Human monoclonal antibodies from transgenic mice.

Since the 1986 regulatory approval of muromonomab-CD3, a mouse monoclonal antibody (MAb) directed against the T cell CD3epsilon antigen, MAbs have become an increasingly important class of therapeutic compounds in a variety of disease areas ranging from cancer and autoimmune indications to infectious and cardiac diseases. However, the pathway to the present acceptance of therapeutic MAbs within the pharmaceutical industry has not been smooth. A major hurdle for antibody therapeutics has been the inherent immunogenicity of the most readily available MAbs, those derived from rodents. A variety of technologies have been successfully employed to engineer MAbs with reduced immunogenicity. Implementation of these antibody engineering technologies involves in vitro optimization of lead molecules to generate a clinical candidate. An alternative technology, involving the engineering of strains of mice to produce human instead of mouse antibodies, has been emerging and evolving for the past two decades. Now, with the 2006 US regulatory approval of panitumumab, a fully human antibody directed against the epidermal growth factor receptor, transgenic mice expressing human antibody repertoires join chimerization, CDR grafting, and phage display technologies, as a commercially validated antibody drug discovery platform. With dozens of additional transgenic mouse-derived human MAbs now in clinical development, this new drug discovery platform appears to be firmly established within the pharmaceutical industry.

Transgenic human lambda 5 rescues the murine lambda 5 nullizygous phenotype.

The human lambda 5 (hu lambda 5) gene is the structural homologue of the murine lambda 5 (m lambda 5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The lambda 5 and VpreB polypeptides together with the Ig mu H chain and the signal-transducing subunits, Ig alpha and Ig beta, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu lambda 5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu lambda 5 gene. High levels of expression of the transgenic hu lambda 5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu lambda 5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu lambda 5 polypeptide on the cell surface and in association with mouse Ig mu and mouse VpreB. Finally, we found that the hu lambda 5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m lambda 5-/- background. Therefore, we conclude that the hu lambda 5 polypeptide can biochemically and functionally substitute for m lambda 5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

CD3-epsilon overexpressed in prothymocytes acts as an oncogene.

BACKGROUND: Upon engagement of the T cell receptor for antigen, its associated CD3 proteins recruit signal transduction molecules, which in turn regulate T lymphocyte proliferation, apoptosis, and thymocyte development. Because some signal transducing molecules recruited by CD3-epsilon, i.e., p56lck and p59fyn, are oncogenic and since we previously found that overexpression of CD3-epsilon transgenes causes a block in T lymphocyte and NK cell development, we tested the hypothesis that aberrant CD3-epsilon signaling leads both to abnormal T lymphocyte death and lymphomagenesis. MATERIALS AND METHODS: Ten independently derived transgenic mouse lines were generated with four different genomic CD3-epsilon constructs. Mice either homozygous or hemizygous for each transgene were analyzed for an arrest in T lymphocyte development and for the occurrence of T cell lymphomas. RESULTS: Aggressive clonal T cell lymphomas developed at very high frequencies in seven mouse lines with intermediate levels of copies of CD3-epsilon derived transgenes. However, these lymphomas were not found when high copy numbers of CD3-epsilon transgenes caused a complete block in early thymic development or when a transgene was used in which the exons coding for the CD3-epsilon protein were deleted. Analyses of a series of double mutant mice, tgCD3-epsilon x RAG-2null, indicated that lymphomagenesis was initiated in lineage-committed prothymocytes, i.e., before rearrangement of the T cell receptor genes. In addition, the transgene coding for the CD3-epsilon cytoplasmic domain and its transmembrane region induced a T cell differentiation signal in premalignant tgCD3-epsilon x RAG-2null mice. CONCLUSION: The nonenzymatic CD3-epsilon protein acted as a potent oncogene when overexpressed early in T lymphocyte development. Lymphomagenesis was dependent on signal transduction events initiated by the cytoplasmic domain of CD3-epsilon.

A candidate gene for the amnionless gastrulation stage mouse mutation encodes a TRAF-related protein.

We report the identification of a new recessive prenatal lethal insertional mutation, amnionless (amn). amn mutant embryos first appear abnormal during the Early Streak stage, between E6.5 and E7.0, when they initiate mesoderm production. Subsequently, the amn mutants become developmentally arrested between the Mid and Late Streak stages of gastrulation and they die and are resorbed between E9.5 and E10.5. While extraembryonic structures, including the chorion, yolk sac blood islands, and allantois appear to develop normally, the small embryonic ectoderm remains undifferentiated and generates no amnion. In addition, the embryonic mesoderm that is produced does not become organized into node, notochord, and somites and there is no morphological evidence of neural induction. Interspecific backcross and fluorescence in situ hybridization analyses map the transgene insertion, and thus the amn mutation, to the distal region of mouse chromosome 12, which has synteny with human chromosome 14q32. A gene encoding a 7.5-kb transcript has been identified at a junction between the integrated transgene and host chromosome 12 sequences that meets three criteria expected of a candidate amn gene. This gene maps to the site of transgene insertion; it is transcribed during gastrulation, and its expression is disrupted in amn mutant embryos. Nucleotide sequencing studies show that the 567 amino acid protein encoded by the 7.5-kb transcript is a member of the newly defined family of putative signal transducing proteins, TRAFs, that associate with the cytoplasmic domains of members of the tumor necrosis factor (TNF) receptor superfamily. Thus, we have named the gene encoding the 7. 5-kb transcript TRAFamn. TRAFamn is identical to a recently reported protein (CD40bp, CAP-1, CRAF1, LAP1) that can bind the cytoplasmic domains of CD40 and the lymphotoxin beta receptor (LTbetaR), both of which are known members of the TNF receptor superfamily. The implications of these findings regarding a possible role for the TNF receptor superfamily during gastrulation are discussed.

High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice.

Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology. We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgG kappa Mabs directed against a human antigen. The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human V kappa region. In addition, the heavy-chain transgene encodes both human mu and human gamma 1 constant regions, the latter of which is expressed via intratransgene class switching. We have used these animals to isolate human IgG kappa Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro. The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels. Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.

Class switching in human immunoglobulin transgenic mice.

We have introduced human germline-configuration heavy and kappa light chain minilocus transgenes into mice that have been engineered so that their endogenous heavy and kappa light chain loci are inactive. The two human transgenes are inserted by pronuclear microinjection, while the two endogenous mouse genes are disrupted by homologous recombination in embryonic stem cells. The resulting animals contain four unlinked genetic modifications and must rely on the introduced transgenes for the development of the B-cell lineage and for the generation of an antibody repertoire. The heavy chain transgene includes both the human mu and the human gamma 1 constant region gene segments, as well as upstream switch region sequences. Although mouse B cells and human B cells exhibit species-specific differences in the induction of gamma isotype expression, the transgenic mouse B cells appear to undergo regulated switching to human gamma 1 both in vivo and in vitro. This observation defines a subset of the heavy chain constant region that is sufficient for class switching, and implies that the human gamma 1 switch region includes a core of sequence that is functionally homologous to those cis-acting regulatory elements that direct mouse gamma switching.

Human antibodies from transgenic mice.

We have used homologous recombination in ES cells to engineer B cell-deficient mice that are incapable of expressing endogenous immunoglobulin heavy and kappa light chain genes. We find that B cell development in these mutant mice can be rescued by the introduction of human germline-configuration heavy- and kappa light-chain minilocus transgenes. The transgenes rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation in response to antigen stimulation; thus recapitulating both stages of the humoral immune response using human, rather than mouse, sequences. The mice can be immunized; and human sequence, antigen specific, monoclonal antibodies can be obtained using conventional rodent hybridoma technology. These animals are also of interest for studying the normal processes of immunoglobulin gene expression. We discuss the example of heavy chain class switching, which has not been previously observed within an autonomous transgene.

A block in both early T lymphocyte and natural killer cell development in transgenic mice with high-copy numbers of the human CD3E gene.

A severe immunodeficiency involving a complete loss of T lymphocytes and natural killer cells was observed in independent lines of transgenic mice containing > 30 copies of the human CD3E gene (pL12). T-cell- natural killer (NK)- mice could also be generated by using a gene fragment pL12 delta 1 (without exons 4A and 5) coding for the CD3-epsilon transmembrane region and its 55-amino acid nonenzymatic cytoplasmic tail. The abnormally small thymus gland in the homozygous transgenic animals, which was approximately 1% the size of a wild-type thymus, contained only a few (2-4%) prethymocytes with a Thy-1+Pgp-1+IL-2R alpha- CD3-4-8- phenotype. In mice with lower copy numbers of the transgene thymocyte development was blocked at the Thy-1+Pgp-1-IL-2R alpha+CD3-4-8- stage, and normal NK activity was detected. Mice generated with high-copy numbers of a transgene pL12 delta 2 (pL12 delta 1 minus exons 6), coding for a truncated protein from which the CD3-epsilon extracellular domain, its transmembrane region, and most of its cytoplasmic region were absent, contained normal numbers of T lymphocytes and NK cells. These transgene effects suggested that recruitment of signal-transduction molecules by the cytoplasmic tail of this protein played an important role in the abrogation of both lineages. Taken together these observations support the notion that T lymphocytes and NK cells stemmed from a common precursor.

Antigen-specific human antibodies from mice comprising four distinct genetic modifications.

Human sequence monoclonal antibodies, which in theory combine high specificity with low immunogenicity, represent a class of potential therapeutic agents. But nearly 20 years after Köhler and Milstein first developed methods for obtaining mouse antibodies, no comparable technology exists for reliably obtaining high-affinity human antibodies directed against selected targets. Thus, rodent antibodies, and in vitro modified derivatives of rodent antibodies, are still being used and tested in the clinic. The rodent system has certain clear advantages; mice are easy to immunize, are not tolerant to most human antigens, and their B cells form stable hybridoma cell lines. To exploit these advantages, we have developed transgenic mice that express human IgM, IgG and Ig kappa in the absence of mouse IgM or Ig kappa. We report here that these mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins. They are also homozygous for targeted mutations that disrupt V(D)J rearrangement at the endogenous heavy- and kappa light-chain loci. We have immunized the mice with human proteins and isolated hybridomas secreting human IgG kappa antigen-specific antibodies.

Human immunoglobulin heavy-chain minilocus recombination in transgenic mice: gene-segment use in mu and gamma transcripts.

We (N.L. and L.D.T.) have introduced a human heavy-chain minilocus into mice transgenically. Constructs contain 2 heavy-chain variable (VH; psi VH3-105 and VH5-251), 10 diversity (D), 6 heavy-chain joining (JH), and either constant (C)mu or C mu and C gamma gene segments. Several founder lines were established and studied before immunization. Seventy heavy-chain transcripts were cloned and sequenced from murine splenic B lymphocytes, and gene-segment use was assessed before and after class-switching. In general, the repertoire was "fetal" in appearance with little evidence of somatic mutation in any gene segment. The two VH gene segments were found rearranged to mu- and gamma-chain C segments, with a preference of VH5-251. We observed a preponderance of the most-J-proximal D gene (DHQ52) segments among the mu transcripts (44%). The JH gene-segment use mimics most patterns seen in human antibodies. Diversification in CDR3 was extensive and included clear examples of D inversions and D-D fusions. These data suggest that a human immunoglobulin minilocus can undergo recombinatorial processes in a manner analogous to that seen in the human fetal/preimmune repertoire. This model, in addition to providing a potential source of human monoclonal antibodies, is ideal for the study of further questions concerning immunoglobulin gene-segment recombination.

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.

Structure of the T-cell antigen receptor: evidence for two CD3 epsilon subunits in the T-cell receptor-CD3 complex.

The T-cell antigen receptor (TCR) consists of heterodimeric glycoproteins (TCR alpha beta or gamma delta) that demonstrate homology with immunoglobulins. Noncovalently associated with the alpha beta (or gamma delta) heterodimer are at least five nonvariant proteins (CD3-gamma, -delta, -epsilon, -zeta, and -eta), which together comprise the TCR-CD3 complex. The stoichiometry of the antigen receptor has been assumed to be either alpha beta gamma delta epsilon zeta zeta or alpha beta gamma delta epsilon zeta eta. In this paper we provide several lines of evidence that support the notion that the mature TCR-CD3 complex on the cell surface contains two CD3-epsilon polypeptide chains. Transfection of two murine T cell-T cell hybridomas with the human DNA encoding CD3-epsilon protein demonstrated that both murine and human CD3-epsilon chains were present within the same TCR-CD3 complex. Analysis of thymocytes isolated from transgenic mice that expressed high copy numbers of the human CD3-epsilon gene showed that the heterologous human CD3-epsilon subunits were coexpressed with murine CD3-epsilon in the same TCR-CD3 complex. Since CD3-epsilon was shown to form disulfide-linked homodimers both in human and murine T cells, the two CD3-epsilon subunits present in the TCR-CD3 complex were in direct contact with one another. The presence of two CD3-epsilon polypeptide chains in close proximity to one another in the TCR-CD3 complex may have important implications for its assembly and its signal transduction mechanisms.

Bovine alpha S1-casein gene sequences direct high level expression of active human urokinase in mouse milk.

We have produced a line of transgenic mice carrying a hybrid bovine alpha S1 casein/human urokinase gene. Bovine alpha S1-casein gene regulatory sequences specifically direct expression of the human urokinase gene in lactating mammary tissue from these mice. Urokinase is a 54 kD protein with 9 disulfide bonds that is normally synthesized in the kidney; however, the casein/urokinase transgenic mice secrete active human urokinase into their milk at concentrations of 1-2 mg/ml. The mice show no other abnormalities. They give birth to, and nurse, normal sized healthy litters. Thus it is possible to produce high concentrations of a large, cysteine rich, non-milk protein in the milk of transgenic animals. This line of transgenic mice provides a model for the eventual production of transgenic farm animals producing high levels of recombinant proteins in their milk.

An enhancer located in a CpG-island 3' to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter.

The gene encoding the CD3-epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3-epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue-specific fashion. The gene was then screened for T lymphocyte-specific cis-acting elements in transient chloramphenicol transferase assays. The promoter (-228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell-specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3-epsilon enhancer did not display sequence similarity with the T cell-specific enhancer of CD3-delta, a related gene co-regulated with CD3-epsilon during intrathymic differentiation. The CD3-epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I-transformed T cell lines were identified in which the CD3-epsilon gene was not expressed, and in which the enhancer was inactive.

Alternatively spliced mRNA encodes a secreted form of human CD8 alpha. Characterization of the human CD8 alpha gene.

We have determined the organization and nucleotide sequence of the gene encoding the human T cell surface glycoprotein CD8 alpha. This gene spans approximately 8 kb and is organized into six exons which encode separate functional domains of the protein. Exon 1 encodes the 5' untranslated region and leader peptide, exon II the Ig V-like region, exon III the hinge-like region, exon IV the transmembrane domain, and exons V and VI the cytoplasmic tail. Alternative splicing that excludes nucleotide sequences from exon IV results in a transcript which encodes a secreted form of the protein. This transcript accounts for approximately 15% of the total CD8 alpha mRNA in human T cell leukemia lines and in normal human tissues. Secreted CD8 alpha protein can be detected in culture supernatants of T cell leukemia lines and PHA-stimulated PBMC by immunoprecipitation with the anti-CD8 alpha mAb OKT8 or with a polyclonal rabbit antiserum specific for the 28 amino acid cytoplasmic domain of CD8 alpha. The secreted CD8 alpha protein forms homodimers; when analyzed by SDS-PAGE, the protein migrates with an apparent molecular mass of 27 or 54 kDa under reducing or non-reducing conditions, respectively. Human secreted CD8 alpha may serve an immunoregulatory role for the interactions of T cells with their targets in vivo.

Mouse brain CD4 transcripts encode only the COOH-terminal half of the protein.

The T-cell surface glycoprotein CD4 is thought to function as a receptor for class II major histocompatibility complex molecules. Human CD4 is also the lymphoid cell receptor for human immunodeficiency virus, the causative agent of acquired immune deficiency syndrome. The observed infection of the central nervous system in acquired immune deficiency syndrome patients raises the possibility that CD4 is also present in nerve tissue and that a cell surface receptor for class II major histocompatibility complex antigens could play a role in central nervous system function. This possibility is reinforced by the detection of unique CD4-related transcripts in mouse and human brain tissue. In this study, the structure of the mouse brain CD4 transcript was determined. It is identical to the last two-thirds of the CD4 message and is capable of encoding a 217-residue protein that would consist of a truncated, 154-residue, cell surface region, together with the complete CD4 transmembrane and cytoplasmic regions. It would not include an amino-terminal hydrophobic leader peptide.

Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the determination of thermodynamic parameters.

Many biologically important proteins bind nonspecifically, and often cooperatively, to single-or double-stranded nucleic acid lattices in discharging their physiological functions. This binding can generally be described in thermodynamic terms by three parameters: n, the binding site size; K, the intrinsic binding constant; omega, the binding cooperativity parameter. The experimental determination of these parameters often appears to be straightforward but can be fraught with conceptual and methodological difficulties that may not be readily apparent. In this paper we describe and analyze a number of approaches that can be used to measure these protein-nucleic acid interaction parameters and illustrate these methods with experiments on the binding of T4-coded gene 32 (single-stranded DNA binding) protein to various nucleic acid lattices. We consider the following procedures: (i) the titration of a fixed amount of lattice (nucleic acid) with added ligand (protein); (ii) the titration of a fixed amount of ligand with added lattice; (iii) the determination of ligand binding affinities at very low levels of lattice saturation; (iv) the analysis of ligand cluster size distribution on the lattice; (v) the analysis of ligand binding to lattices of finite length. The applicability and limitations of each approach are considered and discussed, and potential pitfalls are explicitly pointed out.