Increasing Serum Half-life   See engineering antibodies for pH dependent affinity

Introduction

The Fc region of an antibody interacts with a number of Fc receptors and ligands, imparting an array of important functional capabilities referred to as effector functions. (Lazar (US8,188,231)

FcyRs are expressed on immune system effector cells (e.g., natural killer (NK) cells, neutrophils, monocytes/macrophages, B cells) and include three classes: FcyR1 (CD64), FcyRII (CD32) and FcyRIII (CD16). In humans, the latter two classes are further divided into FcyRIIa and FcyR11b, FcyRIIIs and FcyRIIIb. The receptors differ in their affinity for IgG, e.g., FcyR1 exhibits a high affinity for monomeric IgG1 whereas FcyRII and FcyRIII exhibit a relatively weak affinity for monomeric IgG1 and hence can only interact effectively with multimeric immune complexes. The complexity of teh human FcyR system is amplified by the presence of polymorphic forms of teh receptors. FcyRIIa has tow forms, Arg-131 and His-131, that differ in binding of IgG2. FcyRIIIa has polymorphisms at positions 48 (Leu/His/Arg) and 158 (Phe/Val) although only the latter has been reported to effect binding and biological differences. FcyR-IIIb polymorphic forms NA1 and NA2 differ in rou amino acids. (Presta, “Engineering therapeutic antibodies for improved function” Mediation and Modulation of Antibody Function, 30(4), 2002). 

Improved binding to FcyR

Enhanced affinity of Fc for FcyR has been achieved using engineered glycoform generated by epxression of antibodies in engineered or variant cells lines (Lazar (US8,188,231)

Presta (US 6,737,056) discloses an antibody variant with improved binding to the FcyR having an aa modification at 255, 256, 258, 267, 268, 272, 276, 280, 283, 285, 286, 290…)

Regarding IgG1 variants with improved binding to FcyR, though these variants have not yet been tested in vivo, three possibilities exist. First, if a therapeutic mAb utilizes FcyR bearing cells as part of its mechanism to action (either as cross-linking agent or to elicit ADCC or phagocytosis), then mAbs with improved binding to FcyR could exhibit increased efficacy. Second, IgG variants that have enhanced binding to activating FcyRs (FyR1, RcyRIIa and/or FcyRIIIa) and reduced binding to the inhibitory FcyRIIb might increae the efficacy or a therapeutic mAb even furtehr, suggested by the improved efficacy of Herceptin in FcyRIIb deficient mice. Third, the human proteome contains a Val 138/Phe-158 polymorphism in FcyRIIIa, and human IgG1 binds better to teh FcyRIIa(Val-158) form than it does to the FcyRIIIa(Phe-158) form. (Presta, “Engineering therapeutic antibodies for improved function” Mediation and Modulation of Atnibody Function30(4), 2002)

Improved binding to FcyRIII

Presta (US 6,737,056) discloses polypeptide variants with improved binding to FcyRII having an aa modification at position(s) 298 and/or 333 of the Fc region

–Increased affinity to FcyRIIIa Receptor

Bernett (WO2008/022152) discloses an antibody that binds CD19, including at least one mdofication in the constant region relative to a parent anti-Ced19 antibody, wherein the antibody binds with increased affinity to the FcyRIIIa receptor.

Decreased binding to FcyR

For Reduced binding to FcyRI (CD64)

Presta (US 6,737,056) discloses antibody variants having modifications at any of AA positions 238, 265, 269, 270, 327 or 329 of the Fc region. that display reduced binding to an FcyRI.

For reducing binding to FcyRIII (CD16)

Dall’acua (WO 2006/116260) discloses Fc variants having reduced binding to FcyRIIIA and decreased ability to mediate ADCC.

Presta (US 6,737,056) discloses polypeptide variants with reduced binding to FcyRIII having an AA modification at an AA such as 138, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 461, 434, 435 or 437 of teh Fc region.

Altered Binding to Neonatal Fc Receptor (FcRn)

The IgG-FcRn interaction is pH-dependent, with IgG binding at pH 6 but not at pH 7.2 (the pH of blood). (Presta, “Engineering therapeutic antibodies for improved function” Mediation and Modulation of Atnibody Function30(4), 2002)

The in vivo half-life of an antibody can impact its effector functions. In some embodiemtns, it is desirable to increase or decrease the half-life of an antibody to modify its therapeutic activities. FcRn is a receptor ath is structurally similar to MHC Class I antigen that non-covalently associates with .beta.2-microglulin. FcRn re Law (US 2006/0083736). 

The FcRn interaction site encompasses three spatially close loops comprised of sequences that are distal in the primary amino acid sequence. The central role of Fc hisitidines in hiblding thsi site accounts for teh marked pH dependence (binding at pH 6.0, rlease at pH 7.4) of teh Fc-FcRn interaction (Ward, US 2002/0098193). 

FcRN is known to be a salvage receptor that can protect IgG from lysosomal degradation and maintain a long half-life in vivo, because it binds to IgG within the endosome at acidic pH and is recycled back tot eh cell surface, where it subsequently releases the IgG at neutral pH. Previous studies have demonstrted that Fc engingeering to increase the binding affinity to FcRn at acidic pH improved the endosomal recyling efficiency and prolonged the half-life of the IgG. (Igawa “Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens form circulation” Immunological Reviews 2016, 770, 132-151).

Engineering to Reduce binding to FcRN:

Presta (US 6,737,056) discloses polypeptide variants with reduced binding to FcRN having aa modification at aa position(s) 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447 of the Fc region.

Engineering to Increase binding to FcRn: 

–Engineering of Fc region to increase binding to FcRn (Fc variants)

The region of human IgG1 involved in FcRn binding has been mapped. Alanine substiutions at positions Pro238, Trh256, Thr307, Gln311, Asp312, Glu380, Glu382 or Asn434 of human IgG1 enyhance Fc Rn binding. IgG1 molecuels harboring these substiutions are expected to have longer rum half-lives. Consequently, there modified IgG1 molecules may be able to carry out their effector funcitons and hence exert their therapeutic efficacies, over a longer

Substituting specific IgG1 residues with alanine enhanced binding to FcRn.  Notably, these alterations improved binding only at pH 6.0, but not pH 7.2 and thus would not hinder dissociation of the IgG from FcRn. Hence, the half life of a therapeutic mAbs may be able to be extended by a varying degree dependent on which IgG1 amino acids are altered or in which combination. In some instances, it might be advantageous to decrease or prolong the half life. (Presta, “Engineering therapeutic antibodies for improved function” Mediation and Modulation of Atnibody Function30(4), 2002). 

Chamberlain (US2009/0041770) discloses antibodies with modified Fc regions which results in increase in FcRn affinity and serum half-life. Chamberlain (US 2007/0135620) also discloses IgG variants that is engineered for increased binding affinity to FcRn at lowered pH, such as the pH associated with endsome, e.g., pH 6.0, while maintaining the reduced affinity at higher pH, such as 7.4, to allow increased uptake into endosomes but normal release rates. 

Dall’Acqua (“Properties of human IgG1s engineered for enhanced binding to the neonatal Fc Receptor (FcRn)” J. Biological Chemistry, 281(33), pp. 23514-23524 (2006) disclsoses a triple mutation M252Y/S254T/T256E introduced into the Fc porition fo a humanized anti-respiratory sincytial virus monoclonal antibody which did not affect the ability of the anitbody to bind its antigen and inhibit RSV replication, resulted in a 10 fold increase in its binding to human FcRn at pH 6.0 and was efficiently released form FcRn at pH 7.4 and further consistently exhibited a nearly 4 fold increase in serum half-life in cynomolgus monkeys when compared to the wild type antibody.  Dal’Aqua (US 2007/0122403) also discloses that phage-dervied IgG1 mutants exhibited a significant increases in affinity towards murine FcRn at pH 6.0 but minimal binding to the human receptor at pH 7.4 

Hinton “Engineered human IgG antibodies with longer serum half-lives in primates” J. of Biological Chemistry” 279(8), 2004) discloses that the neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies and further discloses several IgG2 mutants with increased binding affinity to human FcRn at pH 6 which do not bind to human FcRn at pH 7.5. Hinton further discloses that a pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had increased serum half-lives in rehesus monkeys –2 fold longer than the wild-type antibody.  Hinton (US 2008/0287757) also disclsoes that IgG binding to FcRn is known to be pH-dependent such that it binds strongly to FcRn at pH 6.0 but weakly at pH 8.0. Hinton discloses an engineered mutant Fc-fusion protein with longer serum half-liveswith increased binding to FcRn at pH 6.0 while retaining pH depndent rlease form FcRn at pH 8.0. A modified Il-13/Fc-fusion protein with the T250Q/M428L mutation exhibited pH dependent binding to human FcRN. The binding was srgonest at pH 6.0 and progressively diminised as the pH values incrsased to pH 6.5, 7.0. 7.5 and 8.0. A modified LFA-3/Fc fusion protein with a T250Q/M428L mutation also exhibited pH depndent binding to human FcRn. Like the modified IL-13/Fc fusion prtoein, binding was strongest at pH 6.0 and progessively diminised as the pH values increased to pH 6.5, 7.0, 75. and 8.0. 

Huang (US 16/510,416, published as US 2019/0330313) discloses a method for prolonging serum half-life of IgG antibodies by by introducing mutations to the Fc fragment, specifially at positions 254, 308 adn 434. In a particular emobdiment, the amino acids at positions 254, 308 and 434 in the FcRn binding site of the heavy chain constant region are mutation into threonine , proline and alanine respectively. 

Oetjiva (“enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease” International immunology, 18(12), pp. 1759-1769) disclsoes mutants of the humanized monoclonal Herceptin antibody, Hu4D5-IgG1), directed against human epidermal growth factor receptor 2, engineered to have enhanced binding to hFcRn. Two engineered IgG1 mutations (N434A and T307A/E80A/N434A) showed a considerably exnted t/12 in vivo compared with wild-type antibody in mice expressing an hFcRn transgene but not in mice expressing the endogenous mouse FcRn. Administration of mutant with high binding to hFcRn ameliorated arthritis induced by passive transfer with human pathogenic plasma. 69) 

Petkova (International Immunology, 18(12), pp. 1759-17, 2006) discloses  that FcRn plays an essential role in extending the half-life (51/2) of IgG antibodies and IgG-Fc-based therapeutics in the circulation. Petkova disclsoes two engineered IgG1 mutaions (N434A adn T307A/E380A/N434A) to a humanized Herceptin antibody with enhanced in vitro binding to FcRn which showed a considerably extended t1/2 in vivo compared with wild-type antibody in mice expressing an hFcRn transgene

Increasing the serum half life of a therapeutic antibody is another way to improve its efficacy, allowing higher circulating levels, less frequent administration and reduced doses. This can be achieved by enhancing the binding of the Fc region to neonatal FcR (FcRn) which is expressed on the surface of endothelial cells, binds the IgG in a pH dependent manner and protects it from degradation. Several mutations located at the interface between the CH2 and CH3 domains have been shown to increase the half-life of IgG1. (Eon-Duval, WO 2008/025747A1).

–Engineered with substitution(s) Variable Region and Constant region to improved FcRn binding:

A large body of data shows that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. However, the PK of IgG molecules vary widely, even when they share identical Fc domains. For example, a panel of IgG molecuels differing only by 1-5 mutations in CDRs altered binding affinity to FcRn in vitro by up to 79 fold. In addition, higher affinity values trended with faster in vivo clearnace of a set of IgG molecules differing only by 1-3 mutations in human FcRn trasngenic mice. (Piche-Nicholas “Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics” MABS, 10(1) pp. 81-94, 2018). 

Adams (US 2006/0246004) discloses humanized antibodies which bind to CD20 which include mutations in the variable region (D56A in VH-CD4 and S100aR in VH-CDR3. In one embodiment, the antibody further included at least one substiution in the Fc region to improve ADCC or CDC activity. Soluble Fc variants with amino acid alterations in the Fc (e.g., N434W, N434Y..) were assayed in a BIAcore binding assay for their affinity for human, FcRn. The variants showed increase in affinity over wild type Fc of 170 fold, 9 fold at pH 6.0. In constrast, at pH 7.4, affinity for the variants was low. 

Palvivizumab, which is an FDA approved anti-RSV IgG1 antibody was engineered in the variable region to improve the binding affinity to surface RSV to improve the neutralizing potency, and was further engineerd in the constant region to improve FcRn binding affinity at acidic pH to improve the half-life. (Igawa “Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens form circulation” Immunological Reviews 2016, 770, 132-151).

Altered binding to C1Q

Increased binding to C1q

Dall’acua (WO 2006/116260) discloses Fc variants with enhanced binding to C1Q and increased complement dependent cytotoxicity (CDC).