Chapter 3 Therapeutic Antibody
Antibodies are glycoproteins of the immunoglobulin superfamily.Antibodies are produced by plasma cells which are derived from differentiated B lymphocytes of the immune system in response to foreign substances.The basic structure of an antibody molecule such as human immunoglobulin G (IgG) consists of the four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains.One light chain (eitherκorλtype) is linked alongside one H chain(μ, δ, γ, α, ε), while the two H chains are linked together, also by disulfide bonds.There are two immunoglobulin domains in each L chain that are designated (from N-terminal) variable (VL) and constant (CL) domains.Each H chain contains one variable (VH) domain and 3-4 constant (CH)domains (i.e., CH1-CH3 for IgA, IgD, and IgG, and CH1-CH4 for IgM and IgE).The VL and VH domains form an antigen-binding site (paratope), while the constant part of the molecule determines antibody classes or isotypes (IgM, IgD, IgG, IgA, or IgE) and other biological functions,including complement activation and fixation to cell surface receptors (Fc receptors; FcR) (Figure 3.1).Antibodies provide host resistance to invaders, such as microorganisms, by different mechanisms (antibody-mediated immunity), either alone or via cooperation with other humoral and cellular factors of the immune system [1].
Figure 3.1 Basic structure of convention al antibody molecule, such as human IgG .(Please scan the QR code on the Preface to get original color figures.)
Source: Chaisri U, Chaicumpa W.“Evolution of therapeutic antibodies, influenza virus biology, influenza,and influenza immunotherapy”.BioMedResearchInternational, 2018, 2018:9747549.
Monoclonal antibodies (mAbs) are produced by B cells and specifically target antigens.The hybridoma technique introduced by Köhler and Milstein in 1975 has made it possible to obtain pure mAbs in large amounts, greatly enhancing the basic research and potential for their clinical use.Other scientific and technological advances have also enabled the successful translation of mAbs to the clinic.Around the world, at least 570 therapeutic mAbs have been studied in clinical trials by commercial companies, and 79 therapeutic mAbs have been approved by the US FDA and are currently on the market, including 30 mAbs for the treatment of cancer.The increasing importance of therapeutic mAbs is apparent, as mAbs have become the predominant treatment modality for various diseases over the past 25 years.During this time, major technological advances have made the discovery and development of mAb therapies quicker and more efficient.Since 2008, 48 new mAbs have been approved, contributing to a total global market of 61 mAbs in clinical use at the end of 2017, according to the US FDA.Strikingly, a total of 18 new antibodies were granted approval by the US FDA from 2018 to 2019-this number was tallied from information contained on various websites, including the antibody society, the database of therapeutic antibodies, and company pipelines and press releases [2].A list of antibody-based drugs approved by the US FDA is shown in Table 3.1.
Therapeutic polyclonal antibodies (PAbs) obtained from serum/plasma of specifically immunized animals (such as horse, sheep, donkey, camel, goat, and rabbit) have been used in the early days of serum therapy.Nevertheless, adverse side effects including immediate reactions (both IgE and non-IgE mediated), pyrogenicity, and/or delayed serum sickness occur frequently in human recipient.The animal immunization requires repeated and lengthy immunization process before a satisfactory antibody level is reached.Besides, limited amount of the immune serum/plasma(compared to the high demand) is obtained from individual animals at one bleeding time.There is a batch-to-batch variation of the antibody quality as well as a difficulty in eliciting antibodies against low immunogenic but highly toxic substances.Moreover, large animals require large pasture for gazing and roomy shelter.Proper husbandry must be provided in order to keep them in good health and free of infection, particularly zoonosis such as equine encephalitis that may be transmitted to the animal caretakers or the recipients of the antibodies.Invention of hybridoma technology by Köhler and Milstein in 1975 has abolished some limitations of therapeutic antibody production by animal immunization.
Mouse monoclonal antibodies with well-defined target- specificity, high purity, and reproducible quality at the desired amount can be produced by growing an established hybridoma clone (derived from fusion of parental B and mouse myeloma cells) in vitro.Monoclonal antibodies from the hybridoma technology have been used extensively in immunoassays, imaging, and passive immunotherapy of infectious and noninfectious diseases.The first therapeutic mouse monoclonal antibody, that is, muromonab, which is IgG2a specific to CD3 on T cells was approved by US-FDA in 1986 for treatment of allograft rejection.Limitations of the mouse monoclonal antibody include requirement of tissue culture facility, strict aseptic techniques, and expensive culture medium.Hybridoma cultures require frequent subcultures due to rapid depletion of nutrients in the growth medium, deposition and accumulation of apoptotic/necrotic cells, and cell cycle arrest (which can stop dividing, known as senescence stimulated by cell-to-cell contact).The major obstacle in using mouse monoclonal antibodies for human treatments is their immunogenicity.The mouse proteins are foreign to human immune system and human anti-mouse antibody (HAMA) response is elicited which leads to a rapid clearance of the mouse antibody and adverse reactions.Besides, murine monoclonal antibodies are relatively inefficient in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), the activities which are critical for anti-cancers [4, 5].(https://www.daowen.com)
The first attempt to reduce immunogenicity of the therapeutic mouse monoclonal antibody was to replace the mouse Fc fragment or the whole antibody constant regions (CH1-CH3) with the human counterpart by means of genetic manipulations, that is, mutations or engineering (Figure 3.2 and Figure 3.3).The chimeric antibody retains the mouse Fab or Fv (VH-VL) fragments with the same epitopic specificity to the original molecule but the immunogenicity to the human immune system is reduced by ≥70%.Retention of the Fc portion in the chimeric molecules is necessary for maintaining the immune effector activities such as Fc-dependent ADCC or binding to cellular receptor for enhancement of phagocytosis, activation of complement, and clearance of immune complexes.The first chimeric human-mouse monoclonal antibody, that is, rituximab, was approved by US-FDA in 1997.It is mouse IgG1 specific to CD20 for treatment of non-Hodgkin lymphomas.Alternative approach for production of chimeric human-animal antibodies has emerged through the use of humanized-rodent such as OmniRat that carries a chimeric human/rat IgH locus and fully human Igκ or Igλ locus.Mouse antibody fragments including F(ab)′2, Fab, and single-chain antibody variable fragment (scFv, which the VH is linked to the VL by a polypeptide) can be used for human therapy when the effector functions of the Fc are not needed.However, these antibody fragments are still immunogenic in human recipients.To obviate this problem, further reduction of the mouse antibody immunogenicity was performed by molecular grafting all antigen-binding loops (complementarity determining regions, CDRs) of the mouse antibody onto the closest human immunoglobulin framework regions (FRs) (humanization process).
The humanized-mouse antibody retains the antigenic specificity of the parental molecule (provided that the conformation of the antigen-binding fragment is conserved after humanization, protein purification, and refolding).Alternatively, humanization of murine antibody can be done by replacing some surface-exposed residues of the mouse framework regions with those of the human regions while maintaining the CDRs and core residues of the mouse framework (residues that are important for maintaining the affinity, i.e., canonical structure or regions that contact with the antigen).This process is called “Resurfacing”.The first humanized-monoclonal antibody, antithrombolism, was approved by the US-FDA in 1994.Nowadays, many of the licensed therapeutic monoclonal antibodies are in the humanized-mouse format.Production of the humanizedantibodies by CDR grafting or resurfacing is laborious and cumbersome as it has to be done antibody-by-antibody.Sometimes the target binding affinity or specificity of the parental molecule is not maintained.Nowadays, fully human monoclonal antibodies can be produced by using several strategies.Human monoclonal antibodies could be produced from hybrids which were obtained by fusion of either immunized peripheral blood lymphocytes or immune B cells obtained at disease recovery period with human lymphoblastoid or lymphoma cell lines (human hybridomas).Immune B cells derived from immunized or disease convalescing subject can be immortalized by infecting with Epstein-Barr virus (EBV); then the virus transformed-immune B cells are cloned; individual clones grown in vitro similar to the mouse hybridoma culture and the secreted human monoclonal antibodies can be harvested from their culture supernatants.Transgenic animals which their B cells carry human immunoglobulin gene loci produce human antibodies after antigen exposure.Hybridomas secreting human monoclonal antibodies can be obtained from the immune transgenic animals.Currently, several human monoclonal antibodies produced by transgenic animal lines have been approved for use in treatment of various human cancers and many more are in different stages of clinical development for various therapeutic purposes including allergy, autoimmune diseases,cancers, cardiovascular diseases, inflammatory diseases, infectious diseases, and pain [6-9].
Figure 3.2 Schematic overview of antibody humanization from murine antibodies (green domains)to fully huma n antibod ies (orange domains) and assoc iated suffixes.(A) The murine monoclonal antibody.(B) The chimeric monoclonal antibody: variable regions are of murine origin, and the rest of the chains are of human origin.(C) Humanized monoclonal antibody: only includes the hypervariable segments of murine origin.(D) Human monoclonal.CH: domains of the constant region of the heavy chain; CL: constant domain of the light chain; Fab and Fc: fragments resulting from proteolysis; VH:variable domain of the heavy chain; VL: variable domain of the light chain.(Please scan the QR code on the Preface to get original color figures.)
Source: Lu R M, Hwang Y C, Liu I J, Lee C C, Tsai H Z, Li H J, Wu H C.“Development of Therapeutic Antibodies for the Treatment of Diseases”.JournalofBiomedicalScience, 2020, 27(1):1.
Figure 3.3 Approaches for the devel opment of therapeuti c antibodies.(A) The traditional mouse hybridoma technique starts by immunization of mice with desired antigens to trigger an immune response.Harvested splenocytes are fused with myeloma cells to produce hybridoma cells that persistently secrete antibodies.After the screening, selected leads are used to generate chimeric or humanized antibodies.(B) Phage display.A human phage-displayed human antibody library is used to select antigens of interest.After 3-5 rounds of biopanning, immuno-positive phage clones are screened by ELISA; then DNA sequences are analyzed to construct and express human IgGs.(C) Transgenic mouse.Similar to the mouse hybridoma technique or single B cell methods.(D) The single B cell technique.From infected or vaccinated donors, PBMCs are prepared for isolation of suitable B cells by flow cytometry.Following the RT-PCR, VH and VL information of each B cell informs the generation of human mAbs.(Please scan the QR code on the Preface to get original color figures.)
Source: Lu R M, Hwang Y C, Liu I J, Lee C C, Tsai H Z, Li H J, Wu H C.“Development of Therapeutic Antibodies for the Treatment of Diseases”.JournalofBiomedicalScience, 2020, 27(1):1.
Phage display technology invented by Smith in 1985 has made in vitro production of human monoclonal antibodies specific to a desired target possible and relatively simple.Human immunoglobulin gene repertoire that resembles in vivo B lymphocyte pool (or even more diverse)can be generated in vitro by PCR amplification of human immunoglobulin gene amplicons derived from naïve, specifically immunized subjects, or synthetic gene pool and cloned into genome of a display system.Degenerate primers can be used to obtain multiple amplicons from a single template.Currently, many systems are available for the display purpose including yeast, bacteria such as Escherichia coli, mammalian cells, ribosomes, and phages (most commonly used).For constructing a human antibody-phage display library, genes of all human immunoglobulin families and subfamilies coding for diverse human antibody molecules (Fab, scFv, or sdAb) are PCR amplified and cloned into a phagemid vector (plasmid with a phage origin of replication)downstream of the phage gene coding for one of the coat proteins (pIII or pVIII) of the M13 phage.An amber stop codon is inserted between the gene sequences of the antibody and the phage coat.The recombinant phagemids are transformed into a special strain of E.coli that can produce tRNA of the stop codon (called suppressor E.coli).After growing and coinfecting the recombinant phagemid-transformed E.coli with a helper phage (such as M13KO7), the bacteria produce complete phage particles that individually display contiguous antibody-phage coat protein on their surface.Each phage particle resembles a B lymphocyte, which contains antibody coding gene in the genome and concurrently displays the respective antibody on the surface as a fusion partner of one of the phage coat proteins.
The antibody display phage library resembles a pool of B lymphocytes with diverse antigen-binding specificity.In our laboratory, a human scFv phage display library was constructed.Gene sequences coding for human VH and VL families and subfamilies were PCR amplified using the pooled cDNAs as templates and 14 forward and 3 reverse degenerate primers designed from multiple alignments of human functional immunoglobulin genes in the VBASE.The amplified vh and vl sequences were linked together via a polynucleotide linker coding for a polypeptide composed of a triplicate of four glycines and one serine [(G4S)3] by means of spliced overlapped extension PCR (SOE-PCR) to generate DNA sequences (huscfvs) coding for human scFvs(HuscFvs) or VH-peptide linker-VL.The huscfv sequences were cloned into phagemid via the appropriate endonuclease restriction sites at the 5′ and 3′ ends, respectively.The recombinant phagemids were used to transform competent F + E.coli (the E.coli strain that a fertility factor [F]exists autonomously on the F episome [conjugative plasmid] in the cytoplasm; the bacteria express pili on their cellular surface which function in bacterial conjugation; the pili served also as receptors for filamentous phage transduction) by electroporation.The transformed E.coli were grown and cotransfected with helper phage and the complete phage particles were collected from the bacterial culture supernatant.The library has been used to generate human single-chain antibodies (HuscFvs) against several antigens including snake venoms, toxins, viral proteins, and other molecules as well as human proteins [10-13].
Based on differential adsorption on protein G- and protein A- affinity resins, serum of camelidae including one humped (old world) camels (Camelus dromedarius, C.bactrianus), llamas (Lama glama and L.guanicoe), and alpaca (Vicugna pacos) were found to contain three different IgG subclasses including IgG1, IgG2, and IgG3.The IgG1 which is conventional heterodimeric four-chain antibody with two heavy (H) and two light (L) polypeptide chains linked together by disulfide and noncovalent bonds.The IgG2 (46 kDa) and IgG3 (43 kDa) are homodimers of H chains without the L chains; these dimeric H chain antibodies are called “Heavy chain antibody,HCAb”.The two H chains of the HCAb are associated by noncovalent bonds.The H chain does not contain CH1 domain; the hinge region is relatively long compared to that of the conventional four-chain IgG .The antigen-binding site of the HCAb comprises only a variable domain of the H chain, designated variable domain of heavy chain of HCAb or VHH.The VHH is linked to the hinge region followed by the Fc portion.Diagrammatic structure of the conventional IgG (IgG1 of camelids) and the antigen-binding site (VH-VL) in comparison to the HCAb structure and the HCAb antigen-binding site (VHH).Some hydrophobic amino acids in the region of the conventional VH that usually interacted with VL are mutated in the VHH to be more hydrophilic for reducing molecular aggregation.This area is located on immunoglobulin framework region 2(FR2) and contains characteristic tetrad amino acids including F/Y42, E49, R/C50, and G/L52.The tetrad amino acid hallmark is used for distinguishing the camel variable antigen-binding fragment(VHH) from the conventional VH.
The sequence of the complementarity determining region 3 (CDR3) of the VHH is unusually long(16-18 amino acid residues) and longer than that of the human and mouse VH (average of 12 and 9 amino acid residues, resp.).The camelid VH and VHH can be engineered to express as single-domain antibody fragments (sdAb) of ～15-20 kDa which still retain antigen-binding capacity.The sdAb have about 10x lower molecular weight than the IgG and about 2x smaller than the scFv;therefore, they are called “nanobodies or minibodies”.The genes coding for sdAbs can be cloned and expressed in E.coli system with relatively high yield, highly soluble in aqueous environments,and very robust.Because of their small sizes, the recombinant sdAbs are relatively stable to heat.They also have high binding affinity to the target.VHH antibodies have been shown to be potent enzyme inhibitors, as their long CDR3s can penetrate into the active pockets of the enzymes and block directly the respective catalytic activities which the conventional paratope consisting of VH and VL of the conventional four-chain antibody cannot do so.The sdAbs have become attractive therapeutic molecules for cancers, infectious diseases, parasitic infections, envenomation,intoxication, and inflammatory conditions caused by toxic enzymes.Camel VHH (Nanobody®,namely, ALX-0081, Ablynx, SOFINNOVA) specific to von Willebrand factor (anti-vWF) was tested in clinical trial in humans and found to be relatively safe without any untoward reactions in the recipients.Moreover, nanobodies have been used successfully as novel magic bullets for in vitro and in vivo immune-imaging for research and preclinical and clinical applications.A phage library that displays humanized-camel VHs/VHHs was constructed in our laboratory.The library was used subsequently in biopanning for selecting humanized-VHs/VHHs display phage clones that bound to a variety of targets including toxins and viral proteins [10,14-17].
Plasma membrane is a formidable barrier (because of the physicochemical properties) and only selectively allows permeability of certain small molecules (by means of passive diffusion,facilitated diffusion, or carrier proteins/transporters) such as gases, ions, water, sugars, amino acids,nucleosides, fat soluble vitamins.A variety of biomolecules including antibodies are retained extracellularly.Antibodies are thus inaccessible to their intracellular targets such as proteins/enzymes of replicating virus, intracellular bacteria, or toxins that have entered the cells.To circumvent this obstacle, several delivery systems including cationic liposome, polyethyleneimine(PEI), and peptides with cell penetrating capacity, called “Cell penetrating peptides, CPPs”, have been developed for carrying antibodies/antibody fragments and a variety of other cargoes including proteins, drugs, nucleic acids, plasmids, and siRNAs across the plasma membrane into cytosol and also to different subcellular compartments.CPPs have been used as a vehicle for cellular import of therapeutic molecules, both in vitro and in vivo.Examples of CPPs are (1) protein transduction domains (PTDs) such as penetratin (PEN; synonym antennapedia homeodomain peptide of Drosophila melanogaster), HIV-1 Tat peptide: Tat49-57, transportan (a 27 residue-peptide from galanin neuropeptide and mastoparan or wasp venom toxin), and VP-22 peptide of structural protein of herpes simplex virus; (2) amphipathic peptides such as noncytotoxic sweet arrow peptide(SAP) which is a proline-rich motif (VRLPPP), peptide vector named MPG derived from the fusion sequence of HIV-1 gp41, and a hydrophilic domain of SV40 nuclear localization sequence; and (3)other CPP type such as nonaarginine (R9) and poly-lysine.In our laboratory, cell penetrable human scFvs and humanized-camel VHs/VHHs specific to viral proteins and toxins have been prepared by linking the antibody molecules to either penetratin or R9.These fusion proteins readily entered mammalian cells without causing cytotoxicity and bound to their respective intracellular targets.They were safe for mice after injecting repeatedly either intravenously or intraperitoneally at comparable doses to those given to humans for passive immunotherapy [10, 18-20].