Patent Application
20170226199
The invention relates to anti-TNFa antibodies which are engineered to exhibit a pH-sensitive antigen binding. The invention is preferably directed to anti-TNFa antibody adalimumab (Humira®) or biologically active variants and fragments thereof, wherein the original adalimumab antibody or variant or fragment thereof is engineered by modifications of amino acid sequence within the variable regions. Specifically, the invention relates to adalimumab or biologically active variants or fragments thereof, wherein the CDR domains are modified by replacing one or more amino acid residues by histidine residues.
The resulting modified anti-TNFa antibodies elicit improved pharmacokinetic properties with improved antigen-mediated IgG clearance and extended over-all serum half life.
It is believed that therapeutic antibodies (mAbs) at least offer important treatment options for many diseases like inflammatory, autoimmune or oncological disorders. In 2012 there were 40 FDA-approved mAbs on the US market against various targets in oncology and anti-inflammatory disorders with ˜38.5% share within the biologics market. Sales of ˜$24.6 billion manifest the role of therapeutic antibodies as highest earning category of all biologics (Aggarwal, 2009, Aggarwal, 2014).
For therapeutic antibodies different biological outcomes are determined by the interaction profiles with four classes of naturally occurring interaction partners: antigen, neonatal Fc-receptor (FcRn), Fc-receptors (FcγRs), and factors of the complement system (Chan and Carter, 2010). Several strategies have been reported to optimize antibodies that aim for additional or improved functions and specificities. (Beck et al, 2010). Within antibodies there are two structural features that can be addressed for engineering. First, the variable fragment (Fv) that mediates interaction with the antigen, second the constant fragment (Fc) that is involved in antibody recycling or mediates interactions with immune cells.
For different antigens (e.g. cytokines and growth factors) there are multiple mechanisms of action, e.g. blocking of soluble ligands thereby preventing the interaction to its corresponding receptor or blocking of the receptor itself. (Chan and Carter, 2010). A lot of effort has been invested on improving functions towards Fv-engineering that is often valued by increasing specificity and/or binding affinity to respective antigens (Beck et al, 2010). One strategy to elevate antibody efficacy is to enhance the Fv—antigen interaction by affinity maturation approaches. Herein the use of display technologies for screening molecule libraries allows isolation of variants that exhibit superior affinity.
The constant fragment (Fc) and its linked properties can be modulated with altered outcomes for immunity or antibody recycling. Prominent examples for altered Fc-mediated immune functions are enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) that have been addressed to enhance antibody efficacy and to reduce the dosages.
Further strategies have been explored including direct and indirect arming of antibodies or modulation of specificities within multivalent antibodies (Carter 2011).
One important aspect of Fc-function corresponds to its critical role in antibody recycling that determines the long serum half life of human immunoglobulin 1 (IgG1). After cellular absorbtion via fluid phase pinocytosis, the Fc-portion of an IgG1 interacts in a pH-dependent manner with the neonatal Fc-receptor (FcRn) that leads to antibody capture in the acidified endosome (Kuo and Aveson, 2011). From there, antibodies are recycled back to the circulation and therefore can be protected from intracellular catabolism. Different mutational Fc-species with enhanced FcRn binding affinity were generated and tested for increased recycling rates with up to 4-fold extended serum half-life in cynomolgus studies by substituting three amino acids (Dall'Acqua et al, 2006).
Although most antibodies demonstrate highly efficient antigen blocking, there are drawbacks that are not fully addressed in the development process of therapeutic antibodies:
Considering these drawbacks of therapeutic antibodies, there is a need for more efficient molecules that produce therapeutic responses without high dosing and/or frequent administration (Chapparo-Riggers et al, 2012).
One possibility to achieve these goals is the specific engineering of the variable and optionally the constant region of a new or well established and approved antibody. One approach is to develop antibodies that exhibit pH-sensitive antigen binding. It has been shown that rational or combinatorial incorporation of histidines in the binding interfaces of antibodies and other proteins (Sarkar et al., 2002, Chaparro-Riggers et al., 2012, Ito et al., 1992, Igawa et al., 2010, Igawa et al., 2013, Murtaugh et al., 2011, Gera et al., 2012) can be commonly used to engineer pH-dependent binding. The basis for the pH-sensitive binding arises from the histidine's sensitivity to get protonated as a result of lowered pH-values in the microenvironment. More in detail, the histidines need to undergo a pKa-change upon binding in order to get protonated in a physiological pH-range (Murtaugh et al., 2011). Protonation of histidine side chains in binding-interfaces can alter electrostatic interactions or may induce conformational changes that lead to pH-dependent differences in binding affinity (Gera et al., 2012). Balanced electrostatic and non-electrostatic components of the binding equilibrium determine the sensitivity of binding (Murtaugh et al., 2011).
Incorporation of pH-sensitivity into the antigen binding site can increase the number of antigen-binding cycles. Herein, pH-dependent antibodies bind with similar high or reduced sufficient affinity to their antigens at plasma pH (pH 7.4) and show decreased binding at acidic pH (pH 6) (Chaparro-Riggers et al., 2012, Igawa et al, 2010) resulting in a faster and increased dissociation of the antibody from its antigen binding site within the acidic endosome, thereby enabling recycling back to the plasma and reducing antigen-mediated clearance.
During the FcRn-mediated recycling (
The pH-sensitive binding therefore enables the antibody to interact with another antigen and allows the neutralization of multiple antigen molecules per antibody molecule. PH-sensitivity can also increase the half-life of antibodies that address membrane associated targets and internalize and degrade upon e.g. receptor binding. Herein pH-sensitivity can lead to increased half-life, when the antibody gets released during the endosomal acidification.
Several different strategies were published that aim for the engineering of pH-switches in proteins. Histidine (His) scanning by which every single amino acid residue (e.g. within the CDR regions) is mutated to His allows the characterization of single substitution variants and identification of effective mutations. Creation of new variants by combining these substitutions can result in enhanced pH-dependent binding (Murtaugh et al., 2011, Chaparro-Riggers et al., 2012, Igawa et al, 2010). Identification of residues that may contribute to pH-sensitivity upon replacement with histidines by structure-based modeling can help to minimize effort & time that is needed during the histidine scanning approach. Crystal structures are required in order to have a precise idea of residues that are critical for binding and the rational design of pH-switches (Sarkar et al., 2002). Combinatorial histidine scanning library approaches require in vitro screening technologies (e.g. phage display or yeast display) to isolate pH-sensitive variants from a large molecule library. Murtaugh and colleagues designed a llama VHH antibody library by using oligonucleotide-directed mutagenesis thereby allowing every residue within the binding interface to sample both histidine residues and wild-type residues of the parental VHH antibody. Towards screening of a M13-phage display library (diversity ˜1012) isolated variants showed KDs between 35-91 nM at pH 7.4 and a ˜104fold decrease in binding affinity at pH 5.4 (Murtaugh et al. 2011).
Since the recycled free antibody is capable of binding to another antigen, pH-dependent antigen binding would enable a single antibody molecule to repeatedly bind to multiple antigens, in contrast to the conventional approach in which a single antibody can bind to antigen only once.
Therefore, it is a general need to make available antibodies which are more effective with respect to their plasma and serum concentration, which can be achieved by installing pH sensitivity with respect to different cellular action sites of the therapeutic antibody.
The invention provides mutated forms of anti-TNFa antibody adalimumab (Humira®), wherein the mutation consists of one or more substitutions of amino acid residues of the original, non-mutated antibody adalimumab by histidine residues in the complementary determining regions (CDRs) of the heavy and/or light chain variable domains of the antibody. The introduction of histidine residues at defined position in some of the CDRs of adalimumab renders the original antibody much more pH-sensitive or pH-dependent compared to the non-mutated antibody. Interestingly, although the histidine-mutated antibody undergoes a significant loss of binding affinity (up to factor 10 and more of KD) the mutated antibody of the invention is therapeutically significantly more effective compared to its parental non-mutated version, resulting in the possibility to reduce the antibody dosage or the antibody dosing intervals and thus the treatment costs significantly.
It has been shown by the invention that the successful introduction of histidine residues according to the invention by replacement of original amino acid residues in the CDRs of adalimumab or biologically active variants and fragments thereof, does not obey routine rules or mental considerations in view of the desired results regarding degree of pH-sensitivity and binding affinity.
Thus, it was found that histidine-mutated versions of adalimumab are specifically therapeutically effective, if the pH dependent antigen binding results in an antigen dissociation rate (Kdis) ratio pH 6/pH 7 which is 5-20 fold higher compared to the respective Kdis rate ratio of non-mutated adalimumab, and the binding affinity of the mutated adalimumab is 1-25%, preferably 1-15%, more preferably 2-12% of the binding affinity of the non-histidine mutated adalimumab.
Therefore, the invention provides a human anti-TNFa antibody or an antigen binding fragment thereof with pH dependent antigen binding comprising the light and heavy chain variable regions of human antibody adalimumab or a variant thereof with same or similar biological activity, wherein at least one of the CDR domains of the light and/or the heavy chain variable regions is mutated by replacement of one or more amino acids within said CDR domains by a histidine residue, thus generating a mutated adalimumab or adalimumab variant eliciting a pH dependent antigen binding with an antigen dissociation rate (Kdis) ratio pH 6/pH 7 measured by Bioayer Interferometrie (Octet Red), or other comparable methods, which is at least 5, 10, 15 or 10 fold higher compared to the respective Kdis rate ratio of non-mutated adalimumab.
In a further embodiment, the invention provides a respective mutated adalimumab, wherein the mutated antibody or antigen binding fragment thereof has a reduced antigen binding affinity, which is preferably 1-15%, respectively 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% 14% or 15% of the binding affinity of non-mutated adalimumab.
The invention provides human anti-TNFa antibodies deriving from adalimumab, wherein one or more original amino acid residues within the CDRs of parental adalimumab were replaced by histidine residues. Interestingly, the most effective histidine-mutated versions of adalimumab comprise histidine mutations within the variable light chain, especially in the CDR1 and CDR3 domains of the light chain. Preferred histidine-mutated adalimumab versions include further or independently a histidine-mutated CDR3 heavy chain.
Thus the invention provides mutated adalimumab or antigen binding fragment thereof comprising a CDR3 heavy chain sequence selected from the group consisting of:
The invention further provides mutated adalimumab or antigen binding fragment thereof comprising a CDR3 light chain sequence selected from the group consisting of:
HH
YHRAPYT
X
1
H
YHRAPYX2, wherein X1 is Q or H, and X2 is
X
1
X
2
X
3
X
4
RAPYX5, wherein X1 is Q or H, X2 is R or
The invention further provides mutated adalimumab or antigen binding fragment thereof comprising a CDR1 light chain sequence selected from the group consisting of:
The invention further provides mutated adalimumab or antigen binding fragment thereof comprising a CDR2 light chain sequence selected from the group consisting of:
In an embodiment of the invention, the histidine-mutated adalimumab or antigen binding fragment thereof comprises a CDR3 heavy chain sequence as specified above and in the claims, and a CDR1 light chain sequence as specified above and in the claims.
In a further embodiment of the invention, the histidine-mutated adalimumab or antigen binding fragment thereof comprises a CDR3 heavy chain sequence, and a CDR3 light chain sequence as specified above and in the claims.
In a further embodiment of the invention, the histidine-mutated adalimumab or antigen binding fragment thereof comprises a CDR3 heavy chain sequence, a CDR2 light chain sequence, and a CDR3 light chain sequence as specified above and in the claims.
In another embodiment of the invention, the histidine-mutated adalimumab or antigen binding fragment thereof comprises a CDR3 heavy chain sequence, a CDR1 light chain sequence, and a CDR3 light chain sequence as specified above and in the claims.
It was found by the inventors that preferable versions of histidine-mutated adalimumab comprise one of the following light chain variable regions:
It was further found by the inventors that preferable versions of histidine-mutated adalimumab comprise one of the following heavy chain variable regions:
Preferable histidine-mutated adalimumab according to the invention comprises any of the variable light chains as specified above and any one of the variable heavy chain domains as specified above.
A first preferred embodiment of the invention is a respectively histidine-mutated adalimumab comprising the variable heavy chain sequence:
and the variable light chain sequence:
A second preferred embodiment of the invention is a respectively histidine-mutated adalimumab comprising the variable heavy chain sequence:
and the variable light chain sequence:
A third preferred embodiment of the invention is a respectively histidine-mutated adalimumab comprising the variable heavy chain sequence:
and the variable light chain sequence:
The histidine-mutated adalimumab antibodies or variants or fragments thereof can further comprise human heavy and/or light constant regions.
In one embodiment they comprise a human IgG, preferably a human IgG1 (such as specified by SEQ ID NO: 11), or IgG2 heavy chain constant region.
In another embodiment of the invention they comprise human kappa light chain constant region.
In another embodiment of the invention, the histidine-mutated adalimumab versions of the invention comprise a human heavy chain constant region, preferably IgG1, wherein the Fc portion is mutated at one or more amino acid positions by replacement of the original amino acid residues by other natural amino acid residues which mediate (increase or decrease) binding of the antibody to FcRn.
The histidine-mutated adalimumab antibodies of the invention can be conjugated to other molecules by recombinant fusion with other polypeptides or proteins such as cytokine, or by chemical linkage to chemical, preferably cytotoxic entities, preferably via linker molecules to form antibody-drug-conjugates (ADCs). Techniques and methods to produce such antibody fusion proteins or antibody drug conjugates are well established in the art.
The invention also provides pharmaceutical compositions suitable for the treatment of inflammatory, autoimmune or cancer diseases comprising histidine-mutated anti-TNFa antibody adalimumab or a variant or an antigen binding fragment thereof, or a respective antibody-drug conjugate or an antibody cytokine fusion protein together with a pharmaceutically acceptable carrier, diluent or excipient.
The invention finally provides the therapeutic use of such histidine-mutated adalimumab or biologically effective and active variant or fragment thereof, or ADCs or fusion proteins thereof, for the manufacture of a medicament for the treatment of TNFa induced inflammatory, autoimmune or cancer diseases, as specified in detail below.
The histidine-mutated adalimumab versions of the inventions exhibit the following advantageous properties and functions:
TNFα is a cytokine that plays a key role in immune responses by initiating defense responses to e.g. some bacterial infections. It acts in a complex network in normal inflammation and is also involved in Iymhphoid tissue development (Tracey et al., 2008).
Many different cells produce TNFα including macrophages, T cells, mast cells and granulocytes, to name a few. TNFα is a general term that includes soluble (sTNFα) and transmembrane TNFα (mTNFα). Soluble homotrimers are released by cells after proteolytic cleavage of transmembrane TNFα (homotrimers of 26 kDa monomers) by the metalloprotease TNF-alpha-converting enzyme (TACE) (Wajant et al., 2003). Both forms, the sTNFα or mTNFα interact with two distinct receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Both receptors differ in their cellular expression profiles and signaling mechanisms. Complexity of the interaction network increases as mTNFα itself can act either as ligand or receptor to both TNF receptors (Wajant et al., 2003).
During disease, high expression of TNFα triggers and mediates downstream mechanisms that can lead to chronic inflammation and pathogenesis within affected compartments (Tracey et al., 2008). Common consequences of TNFα activities in autoimmune diseases are modulation of immune cell recruitment, cell proliferation, cell death and immune regulation. Other effects like matrix degradation and osteoclastogenesis are more disease specific and may be related to different cell types in the affected tissues (Reviewed in Tracey et al., 2008, Choy & Panayi, 2001; Feldmann, 2002; Schottelius et al., 2004).
TNFα has a particularly important role in the regulation of pathogenic events in e.g. rheumatoid arthritis, crohn's disease and plaque psoriasis and rapidly induces other cytokines (e.g. IL-1(3 and IL-6) (Tracey et al., 2008). In several inflammatory autoimmune diseases the positive clinical outcome upon TNFα-blockade by therapeutic anti-TNFα antibodies has corroborated the link between excessive TNFα exposition and disease pathologies (Aggawal, 2003; Tracey et al., 2008).
Adalimumab (also known by its trademark name HUMIRA®) is a fully human 148 kDa IgG1K monoclonal antibody against TNFα that originally was developed by using phage display (Abbott Laboratories, 2014). Since market launch in 2002, adalimumab generated huge sales that achieved $4.6 billion just on the US market in 2012 (Aggarwal, 2014). Adalimumab binds to TNFα with high sub-nanomolar affinity and thereby blocks the interaction between TNFα and p55 and p75 cell surface receptors TNFR1 or TNFR2 (Abbott Laboratories, 2014).
As adalimumab binds also to mTNFα with high affinity further possible modes of action include apoptosis or cytokine suppression upon reverse signaling mechanisms and CDC or ADCC mediated cell killing (Reviewed in Tracey et al., 2008 and Horiuchi et al, 2010).
The amino acid sequences of Adalimumab (and variants thereof) and its pharmacological and therapeutic properties are disclosed, for example in WO 97/029131.
The sequences which are important in view of the present invention are listed below in detail:
Adalimumab full light chain sequence:
DIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLAWYQQKP
GKAPKLLIYA ASTLQSGVPS RFSGSGSGTD FTLTISSLQP
EDVATYYCQR YNRAPYTFGQ GTKVEIKRTV AAPSVFIFPP
Adalimumab light chain sequence, variable region (VL):
Adalimumab CDR1 sequence of VL
Adalimumab CDR2 sequence of VL
Adalimumab CDR3 sequence of VL
Adalimumab full heavy chain sequence:
EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA
PGKGLEWVSA ITWNSGHIDY ADSVEGRFTI SRDNAKNSLY
LQMNSLRAED TAVYYCAKVS YLSTASSLDY WGQGTLVTVS
SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV
Adalimumab heavy chain sequence, variable region (VH):
Adalimumab CDR1 sequence of VH:
Adalimumab CDR2 sequence of VH:
Adalimumab CDR3 sequence of VH:
Adalimumab human heavy chain IgG1 constant region:
Adalimumab is approved in many countries for a couple of therapeutic treatments, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and Crohn's disease.
Therefore, the histidine-mutated adalimumab versions according to the invention are also applicable in the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and Crohn's disease, but also in the treatment of other diseases which are induced or triggered by TNFa. This may include autoimmune disorders as well as cancer diseases.
Selection of suitable anti-TNFa antibody versions of adalimumab and fragments thereof according to the invention may be achieved by well established and known methods and techniques in the art, such as by histidine substitution via page display libraries or from combinatorial histidine substitution libraries by yeast surface display. Details are provided in the Example section.
The term “antibody” or “immunoglobulin” is used according to the invention in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. Depending on the amino acid sequence of their constant regions, intact or whole antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
Preferred major class for antibodies according to the invention is IgG, in more detail IgG1 and IgG2, most preferably IgG1.
“Antibody fragments” according to the invention comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fv and Fc fragments, diabodies, linear antibodies, single-chain antibody molecules; bispecific and multispecific antibodies formed from antibody fragment(s).
A “whole or complete” antibody according to the invention is an antibody which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.
A “Fc” region of an antibody according to the invention comprises, as a rule, a CH2, CH3 and the hinge region of an IgG1 or IgG2 antibody major class. The hinge region is a group of about 15 amino acid residues which combine the CH1 region with the CH2-CH3 region.
A “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain and has one antigen-binding site only.
A “Fab” fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the heavy chain CH1 domain including one or more cysteine residues from the antibody hinge region.
A “F(ab′)2” antibody according to this invention is produced as pairs of Fab′ fragments which have hinge cysteines between them.
“Single-chain FV” or “scFv” antibody fragments according to the invention comprise the V, and V, domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
The “variable domain” of an antibody according to the invention comprises the framework regions (usually FR1 to FR4) as well as the CDR domains (usually CDR1, CDR2 and CDR3) which are designated as “hypervariable regions”.
The term “hypervariable region” or “CDR” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; and/or those residues from a “hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
If not otherwise pointed out, the amino acid positions within the antibody molecules according to this invention are numbered according to Kabat.
“Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
“Antibody variants” according to the invention include antibodies that have a modified amino acid sequence compared to the parental antibody but have same or changed binding affinity to the targeted antigen. Antibody variants differ from the parental antibody by replacement or deletion or addition of one or more amino acid residues at specific positions within the variable domains, including the CDR domains, and or the constant regions of the antibody, in order to modify certain properties of the antibody, such as binding affinity and/or receptor functions, like ADCC, FcRn binding and the more. The histidine-mutated antibodies of this invention without further modifications are not designated as “antibody variants” according to this invention. Antibody variants according to the invention exhibit a sequence homology of 80-99% compared to the parental antibody, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%, dependent on the specific location of the amino acid residue to be replaced, deleted or added. Antibody variants of adalimumab are, for example, disclosed in
The term “fusion protein” refers to a natural or synthetic molecule consisting of one ore more biological molecules as defined above, wherein two or more peptide- or protein-based (glycoproteins included) molecules having different specificity are fused together optionally by chemical or amino acid based linker molecules. The linkage may be achieved by C—N fusion or N—C fusion (in 5′→3′ direction), preferably C—N fusion. A fusion protein according to the invention is said fusion of an antibody or antibody variant of this invention fused to another protein or polypeptide, preferably a cytokine.
The term “antibody-drug conjugate (ADC)” refers according to the invention to an immunoconjugate composed of an antibody, preferably complete antibody, according to the invention, and a preferably chemical cytotoxic agent. The components are chemically attached to each other by specific linkers. The antibody of the invention (preferably within its heavy chain constant region) may be modified at one or more amino acid positions in order to create a suitable linkage to the linker and/or the cytotoxic payload drug. Method and techniques to generate such ADCs are well known in the art.
The term “Fc receptor” means, according to the invention, receptors for the Fc region of immunoglobulins (FcRs) that link humoral responses to cellular activities within the immune system. Based on their function, two general groups of FcR can be distinguished: those expressed predominantly by leucocytes that trigger antibody effector functions and those that primarily mediate transport of immunoglobulins across epithelial or endothelial surfaces. ADCC is triggered through interaction of target-bound antibodies (belonging to IgG or IgA or IgE classes) with certain Fc receptors (FcRs). ADCC involving human IgG1 is highly dependent on the glycosylation profile of its Fc portion and on the polymorphism of Fcγ receptors. The term “FcRn” means the specific neonatal Fc receptor, which binds binds IgG at acidic pH of (<6.5) but not at neutral or higher pH. The receptor is responsible for extending half-life of IgG antibodies in serum.
The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones, such as vascular endothelial growth factor (VEGF); integrin; thrombopoietin (TPO); nerve growth factors such as NGFβ; platelet-growth factor; transforming growth factors (TGFs) such as TGFα and TGFβ; erythropoietin (EPO); interferons such as IFNα, IFNβ, and IFNγ; colony stimulating factors such as M-CSF, GM-CSF and G-CSF; interleukins such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; and TNF-α or TNF-β.
The term “biologically/functionally effective” or “therapeutically effective (amount)” refers to a drug/molecule which causes a biological function or a change of a biological function in vivo or in vitro, and which is effective in a specific amount to treat a disease or disorder in a mammal, preferably in a human.
The term “pharmaceutical treatment” means a variety of modalities for practicing the invention in terms of the steps. For example, the agents according to the invention can be administered simultaneously, sequentially, or separately. Furthermore, the agents can be separately administered within one or more time intervals between administrations. Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with the relevant agent as described herein, dissolved or dispersed therein as an active ingredient.
As used herein, the term “pharmaceutically acceptable” refers to compositions, carriers, diluents and reagents which represent materials that are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable preparation either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which may enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
The histidine-mutated adalimumab versions according to the invention are suitable for the treatment of the same disorders and diseases as the approved and marketed non-histidine mutated adalimumab (HUMIRA®), which are rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis and chronic plaque psoriasis, wherein the drug is preferably administered by subcutaneous injection.
Like the marketed drug, the histidine-mutated adalimumab according to the invention can be used alone or in combination with other drugs which support the therapy, such as methotrexate, DMARDS, glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and/or analgesics.
The standard dose regimen of HUMIRA® is usually 40 mg every week as single dose. The histidine-mutated adalimumab versions according exhibit, as pointed out earlier, a significantly stronger pH-dependency as HUMIRA®, and thus can be administered in doses which correspond only 10-90%, in detail 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, of the recommended dose of HUMIRA®, dependent on the respective Kdis values of the used histidine-mutated adalimumab versions.
Selection of anti-TNFα antibody fragments from combinatorial histidine substitution libraries by yeast surface display: Based on the heavy and light chains of adalimumab, two antibody libraries were synthesized by Geneart, Regensburg by using pre-assembled trinucelotides building blocks. During the synthesis either parental or histidine residues were sampled whereby sampling of histidines was restricted to the complementary determining regions (CDRs) of the heavy and light chains. Most adalimumab library members carried three histidine residues that were spread over all three CDRs (
Both libraries were separately subcloned into plasmid vectors by gap-repair cloning in the EBY100 yeast strain that allows covalent yeast surface display of antibody Fab-fragments (Boder and Wittrup, 1997). Corresponding parental chains were paired with the heavy or light chain libraries and the two resulting libraries were separately screened by fluorescence activated cell sorting (FACS). Cells that carried pH-sensitive adalimumab variants were subsequently enriched over three rounds of screening by applying a specific staining & selection strategy (not explained here).
Fab-fragments were selected that do reversible high affinity (KD within sub nanomolar ranges) binding to recombinant human TNFα (rhTNFα) at pH 7.4, once after rhTNFα has been released within 30 minutes at pH 6. After three rounds of screening for variants that bind to rhTNFα in pH-dependent manner, sequence analysis of isolated single clones revealed variants that carried specific histidine substitutions patterns (shown in
Variable regions of abundant VH/VL variants as well as parental adalimumab sequences were cloned into vectors that allow expression of full length IgG1 K molecules in mammalian cells (HEK293 & Expi293). All possible combinations of heavy and light chain variants were co-expressed in mammalian cells. Herein 24 heavy chain and light chain variant combinations were expressed as well as 11 IgG species that derived from combinations of the heavy or light chain variants with the corresponding parental chains. Initial Octet Red experiments with immobilized antibodies assessed differential dissociation behavior at pH 6 or pH 7.4 after associating rhTNFα at pH 7.4. Ten variants were selected according to their binding profiles in regard of high affinity binding at pH 7.4 (association/dissociation at pH 7.4) and fast release of the antigen at pH 6 (association at pH 7.4/dissociation at pH 6). For further characterization antibodies were purified via protein-A purification from crude supernatants and finally buffer was exchanged to PBS. Further octet assays revealed differences in binding at pH 7.4 and differences in rhTNFα release at pH 6.
Subsequently three variants (IDs according to
Binding characteristics of several histidine-mutated adalimumab variants were analyzed in Octet Red experiments. Different histidine mutations in heavy and light chains as well as different heavy and light chain variant combinations have been shown to affect binding affinities at pH 7.4 and the dissociation rates at pH 6.
A combination of several mutations including Leu98His in the heavy chain and Tyr32His, Leu33His in the light chain generated PSV#3. Light chain mutations GIn89His, Arg90His and Asn92His generated the light chain of PSV#2. The mutations Arg90His, Asn92His and Thr97His generated the light chain of PSV#1. For both, PSV#1 and PSV#2, mutations of Ser100.bHis and Ser100.cHis generated the heavy chain.
(The sequences were numbered as shown in
Representative sensorgrams of the Octet Red measurements are shown in
4.81E−05
Parental adalimumab binds with high affinity to rhTNFα at pH 7.4 that was also shown in Kaymakcalan et al., 2009. For the three variants the increasing kdis-values result in a decrease in binding affinity (10-24fold decrease in KD), however interaction with TNFα with picomolar binding affinities in the three-diget range still represents very tight binding (
Octet Red measurements were also performed to assess the antibodies' pH-sensitivities. Improved antibody efficacy in context of the FcRn-mediated recycling requires tight binding to TNFα in the circulation at pH 7.4 and its fast release in the acidic endosome. In order to evaluate the release of TNFα within the acidic endosome, dissociation was measured at pH 6 after association of rhTNFα at pH 7.4 (
One additional experiment addressed the ability of PSV#1, PSV#2 and PSV#3 to reversibly bind rhTNFα at pH 7.4, after dissociation at pH 6. To ensure that incubation at pH 6 does not irreversibly change TNFα binding capabilities, two cycles of binding (pH 7.4) and release (pH 6) were performed (
The effect of parental IgG construct and mutants thereof on the PK of human TNFα was investigated in heterozygous transgenic human FcRn mice, line 176, as well as in homozygous line 32 mice. The former line was more suitable to investigate PK differences between administered IgG constructs, while the latter mouse line provided a better FcRn protection, resulting in longer half-lives, closer to what was expected in human. This longer residence of the scavenger allowed better to evaluate the impact of the antibody on the clearance of the TNFα.
Human TNFα and scavenger were administered by SC route in predefined ratios. Plasma concentration profiles of both total scavenger and total hTNFα was investigated. pH-dependent hTNFα binding was expected to result in increased clearance of the cytokine and decreased clearance of the scavenger.
Selected tissues were collected from the mice, in order to investigate distribution of the scavenger and the target cytokine. Parental IgG were used as reference compound in this study.
The in vitro and in vivo data sets were used to establish correlations between:
The correlations were used to build a physiologically-based pharmacokinetic (PBPK) model capable of characterizing and simulating plasma and tissue pharmacokinetics.
| Number | Date | Country | Kind |
|---|---|---|---|
| 14002231.0 | Jun 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/001296 | 6/26/2015 | WO | 00 |
