Therapeutic Antibodies

Manufacturing Development and optimization Antibody engineering THERAPEUTIC ANTIBODIES This infographic will explore advances related to the research and development of therapeutic antibodies for dierent disease applications. WHAT ARE ANTIBODIES? THERAPEUTIC MABS Antibodies (Abs) are Y-shaped glycoproteins that are produced by B lymphocytes in response to the presence of a foreign molecule, like a virus. Antibodies recognize and bind to antigens that are presented by the foreign molecule, triggering an immune response that neutralizes the pathogen. Antibodies bind their target antigen with high specificity. Researchers have leveraged this specificity to create antibodies that can be used as drugs to treat a variety of human diseases. There are several stages involved in the discovery and development of therapeutic mAbs, prior to preclinical and clinical testing: While the majority of therapeutic antibodies approved or in regulatory review are indicated for cancer treatment, other disease areas are being targeted too, such as: The epitope is the part of the antigen that is recognized by the antibody. Bind to single epitope on target antigen nzymes can be used to cleave antibodies into various fragments for dierent purposes. -S-S- -S-SS- S S-S -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- -S-S- -S-SAntigen binding site Antibodies consist of four polypeptides: Two light chains Two heavy chains The variable region diers across antibodies and gives the antibody its specificity for binding to a target antigen. The base of the Y shape, the Fc region, determines the response that is triggered by the antibody–antigen binding. Antibody structure Monoclonal antibodies (mAbs) are derived from one cell clone B cell Homogenous 1998 5 10 15 2000 02 04 06 08 10 12 14 16 18 20 Number of antibody therapeutics Year of first US or EU approval Number of antibody therapeutics granted a first approval in the US or EU each year, 1997-2021: Source: The Antibody Society Cancer Non-cancer Oncology 2% 3% 3% 5% Cardiovascular/ 7% hemostasis Neurological disorders Genetic diseases Opthalmic disorders Musculoskeletal disorders Immunemediated Infectious disorders diseases 45% 27% 8% Primary indications for antibody therapeutics approved – or in regulatory review – in the US or European Union: Antibody discovery Target antigen selection Let’s review key milestones and recent advancements in various stages of this pipeline. TARGET ANTIGEN SELECTION High-throughput multi-omics approaches, which incorporate analysis of the genome, epigenome, transcriptome, proteome and metabolome, can now oer holistic insights into disease pathology – at a single-cell level, in some cases. Genomics Epigenomics Metabolomics Choosing a target antigen requires comprehensive understanding of a disease mechanism, or a disease-specific antibody eect. Transcriptomics Proteomics Transgenic mice have been created that can produce humanized or completely human antibodies: Mouse IgG locus replaced with human DNA sequence Human recombinant antibody Mouse injected with human therapeutic target Target-specific antibody DNA extracted Antibody genes cloned, and mAb expressed from Chinese Hamster Ovary cells 2 Transgenic mice engineered to produce human antibodies A “breakthrough” for therapeutic mAb development Fully human monoclonal antibody Humanized monoclonal antibody Owing to advances in next-generation sequencing, we can now sequence the genes encoding the entire antibody repertoire of an animal. With this knowledge, scientists are able to explore the eects of manipulating this repertoire and discover novel antibodies that may exist in human populations. 1 2 3 Mouse IgG locus intact Humanized recombinant antibody Mouse injected with human therapeutic target Target-specific antibody DNA extracted Complementarity-determining region engineered for high ainity and grafted onto human framework, cloned, and mAb expressed from Chinese Hamster Ovary cells 1 2 3 “These advancements will no doubt allow the recovery of rare antibodies with properties satisfying the demanding design goals of the next generation of therapeutic targets” Chen and Murawsky The seminal hybridoma technique developed by Köhler and Milstein in 1975 shaped the field of antibody development for therapeutic applications. However, mouse mAbs can trigger adverse reactions in humans, such as the “human anti-mouse antibody” (HAMA) response, limiting their utility. Non-human variable domains were combined with human constant domains to manufacture antibodies with 65% human content, a method known as chimerization. Rituximab (Rituxan®) – the first chimeric therapeutic antibody – was approved in 1997 for the treatment of cancer. Over the last few decades, novel technology platforms emerged and have been refined to produce increasingly human-like mAbs (or humanized). 1 Mouse hybridoma technique Antigen Harvest splenocytes B cells that produce antibodies that bind to the antigen Hybridoma screened for antibody production Clonal expansion Mouse mAbs 1975 1997 1980spresent 1980s RITUXIMAB Murine (0% human) Chimeric (65% human) Humanized (>90% human) Fully human (100% human) High Potential for immunogenicity Low The advancement of high-throughput technologies to rapidly sort B cells provides the opportunity to eiciently study antibody repertoires and develop mAbs for public health emergencies, such as infectious diseases. Artificial intelligence (AI) is anticipated to lead to completely in silico approaches for antibody discovery and development, with in vitro and in vivo methods only being required for validation. Peripheral blood mononuclear cells (PBMCs) are isolated from humans that have been infected with a pathogen or vaccinated. Flow cytometry is utilized to sort cells based on their cell surface expression markers. After B cells are isolated, immunoglobulin transcripts are amplified using reverse transcriptase polymerase chain reaction (RT-PCR). Gene transcripts are cloned and expressed in mammalian cell lines to generate recombinant mAbs. 4 Sourcing mAbs from human cells Relies on the robustness of the human immune system. PBMC Sort B cells with labeled antigens PCR, construct VH and VL VHDJHCY1 VKJKCK ANTIBODY CHARACTERIZATION NOVEL THERAPEUTIC MODALITIES BEING EXPLORED… CLINICAL APPLICATION OF THERAPEUTIC ANTIBODIES PRESENT AND FUTURE BISPECIFIC MONOCLONAL ANTIBODY THERAPY CAR TCELL THERAPY ANTIBODY FRAGMENTS ANTIBODY DRUG CONJUGATE THERAPY Chromatography Liquid chromatography-mass spectrometry (LC-MS) High-performance liquid chromatography (HPLC) coupled with MS Regardless of the method used to generate them, critical quality attributes of a therapeutic mAb, such as: Protein structure Post-translational modifications Function at the biomolecular and cellular levels require mAb characterization. Some key analytical techniques adopted here include, but are not limited to: A number of dierent antibody therapeutics currently exist, including: Antibody—drug conjugate (ADC) therapy A targeted antibody has a cytotoxic drug attached. When the antibody binds, it delivers the drug directly to the cell. ADCs oer the potential to reduce systemic side eects of certain drugs. Bispecific mAb therapy Include two types of mAbs directed at dierent sites, either on the same antigen or targeting two dierent antigens. CAR T-cell therapy Gene for a chimeric antigen receptor that targets a marker for a specific cancer is inserted into isolated T cells. When administered back in the body, the T cells can target cancer cells and destroy them. Antibody fragments Antibody engineering is used to develop functional antibody fragments that can be modified in vitro to optimize molecular features, such as size, binding ainity and pharmacokinetics. Antigen-binding fragments (Fabs) account for most antibody fragments in clinical trials. Over recent years, nanobodies – domains found on the heavy-chain antibodies of camelids – have garnered increased research attention. They: Possess high ainity Are highly stable Are eicient to produce Have low immunogenicity Existing mAbs can be limited due to their large size and – sometimes – low stability. Nanobodies may oer a novel antibody-based approach for treating solid tumors and other indications, such as coronavirus infections. bNAbs target a conserved region of the human immunodeficiency virus (HIV) viral envelope. bNAbs have been shown to recognize and block entry of dierent HIV strains, in addition to recruiting immune cells to destroy already-infected HIV cells. They therefore could be utilized as a preventive and therapeutic for HIV. Nanobodies Broadly neutralizing antibodies (bNAbs) Viral envelope Reverse transcriptase RNA Capsid Glycoprotein “With increased understanding of immunobiology and the continued development of molecular biological methods, the possibilities for antibody-based therapeutics are bounded only by the scope of human ingenuity” Goulet and Atkins A bacteriophage is genetically manipulated to possess genes that represent human antibody pools, which are then expressed on the surface of the phage, creating a “library” of antibodies. Most existing display systems present antibodies as fragments and without post-translational modifications, rather than full-length IgG molecules. Conversion to full-length IgG molecules is required, with subsequent optimization in mammalian cells. Overall, this process is low-throughput. Mammalian display libraries present as a potential alternative, as some cell lines could be directly used as the antibody production platform, rather than just as a library. However, it has proven diicult to transfect antibody-encoding genes without introducing multiple copies at dierent sites. Novel genome-editing approaches, like CRISPR-Cas9 technologies, are being used to generate increasingly larger display monoclonal antibody libraries at pre-defined sites in the mammalian cell genome – a step closer to high-throughput antibody engineering. Involves two key stages: Antibody library construction Biopanning 3 Phage display The most widely used in vitro approach for de novo generation of antibody candidates Fully human IgG antibody Antibody encoding genes Antibody phage display libarary Screening of phage on immobilized antigen Wash unbound phage Elution of surface-bound phage Screening Amplify phage for subsequent round Phage proteins Repeat 3-5 rounds to enrich library Site-specific integration of antibody-encoding gene repetoire, using novel gene-editing technologies For several mammalian systems, the library cells can be used directly for antibody production and chaterization. Mammillian antibody display library Target antigen Selection of antigen-specific clones Characterization The phage library is incubated with the immobilized antigen of interest. Unbound phage are then removed via multiple rounds of washing, and bound phages are eluted, amplified and the process repeats for several rounds to enrich the antibody population that binds to the antigen of interest. Advancement KEY ADVANCEMENTS THAT HAVE REVOLUTIONIZED ANTIBODY DISCOVERY AND ENGINEERING