VH gene’s individual codons are codon-optimized to allow efficient expression in non-human mammals. Another aspect is that the individual codons can be used as a template to facilitate somatic hypermutation.
“In an alternative embodiment, the composite IgH sequencing comprises a sequence of mouse DNA beginning at least 3 kb downstream from the promoter for VH genes in the closest cluster D, e.g. VH5-2 through 3? JH4 in germline form and into which the coding sequencings have been replaced all or part by human coding. E.g., coding for mouse VH5-2 is replaced with coding for human VH6-1. The mouse DH coding series has been replaced by human DH coding and the mouse JH coding sections have been replaced by human JH coding. The additional JH genes can be added to the genome by various methods, such as inserting human JH sequences with their cis regulatory element, such recombination signals sequences downstream from the JH4, or completely omitting them.
“In some other embodiments, the entire mouse VH coding sequences can be replaced by VL coding sequences. In some embodiments, the whole DH gene cluster can be of mouse sequence. In other embodiments, the whole DH gene cluster can be of xenogeneic species. Another aspect of the invention is that there are additional VH gene sequences upstream from VH6 coding sequences. This means that all sequences are mouse-specific, except for the coding sequences functional VH gene sequences.
“Another aspect is that the additional VH genes exist in germline configuration. Alternative aspects include additional VH genes with smaller sizes than those in the mouse genome. These units contain upstream regulatory elements, such as binding sites for trans-acting factor binding sites and cis-regulatory element bindings. One aspect states that the unit size is less than 10 kb. Another aspect states that the unit size should be 5 kb or smaller.
“In another aspect, VH genes are chosen from a subset that is functional. The replacing human VH coding sequence is from a functional human VH genetic sequence and the replacement mouse VH coding sequence from a functional mouse VH genetic sequence. Another aspect is that the VH coding sequences for human are chosen from the subset common VH genes within the human haplotypes. Another aspect is that the VH coding sequences for replacing VH coding, DH coding and JH coding sequences reflect a particular allele, such as the most common in human populations. Another aspect is that some or all the VH coding, DH coding, and JH coding sequences being replaced are not from humans. Another aspect is that the individual codons in the VH gene have been codon-optimized to allow efficient expression in non-human mammals. Another aspect is that the individual codons can be used as a template to facilitate somatic hypermutation.”
“In another embodiment, the composite IgH series further comprises 3? The most 3? JH The mouse sequence immediately below mouse JH4 through mouse E? via C? Through C? via C? All the C?3 promoters for mice in germline configuration, with the exception of replacing the CH1 domains for mouse C? C? C? by their human counterparts. Sometimes, the upper mouse hinge regions can be replaced with their human counterparts. The mouse C? The genes are set up in germline configuration, with the exception that their CH1 domains are replaced by human CH1 domains.”
“In certain cases, the mouse’s upper hinge regions can be replaced by human ones. In certain embodiments, the mouse C.3 coding sequences can be replaced by human and mouse CH1 and CH2, respectively. Another embodiment uses the entire germline-configured mouse C3 sequence starting at the promoter and continuing through the intracellular domains to the 3? The untranslated sequence, poly(A), and the promoter upstream of the switch region are replaced with the complete sequences from C1 in germline configuration. Human CH1 replaces mouse CH1 from C1 to effectively replace C?3 by chimeric C1 Some embodiments replace a mouse constant coding sequence with human CH1 or mouse CH2, and intercellular and membrane domains of different mouse constant region isotypes. Other embodiments further modify the sequence of CH2 and CH3 domains to increase binding to Fc receptors.
“In another embodiment, the cell/non-human animal includes a locus encoded with a human Ig lightchain comprising a human Ig?” Variable region. A related embodiment of the Ig light chain locus also includes a human Ig. constant region. The composite Ig? is one embodiment. The sequence includes mouse DNA sequence starting at least 3 kb downstream of the V? promoter. V?3-1 through 3? The mouse J?5 gene is located most proximal to mouse J?1 (V?3-1) through 3? Replaced by human J? Code sequences. Another embodiment uses the sequence J?5 through C? The sequence from J?5 through C? is in mouse and germline configuration. “The coding sequences have been completely or partially replaced by human coding.”
“In another aspect, there is a 3??LCR region downstream of the C? gene. The 3? LCR and RS elements can be used in mouse or germline configurations. Another aspect of the invention is that there are additional V? There are additional V? genes upstream from the coding sequences to human V?4-1. This means that the entire sequence is mouse, except for the coding sequences of functional V?? Replaced with the human V? genes.”
“In another aspect, the additional V?” Genes are in germline configuration. The additional V is another alternative. The genes have a smaller size than the one in the mouse genome. They contain upstream regulatory elements like cis-regulatory element and binding sites for transacting factors. One aspect of the unit size is less than 10 kb. The unit size in another aspect is 5 kb. Another aspect is the V? The V???? genes are chosen from the subset that is most commonly shared among human haplotypes. genes that are common among human haplotypes. V? V? genes and J? Genes are selected to reflect a particular allele, such as the dominant one in human populations. The individual codons for the V? are another aspect. The individual codons of the V? gene have been codon-optimized to allow efficient expression in a non-human mammal. Another aspect is that the individual codons can be used as a template to facilitate somatic hypermutation.”
“In another embodiment, the human Ig Light Chain locus includes all or part of a human Ig?” light chain locus, and an Ig? 3?LCR or a functional fragment thereof. The human Ig? is one embodiment. The entire human Ig’s light chain locus is included in one embodiment. locus. Another embodiment of the human Ig is light chain locus comprises human V? coding sequences and 1-7 J?-C? coding sequence pair, in which the human C? is replaced by syngeneic? C? The human Ig? is another example. light chain locus comprises human V? coding sequences, 1-7 human J? Code sequences and one human C? coding sequence in which the human coding sequences look like a human Ig? locus configuration.”
“In particular embodiments the Ig? 3? LCR or a functional fraction thereof is from a mammal chosen from the group consisting human, non-human primate and rat. One embodiment of the Ig? 3? LCR or a functional segment thereof is human. Ig? is a particular embodiment. 3? LCR or a functional fragment thereof binds NF??b. The Ig? 3? LCR or a functional portion thereof is from mice and has been mutagenized to restore binding of the NF?b. The 3? is used in other embodiments. LCR or a functional segment thereof in the human Ig. locus is an IgG? 3? LCR or functional fragment thereof.”
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