Depression:

Bupropion is an antidepressant drug approved for depression. It has also been used off label for weight loss.  Bupropion is an antidepressant medication that works in the brain. It is approved for the treatment of major depressive disorder (MDD), seasonal affective disorder (SAD), and to help people quit smoking (smoking cessation).

Trintellix is a brand-name prescription antidepressant. It’s used in adults to treat a serious form of depression called major depressive disorder.

Selective Serotonin Reuptake Inhibitors:

Lexapro (escitalopram): Lexapro (escitalopram) is an antidepressant from the group of drugs called selective serotonin reuptake inhibitors (SSRIs) and is used to treat certain types of depression and anxiety. Lexapro works by balancing levels of serotonin in the brain and nerves. Lexapro (escitalopram) is good for treating depression and anxiety. It’s generally well-tolerated and has fewer drug interactions than other antidepressants. 

Escitalopram is a selective serotonin reuptake inhibitor while alprazolam is a benzodiazepine. Both can be used to treat anxiety, but escitalopram is also used to treat major depressive disorder while alprazolam is used to treat panic disorder.

Antihistamines:

Hydroxyzine: is an antihistamine that is also used to treat anziety. 

Benzodiazepines:

Benzodiazepines are central nervous system depressant medications that are often used for treating or managing anxiety disorders or insomnia. These medications have a sedative effect that can help people sleep better and feel less anxious. 

Xanax (Alprazolam): is a prescription drug that’s designed to address panic and anxiety disorders. This sedative is common and comes with warnings about addiction and abuse potential, as well as the risks of mixing it with other substances like alcohol.

Ativan (Lorazapam): is prescribed to reduce anxiety. Like many benzos, this medication works by slowing down brain activity and increasing relaxation. Because of its effects of sedation, there is a significant potential for drug abuse.

Valium (Diazapam): has a variety of approved uses, including treating anxiety, seizures, and alcohol withdrawal. Long-term Valium use can lead to dependence, tolerance, and dangerous withdrawal symptoms if you abruptly stop use.

See also Oxidation and Reduction in Biochemistry

Disulfide Bond Variants 

Disulfide reduced variants have one more disulfide bonding cystein(s) chemically reduced to the free thiol form. This variant can be monitored by HIC or by sizing methodology such as Capillary Electrophoresis with Sodium Dodecyl Sulfate (Harris, US2009/0202546).

Half-antibodies: Occasionally the disulfide bonds between the heavy chain polypeptides of antibodies are not formed, resulting in the formation of an antibody with no interchain disulfide bonds between two pairs of heavy and light chains. These antibodies have been called “half antibodies” (or “Hab”). Certain antibody classes and types are more susceptible to half antibody formation such as IgG4. In both natural and recombinant antibody production, a significant proportion of IgG4 antibodies at least as high as 35% are produced as half antibodies (Brower US 14/668820, published as US 2016/0108127).

Causes of Antibody Reduction in Cell Culture Production Processes:

Reducing enzymes: A very important structural feature of an antibody is the disulfide bonds that link its light and hiavy chains (inter-chain disulfides) together to form a quaternary complex. Under specific conditions, extensive reduction of the antibody’s inter-chain disulfides bonds are observed after harvest operations (centrifugation and filtration) and/or the first purificaiton step (i.e., protein A chromatography). On one such instance, it was estimated that as little as 10% of the antibody remained intact after the protein A step. This antibody reduction is not a unique issue to a specific antibody or a particular cell line. The reduction is reportedly caused by an active thioredoxin (Trx) system or other reducing enzymes with thioredoxin-like activity in the harvested cell culture fluid. The Trx system (i.e., Trx and TrxR) is one of the two intracellular enzymatic systems (the other is the glutaredoxin (Grx) system) that regualtes the cellular redox status and maintains a reducing environment in the cytosol. *Kao “Mechanism of antibody reduction in cell culture production processes” Biotechnology and Bioengineering” (2010). 

During purificaiton of IgG2, high level of fragment in the prcoess intermedaites was detected by HP-SEC. Fruther investigations traced the casue to be antibdoy reduction in the harvested cell culture fluid (HCCF). In addition, when the HCCF was redued, larger increase in aggregate content was observed druing low pH treatment. When purificaiton was initiated immediately after harvest, antibody reduction was not observed in the HCCF and aggregate formation during low pH treatment was minimized. This can be attributed to the shorter exposure time of the antibody to reducing enzymes in teh HCCF. Increasing cystine concentraitons also resulted in lower free thiol levels when the HCF was held. (Chun “Effects of antibody disulfide bond reduction on purificaiton process performance and final drug substance stability” Tiotechn Bioeng, 114(6) (2017). 

Methods to Decrease Reduction of Disulfide bonds

Reduction of the disulfide bonds that internally anchor disulfide bond-containing proteins can occur during manufactering. Reduction of the inter-chain disulfide bonds results in loss of functional antibodies and requires more coplex purification. During mAb process development, fragments and aggregates have to be removed to adequate levels due to their associated risks with increased immunogenicity and unknown effects on drug efficacy (Ahuja, US 2019/0112357)

Using Charged Depth Filter

Hoang (US 15/751,231, published as US 2018/0230180) discloses a method of reducing the perentage of reduced antigen-binding protein molecules using a charged depth filter.

Control of Disulfide bonds and Half-antibody formation using reduction/oxidation (redox) agents:

–-Addition of 2-MEA, reduced gluathione, oxidized glutathione, 2-mercaptoethanol, DTT, cystein, cystein, dithiobutylaine or sodium sulfite or Oxidizing metal ions (Cu2+, F32+, Fe3+, Zn2+)

—-During Cell Harvest

Brower (US 14/668820, published as US 2016/0108127) discloses a process to control the formation of disulfide bonds between polypeptides by contacting the polypeptides with a conditioned solution that includes predetermined solution parameters such as a redox reagent like 2-mercaptoethylamine (2-MEA), reduced glutahione, oxidized glutathione, 2-mercaptoethanol, dithiothreitol (DTT), cystine, dithiobutylamine or sodium sulfite. at a specific time point during the bioprocess. In some embodiments, the specific time point occurs during the step of viral inactivation, adjustment, chromatography, filtration, dilution, concentration or any bioprocess step that is cell free. In other embodimetns, the specific time point occurs during chromatogaphy.  In one embodiment, Brower discloses that the level of half antibody decreased in the presence of 1 mM 2-MEA but that higher levels (25 and 50 mM) results in increassed level of half-antibody and also generated H and L fragments.The level of half antibody also decreased within about 30 minutes in the presence of 2-MEA at 1-3 mM at pH 4.8. 

Chaderjian (“Effect of Copper Sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed” Biotechnol. Prog. 21: 550-3 (2005) discloses that cooper sulfate can at as an oxidizing agent, thereby facilitating disulfide bond formation when added to cell culture production medium. Purified antibody showed that either 50 or 100 uM copper sulfate additions reduce the level of free thiol by more than 10-fold. 

Dillon (WO 2006/047340) discloses a method of producing a IgG antibody by contacting the antibody that has been recombinantly produced by mammalian cells with a reduction/oxidation coupling reagent at a pH of about 5-11, optionally with a chaotropic agent. The method reduces the heterogeneity of the various antibody isotypes. For example, the method can be used to refold IgG4 molecules to decrease the present of IgG4 half molecuels. The redox coupling reagent may be any redox coupling reagents such a reduced glutatione and oxidized glutathione. 

Schotte (WO2010/125187) discloses a method for producing a domain antibody in a host other than E. coli, preferably yeast, by applying conditions that promote the formation of disulfide bridges in the domain antibody, specifically the addition of oxidizing agent, preferably oidizing metal ions such as Cu2+, Fe2+, Fe3+, Zn2+, or removing domain antibodies lacking at least one disulfide bridge or a combination of both. The inventor found that despite the high yield and funcitonality of domain antibodies produced in non-E coli hosts, there is a quantiatively significant fraction of product that represents a structural variant, specifically a product that lacks at least one disulfide bond. 

–—–Addition of Proxides to prevent antibody disulfide bond reduction:

During the manufacturing process, extensive reduction of antibodies has been observed after harvest operation or Protien A chromatography and multiple process parameters correlate to the extent of the reduction. For example, maintaining high levels of dissolved oxygen (DO) during harvest is vital to keep antibody molecuels intact. Mechanical shear forces, which cause cell lysis and cellular components to leak into harvest fluids also significantly contribues to the reduction. Other process parameters such as harvest hold time, media componetents (such as copper ions, cystein/cystine and pH) and termpature also have an effect on the extent of disulfide reduction. The reduciton of antibody products due to enzyme systems may be complete or partial Human IgG contain two H and 2 L chains that are held together by non-covalent interactions as well as inter chain disulfide bonds. There are a total of 12 intra chain disulfide bonds and one disulfide bond between L and H chains in all four IgG subclasses. The nubmer of inter-chain disulfide bonds between the two H chains is 2 for IgG1 and IgG4, 4 for IgG2 and 11 for IgG3. Additionally, the interchain disulfide bonds are more solvent acccessible than intra-chain bonds and the exposed cysteine residues are considered more reactive than non-exposed cysteine residues. Hydrogen proxide can oxidize reducing agents and prevent the reduction of disulfide bonds and prevent the generation of LMW species. There are many peroxide containing compounds, inorganic or oragnic that can be substituted for hydrogen peroxide. Two inorganic forms, sodium percabonate and sodium perborate, can also effectively suppress the reduction of disulfide bonds in a concentraiton dependent manner. (Du, “Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process” MABS, 10(3), pp. 500-510 (2018). 

——-Addition of Peroxides + Red Light:

Color variation due to antibody process related impurities can be a major concern as a problem of lack of process control. One of the main sources of pink/red color in final drug substance has been identified as the vitamin B12-mAb complex.  Cyanocoblamin is the vitamin B12 form typically included in cell culture media as a cofactor required for DNA synthesis. Under cell culture conditions, however, cyanocobalamin is readily converted to hydroxo-cobalamin due to light exposure, with up to 80% converted after 10 d in culture. The red color intensity can be minimized by reducing the medium concentration of vitamin B12. Since disulfide reduction mediated mAb dissocaition can be prevented at the manufacturing scale( with putblished methods, the risk of red color can also be minimimized with these published methods. (Derfus “Red colored IgG4 casued by vitamin B12 form cell culture media combined with disulfide reduction at harvest” mAbs 2014).

The intensity of pink color depends on concentraitons of both free sulfhydryl groups on reduced mAb and hydroxocoalamin, the active form of vitamin B12. Both reactants are necessary to generate pink colr. Thus process control strategy can consider limiting either one or both factors. (Du, “Vitamin B12 association with mAbs: mechanism and potential mitigation strategeis” Biotechn. and Bioeng. 2018, 115: 900-909). 

Tan (US 16/612,175, published as US 20230103511) discloses preventing the reduction of antibodies by adding peroxides (e.g., hydrogen peroxide, sodium percarbonate or sodium perborate) to the harvest solution and inhibiting the conversion of vitamine B 12 (cyanocoalamin (CN-Cbl)) to hydroxocobalamin (HO-Cbl) by culturing cells under red light conditions during manufacture or cell harvest. Tan showed that the generation of pink colr during antibody manufacturing is dependent on concetnrations of both free sulfhydryl groups on reduced antibodies and hydroxocobalamin, the active form of vitamin B12. Both reactants are necessary and neither one alone is sufficient to generate pink color. The active form of vitamin B12 attaches to the free sulfhydryl group of the cystein located at H chain 134, LC 214, HC 321, 367 and 425. The five cysteins residues are distributed among both the Fab and the Fc regions. Cynocobalamin convertion to hydroxocobalamin is inhibited or reduced by replacing white light with red colored light (wavelenghts >600 nm) in the areas where the cell culture media is prepared and sotred and optionally in cell culture manufacturing and harvesting areas. 

–During Chromatography Process

Godavarti (US 7,825,223) discloses purifying an AB binding protein having an Fc region such as an anti-AB antibody by absorbing the antibody to an Fc binding agent such as Protein A, followed by a wash with a divalent cation such as copper, nickel, manganese, to remove impurities. 

Zou (WO 2006/060083) discloses subjecting column isolated preparations of polypetpides with a reduction/oxidation reagent such as cysteins and cystine, reduced and oxidized gluathione, dithiothreitol (DTT), 2-mercaptoethanol, hydrogen peroxide (oxidizer) and dithionitrobenzoate and a chatropic agent and isolating the refolded active protein produced from said contacting. In one embodiment, the redox reagent is cystein/cystine. The chaotropic agent can be urea, SDS or guanidine hydrochloride. In other embodimetns, refolding of the protein can be performed using other redox reagents such as copper. The refolding can occur on a column such as an affinity resin (e.g., Protein A) of a Fc domain containing polypeptide.

Isolation of domain antibodies with oxidizing agent(s)

Schotte (US13/266503 and US2012/0157664) discloses a method of producing a domain antibody in yeast by applying conditions such as addition of oxidizing agents, preferably oxidizing metal ions such as Cu2+, Fe2+, Fe3+ and Zn2+ that promote the formation of disulfide bridges in the domain antibodies and/or removing domain antibodies lacking at least one disulfide bride. The method results in the production of domain antibodies wherein the quality of the domain antibodies is improved with a reduced level of free thiol or its absence. 

Oxidation of Amino Acids:

Oxidation is a common degradation pathway that may occur during the life cycle of prtoeins and peptides. The resulting chemical modificaitons may affect in vitro stability and in vivo biologcial functions. In order to assess the potentail susceptibility of protein products to oxidation, different oxdiation stress conditions have been studies such as hydrogen peroxide (H2O2). , transition metal ions or UV exposure. Scuh treatment often result in a myriad of modificaitons on several amino acid residues, such as histidine, phenylalanine and tyrosine. However, methionine and tryptophan are the residues that are oxidized most cmmonly  and to the highest extent. (Folzer, “Selective oxidation of methionine and tryptophan residues in a therapeutic IgG1 molcules” J of Pharmaceutical Sciences, 2015). 

Recombinant mAbs are constantly exposed to oxidizing environments, including dissolved oxygen, oxygen in teh air and free radicals such as those generated by reactions with metals and impurities form raw materials (e.g., peroxides). Forced oxidation is used to probe residues taht are susceptible to oxdiation and to udnerstand if there is a practical impact on this degradation pathway (e.g., oxidation of a residue in the CDR may affect binding and potency). The most commonly used approaches are incubation of recombinant mAbs with hydrogen peroxide or tert-butyl hydrogen peroxide for methionine residues. (Nowark “Forced degradation of recombinant monocloanl antibodies: A practical guide” mAbs (2017)). 

Oxidation of methionine: 

Although protein oxidation can occur at cysteine, tryptophan, lysine and other amino acids, methionine is often the most susceptible residue to oxidation. The most common product of methionine oxidation is methionine sulfoxide, which is more polar and less hydrophobic than methionine. Susceptible methionines are typically located on the surface of the protein and exposed to the solvent. Met oxidaiton can have adverse effects on proteins, including decreased stability and decreased biological activity. (Pan “Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn” Protein Science, 2008, 18, 424-433).

Folzer, “Selective oxidation of methionine and tryptophan residues in a therapeutic IgG1 molcules” J of Pharmaceutical Sciences, 2015) disclsoes that oxidation levels of Fc methionine was first quantified by prtoein A chromatogrpahy. the mAb species eluted at 23 min. The oxidized species formed upon inucation with oxidant eluted earlier (before 19.8 min) because of the lower affintiy of oxidized Fc methionine to protein A. The incubation of the mAb with 1% H2O2 for 24 hr at 5C as well as the incubation with 2% t-BHP for 120 h at 25C allowed for teh nearly complete oxidation of the methionine residues form teh Fc region. 

Anion exchange and hydrophilic interaction: 

Negatively charged mixed mode ligands below to the cation exchanger mixed mode (for example CaptoAdhere) (Melinda, US 20170058019)

Examples include Capto-QTM which is a strong anion exchange and is a trademark of GE Healthcare Bio-Sciences. 

Polyethyleneimine derivatized polymeric media:

Deorkar (US 2008/0203029) discloses useful mixed mode chromatographic media for bioseparations whcih media polymeric includes particles dervivatized with polyethyleneimine (PEI) which are further funcitonalized with appropriate reactants. Examples of polyyric anyhdride and akly chlorides. In one embodiment, an anion excahnged with mixed primary and secondary and tertiary amine sites is prepared by reaction polyethyleneimine with a polymeric support bearing epoxy or halo groups such as chloro, boromo, iodo groups. Such polymeric support can be any suitable synthetic polymer or natural polymer resin such as poly(meth)acrylate, cellulose.  Resin particle suitable for derviatizaiton with polyethyleneimine includes cellulose, agarose, eopoxidized or halogenated polystyrenes. Examples of suitable functionalizaiton reagents for reaction with the PEI surface derivatized polyemeric resin partciles include anyhdrides such as cyclic carboxylic anyhdrides such as glutaric and succinic anhydrides, unsatured carboxylic anhydrides such as maleic meta-bisulfite, alkyl chorides or anhydrides such as butryl chloride.  Examples of such commerically avaialble resins are Tosoh Biosciences Toyopeark AF-eopxy 650M apoxy activated Sepharose 6B. These materials can be reacted with one of the teminal amino group of polyethyleneimine of various molecular weights through the formation of chemically stable alpha-hydroxy amino groups. In one embodiment, the mixed mode media having weak anion, weak cation and strong cation sites were prepared by reacting the EEI derivtized polymer with an unsaturated carboxylic acid anyhdride followed by sulfunation.

Anion and hydrophobic: 

Polyamines with Hydrophobic Group:

Engstrand (US 2007/0259453) discloses a method of separating antibodies using a multi-modal separation matrix to adsorb undesired compounds. The multi-modal separation matrix includes first groups which are capable of interacting with negatively charged sites of the target compoudns and second groups, which are capable of at least one interaction othern than charge-charge itneraction with said target compounds. In one embodimetn, the first anion-exchanging groups are strong anion exchanges such as qin that the groups remain charged within a wide pH range. In another embodiment, the first groups are weak ion exchanges in that they are charged at certain pH values but may loose charge by a pH switch. The second groups may give electron acceptor-donor interactions (e.g., hydrogen-bonding, dipole-dipole)  and/or hydrophobic and/or hydrophilic interactions. The functional groups may be present on the same ligand or on different ligands. In one embodiment, the the ligands are coupled to the support via their frist groups, such as via amines resulting in quaternary amines. 

Johansson (US Patent No: 6,702,943) discloses a method for removal of a target substance by adsorption to a matrix carrying a pluality of lgidnas taht include anion-exchaning groups and a hydrophobic structure. 

Matsumoto, (US 2015/0344520; see also US Patent Applicaiton No: 16/349,617, published as US 2019/0345194) discloses a chromatography media obtained by adding polyamine to a base media containing porous particles and then modifying amino groups in the polyamine with a hydrophobic group has excellentsalt tolerance and excellent adsorption. In particular, the chromatography media contains a base media haivng porous particles and a polyamine bonded with the base media wherein 20-40% of amino groups in the polyamine is modified with a hydrophobic group. The polyamine is selected from the gorup of polyallylamine, polyvinylamine, chitosan, polysine, polyguanidine and polyornithine. 

Commerical Examples:

–Capto adhere: is a mixed mode resin, a combination of anion exchagne and hydrophobic interaction. It can be operated in flow through mode where the antibody does not bind but the contaminante does bind. It is commercially available from GE Healthcare Life Sciences (Shaban, US 14/809,211). 

Capto adhere is a strong anion exchanger with multimodal functionality that gives a different selectivity compared to traditional anion exhangers. The Capto adhere ligand, N-Benzyl-N-methyl ethanol amine, exhibits many functionalities for interaction. The most pronounced are ionic interaction, hydrogen bonding and hydrophobic interaction. Capto adhere is designed for post Protein A purification of mAbs. Removal of leached Protein A, aggregates, HCS, nucleic acids and viruses is performed in flow through mode. The medium is based on a rigid high flow agarose matrix that allows high flow velocities to be used. (GE Healthcare Life Science, Instructions 28-9064-05AC Affinity Chromatography Capto adhere). 

Commercial examples include Capto-Adhere which is a multimodal anion exchange and a trademark of GE Bio-Sciences. Capto Adhere is a mixed-mode chromatography support which exploits a combination of anion exchange and hydrophobic interaction functionalities (US 8,188,242). Its ligand, N-benzyl-methy ethanol amine, contains anion exchange, hydrophobic and hydrogen bonding interaction groups. It is a strong anion exchanger with a phenyl group for hydrophobic interactions and a hydroxyl group for hydrogen bonding. The Capto adhere ligand (N-Benzyl-N-methyl ethanolamine) exhibits multiple modes of protein-interactive chemistries, including ionic interaction, hydrogen bonding and hydrophobic interaction. The multimodal functionality of the resin confers it with an ability to remove antibody dimers and aggregates, leached prtoein A, host cell proteins (HCP), antibody/HCP complexes, process residuals and viruses. The resin is typically used in flow through mode in the context of production scale polishing step employing operational parameters designed to ahve the mAb pass directly through the column while the contaminants are adsorbed. Nti-Gyabaah US14/355014

Generic conditions for use of multimodal anion exchangers such as CAPTO adhere do not exist. Specific conditions must be developed for each protein. The recomended method development procedure consists of evaluting various combinations of pH and sodium chloride concentration in the hope of identifying conditions that preferentially favor retention of aggregates (US 2011/0166332).

Gagnon (US 2011/0166332 and WO2010030222) discloses a method of separating at least one intact non-aggregated protein (e.g., an IgG antibody) form a liquid preparation by contacting the preparation with a multimodal anion exchanger sucha s CAPTO-adhere in the presence of one or more species of protein excluded zwitterions such as glycine.Boschetti (WO2004/024318) discloses mixed mode ligands which comprises a cyclic group which can be a monocyclic group or a polycyclic group such as a aromatic group that is a cylic hydrocarbon containing only unsaturated carbon bonds to give an aromatic system. While any aromatic group can be used, suitable aromatic groups typically have one, two or three aromatic rings. Illustrative aromatic groups are phenyl and its substituted derivatives such as tolyl and xylyl. Bicycl

–MEP HyperCel is a commercially available example of a combination of anion exchange and hydrophobic interaction functionalities (US 8,188,242). The spearation  is based on its pH dependent ionizable dual mode hydrophobic ligand design. In the purificaiton of a monoclonal antibody, for example, IgG adsorbs to the MEP ligand mianly through hydrophobic interactions under physiological buffer conditions. Desorption is through electrostatic repulsion between positivley charged IgG molecules and ionizable pyridine ring of the ligand when the buffer pH is decreased to close to or below the ligand pKa of 4.8, which the ligand is positively charged. See also “HCIC” under Antibody purification schemes.

Other commercial examples include MEP-Hypercel and Capto Adhere. Ma (US14208043)

Anion exchange + hydrophobic interaction + hydrogen bonding and pi-pi bonding: 

ic aromatic group include fused individual rings and include napthyl. Polycyclic aromatic groups include anthracenyl and phenanthrenyl and groups such as acenanaphthylenyl that contain fused rings of different sizes. If an aromatic group is selected, it is preferred that the group be fused to a heterocylic (saturated to particaly saturated ring include at least one hetero atom such as N, S or O) or heteroaromatic group. Exemplary heterocyclic or heteroaromatic groups include thiazoline, thizolidone, imidazole, imidazoline, thiazole, traizoles, tetrzole, thiadiazole, imidazole, pyridine and morpholine. The ligand includes a linking group that optionally comprises a sulfur ion. The monocyclic or polycylic group is substituted with a sulfate, sulfonate, phospahte, or phosphaonate group. These groups are sufficiently acidic to exist as charged moieites with a large pH range such as from 2-12. The monocylic or polycyclic group is tethtered to the solid support by a linking group which includes a mercapto, ether, or amino containing moeity. The solid support operated via “mixed modes” of interaction. The monocyclic and polycyclic groups have a pK below 4 and thus are negatively charged within the pH ranges used. A biological substance such as an immunoglobuilin, is contacted with the substrate between pH 4-6, in which the range the biological substance bears a net postive or neutral charge.. When the pH is raied above about 8, the biological substance gains a net negative charge, thereby creating an electrostati repulsion between the negatively charged solid substrate and the negativey charged biological substance. Consequently, the biological substance is released and can be isolated. 

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). 

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). 

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. 

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.

See also complement inhibitor targetting under “Pharmacology” and “Drug delivery” See also Human Complement Regulatory Proteins (CRegs) 

See also disease mechanisms of viruses and bacteria for how pathogens use complement regulatory proteins to evade the immune system

Mechanisms of Complement Regulation

The complement system is regulated via a number of interrelated mechanisms. There are two broad mechanisms for inhibition of the complement system; inhibitors of the complement activation pathway (inibitors of C3 convertase formation) and inhibitors of the terminal complement pathway (inhibit MAC formation).

(1) Inactivation of Complement Enzymes: In the first mechanism, CRegs function by inactivating enzymes, such as the C3 and C5 convertases, which are formed during C activation and which are responsible for cleavage of C3 and C5. The first mechanism is generally reversible, facilitating the dissociation of the C3 convertases (i.e., C3b from Bb and C4b from C2a). The dissociation may also involve reversible binding of the antagonist proteins to C3b or C4b components, thus preventing their reassociation.

(2) Interference with MAC: In the second mechanism, CRegs function by interfering with MAC formation. This second mechanism, which is an irreversible inactivation process, results from proteolytic cleavage of the C3 convertase components C3b or C4b by the serine protease factor I. 

Both general regulatory mechanisms, the facilitation of dissociation of C3b and C4b and the inactivation of C3b and C4b through cleavage by factor I, also apply to the inhibition of the alternative pathway C5 convertase (C3bBbC3b) and the classical pathway C5 convertase (C4bC2aC3b).

The proteins encoded by a region of the genome which designated the regulators of complement activation (RCA) gene cluster are involved in both mechanisms.

Various assays/methods can be used to mesasure activities of complement pathway molecules and activaiton of the complement system. (see US Patent No. 6,087,120 and Newell, J Lab Cin Med, 100:437-44, 1982). The two most commonly used techniques are hemolytic assays and immunological assays.

Definitions/General Principals

(1) EGTA blocks the classical C pathway because it chelates Ca++ and thus inactivates C1 (Platts-Mills, J. Immunology, 113(1), 1974).

(2) Amplifcation loops of the AP: The AP pathway involves an amplifcaiton loop utilizing C3b producec by the CP and lectin pathways. Some molecules of C3b generated by the CP C3 convertase are funneled into the AP. Surface bound C3b binds Factor B to yield C3bB, which becomes a substrate for Factor D of the AP. Factor D is a serine esterase that cleaves the Ba fragment, leaving C3bBb bound to the surface of the target cell. C3bBb is stablized by proeprdin, forming C3bBbP, which acts as the AP C3 convertase. This C3 convertase participates in an amplifcaiton loop to cleave many C3 molecules, resulting in the deposition of C3b on the target cell. Some of these C3b bind back to C3bBb to form C3bBb3b, the AP C5 convertase which cleaves C5 into C5a and C5b. C5b binds to the surface of the cell to initiate the formation of MAC. (Fung, 2006/0140939).

Antibody sensitized sRBCs only activate the CP. They do not, by themselves, activate the AP. However, in the presence of sufficient NHS (e.g., 7-10%) activated CP will utilize the amplication loop of the AP. (Bansal, US13/646,286).

(3) Calcium/magnesium: The CP is a calcium/magnesium dependent cascade whereas the AP is a magnesium dependent cascade. With the lectinpathway, Ca++ dependent binding of MBL to a mannan coated surface triggers activaiton of C3. C2 is a single chain plasma protein that is specific for the CP and LP. Membrane bound C4b expresses a binding site which, in the presence of Mg++, binds the proenzyme C2 near its amino termus and present it for cleavage by C1s (for the CP) or MASP-2 (for the LP) to yeild a 30 kD amino terminal fragment, C2b, and 70 kD carboxy terminal fragment, C2a.  (Fung WO01/70818).

Hemolytic Assays/techniques

Hemolytic Assays measure the functional capacity of the entire pathway.

Erythrocyte Lysis Assays: 

Erythrocyte lysis assays are based on the formation of a terminal complement complex on the surface of rabbit red blood cells (rRBC). As a reuslt of the formation of this complex, the rRBCs are lysed. The progressive decrease in light scatter at 700 nm is a direct measure of erythrocyte lysis.  

Assays for the Alternative Pathway (AP)

Rabbit erythrocyte hemolysis:

It is well established that rabbit erythrocytes specifically activate the complement AP, with a resulting lysis of the cells by the C5b-9 complex (Bansal, US 2005/0107319)

Alternative Pathway Isolated from the Classical Pathway: 

It is accepted that AP activation is dependent on Mg++ but not Ca++ (Platts-Mills, J. Immunology, 113(1), 1974). AP activation in Mg++ ions without calcium ions guarantees only the AP activaiton (Bansal, 13/646286). 

—-Rabbits RBCs in 10% NHS/Mg2+:EGTA: Introducing rabbit Erythrocytes (rRBC) into 10% human serum (with Mg2+/EGTA) represent the introduction of a foreign cell surface which initiates the AP cascade. AP activation in Mg++ ions without calcium ions (EGTA is used to chelate the calcium ions to prevent CP activation)guarantees only AP activation. Rabbit RCs can be used to activate the AP in 10% NHS in the presence of Mg2+ in the absence of Ca2+ and in the presence of sufficient NHS concentration . Because the CP requires the presence of Ca2+, the CP will not be active under these conditions. 

Example (anti-properdin): To determine the effects of a MoAb to propderin on alternative pathway (AP) function, increasing amounts of the antibody are added to human serum that is incubated with rabbit erythrocytes in buffer containin EGTA and Mg++. The cells cause AP activation which results in C5b-9 formation and consequent lysis of the cells. The extent of erythrocyte lysis can be monitored by examining the amount of light scattered by intact red blood cells (Polhill, 1978). Human serum + rabbit erythrocytes in buffer containing EGTA and Mg++. These cells cause AP activation, which results in C5b-9 formation and consequent lysis of the cells. (Bansal, US 13/646286)

—-Rabbit erythrocytes + 7.5% NHS + GVB-Mg+++-EGTA: Song (US2010/0263061) disclose incubation of rabbit erythrocytes with 7.5% NHS in GVB with or withoug anti-P antibodies or EDTA. Song shows properdin mAbs which dose dependently inhibited complement mediated lysis of the rabbit red blood cells. Anti-P antibodies and EDTA were used as positive controls for inhibition (EDTA blocks CP complement).

Assays for the Classical Pathway (CP)

The classical CP is typically triggered by immune complexes, for example, an antibody boudn to a foreign particle and thus requires prior exposure to that particle for the generation of specific antibody. (Gupta-Bansal (US 6,333,034). The CP is a calcium/magnesium dependent cascade. C1, the first enzyme complex in the cascade, is a pentamolecular complex consisting of C1q, 2 C1r molcules and 2 C1s molecules. This complex binds to an antigen-antibody complext through the C1q domain to initiate the cascase. (Fung, US 2005/1096394)

CH50 Assay:

The complement system is a group of proteins that wehn activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Inidividual ocmmplement components can be quantified; however this does not provide any informaiton as to the activity of the pathway. The CH50 is a screening assay for the activaiton of the CP and it is sensitie to the reduciton, absence and/or inactivity of any component of the pathway. The CH50 tests the funciotnal capability of serum complement components of the CP to lyse sheep red blood cells pre-coated with rabbit anti-sheep red blood cell antibody (haemolysis). When antibody-coated SRBC are incubated with test serum, the classical pathway of coplement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zerio; if one or mroe components are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the sueprnatant at 540 nm. The amount of complement acitvity is determiend by examining teh capacity of various dilutions of test serum to lyse antibody coated SRBC. (Costabile “Measuring the 50% haemolytic complement (CH50) activity of serum” J Visualized experiments, 2010). 

Classical Pathway isolated from alternative pathway: 

—-Antibody Sensitized Sheep cells in 1% NHS/Ca2+/Mg2+: To measure the functional capacity of the classical pathway, sheep red blood cells coated with hemolysin (rabbit IgG to sheep red blood cells) are used as target cells (sensitized cells). These ag-Ab complexes activate the classical pathway and result in lysis of the target cells when the components are functional and present in adequate concentration. To determine functional capacity of the alternative pathway, rabbit red blood cells are used as the target cell (see US Patent No. 6,087,120). The antibody sensitized sheep cells are used as an activator in 1% normal human serum in the presence of Ca2+/Mg2+. The calcium ionsare required for activation of the CP for the initial trigger of the C1q/C1r/s complexes. CP will not occur in the basence of the calcium ions. Mg2+ is required for AP activation. However, in 1% normal human serum containing Ca2+/Mg2+, only the CP proceeds to completion. Without the requisite levels of NHS which is 10%, the AP pathway will not have a significant presence.  (Bansal, US 13/646286) 

Classical Pathway activation of the Alternative Pathway Via way of the Amplication Loop of the AP

—-Antibody sensitized sheep erythrocytes + 7.5 NHS + GVB-Mg++: Antibody sensitized sheep erythrocytes were incubated with 7.5 NHS in GVB-Mg++ buffer with or without anti-P antibodies or EDTA. Antibody sensitized sheep erythrocytes are another well established assay for the classical pathway complement activation.  Cells completely lysed by hypotonic shock were aslo sued as a control (100% lysis). The degree of lysis was determined by hemoglobin release using a spectrophotometer. Polyclonal anti-P antibody had no effect on human complement mediated lysis of the antibody sensitized sheep erythrocytes. In contrast, EDTA inhibited human complement mediated lysis and was used as a positive control. (Song, 2010/0263061).  Antibody sensitized sRBCs only activate CP. They do not, by themselves, activate AP. However, in the presence of sufficient NHS, activated CP will utilize the amplifcation loop of the AP. (Bansal, US 13/646,286).

—-Antibody sensitized sheep cells in in 10% NHS/Ca2+/Mg2+: For CP and AP action, antibody sensitized sheep red blood cells are used as an activator in 10% ca2+/Mg2+ in human human serum. The difference between the assay above for the CP only is that NHS is 10%. The Ca2+/Mg2+ provides the level of Mg2+ required for AP activation and allows for both CP and AP to be active. Antibody sensitized sRBCs only activate the CP. They do not by themselves activate the AP. However, in the presence of sufficient NHS, activated CP will utilize the amplification loop of the AP. Under these conditions, the C3b produced vial the CP can feed into the AP causing amplificaiton of the AP loop. In other words, the AP has been activated by the CP. (Bansal, US 13/646286)

Assays for the Lectin pathway

 Assays for the lectin pathway include measuring the functional activity of lectin-MASP complext through activation of added exogenous C4 or measuring endogenous activation of C4 after blocking of CP and AP using a high ionic strenght dilution buffer (1M NaCl) Palarasah (US2010/0196927).

Polyanethol sulphonate (PAS) + activation of lectin pathway:

Palarasah (US2010/0196927) discloses a method of determining functional deficiencies in the lectin pathway by diluting a sample with polyanethol sulphonate (PAS) which blocks the CP and AP, activating the lectin pathway and then determining activation of one or more complement factors C3, C4 of one or mroe components of the C5-C9 complex.

Immunologic Assays/techniques: 

Immunologic assays use antibodies agaisnt the different epitopes of the various complement components (e.g., C3, C4, and C5) to detect split product of the complement components (e.g., C3a, C4a, C5a and the C5b-9 terminal complex).

Alternative Pathway: To accomplish this assay one immobilizes LPS onto microtiter wells to activate the AP in diluted serum samples. Can then measure AP components like C5b-9 (MAC).

Particular Complement Components

—-C3b:

Example: LPS + 10% NHS/Mg++: LPS is a specific activator of the AP.  (Bansal, US 13/646286) AP activation generates C3 and C3b as a result of C3 cleavage by the C3 convertase of the AP. AP is acivated in NHS by LPS under conditions that allow activation of the AP. This assay was used to demonstrate whether an anti-properdin antibody would inhibit the foramtion and deposition of C3b which initiates the start of AP. As a way of mechanims, activated and deposited C3b provides high affinity binding to properdin. Properdin-C3b complexes bind factor B and the complex is cleaved by factor D to generate PC3bBb, an AP C3 convertase. As the AP proceeds, C5b-9 complexes are formed and deposited. Polystyrene microtiter plate wells were coated with LPS. Normal human serum (NHS) at 10% in AP buffer was mixed with varying concentrations of an anti-properdin antibody and C3b was detected with rabbit anti-human C3c, properdin was detected with goat anti-human P, Bb was detected with goat anti-human factor Bb and C5b-9 was detected with HRPO-conjugated neo-anti-human C5b-9. The presence of C3b, p and Bb and MAC together was indicative of AP C3 convertase formation, and the antibody was shown to inhibit C3b formation. (Bansal, US8,664,362 and 13/583879; 14/195, 458). 

—-C5 protein and fragments: Methods for determining whether an antibody can block the generation or activity of the C5a and/or C5b active fragments of C5 or binding to complement component C4b or C3b are known in the art (US6355245). 

—-C5a: Methods for measuring C5a activity include chemotaxis assays, RIAs, or ELISas (Ward and Zvaifler (1971) J Clin Invest 50(3): 606-16). 

Classical Pathway:

–CH50 EIA:  The binding of the C1q component of C1 to immune complexes triggers the CP which results in a cascade of enzymatic and non-enzymatic reactions culminating in the formation of terminal complement complexes (TCC). Th CH50 EIA measures the hemolytic complement (CG50) in human serum and allowed detection of a deficiency of one or more complement components C1-C9. It is a tradtional method for measuring the CP. In this lytic assay, antibody sensitized sheep erythroytes (EA) are used to activate the CP and various dilutions of the test serum are used to determine the amount required to give 50% lysis. The assay uses a mAb to a unique neoantigen to caputre the TCC analytic.

Lectin Pathway: Mannan can be as a ligand and coated on plates and then serum is added. Detection of MBL, C4 and C3 can then be performed with mAbs against these components. As both the LP and the CP are calcium dependent and lead to activation of C4, one challenge in devising an assay to assess the functional characterization of the LP is distinguishing its activation from activation of the CP. Using manna, for example, to activate the LP is also likely to activate the CP via anti-annan Ab which is present in huan serum.

Roos (Mol Immunol. 2003; 39: 655-68) describe a lectin pathway assay which uses a mAb to C1q to inhibit CP (blocks CP but allows lectin activation as by mannan to proceed normally) and a mAb to factor D to inhibit AP.

Petersen (J Immunol Methods, 2001, 257: 107-16) escribe blocking CP and AP using a high ionic strenght dilution buffer (1M NaCL) and then measuring endogenous activation of C4. 

Palarasah (US 12/676283) disclose an in vitro method for determining functional deficiencies in the lectin pathway by diluting a sample with a polyanion, preferably a sulphated polyanion and even more preferably polyanethol sulphonate (PAS), which is an inhibitor of the AP and CP, but not the lectin pathway, then activating the lectin pathway and then determining the activation of one or more of the complement factors C3, C4 or one or more of the components of the C5-C9 complex such as with an antibody, wherein a lower level compared to a normal reference level is indicative of a functional deficiency in the lectin pathway.