See also Fc Receptors   See also FcRn recpetor  See also Fc modifications

Increasing the half lives of therapeutics can decrease the frequency of patient inections and allow the administration of low concentrations of the molecule, which can imnimize the toxic side effects. Kamei, Biotechnol Bioeng., 2005, 92(6): 748-60).

One way discussed below to increase antibody half life is to engineer antibody to dissociate from the antigen pH-dependently. This can be done for example by muttion of tyrosine in CDR into histidine.  (“Proprietary Innovative Antibody Engineering Technologies in Chugai Pharmaceutical” 12/18/2012)

Conditional pH binding to FcRn:

The generally accepted model of FcRn mediated IgG homeostasis states that IgG, upon internalization via pinocytosis, binds to FcRn in the acidic endosome and is salvaged via trafficking to the cell surface (pH 7.4) where it is returend to circulation, thereby maintaining high serum levels of IgG It is proposed that specific histidi residues on the IgG Fc and serum albumin (SA) facilitate pH dependent binding by toggling between positively charged and neutral states in different pH environments. In the acidic endosome, postively charged histidine side chains can interact favorably with negatively charge side chains of FcRn; however, this affinity is greatly diminished at the neutral cell surface where IgG and SA are rleased into ciruclation. Borrok (j Biol Chem 2015, 290 (7), 4282-90). 

Increased binding affinity to FcRn at acidic pH and minimal binding at neutral pH:

The guiding engineering principale for generating antibodies with longer half-lives has been to increase binding affintiy to FcRn at acidic pH while maintaining minimal binding at nuetral pH. Borrok (j Biol Chem 2015, 290 (7), 4282-90)

Chamberlain (US2009/0041770) discloses antibodies with modified Fc regions which results in increase in FcRn affinity and serum half-life

Dall’Acua (US 7,670,600) discloses modified IgG constant domains which increases the affinity of the constant domain for FcRn resulting in increased half-lives of the IgG which is advantageous in that less frequent dosing is required for therapeutic purposes. 

Eon-Duval, (WO 2008/025747A1) discloses that 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. 

Kamei, (Biotechnol Bioeng., 2005, 92(6): 748-60) discloses that since the interaction of Fc with the neonatal Fc receptor, FcRn, is reponsible for the long half life of IgG, one approach invovles optimziing this interaction through Fc mutagenesis. Compared to other plasma proteins, IgG has a relatively long half life that reuslts from its propensity for being recycled in the vascular endothelium. Proteins in the bloodstream, including IgG, enter the endothelial cells via endocytosis as the cells sample their environment. When IgG enteres the endosomes of these endotheilial cells, through the neonatal Fc receptor, FcRn, binds to the Fc region of IgG. The binding enhances the recylcing of IgG back to the cell surfac3e and decreases the amount of IgG that is degraded in the lysosomes. Dissociation of the Fc fragment form FcRn at the cell surface then releases IgG back into the bloodstream. Fc has a greater binding affinity for FcRn in the endosome (pH 5-6) than at the cell surface (pH 7.4) becasue a favorable interaction between Fc and FcRn is induced by the protonation of certain histidines. Since binding in the endosome and subsequent dissociation at the cell surface is desirable, a design criterion can be established for further increasing half life through enginiering the Fc fragment. 

Hinton (2005/0032114) teaches a modified antibody of class IgG in which at least one amino acid from the heavy chain constant region is substituted with another amino acid which is different from that present in the unmodified antibody, thereby altering the binding affinity for FcRn and/or the serum half-life in comparison to the unmodified antibody (abstract). Hinton further teaches engineering of mutant antibodies with longer serum half-life by increasing binding  [0333] to FcRn at pH 6.0, while retaining pH-dependent release from FcRn at pH 8.0. Hinton further teaches that the modified antibodies all showed strong binding to human FcRn at pH 6.0, with diminishing binding as the pH values increased to pH 8.0 (¶333). See also therapeutic applications of antibodies and the example there for human hepcidin.  Hinton (WO2004/035752) also teaches modified antibodies having altered FcRn binding affinity and/or serum half-life by virture of the presence of a modified IgG constant domain or FcRn binding protion thereof (preferably the Fc or hinge Fc domain). In one embodiment, the modified IgG in vivo half lives are extended by chainges in aa residues at positions identified to be involved in the interaction of the hinge Fc domain with the FcRn receptro. In preferred embodiments, the contant domain has a higher affinity for FcRn at pH 6 than at pH 7.4.

Correct pH dependent binding of the antibody Fc to FcRn alone may not be enough to achieve longer half-life in all IgGs. for example, Hinton showed that a T250Q, M428L mutant yielded a two fold increase in the half life of an anti-hepatits B virus IgG1. The T250Q,M428L mutation, however, was sown by Eli Lilly not to provide elongated half-life to an anti- TNF-alpha antibody, suggesting that the T250Q,M428L mutatnt may not possesses robust activity with different variable chains. (William Strohl, Current opinion in Biotechnology, 2009, 20 585-691). 

Ward (WO97/34631) discloses antibodies with an increased serum half life through the interaction with FcRn. The antibodies bind to FcRn in  a pH dependent way such that binding affinity is strong at about pH 6-6.5 relative to binding at pH 7.4. This allows the antibody to be salvaged by FcRn at lower pH and released into the essentially neutral pH environment of the serum.

Increased binding at both pH 6.0 and pH 7.4

Borrok (j Biol Chem 2015, 290 (7), 4282-90) discloses engineering the FcRn binding of an Fc variant taht shows ultra high binding affintiy at both pH 6.0 and pH 7.4 and exhibits rapid serum clearance by targeting His-435 and adjacent residues in the CH3 domain. 

Conditional pH binding to Antigen:

Enhancement of binding to antigens at lower pH than higher pH

Datta 9US2005/0260711) discloses modified antibodies which have enhanced binding to tumor antigens in acidic environments.

Eshhar (WO 95/14710) discloses that mutation of the binding site of antibodies with teranitromethan (TNM) exhibit pH dependent binding in that all of them have a loss of binding activity at pH>9 which is recovered at pH<6.

Enhancement of binding to antigens at higher (neutral) pH than at lower (acidic) pH

Edwards (US 2006/0141456) discloses an interesting way to increase stability by selecting peptide sequences that selectively bind to HSA with high affinity in most tissues but interacts only weakly with HSA in a target tissue. As a consequence, such a peptide will be transported with high efficiency and long slow clearance through the blood stream and will be selectively released in the target tissue. Of particular interest were agents that bind to HSA with high affinity at normal pH (about 7.4) but with weaker affinity at lower pH.

Foltz (US12/990137, published as US 2011/0150888) discloses anti-hepcidin antibodies that exhibit differential pH binding such as antibodies that bind to antigen with about 50-1000 fold reduced binding affinity at a pH of about 5.5 or 6 compared to a pH of aobut 7.4. The antibodies exhibit enhanced target antigen clarance. 

Igawa (US13/889512, published as US 2013/0336963) also discloses a method of remvoing an antigen from plasma and increasing the serum half life of an antibody by screening for those antibodies which have imparied antigen binding ability at acidic pH as compared to that at neutral pH. The method invoves screening for binding of the antibody to an antigen such that the antibody has a KD (pH5.8/KD(pH7.4) value of 2-10,000 where the antibody binds to the antigen in plasma in vivo and dissociates from the bound antigen under conditions present in an endosome in vivo. Igawa also discloses a method for producing an antibody (US 13/889,484, published as US 2013/0303396 and US 9868948) and a method of screening for an antibdoy (US 13/595,139, published as US 2013/0011866) by determining binding actvity of the antibody through the ABD at a higher pH 6.7-10 and then determining binding at a lower pH 4.0 to pH 6.5 such that the antibody binds to an antigen in plasma in vivo and dissociates from the bound antigen under conditions present in an endosome in vivo.  Igawa (US 15/952951, published as US 2018/0282719 also discloses a method for producing an antibody that binds at a frist pH in the range of 6.7-10, determining the binding activity of the antibody at a second pH in the range of pH 4-6.5 and slecting the antibody whose anitgen binign activity at the first pH is greater than that at the second pH. Igawa (Nautre Biotechnology 28, 12-3-1207 (2010) discloses making Histidine substitutions to tocilizumab (Actemra) which is an antibody against the IL-6R to rapidly dissociate the antibody from IL-6R within the acidic enviornment of the endosome (pH 6) while maintaining its binding affinity to IL-6R in plasma (pH 7.4).

By applying the technology of Igawa, Fukuzawa (Sci, Rep. 2017, &9)1): 1080) generated an anti-C5 recycling antibody that has significantly longer acting neutralization of plasma C5 than a convention antibody. 

Ito (“the His-probe method: effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interacts at different pH values” FEBS 309 (1), pp. 85-88) discloses mutant antibodies which showed different patters of pH-dependent binding to lysozyme, which depended on the lcoation of the introduced histidine residues. From the data, Ito concluded that binding contstants at higher pH are higher than those at lower pH. They also concluded that binding constants at low ionic strengh are higher than those at higher ionic strench at all pH vlaues tested. The differences between the binding constant at pH 5.2 and those at pH 7.8 at low ionic strengh are larger than those at high ionic strenght. 

Javier (J Biological Chemistry 287(14) pp. 11090-11097, 2012) disclose an anti-PCSK9 (properotein convertase substilisin kexin type 9) antibody with introduced histidines into CDR residues such that it binds tightly to the antigen in the plasma (pH 7.4) and dissocaites in the acidic endosome. This allows the antibody to undergo futher binding cycles and may mitigate target mediated degradation by dissociation of the antigen-antibody complex in the acidic endosome. This prolongs half-life and increased duration of cholesterol lowering through inhibition of endogenous PCSK9 in two species in vivo. PCSK9 has been implicated a a major regulator of plasma LDL-C and has emerged as a promising target for prevention and treatment of coronary heart disease. 

Krailling (US 16/373,293, published as US 2019/0248922) discloses generating antibodies using the HuCAL PLATINUM library which includes the CysDisplay selection technology. The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. The aim was to generate Fab antibodies against human IgM, IgA and IgE that bind at neutral pH but can be eluted form the antigen under mild conditions (eg., pH 4-5). Selection of the antigens using elution under mild conditions resulted in these types of antibodies. 

Lasters (US 2010/0216187 and WO2008/043822) teaches amin acid sequences such as those coding for antibodies that bind to serum proteins such as serum alumin which are subject to recylcing and are subject to a first biological condition such as pH in the extracellular and a second biological ph condition inside the cell such as the endosome and the amino aicd sequences are such that they bind with a different dissociation constant or association constant under the first and second biological conditions. (referred to as “conditional binders” by Lasters. In one embodiment, Lasters discloses that the conditional binder binds to a chosen antigen at a physiological pH (&.2-7.4) at 10-1000x more efficiently than at pH in the endosomal compartment (pH 6-6.5).  

Pons (US 9,029,515) teaches antibodies with pH dependent binding to its antigen such that the affinity for antigen binding at physiological pH (i.e., pH 7.4) is greater than at endosomal pH (i.e., pH 6.0 or 5.5). In other words, the Kd or Koff ratio at pH 5.5/pH 7.4 or at pH 67.0/pH 7.4 is more than between 2, 3, 4, 8, 10, 16, 20, 30 40 or 100 or more.

Yancopoulos (WO 2014/028354) discloses antibodies that bind PCSK9 with greater affinity at neutral pH than at acidic pH by intorducing one or more hisitdine substiutions in one or more complementarity determining regions. Advantages include remaining in ciruclation for prolonged periods of time. 

–Using histidine mutagenesis:

Bartholomew (WO 83/03678) recognized that among a population of hybridomas which secreted antibodies against a specific antigen and the subpopulation of those hwich secrete antibodies having a high affinity for teh antigen, a very much smaller population secreted antibodies which ahve a high affinity for teh anitgen in a first environment, but a much lower affinity in a second environment. Batholomew screened for antibodies that have a higher affinity in a first pH enviornment and a low affinity in a second pH environment. An antibody is considered to exhibit a high affinity when it affinity constant (Ka) is ≥ 10 to the 9 and a low affinity when its Ka is about ≤10 to the 8. Usually the first pH will be near 7.  The screening is done by fusing spleen cells to myeloma cells, cloning the resulting hybridomas and screen those secreting antibody specific for an antigen such as HGH. The hybrodomas produced are further screened to by immobilizeng the antibdoy on a solid support and, after permitting it to bind antigen, measuring the extent of desorption of the antigen that occurs at differnt pH levels. For example, if one wishes to obtain antibodies which exhibit a high affinity at pH 7 adn a low affinity at pH 4, the immobilized antigen is contacted with an excess of antisera at pH 7 and the immobilized antigen washed with a medium at pH 7 to remove antibodies which exhibit a low affinity at pH 7. The immoadsorbent is then eluted at pH 4 to remove antibodies which exhibit a low affinity at pH 4. Barthlomew attributed the change in Ka with changes in pH as being cause by protonation of histidine residues or deprotonation of lysine or possibly tyrosome or arginine residues in either teh antibody, the antigen or both which alters the ability of teh antigen and antibody to complex with each other. 

Histidine has a pKa of about 6 (depending on the surrounding environment) and is utlized in naturally occurring pH dependent protein-protein interactions, such as the interaction between Fc and FcRn. Because of the pKa of the imidazole group, histidine residues are protonated at endosomal acidic pH, a change that destailizes the antibody-antigen interaction in two ways. When the introduced hisitidine resiudes are direclty involved in interacting with the antigen, hisitidine protonation resutls in destabilizing the antibody-antigen interaction directly, and wehn the histidine residues are involved in maintaining teh conformation of teh CDR, hisitidine protonation results in conformational change of the CDR, thereby destablizing the antibdoy-antigen interaciton indirectly. The first histidine engineered pH dependent antibody reproted was derived from tocilizumab, a humanized anti-IL-6R antibody. Tocilizumab was engineered into a pH dependent anti-IL-6 recetpor antibody by introducing foru histidine residues, two in the H chain (positions 27 and 31 in Kabat numbering) and two in the L chains (Positions 32 and 53). A crystal structure of tocilizumab Fab fragment in complex with IL-6R suggested that hisitdine residues at 31 in the H chain and position 32 in the L chain were directly involved in the antibody-antigen interaciton, while histidine residue at position 27 supports the conformation of teh H CDR1. When hisitdine residues are protonated in acidic endosome, the antibdoy-antigen itneraction is destabilized both by the electrostatic repulsion between teh arginine resiudein the IL-6 recetpor and the protonated hisitidine resiude and by the conformational change in the heavy chain CDR1. Display technologies such as phage display can be sued to isoalte an antibody with teh desired property from a large library of antibodies. A naive or synthetic human phage library can be subjected to several rounds of selection based on binding at neutral pH and elution at acidic pH to isolate a clone with a pH dependent antigen binding. (Igawa “Sweeping antibody as a novel therapeutic antibody modality capable of eliminating soluble antigens form circulation” Immunological Reviews 2016, 770, 132-151).

Antibodies with Altered isoelectric point (pI)

(Dahiyat WO 2012/016227) teaches modifying the isolectric point of an antibody by introducing at least 6 amino acid mutations in a constant domain wherein the substituted amino acids have a pI lower than the native amino acid.