Invention for Fc variants with modified binding to FcRn

Invented by Aaron Keith CHAMBERLAIN, Bassil Dahiyat, John R. Desjarlais, Sher Bahadur Karki, Gregory Alan Lazar, Xencor Inc

The Xencor Inc invention works as follows

The present application is for a variant Fc-region that contains at least one modification compared to a wild type human Fc-region, with the modification being selected from a group consisting 434S/434S/428L, 434S/434S/428L and 428L/434S. Numbering follows the EU index.

Background for Fc variants with modified binding to FcRn

Antibodies” are immunological proteins which bind to a specific antigen. Most mammals, such as humans and mice are made up of paired polypeptide chains. The term “immunoglobulin” is used to describe these proteins because each chain is composed of a single immunoglobulin domain (Ig). Each chain consists of two distinct areas, known as the constant and variable regions. The variable light and heavy chains show a significant amount of sequence diversity, and they are responsible for binding to the antigen target. The constant regions have less diversity in sequence and are responsible for binding to several natural proteins that trigger important biochemical reactions. There are five classes of antibodies in humans: IgA, IgD (including subclasses IgA1-IgA2) IgE (including subclasses IgG1, IgG2, IgG3, IgG4, and IgM), and IgG. These antibody classes are distinguished by their constant regions. However, subtle differences can exist in the V-region. IgG antibodies consist of two heavy chains, and two light chains. The IgG Heavy Chain is made up of four immunoglobulins domains, linked from the N- to the C-terminus, in the order VH – CH1 – CH2-CH3, which refers to the heavy-chain constant domains 1, 2, and 3 (also known as VH – C?1 -C?2-C??3, referring respectively to the constant-gamma domains constant gamma domain 1, constant-gamma domain 2 and constant-gamma domain 3). The IgG heavy chain is made up of four immunoglobulins domains, linked in order from N-to-C-terminus. These domains are the heavy chain variable domain, the heavy chain constant domain 1, the heavier chain constant domain 2, and the heavier chain constant domain 3 (also referred to as VH-C?1-C?2-C?3, referring to the heavy chain variable domain),

In IgG a site between the C?2?and C?3?domains on Fc mediates interaction with neonatal receptor FcRn. Binding FcRn allows endocytosed antibodies to be recycled from the endosome to the bloodstream. (Raghavan and al. 1996, Annu Review Cell Dev Biol 12, 181-220; Ghetie and al. 2000, Annu. Rev Immunol 18, 739-766. Both are incorporated entirely by reference. The large size of the full molecule prevents kidney filtration, which results in a favorable antibody serum half life of one to three week. The binding of Fc with FcRn is also important for antibody transport. The binding site of FcRn on Fc is the same site where the bacterial proteins G and A bind. In most cases, the tight binding of these proteins can be exploited to purify antibodies using protein A or G affinity chromatography. The fidelity of the Fc region is therefore important for the clinical properties and purification of antibodies. The structures of the Fc/FcRn complex in rat (Burmeister, et. al. 1994, Nature 372:383-383; Martin, et. al. 2001, Mol Cell 7, 868-877, both incorporated entirely by reference) and the complexes between Fc and proteins A and G, (Deisenhofer 1981, Biochemistry 20, 2361-2370; Sauer Eriksson, et. al. 1995, Structure 3, 265-278; Tashiro The FcRn is also responsible for transferring IgG into the neonatal gut, and the lumens of the intestinal epithelia of adults (Ghetie & Ward, Annu. Rev. Rev.

The importance of certain Fc residues in the binding of FcRn has been demonstrated by studies of the rat and human Fc regions. In the Fc region, the sequences of rat and humans are similar to a degree of 64% (residues 235-443 according to the EU index numbering). See FIGURES. 3, 4, and 5 for the rat/human alignments of Fc, FcRn heavy chain, and FcRn light chain (beta-2-microglobulin). The structure of the rat Fc/FcRn was used to build a model of the Fc/FcRn human complex (Martin et al. 2001, Mol Cell 7, 867-877; incorporated entirely by reference). Both the rat and human sequences contain residues such as H310 or H435 that are crucial for FcRn-binding (Medesan and al., 1997 J. Immunol. 158(5):221-7; Shields et al., 2001, J. Biol. Chem. Chem. The human and rat protein sequences differ in amino acids at many positions. This gives the residues of the human sequence a different environment and, possibly, a different identity than the sequence from the rat. This variability limits the transfer of characteristics from one homologous protein to the other.

In the murine Fc random mutations and phage selection at the T252, T254, T256 sites lead to a T252L/T254S/T256F triple mutant that has a 2.5-fold increased FcRn affinities and 1.5-fold increases in serum half-life. (Ghetie and al., 1997. Biotech. 15(7): 637-664, incorporated entirely by reference). In vivo, mutations that disrupt the Fc/FcRn interactions at positions 253, 310, and 435 lead to a decreased half-life as well (Medesan et al J. Immunol. “1997 158(5) 2211-7 (included by reference).

Mutational Studies in Human Fc?” Some of the residues which are important for binding with FcRn have been studied and have shown an increased serum half life. In human Fc?1, Hinton et al. Mutated three residues to each of the 19 other common amino acids. Hinton et. al. found that a double mutant and some point mutations increased the FcRn-binding affinity (Hinton, et. al. 2004, J. Biol. Chem. 279(8): 6213-6216; Hinton et al. Journal of Immunology, 2006, 176, 343-356 (both are incorporated as references). Two mutations increased the half-lives of monkeys. Shields et al. Shields et. Chem. 276(9) 6591-6604, incorporated entirely by reference).

Dall’Acqua et al. “Dall’Acqua et. 2002, J. Immunol. 169:5171-5180 (included by reference). The DNA sequences that were selected were primarily triple and double mutants. Reference expressed many of the proteins encoded in their selected sequences, and found that some bound to FcRn tighter than wild-type Fc.

The administration as a therapeutic of antibodies and Fc-fusion proteins requires that the frequency of injections is determined by the half-life and clearance characteristics of the protein. In vivo, longer half-lives can allow for more frequent injections or lower dosages. This is obviously advantageous. The Fc domain mutations that have been made in the past have led to proteins with higher FcRn affinity and longer in vivo halves-lives. However, they have not found the best mutations for a longer in vivo HALF-LIFE.

Antibodies are being developed for therapeutic purposes.” Publications that are representative of such therapies include Chamow, et. al. 1996, Trends Biotechnol 14, 52-60, Ashkenazi, et. al. 1997, Curr Opin Immunol 9, 195-200; Cragg, et. al. 1999, Curr Opin Immunol 11, 541-547; Glennie, et. al. 2000, Immunol today 21:403-4410; McLaughlin, et For anticancer treatment, even a small reduction in mortality is considered a success. Certain IgG variations disclosed herein increase the ability of antibodies to limit future growth or destroy, at least partially cancer cells.

The anti-tumor effect of antibodies can be enhanced by their ability to mediate cytotoxic effects such as ADCC. Examples include Clynes et al., 1998, Proc Natl Acad Sci USA 95:652-656; Clynes et al., 2000, Nat Med 6:443-446 and Cartron et al., 2002, Blood 99:754-758, both entirely incorporated by reference.

The majority of engineering studies were conducted in this context. “Human IgG1 was the most common antibody used for therapeutic purposes. IgG1, IgG2, IgG3, IgG4 and other isotypes in the IgG family have different physical, biological and clinical properties. In the art, it is necessary to develop improved IgG1, IgG2, IgG3, IgG4 variations. It is also necessary to design these variants in order to increase the in vivo halves life of native IgG polypeptides and/or improve their binding to FcRn. There is also a need to combine improved pharmacokinetic variants with variants containing modifications to enhance efficacy via altered FcgammaR-binding. This application addresses these and other requirements.

The present application is directed at Fc variants of parent polypeptides that include at least one modification to the Fc region. In different embodiments, variant polypeptides have altered binding to FcRn compared to parent polypeptides. In some variations, the modification may be chosen from: 428L 434M or 434S. The numbering follows the EU Index of Kabat et al.

In another embodiment, Fc includes at least two modifications chosen from the group consisting: 252Y/428L; 428L/434H; 428L/434Y; 428L/434A; 428L/434M and 428L/434S.

In another embodiment, Fc includes at least one variant selected from the following group: M428L/N434S; V308F/M428L/N434S.

The Fc variant can also include at least one modification from the following: 308F/434S; 259I/308F/434S; 307Q/308F/434S; 250I/308F/434S and 308F1319L/434S.

In another embodiment, Fc includes at least one variant selected from the following group:

In another embodiment, the invention also includes a treatment method for a patient who needs said treatment. This involves administering a sufficient amount of a Fc variant described in this document.

In another embodiment of the invention, the method includes increasing the half-life an antibody or an immunoadhesin using a Fc in accordance with the modifications described herein.

The invention also includes Fc variants with enhanced FcRn-binding and additional Fc variations that modulate the effector function.

The present invention discloses novel variants of Fcdomains that have a higher binding affinity to the FcRn. “As noted in this publication, binding to FcRn leads to longer serum retention in vitro.

To increase retention of Fc proteins in vivo the binding affinity needs to be increased at pH 6, while the lower affinity is maintained at pH 7.4. FcRn binding at pH 6 sequesters Fc in endosomes (Ghetie & Ward, 1997, Immunol Today). 18(12), 592-598 (included by reference). The endosomal cavity recycles Fc back to the cell surface. The higher pH (?7.4), once the compartment is opened to the extracellular area, induces the release back of Fc into the blood. In mice, Dall’ Acqua et al. In mice, Dall’ Acqua et al. 2002, J. Immunol. 169:5171-5180 (included by reference in its entirety). It is believed that the increased affinity of FcRn for pH 7.4 prevents the Fc from being released back into the blood. The Fc mutations which increase Fc half-life will, therefore, increase FcRn bind at lower pH levels while still allowing Fc release at higher pH. Histidine’s charge changes in the pH range from 6.0 to 7.4. It is therefore not surprising that His residues are found at key positions in the complex Fc/FcRn (FIG. 6.)

Click here to view the patent on Google Patents.