Day 1 :
Keynote Forum
William George Whitford
GE Healthcare, USA
Keynote: Bioinks for 3D Bioprinting: A Development Parameters Review
Time : 09:35-10:10
Biography:
William George Whitford is a Strategic Solutions Leader for BioProcess at GE Healthcare Life Sciences in Logan, UT with over 20 years experience in biotechnology product and process development. He has joined the company as an R&D Team Leader developing products supporting biomass expansion, protein expression and virus secretion in mammalian and invertebrate cell lines. He has published over 300 articles, book chapters and patents in a number of fields in the biotechnology arena.
Abstract:
Additive manufacturing (or 3D printing) is supporting innovations in many areas. 3D bioprinting is the 3D printing of materials and cells into functional living assemblies or tissues. It is being applied to in vitro analytics and modeling as well as regenerative medicine. Challenges remain in many areas, including in the development of printing fluids and cell culture media employed in the printing process and post printing construct incubation and development. These bioinks have critical nutritional factor, physicochemical and rheological parameters as determined by the cell types, structural matrices and final applications involved. Functional goals of bioinks include maintenance of cell viability, development of cell functionality and control of CD phenotype and differentiation as well as providing structural support and accommodation of matrix requirements. Components of 4D bioprinting, where the deposited mass responds and evolves to its host environment will be addressed.
Keynote Forum
Yu-Chan Chao
Academia Sinica, Taiwan
Keynote: Display of Influenza Virus Hemagglutinin On Baculovirus Envelope for Antibody Production
Time : 10:10-10:45
Biography:
Yu-Chan Chao has received his PhD from University of Arkansas and Postdoctoral training at the Cold Spring Harbor Laboratory and Cornell University. He previously served as the Dean of the College of Life Science, National Chung-Hsing University and as Deputy Director at the Institute of Molecular Biology, Academia Sinica, and he is currently a Professor at this latter institute. He has received three Distinguished Research Awards from the Ministry of Science and Technology, Taiwan and was elected as a Distinguished Research Fellow. He has published more than 70 papers and serves as an Editor for several highly regarded international journals.
Abstract:
Baculoviruses are a group of double-stranded DNA viruses that only infect invertebrates; specifically, lepidopteran insect larvae. They have been approved by many countries as beneficial microbial agents for eradicating insect pests and are also deemed safe for medical applications. Here, we used Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a typical baculovirus as a tool to present the hemagglutinin (HA) of influenza virus onto baculovirus envelope, which we name HA-Bac, as an antigen to immunize mouse for the generation of antibody against hemagglutinin. Anchoring of HA onto the baculovirus envelope required the cytoplasmic domain (CTD) of GP64, a baculovirus envelope protein. HA-Bac can express a GP64-CTD-fused HA on the envelope of baculoviruses and insect cell membranes. Both HA-Bac and cell membrane-anchored HA protein were purified from HA-Bac-infected insect cells. Three mice per individual treatment were injected separately with purified HA, as either 0.06 or 30 μg/injection. In addition, a 1×109/shot of HA-Bac (containing 0.06 μg HA) was also applied to injected mice with or without adjuvant. We found that mice injected three times with 0.06 μg/shot purified HA could not generate detectable antibody but all of the 30 μg/shot HA and HA-Bac injected mice (with or without adjuvant) generated strong antibodies against hemagglutinin. These antibodies interacted with HA, as evidence by Western blot analysis and hemagglutination inhibition assays. The results of these experiments show that presentation of hemagglutinin on the envelope of baculovirus can serve as a convenient, easy-to-purify antigen for stimulating antibody production in animals.
Keynote Forum
Ronald James Christie
MedImmune, USA
Keynote: Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides
Time : 10:45-11:10
Biography:
Ronald James Christie has received his PhD in Chemistry from Colorado State University in 2006 and completed Post-doctorate at the University of Tokyo. He has published more than 25 articles in scientific journals on topics including; drug delivery, materials science and bioconjugation. He is currently a Scientist at MedImmune, working to develop antibody-drug conjugate therapeutics.
Abstract:
Maleimides are often used to link drugs to cysteine thiols for production of antibody-drug conjugates (ADCs). However, ADCs formed with traditional N-alkyl maleimides have variable stability in the bloodstream leading to loss of drug and decreased therapeutic activity. Loss of payload occurs via a retro-Michael reaction that regenerates the maleimide group, which can then react with other thiol species that serve as an off-target sink for liberated ADC drugs. Here, a maleimide based strategy to produce stable ADCs while also maintaining efficient thiol reactivity is presented. The current approach aimed to accelerate thiosuccinimide hydrolysis after thiol-maleimide conjugation to produce a stable thioether bond. The rate of conjugate stabilization (i.e., thiosuccinimide hydrolysis) was measured directly on antibodies using mass spectrometry by observing the addition of water (18 amu) to the antibody conjugate. Conjugate stability was confirmed in both thiol-containing buffer and mouse serum at 37 oC over a period of 7 days. An ADC prepared with monomethyl-auristatin-E (MMAE) comprising stable maleimide chemistry maintained potency towards cultured cancer cells following serum incubation whereas an ADC prepared with MMAE linked through a traditional N-alkyl maleimide lost potency over time. This approach could also be applied to other drug delivery or diagnostic technologies such as polymer conjugates, targeted nanoparticles and biosensors where stable thiol conjugates are critical for performance.
- Autoimmune Antibodies, Antibody Engineering, Antibodies: Infectious Diseases, Auto Antibodies, Monoclonal Antibodies & Monoclonal Antibody Therapy, Antibodies: Medical Applications, Immunotherapy and Immune Checkpoints, Anti-Cancer Antibodies, Antibody Humanization Technologies
Chair
William Whitford
GE Health Care, USA
Co-Chair
Yu-Chan Chao
Academia Sinica, Taiwan
Session Introduction
Ronald James Christie
MedImmune, USA
Title: Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides
Time : 10:45-11:10
Biography:
Ronald James Christie has received his PhD in Chemistry from Colorado State University in 2006 and completed Post-doctorate at the University of Tokyo. He has published more than 25 articles in scientific journals on topics including; drug delivery, materials science and bioconjugation. He is currently a Scientist at MedImmune, working to develop antibody-drug conjugate therapeutics.
Abstract:
Maleimides are often used to link drugs to cysteine thiols for production of antibody-drug conjugates (ADCs). However, ADCs formed with traditional N-alkyl maleimides have variable stability in the bloodstream leading to loss of drug and decreased therapeutic activity. Loss of payload occurs via a retro-Michael reaction that regenerates the maleimide group, which can then react with other thiol species that serve as an off-target sink for liberated ADC drugs. Here, a maleimide based strategy to produce stable ADCs while also maintaining efficient thiol reactivity is presented. The current approach aimed to accelerate thiosuccinimide hydrolysis after thiol-maleimide conjugation to produce a stable thioether bond. The rate of conjugate stabilization (i.e., thiosuccinimide hydrolysis) was measured directly on antibodies using mass spectrometry by observing the addition of water (18 amu) to the antibody conjugate. Conjugate stability was confirmed in both thiol-containing buffer and mouse serum at 37 oC over a period of 7 days. An ADC prepared with monomethyl-auristatin-E (MMAE) comprising stable maleimide chemistry maintained potency towards cultured cancer cells following serum incubation whereas an ADC prepared with MMAE linked through a traditional N-alkyl maleimide lost potency over time. This approach could also be applied to other drug delivery or diagnostic technologies such as polymer conjugates, targeted nanoparticles and biosensors where stable thiol conjugates are critical for performance.
Ronald James Christie
MedImmune, USA
Title: Stabilization of cysteine-linked antibody drug conjugates with N-aryl maleimides
Time : 10:45-11:10
Biography:
Ronald James Christie has received his PhD in Chemistry from Colorado State University in 2006 and completed Post-doctorate at the University of Tokyo. He has published more than 25 articles in scientific journals on topics including; drug delivery, materials science and bioconjugation. He is currently a Scientist at MedImmune, working to develop antibody-drug conjugate therapeutics.
Abstract:
Maleimides are often used to link drugs to cysteine thiols for production of antibody-drug conjugates (ADCs). However, ADCs formed with traditional N-alkyl maleimides have variable stability in the bloodstream leading to loss of drug and decreased therapeutic activity. Loss of payload occurs via a retro-Michael reaction that regenerates the maleimide group, which can then react with other thiol species that serve as an off-target sink for liberated ADC drugs. Here, a maleimide based strategy to produce stable ADCs while also maintaining efficient thiol reactivity is presented. The current approach aimed to accelerate thiosuccinimide hydrolysis after thiol-maleimide conjugation to produce a stable thioether bond. The rate of conjugate stabilization (i.e., thiosuccinimide hydrolysis) was measured directly on antibodies using mass spectrometry by observing the addition of water (18 amu) to the antibody conjugate. Conjugate stability was confirmed in both thiol-containing buffer and mouse serum at 37 oC over a period of 7 days. An ADC prepared with monomethyl-auristatin-E (MMAE) comprising stable maleimide chemistry maintained potency towards cultured cancer cells following serum incubation whereas an ADC prepared with MMAE linked through a traditional N-alkyl maleimide lost potency over time. This approach could also be applied to other drug delivery or diagnostic technologies such as polymer conjugates, targeted nanoparticles and biosensors where stable thiol conjugates are critical for performance.
Azad K Kaushik
University of Guelph, Canada
Title: Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope
Time : 11:25-11:50
Biography:
Azad K. Kaushik has published over 87 research articles and book chapters, and co- edited two books [Molecular Immunobiology of Self-Reactivity (1992) and Comparative Immunoglobulin genetics (2014)]. He is on the editorial boards of several immunology journals and is a Consultant to various international organizations. He was recognized as The Esther Z. Greenberg Honors Chair in Biomedical Research, and Visiting Professor, Oklahoma Medical Research Foundation, USA, in 1998. He received BVSc&AH (Honors) in 1976 and MVSc (1978) from the Faculty of Veterinary Science, Hisar, Haryana, India; followed by Docteur es Science (DSc) in Immunology (1987) from the Pasteur Institute (University of Paris VII), Paris, France. He has been teaching Immunology at the University of Guelph since 1991.
Abstract:
Some bovine antibodies are the largest known to exist in a species because of an exceptionally long CDR3H (up to 61 amino acids). The exceptionally long CDR3H is encoded by an unusually long germline IGHD genes together with insertion of “a” nucleotide rich conserved short nucleotide sequence at the IGHV-IGHD junction. The atypical CDR3H confers unique “knob and stalk” structural architecture where configurational diversity of the knob is generated by variable intra-CDR3H disulfide bridges. The knob is separated by solvent exposed stalk formed by anti-parallel beta strands. Structural features of the bovine antibody with an exceptionally long CDR3H can be exploited for the development of new therapeutics, vaccines and drugs. In this context, I will discuss structural optimization of bovine scFvs to enhance viral neutralization potency. An evidence for subtle influence of framework residues on viral neutralization functions will be presented. I will discuss our recent data on bovine scFv with exceptionally long CDR3H antigenized with B-epitope that induces specific immune response. To this end, we first developed functional scFv with an exceptionally long CDR3H followed by grafting of a B-epitope (gC156) from bovine herpes virus-1 into the CDR3H. The grafted B-epitope in bovine scFv with an exceptionally long CDR3H sustained configuration similar to the native epitope. The antigenized scFv (gC156scFv1H12) induced higher antibody response as compared to free recombinant gC156 fragment in the calves. To conclude, antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing new vaccines for humoral protection against infectious agents.
Ljudmila Stojanovich
Belgrade University, Serbia
Title: Diagnostic challenges in Antiphospholipid Syndrome (APS), or Hughes Syndrome
Time : 11:50-12:15
Biography:
Ljudmila Stojanovich is the Scientific Director and Research Professor at the Bezhanijska Kosa, University Medical Center of Belgrade University in Serbia. Her research interests include Systemic Lupus Erythematosus (SLE), Antiphospholipid syndrome (APS) and vaccination in patients with autoimmune rheumatic diseases. She is an author of three monographs and of about 250 articles on various aspects of autoimmune rheumatic disorders, published in both international and domestic journals and in conference proceedings. She is on the Editorial Boards and Reviewer of numerous journals. She is also a Member of a number international projects, including “the European Forum on Antiphospholipid Antibodies/Catastrophic form”, “Multicenter studies antiphospholipid antibodies, infections and autoimmune diseases”. She is a Mentor to and supervises a number of Post-doctoral students. She has longstanding experience in the clinical management of patients with autoimmune rheumatic diseases, including SLE, APS and allied diseases. She is an Investigator in several clinical trials, including patients with SLE.
Abstract:
Antiphospholipid syndrome (APS) or Hughes syndrome is probably the most important paradigm of systemic autoimmune disease. Although APS is now a well-described difficult to diagnose entity, it took many decades to define the diagnostic criteria. The latest classification criteria for diagnosing APS are the 2006 Sapporo criteria that require the presence of at least one clinical manifestation and one positive laboratory criterion. Following the application of the Sapporo criteria, controversy arose because those criteria identify a more homogeneous group of APS patients at the expense of excluding another, a group collectively referred to as seronegative APS. The need for more guidelines regarding the detection of lupus anticoagulant is now fulfilled by the updated Scientific Standardization Committee guidelines. There are recent studies present on the most promising antibodies of this heterogeneous Antiphospholipid syndrome (APL) family. Nowadays, APS is increasingly recognized as a multisystem disease, the clinical expression of which may include (many non-criteria ARA) cardiac, neurological, hematological, cutaneous and other manifestations. Special attention should be given to secondary APS patients when they are subject to high-risk events: 7-10% of primary APS patients may go on to develop secondary APS with SLE. Despite updates of the diagnostic criteria, the diagnosis of SLE and APS remains difficult.
Biography:
Scott K Dessain is the Co-Founder and CTO of Immunome Inc., and an Associate Professor at the Lankenau Institute for Medical Research, Wynnewood, PA. He has received MD and PhD degrees from Yale University, Postgraduate Medical Training at Brigham and Women’s Hospital and Dana Farber/Partners Cancer Care Boston, MA. His Post-doctoral Training was with Dr. Robert Weinberg, with whom he discovered the scientific basis for the RealMAbs platform. He currently attends in clinical oncology at the Lankenau Medical Center and runs an Immunology Research Laboratory.
Abstract:
Tumor draining lymph nodes contain B-cells that produce antibodies that are reactive with tumor neoantigens. The Immunome RealMAb system directly immortalizes B-cells so that they can grow and maintain antibody secretion indefinitely. RealMAb uses a genetically stabilized fusion partner cell line licensed from the Whitehead Institute in Cambridge, MA. RealMAb produces libraries of thousand of unique, stable cells that secrete high levels of human monoclonal antibodies (mAbs) in their real “native” configurations. The Immunome ScreenMAb system is an integrated, high-throughput platform for screening RealMAb libraries to identify the mAbs that bind novel cancer neoantigens and to assess their potential as anti-tumor therapeutic leads. We are examining B-cell repertoire in lymph nodes from breast cancer patients and non-small cell lung cancer patients and in peripheral blood memory B-cell populations of solid tumor patients who respond to anti-PD1 antibody therapy. We have cloned a variety of human mAbs that specifically recognize human cancer neoantigens. Many of the neoantigens are expressed on multiple tumor cell types indicating diverse breast cancer and lung cancers. Neoantigens selected by the studying the human anti-tumor antibody response includes many that cannot be identified by other methods. The RealMAbs used to identify and characterize the biology of the cancer neoantigens can themselves be used as molecular leads for the creation of novel therapeutics based on recombinant mAbs, antibody-drug conjugates and Chimeric Antigen Receptors for T-cells and NK cells.
Ravi P Sahu
Wright State University, USA
Title: Tumor microenvironment associated immune cells mediate platelet-activating factor induced tumor growth and treatment effectiveness
Time : 12:40-13:05
Biography:
Ravi P Sahu has obtained his Bachelor’s degree in Biology, Physics and Chemistry from Allahabad University at Allahabad and Master’s degree in Biochemistry from Dr. R.M.L. Avadh University at Faizabad, India. His interest in studying the clinical aspects of human diseases began with his doctoral work at Sanjay Gandhi Postgraduate Institute of Medical Sciences at Lucknow, India. He studied the role of genetic mutations in the transcription factor hepatocyte nuclear factor-1 alpha (HNF-1ï¡), mitochondrial genes and autoantibodies against islet antigens in the etiology of early onset type 2 diabetes mellitus. He began his postdoctoral career determining the role of natural compounds in cancer chemoprevention at University of Pittsburgh at Pittsburgh, PA and Texas Tech University Health Sciences Center at Amarillo, TX. His second postdoctoral work at Indiana University was focused on investigating the impact of various pro-oxidative stressors in the pathogenesis of cutaneous immunity and non-melanoma skin cancer. In 2012, he began his independent research career as an Assistant Professor at Indiana University, Indianapolis and in 2015 he moved to Wright State University at Dayton, OH. His long term research goal is to understand the mechanisms of immunomodulatory effects of environmental and therapeutic pro-oxidative stressors in cancer growth and cancer therapeutic responses, particularly melanoma and lung cancer.
Abstract:
The exposure of pro-oxidative stressors modulates host anti-tumor immunity against cancer. Studies from our group have demonstrated that pro-oxidative stressors both from environmental and therapeutic exposures generate the potent lipid mediator with platelet-activating factor (PAF) agonist activity. These PAF agonists mediate systemic immunosuppression and augment the growth of subcutaneously implanted Murine melanoma and Lewis lung carcinoma tumors in a PAF-receptor (PAF-R) dependent manner. Importantly, PAF-dependent enhanced growth of melanoma tumors was mediated via up-regulation of tumor microenvironment associated regulatory T-cells (Tregs) and myeloid derived suppressor cells (MDSCs). Notably, depleting antibodies against Tregs and MDSCs blocked PAF agonists mediated systemic immunosuppression and melanoma tumor growth. As various treatment modalities including chemotherapeutic agents can act as potent pro-oxidative stressors, we demonstrated that chemotherapy generate PAF and other oxidized PAF-R agonists from melanoma cells/tumors in a process blocked by antioxidants. In a murine dual tumor model, our studies demonstrate that intratumoral injection of chemotherapy to one tumor augments the growth of other (untreated) tumors in a PAF-R dependent manner. Importantly, this chemotherapy mediated enhanced growth of secondary tumors was attenuated via systemic supplementation of antioxidants, COX-2 inhibitors as well as depleting Tregs. Interestingly, we demonstrated that perfusates collected from melanoma patients’ post-chemotherapy exhibited significant PAF-R agonistic activity compared to perfusates obtained prior to chemotherapy. Altogether, our studies indicate the importance of the PAF-R mediated pathway in modulating tumor growth and treatment effectiveness and suggest it’s implication as a promising approach for cancer treatment.
Juan Zhang
China Pharmaceutical University, China
Title: Tumor specific antigen targeted antibody fused with MICA stimulates NKG2D mediated immunosurveillance and exhibits potent anti-tumor activity against malignancies
Time : 13:50-14:15
Biography:
Juan Zhang is currently working as an Associate Professor of Microbiology & Biochemical Pharmaceutics. She has obtained her BSc in Microbiological Pharmacy, MSc and Doctor’s degree (PhD) from China Pharmaceutical University. In 2007, she was trained at Department of Chemical Engineering, Imperial College London as a co-supervised PhD Student and a Visiting Scholar at University of California, Los Angles (UCLA) from 2013 to 2014. She was awarded with Sanofi-Aventis Young Scientist Prize in Biological Medicine in 2011 and 333-Project Talent of Jiangsu Province in 2013, Young-and-Middle Aged Leading Academic Professor of Jiangsu Province in 2014. She is an Editorial Board Member of Chinese Journal of Biochemical Pharmaceutics and Youth Member of Jiangsu Society of Biochemistry and Molecular Biology. Currently her research focuses on the discovery of novel antibodies using phage display fully human antibody library and Hybridoma Technology. One of the antibodies she invented was transferred and evaluated in cell therapy. A number of bispecific antibodies she designed exhibit promising anti-cancer efficacy.
Abstract:
MHC class I-related chain A (MICA) is a principal immunoligand of the natural killer (NK) cell receptor NK group 2, member D (NKG2D) and plays a key role in NK cell-mediated immune recognition. Shedding of MICA from tumor cells leads to immunosuppression. To trigger NKG2D-mediated immunosurveillance, we designed antibody fusion protein which consisted of human MICA and antibody component. These bispecific proteins maintained the properties of the parental antibodies. Upon binding to the tumor-associated antigens, the MICA portion was expected to promote the recognition of tumor cells by NK cells and to enhance NK cell-mediated cytotoxicity. In tumor-bearing nude mice, these fusion proteins specifically targeted to relevant tumor tissues, where they effectively recruited NK cells and induced the release of cytokines. The MICA based antibody fusion proteins have attractive potentials for clinical applications and this design provides a new approach for tumor-targeting immunotherapy.
Andrew Tsourkas
University of Pennsylvania, USA
Title: Rapid production of bispecific antibodies using ‘off-the-shelf’ IgG
Time : 14:15-14:40
Biography:
Andrew Tsourkas is a Professor and undergraduate chair of Bioengineering at the University of Pennsylvania in Philadelphia, PA. He has received his Bachelor’s degree in Mechanical Engineering in 1997 from Cornell University, Master’s degree in 1999 from Johns Hopkins University and PhD in Biomedical Engineering from the Georgia Institute of Technology/Emory University joint PhD program in 2002. He has then conducted a two-year Post-doctoral Fellowship in Cellular and Molecular Imaging in the Department of Radiology at Harvard University, before joining Penn in 2004. He is currently the Associate Director for the Center for Targeted Therapeutics and Translational Nanomedicine. He has over 80 peer-reviewed and invited publications and was a recipient of the Wallace H. Coulter Foundation Early Career Award and the National Science Foundation Career Award. In 2015, he was also elected as a Fellow to the American Institute for Medical and Biological Engineering. He is an Inventor on over a dozen patents and has founded four biotechnology companies.
Abstract:
All current bispecific antibody platforms require antibody engineering and cloning up front to generate each new formulation. While made to order genes, advanced expression systems and new high efficiency cloning techniques can simplify and accelerate this process; the final products will frequently exhibit a loss of specificity, aggregation, light chain swapping, heterogeneity, etc. For many academic labs, this creates a barrier to entry into the field of bispecifics and for industry this limits the throughput of bispecific antibody production. Recently, we developed a simple method to site specifically and covalently attach an anti-CD3 single chain variable fragment (scFv) to any off-the-shelf, full length human Immunoglobulin G (IgG). This approach to making bispecific antibodies does not require antibody engineering, cloning or modifications. As a result, we can prepare libraries of bispecific antibodies with high purity in just a few hours. As proof of principal, we created a small library of eight bispecific antibodies simultaneously. IgG binding affinity was not affected by attachment of scFv to each heavy chain and T-cell mediated cell lysis assays confirmed potencies that were comparable to other bispecific antibody platforms.
Cenk Suphioglu
Deakin University, Australia
Title: Mimtags: The usage of phage display technology tp produce novel protein-specific probes
Time : 14:40-15:05
Biography:
Cenk Suphioglu has completed his PhD from the University of Melbourne in 1994 and has over 20 years of research experience. He has more than 70 publications and several patents to his credit. He is currently an Associate Professor of Biomedical Science and Head of the Neuro Allergy Research Laboratory (NARL).
Abstract:
In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: Epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesized, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterized had identical peptide sequence motifs. Only one was synthesized for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterize mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies.