Larry Green

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (κ) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 Vκ genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Antigen–specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and κ light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen–specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

Fully Human Monoclonal Antibodies to Hepatocyte Growth Factor with Therapeutic Potential against Hepatocyte Growth Factor/c-Met–Dependent Human Tumors

c-Met is a well-characterized receptor tyrosine kinase for hepatocyte growth factor (HGF). Compelling evidence from studies in human tumors and both cellular and animal tumor models indicates that signaling through the HGF/c-Met pathway mediates a plethora of normal cellular activities, including proliferation, survival, migration, and invasion, that are at the root of cancer cell dysregulation, tumorigenesis, and tumor metastasis. Inhibiting HGF-mediated signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of human tumors. Toward this goal, we generated and characterized five different fully human monoclonal antibodies that bound to and neutralized human HGF. Antibodies with subnanomolar affinities for HGF blocked binding of human HGF to c-Met and inhibited HGF-mediated c-Met phosphorylation, cell proliferation, survival, and invasion. Using a series of human-mouse chimeric HGF proteins, we showed that the neutralizing antibodies bind to a unique epitope in the beta-chain of human HGF. Importantly, these antibodies inhibited HGF-dependent autocrine-driven tumor growth and caused significant regression of established U-87 MG tumor xenografts. Treatment with anti-HGF antibody rapidly inhibited tumor cell proliferation and significantly increased the proportion of apoptotic U-87 MG tumor cells in vivo. These results suggest that an antibody to an epitope in the beta-chain of HGF has potential as a novel therapeutic agent for treating patients with HGF-dependent tumors.

Antibody discovery: the use of transgenic mice to generate human monoclonal antibodies for therapeutics.

Technical advances made in the 1980s and early 1990s resulted in monoclonal antibodies that are now approved for human therapy. Novel transgenic mouse strains provide a powerful technology platform for creating fully human monoclonal antibodies as therapeutics; ten such antibodies have entered clinical trials since 1998 and more are in preclinical testing. Improved transgenic mouse strains provide a powerful technology platform for creating human therapeutics in the future.

Production of Antigen-Specific Human Antibodies from Mice Engineered with Human Heavy and Light Chain YACsa

Our paper describes the introduction of large fragments of both the human heavy and light chain Ig genes into the mouse germline to create a mouse strain capable of producing a broad repertoire of antigen-specific, fully human antibodies. The human immunoglobulin gene sequences were functional in the context of the mouse machinery for antibody recombination and expression, either in the presence or absence of functional endogenous genes. This was demonstrated by their ability to undergo diverse rearrangement, to be expressed at significant levels, and to exclude expression of mouse immunoglobulins irrespective of their copy number or site of integration. The decrease in susceptibility to influence by adjacent genomic sequences may reflect the greater size, variable gene content, or structural integrity of the human Ig YACs and/or the presence of unidentified but important regulatory elements needed for optimal expression of the human immunoglobulin genes and their correct regulation. Our results show that mouse B cells coexpressing human heavy and kappa chains, upon immunization, can produce antigen-specific, fully human antibodies. Furthermore, the human heavy and kappa chain YACs induced differentiation and maturation of the growth-arrested B-cell lineage in mice with inactivated endogenous Ig genes, leading to the production of a diverse repertoire of fully human antibodies at levels approaching those in normal serum. These results suggest the potential value of these mice as a source of fully human antibodies for human therapy. Furthermore, it is expected that such mice would lack immunological tolerance to and thus readily yield antibodies to human proteins, which may constitute an important class of targets for monoclonal antibody therapy. Our findings suggest that the introduction of even larger portions of the human heavy and light chain loci, which should be achievable with the ES cell-yeast spheroplast fusion technology described, will result in strains of mice ultimately capable of recapitulating the full antibody repertoire characteristic of the human humoral response to infection and immunization. The present and future mouse strains may prove to be valuable tools for studying the molecular mechanisms and regulatory sequences influencing the programmed assembly and expression of human antibodies in the normal immune response, as well as the abnormal response characteristic of autoimmune disease and other disorders. The strategy we have described for the introduction of large segments of the human genome into mice in conjunction with the inactivation of the corresponding mouse loci may also have broad applicability to the investigation of other complex or uncharacterized loci.

Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

Development of a new fully human anti-CD20 monoclonal antibody for the treatment of B-cell malignancies

SummaryDespite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin’s lymphomas, there is a recognized need to develop new agents with improved efficacy. Towards this end, using XenoMouse® technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to rituximab, in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcγRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.

Reply to Barroso-Sousa R et al.

G-CSF prophylaxis is routinely recommended for every cycle of dose-dense chemotherapy regimens such as ddAC-T [1e3]. In a small, retrospective, single-institution study, Barroso-Sousa et al. described G-CSF use during the T portion of ddAC-T: 87% of patients (n 1⁄4 135) received G-CSF during 1 cycle of T, 13% (n 1⁄4 21) did not. In contrast with previous studies showing G-CSF benefit during T [4,5], Barroso-Sousa reported no significant differences in dose delays or treatment completion. However, as the authors discussed, this study was limited by a small sample size, which precluded the ability to accurately compare outcomes. In addition, appropriate G-CSF use could not be adequately evaluated, as G-CSF use was at the physicians discretion and patient risk factors were not thoroughly assessed or controlled for. Furthermore, while high relative dose intensity (RDI) is associated with improved clinical outcomes in patients with early-stage breast cancer [6], the effect of G-CSF use on chemotherapy dose reductions and RDI were not described by Barroso-Sousa et al. We appreciate the hypothesis-generating data in this study. Given the limitations of this retrospective study, we look forward to the results of the prospective trial currently underway (NCT02698891) to describe baseline myelotoxicity associated

Biologics, Pharmacovigilance, and Patient Safety: It’s All in the Name

UNLABELLED The most appropriate naming convention for biologics and biosimilars has been an area of significant debate. The ultimate decision will have an impact on patient safety, pharmacovigilance program effectiveness, and, potentially, the overall adoption of biosimilars in the United States. This article reviews some of the advantages and disadvantages of various naming approaches. For clarity in communication, optimal pharmacovigilance, and patient safety, it is recommended that biosimilars be named with a common USAN (United States Adopted Name) with the reference product, along with a suffix that is memorable, such as one associated with the original manufacturer of the product. This approach supports the FDAs mission of protecting patient safety and public health, while minimizing the possibility of inadvertent switching of products and facilitating effective pharmacovigilance. DISCLOSURES No funding supported the writing of this article. Stevenson reports consulting fees from Amgen, Inc., AbbVie, and Pfizer and is employed by Visante. Green is employed by and owns stock in Amgen, Inc. Article concept was contributed primarily by Stevenson, along with Green. The manuscript was written and revised primarily by Stevenson, along with Green. Both authors contributed equally to data interpretation.