Monoclonal Antibodies: Recent Development in Drug Delivery (PDF)

C H A P T E R 4 Monoclonal antibodies: recent development in drug delivery Sumel Ashique1, Prathap Madeswara Guptha2, Jovita Kanoujia2, Ashish Garg3, Afzal Hussain4,...

C H A P T E R 4 Monoclonal antibodies: recent development in drug delivery Sumel Ashique1, Prathap Madeswara Guptha2, Jovita Kanoujia2, Ashish Garg3, Afzal Hussain4, S. Mohana Lakshmi2 and Neeraj Mishra2 1 Department of Pharmaceutics, Pandaveswar School of Pharmacy, Pandaveswar, West Bengal, India 2Amity Institute of Pharmacy, Amity University Madhya Pradesh (AUMP), Gwalior, Madhya Pradesh, India 3Department of Pharmaceutics, Guru Ramdas Khalsa Institute of Science and Technology (Pharmacy), Jabalpur, Madhya Pradesh, India 4Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia 4.1 Introduction Monoclonal antibody (mAbs) are manufactured by cloning a distinctive white blood cell and having a unique affinity toward the specific epitope. Therapeutic mAbs are basi- cally gamma-immunoglobulin (or IgG) isotype in nature. mAbs are prepared by a unique clone of B cells and myeloma cells and mainly attack the specific antigens. mAbs are com- posed of four polypeptide chains (two similar heavy chains and two alike light chains) having a molecular weight of B150 kD. mAbs are classified into five types (e.g., IgA, IgG, IgD, IgM, and IgE), and the most active therapeutics is IgG, which has the greater site- specific quality and affinities from nano to picomolar. The chains are bound together by disulfide linkages and fold to form a “Y”-shaped tetramer (Fig. 4.1A). Scientists Köhler and Milstein successfully developed hybridoma in 1975 to produce pure mAbs in bulk quantity, considerably increasing the research and significant clinical utilization, and depending on their inception, mAbs are classified into four types [e.g., murine, chimeric, humanized, and human (Fig. 4.1B)]. Because of advancements in scientific and technologi- cal advances, mAbs have successfully been introduced into different health care centers. At present, in the world, around 570 therapeutic mAbs have been developed in clinical trials by various trading companies , and around 79 therapeutic mAbs have been Molecular Pharmaceutics and Nano Drug Delivery DOI: https://doi.org/10.1016/B978-0-323-91924-1.00014-9 79 © 2024 Elsevier Inc. All rights reserved. 80 4. Monoclonal antibodies: recent development in drug delivery FIGURE 4.1 (A) Basic structure of mAbs and (B) classification of mAb murine, chimeric, humanized, and human. approved by the United States Food and Drug Administration (US FDA) and now are available on the market (Table 4.1). The first monoclonal antibody named Orthoclone OKT-3 (muromonab-CD3) (murine originated) was approved in the year 1985, by the FDA for applying clinically in humans as an antirejection agent. The significant upgrowing of therapeutic mAbs has been developed for several disease conditions in the last three decades. Currently, the chief technical developments have made the advancement of mAb therapies faster and more effective. Antigen-specific tar- geting offers efficient therapeutic treatment, and the incidence of molecular targeting ther- apy is indicating the progression of a novel initiation of bioactives. However, there is one limitation: a majority of the approved therapeutic mAbs have selectivity toward the pri- mary assembly of definite antigens, though all proteins have original inherent structures for their own well-defined response. mAbs mediate their functions by different types of direct or indirect effects by binding with cell surface receptors, membrane-associated proteins, growth factors, and so forth. By binding to the targeted antigen, antibodies can alter the cells also [5,6]. Antibody-based therapeutics have shown minimum adverse effects because of their greater selectivity; thus, therapeutic mAbs are considered as the potent category of new bioactives used currently. To overcome the issues of inhibited immunogenic viewpoint and efficiency, during the preparation of mAbs for a long time, researchers planned to use several methods to change rodent antibodies into structures similar to human antibodies, without changing targeting features. For active targeting, physically or covalently conjugated ligands, such as mAb and their components, are con- stantly utilized and evaluated for targeted delivery designs in various chronic diseases also (Table 4.2) (Table 4.3 and Table 4.4). Molecular Pharmaceutics and Nano Drug Delivery TABLE 4.1 List of recently Food and Drug Administration approved antibody drug conjugate. Approval Drug Maker Condition Indication Route Does Target year Tisotumab Seagen & Genamb Recurrent or metastatic cervical cancer Tivdak i.v. 40 mg/vial Tissue factor 2021 vedotin Loncastuximab ADC Therapeutics Large B-cell lymphoma Zynlonta i.v. 10 mg/vial CD19 2021 tesirin Belantamab GlaxoSmithKline Adult patients with relapsed or refractory Blenrep i.v. 100 mg/vial BCMA 2020 mafodotin (GSK) multiple myeloma Trastuzumab AstraZeneca/Daiichi Treatment of adult patients with Enhertu i.v. 100 mg/vial HER2 2019 deruxtecan Sankyo unresectable or metastatic HER2-positive breast cancer who have received two or more prior anti-HER2-based regimens in the metastatic setting Enfortumab Astellas/Seattle Adult patients with locally advanced or Padcev i.v. infusion 20/30 mg/vial Nectin-4 2019 vedotin Genetics metastatic urothelial cancer who have received a PD-1 or PD-L1 inhibitor, and a Pt-containing therapy Polatuzumab Genentech, Roche Relapsed or refractory (R/R) diffuse large B- Polivy i.v. infusion 140 mg/vial CD79 2019 vedotin cell lymphoma (DLBCL) Moxetumomab Astrazeneca For the treatment of adult patients with Lumoxiti i.v. infusion 1 mg/vial CD22-directed 2018 pasudotox relapsed or refractory hairy cell leukemia cytotoxin Inotuzumab Pfizer/Wyeth Relapsed or refractory CD22-positive B-cell Besponsa i.v. infusion 0.9 mg /vial CD22- 2017 ozogamicin precursor acute lymphoblastic leukemia directed antibody-drug Trastuzumab Genentech, Roche HER2-positive metastatic breast cancer (mBC) Kadcyla i.v. infusion 100 mg/vial HER2- 2013 emtansine following treatment with trastuzumab and a positive, maytansinoid metastatic breast cancer 82 4. Monoclonal antibodies: recent development in drug delivery 4.2 The molecular mechanisms of therapeutic antibody The efficiency of mAb therapy can be determined via various mechanisms, such as direct effects of the antibody on cell action, for example, by hindering or the initiation of signaling mechanisms transported by the target molecule, in some cases initiating apoptosis directly. The additional significant mechanism is the targeting of the immune effector process to the tumor. The mAb can also be used for targeting payloads to the cell of interest. One of the novel findings in mAb therapy includes using the specificity of a mAb for a tumor-linked target in the development of chimeric antigen receptor (CAR) T-cell therapies. Various treatment strategies applying unmodified mAbs have shown to be efficient in the manage- ment of several cancers, which acts by binding to the antigen on cancer cells or an antigen on other cells or proteins or by initiating the immune response process [11,12]. 4.3 Advantages and disadvantages of mAbs mAbs are now the highly effective biotherapeutics for the treatment of a variety of cancers and autoimmune diseases. They have various benefits over other antineoplastic bioactives, including enhanced efficacy, safety, and lower toxicity, as well as less side effects and a higher patient survival rate. Antibodies have a biorecognition mechanism designed to be able to improve the immune response by preventing intracellular and extracellular threats. As a result of its specificity and accuracy, it is reminiscent of the "Ehrlich’s magic bullet" concept, which involves addressing a particular infection for clinical intervention while limiting collateral harm to the patient. Surprisingly, it may provoke an antibody reaction and has been extensively utilized in microbiological investigation, diagnosis, and, more importantly, for therapy purposes. A mAb is a type of antibody that has a greater affinity for specific antigens and is increasingly used as a targeted treatment for a variety of diseases. By establishing an "Ab-drug combina- tion", the mAbs may specifically deliver the bioactive to the designated target. This method has a promising future because of the high specificity with which they adhere to specific anti- gens. Although there have been promising studies, this strategy faces various obstacles that prevent its widespread implementation as a therapy. There are a few limitations to clinical mAbs, including limited pharmacokinetic statistics and tissue reachability, as well as decreased immune system interactions, and these deficiencies highlight the need for additional research. Costs of production: mAbs are huge (150 kDa) "multimeric proteins" that were gener- ated passively using complex eukaryotic apparatus. Thus, the production of therapeutic monoclonal antibodies needed a large number of mammalian cells accompanied by numerous refinement steps, resulting in prohibitively expensive production expenditures and limiting the clinical mAb’s use [17,18]. 4.4 Pharmacokinetics versus tumor targeting In mouse xenograft models, mAbs controlled against malignant cells antigens are highly retained in the systemic circulation, with less than 20% of the injected generally reacting Molecular Pharmaceutics and Nano Drug Delivery TABLE 4.2 List of Therapeutic mAb approved by Food and Drug Administration for treatment of chronic diseases. Brand Approval MAb Name Company Target Format Technology Condition year Alemtuzumab Campath, Berlex Inc./Genzyme CD52-protein present on Recombinant Hybridoma B-cell chronic lymphocytic 2001 Lemtrada Corp./Millennium the surface of mature humanized leukemia Pharmaceuticals Inc. lymphocytes immunoglobulin G1-IgG1 Adalimumab Humira AbbVie Inc. inactivating tumor Human IgG1 Phage Rheumatoid arthritis 2002 necrosis factor-alpha display (TNFα) Ibritumomab Zevalin Biogen Inc./Schering CD20 Murine IgG1 Hybridoma Non-Hodgkin Lymphoma 2002 Tiuxetan AG/Spectrum Pharmaceuticals Inc Omalizumab Xolair Roche, F. Hoffmann-La IgE Human IgG1 Hybridoma Asthma 2003 Roche, Ltd. Cetuximab Erbitux Bristol-Myers Squibb Binds epidermal-growth- Chimeric IgG1 Hybridoma Advanced bowel cancer 2004 factor-receptor Bevacizumab Avastin Roche, F. Hoffmann-La VEGF-A (vascular (IgG1) Hybridoma Brain tumor 2004 Roche, Ltd./Genentech endothelial growth Inc factor-VEGF) Natalizumab Tysabri Biogen Inc./Elan ITGA4 Humanized IgG4 Hybridoma Multiple sclerosis 2004 Pharmaceuticals International, Ltd. Panitumumab Vectibix Amgen EGFR Human IgG2 Transgenic Colorectal cancer 2006 mice Ranibizumab Lucentis Ovartis Pharmaceuticals VEGF-A Humanized IgG1 Hybridoma Macular degeneration 2006 Corp Fab Eculizumab Soliris Alexion C5-gene complement Humanized Hybridoma prevents the breakdown of 2007 Pharmaceuticals Inc component IgG2/4 RBC in adults with paroxysmal nocturnal hemoglobinuria (PNH) (Continued) TABLE 4.2 (Continued) Brand Approval MAb Name Company Target Format Technology Condition year Ofatumumab Arzerra Genmab A/S CD20- A protein found Human IgG1 Transgenic Chronic lymphocytic leukemia 2009 /GlaxoSmithKline/ on B cells (a type of mice Novartis. white blood cell) Ipilimumab Yervoy Bristol-Myers Squibb/ CTLA-4(cytotoxic T- Human IgG1 Transgenic Metastatic melanoma 2011 Medarex lymphocyte-associated protein 4) Pertuzumab Perjeta Roche, F. Hoffmann-La HER2 Humanized IgG1 Hybridoma Breast cancer 2012 Roche Ramucirumab Cyramza Eli Lilly/ImClone VEGFR2 Human IgG1 Phage Gastric cancer 2014 Systems Inc display Nivolumab Opdivo Bristol-Myers Squibb/ Programmed cell death Human IgG4 Transgenic Melanoma, non-small cell lung 2014 Ono Pharmaceutical protein 1 mice cancer Co., Ltd Daratumumab Darzalex Genmab A/S/Janssen CD38-protein Coding Human IgG1 Transgenic Multiple myeloma 2015 Biotech Inc gene mice Atezolizumab Tecentriq Roche, F. Hoffmann-La PD-L1 Humanized IgG1 Hybridoma Bladder cancer 2016 Roche, Ltd./Genentech Inc Inotuzumab Besponsa Wyeth CD22 Humanized IgG4 Hybridoma Acute lymphoblastic leukemia 2017 ozogamicin Pharmaceuticals/Pfizer Durvalumab Imfinzi MedImmune/ PD-L1-programmed Human IgG1 Transgenic Bladder cancer 2017 AstraZeneca death-ligand 1 mice Moxetumomab Lumoxiti MedImmune/ CD22 Murine IgG1 Phage Hairy cell leukemia 2018 pasudodox AstraZeneca dsFv display Tisotumab TIVDAK Tissue factor Disrupting agent Tissue Factor Human Cervical cancer 2021 vedotin monomethyl auristatin E (TF) specific IgG1 ADC human IgG1 Dostarlimab Jemperli PD-1 binds to the PD-1 Humanized IgG4 Humanized Endometrial cancer 2021 receptor and blocks its monoclonal interaction with PD-L1 antibody and PD-L2 TABLE 4.3 Top 10 best-selling monoclonal antibody drugs in 2021. 2021 worldwide Rank Generic name Brand Name Manufacturer Condition/pharmacology class Dosage form/strength sales (US $million) 1 Adalimumab Humira AbbVie Rheumatoid and psoriatic 40 mg/0.8 mL in a single-use 19,963 arthritis, ankylosing spondylitis, prefilled glass syringe Crohn’s disease, ulcerative colitis 2 Pembrolizumab Keytruda Merck Anti PD1-mAb 100 mg/4 mL (25 mg/mL) 16,825 solution in a single-dose vial 3 Lenalidomide Revilmid Beigene Multiple myeloma, Capsules: 2.5, 5, 10, 15 and 12,710 Immunomodulator 25 mg 4 Apixaban Eliquis Pfizer Factor Xa inhibitor anticoagulant 2.5 and 5 mg orally twice daily 10,546 5 Aflibercept Eylea Regeneron Age-Related Macular 40 mg/mL solution for intra 8,872 Pharmaceuticals/Bayer Degeneration vitreal injection 6 Nivolumab Opdivo Ono Pharmaceutical Anti-PD1 mAb 40, 100, and 240 mg/24 mL 8,759 solution in a single-dose vial 7 Ustekinumab Stelara Johnson & Johnson Human interleukin-12 and -23 45 mg/0.5 mL or 90 mg/mL in 8.445 antagonist- plaque psoriasis, a single-dose prefilled syringe active psoriatic arthritis 8 Bictegravir, Biktarvy Gilead Sciences HIV INSTI/NTRI-nucleotide One tablet: 50 mg of bictegravir emtricitabine, and reverse transcriptase inhibitors 200 mg of emtricitabine 25 mg tenofovir of tenofovir alafenamide alafenamide 9 Ibrutinib Imbruvica AbbVie/Johnson & BTK inhibitor Capsules: 70 and 140 mg 7,607 Johnson Tablets: 140 mg, 280, 420, and 560 mg 10 Rivaroxaban Xarelto Bayer/Johnson & Factor Xa inhibitor anticoagulant Tablets: 10, 15, and 20 mg 7,605 Johnson TABLE 4.4 List of US Food and Drug Administration approved human mAbs. Approval US FDA Ref S. No Antibody Brand Name Manufacturer Target Technology Dosage form/strength year ID 1. Adalimumab Humira Abbott Laboratories TNFα Phage display 40 mg/0.8 mL in a single-use 2002 Reference ID: prefilled glass syringe 4359269 2 Panitumumab Vectibix Amgen EGFR Xeno Mouse Single-use vials (20 mg/mL): 2006 Reference ID: 100 mg/5 mL, 200 mg/10 mL, 3618836 400 mg/20 mL 3 Ustekinumab Stelara Johnson & Johnson IL-12 HuMabMouse Injection: 45 mg/0.5 mL or 2009 Reference ID: 90 mg/mL in a single-dose 3990240 4 Canakinumab Ilaris Novartis IL-1β HuMabMouse single-use 6-mL, glass vial 2009 Reference ID: containing 180 mg of ILARIS as a 3109737 lyophilized powder for reconstitution 5 Ofatumumab Arzerra GlaxoSmithKline CD20 HuMabMouse 100 mg/5 mL single-use vial 2009 Reference ID: (Genmab) 3490691 6 Denosumab Prolia, Xgeva Amgen RANKL XenoMouse 60 mg in a 1 mL solution 2010 Reference ID: 3324257 7 Belimumab Benlysta GlaxoSmithKline BCAF Phage display 120 mg per vial, 400 mg per via 2011 Reference ID: 3109129 8 Ramucirumab Cyramza Eli Lilly VEGFR2 Phage display 100 mg/10 mL (10 mg/mL) or 2014 Reference ID: 500 mg/50 mL (10 mg/mL) solution 4615930 in a single-dose vial 9 Nivolumab Opdivo Bristol-Myers PD-1 HuMabMouse 40 mg/4 mL, 100 mg/10 mL, and 2015 Reference ID: Squibb 240 mg/24 mL solution in a 4844290 single-dose via 10 Alirocumab Praluent Sanofi and PCSK9 Veloclmmune 75 mg/mL or 150 mg/mL solution 2015 Reference ID: Regeneron Mouse in a single dose 3797282 11 Daratumumab Darzalex Johnson & Johnson CD38 HuMabMouse 100 mg/5 mL solution in a single- 2015 Reference ID: (Genmab) dose vial 4016996 400 mg/20 mL solution in a single-dose via 12 Necitumumab Portrazza Eli Lilly (ImClone) EGFR Phage display Injection: 800 mg/50 mL (16 mg/mL) 2015 Reference ID: solution in a single-dose via 3851135 13 Secukinumab Cosentyx Novartis IL-17α XenoMouse Injection: 50 mg/mL solution in a 2015 Reference ID: single-use 3874646 14 Atezolizumab Tecentriq Roche, F. Hoffmann- PD-L1 Phage display Injection: 40 mg/14 mL (60 mg/mL) 2016 Reference ID: La Roche, Ltd./ and 1200 mg/20 mL (60 mg/mL) 4872883 Genentech Inc. solution in a single-dose vial 15 Avelumab Bavencio Pfizer PD-L1 Phage display 200 mg/10 mL (20 mg/mL) solution 2017 Reference ID: in single-dose vial. 4171112l 16 Dupilumab Dupixent Sanofi and IL-4R Veloclmmune Injection: 300 mg/2 mL solution in a 2017 Reference ID: Regeneron Mouse single-dose 4075926 17 Guselkumab Tremfya Jassen Biotech IL-23 Phage display Injection: 100 mg/mL in a single- 2017 Reference ID dose prefilled syringe. 4123919 18 Erenumab Aimovig Novartis and CGRPR XenoMouse Injection: 70 mg/mL solution in a 2018 Reference ID: Amgen single-dose 4264882 19 Emapalumab Gamifant NovImmmune IFNγ Phage display Injection: 10 mg/2 mL (5 mg/mL) 2018 Reference ID: solution in a single-dose vial 4352133 50 mg/10 mL (5 mg/mL) solution in a single-dose vial 20 Moxetumomab Lumoxiti MedImmune/ CD22 Phage display Injection:1 mg lyophilized cake or 2018 Reference ID: pasudodox AstraZeneca powder in a single-dose vial 4320135 21 Ravulizumab Ultomiris Alexion C5; Recombinant Injection: 300 mg/30 mL (10 mg/mL) 2019 Reference ID: Pharmaceuticals humanized DNA in a single-dose vial 4367173 IgG2/4 technology 22 Isatuximab Sarclisa Sanofi-aventis U.S. CD38 Chimeric Injection: 100 mg/5 mL (20 mg/mL) 2020 Reference ID: LLC IgG1 solution in single-dose vial 4568826 500 mg/25 mL (20 mg/mL) solution in single-dose vial 23 Dostarlimab Jemperli GlaxoSmithKline PD-1, Humanized Injection: 500 mg/10 mL (50 mg/mL) 2021 Reference ID: Endometrial IgG4 solution in a single-dose via 4783636 cancer 88 4. Monoclonal antibodies: recent development in drug delivery with the tumor, which is the most difficult portion encountered by mAbs. Large spe- cific antibodies, which firmly bind their antigen during the first attack at the tumor’s mar- gin, cannot penetrate deeper within the cancer until enough antigen molecules in the periphery have been saturated. Therapeutic antibodies are also restricted by the affinity of these molecules for inhibitory Fc receptors, including FcRIIb, B-cells, macrophages, den- dritic cells, and neutrophils that express it. Over 99% of the cells will not survive because the process of fusing prevents the production of antibodies directed against spe- cific antigens. A higher pH level can alter the surface charges of antibodies, causing them to buildup and cause an increase in viscosity due to electrostatic attraction, especially at higher concentrations. mAbs confront the same hurdles as all proteinaceous treat- ments due to their protein structure , namely, the drug stability issue. It has been reported that a number of structural, colloidal, and chemical factors can hinder the devel- opment of good mAb formulations (such as oxidation, isomerization, deamidation, clump- ing, degradation, and fragmentation) [23,24]. As mAbs come into interaction with a wide range of temperatures, moisture, ionic strength, and stress levels, their structure changes, primarily in the hypervariable region, resulting in a series of unwanted products, many of them inhibiting the activity and enhancing the immunogenicity of the protein, potentially putting patients at risk. Therefore, it is very challenging to find optimal adjuvants to prevent destabilization of mAbs. Furthermore, mAbs suffer other significant restrictions due to ambiguity and a significant reduction in their capacity to bypass biological block- age. Poor collaboration and high clinical adaptation costs are the two major impediments to mAb use universally. The viscosity and injection pressure for subcutaneous administra- tion are influenced by the concentration of mAb, which results in a shorter half-life in low molecular weight compounds and changes in immunological reactions. 4.5 Approaches for developing targeted monoclonal antibodies Human, humanized, hybrid, and mouse antibodies represent 51%, 34.7%, 12.5%, and 2.8% of all mAbs in clinical utilization, respectively; in therapeutic applications, human and humanized mAbs have become the dominant modality. In this segment, we address antibody humanization and methodological frameworks for generating completely human Abs (phage display, transgenic mice, and single B cell antibody isolation). 4.5.1 XenoMouse hybridoma technology Gene-targeted removal of the mouse immunoglobulin genes (heavy and light chain) has initially been used to immobilize these loci within embryonic stem (ES) cells in order to create homogeneous animals for the requisite alterations (Fig. 4.2). Mice resulting from hybridizing were in animals homozygous for these deletions being incapable of producing mouse immunoglobulin. Then, either human heavy- or light-chain DNA-carrying yeast artificial chromosomes were inserted into ES cells. Genetic recombination of mouse result- ing from these ES cells caused in transgenic animals that produced both human and murine antibodies (Abs). Genetic recombination mouse unable of producing mouse Molecular Pharmaceutics and Nano Drug Delivery  4.5 Approaches for developing targeted monoclonal antibodies 89 FIGURE 4.2 Hybridoma technology for mAb production. immunoglobulin with genetically-engineered mice (carrying both human and mouse Abs) resulted in the Xeno Mouse strain, which carries only human Abs and is incapable of pro- ducing mouse antibodies. Antibody-producing B cells taken from an immunized XenoMouse’s spleen are utilized to create hybridomas, in which the B cells merge with an immortal cell line. Finally, the hybridoma technique is used to generate completely human mAbs. The results of this technology are handy in terms of production since each hybrid- oma may produce larger amounts of identical totally human Abs that can be cultivated indefinitely and screened to detect antibodies of the desired specificity and sensitivity, besides targeting activities. 4.5.2 Phage display for the production of human mAbs The first and most commonly utilized approach for in vitro antibody selection was phage display (Fig. 4.3). The surface of peptides should be able to display these peptides of bacter- iophages. George P. Smith fused external peptides with pIII, a coat protein of bacteriophage Molecular Pharmaceutics and Nano Drug Delivery 90 4. Monoclonal antibodies: recent development in drug delivery FIGURE 4.3 Transgenic animals, phase display, and single B cell approaches utilized in the production of mAb. M13 using recombinant DNA techniques in 1985. In order to identify mAbs from phage display libraries, an antibody library must be created. Variable heavy and variable light polymerase chain reaction (PCR) products, representing the repertoire of Ig genes, are inte- grated into a phage display vector (phagemid). Reverse transcription of high-quality mRNA is carried out from human peripheral blood mononuclear cells (PBMCs) into cDNA. Following that, the chain-region genes of VH and VL are replicated using specialized pri- mers to replicate all transcribed variable areas within the Ig repertoire. Fab fragments can be found in phage-displayed libraries, while scFvs are composed of two domains linked by a flexible linker. A phage coat protein containing antibody Fab fragments showed increase; their structure is stable, and they can be converted into whole IgG antibodies with- out affecting their function. Apparently, rearranged V genes of IgM repertoires are used to create naive antibody libraries. The human antibody germline is closely related to the sequences of the naive libraries since each sequence is generated from human B cells. This means that immunogenicity risks are very low. Today, practically all publicly available marketable collections are constructed on very varied nonimmunized gene repertoires, allowing the Abs selected for detection a potentially infinite number of targets. Phage display provides the benefit of allowing researchers to customize essential properties of Molecular Pharmaceutics and Nano Drug Delivery  4.5 Approaches for developing targeted monoclonal antibodies 91 effective antibody medicines (e.g., affinity, specificity, cross-reactivity, and stability). A total of nine human antibodies derived from phage display are now in use, because US FDA has approved the use of this medication to treat humanoid diseases, confirming the technique’s dependability as a foundation for antibody development. 4.5.3 Transgenic mice that produce human monoclonal antibodies Transgenic mice (Fig. 4.3) offer a dependable platform for antibody drug development. Transgenic animals have various benefits over conventional human antibody manufactur- ing systems, including less requirement for humanization, more variety, a clonal selection approach to optimize antibody affinity through in vivo maturation. Human antibodies were first produced in transgenic mice by Alt et al. in 1985, when they recommended inserting a human antibody gene into the mouse genome. This concept was novel and pro- vided a fresh path for the advancement of human antibody production. A human heavy chain construct was cloned for the first time by Brüggemann et al. in 1989, which had two different VH genes for diversity segments (D) coupled to the human heavy chain con- necting clusters (CH) and the constant region. This transgenic strain might possibly be used to create hybridomas of human IgM antibodies. Several murine Ig knockout mouse strains were developed. Chen et al. used gene-targeted deletion to knockout murine JH and JK genes, inactivating mouse Ig. Human IgH and IgL transgenic mice were then mated with murine IgH and IgL knockout mice to establish lines capable of producing more varied human antibodies. Longberg et al. created the first human Ig transgenic mouse strain, HuMab-Mouse , in 1994. Green et al. used yeast spheroplast-ES cell fusion to successfully introduce human IgK (170 kb) and IgH (220 kb) genome YACs into mouse ES cells. Mendez et al. subsequently established XenoMouse by crossing human YACs with murine IgH and IgL knockout mice and introducing them into the mouse ES cells, resulting in larger human IgK (*700 KB) or IgH (* 1 Mb) YACs. Unlike mouse antibodies, XenoMouse only expresses human antibodies. 4.5.4 Antibody technique based on single B cells It is well-known that body’s immune system responds robustly to antibodies, which are highly precise, counteracting, and self-tolerating. Antibodies that can be used as therapeu- tics via the standard hybridoma process or utilizing transgenic mice might cause severe immunogenic responses, in addition to long-term vaccination protocols and screening [such as human antimouse antibodies (HAMAs)]. Human B cells were immortalized with Epstein Barr virus to overcome these challenges. Micromanipulation, laser cap- ture microdissection, and fluorescence-activated cell sorting (Fig. 4.3) can be used to isolate single B cells from either PBMCs or lymphoid tissues. Further, each Ig heavy chain and its corresponding light chain must be cloned directly besides single B cell sorting. By using nested or semi-nested reverse transcription-PCR, the variable heavy and light chains of each identified B cell are amplified. Forward primers are usually targeted at the variable leader sequences of IgH and IgL, whereas reverse primers complement the Ig con- stant region. The VH and VL recoveries might be increased by adjusting different Molecular Pharmaceutics and Nano Drug Delivery 92 4. Monoclonal antibodies: recent development in drug delivery primer-set compositions. In order to generate recombinant mAbs, a mammalian cell is then used to express the cloned genes. A profile of each generated mAb is determined after it has been detected that it is reactive. The use of microarray chips and microengrav- ing techniques is notable for screening secreted mAbs with high throughput [46,47] and evaluating their ideal reactivity. Using the immunospot array test on a chip and a cell-based microarray chip technology and by covering a chip with an anti-Ig antibody, a chip can trap secreted antibodies and is therefore used to detect and recuperate particular antibody-secreting cells. Human mAbs have previously been produced for bacterial, parasitic, virus-infected, or autoimmune ill- nesses using the single B cell approach. A yeast infection can be treated with monoclonal anti-Candida mAb antibodies developed by single human B cells, which can boost protec- tion against disseminated candidiasis through phagocytosis. Antiviral mAbs have also been successfully produced using the single B cell method. Several studies have reported identifying dengue-neutralizing antibodies in memory B cells cultured from human anti- gens and identifying antigen-specific B cells in the peripheral blood of DENV-immune individuals. It was observed that rotavirus antibody gene repertoires in naive and memory B cell subsets were analyzed with the single B cell method and that human rotavirus-specific mAbs could be generated by isolating single B cells from the intestinal tract mucosal. A single human B cell method plus mAbs for bacterial and viral infec- tions has also resulted in antibodies against the complement factor H that are therapeutic for cancer. 4.5.5 Humanization of mAbs The humanization of mouse mAbs is considered on bulk quantities due to the ease of obtaining, low cost, and rapid development of mouse mAbs. Typical nonhumanized murine mAbs have a number of limitations as therapy, such as prompt HAMAs when patients are medicated with them. When humanized mAbs are used to reduce the immu- nogenicity of murine antibodies, they can effectively show effector responses. 4.5.6 Generation of humanized mAbs In humanized mAbs, only the complementary-determining region (CDRs) of the light and heavy chains are murine, which were authorized in clinical trials in early 1988. CDR grafting is a well-known approach for producing humanized mAbs that was first proposed by Gregory P. Winter in 1986. Nonhuman CDR sequences are added to human framework sequences using this method, allowing the antibody to maintain its target-specific antigen response. It was the development of daclizumab in 1997, which attaches to the IL-2 receptor and inhibits transplant rejection , which was the first CDR-grafted humanized monoclonal antibody to be approved by the FDA. Various approaches have been developed to measure the quality of human mAbs in changing environments. An assessment of antibody sequence humanness known as the H-score has Molecular Pharmaceutics and Nano Drug Delivery  4.5 Approaches for developing targeted monoclonal antibodies 93 been developed by Abhinandan and Martin , which compares subset human variable region sequence databases with mean value specifications. mAb-based drugs are clinically more effective when humanized antibodies are used. It has created a new era in which mAbs can be engineered to have a greater range of potential uses in the biomedical field due to this complex control over antibody sequences. Recent developments have seen most mAbs used to treat various diseases being chimeric or humanized. 4.5.7 Human and humanized mAbs There is already a plethora of antibodies that are therapeutic on the market for the treatment of a variety of medical problems, including immune-mediated chronic inflam- mation. Numerous clinical systems have been implemented, and the insufficiency of head-to-head comparative trials in distinguishing the relative clinical efficiency and safety profiles of one monoclonal antibody vs another can be a difficult problem. The ability to determine whether a monoclonal antibody is entirely human or humanized is one distinguishing feature of dermatologists when explaining clinical trial data. This finding illustrates the differences and similarities between totally human and humanized monoclonal antibodies in terms of nomenclature, engineering, and therapeutic prospects. While there are several variances among different types of monoclonal antibodies, recent research indicates that this categorization has little bearing on the overall clinical efficacy and safety profiles of a specific treatment. It is obvious from the molecular insights pro- vided in this perspective that any monoclonal antibody, whether humanized or not, will have the same effect on cells, and must be examined separately for its medical influence in terms of safety and efficiency. Monoclonal antibodies will continue to be a potential treatment option for a wide range of disorders with dermatological consequences in the future. 4.5.8 Stereospecific monoclonal antibodies Moreover, therapeutic mAbs can also be developed by one of the valuable approaches, in which mAbs are selected due to their greater affinity and selectivity toward definite antigens (Ag). It was discovered that mAbs can recognize two kinds of epitopes with great accuracy (linear epitope present on protein’s primary structures and conformational epi- topes present on protein’s secondary and tertiary structures). As a result, identifying pro- tein conformational configurations leads to increased specificity and affinity for therapeutic treatments. Usually, linear epitope-specific mAbs accept 2D configuration. However, stereospecific mAbs can recognize 3D molecule configurations. Because of their specificity and selectivity for target antigens, mAbs can be utilized as bioactives, and con- formational epitope-specific mAbs have been demonstrated to be superior to linear epitope-specific mAbs. Because proteins are full conformational assemblies in nature, ste- reospecific antigen identification brings up new treatment options. Molecular Pharmaceutics and Nano Drug Delivery 94 4. Monoclonal antibodies: recent development in drug delivery FIGURE 4.4 Novel drug delivery sys- tems for mAbs. 4.6 Novel drug delivery systems for mAbs Though mAbs have had a lot of success for therapeutic purposes over the last 40 years, some constraints such as deficient pharmacokinetics, poor pharmacodynamics, and sys- temic toxicity have limited their prospective usage. Advanced research is being directed toward numerous new techniques, including changed Fc function antibodies, bispecific antibodies, intrabodies, and antibody fragments, to achieve superior pharmacokinetics, improved selectivity, and increased efficiency. In clinical application, both patients as well as health professionals need additional competent and more secured mAbs to initia- tion improved concurrence. The following section discusses a variety of novel drug delivery approaches that have been used to solve obstacles encountered during the devel- opment of mAb-based medicines (Fig. 4.4). 4.6.1 Nanoparticles Nanoparticles (NPs) are a type of particulate delivery system that come in sizes ranging from 10 to 1000 nm and are widely used in pharmaceutical drug delivery systems. There are numerous polymers accessible for creating NPs, one of which being poly lactic- co-glycolic acid (PLGA), which is a popular biodegradable polymer. This polymer hydro- lysates into glycolic acid and lactic acid, which are easily metabolized in-vivo. Because the EMA and FDA have approved this polymer for a variety of uses in the development of drug delivery systems, PLGA-conjugated NPs are frequently employed in clinical trials. When compared to free anti-OX40 mAbs, PLGA-based NPs loaded with mAbs are able to target tumor necrosis factor (TNF) receptor OX40, which is predominantly expressed on activated T cells, and are found to be more efficient. Molecular Pharmaceutics and Nano Drug Delivery  4.6 Novel drug delivery systems for mAbs 95 Furthermore, it can persuade the development of cytotoxic T lymphocytes (CTLs), cyto- kine development, and tumor antigen-specific cytotoxicity. It was also reported that mAb fragments (3D8 scFv) were loaded into PLGA-NPs, resulting in increased cellular uptake via cellular endocytosis. Self-analogous bevacizumab NPs were shown to be three times more readily taken up in A549 cells than in MRC-5 cells. Cancer cells prefer to take up uncoated mAb-NPs over normal cells. 4.6.2 Microspheres Microspheres are being evaluated as a viable drug delivery carrier for IgGs or monoclonal antibodies, as they enable consistent therapeutic release and extended stability in the systemic circulation. IgG-MPEG-PCL-MPEG microspheres (6 m) were developed utilizing the double emulsion solvent evaporation technique, using copolymer of poly(ethylene glycol)-poly(-capro- lactone) (PEG-PCL). At the time of irradiation, it was found that IgG-MPEG-PCL-MPEG micro- spheres had lower IgG destabilization rates and higher entrapment efficacy than IgG-PCL microspheres. The s/o/w (solid-in-oil-in-water) method was established and optimized to prepare microspheres without compromising the stability issues of IgG or mAb. The combina- tion of the s/o/w emulsion method and spray-drying leads to successful entrapment of IgG microparticles into the PLGA microsphere core. This new combination process resulted in improved entrapment efficiency (60% w/w) and drug loading (6% w/w). The full length anti-human TNF-α mAb-entrapped microspheres of PLGA were positively prepared by the s/o/s process with capability to load bioactives as well as single mAbs in microspheres. The combination of mesenchymal stem cells and anti-BMP2 mAbs, as well as their entrapment in alginate microspheres, significantly boosted the osteogenesis associated with human bone marrow mesenchymal stem cells. Ethylene-vinyl acetate copolymer (EVAc) was used to load IgG in the form of dry powder and characterized for efficient polymer-fabricated antigen delivery strategy. EVAc microspheres can also be used for topical administration, sustained release injectables, and extended-release IgG. Another example of polymer-based vaginal delivery system was a vaginal ring, which delivers an Ab in the mouse model. Direct, local delivery of Abs by applying aqueous gels made up of carboxymethyl cellulose may be effective antiinfective technique after surgical proce- dures. Abs have been delivered to the CNS for a long time using a hyaluronic acid-bound Ab hydrogel with a covalent link. 4.6.3 Hydrogels Using a novel drug-loaded hydrogel system, the stability of epidermal growth factor was successfully enhanced. The porous architecture and higher water content are ideal for encapsulating mAbs. Furthermore, three dimensional structures of hydrogel were capable of entrapping the mAbs in complex networking of hydrogel networks. For the construction of therapeutically safe hydrogels capable of prolonged release of mAbs, a lot of research has been done on both natural materials (chitosan) and synthetic polymers (PLGA-PEG-PLGA). mAb delivery can be improved by applying the thermosensitive hydrogels with the character of transforming from solution to gel at body temperature. A modified Molecular Pharmaceutics and Nano Drug Delivery 96 4. Monoclonal antibodies: recent development in drug delivery biocompatible triblock copolymer of poly(2-ethyl-2-oxazoline)-bpoly(ε-caprolactone)-b-poly(2- ethyl-2-oxazoline) (PEOz-PCL-PEOz) was designed to prepare a biodegradable thermosensitive hydrogel, and it was able to delay the release of bevacizumab for several days (20 days). When delivered through subcutaneous injection, a trastuzumab-loaded hydrogel containing vitamin E-functionalized polycarbonate and poly(ethylene glycol) triblock copolymers was found to be effective in the treatment of breast cancer. 4.6.4 Other delivery systems The degradation of mAbs during prolonged release has been measured using several innovative mAb-based delivery techniques. Using electrostatic forces (pH 7.4 solution), bevacizumab-loaded nanosized mesoporous silica (SiO2) was created and found to have a delayed release of up to 30 days with protected functionality of released molecules. 4.7 Formulation challenges of mAbs The structural changes in mAbs can happen at any stage of the development steps due to the presence of the tertiary structure of biologics. These biologics are highly susceptible to physical stress, which can cause alterations in mAbs throughout the process of protein synthesis, processing, and storage. Structural modifications can be linked to the presence of nonphysiological conditions during processing, which may cause structural alternatives to adapt in the final formulation. This is a significant constraint and hurdle in biopharma- ceutical development, which is distinct from the creation of small molecule medicines, where such issues do not arise; nonetheless, pressures such as buffer selection, evolving processes, and container selection might raise this issue. Furthermore, any finished mAb-dependent biopharmaceutical formulations containing nonnative proteins should be overlooked. While taking protein solutions from vials, accumulation may result in nonuni- form dosage [72,73]. Because of their proteinaceous composition, mAbs suffer stability concerns when exposed to pH, higher temperatures, stress and humidity. The formu- lations rich in concentration are facing the problem of concentration-based decomposition of accumulated mAbs. The development of highly viscous concentrated mAb is mostly dependent on molecular interactions and protein self-association. Typical difficul- ties encountered during the development of mAb-based therapeutics as well as mAbs are constrained to intravenous and subcutaneous administration of lyophilized and liquid for- mulations. The main issue is mAb stability, which has a detrimental impact on yield due to product loss due to buildup in downstream processing steps. Unwanted byproducts created during manufacturing procedures can accumulate in HEK 293 exposed mAbs. Immunogenic mAbs are made using hybriddoma technology, and CHO-expressed mAbs have an immunogenic glycosylation profile. Biologics with instability issues may produce less therapeutic response or sometimes adverse events too. The buildup of IFN, for example, can affect the production of neutralizing anti-drug Abs (NAb) capable of block- ing IFN receptor binding, lowering clinical efficacy. Molecular Pharmaceutics and Nano Drug Delivery  4.8 Future trends of mAbs 97 4.8 Future trends of mAbs The approach of therapeutic antibodies is currently becoming an eye-catching option in the therapeutic purpose. Though there is still important growth likely for the thera- peutic antibody field. Traditionally, antibodies are utilized to manage the cancer, autoim- mune disorders, and infectious disorders. The mAbs may offer a potent therapeutic approach if the molecular mechanisms of a disease can be clearly described and the dis- tinct proteins can be linked in pathogenesis. For example, anti-CGRP receptor antibodies such as erenumab, galcanezumab, or fremanezumab have been progressed for the man- agement of migraine. Anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) antibo- dies (evolocumab or alirocumab) are used for the treatment of hypercholesterolemia. Anti-fibroblast growth factor 23 (FGF23) antibody (burosumab) is utilized to treat X- linked hypophosphatemia. Anti-IL6R antibody (sarilumab and tocilizumab) can be used to treat rheumatoid arthritis. Anti-factor IXa/Xa antibody (emicizumab) is a promising treatment for hemophilia A. Anti-von Willebrand factor antibody (caplacizumab) is con- sidered for the treatment of thrombotic thrombocytopenic purpura, and other antibodies will be accepted for new considerations in the future. Therapeutic mAbs can readily be classified into two broad categories. In the first one, the naked antibody is directly applied for disease therapy. Cancer treatments from this category may work by various mechanisms, such as mediated pathways like ADCC/CDC, direct targeting of cancer cells to induce apoptosis, targeting the tumor microenvironment, or targeting immune checkpoints. In altered pathways, the antibody kills cancer cells by recruiting natural killer cells or other immune cells. Currently, various novel methods have been employed to improve the therapeutic efficacy of ADCC or CDC, like antibody Fc point mutations or alteration of glycosylation to enhance the cancer cell killing rate [76,77]. The direct induction apoptosis in cancer cells has traditionally been the considered mechanism for therapeutic antibodies. For targeting the tumor microenvironment, antibodies can reduce tumorigenesis by targeting factors linked in cancer cell progression. Findings have mea- sured the synergistic actions of antibodies and chemotherapeutic bioactives, radiother- apy, and other biologic components, which will greatly benefit the further progression of antibody drugs. Thereafter, the identification of novel biomarkers may augment the effi- ciency and specificity of antibody-based therapy for human disorders. In the second type of antibody therapeutics, additional alterations are made to the antibody in order to advance its therapeutic efficacy. To create an immunocytokine, a selected cytokine is fused to an antibody to improve the delivery specificity. Antibody drug conjugates (ADCs) are made up of an antibody that targets a cancer-specific marker combined to the small molecule drug; the antibody developments the delivery to the target site, attractive the efficiency of the small molecule while decreasing side effects by inhibiting nonspecific toxicity to nontarget tissues [78,79]. The antibody may also be fused to a radionuclide for direct radiotherapy specifically to the tumor site [80,81]. Antibody- engaged effector cell functions may increase the therapeutic potential of bispecific anti- bodies. With regard to immunoliposomes, the coupling site of the antibody (Fab) is cleaved from the constant area and eventually conjugated to different nanodrug delivery systems like liposomal drugs to provide more specific targeting (Fig. 4.5). Molecular Pharmaceutics and Nano Drug Delivery 98 4. Monoclonal antibodies: recent development in drug delivery FIGURE 4.5 Molecular mechanism of therapeutic monoclonal antibody (mechanism of action of mAb ther- apy). (A) Naked mAb can function through various mechanisms, including antibody-dependent cellular cytotox- icity, using immune effector cells such as natural killer cells. Complement-mediated cytotoxicity (CMC) functions through the membrane attack complex and by inhibition of the receptor dimerization, inducing an apoptotic sig- nal or through effecting target cells by blocking the binding of ligands. Immune checkpoint inhibitors, including PD-1, function by blocking the T-cell-inhibitory receptors and CTLA-4, which function through activation of the T-cell function. (B) Conjugated mAb includes payload antibody for delivery of specific drugs or chemotherapy agents directly to the tumor cells, radioimmunoconjugates to deliver radioisotopes to the cancer cells, immunotox- ins to deliver highly toxic drugs to the target cells, and siRNA particles to downregulate a target gene. (C) CAR T-cell therapy uses a genetically engineered T cell to target a specific antigen on tumor cells. (D) Bispecific mAbs consists of two arms, with one arm recognizing cancer cells and the other activating antigens on immune effector cells including CD3. Molecular Pharmaceutics and Nano Drug Delivery  References 99 4.9 Conclusion There is a diverse variety of mAbs currently on the marketplace that have been exten- sively employed in clinics for diagnosis, treatment, and prognosis. Biotechnology-derived bioactives, such as antibody-dependent therapeutics, are on the forefront of innovation when it comes to treating a wide range of clinical conditions. In addition to conduct clini- cal studies, substances must be developed into stable formulations that may be utilized for targeted delivery. Since the mAbs have shown excellent clinical and commercial results, several scientists and pharmaceutical industry are now focusing on additional mAbs for delivery strategies. An enhancement in the efficiency of antibody therapeutics may be achieved by the use of transgenic organisms and "second-generation human chimeric mice." In the global pharmaceutical sector, the continual processing and refining of trans- genic animals gives new avenues for the creation of antibody drugs. Because of the tre- mendous breakthroughs in the field of antibody medications, the use of mAbs has evolved as a crucial category of pharmacological compounds for a wide range of human conditions such as metabolic disorders, immunological conditions, infection, malignancies, and brain disease. References Kohler G, Milstein C. 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