Intracellular Antibodies for Drug Discovery and as Drugs of the Future.

The application of antibodies in cells was first shown in the early 1990s, and subsequently, the field of intracellular antibodies has expanded to encompass antibody fragments and their use in target validation and as engineered molecules that can be fused to moieties (referred to as warheads) to replace the Fc effector region of a whole immunoglobulin to elicit intracellular responses, such as cell death pathways or protein degradation. These various forms of intracellular antibodies have largely been used as research tools to investigate function within cells by perturbing protein activity. New applications of such molecules are on the horizon, namely their use as drugs per se and as templates for small-molecule drug discovery. The former is a potential new pharmacology that could harness the power and flexibility of molecular biology to generate new classes of drugs (herein referred to as macrodrugs when used in the context of disease control). Delivery of engineered intracellular antibodies, and other antigen-binding macromolecules formats, into cells to produce a therapeutic effect could be applied to any therapeutic area where regulation, degradation or other kinds of manipulation of target proteins can produce a therapeutic effect. Further, employing single-domain antibody fragments as competitors in small-molecule screening has been shown to enable identification of drug hits from diverse chemical libraries. Compounds selected in this way can mimic the effects of the intracellular antibodies that have been used for target validation. The capability of intracellular antibodies to discriminate between closely related proteins lends a new dimension to drug screening and drug development.

Selection of human single domain antibodies recognizing the CMYC protein using enhanced intracellular antibody capture.

Protein functions that are mediated by interaction with other proteins (protein-protein interactions, PPI) are important for normal cell biology and also in disease. Molecules that can interfere with PPI are required as laboratory tools to dissect function, as lead drug surrogates for target validation and as templates for drug discovery. We describe enhanced developments to Intracellular Antibody Capture (IAC) technology that can select antibody fragments able to interact with targets in cells. This is illustrated by the isolation of single heavy chain variable region domains binding to the basic-helix-loop-helix and leucine zipper region of the CMYC oncogenic protein. The enhanced IAC (eIAC) methodology deploys screening in yeast cells of a single diverse library initially with randomization only of CDR3. Further sequential randomization of CDR2 and CDR1 of three independently selected anti-CMYC clones illustrates an in vivo affinity maturation process. This concise eIAC approach facilitates the rapid selection of antibody fragments to explore the proteome interaction spectrum of mammalian cells and disease targeting.

Conformational flexibility of the oncogenic protein LMO2 primes the formation of the multi-protein transcription complex.

LMO2 was discovered via chromosomal translocations in T-cell leukaemia and shown normally to be essential for haematopoiesis. LMO2 is made up of two LIM only domains (thus it is a LIM-only protein) and forms a bridge in a multi-protein complex. We have studied the mechanism of formation of this complex using a single domain antibody fragment that inhibits LMO2 by sequestering it in a non-functional form. The crystal structure of LMO2 with this antibody fragment has been solved revealing a conformational difference in the positioning and angle between the two LIM domains compared with its normal binding. This contortion occurs by bending at a central helical region of LMO2. This is a unique mechanism for inhibiting an intracellular protein function and the structural contusion implies a model in which newly synthesized, intrinsically disordered LMO2 binds to a partner protein nucleating further interactions and suggests approaches for therapeutic targeting of LMO2.

Interfering with RAS-effector protein interactions prevent RAS-dependent tumour initiation and causes stop-start control of cancer growth.

RAS mutations are the most common gain-of-function change in human cancer and promise to be a critical therapy target. As a new approach, we have used a surrogate to drug the 'undruggable' (that is, RAS-effector protein-protein interactions inside cancer cells) in pre-clinical mouse models of RAS-dependent cancers. Using this novel reagent, we have specifically targeted RAS signalling in a transgenic mouse model of lung cancer by directly blockading RAS-effector interactions with an antibody fragment that binds to activated RAS, and show that the interaction of RAS and effectors, such as phosphoinositide 3-kinase and RAF, is necessary for tumour initiation. Further, interference with oncogenic RAS-effector interactions result in control of tumour growth in human cancer cells but, crucially, does not necessarily cause tumour regression. These findings support the concept that ablating RAS-dependent signalling in cancer will have chemo-preventive effects that confer a chronic state in cancer and suggest that mutant RAS-targeted therapies may require conjoint targeting of other molecules and/or current cancer therapeutic strategies (for example, radiotherapy and chemotherapy) to be curative. In this context, our findings suggest that the oncogene addiction model is not universally correct in its central thesis that cancer cell death is inevitable after loss of oncogenic protein function.

Microdissection molecular copy-number counting (microMCC)--unlocking cancer archives with digital PCR.

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

An antibody inhibitor of the LMO2-protein complex blocks its normal and tumorigenic functions.

The LIM-domain protein LMO2 is a T-cell oncogenic protein first recognized by gene activation through chromosomal translocations, but it is also responsible for leukaemias arising as secondary, adverse effects in an X-SCID gene therapy trial. There are no specific reagents currently available to analyse the LMO2 multiprotein complex or to combat LMO2-dependent leukaemias. Accordingly, we have isolated an anti-LMO2 single chain Fv antibody fragment to determine if intracellular interference with LMO2-protein complexes can avert LMO2-dependent functions in normal and cancer settings. The anti-LMO2 single chain Fv, obtained using Intracellular Antibody Capture (IAC) technology, is specific for LMO2 among the LIM-only protein family and binds LMO2 through the third and fourth LIM fingers. Using vector-mediated expression of anti-LMO2 scFv, we show inhibition of Lmo2-dependent erythropoiesis but not endothelial development. We also demonstrate inhibition of Lmo2-dependent leukaemia in a mouse T-cell tumourigenesis transplantation assay with retroviral-mediated expression of anti-LMO2 scFv. Our studies establish that interference with the LMO2 multiprotein complex inhibits both normal and tumourigenic roles. The antibody fragment is a tool for dissecting LMO2 function in haematopoiesis and leukaemia and is a lead for development of therapeutics against LMO2-dependent T-ALL.

MLL gene fusions in human leukaemias: in vivo modelling to recapitulate these primary tumourigenic events.

Recurrent reciprocal chromosomal translocations are frequently found in leukaemias and sarcomas as initiating events in these cancers. Mouse models of chromosomal translocations are not only important for the elucidation of the mechanism of these factors underlying the disease but are also important pre-clinical models for assessing new drug combinations, developing new rational therapeutic strategies based on new drugs and testing novel macromolecular drugs. We describe three technologies for creating chromosomal translocation mimics in mice, applied specifically to understand how the MLL-fusions contribute to leukaemia. An important finding of this work is that the lineage of the tumours can be controlled by the MLL-protein fusion. The translocation mimic methods can be applied to any human reciprocal chromosomal translocation.

Leukaemia lineage specification caused by cell-specific Mll-Enl translocations.

Chromosomal translocations involving the Mixed-Lineage Leukaemia (MLL) gene underlie many human leukaemias and MLL rearrangements are found in both acute myelogenous and acute lymphoblastic leukaemias. To assess the functionally relevant haematopoietic cell contexts for MLL fusions to be tumorigenic, we have generated different lines of mice in which de novo Mll-associated translocations occur. In these models, reciprocal chromosomal translocations occur by means of Cre-loxP-mediated recombination (translocator mice) in different cells of the haematopoietic system (namely haematopoietic stem cells, semi-committed progenitors or committed T or B cells). Translocations between Mll and Enl cause myeloid neoplasias, initiating in stem cells or progenitors while no tumours arose when the translocation was restricted to the B-cell compartment. Despite the absence of tumorigenesis, Mll-Enl translocations did occur and Mll-Enl fusion mRNA was expressed in B-cell-restricted translocators. A permissive cellular environment is therefore required for oncogenicity of Mll-associated translocations since the occurrence of Mll-Enl does not promote unrestricted proliferation in all haematopoietic cellular contexts, consistent with a specific instructive role of the MLL-fusion proteins in leukaemogenesis.

A conditional model of MLL-AF4 B-cell tumourigenesis using invertor technology.

MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukaemia and is associated with a poor prognosis. MLL-AF4 is thought to be required in haematopoietic stem cells to elicit leukaemia and may be involved in tumour phenotype specification as it is only found in B-cell tumours in humans. We have employed the invertor conditional technology to create a model of MLL-AF4, in which a floxed AF4 cDNA was knocked into Mll in the opposite orientation for transcription. Cell-specific Cre expression was used to generate Mll-AF4 expression. The mice develop exclusively B-cell lineage neoplasias, whether the Cre gene was controlled by B- or T-cell promoters, but of a more mature phenotype than normally observed in childhood leukaemia. These findings show that the MLL-AF4 fusion protein does not have a mandatory role in multi-potent haematopoietic stem cells to cause cancer and indicates that MLL-AF4 has an instructive function in the phenotype of the tumour.

The versatile mixed lineage leukaemia gene MLL and its many associations in leukaemogenesis.

The marked association of abnormalities of chromosome 11 long arm, band q23, with human leukaemia led to the identification of the 11q23 gene called MLL (or HTRX, HRX, TRX1, ALL-1). MLL can become fused with one of a remarkable panoply of genes from other chromosome locations in individual leukaemias, leading to either acute myeloid or lymphoid tumours (hence the name MLL for mixed lineage leukaemia). The unusual finding that a single protein could be involved in both myeloid and lymphoid malignancies and that the truncated protein could do so as a fusion with very disparate partners has prompted studies to define the molecular role of MLL-fusions in leukaemogenesis and to the development of MLL-controlled mouse models of leukaemogenesis. These studies have defined MLL-fusion proteins as regulators of gene expression, controlling such elements as HOX genes, and have indicated a variety of mechanisms by which MLL-fusion proteins contribute to leukaemogenesis.

Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules.

The use of antibodies in medicine and research depends on their specificity and affinity in the recogniton and binding of individual molecules. However, these applications are limited to the extracellular targets. Advances in antibody engineering has allowed the manipulation of the antibody segments containing the antigen-binding regions and generation of small fragments that can be stably expressed in cells. These entities are called intracellular antibodies or intrabodies and have being successfully applied, mainly in the scFv format, to inhibit the function of intracellular target proteins in specific cellular compartments. As new techniques to select and isolate intrabody fragments have been developed, intrabodies are beginning to be used to interfere with the function of a greater number of relevant disease targets. Just as monoclonal antibodies are opening a new era in human therapeutics, intrabodies promise a new prospective for antibody tools for therapy and research. Their varied mode of action gives intrabodies great potential in different approaches in the treatment of human diseases, as well as in the area of functional genomics for characterisation of novel gene products and subsequent validation as potential drug targets. While techniques for identifying functional intrabodies have improved, there are still many significant problems to be overcome before intrabodies can actually be used in treatment of diseases such as cancer, AIDS or neuro-degenerative disorders.

Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice.

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene.

Chromosome 3 allele loss in preinvasive bronchial abnormalities and carcinogen-exposed, histologically normal bronchial epithelium indicates that it is an early, possibly the first, somatic genetic change in lung tumor development. Candidate tumor suppressor genes have been isolated from within distinct 3p regions implicated by heterozygous and homozygous allele loss. We have proposed that DUTT1, nested within homozygously deleted regions at 3p12-13, is the tumor suppressor gene that deletion-mapping and tumor suppression assays indicate is located in proximal 3p. The same gene, ROBO1 (accession number ), was independently isolated as the human homologue of the Drosophila gene, Roundabout. The gene, coding for a receptor with a domain structure of the neural-cell adhesion molecule family, is widely expressed and has been implicated in the guidance and migration of axons, myoblasts, and leukocytes in vertebrates. A deleted form of the gene, which mimics a naturally occurring, tumor-associated human homozygous deletion of exon 2 of DUTT1/ROBO1, was introduced into the mouse germ line. Mice homozygous for this targeted mutation, which eliminates the first Ig domain of Dutt1/Robo1, frequently die at birth of respiratory failure because of delayed lung maturation. Lungs from these mice have reduced air spaces and increased mesenchyme, features that are present some days before birth. Survivors acquire extensive bronchial epithelial abnormalities including hyperplasia, providing evidence of a functional relationship between a 3p gene and the development of bronchial abnormalities associated with early lung cancer.

Chromosomal translocation master genes, mouse models and experimental therapeutics.

Molecular biologists have elucidated general principles about chromosomal translocations by cloning oncogenes or fusion genes at chromosomal translocation junctions. These genes invariably encode intracellular proteins and in acute cancers, often involve transcription and developmental regulators, which are master regulators of cell fate (e.g. LMO2 which is involved in acute leukaemia). Chromosomal translocations are usually associated with specific cell types. The reason for this close association is under investigation using mouse models. We are trying to emulate the cell-specific consequences of chromosomal translocations in mice using homologous recombination in embryonic stem cells to generate de novo chromosomal translocations or to mimic the consequence of these translocations. In addition, chromosomal translocation genes and their products are important targets for therapy. We have designed new therapeutic strategies which include antigen-specific recruitment of endogenous cellular pathways to affect cellular viability and a novel structured form of antisense to ablate the function of fusion mRNAs. We will evaluate these procedures in the mouse models of chromosomal translocations and the long term aim is to perfect rapid procedures for characterizing patient-specific chromosomal translocations to tailor therapy to individual patients.

T cell tumorigenesis in Lmo2 transgenic mice is independent of V-D-J recombinase activity.

The LMO2 gene is involved in T-cell acute leukaemia (T-ALL) in children with chromosomal translocations t(11;14)(p13;q11) or (7;11)(q35;p13). Transgenic expression of Lmo2 in T cells results in clonal tumours with long latency indicating that mutations in other genes are required for the development of overt tumours. RAG V-D-J recombinase can mediate genetic transposition and thus might create the secondary mutations necessary for T-ALL. Tumour development was compared in Lmo2 transgenic mice in the presence or absence of the Rag1 gene. No difference was observed in the rate of tumour formation nor in tumour histology in Lmo2-transgenic mice with or without Rag1. We conclude that, in this model, RAG recombinase is not a major mediator of mutations needed for T cell tumorigenesis and that antigen binding to alpha-beta or to gamma-delta T cell receptor does not play a role in tumorigenesis. The driving force behind the mutational process involved in this transgenic model remains obscure.

Mouse models of human chromosomal translocations and approaches to cancer therapy.

Cancer arises because of genetic changes in somatic cells, eventually giving rise to overt malignancy. Principle among genetic changes found in tumor cells are chromosomal translocations which give rise to fusion genes or enforced oncogene expression. These mutations are tumor-specific and result in production of tumor-specific mRNAs and proteins and are attractive targets for therapy. Also, in acute leukemias, many of these molecules are transcription regulators which involve cell-type-specific complexes, offering an alternative therapy via interfering with protein-protein interaction. We are studying these various features of tumor cells to evaluate new therapeutic methods. We describe a mouse model of de novo chromosomal translocations using the Cre-loxP system in which interchromosomal recombination occurs between the Mll and Af9 genes. We are also developing other in vivo methods designed, like the Cre-loxP system, to emulate the effects of these chromosomal abnormalities in human tumors. In addition, we describe new technologies to facilitate the intracellular targeting of fusion mRNAs and proteins resulting from such chromosomal translocations. These include a masked antisense RNA method with the ability to discriminate between closely related RNA targets and the selection and use of intracellular antibodies to bind to target proteins in vivo and cause cell death. These approaches should also be adaptable to targeting point mutations or to differentially expressed tumor-associated proteins. We hope to develop therapeutic approaches for use in cancer therapy after testing their efficacy in our mouse models of human cancer.

The T cell oncogene Tal2 is necessary for normal development of the mouse brain.

Transcription factors are commonly involved in leukemia by activation through chromosomal translocations and normally function in cell type(s) that differ from that of the tumor. TAL2 is a member of a basic helix-loop-helix gene family specifically involved in T cell leukemogenesis. Null mutations of Tal2 have been made in mice to determine its function during development. Tal2 null mutant mice show no obvious defects of hematopoiesis. During embryogenesis, Tal2 expression is restricted to the developing midbrain, dorsal diencephalon, and rostroventral diencephalic/telencephalic boundary, partly along presumptive developing fiber tracts. The null mutant mice are viable at birth but growth become progressively retarded and they do not survive to reproductive age. Tal2-deficient mice show a distinct dysgenesis of the midbrain tectum. Due to loss of superficial gray and optical layers, the superior colliculus is reduced in size and the inferior colliculus is abnormally rounded and protruding. Death is most likely due to progressive hydrocephalus which appears to be caused by obstruction of the foramen of Monro (the connection between the ventricles of the forebrain). Thus, in addition to its oncogenicity when ectopically expressed, Tal2 normally plays a pivotal role in brain development and without this gene, mice cannot survive to maturity.