mAb Therapeutics Lecture Notes PDF

mAb therapeutics  A full one-year course Top 10 Drugs by Sales in 2023 of Herceptin treatment costs about US$70,000:...

mAb therapeutics  A full one-year course Top 10 Drugs by Sales in 2023 of Herceptin treatment costs about US$70,000: pembrolizumab Patients in early-stage HER2-positive breast adalizumab cancer should remain on Herceptin (trastuzumab) treatment for one year. Roche says that over 1.2 million women have received Herceptin. dupilumab ustekinumab daratumumab nivolumab Nature Reviews: Drug Discovery | BIOBUSINESS BRIEFS 08 March 2024 3 Indications of mAb Therapeutics CAGR: Compound Annual Growth Rate 2 ( mamune diseases 4 Cancer are the main farget  Monoclonal Antibodies (mAbs) Compared with traditional chemotherapy and radiotherapy approaches, monoclonal antibodies have become the most successful cancer treatments in the past 20 years. highly vorrrable Terms often used Antigen – any substance capable of eliciting an adaptive immune response Epitope – region of the antigen 1 - highly conserved recognized by an antibody Antibody – immunoglobulin secreted by B cells Monoclonal antibodies (mAbs) – antibodies secreted from a single B cell, Each c B ell generate aspecific antibody , Paratope – region of the antibody that site L: light chain; H; heavy chain; V: variable domain; C: constant binds the epitope Basically antigem binding. domain; CDR: complementarity determining region. There are two types of light chain in humans: kappa (κ) chain (on scFv - single-chain variable fragment chromosome 2) and lambda (λ) chain (on chromosome 22). Monoclonal antibodies (mAbs) represent a single B lymphocyte generating antibodies to one specific epitope and have identical paratopes. mAbs are typically bivalent molecules, capable of binding two similar antigens. 5  Monoclonal Antibodies (mAbs) Compared with traditional chemotherapy and radiotherapy approaches, monoclonal antibodies have become the most successful cancer treatments in the past 20 years. Terms often used Antigen – any substance capable of eliciting an adaptive immune response variable reigion Epitope – region of the antigen Fror Light α heavy recognized by an antibody Fusion chain Antibody – immunoglobulin secreted Protein by B cells antibochy " " mina Monoclonal antibodies (mAbs) – antibodies secreted from a single B cell, Paratope – region of the antibody that L: light chain; H; heavy chain; V: variable domain; C: constant binds the epitope domain; CDR: complementarity determining region. There are two types of light chain in humans: kappa (κ) chain (on scFv - single-chain variable fragment chromosome 2) and lambda (λ) chain (on chromosome 22). Monoclonal antibodies (mAbs) represent a single B lymphocyte generating antibodies to one specific epitope and have identical paratopes. mAbs are typically bivalent molecules, capable of binding two similar antigens. 6 7 Monoclonal antibodies (mAbs) were first generated using a hybridoma technology: spleen cells from immunized mice are fused with the myeloma cells, and reported in 1975. cells mixture of B. polychonal antibody , - trans Form into mongclonal on - antibody cell lines. 8  Evolution of Monoclonal Antibodies Previously, monoclonal antibodies were typically made entirely from mouse cells. One problem with this is that the human immune system will see these antibodies as foreign (because they’re from a different species) and will mount a response against them. In the short term, this can sometimes cause an Nomenclature of monoclonal antibodies immune response. In the long term, it means that the replace antibodies may only work the the Constant first time they are given; after Region with ruman that, the body’s immune system is primed to destroy tibody an- less immune them before they can provide Response treatment. 1B cell ) Sequence , get circulating - 9 一 一 Evolution of therapeutic antibody technology and progress to the clinic (FDA approval) In general, FDA-approved mAbs have emerged between 10 and 12 years after the date that the new technologies were reported in the scientific Panitumumab literature. 12 2006 10 How to generate human-mouse chimeric & humanized mAbs? CDR-grafting (grafting of the rodent CDRs into human antibody frameworks) can be used to generate humanized antibodies, that is humanize non-human antibodies (antibodies raised, for example, in a mouse or rat host towards a human target molecule) by the modification of the complementarity-determining regions (CDRs) to increase their similarity to human antibody variants. 11 How to get human humanised Expresshuman antibody Mo heavy and light chain variable region sequences to generate fully human cells Immortolise monoclonal Epstein-Barr virus -四 hyrmphoauytes antibodies Maintaining & _ culture Bcell is veny expensive 12 Find the 1B cell 7 Sequence pIdentiFy the gene { much lower Cost Easier π , mass ← Express in vector to procduce. production  Development of recombinant Chinese hamster ovary (CHO) cell lines for antibody production Chinese hamster ovary (CHO) cells are the predominant workhorses for large-scale stable expression of human glycoproteins, including mAbs. CHO cells can be genetically manipulated and grown either as adherent cells or in suspension. 13  In vitro production in Chinese hamster ovary (CHO) cells is the preferred method of mAb manufacturing MCB Manufacturing Working Cell Bank (MWCB) The mAb process starts with vial thaw and expansion of cells through a series of inoculum steps. The cells (mainly CHO cells) are then further expanded in a series of seed bioreactors before transfer to the production bioreactor where the mAb is expressed into the medium. Centrifugation and a series of filtration steps are then used to harvest the cell culture broth from cells and cell debris. Yields beyond 5 g/l can be achieved. 14  Modifying Monoclonal Antibody Structure glycosilation can contribute Post translation al nodificatio n specificity smaller X with - binding specificity. Generate antibody fragments 15 场 Antibody are Very large protein Hardto penetrate deep into the tissue  Antibody Fragments Many new mAb entities are Ab-fragments. Three Ab-fragment-based biotherapeutics have been approved by FDA. Bispecific F(ab)2 Trispecific F(ab)3 single-chain single-domain variable fragment antibody fragment Ab-fragments with reduced size can penetrate tissues more efficiently. Many Ab-fragments eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells (such as NK cells macrophages, dendritic cells, neutrophils, and B cells) 16 一 Single chain antibodies from camelids  Single chain antibodies from camelids shaagleahainproduntibody they only haretwo heavy Chain https://www.chemistryworld.com/features/the-incredible-antibodies-of-sharks-llamas-and-camels/4015629.article  Camelid ' antibodyare verystable s a Easy toexpress ( Antigen bindins site region of camelids single chorim Atb) https://doi.org/10.1186/s40364-021-00332-6  Action Mechanisms of Therapeutic Antibodies bringeffector - cellogetall ADC: Antibody-drug conjugate Concentrate the dug locally J Toxicol Pathol 2015; 28: 133–139 20  mAbs for Targeted Cancer Therapy Most mAb therapeutics are developed as treatments for cancer or immunological diseases §Block growth signals. Trastuzumab (Herceptin) interferes with the HER2/neu receptor in breast cancers. Cetuximab (Erbitux) attaches to epidermal growth factor on cancer cells that accept growth signals. §Make the cancer cell more visible to the immune system. Rituximab (Rituxan) attaches to a specific protein (CD20) only found on B cells and lymphomas, triggering NK cell- mediated ADCC to destroy cancer cells. §Deliver radiation or toxic drug to cancer cells. After combining a radioactive particle or toxic drug, a monoclonal antibody can be delivered to kill cancer cells. Ibritumomab (Zevalin) and Gemtuzumab (Mylotarg) are approved to treat non-Hodgkin's lymphoma, leukemia. §Bispecific antibodies (BsAbs) recognize two different epitopes. This dual specificity opens up a wide range of applications, including redirecting T cells to tumor cells, blocking two different signaling pathways simultaneously, dual targeting of different disease mediators, and delivering payloads to targeted sites. Most monoclonal antibodies usually cannot penetrate the cell’s plasma membrane and are directed against targets that are outside cells or on the cell surface.  Bispecific T Cell Engager (BiTE) use ScFu to Link Tcell to tumour cells Two short chain variable fragments (scFv) joined by a flexible linker are all parts of a single polypeptide. When the CD3 targeted scFv has bound CD3 and the other scFv has bound its target, the T cell becomes activated. Cell-mediated cytotoxicity directed at the tumor cell follows. 22 How do anti–TNF mAbs work? First FDA approved antibody Generic name: Adalimumab Trade name: Humira Psoriasis is a chronic inflammatory immune- mediated skin disease that affects 1%-3% of general population. High levels of proinflammatory cytokines, including tumour necrosis factor (TNF), have been detected in psoriatic skin lesions. After subcutaneous injection, Humira can specifically bind to TNF and neutralize the biological function of TNF by blocking its interaction with the Adalimumab (trade name Humira) is a recombinant, fully human cell surface TNF receptors. IgG1 monoclonal antibody. The drug is produced by recombinant DNA technology in a mammalian cell expression system. 23  How do anti–PD1/PD-L1 mAbs work? Anti–PD1 antibody Keytruda is a humanized antibody used in PD1 immunotherapy of cancers including treat melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma, PDL1 and stomach cancer. It is given by slow injection into a T-Cell Cancer Cell T-Cell Cancer Cell vein. PD-L1 is frequently expressed on tumor cells and tumor-infiltrating immune cells within the tumor microenvironment PD-L1 binding to the PD-1 receptor, which is expressed on activated T cells, induces T-cell exhaustion, a state of ineffective T-cell activity too much PD L1 exhaust Tcells - Inhibiting the PD-L1 to PD-1 interactions of the T cell and the tumor cell may by PD1 mAbs restore the T cell's cytotoxic antitumor activity. 2018 Nobel Prize in medicine awarded to cancer immunotherapy: The PD-1/PD-L1 and CTLA-4 immune checkpoints 24 一  Major FDA Approvals of PD-1/PD-L1 Inhibitor mAbs - Another type of cancer immunotherapy works by "unblocking" T-cells so they are able to carry out a full attack. This is done through the use of monoclonal antibodies, one of which is called anti-CTLA-4 (brand name ipilimumab), and another called anti-PD-1. In a study testing anti-PD-1 in nearly 300 people with different types of cancer, tumors shrank by at least half in 31 percent of patients with melanoma, and 29 percent of patients with kidney cancer. Ø The first checkpoint inhibitor in the market (approved in March 2011) is Yervoy (ipilimumab) from Bristol Myers Squibb targeting CTLA4. But concerns were about strong autoimmune reactions to the drug in up to 20% of patients. Ø The first PD-1 inhibitor, Keytruda, had impressive effects on patients with metastatic melanoma that were unresponsive to standard therapies. Ø In 2017 the FDA approved Keytruda, anti-PD1 antibody, for any unresectable or metastatic solid tumor with microsatellite instability. 25 PD-1 Immune Checkpoint Inhibitor mAbs in Clinical Trials There are 2,250 active trials testing anti- PD-1/PD-L1 agents as of September 2018, compared with 1,502 trials in September 2017, an increase of 748 trials in one year. Anti–PD1/PDL1 therapy rapidly becoming standard of care for various types of cancers. 26  Anatomy of Antibody-Drug Conjugate (ADC) Linker stable in circulation Antibody targeted Linker biochemistry to tumor Acid labile (hydrazone) Enzyme dipeptides (cleavable) Human or humanized monoclonal Ab (IgG1) Thioether (uncleavable) mAb with Fc modifications Hindered disulfide (uncleavable) (modulate ADCC, CDC Site of conjugation activity) Fc, HC, LC An alternative to using a full length or Fc modulated Ab, is to use a Very potent fragments of the mAb (such as Fabs). chemotherapeutic drug Tubulin polymerization inhibitors Maytansines (DM1, DM4) Auristatins (MMAE, MMAF) DNA damaging agents Calicheamicins Duocarmycins Anthracyclines (doxorubicin) 27 Improving Therapeutic Window by ADC ADCs can selectively deliver a potent cytotoxic drug to tumor cells via tumor- specific and/or over-expressed antigens Increase drug delivery to tumor p Increase site concentration - , Reduce normal tissue drug exposure Chemotherapy ADC TOXIC DOSE (MTD) DRUG DOSE TOXIC DOSE (MTD) Therapeutic Window Therapeutic Window EFFICACIOUS DOSE (MED) EFFICACIOUS DOSE (MED) MTD: Maximum tolerated dose; MED: Minimum Efficacious Dose 28 ADC is More Efficacious than Free Cytotoxin in Mice MMTV-HER2 Fo5 mammary tumor 1500 (HER2-positive, trastuzumab-insensitive) Vehicle DM1 Trastuzumab-mertansine 15 mg/kg, 817 µg/m 2 Mean Tumor Volume (mm3 ) Trastuzumab 15 mg/kg 1000 Trastuzumab 15 mg/kg + Free DM1 817 µg/m2 Free DM1 817 µg/m2 +/- SEM Free DM1 (near MTD) 1947 µg/m2 Free DM1 500 (cytotoxin) T-DM1 (ADC) 0 0 5 10 15 20 25 30 IV Dosing Day Parsons et al, AACR (2007); Modified from S. Spencer DM1: Emtansine, inhibits the assembly of microtubules by binding to tubulin, destroying cancer cells T-DM1: Trastuzumab emtansine  Can administered larger amount of Dugs ADC is Better Tolerated than Free Cytotoxin in Rats Single IV dose; rats T-DM1 (2040 µg DM1/m²) (% change from baseline) Body Weight Free DM1 (2400 µg DM1/m²) Early mortality (100%) Time (Day) T-DM1: Trastuzumab emtansine was approved by FDA in February 2013 for the treatment of people with HER2-positive metastatic breast cancer 30 Summary of the Lecture § mAb Basics & Market Analysis § Antibody Production § mAb Function Mechanisms: - PD1/PDL1 antibodies - ADC 31