# Patent

## Title
Transgenic animals expressing chimeric antibodies for use in preparing human antibodies

## Abstract

The invention provides transgene constructs for expressing chimeric antibodies, and transgenic non-human host animals carrying such constructs, wherein the chimeric antibodies comprise human variable regions and constant regions of the non-human transgenic host animal. The presence of immunoglobulin constant regions of the host animal allows for generation of improved antibodies in such transgenic host animals. Subsequently, the chimeric antibodies can be readily converted to fully human antibodies using recombinant DNA techniques. Thus, the invention provides compositions and methods for generating human antibodies in which chimeric antibodies raised in vivo in transgenic mice are used as intermediates and then converted to fully human antibodies in vitro.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of Ser. No. 12/295,557, filed Jan. 28, 2009, which is a national phase of International Application No. PCT/US07/008231, filed Mar. 30, 2007, which claims priority to U.S. provisional application Ser. No. 60/744,104 filed Mar. 31, 2006, the contents of which are hereby incorporated in their entirety.


SEQUENCE LISTING
The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Feb. 17, 2011. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as “Sequence Listing 077375_0926_ST25.txt”, is 2,383 bytes and was created on Feb. 16, 2011. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.
BACKGROUND OF THE INVENTION
Antibodies have proven to be effective therapeutic agents in humans for the treatment of a wide variety of disorders, including cancer, autoimmune diseases and infectious diseases. Although originally mouse monoclonal antibodies were tried as therapeutic agents, they generally proved to be unsuitable for use in humans due to the occurrence of a human anti-mouse antibody (HAMA) response. Rather, antibodies composed in part or entirely of human antibody amino acid sequences currently are the antibody agents of choice for use in humans. Of the numerous antibodies approved by the FDA for use in humans or currently in clinical trials, certain antibodies contain mouse variable regions linked to human constant regions and typically are referred to as chimeric antibodies. Others contain mouse CDRs within human framework and constant regions and typically are referred to as humanized antibodies. Still others are composed entirely of human-derived sequences (i.e., fully human variable and constant regions) and typically are referred to as human antibodies.
A number of approaches are known in the art for preparing human antibodies. In one type of approach, a library of human immunoglobulin sequences is screened on a display system (e.g., bacteriophage) with an antigen of interest to select antibody sequences having the desired antigenic specificity (see e.g., U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.). Since this approach is carried out in vitro, the human antibody sequences do not undergo affinity maturation or somatic mutation during the selection process, which may result in antibodies of lower affinity as compared to antibodies generated in vivo.
Thus, in another type of approach, mice whose genomes have been modified to contain human immunoglobulin sequences are used to raise antigen-specific antibodies by immunization with an antigen of interest. Such mice carry unrearranged human immunoglobulin genes (variable and constant regions) on transgenes and/or transchromosomes, which genes undergo apparently normal rearrangement and isotype switching in the mice. Moreover, somatic mutation occur during the maturation of the antibody response in these mice.
One example of such a mouse is the HuMAb Mouse® (Medarex, Inc.), which contains human immunoglobulin transgene miniloci that encode unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGκ monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93 and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparation and use of HuMab mice, and the genomic modifications carried by such mice, are further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby specifically incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 by Korman et al.
An alternative transgenic mouse system for expressing human immunoglobulin genes is referred to as the Xenomouse (Abgenix, Inc.) and is described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al. Like the HuMAb Mouse® system, the Xenomouse system involves disruption of the endogenous mouse heavy and light chain genes and insertion into the genome of the mouse transgenes carrying unrearranged human heavy and light chain immunoglobulin loci that contain human variable and constant region sequences.
Other systems known in the art for expressing human immunoglobulin genes include the KM Mouse® system, described in detail in PCT Publication WO 02/43478 by Ishida et al., in which the mouse carries a human heavy chain transchromosome and a human light chain transgene, and the TC mouse system, described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727, in which the mouse carries both a human heavy chain transchromosome and a human light chain transchromosome. In each of these systems, the transgenes and/or transchromosomes carried by the mice comprise human immunoglobulin variable and constant region sequences.
U.S. Pat. No. 6,596,541 provides a prophetic example of a homologous recombinant mouse that expresses chimeric antibodies having human variable region sequences linked to mouse constant region sequences. In the example, the mouse heavy chain locus variable region (V-D-J segments) is precisely replaced with the human heavy chain V-D-J counterpart via a multi-step process. First, a large genomic fragment (greater than 20 kb) spanning the human immunoglobulin variable gene segments of interest is obtained and bacterial recombination is used to prepare a large targeting vector for use in eukaryotic cells (LTVEC) that includes homology arms totaling greater than 20 kb. The homology arms contain sequences from the endogenous mouse immunoglobulin locus. The LTVEC is then introduced into mouse embryonic stem cells. A cell in which homologous recombination has occurred between the LTVEC and the endogenous mouse immunoglobulin locus is identified using a quantitative assay to detect modification of allele (MOA) in the ES cells. No actual mice expressing chimeric antibodies, however, are described or characterized.
SUMMARY OF THE INVENTION
In this invention, transgenic animals expressing chimeric antibodies, comprising human variable regions and non-human constant regions, are described and characterized. In particular, the constant regions are those of the host animal (e.g., in mice, the constant regions are mice constant regions). The animals of the invention can be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are easier to prepare and select than approaches using homologous recombination. Moreover, the expression of the human Ig variable regions linked to the host animal Ig constant regions in the transgenic animals of the invention is thought to allow for improved trafficking and development of B cells and antibodies in vivo such that improved antibodies can be obtained in these animals, as compared to transgenic animals that express human Ig variable regions linked to human Ig constant regions. Such improvements in the antibodies can include, for example, increased somatic mutations, improved association with endogenous mouse accessory proteins, and improved binding to mouse Fc receptors in vivo.
Moreover, the chimeric antibodies can readily be converted to fully human antibodies by isolation of the sequences encoding the human V regions and linkage of these sequences to human constant region sequences using standard recombinant DNA technology in vitro. Thus, the invention provides a means to obtain improved human antibodies, suitable for use in therapy, through the use of a chimeric antibody intermediate raised in vivo in transgenic animals.
In the animals of the invention, a transgene comprising unrearranged human immunoglobulin variable region sequences and at least one host animal constant region sequence (e.g., an IgM constant region) is prepared and inserted into the genome of the host animal (e.g., by pronuclear microinjection into a zygote of the host animal). When inserted into the genome of the host animal, the transgene construct undergoes rearrangement and expresses chimeric antibodies in the non-human host animal, the chimeric antibodies comprising a human variable region and a constant region of the non-human host animal. Moreover, as demonstrated herein, the inserted transgene is capable of undergoing trans-switching in the host animal with endogenous constant regions such that chimeric antibodies of different isotypes are obtained in the animals.
Accordingly, in one aspect, the invention pertains to a transgene construct comprising a plurality of unrearranged human immunoglobulin (Ig) variable region sequences operatively linked to at least one immunoglobulin (Ig) constant region sequence of a non-human host animal, wherein the transgene construct undergoes rearrangement in the non-human host animal and expresses chimeric antibodies in the non-human host animal. The chimeric antibodies comprise a human variable region and a constant region of the non-human host animal. In a preferred embodiment, the plurality of unrearranged human Ig variable region sequences are heavy chain variable region sequences. Alternatively, the plurality of unrearranged human Ig variable region sequences can be light chain variable region sequences.
In a preferred embodiment, the construct comprises heavy chain variable region sequences comprising V-D-J sequences. For example, the construct can comprise, in 5′ to 3′ direction, a plurality of human VH regions, a plurality of human D segments, a plurality of human JH segments, a J-μ enhancer from a non-human host animal, a μ switch region from a non-human host animal and a μ constant region from a non-human host animal. In one embodiment, the construct comprises four human VH regions, 15 human D segments and six human JH segments. A preferred transgene construct is a 9B2 transgene construct.
As demonstrated herein, transgene constructs comprising human heavy chain V-D-J sequences linked to a μ constant region of the non-human host animal are capable of undergoing trans-switching with an endogenous constant region of the non-human host animal when the transgene construct is integrated into the genome of the non-human host animal such that chimeric antibodies of more than one isotype can be raised in the host animal. In a particularly preferred embodiment, the invention provides a transgene construct which comprises, in 5′ to 3′ direction, a plurality of human VH regions, a plurality of human D segments, a plurality of human JH segments, a mouse J-μ enhancer, a mouse μ switch region and a mouse μ constant region, wherein the transgene construct, when integrated into a mouse genome, undergoes trans-switching with an endogenous mouse γ constant region such that chimeric antibodies comprising human V regions and mouse constant regions of IgM and IgG isotype are produced in the mouse.
Although the presence of the μ constant region in the transgene construct has been shown to be sufficient for trans-switching to occur, in certain embodiments it may be preferable to include more host animal constant regions in the transgene construct itself, such that both trans-switching and cis-switching can occur to generate antibodies of different isotypes. Thus, in certain embodiments, the transgene construct can include, for example, a γ constant region from the non-human host animal or an α constant region from the non-human host animal. Alternatively, the transgene construct can comprise all Ig constant regions of the non-human host animal (i.e., the transgene construct comprises the entire constant region of the non-human host animal).
In another aspect, the invention pertains to transgenes for expressing chimeric antibodies in which the unrearranged human Ig variable region sequences are human light chain variable region sequences, such as human kappa V-J sequences, linked to light chain constant region sequences from the non-human host animal. For example, in one embodiment, the construct comprises, in 5′ to 3′ direction, a plurality of human Vκ regions, a plurality of human Jκ segments, a J-κ enhancer from a non-human host animal and a Cκ coding region from a non-human host animal.
Another aspect of the invention pertains to transgenic non-human host animals comprising one or more transgene constructs of the invention, wherein the animal expresses chimeric antibodies comprising human Ig variable regions and non-human host animal constant regions. Preferably, the transgene undergoes trans-switching and the animal expresses chimeric antibodies comprising human Ig variable regions and non-human host animal Ig constant regions of at least the IgM and IgG isotypes. Preferred non-human host animals are mice, although other animals suitable for transgenesis are also encompassed by the invention. Moreover, preferably the endogenous immunoglobulin loci are inactivated in the non-human host animal, for example by homologous recombination. In a preferred embodiment, an endogenous heavy chain locus of the transgenic host animal is inactivated by disruption of the JH region. In another preferred embodiment, an endogenous light chain locus of the transgenic host animal is inactivated by disruption of the Jκ region.
Yet another aspect of the invention pertains to a method of making a chimeric antibody specific for an antigen of interest. The method comprises immunizing a transgenic non-human host animal of the invention, which comprises a transgene construct of the invention, with the antigen of interest and obtaining from the animal a chimeric antibody specific for the antigen of interest. For example, hybridomas expressing chimeric antibodies can be prepared from the immunized host animal using standard techniques. In a preferred embodiment, the method further comprises isolating from the animal nucleic acid encoding the chimeric antibody, replacing nucleic acid encoding the non-human host animal Ig constant region with nucleic acid encoding a human Ig constant region to thereby convert the chimeric antibody to a human antibody and expressing the human antibody. In certain embodiments, the human antibody exhibits higher affinity toward the antigen of interest than the chimeric antibody.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of the 9B2 transgene construct and of the three fragments comprising the HCo26 transgene constructs aligned above the mouse genomic immunoglobulin locus.



DETAILED DESCRIPTION OF THE INVENTION
This invention involves the use of a non-human transgenic animal that expresses chimeric antibodies as a host to raise a chimeric antibody to an antigen of interest, followed by conversion of the chimeric antibody to a fully human antibody. The chimeric antibodies expressed in the transgenic non-human host animal comprise human variable regions linked to constant regions of the non-human host animal. The invention pertains to transgene constructs, non-human transgenic host animals carrying such transgene constructs and methods of using such host animals to raise chimeric antibodies, which then can further be converted to fully human antibodies.
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
As used herein, the term “chimeric antibody” refers to an antibody in which at least one of the antibody chains (heavy or light) comprises variable regions sequences from one species (e.g., human) and constant region sequences from another species (e.g., mouse). The term “chimeric antibody” is intended to encompass antibodies in which: (i) the heavy chain is chimeric but the light chain comprises V and C regions from only one species; (ii) the light chain is chimeric but the heavy chain comprises V and C regions from only one species; and (iii) both the heavy chain and the light chain are chimeric.
As used herein, the term “transgene construct” refers to a nucleic acid preparation suitable for introduction into the genome of a host animal. A “transgene construct” of the invention can comprise a single piece of nucleic acid (such as the 9B2 transgene) or multiple pieces of nucleic acid (such as the HCo26 transgene). When the transgene construct comprises multiple pieces of nucleic acid, the individual pieces making up the transgene construct preparation contain overlapping sequences such that when they are introduced into the genome of the host animal, they recombine to create a contiguous transgene (see, for example, the further description of the HCo26 transgene herein).
As used herein, the term “isotype switching” refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to another Ig class through a recombination process mediated by switch sequences.
As used herein, a “nonswitched isotype” refers to the isotypic class of the heavy chain that is produced when no isotype switching has taken place. The CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene (e.g., the IgM isotype).
As used herein, the term “switch sequence” or “switch region” refers to those DNA sequences, known in the art, that are responsible for switch recombination resulting in Ig class switching.
As used herein, the term “trans-switching” refers to isotype switching that involves recombination between one switch region and another switch region located on a different chromosome, such as recombination between a transgene switch region and an endogenous switch region located on a different chromosome than the chromosome that harbors the transgene. In particular, it refers to recombination between a transgene switch region and a switch region of the endogenous Ig constant region of the non-human transgenic host animal.
As used herein, the term “cis-switching” refers to isotype switching that involves recombination between one switch region and another switch region located on the same chromosome, such as recombination between two switch regions within a transgene or between two switch regions of the endogenous Ig locus, such as switch recombination between a μ constant region and a γ constant region carried by the same transgene or by the endogenous Ig locus.
As used herein the term “unrearranged” or “germline configuration” in reference to an immunoglobulin V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.
As used herein, the term “rearranged” in reference to an immunoglobulin V segment refers to the configuration wherein the V segment is positioned immediately adjacent to a D-J or J segment so as to encode essentially a complete VH or VL domain, respectively.
As used herein, the term “a plurality of unrearranged immunoglobulin (Ig) variable region sequences” is intended to refer to constructs that contain more than one heavy or light chain variable region segment in an unrearranged configuration.
As used herein, the term “operatively linked” is intended to describe the configuration of a nucleic acid sequence that is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the sequence. With respect to the joining of two protein coding regions, operatively linked means that the nucleic acid sequences being linked are contiguous and in reading frame. For switch sequences, operatively linked means that the sequences are capable of effecting switch recombination.
I. Transgene Constructs for Expressing Chimeric Antibodies
The transgene constructs of the invention comprise a plurality of unrearranged human immunoglobulin (Ig) variable region sequences operatively linked to at least one immunoglobulin (Ig) constant region sequence of a non-human host animal, wherein the transgene construct undergoes rearrangement in the non-human host animal and expresses chimeric antibodies in the non-human host animal, the chimeric antibodies comprising a human variable region and a constant region of the non-human host animal. In this context, the term “at least one Ig constant region sequence” is intended to mean a sequence encoding at least one constant region isotype sequence, such as an IgM constant region sequence or a kappa constant region sequence. The invention encompasses transgene constructs encoding chimeric heavy chain sequences or encoding chimeric light chain sequences. When integrated into a non-human transgenic host animal, the variable regions of the transgene constructs undergo rearrangement such that functional heavy chain or light chain variable regions are created. Moreover, in certain embodiments, the integrated transgene construct undergoes trans-switching such that chimeric antibodies of more than one isotype are made in the transgenic non-human host animal. When the transgene construct encodes chimeric heavy chain sequences, it comprises at least the IgM constant region of the non-human host animal and may contain additional host constant regions although, as demonstrated herein, the presence of the host IgM constant region alone, even without the known 3′ IgH enhancers, is sufficient to secure maturation of the B cells and achieve trans-switching.
Accordingly, in one aspect, the plurality of unrearranged human Ig variable region sequences in the transgene construct are heavy chain variable region sequences. In particular, such heavy chain constructs typically comprise unrearranged human heavy chain V-D-J sequences. Such heavy chain constructs also contain at least one Ig constant region sequence of a non-human host animal and typically also contain regulatory sequences including enhancers and switch sequences. Thus, in a preferred embodiment, the transgene construct comprises, in 5′ to 3′ direction, a plurality of human VH regions, a plurality of human D segments, a plurality of human JH segments, a J-μ enhancer from a non-human host animal, a μ switch region from a non-human host animal and a μ constant region from a non-human host animal. In a particularly preferred embodiment, the construct comprises four human VH regions, 15 human D segments and six human JH segments, as described further in the Examples. A preferred but non-limiting example of a heavy chain construct of the invention is the 9B2 transgene.
Preferably, the heavy chain construct of the invention is capable, when integrated into a non-human host animal genome, of undergoing trans-switching with an endogenous constant region of the non-human host animal. As demonstrated herein, the presence in the transgene construct of the μ switch region and the μ constant region of the non-human host animal is sufficient to allow for trans-switching to occur between the integrated transgene and endogenous constant region sequences in the host animal. Thus, a heavy chain construct of the invention that comprises only one host animal constant region sequence, μ, can nevertheless lead to the generation of chimeric antibodies of multiple isotypes (e.g., IgM and IgG) in the host animal as a result of trans-switching.
Thus, another preferred transgene construct of the invention comprises, in 5′ to 3′ direction, a plurality of human VH regions, a plurality of human D segments, a plurality of human JH segments, a mouse J-μ enhancer, a mouse μ switch region and a mouse μ constant region, wherein the transgene construct, when integrated into a mouse genome, undergoes trans-switching with an endogenous mouse γ constant region such that chimeric antibodies comprising human V regions and mouse constant regions of IgM and IgG isotype are produced in the mouse.
While the presence of the μ constant region (and associated switch sequences) alone may be sufficient for the generation of multiple isotypes of chimeric antibodies, in certain instances it may be desirable to include more than one host animal constant region sequence in the heavy chain transgene construct such that both trans-switching and cis-switching can occur. Accordingly, in various embodiments, a heavy chain transgene construct of the invention may comprise one, two, three or more constant regions of the non-human host animal. For example, in another embodiment, in addition to the μ constant region, the construct can further comprise a γ constant region (and associated switch sequences) from a non-human host animal. For example, when a mouse is used as the transgenic non-human host animal, the heavy chain transgene construct can include murine μ coding sequences (and associated switch sequences) and, additionally, can include one or more of the murine γ1, γ2a, γ2b and γ3 coding sequences (and associated switch sequences). In yet another embodiment, the construct can comprise all immunoglobulin (Ig) constant regions of the non-human host animal (i.e., the construct contains the entire C region of the non-human host animal, encompassing the μ, δ, γ, α, and ε constant sequences).
In another aspect, a transgene construct of the invention comprises a plurality of unrearranged human light chain variable region sequences, linked to a light chain constant region sequence of the non-human transgenic host animal. In particular, such light chain constructs typically comprise unrearranged human light chain V-J sequences, either kappa V-J sequences or lambda V-J sequences. In a preferred embodiment, the chimeric construct is a chimeric kappa light chain construct comprising, in 5′ to 3′ direction, a plurality of human Vκ regions, a plurality of human Jκ segments, a J-κ enhancer from a non-human host animal (e.g., the mouse κ internal enhancer) and a Cκ coding region from a non-human host animal.
In another embodiment, the chimeric light chain construct can be a lambda light chain construct. In both the mouse and human Ig loci, there is a cluster of multiple Vλ genes followed by repeated clusters of Jλ-Cλ units. Thus, in one embodiment, a chimeric lambda chain transgene can be constructed by linking a plurality of human Vλ regions to a plurality of human Jλ-non-human Cλ combination units. Alternatively, one can select a single human Jλ-non-human Cλ placed downstream of a plurality of human Vλ regions. Another possible configuration is to place a plurality of human Vλ regions upstream of the endogenous non-human Jλ-Cλ clusters, which would lead to chimeric antibodies comprising a human Vλ region linked to a non-human Jλ region and a non-human Cλ region.
The transgene constructs of the invention can be prepared using standard recombinant DNA techniques. Cloning vectors containing polylinkers are useful as starting vectors for insertion of DNA fragments of interest. Non-limiting examples of such suitable cloning vectors are described in the Examples. Plasmids or other vectors (e.g., YACs) carrying human unrearranged heavy chain or light chain immunoglobulin sequences have been described in the art (see e.g., U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; and U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963, all to Kucherlapati et al.) Such plasmids and other vectors can be used as the source of human unrearranged heavy chain or light chain variable regions to be included in the transgene constructs of the invention. Additionally or alternatively, suitable human Ig variable region DNA can be obtained from genomic libraries using standard techniques. DNA encoding constant region sequences of the non-human host animal, including the coding sequences of the constant region and the associated enhancer and switch regions, similarly can be obtained from genomic libraries using standard techniques. For example, the B6 BAC library (Invitrogen) of murine genomic DNA can be used as the source of murine constant region sequences for inclusion in the transgene constructs of the invention.
In addition to the variable and constant coding regions, cis-acting regulatory sequences typically are needed for chromatin accessibility, proper V(D)J recombination, class switching, high levels of antibody expression and other locus control functions. Promoter regions located near the V(D)J gene segments may play a role in chromatin accessibility and V(D)J recombination. Intronic enhancers between the JH and IgM coding region and Jκ and kappa coding regions have been identified. Additionally, downstream 3′ DNase hypersensitivity regions have been identified, which make up the IgH locus control region (LCR). Class switch recombination is dependent on promoters and sterile transcripts of switch regions upstream of the different heavy chain constant regions. An origin of replication has also been identified downstream of the 3′ DNase hypersensitive sites which may demark a boundary for the IgH locus. Preferably, the constructs of the invention contain at least the promoter regions located near the V(D)J gene segments, one or more operative switch regions and an intronic enhancer. Inclusion of all genomic DNA from the 5′ intronic enhancers through downstream 3′ LCRs, and possibly beyond, may be preferred (but not essential) for high levels of Ab generation, development and maturation.
The appropriate genomic DNA fragments from the human Ig variable regions and from the non-human host animal Ig constant regions are then operatively linked through ligation into a cloning vector, followed by characterization of the vector (e.g., by restriction fragment analysis or sequencing or the like) to ensure proper arrangement of the genomic fragments. A non-limiting example of the creation of a heavy chain transgene construct of the invention is described in detail in Example 1.
To prepare the transgene construct for microinjection or other technique for transgenesis, the transgene construct can be isolated from the vector in which it is carried by cleavage with appropriate restriction enzymes to release the transgene construct fragment. The fragment can be isolated using standard techniques, such as by pulse field gel electrophoresis on an agarose gel, followed by isolation of the fragment from the agarose gel, such as by β-agarase digestion or by electroelution. For example, the agarose gel slice containing the transgene construct fragment can be excised from the gel and the agarose can be digested with β-agarase (e.g., from Takara), using standard methodology.
II. Preparation and Characterization of Transgenic Non-Human Host Animals
Another aspect of the invention pertains to a transgenic non-human host animal that comprises one or more of the transgene constructs of the invention (i.e., the transgene construct(s) is integrated into the genome of the host animal), such that the animal expresses chimeric antibodies comprising human Ig variable regions and non-human host animal Ig constant regions. Preferably, the transgene construct undergoes trans-switching and the animal expresses chimeric antibodies comprising human Ig variable regions and non-human host animal Ig constant regions of at least IgM and IgG isotypes.
The transgenic non-human host animals of the invention are prepared using standard methods known in the art for introducing exogenous nucleic acid into the genome of a non-human animal, Preferred non-human animals are mice, although other animal species that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response may also be used. Examples of such species include but are not limited to rats, rabbits, chickens, goats, pigs, sheep and cows.
A preferred method for preparing the transgenic non-human animal, in particular a transgenic mouse, is that of pronuclear microinjection. This technology has been known for over twenty years and is well established (see e.g., Wagner, T. E. et al. (1981) Proc. Natl. Acad. Sci. USA 78:6376-6380; U.S. Pat. No. 4,873,191 by Wagner and Hoppe). In general, the method involves introducing exogenous genetic material into the pronucleus of a mammalian zygote (e.g., mouse zygote) by microinjection to obtain a genetically transformed zygote and then transplanting the genetically transformed zygote into a pseudopregnant female animal. The embryo is then allowed to develop to term and the genome of the resultant offspring is analyzed for the presence of the transgenic material. Southern blot analysis, PCR or other such technique for analyzing genomic DNA is used to detect the presence of a unique nucleic acid fragment that would not be present in the non-transgenic animal but would be present in the transgenic animal. Selective breeding of transgenic offspring allows for homozygosity of the transgene to be achieved.
Although the preferred embodiment of the invention comprises transgenic mice prepared by pronuclear microinjection, the invention encompasses other non-human host animals, including but not limited to rats, rabbits, pigs, goats, sheep, cows and chickens. Techniques for creating transgenic animals of each of these species have been described in the art.
For example, preparation of transgenic rats is described in Tesson, L. et al. (2005) Transgenic Res. 14:531-546, including by techniques such as DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis. Methods of transgenesis in rats are also described in Mullin, L. J. et al. (2002) Methods Mol. Biol. 180:255-270.
Preparation of transgenic rabbits is described in, for example, Fan, J. et al. (1999) Pathol. Int. 49:583-594; Fan, J. and Watanabe, T. (2000) J. Atheroscler. Thromb. 7:26-32; Bosze, Z. et al. (2003) Transgenic Res. 12:541-553.
Preparation of transgenic pigs is described in, for example, Zhou, C. Y. et al. (2002) Xenotransplantation 9:183-190; Vodicka, P. et al. (2005) Ann. N.Y. Acad. Sci. 1049:161-171. Alternative transgenesis techniques to pronuclear microinjection in pigs include adenovirus mediated introduction of DNA into pig sperm (see e.g., Farre, L. et al. (1999) Mol. Reprod. Dev. 53:149-158) and linker-based sperm-mediated gene transfer (Chang, K. et al. (2002) BMC Biotechnol. 2:5).
Preparation of transgenic goats is described in, for example, Ebert, K. M. et al. (1991) Biotechnology (NY) 9:835-838; Baldassarre, H. et al. (2004) Reprod. Fertil. Dev. 16:465-470. Somatic cell nuclear transfer in goats is described in, for example, Behboodi, E. et al. (2004) Transgenic Res. 13:215-224.
Preparation of transgenic sheep is described in, for example, Ward, K. A. and Brown, B. W. (1998) Reprod. Fertil. Dev. 10:659-665; Gou, K. M. et al. (2002) Shi Yan Sheng Wu Xue Bao 35:103-108
Preparation of transgenic cows is described in, for example, Donovan, D. M. et al. (2005) Transgenic Res. 14:563-567. Gene transfection of donor cells for nuclear transfer of bovine embryos is described in, for example, Lee S. L. et al. (2005) Mol. Reprod. Dev. 72:191-200.
The state of the art in the preparation of transgenic domestic farm animals is also reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.
Preparation of transgenic chickens is described in, for example, Pain, B. et al. (1999) Cells Tissues Organs 165:212-219; Lillico, S. G. et al. (2005) Drug Discov. Today 10:191-196. Use of retroviral vectors in the preparation of transgenic chickens is described in, for example, Ishii, Y. et al. (2004) Dev. Dyn. 229:630-642.
The transgenic non-human animals of the invention may comprise an Ig heavy chain transgene construct for expressing chimeric antibodies or an Ig light chain transgene construct for expressing chimeric antibodies, or both a light and a heavy chain construct for expressing chimeric antibodies. Typically, to create animals that carry more than one transgene, animals carrying individual transgenes are prepared and then cross-bred to create animals carrying more than one transgene. Animals that inherit both transgenes can be identified and selected by standard techniques for analysis of genomic DNA in the animals. Moreover, an animal of the invention carrying a transgene construct for expressing one chimeric Ig chain (e.g., a chimeric heavy chain) can be cross-bred with an animal that carries a transgene that expresses a non-chimeric form of the other Ig chain (e.g., a fully human light chain). In such animals, the antibodies expressed comprise one chimeric chain (e.g., a chimeric heavy chain) and one non-chimeric chain (e.g., a non-chimeric, fully human light chain). Such animals are also encompassed by the invention and suitable for use in raising chimeric antibodies against an antigen of interest. See the Examples for a further description of a transgenic animal expressing a chimeric heavy chain transgene construct and a fully human light chain transgene construct, wherein the chimeric heavy chain transgene construct undergoes trans-switching to produce antibodies of multiple isotypes (e.g., IgM and IgG) in the animals.
The non-human transgenic host animals of the invention express chimeric antibodies, in which at least one chain of the antibody comprises a human variable region sequence and a constant region sequence of the host animal. The non-human transgenic host animals of the invention do not express any fully human antibodies (comprising both a fully human heavy chain and a fully human light chain) since they do not carry both human constant region sequences (human CH and human CL) in their genome. Thus, the antibody repertoire of these animals differs from that of the HuMab Mouse® (e.g., as described in PCT Publication WO 94/25585). Although the Ig transgenes in the HuMab Mouse® are described in WO 94/25585 as being capable of undergoing trans-switching with endogenous mouse Ig constant regions to create chimeric antibodies, the Ig transgenes in the HuMab Mouse® contain human variable region sequences and human constant region sequences and thus, in addition to possibly expressing chimeric antibodies, these mice also express a repertoire of fully human antibodies. In contrast, the transgenic non-human host animals of the present invention only express a repertoire of chimeric antibodies, without the additional presence of fully human antibodies in the animals.
Furthermore, the presence of constant regions of the non-human host animal in the chimeric antibodies expressed by the host animals is thought to allow for improved B cell and antibody development in vivo, as compared to transgenic animals that express antibodies having constant regions of another species (e.g., human constant regions). For example, the chimeric antibodies that contain host animal Fc regions are thought to associate better with endogenous host animal accessory proteins (e.g., Igα/Igβ or other signaling molecules) for more natural receptor signaling leading to more normal B cell development and higher antibody production. Moreover, the chimeric antibodies are thought to bind better to host Fc receptors, leading to increased recirculation and antigen presentation and, thus, more normal immune responses. Still further, the presence in the transgene construct of host animal regulatory sequences in the host animal-derived constant region is thought to lead to improved genetic regulation of antibody expression. Thus, such an environment in which there is more normal B cell development, more serum antibodies, improved genetic regulation of antibody expression and better germinal center formation should lead to a more diverse, and possibly higher affinity, antibody population with appropriate somatic mutations.
Thus, in certain embodiments, chimeric antibodies raised in the non-human transgenic host animals of the invention may exhibit increased somatic mutations as compared to fully human antibodies raised in non-human transgenic host animals. The presence of a constant region of the non-human host animal in the chimeric antibodies of the invention can also afford the advantage that the antibody may possess additional effector functions (e.g., ADCC, complement fixation) in the host animal species that would not be present with antibodies having human constant regions. Thus, a chimeric antibody raised according to the invention may be amenable for use in particular animal models of disease in which a fully human antibody may not be suitable for use. For example, chimeric antibodies raised according to the invention that comprise murine constant regions may be amenable for use in mouse models of disease that involve murine effector functions mediated by the murine Fc region.
In a preferred embodiment, a non-human transgenic host animal of the invention has one or more of its endogenous immunoglobulin loci inactivated. It is preferable that the endogenous Ig loci be inactivated so that endogenous host animal antibodies are not expressed in the animal together with the expression of the chimeric antibodies. In particular, inactivation of the endogenous Ig loci prevents interference from endogenous antibodies and simplifies the detection of the chimeric antibodies. Accordingly, in one embodiment, a transgenic host animal (e.g., mouse) of the invention has at least one endogenous heavy chain locus inactivated and more preferably has both endogenous heavy chain loci inactivated. Additionally or alternatively, the transgenic host animal (e.g., mouse) of the invention has at least one endogenous light chain locus inactivated and more preferably has both alleles of the kappa loci or the lambda loci, or both, inactivated. In the most preferred embodiment, the host animal has a homozygous inactivation of the endogenous Ig heavy chain locus and a homozygous inactivation of the endogenous Ig kappa light chain locus. When the kappa locus is inactivated, it is not essential to disrupt the lambda locus; however, if a chimeric lambda light chain transgene is to be used, it is desirable to inactivate the endogenous lambda locus.
The endogenous Ig loci are preferably inactivated by homologous recombination using a targeting vector that inserts an exogenous sequence into the endogenous Ig locus such that expression of the endogenous Ig genes is disrupted. Preferred regions for insertion are the JH and Jκ regions. Once a host animal having a heterozygous disruption of a heavy chain or light chain locus is obtained, the animal can be bred to homozygosity for the disruption by standard breeding techniques. A mouse strain having a homozygous disruption of the endogenous heavy chain locus at JH has been previously described in the art (see e.g., Examples 10 and 11 of U.S. Pat. No. 5,545,806), as has a mouse strain having a homozygous disruption of the endogenous kappa light chain locus at Jκ and Cκ (see e.g., Example 9 of U.S. Pat. No. 5,545,806). Such mice can be used as breeding partners with mice carrying one or more transgenes of the invention to achieve a mouse strain carrying one or more transgenes and having its endogenous Ig loci inactivated. Such mouse strains are described in further detail in the Examples.
III. Preparation of Chimeric Antibodies in Transgenic Non-Human Animals
Another aspect of the invention pertains to method of making a chimeric antibody specific for an antigen of interest. The method comprises immunizing a transgenic non-human host animal of the invention with the antigen of interest and obtaining from the animal a chimeric antibody specific for the antigen of interest.
Thus, to prepare chimeric antibodies in a transgenic non-human animal of the invention, first the animal is immunized with an antigen of interest. For example, immunization techniques that have previously been used to raise fully human antibodies in transgenic mice carrying human Ig heavy and light chain transgenes (such as the HuMab Mouse® or the Xenomouse) can similarly be used to raise antibodies in the animals of the invention. Immunization techniques previously used in the HuMab Mouse® are described in, for example, Lonberg, N. et al. (1994) Nature 368:856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851 and PCT Publications WO 98/24884 and WO 01/14424. Preferably, mice are 6-16 weeks of age upon the first infusion of antigen and a purified or recombinant preparation of antigen (e.g., 5-50 μg) is used to immunize the mice intraperitoneally. Typically, multiple animals (e.g., between 6 and 24 animals) are immunized for each antigen.
Cumulative experience with various antigens in the HuMab Mouse® have shown that the transgenic mice respond well when initially immunized intraperitoneally with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (up to a total of 6) with antigen in incomplete Freund's adjuvant. However, adjuvants other than Freund's have also been found to be effective and can be used additionally or alternatively. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by ELISA and mice with sufficient titers against the antigen of interest can be used to prepare monoclonal antibodies. Mice can be boosted intravenously with antigen three days before sacrifice and removal of the spleen and/or lymph nodes.
Chimeric antibodies, as a polyclonal mixture, can be obtained from the host animal or, more preferably, monoclonal antibodies can be prepared using B cells obtained from the host animal. Monoclonal antibodies can be prepared and selected by one of a variety of suitable methods known in the art including, but not limited to, (i) hybridoma generation (discussed further below), (ii) PCR amplification of antibody genes directly from B cells of obtained from the host animal (see e.g., Babcook, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848) and (iii) phage display of an antibody library prepared from B cells of the host animal, followed by screening of the phage display library for a monoclonal antibody of interest (see e.g., PCT Publication WO 01/25492)
In a preferred embodiment, hybridomas producing chimeric monoclonal antibodies of the invention are generated. To generate such hybridomas, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line (e.g., P3X63-Ag8.653 (ATCC, CRL-1580) or SP2/0 (ATCC, CRL-1581)). Cells can be fused using techniques well established in the art, such as chemically-mediated fusion (e.g., with PEG) or electrofusion. Once extensive hybridoma growth has occurred, individual wells can be screened by ELISA For example, in animals expressing a chimeric Ig heavy chain transgene and a fully human kappa light chain transgene, wells can be screened by ELISA for expression of antibodies comprising mouse IgM and human kappa or mouse IgG and human kappa. Using similar techniques, the supernatants can be tested for the presence of mouse IgG that binds to the antigen used for immunization. Hybridomas positive for an antibody of interest can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody material in tissue culture medium for further characterization.
IV. Conversion of Chimeric Antibodies to Fully Human Antibodies
In the methods of the invention, once a chimeric antibody of interest has been raised in the transgenic non-human host animal, the method can further comprise isolating from the animal, or a B cell from the animal, a nucleic acid encoding the chimeric antibody and replacing nucleic acid encoding the non-human host animal Ig constant region with nucleic acid encoding a human Ig constant region to thereby convert the chimeric antibody to a human antibody and expressing the human antibody.
Thus, once a chimeric antibody of interest has been identified, it can be converted to a fully human antibody using standard recombinant DNA techniques. For example, DNA encoding the variable region (light or heavy chain) from the chimeric antibody can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNA can be inserted into an expression vector such that the variable region sequences are operatively linked to a human constant region sequence, as well as to transcriptional and translational control sequences. An antibody heavy chain gene and an antibody light chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.
The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). An expression vector can be used that already encodes heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. A non-limiting examples of suitable expression vectors for expressing fully human antibodies the pIE family of vectors as described in U.S. Patent Application No. 20050153394 by Black. Preferred constant region isotypes present in the expression vectors include human IgG1 and IgG4 constant regions for the heavy chain and the human kappa constant region for the light chain.
In the context of antibody expression vectors, the term “operatively linked” is intended to mean that an antibody variable region is ligated into the expression vector such that the coding sequences of the variable region are in-frame with the coding sequences of the constant region. Moreover, the variable and constant regions are positioned within the vector such that the transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Additionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
In addition to the antibody chain genes, the recombinant expression vectors carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or βglobin promoter. Still further, regulatory elements can be composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al (1988) Mol. Cell. Biol. 8:466-472).
In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
Other sequences that can be included in the expression vector include those that enhance expression of the antibody genes in stable transfectants, such as sequences that alter chromatin structure to prevent silencing of the transfected gene. A preferred example is a UCOE (ubiquitous chromatin opening element), which can enhance expression of transfected sequences irrespective of their site of integration in a stable transfectant.
For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).
Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
The conversion of a chimeric antibody of the invention to a fully human antibody is described in further detail in Example 6. As demonstrated in that example, conversion of the chimeric form to the fully human form maintained the binding properties of the antibody toward its target antigen. In certain instances (such as in Example 6), the fully human form of the antibody may even exhibit improved binding toward its target, such as higher affinity toward its target than the chimeric form. Thus, in certain embodiments of the method for converting the chimeric antibody to a human antibody, the resultant human antibody exhibits higher affinity toward the antigen of interest than the original chimeric antibody.
EXAMPLES
In the following examples, certain plasmids, homologous recombinant mice and transgenic mice that have been previously described were used as starting materials to create additional transgenes and transgenic mouse strains.
Plasmids and DNA Clones:
The pGP1 and pGP2 are general cloning vectors whose construction is described in U.S. Pat. No. 5,545,806 (see in particular Example 4 and FIGS. 7 and 8). Both are pBR322-derived plasmids that have been modified to contain large polylinkers. In pGP1, the polylinker is flanked by rare cutting NotI sites for building large inserts. pGP2 was derived from pGP1 and includes an additional SfiI site located between the MluI and SpeI sites in the polylinker. pGP1b and pGP2b are similar pBR322-derived cloning vectors. The construction of pGP1b also is described in U.S. Pat. No. 5,545,806 (see, in particular, Example 12), whereas the construction of pGP2b is described in U.S. Pat. No. 5,625,126 (see, in particular, Example 36 and FIGS. 77A and 77B).
The pHC2 plasmid also is described in U.S. Pat. No. 5,545,806 (see in particular Example 12 and FIGS. 25 and 31). pHC2 contains four functional human immunoglobulin heavy chain variable (VH) regions, 15 human D segments, all six human JH segments, the human J-μ enhancer, human μ switch region, all of the human μ coding exons, and the human γ1 constant region, including the associated switch region and sterile transcript associated exons, together with 4 kb flanking sequencing upstream of the sterile transcript initiation site contained on a NotI fragment.
The RP23-109B20 (B20) genomic C57BL/6J mouse DNA clone was obtained from Invitrogen and contains mouse JH regions through mouse IgG2a coding regions. The RP23-116C22 (C22) genomic C57BL/6J mouse DNA clone was also obtained from Invitrogen and contains mouse IgG2b coding regions and approximately 200 kb downstream of the mouse IgG2b coding regions.
Homologous Recombinant Mice:
Mice in which the endogenous immunoglobulin heavy chain locus and/or the endogenous immunoglobulin light chain locus have been disrupted by homologous recombination have been previously described. In these examples, mice were used in which: (i) the murine light chain JK regions were deleted and replaced with the neoR gene (referred to herein as the “JKD” genotype); (ii) the murine heavy chain Cμ region was disrupted by insertion of the neoR gene in the opposite reading frame of the Cμ gene (referred to herein as the “CMD” genotype); and/or (iii) the murine heavy chain JH region was deleted and replaced with the neoR gene (referred to herein as the “JHD” genotype). Construction of mice carrying the JHD modification is described in Examples 10 and 11 of U.S. Pat. No. 5,545,806. Construction of mice carrying the CMD modification is described in Example 1 of U.S. Application No. 20020086014. Construction of mice carrying the JKD modification is described in Example 9 of U.S. Pat. No. 5,545,806.
Transgenic Mice:
Mice carrying an unrearranged human light chain immunoglobulin transgene, comprising multiple Vκ regions, Jκ regions and the entire human kappa constant region, have been described previously. In these examples, mice were used that carry the KCo5 light chain transgene, which was created by co-injection of a human kappa light chain minilocus and a YAC clone comprising multiple human Vκ segments. Construction and characterization of mice strains carrying the KCo5 light chain transgene are described in detail in Example 38 of U.S. Pat. No. 6,255,458. Through breeding with the homologous recombinant mice strains described above, additional mice strains were obtained in which (i) the KCo5 transgene was present (KCo5+); (ii) the endogenous light chain gene had been disrupted (JKD+/+) and (iii) the endogenous heavy chain gene had been disrupted (either JHD+/+ or CMD+/+).
Example 1
Construction of a Chimeric Transgene for Expressing Chimeric Antibodies Comprising Human VH and Mouse CH Regions
A transgene construct was prepared that contained unrearranged human heavy chain VDJ segments linked to the mouse Jμ enhancer region, μ switch region and μ coding region, as follows.
Intermediate Vectors
The intermediate vector pGP2-1 was constructed by digesting the previously described pGP2b with NotI, releasing the polylinker and ligating with a synthetic linker constructed by annealed overlapping oligos DMTM12 (5′-GGCCGCACGCGTGTCGACTC-3′) (SEQ ID NO: 1) and DMTM13 (5′-GCCGAGTCGACACGCGTGC-3′) (SEQ ID NO: 2). The resulting pGP2-1 plasmid then has a polylinker with a NotI site followed by a MluI, and a Sail site. The orientation and linker sequence were confirmed by sequencing.
The intermediate vector pIM-m2 was constructed by digesting the previously described pGP1b with NotI, releasing the polylinker and ligating with a synthetic linker constructed by annealed overlapping oligos DMTM39 (5′-GGCCGCATTCGCCGGCTAACGGCGCCTA TAACGAGTTC-3′) (SEQ ID NO: 3) and DMTM40 (5′-GGCCGAACGGCTTATAGGCGCCG TTAGCCGGCGAATGC-3′) (SEQ ID NO: 4). The resulting pIM-m2 plasmid then has a polylinker with a NotI site followed by a NgoMIV site, a Nan site, a MluI site.
The intermediate vector pIM-m3 was constructed by digesting the previously described pGP1b with XhoI and HindIII, releasing part of the polylinker and ligating with a synthetic linker constructed by annealed overlapping oligos DMTM37 (5′-TCGAGGCCGGCATGATAG GCGCCGTCGACA-3′) (SEQ ID NO: 5) and DMTM38 (5′-AGCTTGTCGACGGCGCCTA TCATGCCGGCC-3′) (SEQ ID NO: 6). The resulting pIM-m3 plasmid then has a polylinker with these sites: NotI-XhoI-NgoMIV-NarI-SalI-HindIII-NotI.
Construction of Chimeric Transgene
pHC2 has a unique MluI restriction site located downstream of the most 3′ human JH segment and 5′ of the human J-μ enhancer. The approximately 44 kb NotI-MluI fragment from pHC2, containing four functional human variable regions, 15 human D segments and all six human JH segments, was isolated and cloned into the intermediate vector pGP2-1. The new plasmid (phVDJ2) was screened by observing an approximate 44 kb fragment released by NotI and MluI digestion and southern blot hybridization to a probe just 5′ of the human J-μ enhancer.
To isolate the mouse J-μ enhancer, mouse μ switch region, all of the mouse μ coding regions and the mouse δ coding regions, the B20 BAC was digested with NgoMIV and NarI. The resulting 40 kb fragment was isolated by pulse field gel electrophoresis (PFGE) and cloned into pIM-m2. The resulting plasmid (pIM-m2-mED) was screened by the appearance of a PCR fragment using primers specific for mouse IgM and by observing an approximate 40 kb fragment released by NgoMIV and Nan digestion. Furthermore, the mouse μ switch region was checked by southern blot to be full length (as compared to the starting B20 BAC) as part of this region often becomes deleted.
To isolate the mouse J-μ enhancer, mouse μ switch region, all of the mouse μ coding regions, pIM-m2-mED was digested with XhoI (located 3′ of the mouse IgM coding region) and NarI and ligated with a synthetic linker constructed by annealed overlapping oligos DMTM72 (5′-TCGACTCCGCGGTTTAAACTGG-3′) (SEQ ID NO: 7) and DMTM73 (5′-GGCGCC AGTTTAAACCGCGGAG-3′) (SEQ ID NO: 8). The new resulting plasmid (pIM-m2-mEM) contained the mouse J-μ enhancer, mouse μ switch region, all of the mouse μ coding regions on an approximate 13 kb NgoMIV-NarI fragment with no internal XhoI or SalI sites. pIM-m2-mEM was screened by the appearance of a PCR fragment using DMTM73 and DMTM76, which is specific for a region 5′ of the XhoI site and points downstream towards the newly inserted linker.
The approximate 13 kb NgoMIV-NarI fragment, containing the mouse J-μ enhancer, mouse μ switch region and all of the mouse μ coding regions, was then cloned into pIM-m3 to create pIM-m3-mEM, which adds a 5′ XhoI site and a 3′ SalI site to the fragment. pIM-m3-mEM was screened by observing an 13 kb fragment released by NgoMIV and NarI digestion and by XhoI and SalI digestion.
The final phVDJ2-mEM construct was then constructed by ligating the 13 kb XhoI-SalI fragment, containing the mouse J-μ enhancer, mouse μ switch region and all of the mouse μ coding regions, from pIM-m3-mEM into the SalI site 3′ of the human VDJ region in phVDJ2. Clones were checked for directional cloning by the production of a PRC product from DMTM79 (5′-GCTGGAAAGAGAACTGTCGGAGTGGG-3′) (SEQ ID NO: 9), which anneals just downstream of the human JH region pointing downstream, and DMTM80 (5′-CCAAAGTCCC TATCCCATCATCCAGGG-3′) (SEQ ID NO: 10), which anneals to the mouse J-μ enhancer and points upstream. Furthermore, the final clone of phVDJ2-mEM (called 9B2, illustrated schematically in FIG. 1) was checked by southern blot to contain the full length mouse μ switch region (as compared to the starting B20 BAC). The final construct thus contains the human VDJ regions of pHC2 upstream of the mouse J-μ enhancer, mouse μ switch region, and all of the mouse μ coding regions on an approximately 57 kb fragment.
Example 2
Preparation and Screening of Transgenic Mice
The approximately 57 kb NotI-SalI fragment from clone 9B2 of phVDJ-mEM (described in Example 1) was released from the vector and isolated by PFGE. An agarose gel slice with the 9B2 insert was excised and the agarose was digested with β-agarase (Takara) according to the manufacturer's protocol. The 9B2 fragment was micro-injected (by standard methods) into fertilized oocytes. DNA was injected into F1 mice of wild type (JHD−/− CMD−/−, KCo5−)×KCo5 mice (JHD−/−, CMD+/+, JKD+/+, KCo5+/+). Potential founder mice were screened for the 9B2 transgene by PCR with DMTM79 and DMTM80 using tail DNA as template. There were 4 resulting founder mice (9B2-52, 9B2-56, 9B2-58, and 9B2-65) on the JHD−/−, CMD+/−, JKD+/−, KCo5+/− strain background.
Since mice of the CMD+/− genotype still carry the endogenous mouse heavy chain J region, it was desirable to breed these founder mice with mice carrying the JHD deletion to obtain mice of the genotype CMD−/−, JHD+/+. Thus, founder mice positive for the 9B2 transgene were then bred to JHD (KCo5) mice (JHD+/+, CMD−/−, JKD+/+, KCo5+/+ mice) and genotyped for 9B2, JHD, CMD, JKD and KCo5.
To generate 9B2 transgenic animals on the appropriate strain background: (9B2+, JHD+/+ (CMD−/−), JKD+/+, KCo5+), founder offspring were selected in part by their genotype (how similar they were to the final desired strain configuration) and if they were shown to have any pre-immune mouse IgG, or seemingly elevated post immunization mouse IgG levels. Offspring that carried either JHD+/+ (knock out for mouse JH region) or JHD+/− and CMD+/− (functionally knocked out for mouse heavy chain production) on a JKD+/+ and KCo5+ background were pre-immune titered for total mouse IgG/human kappa and mouse IgM/human kappa. The mice were then challenged with Tetanus Toxiod (TT) at 1-2 week intervals, with 50 μg of TT and 25 μg of Keyhole Limpet Hemocyanin (KLH) in 100 μl total volume of RIBI adjuvant, and titered for mouse IgG/human kappa, mouse IgM/human kappa and TT specific mouse IgG levels 10 days post the final immunization. Breeding continued for each founder line in this manner until 9B2+, JHD+/+ (CMD−/−) JKD+/+ strains were achieved.
Four founder lines carrying the 9B2 transgene were achieved, 9B2-52, 9B2-56, 9B2-58 and 9B2-65. Each 9B2 founder line transmits the transgene in a Mendelian manner and the transgene is not linked to the sex chromosomes, or any obvious physical coat coloration genes.
Example 3
Characterization of Transgenic Mice Expressing Chimeric Antibodies
In this example, the antibody responses of the four transgenic mice founder lines described in Example 2 to tetanus toxoid (TT) and Interferon-α (IFN-α) were examined to identify mice expressing high levels of antibodies in which a human kappa light chain was paired with a heavy chain having a mouse IgG constant region, indicating that the heavy chain used in the antibody was derived from the transgene construct that had undergone rearrangement and trans-switching with the endogenous mouse IgG constant region.
Tetanus Toxoid Responses
The antibody responses to TT for the four 9B2 mouse lines were examined as follows. Five or six mice of each strain, consisting of four to five transgenic positive mice and at least one non-transgenic (ntg) mouse for use as a negative control, were challenged weekly with TT at 50 μg of TT in 100 μl total volume of RIBI adjuvant for four weeks. Sera were titered for mouse IgG/human kappa, mouse IgM/human kappa and TT-specific mouse IgG levels 10 days after the final immunization. Other controls were the HC2 HuMab strain (carrying a fully human heavy chain transgene, pHC2 where the human VH, D and JH gene segments are the same as in the 9B2, and a fully human light chain transgene) and the B6 wild-type mouse strain.
Table 1 below summarizes the titer levels for each mouse within the cohort, along with the non-transgenic controls, the HC2 strain and the B6 strain. The results shown for the 9B2 strains are the pre-immunization (naive) serum levels of total mouse IgG/human kappa antibodies in μg/ml (column 3), the preimmune levels of total mouse IgM/human kappa antibodies in μg/ml (column 4), the post-immunization serum levels of total mouse IgG/human kappa antibodies in μg/ml (column 5), the post-immunization serum levels of total mouse IgM/human kappa antibodies at lowest titer dilution at 3× background (column6) and TT specific/mouse gamma antibodies in μg/ml (column 7). The appropriate analogous results are also shown for the HC2 and B6 strains.







TABLE 1





Serum Titers of TT Immunized Transgenic Mice

















post 4 IMS












preimmune

hK/mM















Mouse
mG/hK
hK/mM
mG/hK
(titer-
TT/mG


Line
ID#
(μg/ml)
(μg/ml)
(μg/ml)
3xbkgd)
(μg/ml)





9B2-52
98683
5.2
14.5
21.3
1620
7.7



98684
0.6
9.8
2.6
1620
0.3



98929
1.9
54.3
40.5
14580
34.6



98930
1.4
21.5
16.1
4860
0.0



98931
0.0
17.0
0.2
4860
0.0



99746-
0.0
0.0
0.0
0
0.0



ntg


9B2-56
92986
0.2
317.7
0.5
43740
0.0



93382
0.4
199.3
1.3
43740
0.0



98693
1.6
164.5
2.4
43740
0.3



98694
0.4
183.7
0.2
43740
0.2



98695
0.2
138.4
0.5
14580
24.3



98697-
0.0
0.5
0.0
0
0.0



ntg


9B2-58
96339
1.9
103.9
56.8
14580
48.1



94744
0.6
94.9
6.9
14580
13.6



96042
0.9
230.0
15.0
43740
4.6



97942
1.7
194.9
93.2
43740
24.2



97944
2.5
137.4
163.9
43740
67.7



97945-
0.0
0.0
0.0
0
0.0



ntg


9B2-65
99770
1.1
11.8
3.5
4860
0.0



99771
6.0
11.8
21.2
4860
0.5



99775
18.9
18.7
36.0
60
0.3



99777
2.3
21.7
5.6
1620
0.0



99776
0.1
0.0
0.0
0
0.0



99778-
0.2
0.0
0.0
0
0.0



ntg







hG/hk
hk/hM
hG/hk
hk/hM
TT/hG




(μg/ml)
(titer)
(μg/ml)
(titer)
(μg/ml)





HC2
98679
100.5
1620
56.9
1920
0.2



98680
31.4
1620
57.1
4860
2.4



98681
23.6
1620
84.5
4860
0.6







mG/mK
mK/mM
mG/mK
mK/mM
TT/mG




(titer)
(titer)
(titer)
(titer)
(μg/ml)





B6
99898
180
43740
14580
43740
72712.1



99899
180
43740
14580
43740
147832.0



99900
180
43740
14580
43740
38433.8









As expected, non-transgenic mice from each line do not express any mouse IgM/human kappa or mouse IgG/human kappa antibodies pre or post immunization. In the transgenic mice, expression of mouse IgM/human kappa antibodies is believed to be a result of rearrangement of the transgenic human VDJ segments to form a functional V region and splicing to the downstream transgenic mouse IgM constant region. Furthermore, in the transgenic mice, mouse IgG/human kappa antibodies are believed to be trans-switched antibodies containing rearranged transgenic human VDJ regions trans-switched to endogenous mouse IgG constant regions.
As the results in Table 1 demonstrate, lines 9B2-52 and 9B2-56 have low levels of naïve mouse IgG (trans-switched antibodies) and mouse IgM (derived from the transgene), whereas lines 9B2-58 and 9B2-65 contained higher levels of mouse IgG antibodies in naïve serum. Furthermore, lines 9B2-58 and 9B2-65 had elevated serum titers of mouse IgG/human kappa antibodies after immunization. All the mice tested for line 9B2-58 expressed TT-specific mouse IgG, whereas the results were more variable for the other lines, with some of the tested mice expressing TT specific mouse IgG post immunization and others not. The HC2 HuMab strain also showed variability in the TT-specific responses of individual mice. In some mice, levels of TT specific mouse IgG in sera were higher than levels of total mouse IgG/human kappa in sera. It is thought that this may represent sera containing TT specific mouse IgG paired with an endogenous mouse lambda light chain.
Splenocytes from one TT immunized 9B2-58 mouse were fused via electrofusion and TT specific hybridomas were produced. The hybridomas were stable and 12 hybridomas making anti-TT antibodies were initially isolated. Further cDNA sequence, protein characterization and BIACORE analysis were performed on antibodies from nine of the hybridomas. Supernatants from the hybridomas were used in a BIACORE experiment to determine affinity to TT, as well as on-rates and off-rates. For comparison, a fully human anti-TT antibody (raised in a HuMab Mouse® and expressed in CHO cells) was used (referred to as TT hu IgG), as well as a recombinantly-produced chimeric mouse IgG/human kappa antibody (expressed in CHO cells) which contains the same human VDJ and human kappa chain as the TT hu IgG. Results of the affinity, on-rate and off-rate comparison are shown in Table 2 below, Hybridoma clone names are in column 1 while BIACORE data are in columns 2-4.







TABLE 2







BIACORE Analysis of Antibodies from TT Immunized Transgenic Mice













Affinity
On-rate
Off-rate



Clone Name
(nM)
(1/Ms) × 104
(1/s) × 104
















43H7C10
22.4
3.9
8.8



43C4E8
23.1
4
9.2



41A7B2
29
3.8
1.1



7G7A9
141
3.3
46



14D6G4
100
3.4
34



24F10A8
122
3.3
40



40G8F2
134
3.2
43



50C1G4
139
2.9
40



49A1A6
293
0.18
5.4



hu anti-TT
52
1.9
9.7



chi anti-TT
82
2.1
17










This data demonstrates that the chimeric anti-TT antibodies raised in the transgenic mice have comparable affinities, on-rates and off-rates as the fully human anti-TT antibody and the recombinantly-created chimeric antibody made from the fully human anti-TT antibody. In fact, several of the chimeric antibodies raised in the 9B2-58 mouse have higher affinities than the one fully human anti-TT antibody studied.
IFN-α Responses
To examine the antibody responses of the transgenic mice to IFN-α, six to thirteen mice (five to eleven transgenic mice from each strain, and at least one non-transgenic mouse for use as a negative control) were challenged weekly with 20 μg of IFN-α in 100 μl total volume of RIBI adjuvant. Serum was titered for mouse IgG/human kappa, mouse IgM/human kappa and IFN-α-specific mouse IgG levels 10 days post four and seven immunizations.
Table 3 below summarizes the titer levels for each mouse within the cohort, along with the non-transgenic controls. The results shown are the pre-immunization (naive) serum levels of total mouse IgG/human kappa antibodies in μg/ml (column 3), the preimmune levels of total mouse IgM/human kappa antibodies in μg/ml (column 4), the post-4 immunization serum levels of total mouse IgG/human kappa antibodies in μg/ml (column 5), the post-4 immunization serum levels of total mouse IgM/human kappa antibodies in μg/ml (column 6), IFN-α specific/mouse gamma antibodies at lowest titer dilution at 3× background (column7), the post-7 immunization serum levels of total mouse IgG/human kappa antibodies in μg/ml (column 8), the post-7 immunization serum levels of total mouse IgM/human kappa antibodies in μg/ml (column 9) and IFN-α specific/mouse gamma antibodies at lowest titer dilution at 3× background (column10).







TABLE 3







Serum Titers of IFN-α Immunized Transgenic Mice











Preimmune
post 4 IM
post 7IM


















Total
Total
Total
Total
INFA/mG
Total
Total
INFA/mG




mG/hK
hK/mM
mG/hK
hK/mM
(titer-
mG/hK
hK/mM
(titer-


line
mouse
(μg/ml)
(μg/ml)
(μg/ml)
(μg/ml)
3Xbkgd)
(μg/ml)
(μg/ml)
3Xbkgd)



















9B2-
82441
1.6
107.4
4.2
130.3
60
11.2
117.7
0


52
82444
1.0
64.8
DM



84028
1.6
107.7
7.7
106.7
540
8.2
47.6
0



84029
1.4
77.5
1.1
51.9
20
1.2
40.3
0



84184
6.8
25.1
35.8
54.7
1620
22.8
49.9
1620



84030-
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0



ntg


9B2-
87314
0.6
174.4
1.9
111.9
540.0
2.6
268.7
0.0


56
87315
0.0
139.2
0.2
80.1
180.0
0.2
238.1
0.0



87316
0.1
519.9
0.2
164.7
180.0
0.1
559.0
0.0



87320
0.0
162.2
0.2
169.6
0.0
0.3
344.0
0.0



87321
0.9
165.3
4.1
385.1
0.0
0.2
720.6
0.0



89425-
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0



ntg


9B2-
78744
2.9
64.9
66.7
264.5
4860.0
178.4
47.4
43740.0


58
78751
0.9
170.6
96.2
326.9
4860.0
4.5
126.1
4860.0



78754
5.3
132.6
9.6
381.4
1620.0
181.6
494.5
14580.0



81198
5.8
94.2
24.1
243.2
4860.0
16.9
168.4
43740.0



80620
7.8
169.7
27.5
360.8
14580.0
444.5
1043.1
43740.0



80623
2.8
139.4
17.2
368.3
4860.0
86.4
166.5
14580.0



80624
9.8
133.6
323.6
380.0
43740.0
1694.6
45.9
43740.0



80460
5.8
89.5
24.0
0.8
4860.0
DM



80461
3.1
217.2
12.8
385.6
4860.0
45.6
679.7
14580.0



80468
9.3
62.7
40.1
151.1
1620.0
176.1
115.5
43740.0



80473
1.8
102.7
16.5
159.5
1620.0
38.3
368.7
43740.0



78752-
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0



ntg



80621-
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0



ntg














9B2-
97143
1.0
12.1
5.1
5.8
0.0
not done


65
97955
263.2
19.5
68.7
14.1
0.0



97957
27.7
15.4
72.8
12.5
0.0



97959
2899.0
19.9
1878.0
15.8
50.0



97961
136.3
29.1
32.8
18.1
100.0



97144-
0.0
0.0
0.0
0.0
0



ntg





DM = dead mouse






Again as expected, non-transgenic (ntg) mice from each line do not express any mouse IgM/human kappa or mouse IgG/human kappa antibodies pre or post immunization. In the IFN-α immunized transgenic mice, expression of mouse IgM/human kappa antibodies is believed to be a result of rearrangement of the transgenic human VDJ segments to form a functional V region and splicing to the downstream transgenic mouse IgM constant region. Furthermore, in the transgenic mice, mouse IgG/human kappa antibodies are believed to be trans-switched antibodies containing rearranged transgenic human VDJ regions trans-switched to endogenous mouse IgG constant regions.
Similar to the results for the TT responses, lines 9B2-52 and 9B2-56 have low levels of naïve mouse IgG (trans-switched antibodies) and mouse IgM (derived from the transgene), whereas lines 9B2-58 and 9B2-65 contained higher levels of mouse IgG antibodies in naïve serum. Furthermore, lines 9B2-58 and 9B2-65 had elevated serum titers of mouse IgG/human kappa after four immunizations. Again, in general, all mice of line 9B2-58 expressed high IFN-α specific mouse IgG, while only some mice of the others lines expressed IFN-α specific mouse IgG post immunization.
Example 4
Analysis of Somatic Mutations in Chimeric Antibodies
In this example, the number of somatic mutations that occurred in anti-TT chimeric antibodies from the 9B2-58 transgenic mouse strain was analyzed, as well as the number of somatic mutations that occurred in chimeric antibodies from HuMab mice (which express both chimeric and fully human antibodies).
9B2-58 Transgenic Mice
Based on cDNA sequencing, the antibodies made by the anti-TT hybridomas generated from immunization of 9B2-58 transgenic mice, as described in detail in Example 3 above, were further characterized for their VH and JH usage and for which heavy chain isotype was present. Additionally, the number of somatic mutations occurring at the DNA level and at the amino acid level was determined for the VH segment only (not the D or JH segments). The results are summarized in Table 4 below:







TABLE 4







Sequence Analysis of Antibodies from TT Immunized 9B2 Mice

















SM
SM
HC



Clone Name
VH
JH
(DNA)
(AA)
Isotype


















43H7C10*
3-30.3
4
15
9
mIgG2b



43C4E8*
3-30.3
4
18
8
mIgG2b



41A7B2*
3-30.3
4
15
8
mIgG2b



7G7A9**
3-30.3
4
5
3
mIgG2b



14D6G4**
3-30.3
4
5
3
mIgG2b



24F10A8**
3-30.3
4
5
3
mIgG2b



40G8F2**
3-30.3
4
5
3
mIgG2b



50C1G4
4-34
4
17
7
mIgG1



49A1A6
4-34
4
17
7
mIgG1



hu anti-TT
3-33
4b
13
10
h IgG1



chi anti-TT
3-33
4b
13
10
mIgG2a







SM = somatic mutations;



HC = heavy chain



*indicates antibodies determined to share the same light chain



**indicates antibodies determined to share the same light and heavy chains






With regard to VH region usage, it should be noted that although the 9B2-58 transgenic strain comprises different VH regions than those in the HuMab mouse used to raise the human anti-TT antibody, the 3-30.3 and 3-33 VH regions are similar, differing by only 2 amino acids. Thus, for several of the hybridomas, TT immunization selected similar VH regions in the 9B2-58 transgenic strain as the VH region used in a human anti-TT antibody raised in the HuMab mouse.
With regard to heavy chain isotype determination, since the transgene inserted into the 9B2-58 strain contains only the mouse IgM constant region, all of the chimeric antibodies that contain a human variable region and a mouse IgG2b or IgG1, as observed herein, were produced through trans-switching from the transgene to various endogenous mouse constant regions.
With regard to somatic mutations, at the DNA level, several of the antibodies from the 9B2-58 transgenic strain displayed greater numbers of somatic mutations than the human anti-TT antibody from the HuMab mouse. Moreover, chimeric antibodies with higher affinities for TT also contained more somatic mutations than other anti-TT antibodies identified from the same transgenic mouse. Although at the amino acid level, the chimeric antibodies from the 9B2-58 transgenic strain did not exhibit more somatic mutations than the human anti-TT antibody from the HuMab mouse (due to the degeneracy of the genetic code), the increased number of somatic mutations observed at the DNA level for several of the chimeric antibodies derived from the 9B2-58 transgenic strain supports the position that expression in transgenic mice of chimeric antibodies that contain a host constant region can lead to increased somatic mutations rates in the chimeric antibodies.
HuMab Mice
The data described above for the 9B2-58 strain with regard to somatic mutations is consistent with observations that have been made about somatic mutation rates in the HuMab mouse, which express fully human antibodies but which also may express chimeric antibodies due to trans-switching between the human heavy chain transgene and the endogenous mouse constant region.
For example, HuMab mice (HCo12/7 and HCo12 mice) were immunized with a cell surface receptor and the spleens were isolated and fused according to standard hybridoma techniques. Eight anti-receptor antibodies were isolated from the fusion mixture and sequenced. Three of the antibodies were chimeric in nature, consisting of human VDJ regions linked to a mouse IgG2b constant region. The remaining five antibodies were fully human antibodies. cDNA sequence analysis of all the heavy chain variable regions showed that the chimeric and human antibodies used different variable region recombinations, and the chimeric antibodies had higher somatic mutations at the DNA level than the fully human antibodies and correlating higher amino acid differences. As for the 9B2-58 mice described above, the somatic mutation quantitation was determined for the VH segment only (not the D or JH segments). The results are summarized below in Table 5:







TABLE 5







Sequence Analysis of Human and Chimeric Antibodies from


HuMab Mice


















SM
HC


Clone Name
VH
DH
JH
SM (DNA)
(AA)
Isotype
















Chimeric 1
3-23
3-9
4a
16
12
mIgG2b


Chimeric 2
3-23
3-9
4b
16
13
mIgG2b


Chimeric 3
3-23
3-9
4b
16
12
mIgG2b


Human 1
  3-30.3
nd
4b
8
6
hIgG1


Human 2
  3-30.3
nd
4b
7
7
hIgG1


Human 3
3-33
 3-10
3b
2
2
hIgG1


Human 4
3-33
 7-27
4b
2
2
hIgG1


Human 5
3-33
5-5
3b
6
4
hIgG1





nd = not determined,


SM = somatic mutation;


HC = heavy chain






In another set of experiments, HuMab mice (HCo12/7) immunized with a soluble cytokine and the spleens were isolated and fused according to standard hybridoma techniques. Six anti-cytokine antibodies were isolated from the fusion and sequenced. Five of the antibodies were chimeric in nature consisting of human VDJ regions linked to a mouse IgG2a and IgG2b constant region. The remaining antibody was fully human antibodies. cDNA sequence analysis of all the heavy chain variable regions showed that the chimeric antibodies and human antibody used similar and different variable region recombinations and that the chimeric antibodies again had higher somatic mutations at the DNA level than the fully human antibody and correlating higher amino acid differences. Furthermore, the chimeric antibody #5 had a higher blocking efficiency than the fully human antibody #1 in functional blocking assays. The results are summarized below in Table 6:







TABLE 6







Sequence Analysis of Human and Chimeric Antibodies from


HuMab Mice

















SM
SM



Clone Name
VH
DH
JH
(DNA)
(AA)
HC Isotype
















Chimeric 1
  3-30.3
nd
4b
21
13
mIgG2b


Chimeric 2
  3-30.3
7-27
4b
8
7
mIgG2a


Chimeric 3
1-69
nd
4b
9
6
mIgG2a


Chimeric 4
1-69
nd
4b
9
6
mIgG2a


Chimeric 5
1-69
nd
4b
25
15
mIgG2a


Human 1
1-69
nd
4b
7
7
hIgG1





nd = not determined,


SM = somatic mutation;


HC = heavy chain






Thus, the results from the experiments described above for HuMab mice immunized with two different antigens support the position that expression in transgenic mice of chimeric antibodies, that contain a host constant region, can lead to increased somatic mutations in the chimeric antibodies, as compared to fully human antibodies expressed in the transgenic mice.
Example 5
Generation of a Second Transgene for Expression of Chimeric Antibodies
In this example, another transgene for expressing chimeric antibodies in mice was created, referred to as HCo26. The HCo26 transgene differs from the 9B2 transgene described in Example 1 in that the 9B2 transgene contains only the mouse IgM constant region whereas the HCo26 transgene contains all of the mouse constant region coding sequences.
HCo26 consists of three imbricate DNA fragments that contain unrearranged human V, D and J regions and the mouse IgH locus from the 5′ Eμ enhancer through the identified 3′ DNase hypersensitive sites and origin of replication (Zhou, et al. (2002) Proc. Natl. Acad. Sci. USA 99:13693-13698). The construction of the HCo26 transgene is illustrated schematically in FIG. 1.
The first of the three fragments is the approximately 57 kb NotI-SalI fragment from clone 9B2 of phVDJ-mEM (described in Example 1) containing unrearranged human heavy chain VDJ segments linked to the mouse J-μ enhancer region, μ switch region and μ coding region. Another fragment consists of the approximately 140 kb NgoMIV-NotI fragment of the previously described B20 BAC, which contains the mouse J-μ enhancer, mouse μ constant coding regions and the intervening mouse genomic regions through the mouse γ2b coding constant region. As the mouse J-μ enhancer and mouse μ switch regions make up part of the 9B2 fragment, these regions are part of the overlapping regions of 9B2 and the B20 fragment. Another fragment consists of the approximately 180 kb NotI-NotI fragment of C22 BAC, which consists of the mouse γ2b coding constant region and the intervening mouse genomic regions through the DNase hypersensitive sites 3′ of the mouse a coding regions to the endogenous NotI site found in the C22 BAC fragment. The mouse γ2b coding regions make up the overlapping regions of the B20 and the C22 BAC fragments.
The approximately 57 kb NotI-SalI fragment from clone 9B2 of phVDJ-mEM was released from the vector, as was the approximately 140 kb NgoMIV-NotI fragment of the B20 BAC and the approximately 180 kb NotI-NotI fragment of C22 BAC. Each was then isolated by PFGE. An agarose gel slice with each of the fragments was excised and the agarose was digested with β-agarase (commercially obtained from Takara) according to the manufacturer's protocol. The three fragments were then mixed to a 1:1:1 stoichiometry and the mixture is referred to as HCo26. The HCo26 DNA mixture was then micro-injected (by standard methods) into fertilized oocytes. DNA was injected into JHD (KCo5) (JHD+/+ CMD−/−, JKD+/+, KCo5+) mice. Potential founder mice were screened for the 9B2 transgene by PCR with DMTM79 and DMTM80 using tail DNA as template. Two different founder mice, HCo26-05 and HCo26-16, tested positive for the 9B2 transgene on the JHD+/+, CMD−/−, JKD+/+, KCo5+ strain background. Furthermore, Southern Blot analysis of genomic DNA from the HCo26-05 mouse had 2 positive hybridizing BamHI bands with an approximately 1 kb probe at the 3′ end of the C22 fragment. One band represents the 3′ end of the endogenous mouse IgH locus, while the other represents the integration of the C22 fragment into the mouse genome in a random manner.
Founder mice positive for the HCo26 transgenes were then bred to JHD (KCo5) mice (JHD+/+, CMD−/−, JKD+/+, KCo5+/+ mice) and genotyped for 9B2, JHD, CMD, JKD and KCo5. HCo26-05 founder transmitted the HCo26 transgene to several offspring.
These HCo26 founders are crossed to JHD (KCo5) mice to generate distinct stable HCo26 lines. HCo26 positive mice are tested for pre-immune levels of mouse IgM/human kappa and mouse IgG/human kappa as described in the examples above. Furthermore, HCo26 mice can be immunized with TT or other antigens and titered for mouse IgG/human kappa and antigen+/mouse IgG levels as described in the examples above.
Example 6
Conversion of Chimeric Antibodies to Fully Human Antibodies and Comparison Thereof
To convert a chimeric antibody to a fully human antibody, cDNA of the variable regions (comprising the rearranged human VDJ segments) of the heavy chain and light chain are isolated and sequenced. Total RNA is obtained from hybridoma cell pellets secreting the desired antibody by utilizing Qiagen RNeasy Mini Kit. cDNA is then prepared using the 5′ RACE protocol utilizing Clontech SMART RACE cDNA Amplification Kit. Variable regions of each antibody are then amplified using a 3′ primer specific for the mouse constant region paired with the 5′ RACE universal primer mix. PCR products containing the variable regions are then cloned into the Invitrogen TOPO TA DNA sequencing vectors. Minipreped DNA samples are prepared and DNA sequenced. DNA sequences are then trimmed to include only the variable region of the desired antibody. Variable regions of the antibody are then matched to the germline human V(D)J regions used to generate the 9B2 mice to ensure they are of transgene origin and thus of human origin Variable regions are also compared with mouse variable regions to rule out any mouse derived variable regions. cDNA sequences are compared with the N-terminal amino acid sequencing and mass spec analysis of the desired antibody to ensure the correct cDNA sequence is obtained.
Once the correct cDNA sequence is obtained, primers are synthesized to independently PCR the coding regions of the heavy and light chain variable regions. Furthermore, appropriate restriction sites are added in frame to the coding regions to allow for cloning of the coding variable regions directly into expression vectors that already encode for a human constant region. Thus, a variable region (light or heavy chain) is inserted into an expression vector such that the appropriate variable region sequences are operatively linked to the appropriate human constant region sequence, as well as to transcriptional and translational control sequences. An antibody heavy chain gene and an antibody light chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. An expression vector can be used that already encodes heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Non-limiting examples of suitable expression vectors for expressing fully human antibodies include the pIE family of vectors as described in U.S. Patent Application No. 20050153394 by Black.
For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
The general methodology described above for converting a chimeric antibody to a fully human antibody was used to convert an anti-TT mAb raised in the 9B2-58 strain of transgenic mouse to a fully human antibody. More specifically, the heavy chain and light chain variable regions of the anti-TT antibody 43H7C10 (described in Example 4) (a chimeric antibody consisting of a human VDJ variable region operatively linked to a mouse gamma 2b constant region and paired with a fully human Kappa light chain) were cloned into expression vectors such that the human heavy chain variable region and light chain variable region sequences were operatively linked to sequences encoding a human gamma 1 constant region and a human Kappa constant region, respectively, to allow for the preparation of a recombinant fully human 43H7C10 antibody. The 43H7C10 human heavy chain and light chain variable region sequences also were cloned into expression vectors such that they were operatively linked to sequences encoding the mouse gamma 2a constant region and human Kappa constant regions, respectively, to allow for the preparation of a recombinant chimeric 43H7C10 antibody. Expression vectors encoding the recombinant fully human 43H7C10 antibody and the recombinant chimeric 43H7C10 antibody were transfected by standard techniques into CHO cells and both forms of the 43H7C10 antibody protein was produced independently and purified using standard protein purification methods. The two purified antibody proteins were then used in a BIACORE experiment to determine and compare the affinity to TT, as well as on-rates and off-rates, by standard methodologies. For comparison, 1D6 (a fully human anti-TT antibody originally raised in a HuMab Mouse®) was also produced as a fully human antibody as well as a recombinant chimeric mouse IgG/human kappa antibody (both expressed in CHO cells). Results of the affinity, on-rate and off-rate comparison are shown in Table 7 below.







TABLE 7







Binding Kinetics of Chimeric vs. Human Anti-TT Antibodies












Antibody

On-rate
Off-rate


Clone Name
form
Affinity (nM)
(1/Ms) × 104
(1/s) × 104














43H7C10
Chimeric
17.9
8.9
15.9


43H7C10
Fully human
11.7
4.8
5.6


1D6
Chimeric
66.9
29.1
19.4


1D6
Fully human
46.3
25.1
11.6









This data demonstrates that a chimeric anti-TT antibody raised in a transgenic mouse of the invention can be recombinantly reconfigured to a fully human antibody form and still maintain its binding properties toward its target antigen. In fact, the recombinant fully human form of the antibody exhibits somewhat higher binding affinity for the target antigen than the recombinant chimeric form. Furthermore, both the chimeric and fully human recombinant forms of the 43H7C10 antibody exhibit higher binding affinity for the target antigen than the fully human 1D6 mAb raised in a HuMab Mouse®, thereby demonstrating that the transgenic mice of the invention can allow for the preparation of a fully human antibody against a target of interest that has higher affinity for the target than a fully human antibody raised in a HuMab Mouse®.

## Claims

1. A transgene construct comprising an unrearranged human heavy chain immunoglobulin (Ig) variable region sequence operatively linked to a mouse immunoglobulin (Ig) constant region sequence, wherein said constant region sequence comprises, in 5′ to 3′ direction, an approximately 140kb NgoMIV-NotI fragment of the B20 bacteria artificial chromosome (BAC) and an approximately 180kb NotI-NotI frag of the C22 BAC, and wherein, when integrated into a mouse genome, said transgene construct undergoes rearrangement and the mouse expresses chimeric antibodies, wherein the chimeric antibodies comprise a human heavy chain variable region and a mouse heavy chain constant region.

2. The construct of claim 1, wherein the unrearranged human heavy chain variable region sequence comprises human heavy chain V segment sequences, human heavy chain D segment sequences, and human heavy chain J segment sequences.

3. The construct of claim 2, which comprises, in 5′ to 3′ direction, a plurality of human heavy chain V segment sequences, a plurality of human heavy chain D segment sequences, a plurality of human heavy chain J segment sequences, and said mouse constant region sequence.

4. The construct of claim 3, which comprises four human heavy chain V segment sequences, 15 human heavy chain D segment sequences and six human heavy chain J segment sequences.

5. The construct of claim 4, which comprises a Hco26 transgene.

6. A transgenic mouse whose genome comprises the transgene construct of claim 1, wherein the mouse expresses chimeric antibodies comprising human Ig variable regions and mouse Ig constant regions.

7. A method of making a chimeric antibody specific for an antigen of interest comprising immunizing the transgenic mouse of claim 6 with the antigen of interest and obtaining from the mouse a chimeric antibody specific for the antigen of interest.

8. The method of claim 7, further comprising isolating from the mouse a nucleic acid encoding a chimeric immunoglobulin (Ig) heavy chain and replacing nucleic acid encoding the mouse Ig heavy chain constant region with nucleic acid encoding a human Ig heavy chain constant region to convert the nucleic acid encoding the chimeric Ig heavy chain to a nucleic acid encoding a human Ig heavy chain, and expressing the human Ig heavy chain.