Jasbir Sandhu

Problems of Delivery of Monoclonal Antibodies Pharmaceutical and Pharmacokinetic Solutions

SummaryMonoclonal antibodies to tumour-associated antigens have great theoretical potential for the specific targeting of radioactivity and anti-neoplastic agents to tumours. The clinical success of monoclonal antibody—based cancer diagnosis and therapy depends, however, on solving a number of pharmacokinetic delivery problems. These include: (i) slow elimination of monoclonal antibodies from the blood and poor vascular permeability; (ii) low and heterogeneous tumour uptake; (iii) cross-reactivity with normal tissues; (iv) metabolism of monoclonal antibody conjugates; and (v) immunogenicity of murine forms in humans.As a result of extensive pharmaceutical and pharmacokinetic research conducted over the past 10 to 15 years, several potential solutions to these delivery problems have been identified. Blood concentrations of antibody conjugates may be reduced through regional administration, the use of antibody fragments, interventional strategies and various pre-targeting techniques. Tumour uptake may be increased through administration of higher doses, or the use of agents to increase tumour vascular permeability. Tumour retention of antibody conjugates may be improved by inhibition of metabolism, by using more stable linkage chemistry. Alternatively, normal tissue retention may be decreased through the use of metabolisable chemical linkages inserted between the antibody and conjugated moiety.Very small antigen-binding fragments and peptides that exhibit improved tumour penetration and more rapid elimination from the blood and normal tissues have been prepared by genetic engineering techniques. Chimeric (mouse/human) and human monoclonal antibodies have been developed to circumvent the problem of immunogenicity. Future research will continue to be focused on improvements in the design of monoclonal antibodies for tumour targeting, with the ultimate goal of finally uncovering the ‘magic bullet’ envisioned by Paul Ehrlich almost a century ago.

Protein Engineering of Antibodies

This article reviews the technical advances in antibody engineering and the clinical applications of these molecules. Recombinant DNA technology facilitates the construction and expression of engineered antibodies. These novel molecules are designed to meet specific applications. Although genomic and cDNA cloning have been used widely in the past to isolate the relevant antibody V domains, at present, the PCR-based cloning is the preferred system. Bacterial and mammalian expression systems are used commonly for the production of antibodies, antibody fragments, and antibody fusion proteins. A range of chimeric antibodies with murine V domains joined to C regions from human and other species have been produced and found to exhibit the expected binding characteristics and effector functions. Humanized antibodies have been developed to minimize the HAMA response, and bifunctional immunoglobulins are being used in tumor therapy and diagnosis. Single chain antibodies and fusion proteins with antibody specificities jointed to nonimmunoglobulin sequences provide a source of antibody-like molecules with novel properties. The potential applications of minimal recognition units and antigenized antibodies are described. Combinatorial libraries produced in bacteriophage present an alternative to hybridomas for the production of antibodies with the desired antigen binding specificities. Future developments in this field are discussed also.

A human model of xenogeneic graft-versus-host disease in SCID mice engrafted with human peripheral blood lymphocytes.

Despite previous attempts there is currently no suitable animal model available for xenogeneic graft versus host disease (XGVHD) mediated via human immunocompetent cells. Recently, we have developed an efficient protocol to engraft SCID mice with human peripheral blood lymphocytes (Hu-PBLs). The engraftment efficiency is extremely high, such that 100% of Hu-PBL-SCID mice die of XGVHD within 4 weeks after engraftment with Hu-PBLs (3-5 x 10(7) cells). A series of experiments was performed to investigate the mechanisms involved in the severe XGVHD. The results suggest that XGVHD was induced by human CD4+ T cells, antixenogeneic (antimouse) antibodies, and lymphokines. The SCID mouse model will be extremely valuable for the evaluation and development of immunosuppressive agents and transplantation protocols for human XGVHD.

The use of SCID mice in biotechnology and as a model for human disease.

The use of SCID (severe combined immunodeficient) mice in medical research and biotechnology has increased tremendously in recent years. This review outlines the major characteristics of these animals and the impediments that they pose to the engraftment of human cells and tissues. The development of our SCID mice pretreatment protocol (anti-asialo GM1 antisera and radiation) is described, and the results of xenotransplantation studies of human cells and tissues in these pretreated animals are outlined. Wherever possible, data from transplantation studies (of human tissues and cells) in pretreated and nonpretreated animals are compared. The potential of our pretreated SCID mice for medical research and biotechnology is discussed.

Production of Human Monoclonal Antibodies in SCID Mouse

The advent of hybridoma technology for the production of mouse monoclonal antibodies (MoMAbs) has made it possible to obtain large quantities of antibodies with defined antigen specificity (26). Since then, antibody immunotherapy has become a reality. However, actual clinical application, particularly for therapeutic purposes, has been hampered by the immunogenicity of MoMAbs, known as human anti-mouse antibody (HAMA) response (20). To solve this problem, several antibody engineering techniques have been developed to generate MAbs more suitable for clinical use. These methods include the construction of mouse/human chimeric Abs (7) and humanized MoMAbs (45). However, these antibody engineering methods are laborious and costly. Human monoclonal antibodies (HuMAbs) would be ideal for clinical purposes. Thus, the development of an efficient method to generate HuMAbs with high affinity would be a significant advance in the fields of biotechnology and medicine. We have recently developed a very efficient method to engraft human peripheral blood lymphocytes (Hu-PBL) in severe combined immunodeficient (SCID) mouse (43). The human immune response could be optimized in the Hu-PBL-SCID mouse model, and human antibodies of desired specificity could be generated. In this review we describe a method to obtain HuMAbs with high antigen binding affinity by optimizing the human immune response to specific antigens in the Hu-PBL-SCID mouse model.

Effect of interleukin-6 secreted by engineered human stromal cells on osteoclasts in human bone

The effect of elevated human IL-6 (hIL-6) production by human bone marrow (Hu-BM) stromal cells on osteoclasts in human bone was examined. Human bone was implanted into nonobese diabetic mice with severe combined immunodeficiency (Hu-Bone-NOD/SCID mice). Immunohistochemistry of bone implants and mouse spleens (at 20 weeks), showed human CD45+ cells, B cells, and macrophages in both tissues. Thus, Hu-BM cells survive human bone transplantation and infiltrate mouse tissue. Bone implants had 75 +/- 12% (mean +/- SD) human CD45+ cells, and 9 +/- 4% mouse hematopoietic cells. A retrovirus vector containing the human IL-6 gene was used to transduce Hu-BM stromal cells (IL-6/stromal) and the PA317 cell line (IL-6/PA317). IL-6/ stromal cells (secreting, on average, 17 microg of hIL-6/10(6) cells per 24 h) were injected directly into human bone implants in Hu-Bone-NOD/SCID mice. IL-6/PA317 cells (secreting 16 microg/mL of hIL-6/10(6) cells per 24 h) were injected intraperitoneally into Hu-Bone-NOD/SCID mice. Analyses of sera from both groups of animals showed elevated levels of IL-6. However, only bone implants engrafted with IL-6/stromal cells had a statistically significant increase in osteoclast-lined mineralized trabecular bone surface (BS). Thus, a high concentration of serum hIL-6 in Hu-Bone-NOD/SCID mice alone does not increase osteoclast-lined BS in bone implants. Most importantly, it is the type of human BM cell that secretes the high levels of hIL-6 that is most critical.

Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone

Abstract: Background/aims: Recent advances in molecular cloning of hepatitis C virus (HCV) have enabled us to apply some available HCV molecular clones to experimental studies. However, these investigations have been restricted to chimpanzee models or ‘isolated hepatocytes’ from tree shrews. In this study, we engrafted ‘human liver tissue’ into immunodeficient mice and investigated HCV infection using an infectious molecular clone.

Physiologic human T-cell responses to OKT3 in the human peripheral blood lymphocyte-severe combined immunodeficiency mouse model

BACKGROUND Our goal was to study physiologic responses of human T lymphocytes to OKT3 in the human peripheral blood lymphocyte-severe combined immunodeficiency (hu-PBL-SCID) mouse model. METHODS SCID mice were pretreated with anti-asialo-GM1 (alpha-ASGM1) and radiation, then engrafted with human peripheral blood lymphocytes (PBLs). Seven to 14 days after engraftment, when most human T cells in the spleen of these mice are CD3+/CD4+ and CD3+/CD8+, mice were treated with OKT3 or control antibody. Mice were killed for histopathologic examination, for flow cytometric assessment of the engrafted human lymphocytes, and for analysis of human tumor necrosis factor-alpha serum levels. RESULTS Intravenous injection of 5 microg of OKT3 resulted in early antigenic modulation of engrafted human T lymphocytes, with the emergence of CD3-/CD4+ and CD3-/CD8+ cells in the spleen of hu-PBL-SCID mice. There was an increase in the serum concentration of human tumor necrosis factor-alpha within 4 hr after OKT3 injection, suggesting early T-cell activation. Antigenic modulation and activation of the human lymphocytes in the spleen was followed by their depletion within 24 hr. This human T-cell response to OKT3 in hu-PBL-SCID mice is analogous to the response in humans treated with OKT3 and in BALB/c mice injected with an anti-murine CD3 monoclonal antibody. Graft-versus-host disease in the mice was abrogated by OKT3 treatment, and OKT3-treated mice lived longer than controls. Histopathologic studies showed clearance of lymphocytic infiltration in the liver and lungs of OKT3-treated mice. CONCLUSIONS These findings provide further evidence of functional human immune T cells in the hu-PBL-SCID mouse. This model may have useful applications in the study of transplantation immunology.

Human Gene Therapy

Human gene therapy and its application for the treatment of human genetic disorders, such as cystic fibrosis, cancer, and other diseases, are discussed. Gene therapy is a technique in which a functioning gene is inserted into a human cell to correct a genetic error or to introduce a new function to the cell. Many methods, including retroviral vectors and non-viral vectors, have been developed for both ex vivo and in vivo gene transfer into cells. Vectors need to be developed that efficiently transfer genes to target cells, and promoter systems are required that regulate gene expression according to physiologic needs of the host cell. There are several safety and ethical issues related to manipulating the human genome that need to be resolved. Current gene therapy efforts focus on gene insertion into somatic cells only. Gene therapy has potential for the effective treatment of genetic disorders, and gene transfer techniques are being used for basic research, for example, in cancer, to examine the underlying mechanism of disease. There are still many technical obstacles to be overcome before human gene therapy can become a routine procedure. The current human genome project provides the sequences of a vast number of human genes, leading to the identification, characterization, and understanding of genes that are responsible for many human diseases.