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Immunotherapy for treating Methamphetamine AbuseImmunotherapy for treating Methamphetamine Abuse

IntroductionIntroduction

Traditional treatment for addiction and its challengeTraditional treatment for addiction and its challenge

(+)-Methamphetamine (METH) abuse are mediated via multiple neurotransmitter sites. Medications that target single site of action (e.g., the dopamine transporter) have proven ineffective and in some cases addictive. No medication has been approved by the Food and Drug Administration for the treatment of methamphetamine abuse. Traditional treatment regimes begin with medical detox, during which the substance is slowly weaned out of the user’s body. Because there are no specific pharmacological therapies for excessive methamphetamine use, supportive care is aimed to manage symptoms instead of removing causative agent. Patients are left susceptible to potential neurotoxic effects of methamphetamine[1]. Post detox long-term behavioral modification programs aims at reconstructing positive behaviors and learning new and drug-free ways to cope with life stressors that trigger the desire to use meth, however, to protect patients against relapse remains a profound challenge and there is great need for outcome-based treatment research. Anti-METH mAb reside in the blood and extracellular fluid compartments. It could act as a pharmacokinetic antagonist and alter distribution of METH by high affinity binding and reduce its amount and delivery to its site of action in the brain and other vulnerable organs (heart), therefore favorably alter its metabolism and eliminate the drug.[1]. A monoclonal antibody (mAb) with high affinity binding to METH and a long acting duration (2-3 weeks) could provide a novel therapeutic strategy for patients who have serious problems with compliance [2] therefore will increase the probability of a success treatment.

Two major approaches to developing drug-specific immunotherapies: active and passive immunizationTwo major approaches to developing drug-specific immunotherapies: active and passive immunization

Active immunizationActive immunization

Active immunization involves conjugating a drug-like hapten to a carrier protein and using traditional immunization approaches to generate a specific immune response over time[3].Active vaccines against METH have shown potential for reducing CNS effects such as horizontal locomotion and self administration in animal models[4]. Vaccine has shown to attenuate methamphetamine-Induced disruptions in thermoregulation and activity in Rats[5]. It has also shown to protect rats from METH-induced impairment of behavioral responding for food[6].

Furthermore, it is important to determine whether or not drug use would interfere with antibody generation during the immunization period because it is unlikely that most drug abusers will abstain from drug abuse during the recovery process. In animals immunized against (+)METH, anti-(+)METH antibody titers and affinity for (+)METH were the same, regardless of whether or not they were repeatedly challenged with (+)METH [7].This indicates chronic drug use would less likely to interfere with antibody generation during active immunization.

Passive immunotherapyPassive immunotherapy

Passive immunotherapy involves treatment with carefully selected, preformed monoclonal antibodies or antibody fragments against a drug of abuse[3] .These mAbs are generated by first vaccinating a host animal and then creating mAb-secreting hybridoma cell lines, or, alternatively, by recombinant DNA methods that utilize phage, yeast, or ribosome display[3]. If needed, these mAbs are converted to a human-compatible form(e.g., chimeric, humanized, or fully human immunoglobulin)[3].

There are currently two forms of passive mAbs in preclinical testing: a long-acting intact immunoglobulin G (IgG) (150kDa) form for treating addiction and overdose, and an extremely short-acting single chain variable fragment (scFv; 27kDa) for treatment of overdose.[3] The long half-life of IgG is due partly to the ability of the constant region of the antibody to be salvaged from catabolism by the neonatal Fc receptor (FcRn)[8].

Combining active immunization with monoclonal antibody therapyCombining active immunization with monoclonal antibody therapy

An anti-METH mAb could be used in combination with a METH-conjugate vaccine (MCV). The benefits would include immediate onset of action (from the mAb), timely increases in the immune responses (from the combined therapy) and duration of antibody response that could last for months (from the MCV).[9]


Materials and methodsMaterials and methods

ChemicalsChemicals

[(+)-3H]METH and [(+/-)-3H]AMP were used for synthesizing haptens.

Synthesis of METH-like haptensSynthesis of METH-like haptens

Because drugs of abuse are too small to generate an immune response on their own, a critical step toward making an effective drug-specific vaccine is to synthesize a hapten that maintains the chemical and structural properties of the original drug and has an added, carefully placed chemical linker with a distal moiety of a functional group that can be easily conjugated to a larger carrier antigenic protein.[3]. The complete synthesis (+)-METH P6 was reported 2001[7] . The chemical structures of other stereospecific (+) haptens were reported 2007[10].


Antigen synthesisAntigen synthesis

Carrier proteins, such as keyhole limpet hemocyanin or modified bacterial toxoids that are safe for use in humans. Ovalbumin, bovine serum albumin, or many others are used in animal models[3]. Bovine serum albumin was reported to covalently bound to (+)-METH P6 and ovalbumin to (+)METH MO10 to produce mAb6H4 and mAb4G9 respectively[10] by two-step carbodiimide procedure[11] which forms a peptide bond between the carboxyl group of the hapten linker arm and free amino groups of lysine side chains in the respective proteins[10].

At the end, all antigens were purified on a gel filtration column in a phosphate-buffered saline after dialysis[1].

Immunization, Screening, and Hybridoma GenerationImmunization, Screening, and Hybridoma Generation

Initial subcutaneous immunization of 100 micrograms of the (+)-METH P6 antigen emulsified with adjuvant was followed with monthly boosts of 50 microgram dosage. For all other antigen immunizations, dosage and boost intervals have also been reported[10]. Serum samples were taken via tail bleed periodically to measure IgG titers by enzyme-linked immunosorbent essay (ELISA)[10]. Wells were coated with the original hapten that conjugated to a different protein to avoid selecting carrier protein-reactive antibodies[10]. The mouse with the highest anti-METH serum titer was chosen for monoclonal antibody production[1].(cell line [10]). After fusion of mouse B-cells with a myeloma cell line, hybridomas which has both the antibody-producing ability of the mouse B-cell and exaggerated longevity and reproductivity of the fusion partner myeloma were identified by ELISA and sub cloned to monoclonality[12]. To generate large amounts of monoclonal antibody, mice were injected with hybridoma cells and ascites fluid that contained high concentration of IgG was collected after next several weeks[12].


Mouse-hybridoma isotyping kit is used to decide the IgG isotype and light chain identity[10]. In the case of mAb6H4, it was determined to be immunoglobin G1 WITH A kappa light chain[1].

Production, purification and formulationProduction, purification and formulation

To produce gram quantities for pharmacokinetics studies, the hybridoma cell line was grown in a Cell-Pharm System 2500 hollow-fiber bioreactor [1] and the similar method has been previously described[13]or to be produced in a Biostat B 10 liter bioreactor[10]. Antibody purification was performed with an Index-100 cation exchange column[1]. Similar method has been previously described [14],or they were purified by affinity chromatography using protein G-Sepharose[10], or both[10].

After purification, all antibodies were concentrated, and buffer was exchanged into 15mM sodium phosphate containing 150mM sodium chloride[10] as previously described[15].


Pharmacokinetics, pharmacodynamics and metabolism of anti-METH antibody mAb6H4Pharmacokinetics, pharmacodynamics and metabolism of anti-METH antibody mAb6H4

For a mAb therapeutic to have the broadest medical applicability, it should have high affinity and also specificity for members of this drug class. [(+)-METH,(+)-AMP, and (+)-MDMA].First, these stimulant drugs have similar or overlapping effects. In particular, (+)-AMP is both a pharmacologically active metabolite of (+)-METH and a frequently used drug of abuse that could be substituted for (+)-METH. In addition, the minus isomers of these drugs could potentially be purposely taken by drug abusers to neutralize mAb medications with high affinity binding for both plus and minus stereoisomers. In a related way, there are many structurally similar compounds like ephedrine and pseudoephedrine that could be used to lessen the efficacy of mAb therapies if the mAb is not highly specific for (+)-METH-like structures.[10]

Radioimmunoassay determination of mAb6H4 cross-reactivity and dissociation constantsRadioimmunoassay determination of mAb6H4 cross-reactivity and dissociation constants

Screening for anti(+)METH IgG response was conducted by radioimmunoassay (RIA)[10] in which radio labelled [(+)-3H] METH is competing with unlabelled (+)-METH and (+)-AMP for the binding site of Abs in a manner similar to what has been previously described[16]. An IC50 value was determined for each compound after fitting a sigmoidal curve to the data points[1][10].

Anti-(+)METH mAb6H4 was reported with Kd = 11 nM[1], having <.01% cross-reactivity with almost all compounds tested (including (-)-METH, (+/-)-AMP,(+/-)-MDMA,4-OH METH, Pseudoephedrine, Ephedrine, Dopamine, Norepinephrine, Serotonin, Epinephrine)[1] except for (+)-methylenedioxymethamphetamine (MDMA) which has just slightly higher relative affinity than METH (9 nM to 11nM)[1]. It was also reported stereospecific[1], having an approximately 100 times higher relative affinity for the plus form than the minus forms of these substances[1]. No significant cross activity has been observed in the test for other compounds[1] including methylenedioxyamphetamine, (+)-norpseudoephedrine, L-phenylephrine, (+)-phenylpropanolamine, beta-phenylethylamine, and tyramine[1].


Effect of mAb6H4 on METH and AMP pharmacokineticsEffect of mAb6H4 on METH and AMP pharmacokinetics

Clinical developmentClinical development

Pharmacological effects of two anti-methamphetamine monoclonal antibodies mAb4G9 and mAb7F9Pharmacological effects of two anti-methamphetamine monoclonal antibodies mAb4G9 and mAb7F9

Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9Human-mouse chimeric monoclonal antibody(mAb) Ch-mAb7F9

IgG2, Kappa;METH KD= 7nM

Preclinical characterization of Ch-mAb7F9 for human usePreclinical characterization of Ch-mAb7F9 for human use

Cross reactions in vitro ligand binding studiesCross reactions in vitro ligand binding studies

It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. [2]

Isothermal titration calorimetry potency studiesIsothermal titration calorimetry potency studies

Binding is efficient. [2]

Pharmacokinetics studies in ratsPharmacokinetics studies in rats

METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition. [2]

Human StudyHuman Study

Phase 1 Study: First Human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteersPhase 1 Study: First Human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers

Serum ch-mAb7F9 concentrationSerum ch-mAb7F9 concentration

Immunogenicity analysesImmunogenicity analyses

IgG pharmacokinetic parametersIgG pharmacokinetic parameters

half life 17-19 d, volume of distribution of 5-6 L in the 3 highest dose groups [17]

Human anti-chimeric antibody responseHuman anti-chimeric antibody response

Four(12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study (147 d);however, this response did not appear to be dose related.[17]

Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamineDevelopment and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine

Materials and MethodsMaterials and Methods

ScFv6H4 CloningScFv6H4 Cloning

Sub-cloning and Large-Scale ExpressionSub-cloning and Large-Scale Expression

Production and purificationProduction and purification

Determination of Kd Values using bead-based radioimmunoassayDetermination of Kd Values using bead-based radioimmunoassay

Pharmacokinetic Studies of METH and scFv6H4 in RatsPharmacokinetic Studies of METH and scFv6H4 in Rats

Structural highlights from crystal structure of scFv6H4Structural highlights from crystal structure of scFv6H4

Drag the structure with the mouse to rotate
scFv6H4 free form
4laq
Ligands: , ,
Gene: IgG (LK3 transgenic mice)
Activity:
Related: 4lar, 3gkz, 4las


Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT
Coordinates: save as pdb, mmCIF, xml




Drag the structure with the mouse to rotate
scFv6H4:(+)METH complex
3gkz
Ligands:
Gene: IgG (LK3 transgenic mice)
Activity:
Related: 4lar, 4laq, 4las


Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT
Coordinates: save as pdb, mmCIF, xml




Aromatic-Aromatic Interaction: A Mechanism of Protein Structure StabilizationAromatic-Aromatic Interaction: A Mechanism of Protein Structure Stabilization

The entrance of the binding pocket is lined with seven amino residues, one residue from each of them H1, H2, and H3 loops, 3 from the L3 loop, one from the beta-strand-3c of the heavy chain. These aromatic residues form a hydrophobic barrel around the aromatic portion of METH.



Hydrophilic interactions of METHHydrophilic interactions of METH

Water molecules in the binding cavityWater molecules in the binding cavity

Drag the structure with the mouse to rotate
scFv6H4:(+)AMP complex
4lar
Ligands: ,
Gene: IgG (LK3 transgenic mice)
Activity: ,
Related: 3gkz, 4laq, 4las


Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT
Coordinates: save as pdb, mmCIF, xml



}}

Structure optimization by point mutationsStructure optimization by point mutations

MethodsMethods

Saturation equilibrium dialysis to determine KdSaturation equilibrium dialysis to determine Kd

Inhibition equilibrium dialysis to determine IC50 values for AMPInhibition equilibrium dialysis to determine IC50 values for AMP

Crystallization and structure highlights of scFv-S93T:METH complexCrystallization and structure highlights of scFv-S93T:METH complex

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuseDevelopment and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

ReferencesReferences

[18] [11] [16] [12] [1] [7] [13] [14] [15]

[3] [8] [19] [20] [10] [21] [22] [23] [24] [5] [6] [25] [26] [27] [2] [9] [17] [4] [28] [29]

  1. ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 DOI:/10.1016/S0014-2999(03)01313-X
  2. ↑ 2.0 2.1 2.2 2.3 2.4 Stevens MW, Tawney RL, West CM, Kight AD, Henry RL, Owens SM, Gentry WB. Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use. MAbs. 2014 Mar-Apr;6(2):547-55. doi: 10.4161/mabs.27620. Epub 2013 Dec 23. PMID:24492290 doi:http://dx.doi.org/10.4161/mabs.27620
  3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 Peterson EC, Owens SM. Designing immunotherapies to thwart drug abuse. Mol Interv. 2009 Jun;9(3):119-24. doi: 10.1124/mi.9.3.5. PMID:19592672 doi:http://dx.doi.org/10.1124/mi.9.3.5
  4. ↑ 4.0 4.1 Miller ML, Aarde SM, Moreno AY, Creehan KM, Janda KD, Taffe MA. Effects of active anti-methamphetamine vaccination on intravenous self-administration in rats. Drug Alcohol Depend. 2015 Aug 1;153:29-36. doi: 10.1016/j.drugalcdep.2015.06.014., Epub 2015 Jun 19. PMID:26118833 doi:http://dx.doi.org/10.1016/j.drugalcdep.2015.06.014
  5. ↑ 5.0 5.1 Miller ML, Moreno AY, Aarde SM, Creehan KM, Vandewater SA, Vaillancourt BD, Wright MJ Jr, Janda KD, Taffe MA. A methamphetamine vaccine attenuates methamphetamine-induced disruptions in thermoregulation and activity in rats. Biol Psychiatry. 2013 Apr 15;73(8):721-8. doi: 10.1016/j.biopsych.2012.09.010., Epub 2012 Oct 23. PMID:23098894 doi:http://dx.doi.org/10.1016/j.biopsych.2012.09.010
  6. ↑ 6.0 6.1 Ruedi-Bettschen D, Wood SL, Gunnell MG, West CM, Pidaparthi RR, Carroll FI, Blough BE, Owens SM. Vaccination protects rats from methamphetamine-induced impairment of behavioral responding for food. Vaccine. 2013 Sep 23;31(41):4596-602. doi: 10.1016/j.vaccine.2013.07.038. Epub, 2013 Jul 29. PMID:23906885 doi:http://dx.doi.org/10.1016/j.vaccine.2013.07.038
  7. ↑ 7.0 7.1 7.2 Byrnes-Blake KA, Carroll FI, Abraham P, Owens SM. Generation of anti-(+)methamphetamine antibodies is not impeded by (+)methamphetamine administration during active immunization of rats. Int Immunopharmacol. 2001 Feb;1(2):329-38. PMID:11360933
  8. ↑ 8.0 8.1 Lobo ED, Hansen RJ, Balthasar JP. Antibody pharmacokinetics and pharmacodynamics. J Pharm Sci. 2004 Nov;93(11):2645-68. doi: 10.1002/jps.20178. PMID:15389672 doi:http://dx.doi.org/10.1002/jps.20178
  9. ↑ 9.0 9.1 Hambuchen MD, Carroll FI, Ruedi-Bettschen D, Hendrickson HP, Hennings LJ, Blough BE, Brieaddy LE, Pidaparthi RR, Owens SM. Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti-Methamphetamine Antibody Response. J Med Chem. 2015 Jun 11;58(11):4665-77. doi: 10.1021/acs.jmedchem.5b00220. Epub, 2015 May 22. PMID:25973614 doi:http://dx.doi.org/10.1021/acs.jmedchem.5b00220
  10. ↑ 10.00 10.01 10.02 10.03 10.04 10.05 10.06 10.07 10.08 10.09 10.10 10.11 10.12 10.13 10.14 10.15 Peterson EC, Gunnell M, Che Y, Goforth RL, Carroll FI, Henry R, Liu H, Owens SM. Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse. J Pharmacol Exp Ther. 2007 Jul;322(1):30-9. doi: 10.1124/jpet.106.117150. Epub, 2007 Apr 23. PMID:17452421 doi:http://dx.doi.org/10.1124/jpet.106.117150
  11. ↑ 11.0 11.1 Davis MT, Preston JF. A simple modified carbodiimide method for conjugation of small-molecular-weight compounds to immunoglobulin G with minimal protein crosslinking. Anal Biochem. 1981 Sep 15;116(2):402-7. PMID:7316174
  12. ↑ 12.0 12.1 12.2 Valentine JL, Arnold LW, Owens SM. Anti-phencyclidine monoclonal Fab fragments markedly alter phencyclidine pharmacokinetics in rats. J Pharmacol Exp Ther. 1994 Jun;269(3):1079-85. PMID:8014850
  13. ↑ 13.0 13.1 Valentine JL, Mayersohn M, Wessinger WD, Arnold LW, Owens SM. Antiphencyclidine monoclonal Fab fragments reverse phencyclidine-induced behavioral effects and ataxia in rats. J Pharmacol Exp Ther. 1996 Aug;278(2):709-16. PMID:8768722
  14. ↑ 14.0 14.1 Hardin JS, Wessinger WD, Proksch JW, Owens SM. Pharmacodynamics of a monoclonal antiphencyclidine Fab with broad selectivity for phencyclidine-like drugs. J Pharmacol Exp Ther. 1998 Jun;285(3):1113-22. PMID:9618414
  15. ↑ 15.0 15.1 McMillan DE, Hardwick WC, Li M, Owens SM. Pharmacokinetic antagonism of (+)-methamphetamine discrimination by a low-affinity monoclonal anti-methamphetamine antibody. Behav Pharmacol. 2002 Sep;13(5-6):465-73. PMID:12394422
  16. ↑ 16.0 16.1 Owens SM, Zorbas M, Lattin DL, Gunnell M, Polk M. Antibodies against arylcyclohexylamines and their similarities in binding specificity with the phencyclidine receptor. J Pharmacol Exp Ther. 1988 Aug;246(2):472-8. PMID:2457075
  17. ↑ 17.0 17.1 17.2 Stevens MW, Henry RL, Owens SM, Schutz R, Gentry WB. First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers. MAbs. 2014;6(6):1649-56. doi: 10.4161/19420862.2014.976431. PMID:25484042 doi:http://dx.doi.org/10.4161/19420862.2014.976431
  18. ↑ Burley SK, Petsko GA. Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science. 1985 Jul 5;229(4708):23-8. PMID:3892686
  19. ↑ Kosten T, Owens SM. Immunotherapy for the treatment of drug abuse. Pharmacol Ther. 2005 Oct;108(1):76-85. doi: 10.1016/j.pharmthera.2005.06.009. PMID:16023218 doi:http://dx.doi.org/10.1016/j.pharmthera.2005.06.009
  20. ↑ Hendrickson H, Laurenzana E, Owens SM. Quantitative determination of total methamphetamine and active metabolites in rat tissue by liquid chromatography with tandem mass spectrometric detection. AAPS J. 2006 Nov 22;8(4):E709-17. doi: 10.1208/aapsj080480. PMID:17233534 doi:http://dx.doi.org/10.1208/aapsj080480
  21. ↑ Peterson EC, Laurenzana EM, Atchley WT, Hendrickson HP, Owens SM. Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine. J Pharmacol Exp Ther. 2008 Apr;325(1):124-33. doi: 10.1124/jpet.107.134395. Epub , 2008 Jan 11. PMID:18192498 doi:http://dx.doi.org/10.1124/jpet.107.134395
  22. ↑ Gentry WB, Ruedi-Bettschen D, Owens SM. Development of active and passive human vaccines to treat methamphetamine addiction. Hum Vaccin. 2009 Apr;5(4):206-13. Epub 2009 Apr 20. PMID:19276653
  23. ↑ Celikel R, Peterson EC, Owens SM, Varughese KI. Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse - methamphetamine and 3,4 methylenedioxy-N-methylamphetamine. Protein Sci. 2009 Sep 16. PMID:19760665 doi:10.1002/pro.244
  24. ↑ Nanaware-Kharade N, Gonzalez GA 3rd, Lay JO Jr, Hendrickson HP, Peterson EC. Therapeutic anti-methamphetamine antibody fragment-nanoparticle conjugates: synthesis and in vitro characterization. Bioconjug Chem. 2012 Sep 19;23(9):1864-72. doi: 10.1021/bc300204n. Epub 2012 Aug , 28. PMID:22873701 doi:http://dx.doi.org/10.1021/bc300204n
  25. ↑ Peterson EC, Celikel R, Gokulan K, Varughese KI. Structural characterization of a therapeutic anti-methamphetamine antibody fragment: oligomerization and binding of active metabolites. PLoS One. 2013 Dec 5;8(12):e82690. doi: 10.1371/journal.pone.0082690. eCollection, 2013. PMID:24349338 doi:http://dx.doi.org/10.1371/journal.pone.0082690
  26. ↑ Thakkar S, Nanaware-Kharade N, Celikel R, Peterson EC, Varughese KI. Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering. Sci Rep. 2014 Jan 14;4:3673. doi: 10.1038/srep03673. PMID:24419156 doi:http://dx.doi.org/10.1038/srep03673
  27. ↑ Laurenzana EM, Stevens MW, Frank JC, Hambuchen MD, Hendrickson HP, White SJ, Williams DK, Owens SM, Gentry WB. Pharmacological effects of two anti-methamphetamine monoclonal antibodies. Supporting data for lead candidate selection for clinical development. Hum Vaccin Immunother. 2014;10(9):2638-47. doi: 10.4161/hv.29707. Epub 2014 Nov, 1. PMID:25483484 doi:http://dx.doi.org/10.4161/hv.29707
  28. ↑ Hay CE, Gonzalez GA 3rd, Ewing LE, Reichard EE, Hambuchen MD, Nanaware-Kharade N, Alam S, Bolden CT, Owens SM, Margaritis P, Peterson EC. Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse. PLoS One. 2018 Jun 29;13(6):e0200060. doi: 10.1371/journal.pone.0200060., eCollection 2018. PMID:29958300 doi:http://dx.doi.org/10.1371/journal.pone.0200060
  29. ↑ Vidarsson G, Dekkers G, Rispens T. IgG subclasses and allotypes: from structure to effector functions. Front Immunol. 2014 Oct 20;5:520. doi: 10.3389/fimmu.2014.00520. eCollection, 2014. PMID:25368619 doi:http://dx.doi.org/10.3389/fimmu.2014.00520

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