Published: 27 April 2023

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Diseases of the CNS remain the world’s leading cause of disabilities, accounting for more hospitalization and prolonged care than almost all other diseases combined. Scuh CNS diseases include brain tumours, HIV encephaopathy, epielpsy, verebrovascular diseases and neurodegnerative disorders. The incidence of CNS disorders in human increases with age. Being the most delicate organ of the body, the brain is protected against potentially toxic substances by the blood-brain barrier (BBB); however, the BBB also prevents the penetraiton of most moleules that have potential activity in the CNS. 98% of small molecules are not able to cross the BBB and 100% of large molecuels cannot cross the BBB. (Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

The highly restrictive nature of the BBB results in the majority of small polar molecules being incapable of dif- fusing into the brain (Figure 3C). These molecules include glucose, amino acids, nucleosides and many other mol- ecules. Given the essential roles these molecules play in cell growth and metabolism, it is essential for them to be transported across the BBB. This is achieved through the presence of carrier mediated transport proteins pre- sent on both the luminal and abluminal membranes of the endothelial cells of the BBB. (Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

Present at all levels of the vascular tree, the BBB is com- posed of endothelial cells lining the brain vasculature cou-pled with surrounding astrocytes and pericytes. Forming the morphological basis of the BBB, brain endothelial cells are phenotypically dissimilar to those of the peripheral circulation (Bernacki et al, 2008). Brain endothelial cells are characterised by their highly polarised tight junctions connecting them to adjacent cells. (Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

The highly polarised nature of the BBB endothelial cells and the overall tightness of the structure, restricts the trans- port of molecules greater than 500 daltons into the brain (Daneman, 2012). This results in the prevention of an over- whelming majority of small molecules from crossing the BBB (Gabathuler, 2010). In addition to this, the few drugs capable of crossing the barriers are actively pumped back out by protein efflux transporters present on the endothe- lial cells membranes. (Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

Given the significant need to increase cytotoxic therapy accumulation in metastatic brain tumours, there are numerous strategies to enhance delivery which could be investigated. These approaches include hijacking recep- tor mediated transport mechanisms present on the BBB endothelium, bypassing the BBB through local delivery of therapeutic agents, the delivery ochemotherapeutics simultaneously with drug transport inhibitors and disruption of the barrier. Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

Viral Vector Delivery:

Adeno-Associated Viruses: (AAV): To circumvent the blood brain barrier, most genomic medicines rely on direct intracranial injection of viral vectors encoding the transgene of interest. Viral vectors, such as recombinant adeno-associated virus (AAv) have had great success in gene therapy and are less immunogenic than most viral vectors; however, they require re-manufacturing for each target and are hindered by costly producton scale-up. In addition, AAv has a limited DNA packaging capacity and is assocaited with immunogenicity in the brain from both the vector and expression of foreign transgenes. Although the brain has been considered an immune privileged site, green fluorescent prtoein can induce a strong inflammatory response and neuronal cell death 3 weeks after injection with AAV serotype 9. In addition, Cas9 specific immune response have been elicited following AAV delivery in mice and pre-existing cellualr and humoral immunity ot Cas9 and AAVs are documented in humans. (Stah. “Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs” Molecular Therapy).

Sangamo Therapeutics (engineered AAV capsids with the ability to penetrate the CNS)

Receptor-Mediated Transport:

Receptor-mediated transport (RMT) is how large endogenous molecules, such as neuropeptides (insulin, transferrin or leptin) corrs the BBB. Insulin uptake is for example mediated by the insulin receptor, transferrin uptake is mediated by the transferrin receptor (Tfr), insulin-like growth factor (IGF) upstake is medaited by the IGF recptor and leptin uptake is medated by the leptin receptor. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

Designing genetically engineered fusion proteins to make use of the RMT to transport molecuels across the BBB is one approach for brain targeting. These proteins are prepared by fusing human transferrin to mouse-human chimeric IgG3 at 3 positons. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

The human insulin receptor (HIR), the most potent molecular trjan hourse in the BBB, is active in humans and rehesus monkeys. Brain drug delivery in the rhesus monkey was acheived by attaching a murine 83-14 mAb to the HIR. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),

Fusion constructs:

Stah. (“Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs” Molecular Therapy) disclsoes enablement of self-devliery of the Cas9 ribo nuclear prtoeins. Four repeats of the positively charged Simian vacuolating virus 40 nuclear localizaiton sequences (SV40-NLS) were fused to the N temrinus along with two repeats to the C terminus of Cas9. Using a single guid to turn on the tdTomato reported form the lox-stlop-lox Ai9 mouse, they reproted edited striatal volcume of about 1.5 nm.

Antibody-Mediated Transport:

Given the strong expression of the TfR on the BBB, numer-ous therapeutics have been generated in an attempt to improve the therapeutic treatment of Alzheimer’s disease and glioblastoma (Xu et al, 2011; Yu et al, 2011). In 2011, Xu et al reported the anti-tumour effects of an anti-Tf mono-clonal antibody on glioma cells in vitro, both in combination with a chemotherapeutic drug and alone (Xu et al, 2011). Interestingly, results from this study showed that the anti- body alone had an anti-proliferative effect through induced S phase accumulation and apoptosis, and when used in combination with the chemotherapeutic the effect was fur- ther enhanced, which suggested that combination therapy was more effective. Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

Olson (WO 2018/119001) discloses peptide-antibody fusions/conjugates for targeting therapetuic agents in the CNS or across the blood brain barrier. Olson teaches that the peptide or the antibody of the complex homes or targets to the crebrospinal fluid or hippcampus.

Sengupta (US 2017/0014528) discloses targetd drug delivery conjugates that include a targeting moiety linked ot a drug via a molecule haivng affinity for the targeting moeity (affinity ligand). Typically the conjugate includes a targeting ligand and a molecule of interest such as a therapetuic agent. The tareting ligand and molecule of itnerest are linked to each other via an affintiy ligand. The affintiy ligand is further covalently or non-covalently linked to a drug or therapetuic agent.

Antibodies targetting Transferrin receptor (Tfr):

Antibodies targeting the TfR which is highly expressed by endothelial cells that mkae up the BBB have been reported to cross the BBB, but whether endocytosis of anti-TfR antibodies into the brain vasculature can lead to antibody release and accumulation in the brain parenchyma remains controversial. (Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target” 2011).

Dennis, (“Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target” 2011) disclsoes a bispecific therapeutic antibody with a low affinity for TfR and a high affinity for the enzyme beta-secretase, an Alzheimer’s disease target.

Bohrmann (US 2019/0276530) discloses a blood brain barrier shuttle that includes a brain effector entity, a linker and one monovalent binding entity which binds to a blood brain barrier reeptor, wherein the linker couples the effector entity to the monovalent binding entity which binds to the blood brain barrier receptor. In particular embodiments, the monovalent binding entity which binds to the blood brian barrier receptor includes a blood brain barrier receptor ligand, scFv, Fv, sFab, VHH. In a particular emobidment, the monovalent binding entity is a scFab directed agasint the transferrin receptor.

Aptamers as Drug Delivery Vehicles:

Aptamers are single stranded DNA or RNA oligonucleotides that bind with high affinity to specific target molecules by folding into unique three-dimensional structures. The process of aptamer selection involves repeated steps of incubation of a high complexity library with a target of interest. Franciscis “Aptamer-Mediated Targeted Delivery of Therapeutics: An Update” Pharmaceuticals, 2016)

As aptamers function by molecular recognition, they can be developed for therapeutic applications with the same intended function as antibodies, such as drug delivery vehicles. Though analogous to their protein counterparts in regards to target recognition and application, aptamers possess numerous key advantages over antibodies and antibody fragments, the main being their size, pro- duction process and cost, stability and nucleic acid structure Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).

Aptamers have been covalently or physically functionalized with therapeutic compounds including chemotherapeutics, toxins, and therapeutic functionalized with therapeutic compounds including chemotherapeutics, toxins, and therapeutic oligonucleotides (siRNAs, miRNAs or antimiRs) and many methods of aptamer functionalization oligonucleotides (siRNAs, miRNAs or antimiRs) and many methods of aptamer functionalization have been proposed as valid means to improve the specificity of nanoparticles. These approaches have been proposed as valid means to improve the specificity of nanoparticles. Franciscis “Aptamer-Mediated Targeted Delivery of Therapeutics: An Update” Pharmaceuticals, 2016)

Against Transferrin Receptor:

Wilner, (W O 2013/163303) discloses nuclease-stabilized aptamers which bind the human transferrin receptor and are readily internalized by cells which provides a means to specifically deliver cargoes to human cells which express this receptor.

Rossi (WO 2019/033051) discllsoes ribonucleic acid compound that includes RNA sequence capable of binding to a transferrin receptor (TfR). The compound may further include a compound moeity such as a therapeutic moiety attached to the RNA sequence. The compound/therapeutic moeity can be covalently attached to the RNA sequence. The therapeutic moiety can be an anticancer therapeutic moeity. Anticancer agents include monoclonal anitobdies such as anti-CD20, anti-HER2, anti-CD52. The ribonucleic acid compounds may be used to deliver compound moieties or therapetuic agents. In some embodimetns the nucleic acid compunds include an additional aptamer molecule such that a bispecific apatamer is formed. Preferably the additional aptamer does not target and/or bind to TfR.

Tan “Enhanced in vivo blood-brain barrier penetration by circular Tau-Transferrin receptor bifuncitonal aptamer for Tauopathy therapy” J. American Chemical Society, 2020, 142, 3862-3872) discoses a circular Tau-TfR bifunctional aptamer.

Published: 23 April 2023

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The effector functions mediated by the antibody Fc region can be divided into (1) effector functions that operate after binding of antiobdy to an antigen (these funcitons involved the participation of the complement cascade or Fc receptor (FcR) bearing cells) and (2) effector functions that operate independently of antigen binding (thse functions ocnfer persistence in the circualtion and the ability to transfer across cellular barriers by transcytosis. Presta (US 6,737,056).

Modifying effector functions can be acheived by engineering the Fc region to either improve or reduce binding of FcyRs or the complement factors. When designing an IgG for a particular function, one must consider not only the human IgG isotype but also which of the FcyR would be the preferential target, the immune cell types epxressing the target receptor(s) and the differential binding of the various polymorphs (Presta, 2008, Curr. Opin. Immunol. 20, 460-470).

Numerous studies have shed light on the effector functions of antibodies as important mechanisms of action of therpaeutic antibodies in addition to theri binding affintiy and specificity for targets, in particular antibody-dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and a long half-life/clearance rate. Each of these efector functions is primarily triggered through direct interaciton of the Fc domain of the antibody with its corresponding ligands: ACC through interaction with the Fc gamma receptor IIIa (FcyRIIIa), CDC through interaction with the series of soluble blood rpteoins that constitute the antibody-dependent complement activaiton pathway (e.g., C1q, C3 and C4) and serum persistance through itneraction with the neonatal Fc receptor (FcRn). Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

A number of preclinical studies have suggested that ADCC is a major mechanism of action of antitumor antibodies, such as rituximab. The importance of ADCC has also been recognized in clinical settings, as evidenced by significant correlation between FcyRIIIa funcitonal polymorphisms and clinical outcomes of multiple therapeutic antibodies. (Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

Antibody Engineering for Increased Immunogenicity:

While binding of an antibody to the requisite antigen has a neutralizing effect that might prevent the bidning of foreign antigen to its endogenous target (e.g., reeceptor or ligand), binding alone may not remove the foreign antigen. To be efficient in removing and/or destruction foreign antigen, an antibody should be endowed with both high affinity binding to its antigen, and efficient effector functions. The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody-dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)).

Several antibody effector functions are mediated by Fc receptors (FcRs) which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcepisolonR, for IgA as FcalphaR and so on. Three subclasses of FcyR have been identified: FcyR1 (CD64), FcyRII (CD32) and FcyRIII (CD16). Because each FcyR subclass is encoded by two or three genes, and alternative RNA splicing leads to multiple transcirpts, a broad diversity in FcyR isoforms exists. The three genes encoding the FcyRI subclass (FcyRIA, RcyRIB and FcyRIC) are clusted in region 1q21.1 of the long arm of chromosome 1; the genes encoded FcyRII isoforms (FcyRIIA, FcyRIIB FcyRIIIB) are all clustered in region 1q22. Tehse different FcR subtypes are expressed on different cell types. For example, in humans, FcyRIIIB is ofund only on neutrophiles, whereas FcyRIIA is found on macrophages, monocytes and NK cells and a subpopulation of T cells. Notably, FcyRIIIA is the only FcR present on NK cells, one of the cell types impolicated in ADCC. (Presta (US 6,737,056)),

Enhancing ADCC activity by modifying the amino acid sequence of the Fc domain has been extensively studies, mainly through the random mutational analysis of human IgG1 Fc. For example, Fc domain variants with up to three mutations (S298A, E333A and K334A) which improved binding to FcyRIIIa and enhanced capacity for ADCC. (Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

Antibodies such as Xencar: CmAb and MacroGenics by engineering of amino acid in the Fc region as been done to develop more potent ADCC (“Proprietary Innovative antibody engineering technologies in Chugai Pharmaceutical”, 12/18/2012).

Presta (US 6,737,056) discloses a varaint of a parent polypetpide that includes an Fc region which meidates ADCC in the presence of human effector cells more effectively or binds an Fc gamma recetpor (FcyR) with better afifnity that includes an amino acid modificiation (e.g., a substitution) at any one of amino acid positions256, 290, 298, 312, 326, 330, 333, 334, 360, 378 or 430 of teh Fc region.

Exchanging Fc isotypes:

One study showed that exchanging the human IgG1 of an antibody with a murine IgG2a significantly improved ADCC using mouse effector cells and significantly better anti-tumor activity in a mouse model (Lutterbuese, Cancer Immunol Immunother 2007, 56, 459-468).

Another approach for enhancing CDC actvity is engineering of the H chain by shuffling IgG1 and IgG3 sequences within the H constant region. Several variant H constant regions, screened form a set of IgG1/IgG3 mixed sequences showed unexpectedly strong C1q binding and CDC activity that exceeded the levels observed for either parental IgG1 or IgG3. (Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

Antibody engineering for Reduced Immunogenicity:

Select appropriate isotypes: There are several strategies that can be used in the design of antiboides that avoid Fcgamma receptor interactions. For monoclonal antibodies, one approach is to select the human y4IgG isotype during construction of a humanized antibody. The y4IgG isotype does not bind Fcy receptors. Alternatively, a monoclonal antibody agent can be genetically enineered that lacks the Fc region, including for example single chain antibodies and antigen-binding domains. Yet another approach is to chemically remove the Fc region of a monoclonal antibody using proteolytic enzymes thereby generating antigen-binding antibody fragments such as Fab or F(ab)2 fragments. (Gupta-Bansal (US 2003/0198636).

Remove sugars: Another approach to reduce effector function is to remove sugars that are linked to particular residues in the Fc region, for rexample, by deleting or altering the residue the sugar is attached to, removing the sugars enzymatically, by producing the antibody in cells cultured in the presence of a glycosylation inhbitor, or by expressing the antibody in cells unable to glycosylate proteins. However, the forgoing approaches have residual effector function both in the form of complement-dependent cytolytic activity and Fc receptor binding. (Taylor, US 2007/0048300)

Taylor, US 2007/0048300) discloses a method for prodcing aglycosylated antibodies suitable as therapeutics because of their reduced effector function, by introducing an amino alteration at a first amino acid residue position which results in the reduced glycosylation of the antibody at a different or second amino acid residue position. In one embodiment, the preferred amino acid residue is of sufficient steric bulk and charge such that the residue inhibits glycosylation at a second amino acid position. Such amino acids include lysine, arginine and throsine. In another embodiment, the polypeptide has a first amino acid residue and second amino acid residue that are near or within a glycosylation motif, for example, an N-linked glycosylation motif tht contains the amino acid sequence NXT or NXS. In a particular embodiment, the polyeptide has a first amiho acid 299 and the second amino acid is 297, according to the Kabat numbeirng. In a particular embodiemnt, the amino acid substitution is T299C or T299A.

Exchanging Fc isotypes: Under certain cirumstances, abrogating or diminishing effector functions may be required. Abrogation of effector function has to some extent already been supplied by nature in the form of human IgG2 and IgG4 that exhibit decreased susceptibility to ADCC and CDC.

Alter the Fc region: There are several known ways to reduce the effector function of an antibody while retaining the other valuable attributres of the Fc region (US 2007/0048300A1) . One approach is to replace amino acid residues in the Fc portion (US 5,648,260 and 5,624,821). Another approach to reduce effector function is to remove sugars that are linked to partciular residues in the Rc region.

Moore (US 2006/0275282) describes antibodies and Fc fusion proteins with reduced immunogicity such as having reduced ability to bind one or more human class II MHC molecules.

Presta (US 6,737,056) discloses variants with reduced binding to an FcyRII having amino acid modification at any one of positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 33, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fc region. The varaint may display reduced binding to an FcyrIII with modificaiton at positions 238, 239, 248, 249, 252, 254…

Modification in Fc for Altered binding to FcRn:

FcRn is structurally similar to major histocompatbility complex (MHC) and consists of an alpha chain nocovalently bound to beta2-microglobulin. The binding site of human and murine antibodies for FcyR has been mapped to the “lower hinge region” consisteng of residues 233-239 (EU index).

Modification for increased binding to FcRn: Another direction in the improvement of the therapeutic eficiency of antibodies might be the further prolongation of their long in vivo half-lives. With this aim, there have been extensive studies attempting to introduce mutations in the Fc domain that render antibodies capable of more strongly binding the neonatal Fc receptor (FcRn). Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

Presta (US 6,737,056) discloses a polypeptides varaint with increased binding to FcRn that incldues modification at any of the positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 of the Fc region.

Modificaiton for decreased binding to FcRn:

Presta (US 6,737,056) discloses a polypeptides variant with reduced binding to an FcRn that includes an amino acid modificaiton at any of 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447 of the Fc region.

Modification for C1Q binding:

C1q and two serine proteases, C1r and C1s, form the complex C1, the first component of the complement dependent cyytoxicity (CDC) pathway. F1q is a hexavalent molecule with a MW of about 460k and a structure liikened to a bouqet of tulips in which six collagenous stalks are connected to six globular head regions. To activate the complement cascade, it is necessary for C1q to bind to at least two molecules of IgG1, IgG2 or IgG3 (the consensus is that IgG4 does not activate complement), but only one molecule of IgM, attached to the antigenic target. Presta (US 6,737,056)

Glu318, Lys320 and Lys322 has been reported as forming the binding site to c1q. The residue Pro331 has also been implicated in 1q binding. Presta (US 6,737,056)

It has also been proposed that the ability of IgG to bind C1q and activate the complement cascade also depends on the presence, absence or modificaiton of the carbohydrate moeity positioned between the two CH2 domains Presta (US 6,737,056).

Engineering of glycosylation in the Fc region; De-fucosylated antibodies :

One IgG molecule contains two N-linked oligosacharide sites in the Fc region. The general structure of N-linked oligosacharide on IgG is complex-type, characterized by a mannosyl-chitobiose core (Man3GlcNAc2-Asn) with or without bisecting GlcNAc/l-fucose (Fuc) and other cahin variants including the presence or absence of Ga1 and sialic acid. In addition, oligosaccharides may contain zero (G(0) one (G1) or two (G2) Gal. Recent studies have shown that engineering the oligosaccharides of IgGs may hield optimized ADCC. (Shinkawa, J. Biological Chemistry, 278(5), (2003)

Removing fucose in the Fc region has been used to increase binding affinity to FcyRIIIA. This has been done with Roche/Glycart: Glycomab and Kyowa Hakko Kirin/BioWa: Potelligent (“Proprietary Innovative antibody engineering technologies in Chugai Pharmaceutical”, 12/18/2012).

Published: 9 April 2023

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A main discrimination of recombinant bispecific antibodies is format and in particular the presence or absecne of an Fc region. Bispecific antibodies (biSpAbs) with no Fc will lack Fc mediated efector functions. (Kontermann “The making of bispecific antibodies, 9(2), 2017, 182-212)

Fc-less bispecific antibody formats:

Diabodies (scFv dimers): Diabodies are scFv dimers in which each chain consists of a variable heavy (VH) domain connected to a variable light (VL) domain using a peptide linker that is too short to permit paring betwen domains on the same chain. Consequently, pairing occurs between complementary domains of two different chains, creating a stable non-covalently bound dimer with two binding sites.

In the diabody format, the two variable domains are connected by a short linker that is usually 5 resiues, e.g., GGGS. Because the linker lenght is substantially shorter than that required to allow intrachsin assemble of the antigen-bnidng site, which would result in a ScFV, two chains dimerize in a head-to-tail orientation resulting in a compact molecule with a molecular mass similar to tandem scFv. (aobut 5 kDA). Expressing tow chains within the same cell, with either configuration vHA-VLB and VHB-VLA (A and B representing two different specificites) or VLA-VHB and VLB-VHA results in bispecific heterodimers with correct paring of the corresponding variable domains. (Kontermann “The making of bispecific antibodies, 9(2), 2017, 182-212)

Single-chain Fv (scFv)/Single domains specific to different targets genetically fused by peptide linkers: (e.g., WO2008/096158; WO/2007/112940). For reviews, see Enevery Curr Opin Biotechnol, 2009, 29)4), 405-11).

An scFv is an antibody fragment in which VH and VL domains are joined by a flxible linker that forces them to assemble in a stable manner despite the limited interaction surface. (Fischer “Bispecific antibodies: molecules that enable novel therapeutic strategies” Pathobiology, 2007; 74, 3-14).

Sgine-chain variable fragments (scFvs) are minimalist forms of a functional antibody, generated by fusing variable domains of the IgG heavy chain (VH) and light chain (VL) through a flexible polypeptide linker. ScFv molecuels have a MW in the range of 25 kDa, with a single antigen binding site that is cimprised of components from each arm of the antibody. (Wang, Design and Production of Bispecific Antibodies” Antibodies, 2019).

Single-domain fusions: Rather than connecting antigen binding sites in a tandem arrangement, single-domain antibodies such as VH or VL domains, VHH and nanobodes can be used to make bispecific molecules. Inclusion of two or more single domain antibodies will result in bivalent, trivalent or even multivalent molecules with one or more specificites. For example, two VHH domains were fused thorugh a long hinge sequence dervied form teh upper hinge of the llama IgG2a, selected for its protease resistance and flexibility. (Kontermann “The making of bispecific antibodies, 9(2), 2017, 182-212)

Bispecific T cell engager antibodies (BITE®): are recombinant protein constructs made from two flexibly linked antibody derived binding domains. One binding domain is specific for a selected tumor-assocaited surface antigen on target cells; the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. By the design, these antibodies are uniquely suited to transiently connect T cells with target cells and, at the same time, potently activate the inherent cytolytic potential of T cells against target cells. (Battel, WO @016/016859). ‘s binding activity wen assembled. The short flexible linker connecting the two scFvs enables free rotation of the two arms, which is vital for flexible interaction with targeted receptors on the two opposing cells membranes (cytotoxic T cell and Tumor cell) and the subsequent induction of T cell activation. (Wang, Design and Production of Bispecific Antibodies” Antibodies, 2019).

BiTE molecuels ahve been extensively applied in cancer immunotherapy for re-targeting of T cells to tumor cells. They employ scFv fragments form two different mAbs connected by a peptide linker, enabling them to retain each antibody.

–approved BITEs:

Blinatumomab, a bispecific T-cell engager (BiTE) is an example of a formal lacking an Fc region. It is based on the two single-chain variable fragments joined via flexible linker. Blinatumomomab was approved in 2014 for the treatment of Philadelphia chromosome-negative precursor B cell ALL. (Tustian, Biotechnol. Prog., 2018, 34(3)) The FDA and EMA ahve granted approval for blinatumomab, a CD19-CD3-bispecific T cell engager (BiTe)(Dietz “Programmable-multispecific DNA-origami-based T cell engaers” Natura nanotechnology, 2023)

Dual-affinity re-targeting proteins DARTS): A DART is composed to two Fv fragmetns, with two unique antigne-bnidng sites fomred when two Fc fragmetns hererodimerize. Specifically, Fv1 consists of a VH form antibody A and a VL from antibody B, while Fv2 is made form a VH from antibody B and VL from antibody A. Unlike BiTE antiboides which are connected by a polypetpide linekr, this combination allows dART to mimic natural interation within an IgG molecule. Compared to a BiTE, DART molecules are able to retin potency for both in vitro and in vivo adminsitraiton as well but can be produced at scale with lower aggregation rates. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019).

Published: 2 April 2023

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Categories: drug delivery

Antibody-Drug Conjugates (ADCs) are designed to bind to cell surface receptors and get internalized thorugh receptor medaited endocytosis, then trafficked into endosomes and ultimately lysosomes. Following internalization, ADC antigen complexes are fused with endosomes, which break them up for receptor recyling and transport ADs to lysosomes. Finally, an ADC molecule undergoes lysosomal degradation through a number of pathways to release its cytotoxin, which then binds to its target. (Chamow “Manufacturing challenges of therapeutic antibody -drug conugates, BioProcess Internaitonal, September 2023).

Preparation of Antibody-Cytotoxin Conjugates

In general, antibody cytotoxin conjugates are prepared by a multi-step process. First, antibody is covalently attached to a linker in a modification reaction and unreacted components and reaction products separated from antibody linker conjugate by desalting. Next, purified antibody-linker conjugate is reacted with modified cytotoxin to form the antibody-cytotoxin conjugate. The conjugate is purified from unreacted components, solvent and reaction products as by, for example, size exclusion chromatograph. For more information on purification of antibodies see antibody purification. Mazzola (US 2005/0031627)

The majority of ADCs are built on IgG1 scafolds. These complex about 150 kDa biomolecuels contain mltiple native sites for connjugation and can be modified to include additional reactive sites. (Own “Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry” AAPS Jouranl, 17(2), 2015).

Dai (US 2011/0166319) discloses a prcoess for preapring an atnibody chemically coupled to a drug which cindlues the steps of frist covalently attaching a linker ot the antibody, an optional purificaiton step, conjugated a drug to the antibody-linker and a subsequent purificaiton step, and optional holding steps.

Kaiser (US Patent Application No: 16/631,759, published as US 2020/0164338) discloses a method of prdoucing an antibody drug conjugation which includes the steps of pumpinng an antibody solution and a linker solution into a mixing device and then into a residence time device, followed along with a connection to the next unit operation, followed by conjugation of the drug.

Chemical Modifications & Linking Sites on Antibodies:

Chemical modificiations for the functionalization of antibodies include modification with an ezyme such as alkaline phosphatase or peroxidase (HRP), iodation or addition of a chelating compound for radioisotopes and modification with a low molecular compound such as biotin. These modification are typically performed via lysine amino group, a cystein thiol group and an activated carboxyl group. These modificaitons are specific for the functional groups, but are not site-specific. Thus, the problems include reduction in the activity of antibodies due to the modification or the like of the antigen-bnding sties of the antibodies, and difficult control of the number of compounds to be bound. For ADCs, anticancer agents are bound to antibodies in a site-nonspecific manner. In order to overcome these problems, antibody modificaiton has been practiced using antibodies haivng a particular site specificaly introduced functional group. For example, a particular non natural amino acid or free cysten can be introduced by genetic manipulation. Also, a glutamine can be intorduced using transglutaminase (TG). (Ito US 2018/0141976).

Antibody-drug conjugates (ADCs) are a subclass of biotherapeutics designed to faicilitate the targeted delivery of potent cytotoxic drugs to cancer cells. Depending on the conjugation chemistry, different types of ADCs can be constructed such as cystein-conjugated, lysine conjugated or site-specific ADCs. At a molecular level, all ADC molecuels bear complex chemical structures, combining the molecular characteristics of small molecule drugs with those of large molecule mAbs. In addition, the conjugation reaciton employed increases the complexity of ADC samples by producing a mixture of ADC molecules that are heterogeneous with respect to different drug to antibody ratios (DARs) and also the fact that conjugation is typically a random process becasue there are many surface accessible lysine residues. (Chen, “in-depth stuctural characterization of Kadcyla (ado-trastuzumab emtansine) and its biosimilar candidate” MABS 2016, 8(7), 1210-1223).

Although many drugs are amenable to direct conjugation to an antibody scaffold, heterobifunctional linkers often facilitate ADC bioconjugation. Linkers provide a functional handle for efficient conjugation to antibodies. Reactive handles often fetured in linker chemistry are grouped based on teh site of conjugation. N-hydroxysuccinimide esters are the most common choice for functionalizing amines, especially when coupling to E-lysine residues. For conjugation to cysteines, thiol-reactive maleimide is the most applied handle, alhtough it is also possible to create a disulfide bridge by oxidaiton with a linker bearing a sulfhydrl group. Aldehyde or keto functional groups such as oxidized sugar groups or pAcPhe unnatural amino acids can be reacted with hydrazides and alkoxyamines to yield acid-labile hydrozones or oxime bonds. In addition, hydrozine can be couple with an aldehyde via HIPS ligation to generate a stable C-C linkage. The mechanism of drug release is an important consideration in linker selection. Non-cleavable linkers rely on degradation of the scaffold within the lysosome after internalization. Alternatively, cleavage linkers respond to physicologic stimuli such as low pH, high clutathione concentrations and proteolytic clevage. Several non-clevable alkyl and polymeric linkers have been explored in ADC development. Non-clevable linkers require mAb degradation within the lysosome after ADC internalization to release active drug. Clevage linkers are popular in the ADC clinical pipeline with acid sensitive linkers such as hydrazones and silyl ethers. Hydrozones are easily synthesized and have a plasma half-life of 183 h at pH 7 and 4.4 h at pH 5, suggesting that they are selectively clevable under acidic conditions such as those found in the lysosome. Enzymatically clevage linkers are gaining significant attention due to superior plasma stability and release mechanism. The most popular enzymatic clevage sequence is the dipeptide valine-citrulline, combined with a self immolative linker p-aminobenzyl alcohol. Clevage of an amide linked PAB triggers a 1,6-elimination of carbon dioxide and concomitant rlease of the free drug in parent amine form. (Owen, “Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry” AAPS Journal, 17(2), 2015).

At N-terminal Amino Acid Residue:

Kim (EP 3015116) discloses an antibody-drug conjugate having a drug conjugated to an N-temrinal amino acid residue of a H or L chain of an antibody. Examples of the N-temrinus include not only aino acid residues at the distal end of th N-temrinus but also residues near the N-temrinus. Exampels of the reactive gorup capable of crosslinking with the alpha-amine group include any reactive groups known in the arm which can crosslink with the N-temrinal alpha-amine group of the H or L chain and include isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride.

At cystein/Lysine/Glutamine residues:

In theory, bioconjugation is possible via most amino acids. Nevertheless, the nucleophilic primary amine of lysine and reactive thiol of cysteine are the most commonly used amino acids inthe bioconjugation of antibodies. Lysine is one of the most commonly used amino acid residues for linking substrates to antibodies, becasue they are usually exposed on the surface of the antibody and thus easily accessible. Alkylation and acylation are the most important reaction with the nucleophilic E-amine. Antibodies can ontain up to 80 lysine residues and as a result, conjugaiton wa lysines inevitably leads to different number of substrates per antibody and antibodies with teh same number of substrates attached at idfferent sites. Furthemore, modifed lysines in proximity to the antibody-bnding site may influence the interaction of the IgG with the corresponding antigen. (In contrast to lysines, the number of cysteine residues within the sequence of IgGs is much lower. Tehre are only 4 interchain disulfide bonds that can be targeted as potential conjugation sites and thus the heterogeneity of immunoconjugates can be dramatically reduced. Moreover, these cysteines form covalent disulfide bonds to stabilize the tertiary sturcture of teh antibody and thus under non-reducign conditions are not reactive. Schibli “Antibody Conjugates: form heterogeneous populations to defined reagents” Antibodies 2015, 4, 197-224).

Cysteine based conjugation methods rely on a specific reaction between cysteine residues of the antibody and a thiol reactive functional group intalled on the payload. Amide coupling is a major ADC conjugation ethod connecting a payload and solvent accessible lysine residues on the antibody using linkers containing activated carboxylic acid esters. Amide coupling of an amine and an activated carboxylic acid is one of the most reliable high yielding chemical conversions in oranic synthesis. However, there are about 80 lysine residues on a typical antiboy and about 10 reisdues are checmially accessible. Thus, this conjugaiton modality often gives multiple ADC species with variable DARs and conjugation sites. (Tsuchikama, Protein Cell, 2018, 9(1)): 33-46.)

Employing the thiols of interchain cysteine residues in monoconal antibodies as attachment sites for drug molecules if one of the most used conjugation methods. In a human IgG1, there are four interchain disulfide bonds that can be used as potential conjugation sites. The four interchain disulfide bonds can be reduced by tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DT) which results in eight thiol gorups that are available for conjugating drug molecules. Through this method, different drug antibody ratio (DAR conjugates will be obtained when targeting typical DARs of 2-04. Amines of the antibodies can also react with the carboxyls that dervied form the drugs in the effect of the NHS to give antibdoy drug conjugates. Amines of lysines are commonly used for linking drugs to antibodies becasue lysines are usually exposed on the surface of the antibodies and thus easily accessible. Antibodies contain up to 80 lysines and as a resutl, conjugation through lysine residues inevitably leads to heterogenicity such as different number of drugs per antibody and antibodies with the same number of drugs attached at different sites. (Yao “Methods to design and synthesize antibody-drug conjugates (ADCs) International J. of Molecular Sciences, 17, 194, 2016).

The traditional linking sites in the preparation of protein conjugates are primary amines on lysines. The epsilon amino group in lysine reside can be easily acylated. However, depending on the number and relative accessibility of lysine residues on the protein surface, the random acylation process would generally result in a heterogeneous mixture of conjugates. These challenges have led to the use of thiols for mAb conjugation, as the number of thios is limited and fixed in the different subtypes of IgGs. The commercial antibody products are primarily IgG1s, which possess 4 inter and 12 intra chain disulfide bonds. Drugs can be linked to inter chain disulfides following reduction. Nonethelss, the use of disulfides for conjugation may still yield a hterogeous populaiton of conjugated products that contains positional isomers. Goswami (Antibodies 2031, 2, 452-500)

Cytotoxic drugs are generally conjugated to antibodies either thorugh lysine side-chain amines or through cysteine sulfydryl groups activated by reducing interachain disulfide bonds. Both of these procedures yield heterogenous products containing a mixture of species with different molar ratios of drug to antibody, linked at different sites, each with distinct in vivo pharmaokinetic, efficacy and safety profiles. To limit the potential liabilities associated with such conjugation methods, Junutula engineered reactive cysteine residues at specific sites in anitobodies, termed THIOMABs”, to allow drugs to be conjugated with defined stoichiometry without disruption of interchain disulfide bonds. For conjugation to covalently attach thiol reactive probes to engineered cysteines, first, the cysteine and glutathione adducts were removed from the THIOMABs by partial reduction followed by diafiltration. This partial reduciton also disrupts interchain disulfide bonds but not intrachain disulfide bonds. The interhain disulfide bonds were allowed to reform by air oxidation or by accelerated oxdiation using CuSO4 of dehydro-ascorbic acid. After this treamtnet, THIOMAB variants were then conjugated with biotin-PPO-maleimide. (Junutul “Site-specific conjugaiton of a cytotoxic drug to an antibody improved the therpaeutic index” Nature biotechnology, 24(8), 2008).

Hu (WO 2015/1091883) discloses an Fc cotnaining polypetpide such as an antibody which includes an N-glycosylated Fc region that includes an acceptor gluamine residue flanked by an N-glycosylation site and where the conjugate moiety is conjugated to the Fc containing plypeptide via the acceptor glutamine residue. The term “acceptor glutamine residue” refers to an amino acid that under suitable conditions is recognized by a transglutaminase (TGase) and can be crosslinked to a conjugate moeity that includes a donor amino group by a TGase through a reaction between the glutamine and donor amino groups (such as lysine or a structurally related primary amine such as amino pentyl group). The conjugation between the conjugation moeity and the acceptor glutamine residue is carried out by conjugating the amine donor group of the conjugation moeity or the small molecule handle to the acceptor glutamine residue. Thus, any conjugate moeity containing an amine donor group can be directly conjugated to the Fc bontianing polypetpide. Any conjugate moeity not containing an amine donor group can be indirectly conjugated to the Fc containing polypeptide via a small molecule handle which cotnains an amine donor group. For example, the conjugate moeity can include an amine donor group (e.g., primary amine-NH2), an optional linker, and an active moeity (e.g., a small molecule) The conjugate moeity can also be a polypeptide or a biocompatible polymer containing a reactive Lys. The amine donor group such as a primary amine (-NH2) provides a substrate for transglutaminase to allow conjugation of the agent moetiy ot the Fc contiaining polyeptide via the acceptor glutamine. Accordingly, the linkage between the donor glutamine and the amine donor group can be of –CH2-CH2–CO–NH–. In some embodiments, the Fc containing polypetpide and the conjugate moeity are linked throguh a cleavable linker. In some embodiments, the conjugate moeity includes an active moeity that is a cytotoxic agent. In some embodimetns, the N-glycosylation site and the acceptor glutamine resiude are 5 or less amino acid residues apart.

Shen (Nature biotechnology, 30(2), 2012) discloses generation of antibodies with cystein residues engineered into the IgG H chain that provide reactive thiols for conjugation to the auristatin and maytansine classes of cytotoxic drugs.

Thiol maleimide linkage has been widely used in the synthesis of ADCs because of its high slectivity, rapid reaciton kinetics and compatibility with aqueous reaciton conditions. For example, Bentuximab vedotin is prepared by reducing the endogenous antibody cystines, followed by conjugation of cysteine thiols with a maleimide-funcitonalized monomethyl auristatin D derviative. (pone, “understanding how the stbiliyt of the thiol-maleimide linkage impacts the pharmacokinetics of Lysine-linked antibody-maytansinoid conjugates). Biconjugate Chemistry, 2016).

Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics) are produced. by conjugation to surface exposed lysines or partial disulfide reduction and conjugaiton to free cysteines, respectively. These stochastic modies of conjugaiton lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. (Zimmerman, “Production of stie-specific antiboy-drug conjugates using optimized non-natural amino acids in a cell free expresison system). 2014).

Matsuda (US 2021/0139541) discloses compounds having an affinity substance to an antibody and a bioorthogonal functional group of the formula A-L-E-B where B is a bioothogonal functional gruop, E is a divalent group that includes an electrophilic group coupled with a leaving group and having ability to reactive with a nucleophilic group in the antibody, L is a divalent group that incldues a leaving rup and A is an affinity substance to the antibody.

–Ezyme-directed Modification:

Chilkoti “A noncanonical function of sortase enables site-specific conjugation of small molecules to Lysine residues in proteins” Angew, Chem. Int. Ed. 2015, 54, 441-445) disclsoes that the enzyem sortase A can be used to conjugate small molecules to a mAb with an ezquisite level of control over the site of conjugation. Sortase A recognizes the primary sequence LPXTG (where X is any amino acid) in a protein and cleaves the peptide bond between threonine and glycine, forming a stable intermedaite that joins the catalytic thiol in Sortase A to teh carboxyl group of threonine in a thioster bond. Chilkoti cloned the 4D5 mAb against human epidermal growth factor receptor 2 (Her2) and genetically modifed it to contain a pilin domain at the carboxy termini of its H chain. The recombinant antibody was incubated with SortageA-ELP and biotin-LPETGRAGG peptide overnight. Only the anti-Her2 H chain modifed to contain the pilin domain was biotinylated by SortageA-ELP.

–Affinity Guided Conjugation:

Affinity guided conjugation strategies invovle the incorporation of a scaffold that enables non-covalent interactions to occur between the affinity reagent and the target protein. This, in turn, increased local concetnraiton ofa.reactive moeity to a particular site, allowing for site-specific modificaitons to take place. Chilkoti “A noncanonical function of sortase enables site-specific conjugation of small molecules to Lysine residues in proteins” Angew, Chem. Int. Ed. 2015, 54, 441-445)

Small domains that bind with high affinity to a conserved sequence in teh Fc domain of antibodies are used to form non-covalent antibody conjugates. This approach does not require any modificaiton of the antibody. Among numerous Fc binding domains, teh ZZ domain –a dimer of the modified immunoglobulin binding site of protein A is the most widely used. Its high affintiy interaciton with the Fc part of antibodies has been exploited to by genetically fusing the ZZ domain to Pseudomonas exotoxin A. Incubaton with a antibody yielded hihgly toxic non-coavlently coupled ADCs. Schibli “Antibody Conjugates: form heterogeneous populations to defined reagents” Antibodies 2015, 4, 197-224).

Ito (US 2018/0141976 and EP 3299383) dicloses an IgG binding peptide having an amino acid sequence of 13-17 amino acid residues having the formula (X1-3)-C-(X2)-H-(Xaa1)-G-(Xaa2)-L-V-W-C-(X1-3). Xaa1 is a protein constituting amino acid such as a lysine residue, a cysteine residue, an aspartic acid or a glutamic acid residue or a non-protein constituting amino acid such as diaminopropionic acid or 2-aminosuberic acid. It is preferred that Xaa1 is modified with a cross-linking agent. The cross-linking agent is a chemical substance for linking the IgG binding peptide to IgG Fc via a covalent bonds. This can be a compound having at least two sites capable of binding to the desired amino acids (such as Lysine, cysteme). Examples include cross-linking aagents containing two or more succinimidyl grups such as DSG and DSS, cross-linking agents containing two or more imidic acid moieties such as DMA. It is also preferred that the peptide has no or little the same residue as Xaa1 (e.g., has only one or two same residues as Xaa1) in the sequence. Two cycteins in the peptide can form a disulfide bond to form a cyclic peptide. Alternatively, sulfide groups in the two cysteine residues on the other sides of the peptide may be linked via linker. The peptide can then be mixed with IgG causing a cross-linking reaction of the petide with the cross-linking agent with the IgG. This eliminates the need of altering the sequence of the antibody molecule. The IgG binding petpide can be modified with additional functional substances such as a labelling agent or an additional drug. The cross linking to IgG can occur site specifically between the amino acid residues Xaa1 of the IgG bidning peptide and Lys248 or Lys246.

Yamada (US Patent Applicaiton No: 16/663,791, published as US 2020/0190165) discloses an antibody which includes lysine residue(s) in a target region and 5 or more lysine residues in a non-target region and at least one functional substance which is a drug, a label or stabilizer which binds to the lysine residue(s) in the target reigon with 30% or more regioselectivity through a linker which does not include a peptide portion. The antibody is prepared by preparing a compound haivng the formula A-L-B-R where A is an affinity substance to a soluble protein, L is a cleavable linker which is a divalent group comprising a cleavable porition, B is a divalent group that includes a bioorthogonal functional group or a divalent group with no bioorthogonal functional group and R is a reactive group to a soluble protein such as an antibody. The reactive group can be specific to a side chain of a lysine residue. The bioorthogonal functional groups can bind to the soluble protein via a side chain of a lysine, tyrosine or tryptophan residue. First a soluble protein (T) such as an antibody assocaites with the compound through an affinity substance (A) to the soluble protein. Next, the compound reacts with a side chain of a specific amino acid residue (e.g., a side chain of a lysine resiude) in a target region present near an association site of the affinity substance and the coluble protein through a reactive gorup (R) (e.g., an activated ester) to form a conjugate between the compound and the soluble protein. The cleavable portion enables relase of the affinity substance (polypeptide) to an antibody.

Published: 17 January 2023

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Categories: Patent Law Cases

SPIO refers to superparamagentic iron oxide particles. They are commonly made of maghemite (Fe2O3) or magnetite (Fe3O4) having crystal-containing regions of unpaired spins. Those magnetic domains are disordered in the absence of a magnetic field. When a field is applied such as while takng an MRI, the magnetic domains align to create a magnetic moment much greater than the sum of the individual upaired electrons without resulting in residual magnetization of the particles. When injected into the blood stream, USPIO nanoparticles are taken up by macrophages and accumulate in inflamed tissues. Their iron moiety negatively enhances signal attentuation on T2-weighted images and their relative concentrations can be assessed by decreased T2-signal intensity or, more precisely, by decreased spin spin T2 relaxation time (US13/148028). The imaging capability of SPIOs is not from the SPIO intrinsically, but through their influence on longitudinal and transverse relaxation of the surrounding nuclei. In order to acheive active targeting of SPIO against specific biomolecules, it is necessary to first conjugate targeting agents onto the SPIO durface directly or onto its hydrophilic coating. An advantage of hainvg a polymer coating is that it can usually be modified to possess a variety of reactive moieties (i.e., amines, sulfydryls, carboxyls) which subsequently allow for more control over conjugation.

Because they are superparamagnetic and because they are taken up by phagocytic cells, iron oxide particles are used as a magnetic resonance (MR) contrast agent for the exploration of the mononuclear phagocytic system. After intravenous administration of conventional superparamagentic iron oxide particles of 30-1,000-nm diameter, all of the agents are cleared from the blood within minutes, rapdily accumulating in the cells of the mononuclear phagocytic system (MPS) of liver and spleen. Ultrasmall SPIO (USPIO) particles, however, have a longer blood half-life. Weissleder (Radiology, 1990, 175, 489-493) disclose an intravenous USPIO that is not immediately recognized by the MPS of liver and spleen and thus has a longer blood half life. The small size and prolongation of the plasma half-life enabled this agent to cross the capillary wall and have more widespread tissue distribution, including uptake by the MPS of lymph nodes and bone marrow.

CR2-SPIO/USPIO nanoparticles:

Serkova (“Renal Inflammation: Targeted Iwon Oxide Nanoparticles for Molecular MR Imging in Mice” Radiology, 255(2), 2010) disclose a recombinant protein containing the C3d-binding region of complement receptor type 2 (CR2) conjugated to the surface of an SPIO nanoparticle. Using a mouse model for lupus nephritis, they showed that after injection of the constructs into the mice and MR imaging, a significant reduction in T2 weighted MR imaging signal and T2 relaxation time was confirmed in nephritic kidneys of the mice (a significant accumulation of targeted iron oxide with a subsequent decrease in T2 relaxation times was noticed in the cortex and outer and inner medulla of the kidneys).

USPIO have been used to detect renal inflammation in numerous animal studies as well as to detect macrophage infiltration in glomerulonephritis and renal allograft rjection in humans. Thurman (US 13/148028; see also Sargsyan, Kidney International (2012) 81, 152-159) disclose conjugating superparagmentic iron oxide (SPIO) particles and ultrasmall SPIO particles conjugated with complement receptor type 2 (CR2)-Fc which can be used as negative contrast agents for MRA for the detection of intra-renal C3b/iC3b/C3d deposits in the kidneys of mouse models for lupus nepthritis.

Published: 8 January 2023

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Categories: Biotechnology

Introduction:

RNA interference (RNAi) is an endogenous cellular process in which double-stranded small interfering RNA molecules (siRNAs) bind to a specific mRNA target and trigger its degradation, resulting in reduced protein levels in the cell. This gene silencing method allows researchers to study the funciton of proteins within biologcial pathways by transiently removing them and analyzing the impact on cellular function.

Gene transcription control is also mediated using other endogenous RNA molecules, such as microRNAs (miRNAs) and small nuclear RNAs (snRNAs).

While RNA i has proen to be an effective tool for transient gene control, the discovery of CRISPR has revolutionized the field of permant gene editing. The CRISPR-Cas9 system can be used in various gene editing applicaitons and as gene knock-out thorugh the creation of stop condons or splice site variants. CRISPR is often used to validate gene function data that was initially obtained using RNAi approaches. CRISPR relies on the creation of a DNA double strand break and homology directed repair or the less desirable nonhomologus end joining for egene editing to occur.

RNA interference (RNAi):

The term RNA interference (RNAi) was coined after the discovery that injection of dsRNA into the nematode Caenorhabditis elgegans leads to specific silencing of genes highly homologous in sequence to the delivered dsRNA. RNAi was also observed subsequently in insects and other animals. The natural function of RNAi appears to be protection of the genome againsnt invasion by mobile genetic elements such as transposons and viruses, which produce aberant RNA or dsRNA in the host cell when they become active. Specific mRNA degradation prevents transposon and virus replication. DsRNA triggers the specific degradation of homologous RNAs only within the region of identity with dsRNA. (Tuschl “RNA interference is mediated by 21- and 22-nucleotide RNAs” Genes & Development, 2001).

RNA interference (RNAi) is used by eukaryotic cells in a variety of organisms like fungi, plants, worms, mice and probably humans. In plants, RNAi protects cells against RNA viruses. In other types of organisms it may protect against the proliferation of transposable elements that replicate via RNA intermediates. The presence of free, double stranded RNA triggers RNAi by attracting a protein complex containing an RNA nuclease and an RNA helicase. This complex cleaves the double stranded RNA into small fragments. The bound RNA fragments then direct the enzyme complex to other RNA molecules that have complementary nucleotide sequences which can be single or double stranded and the enzyme degrades these as well. In this way, introduction of a double stranded RNA molecule can be used to inactivate specific cellular mRNAs. Thus, RNA interference is typically a two step process. In the first step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering FNAs (siRNAs), probably by the action of Dicer, a member of the RNase III family of double-strand-specific ribonucleases, which cleaves double stranded RNA in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3′ overhangs. In the second step, siRNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex (RISC). An ATP dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC (containing a single siRNA and an RNase) then targets the homologous transcript by base pairing interactions and typically cleaves the mRNA into fragments of about 12 nucleotides, starting from the 3′ terminus of the siRNA.

siRNAi:

siRNAs are double stranded RNAs with a MW of about 13kDa which suppress protein translation by recruiting RISC to mRNA via Watson-Crick base pairing. Through the action of the catalytic RISC protein Ago2, a member of the Argonaute family, the target mRNA is cleaved. Alternativley, other Ago proteins (Ago 1, Ago3 and Ago4) catalyse endonuclease mediated nonspecific mRNA degradation by localizing the bound mRNA in processing (P) bodies. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022))

siRNA mediated gene silencing has been used safely in humans. These double-stranded RNAs with a MW of about 13kDa suppress protein translation by recruiting RISC to mRNA via Watson-Crick base pairing. Through the action of the catalytic RISC protein Ago2, a member of the Argonaute family, the target mRNA is cleaved. Alternatively, other Ago proteins (Ago1, Ago3 and Ago4) catalyse endonuclease-mediated nonspecific mRNA degradation by localizing the bound mRNA in processing (P)-bodes. siRNA can reduct the expression of any protein-coding gene and has been approved by the FDA and EMA in the form of drugs such as patisiran, which is used to treat hereditary transthyretin-mediated amyloidosis (hATTR), givossiran, which is used to treat acute hepatic pophyria, lumasiran, which is used to treat primary hyperoxaluria type 1 and inclisiran, which is used to treat hypercholesterolaemia. Given that siRNA intefers with mature mRNA, it requires only cytoplasmic delivery, which is easier to acheive than nuclear delivery. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022).

Antisense Oligonucleotides (ASOs):

Companies: Sarepta Therapeutics

ASOs are a second class of RNA therapeutics, and are oligonucleotides with a MW of 6-9 kDa. ASOs have the same manufacturing advantages as siRNA and have been approved by the FDA to treat familial hypercholesterolaemia, hATTR amyloidosis with polyneuropathy, specific subtypes of Duchenne muslar dystrophy, and infantile-onset spinal musclular atrophy. ASOs can act throguh three mechanisms of action. First, similar to siRNAs, ASOs bind mRNA via Watson-Crick base pairing, but unlike siRNAs, the ASO DNA-RNA heteroduplex recruits RNase H1 rather than RISC. RNase H1 dependent ASOs are also known as gapmers and lead to cleavage of the target RNA. Second, ASOs can also interfer with splicing machinery by interacting with pre-mRNA, thereby promoting alternative splicing, and increasing target protein expression. Thus, unlike siRNA, which silences target genes, ASOs can be used to increase protein activity in diseases including Duchenne muscular dystrophy and spinal muscular atoprhy. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022).

MicroRNA (miRNAs):

MicroRNAs (miRNAs) recruit RISC to complementary mRNA sequences, thereby facilitating targeted RNA interference. As a result, miRNA mimics, which are desigend to increase antive miRNA activity, and ant8-miRNAs or antago-miRNAs, which inhibit miRNA activity, have been studies in animal models and used in clincial trials. Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

In 2001, several groups used a cloning method to isolate and identify a large group of miRNA from C. elegans, Drosophila and humans. Several hundeds of miRNAs have been identified in plants and animals which do not appear to have endogenous siRNAs. Thus, while similar to siRNAs, miRNAs are distinct. miRNAs thus far observed have been about 21-22 nucleotides in lenght and they arise from longer precursors, which are transcribed from non-protein-encoding genes. The precursors form structures that fold back on themselves in self-complementary regions; they are then processed by the nuclease Dicer in animals or DCL1 in plants. miRNA molecules interrupt translation through precise or imprecise base-pairing with their targets. Studies have shown that expression levels of numerous miRNAs are associated with various cancers (US 2009/0175827).

Animal cells ahve been shown to express a range of about 22 nucleotide noncoding RNAs termed micro RNAs (miRNAs). The human mir-30 miRNA can be excised from irrelvent, endogenously transcribed mRNAs encompassing the predicted 71 nucleotide mir-30 precursor. One common feature of miRNAs is that they all reside within a putative arm of a predicted aobut 70 nt precursor RNA stem-loop. Dicer, an RNase III-tye enzyme, is beleived to be important for the processing of these miRNA precursors into teh about 22 nt mature miRNAs. (Dicer is also involved in siRNA production form longer dsRNAs above). Expression of the mir-30 miRNA specifically blocks the translation in human cells of an mRNA containing artifical mir-30 target sites. Similalary designed miRNAs can also be excised from transcripts encompassing artificial miRNAs precursores and can inhibit the epxression of mRNAs containg a complementary target site. This approach offers a number of important advantages when contrasted with siRNAs including transfection of miRNA expression plasmids in simple and inexpensive which can result in continous miRNA production. Inhibitor miRNAs could aslo be expressed using viral vectors thaus allowing the production of miRNAs in primary cells or in other cells that are not readily transfectable with syntehtic siRNAs. As the inhibitory RNA is expressed as part of an mRNA, it should also be possible to use regulatable promoters to control miRNA production. (Zen, Molecular Cell, 9, 1327-1333, June 2002).

It is thought that bout 30% of the total genes of the human genome are regulated by miRNAs. The miRNAs are generated through transcription of individual genes in the non-coding regions. The miRNA is transcribed from a pri-miRNA which is a precursor transcribed in the nucleus by RNA polymerase II. The pri-miRNA is cleaved by the RNase III enzyme called Drosha (dsRNA-specific ribonuclease) to produce a pre-miRNA having a hairpin loop structure. The hairpin loop of the pre-miRNA is exported out of the nucleus by the protein exportin-‘5 and Ran-‘GTP, which serve as cofactors, and processed into a miRNA duplex about 22 nucleotides in lenght by the action of teh RNase III enzyme Dicer and TRBP (transactivation-‘responsive RNA binding protein). The miRNA duplex binds with RISC (RNA-‘induced silencing complex) and regulates genes by cleaving mRNAs or preventing translation.

Various kinds of miRNAs and target genes regulated thereby may be useful in predicting the mechanisms of various diseases. Since abnormally increased or decreased miRNA expression is observed in various diseases such as cancer, diabetes and cardiovascular diseases, the miRNA is recognced as a biomarker for diagnosing and predicting diseases.

One phase I clinical trail investigated the use of MRX34, which uses liposomes to deliver a double stranded miRNA-34a mimic, for the treatment of advanced solid tumours. In a phase II clinical trail, the anti-miRNA-122 miravirsen, which binds miRNA-122 and leads to its subsequent inactivity, was tested for the treamtent of hepatitis C. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

Small Nuclear RNAs (snRNAs):

snRNAs contribute to pre-mRNA splicing regulation rather than binding to mRNA and cuasing degradation. These RNA molecuels are also avialbe as synthetic gene modulators and are able to up or down regulate protein expression transienty.

Published: 8 January 2023

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Categories: Protein Purification

See also purification of bi-specific antibodies under particular antibodies purified

In general, IgG type bispecific antibodies are composed of two types of H chains (an H chain for antigen A and an H chain for antigen B and two types of L chains a L chain for antigen A and a L chain for antigen B). When such IgG type bispecific antibodies are expressed, 10 types of combinations are possible as combination of H2L2 since two types of H chains and two types of L chains are expressed. Among these, there is one type of combination that has the desired binding specificity for antigeb A on one arm and antigen B on the other arm. Consequently, in order to aquire the desired bispecific antibody, it is necessary to purify one type of antibody of interest from among ten types of antibodies, which is extremely low in efficiency and difficult. (Kuramochi (US 2014/0370020)

Since in bispecific antibodies the two H chains as well as the 2 L chains are different and can randomly associate, expression of these four chains leads to the formation of 10 different antibody variants. Correct H chain association resulting in a heterodimeric Fc can be enforced wuing KiH technology by introducing a builky tryptophan (Trp) resiude in one Fc fragment and forming a corresponding cavity on the other Fc fragment that can accommodate the Trip residues. More recently, multip alternative approaches to enable correct H chain association have been described such as relying on charge interactions. Although kiH technology was developed in the late 1990s, enabling correct L chain assocaition remained a major problem, and the only approach to achieve this at the time relied on the sue of common L chains for both specificities. However, the use of a common light chain required the de novo identificaiton of the corresponding antibody pairs, which can be challenging and/or time consuming depending on the desired target, and restrict the availabity and diversity of antibodies that can be sued; those methods allowing the generation fo bispecific antibodies from pre-existing antibody pairs were highly desired. (Keiine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Common Heavy chain with two light chains:

lamda/kappa bodies (common heavy change, one Kappa and one lambda):

Fischer (US2014/0179547) discloses the generation of bispecific antibodies where two antibodies having different specificties sharing the same variable heavy chain domain but different variable light chain domains are isolated. The variable heavy chain domain is fused to the constant region of a heavy chain, one light chain variable domain is fused to a Kappa constant domain and the other variable light chain domain is fused to a Lambda constant domain. Preferably, the light chain variable domain fused to the Kappa constant domain is of the Kappa type and the light chain variable domain fused to the Lambda constant domain is of the Lambda type. However, it is also possible to generate hybrid light chains so that two variable light chain domains of the same type can be used to generate bispecific antibodies. The three chains are co-expressed in mamalian cells leading to the assembly and secretion in the supernatant of a mixture of 3 antibodies; two monospecific antibodies and one bispecific antibody carrying two different light chains. The antibody mixture is purified using standard chromatography techniques used for antibody purification. See antibody purification

CrossMab Technology:

Introduction:

A problem with KiH technology remains correct light chain associrion. While a common light chain can be used, a common light chain requires the de novo identification of the corresponding antibody pairs, which can be challenging and/or time-consuming depending on the desired target and restricts the avaiability and diversity of antibodies that can be used. CrossMab technology enables this correct light-chain association in bispecific antibodies. By incorporating the original heavy chain VH-CH1 domain in the Fab of the second specificity of the bispecific antibody as the novel “lgiht chain” and the original light chain VL-CL domain for the novel “heavy chain” by fusing them to the hing region of the Fc fragment, correct lgiht-chain association can be enfored. The Crossmab technology has evolved in the past decade into one of the most mature, versatile and broadly applied technologies in the field for teh generation of various bispecific antibody formats. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Roche developed the CrossMab approach as a possibility to enforce correct light chain pairing in bispecific hterodimeric IgG antibodies, when combining it with the KH technology. In this format, one arm of the intended bispecific antibody is left untouched. In the second arm, the whole Fab region, or the VH-VL domains or the CH1-CL domains are exchanged by domain crossover between the H and L chain. As a consequence, the newly formed “crossed” L chain does not associated with the (normal, i.e., not-crossed) H chain Fab reigon of the other arm of the bipsecific antibody any longer. Thus, the correct L chain aossication can be enfored by this minimal change in domain arrangement. (Schlothauer, WO 2016/071377)

Two problems must be solved to produce the desired bispecific antibody exclusively: effective induction of heterodimerization of the two heavy chains and discrimination between the two light- chain/heavy-chain interactions. The former can be overcome by introducing large amino acid side chains into the CH3 domain of one heavy chain that fit into an appropriately designed cavity in the CH3 domain of the other heavy chain [the “knobs into holes” (KiH) The latter problem is more difficult to address, because a total of four possible pairings of heavy and light chains remain, with only one of which represents the desired compound. To solve this, the heavy chain 1 and light chain 1 are unmodified. On the opposite side, a new “heavy” chain 2 consisting of an Fc part and the Fab of the original light chain. As the new “light” chain 2 the heavy chain domains VH and CH1 are used. Because the sequence of the modified heavy chain now is crossing over between light- and heavy-chain domains, the term “crossover” for this kind of domain interchange and the term “CrossMab” for antibodies based on this technology. Hetero- dimerization of the two heavy chains is achieved by using the KiH method. As a consequence of this domain rearrangement, associations between unrelated partners can no longer occur. The new “light” chain 2 on the crossover side consists of heavy-chain domains only; thus it cannot assemble with the remaining original heavy chain 1. On the other hand, the original light chain 1 on the unmodified side cannot interact with the new “heavy” chain 2 on the crossover side, because both partners contain the same light- chain heterodimerization interfaces, which do not interact. A bispecific antibody (Fig. 1C) with correct light-/ heavy-chain pairing in both Fabs and almost no deviation from the original IgG is thus obtained. (Klein, “Immunoglobulin domain crossover as a generic approach for the prouciton of bispecific IgG antibodies”. PNAS, July 5, 2011, 108(27).

Applications:

–Autoimmune diseases:

Bispecific CrommMab based antibodies have been generated with the goal of treating autoimmune diseases. Fischer showed that combined inhibition of TNFalpha and IL-17 was more effective in inhibting the development of inlammation and bone and carilage destruction in arthritic mice compared to the respective monotherapies. For this purpose bispecific TNFalpha/IL-17 1+1 and 2+2 CroossMab (HC1-CL) antibodies were rpepared that showed superior efficacy in blocking cytokine ahd chemokine resosnes in vitro. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

–Cancer therapy and opthalmology:

For many years, anti-angiogenesis approaches blocking the vascular endothelial growth factor-A (FEGF-A) have been a major area of targeted cancer therapy. One of the first IgG based antibodies and the first bispecific CrossMab to enter clinical trails, in 2012, was the hterodimeric 1+1 VEGF/Ang-2 CrossMab (HC1-CL) vanuucizumab (RG7221) tarteting the pro-angioneic ligands VEGF-A and angiopoietin-2 (Ang-2), which are invovled in tumor angioenesis. VEGF and Ang-2 ahve also been shown to play an important role in ocular angionesis in diseases liek wet age related macular degerenation a. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Another major field in targeted cancer therpay has been and continues to be apoptosis induction through death receptor (DR) signlaing. As conventional DR5 anitobdies have not been successful in clinical trails, approaches for tumor targeted DR5 agonism have been persued. Expression of the fibroblast activaiton prtoein (FAP) on tumor fibroblasts is found in the majority of solid tumors, making fAAP an attractive antigen for tumor targeting. Based on this rationale, FAP targeted bispecific antibodies and fusion proteins have been created using CrossMab technology that rely on FAP binding with one moiety to induce, with their second moeity, hyper-clustering of TNF receptor superfamily members like DR5 for apoptosis induction. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

With the advent of cancer immunotherpay and checkpoint inhibitor antibodies during the past decase, the development of bispecific antibodies for immunotherapy has attracted substantial attention in industry and academia, wehreas the interest in anti-angioenic and pro-apoptotic therapies has declined. In this context, bispecific monovalent dual checkpoint inhibitory PD-1 antibodies co-targeting the checkpoint inhibitory receptors TIM-2 or LAG-3 ahve been designed based on a bispecific 1+1 CrossMab (VH-VL+/-) format, allowing avidity mediated selectivity gain and thus enhanced selectivity for PD-1+ nad PD-1+TIM=3+/LAG-3+ double positive T cells. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Many of bispecific antibodies currently being developed are bispecific T-cell engarers. One of the first IgG based and Roche’s firt, T cell bispecific antibody (TCB) to enter clinical trails was the heterodimeric and invalent CEA/CD3E 2+1 TCB bisatamab. It is a hterodimeric CEA/CD3e bispecific antibodyin the 1+1 CrossMab (CH1-C2) format to whcih a single additional Fab targeting CEZ is fused to the N-terminus of the nokb containg H chain. (Kleine “Ten years in the making: applicaition of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

The msot advanted 2+1 T cell bispecific antibody is glofitamab which, in constrat to cibisatamab, is based on a 2+1 CrossMab (VH-VL) formal with charge interactions using vriable regions dervied from obinutuzumab. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Central nervous system diseases:

Th treatment of CNS diseases with mAbs is hampered by the low penetration of antibodies thorugh the blood brain barrier. To overcome this limitation, Nieoether generated transferrin receptor targeted bispecific antibodies that allowed delivery of these antibodies through the blood brain barrrier and showed improved brain exposure and prevented plaque formation. Using this approach, BS-GANT was generated based on the amyloid-beta antibody gantenerumab as a trivalent C terminally fused amyloid-beta-TfR 2+ 1 bispecific antibodies in a 2+1 CrossMaby (VH-VL=/-) format with charges. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

–Viral Infections:

The appliction of CrossMab technology ahs become popular for the geenraiton of bispecific and multispecific antibodies targeting various viruses. Curing the past years, multiple highly potent bispecific antibodies targeting HIV-1 have been generated using CrossMab technoloyg for the prevention and treamtent of HIV-1. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Introduction of difference in the charges to Heavy chains:

If two heavy chains and two light chains are expressed, there are 10 possible heavy chain and 10 possible light chain combinations. Accordingly, introducing difference in the charges of two heavy chains has been used to facilitation purification of BiAb. (“Proprietary Innovative Antibody Engineering Technologies in Chugai Pharmaceutical” 12/18/2012).

Knobs-into-holes strategy:

The homodimerization of the two heavy chains in an IgG is mediated by the interaction between the CH3 domains alone. Heavy chains were first engineered for heterodimerization in the 1990s using a “knowbs-into-holes” strategy. Starting fromm the a “knob” mutation (T366W) that disfavors CH3 homodimerization, compensation “hole” mutations (T366S, L368A, and Y407V) were identified by phage display providing efficient pairing witht the “knob” while disfavoring homodimerization. (Spies, Molecular Immunology 67 (2015) 95-106).

Bascially, the concept relies on modifications of the interface between the two CH3 domains of the two H cahins of an antibody where most interactions occur. A bulky residue is introduced inot the CH# domain of one antibody H chain and acts similarly to a key (“knob”). In the other H chain, a “hole” is formed that is able to accommodate this builky residue, mimicking a lock. The resulting heterodimeric Fc region can be further stabilized by the introduction of artificial disulfide bridges. Notably, all KiH mutations are buried within the CH3 domains and not “visible” to the immune system. Correct H chain assocaition with hterodimierizaiton yield above 97% can be acheived by introducing xix mutaitons: S354C, T366A in the “knob” H chain and Y349C, T366S, L368A and Y407V in the “hole” H chain. (Schlothauer, WO 2016/071377).

In the knobs in holes technology, one or more small amino acid side chains from the interfact of the first antibody molcule are replaced with larger side chains (e.g., tyrosine or tryptophan) (knobs or protuberances). The proteuberance may exist in the original interface or may be introduced synthetically. Compensatory “cavities” (holes) of identical or similar size to the large side chains are created on the on the interact of the second antibody molecule by replacing large amino acid side chainswith smaller ones. (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-proudcts such as homodimers. (Giese, US 16/221,369 published as US 2019/0256556)

Schlothauer (WO 2016/071377) disclsoes variant Fc regions that specifically bind to SpA and that do or do not bind to human FcRn. Thesse variant Fc region contain specific amino acid mutations in the CH2 domain whereas the CH3 domain is not changed with respect to protoein A binding. The mutations when used in the hole-chain of a heterodimeric Fc regioulfide brdige(s) and wherein the CH3 domain of the first and second polypeptide both bind or both do not bind to protein A and allow for the purification of the heterodimeric Fc region, i.e., the separation of the heterodimeric Fc region from the homodimeric Fc region by-produce (hole-chain-hold-chain dimer). Thus in one embodiment, the heterodimeric polypetide includes a first polypeptide which includes in N to C terminal direction at least a pottion of the hinge region, which includes one or more cysteine residues, a CH2 domain, a CH3 domain and a second polypetide that includes in the N to C terminal direction at least a protion of the hinge resgion which includes one or mroe cysteine residues, a CH1-domain and a CH3 domain, wherein the first polypeptide includes the mutations Y349C, T366S, L368A and Y407V (hole chain) and the second polypetide includes the mutations S354C and T366W (knob-chain) and wherein the first polypetpide (hole-chain) icludes the mtuations I253A or I253G and L314A or L314G or L314D, wherein the first and second polypetpidds are connected by dis

Introduction of + charged amino acids into H chain CH3 and – charged amino acids into L chain CH3:

Zhang (WO 2017/034770) discloses modifying antibody CH3 domain to promote heterodimerization between a first CH3 polypeptide and a second CH3 harboring polypeptide. In particular, at least two positively charged amino acids such as arginine, lysine or histidine are introduced into the first polypeptide by amino acid substitution to increase the positive charge while at least 2 negatively charged amino acids such as aspartic acid or glutamic acid are introduced into the seocnd polypeptide to increase negative charge of the polypeptide chain. Those different modifications at the itnerfact of two complementary CH3 omains presumably provides much stronger electrostatic attractions between those two chain to favor heterodimer formation rather than homodimer formation. In addition, cysteine molecules may be intoduced togehter wtih the charged amino acids to the appropriate position of both interfaces of the two complementary CH3 domains to allow inter-chain disulfide bond formation and further strenghten the heterodimer formation. In cerain embodiments, the molecule may be a bispecific, trispecific or qudrospecific antibody.

Modification of H chain CH1 and L chain constant region;

Kuramochi (US 2014/0370020) discloses regulation of the association of the H and L chain such that in one eobmdiment the amino acids repel electrically or in a separate embodiment the amino acids do not repeal electrially. By making amino acid residues at given locations in the H constant region (CH1) and L chain constant region of a desired bomination that do not mutually repel electrically, a desired combination of H and L chain can be foremd by suing the attractive force of the electric charges. Amino acids that do not mutually repeal electrially include amino acids in which one of the amino acids is a positively charged amino acid and the other is a negatively charged amino acid.

Modifying AAs of first and/or second polypeptides to create differences in isoelectric point:

Igawa (US 2009/0263392) discloses modifing both or either one of a nucleic acid encoding the amino acid residue of the first polypetpide and a nucleic acid encoding the amino acid residues of the second polypeptide, usch that the difference between the isoelectirc point of the rift and second polypeptide will be incdreased. The modification results in peaks of the homomultimer for the first polypeptide and second polypeptide and heteromultimer of the first polypeptide and the second polypeptides that can be seaprated.

Common Light chain with two Heavy Chains:

If two heavy chains and two light chains are epxressed, there are 10 possible H and 10 possible L chain combination. But if a common light chain is used for two H chains, combinations become only three. (“Proprietary Innovative Antibody Engineering Technologies in Chugai Pharmaceutical” 12/18/2012)

A common light chain strategy was applied to assemble IgG like bispecific antibodies which can be combined with the knobs-into-holes approach. The mechanism of a common light chain is based on the fact that antibodies discvoered form phase display screening against diverse antigens often share the same VL domain, reflecting the very limited size of the L chain repertoire in the phage library. One of the advantages of the common L chain format is that it allows the use of methods that simplify the antibody engineering and the purificaiton process in industrial production. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019)

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Categories: diseases and disorders

The term thromobitic microangiopathy (TMA) encompasses a group of conditions that are defined by, or result from, a similar histopathological lesion. Hemolytic uremic syndrome HUS), thrombotic thrombocytopenic purpura (TTP) and several other conditions are assocaited with TMA. (Kplan, Pedian ephtrol (2008) 23: 1761-1767). Other notable causes for TMA iclude infection (e.g., bloody diarrhea associated with E. coli infection), medications (e.g., quinine, bevacizumab), connective tissue dsieases (e.g., systemic lupus erythematosus, antiphospholipid anitobdy syndrome, scleroderma), cancer, vasculitis, pregnancy, malignant hypertension, organ transplant and metabolic disorders. uraaemic syndrom (HUS) and thrombotic thrombocytopenic purpra (TT) are the clincial entitles comprising TMA, with predominantly renal manifestrations in the former, while the latter more often present with systemic and neurological findings. There are no standard laboratory values that define TMA, but the clinical traid of renal failure, thrmobocytopenia and microangiopathic haemolytic anaemia is considered the hallmark of TMA syndromes. Nephrol Dial Transplant (2006) 21: 3038-3045).

Thrombotic microangiopathy (TMA) is a microvascular occlusive disorder characteried by preedominantly platelt thrombi in the renal and/or systemic circulations. Haemolytic

Hemolytic Uremic Syndrome (HUS): See Kidney Diseases

Another major cause for TMA is atypical hemolytic uremic syndrome (aHUS), a discorder caused by dysregulation of a prt of the complement system. About 50% of aHUS pateints are found to have ither a genetic mutation in the complement system or an aut-antibody that inteeres with the reulation of complement.

Hemolytic uremic syndrome (HUS) is characterized by the triad of thrombocytopenis, Coomb’s test negative microaniopathic hemolytic anemia, and acute renal failure. HUS is classified as eitehr D+ when it is associated with a preceding diarrhoeal illness, which in most people is caused by infection with E. coli 0157, or less commonly nondiarrhoeal associated (D_) (also called “atypical”). D- HUS may be sporadic or familial. Mutations have been reproted in the complement regulatory protein factor H in both sporadic and familial HUS. (Goodship, PNAS, 100(22), October 28, 2003).

Immune Thrombocytopenic Purpura (ITP)/ Thrombotic Thrombocytopenic Purpura (TTP):

ITP refers to a rare disease characterized by microangiopathic hemolytic anemia, causing blood clots to form in small blood vessels throughout the body. These clots can cause serious problems if they block blood vessels and limit blood flow to the brain, kidneys or heart. Blood clots form when blood cells called platelets clump together. ITP is an autoimmune disease characterized by platelet clearance mediated by pathogenic platelet-specific antibodies. Autoantibody-coated platelets induce Fc receptor mediated phagocytosis by macrophages, primarily but not exclusively in the spleen. Thus the spleen is the key organ in the pathophysiology of ITP not only because platelet autoantibodies are formed in the white pulp but also because macrophages in the red pup destroy immunoglobulin coated platelets. If bone marrow megakaryocytes cannot increase production and matain a normal number of circulating platelets, thrombocytopenia and purpura develop. In the US, the annual incidence of chronic ITP is stimated to be about 6.6 cases per 100k persons. In adults, women are affectied about 3 times more frequently than men. Adults may be affected at any age, but most cases are diagnosed in women aged 30-40 years. Onset ina patient older than 60 years is uncommon, and a search for other causes of thrombocytopenia is warranted. The disease is characterized by reduced blood platelets, which cause visible skin blemishes from bleeding or brusing. Symptoms can include bleeding, red dots on the skin, red dots on the mouth membranes, purplish mouth membrane areas, bleeding nose, bleeding gum, digestive bleeding, urinary bleeding and brain bleeding. To establish a diagnosis of ITP, other causes of thrombocytopenia are excluded, such as leukemia.

Thrombotic thrombocytopenic purpura (TTP) is due to low activity of a protein called ADAMTS13. Some individuals are born with a mutation in the gene for ADAMTS13, although most affected pateitns have an acquired auto-antibody that blocks the activity of ADAMTS13.

Causes/Pathophysiology:

Role of complement system:

(Hughes Am J Physiol Renal Physiol, 278: F747-F757, 2000) discloses that in an immune-meidated thromobtic microangiopathy model both CVF treatment and C6 deficiency reulsted in 10-25 fold reudction in the number of apoptotic cells. The results indicated that complement is the principal inducer of endothelial cell apoptosis in antibody-mediated glomerulenphritis and that this effect is mediated primarily by sublytic C5b-9. The data also strongly suggests that the C5b-9 membrane attack complext may play an important role in the induction of endothelial cell apoptosis in vivo, particularly in diseases associated with antiendothelial antibodies such as systemic lupus erhematosis, scleroderma, hemolytic uremic syndrome and the systemic vasculitides.

Mutations have been reproted in the complement regulatory protein factor H in both sporadic and familial HUS with mutations identified in 10-20% of cases studied. (Goodship, PNAS, 100(22), October 28, 2003)

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Categories: immunology

See also specific diseases for references to CRegs and Drug Targetting under Pharmacology

CRegs for the Treatment of Diseases

CR1: Levin (US 6,169,068) teaches a method for treating diseases involving complment by pulmonary administration of complement inhibitor proteins such as a soluble complement receptor type 1 (sCR1).

CD46 (MCP): Sweigard teaches that adenovirus mediated delivery of CD46 attenuates the AP pathway and has implications for age related macular degeneration (Gene Therapy 18, 613-621 (2011).

CD59:

–AMD: Bora discloses that a recombinant membrane targeted form of CD59 inhibits the growth of choroidal neovascular complex in mice (J Biol Chem 285, 2010, 33826-33833). Cashman also discloses that a non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration (PLOS ONE, e 19078, 6(4) 2011, 1-9).

Ramo evaluates adenovius delivered human CD59 as a potential therapy for AMD in a model of human membrane attack complex formation on murin RPE (Invest Opthalmol Vis Sci 49(9): 4126-4136 (2008).

–Atherosclerosis: Wu teaches that CD59 protects against atherosclerosis by restricting the formation of complement membrane atttack complex (Circ Res 104: 550-558), 2009).

CReg Fusion Constructs for the Treatment of Diseases

–CD55 (DAF) and CD46 (MCP) fusion proteins: have been shown to be more effective thatn DAF, MCP or DAF and MCP at inhibiting C3 deposition via the alternative pathway. Chimeric proteins in which MCP, DAF and MCP-DAF hybrids have also been produced with cell surface loalizing domains which target the molecules to cell surfaces, thereby increasing the concentraiton of these molecules on cell surfaces where they can act to inhibit complement-mediated cell lysis. MCP-DAF hybrids may contain the entire sequence of both molecules or may contain just the SCR regions form each MCP and DAF. (Innis, WO 96/34965). Ko (US 5,679,546 and 5,851,528) and Wijesuriya (US 2006/0154336) also disclose chimeric genes and proteins which express the biological activities of both MCP and DAF. The proteins are referred to as “Complement Activation Blocker” (CAB) proteins.

–CD46 and CD55 and CD59 fusion construct: Kumar-Singh (US 13/692734) teaches a construct having the complement regulatory domains of each of CD46, C55 and CD59 with the native secretory signal of CD59 at the N-terminus. The SCR domains of CD46 and CD55 are involved in complement control and the serine threonine proline (STP) region is heavily glycosylated. The majrority of amino acids of the STP region were removed and the amino acid sequences of the full-lenght SCR doamins for each regulator was retained. Glycine linkers were added to separate each complement regulatory domain.

–CR2 fusion constructs with CRegs: (see Complement Receptor Drug Targetting under Pharmacology)

–DAF-CD59: Fodeor (5627264) teaches chimeric complement inhibitors having C3 inhibitory activity and a second functional domain having C5b-9 inhbitory activity such as DAF-CD59 fusion consructs.

–Antibody-CD59 constructs (see also drug delivery under pharmacology): Zhang (J. Clinical Invest. 1999, 103(1)) teaches antibody-CD59 chimeric fusion proteinswhich provided targeted cells with effecitve protection from complement mediated lysis.

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Categories: Patent Law Cases

See also Applications of Cregs:

Complement Receptor 1 (CR1; CD35; C3b/C4b receptor): is present on erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells and glomerular pdocytes. CR1 specifically binds C3b, C4b, and ic3B. A soluble form of the receptor has been found in plasma that has ligand binding activity and the same MW as membrane-associated CR1. CR1 binds C3b and C4b that have covalently attached to immune complexes and other complement activators, and the consequences of these interactions depend upon the cell type bearing the receptor. CR1 can also inhibit the classical and alternative pathway C3/C5 convertases and act as a cofactor for the cleavage of C3b and C4b by factor I, indicating that CR1 also has complement regualtory functions in addition to serving as a receptor (Fearon, 5,472,939). CR1 possesses decay-accelerating activity for both C3 and C5 convertases in both the classical and alternative pathways, as well as factor I cofactor activity for the degradation of both C3b and C4b.

Factor H: is a glycoprotein that circulates in high concentrations and is a potent inhibitor of the AP pathway. Factor H competes with factor B for binding to C3b. Binding of C3b to Factor H also leads to degradation of C3b by factor I to the inactive form C3bi (also designated iC3b), thus exerting a further check on complement activation. Several regions within the factor H protein bind to anionic surfaces such as membranes rich in heparin sulfate or sialic acid, as well as to C3b on the surface.

The ability of factor H to discriminate between host cells and invasive pathogens has been attributed to binding of factor H to negatively charged molecules such as sialic acid and glycosaminoglycans that are displayed on the surface of host cells (S. Meri et al., Proc. Nat’l Acad. Sci. USA (1990) 87: 3982-3986. Distinct tissues express different combinations of the membrane boudn complement inhibitors and factor H likely has different affinities for various cell types.

–Structure: FH is a single polypeptide chain plasma glycoprotein composed of 20 repetitive SCR domains of about 60 amino acids arranged in a continous fasion like a string of 20 beads. FH has at least 3 distinct binding domains for C3b, which are located within SCR 1-4, SCR5-8, and SCR 19-20. Each site of factor H binds to a distinct region within the C3b protein. The N-terminal sites bind to native C3b; the second site, located in the middle region of FH, binds to the C3c fragment and the site located within SCR19 and 20 binds to the C3d region. In addition, FH contains binding sties for heparin, which are located within SCR7, SCR5-12 and SCR20 and overalp with that of the C3b binding site. Structural and functional analyses have shown that the doamins for the complement inhibitory activity of FH are located within the first 4 N-temrinal SCR domains (Gilkeson, WO/2007/14/9567).

Knoell (EP1336618) discloses that complement regulation is generally considered to be species specific and that rat FH is not interchangeable with human FH. This fact supports the unpredictability of taking sequences sharing sequence homology and expecting those sequences to have similar complement regulatory activity in different species.

Thurman (US 13/120125, now US 8937046) disclose that factor H binds to annexin A2 in post ischemic kidneys and that mice that do not express annexin A2 develop more severe injury after renal I/R.

Membrane Cofactor Protein (MCP; CD46): Mature human CD46 protein is composed of 392 amino acids and is found in leukocytes, platelets, epithelial cells, sperm cells and fibroblasts. Numerous transcript variants encoding different isoforms have been identified. MCP has four SCR sequences and serine/threonine enriched region in which heavy O-linked glycosylation occurs. MCP has a transmembrane and cytoplasmic domain. MCP works by binding to the C3b and C4b present on the cell surface thereby targeting the protien for degradation by factor I, a plasma protease, and thereby destroying any C3 or C4 convertase activity. Thus, MCP is said to have “cofactor activity”. Because MCP is localized on the cell surface, it protects only the cells on which it is present and therefore is said to act in an intrinsic manner. Portions of the mature MCP sequence can be deleted and yet the protein retains complement inhibiting activity. Examples of portions that can be deleted include the cytoplasmic tail and the transmembrane domain. (WO 96/34965).

Decay Accelerating Factor (DAF; CD55): DAF controls the early part of the complement system by regulating the activity of the C3 and C5 convertases. DAF is composed of 4 SCRs plus a serine/threonine-enriched domain that is capable of extensive O-linked glycosylation. DAF is attached to cell membranes by a glycosyl phosphatidyl inositol and through its ability to bind C4b and C3b, it acts by dissociating the C3 and C5 convertases in both the classical and alternative pathways. The human CD55 protein is uniformly GPI-anchored which is referred to as DAF-GPI or GPI-DAF or CD55a. Mice possess two closely related genes, termed decay-accelerating factor 1 (DAF1) and decay-accelerating factor 2 (DAF2), also referred to as CD55b. These genes share 93% identity in their coding regions.

As with MCP, DAF regulates complement in an intrinsic manner, thus protecting only the cells on which DAF is located. Portion of DAF such as the GPI-anchor domain can be deleted and yet the protein retain complement inhibiting activity (WO 96/34965).

CD59: is a membrane inhibitor of complement that inhibits MAC formation by binding C8 and/or C9, and inhibiting C9 polymerisation during MAC formation. CD59 appears to function by competing with C9 for binding to C8 in the C5b-8 complex, thereby decreasing the formation of the C5b-9 membrane attack complex. Like DAF, CD59 is anchored in the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, which is attached to an asparagine at amino aicd 102.

CD59 is a 18-20 kDa glycosyl phosphatidylinositol (GPI)-anchored membrane glycoprotein. The CD59 antigen has been well characterized by amino acid analysis and NMR. It consists of 128 amino acids, of which the first 25 comprise a signal siequence. Tehre are 10 cysteine resiudes, which result in a tightly folded molecule. The asparagine resiude at position 18 is known to be N-glycosylated, while the asparagine resiude at position 77 is linked to the GPI anchor. The C-terminuys residues are characteristic of GPi anchored proteins Young, (US 2006/0140963).

Analysis of the physical association of CD59 with components of MAC suggested that separate bidning sites for CD59 are contained within the alpha chain of human C8 and C9. The complement inhibitory activity of CD59 is species selective and is most effective towards C9 direvied from human or other primate plasma. (Sims (US2003/0166565).

Vitronectin (complement S-protein): is a multifunctional gyocoprotein that inhibits complement mediated cytolysis at two identified stages of terminal complement complex formation: blocking of C5b-7 membrane binding and prevention of C9 polymerization (Sheehan, Clin Exp Immunol. 1995, 101(1) 136-41). Vitronectin occupies the metastable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed SC5b-7 is unable to insert into cell membranes. Vitronectin has also been shown to inhibit C9 binding to the terminal complement complex thereby directly affecting lytic pore formation.

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Brain Drug Delivery

Altered Effector Function

BispAb Formats

Conjugation & Linkers

Ultrasmall Superparamagnetic Iron Oxide Nanoparticles

RNA Interference

Engineering of BiAbs to Facilitate Production/Purification

Thrombotic microangiopathies (TMA)

Applications of Complement Regulators

Regulation of the complement system