Patent Application
20200247897
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 21, 2020, is named GMI_163US_Sequence_Listing.txt and is 146,215 bytes in size.
The present invention relates to pharmaceutical compositions comprising antibodies of an IgG isotype having a mutation in the Fc region that enhances hexamerization of IgG antibodies after cell-surface antigen binding. The invention also relates to methods for preparing pharmaceutical compositions of the invention and the uses of such compositions.
IgG antibodies can organize into ordered hexamers on cell surfaces after binding their target antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Mutations have been identified that enhance hexamer formation and complement activation by IgG antibodies against a range of targets on cells from hematological and solid tumor indications (de Jong et al. 2016 PLoS Biol 14(1): e1002344, WO2013/004842, WO2014/108198). IgG backbones e.g. IgG1 having mutations at specific positions in the Fc region conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. The mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis (de Jong, supra).
DR5, also known as death receptor 5, Tumor necrosis factor receptor superfamily member 10B, TNFRSF10B, TNF-related apoptosis-inducing ligand receptor 2, TRAIL receptor 2, TRAIL-R2 and CD262, is a cell surface receptor of the TNF receptor superfamily that binds tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mediates apoptosis. DR5 is a single-pass type I membrane protein with three extracellular cysteine-rich domains (CRDs), a transmembrane domain (TM) and a cytoplasmic domain containing a death domain (DD). In the absence of ligand, DR5 exists in the cell membrane either as monomer or as pre-assembled complexes of two or three receptors through interactions of the first cysteine-rich domain, also known as pre-ligand assembly domain (PLAD) (Wassenaar et al., Proteins. 2008 Feb. 1; 70(2):333-43; Valley et al., J Biol Chem. 2012 Jun. 15; 287(25):21265-78; Sessler et al., Pharmacol Ther. 2013 November; 140(2):186-99). A Crystal structure of TRAIL in complex with the DR5 ectodomain showed that TRAIL binds to CRD2 and CRD3 in the extracellular domain of DR5 in a complex containing a trimeric receptor and a trimeric ligand (Hymowitz et al., Mol Cell. 1999 October; 4(4):563-71). The DR5 trimers can further cluster into higher-order receptor aggregates in lipid macrodomains, so-called lipid rafts (Sessler et al., Pharmacol Ther. 2013 November; 140(2):186-99). In the ligand-bound conformation, the cytoplasmic death domain-containing adaptor protein FADD associate with the intracellular DD surface of the oligomerized DR5 molecules and engage initiator caspases caspase-8 and caspase-10 to form the death-inducing signaling complex (DISC).
Based on the sensitivity of cancer cells to TRAIL-mediated apoptosis, numerous agents were developed to activate this pathway to induce apoptosis selectively in cancer cells. Human recombinant TRAIL (hrTRAIL), is being developed as dulanermin, and a series of conventional (monospecific, bivalent) anti-DR5 antibodies have been developed and tested in the clinic (reviewed in Ashkenazi et al., Nat Rev Drug Discov. 2008 December; 7(12):1001-12; Trivedi et al., Front Oncol. 2015 Apr. 2; 5:69): DR5 antibodies include lexatumumab (HGS-ETR2), HGS-TR2J, conatumumab (AMG655), tigatuzumab (CS-1008), drozitumab (Apomab) and LBY-135. Clinical studies with these compounds demonstrated that DR5 antibodies were generally well tolerated but failed to show convincing and significant clinical benefit. Efforts to enhance the efficacy of DR5 targeting antibodies mainly focus on (i) improving the sensitivity of cancer cells to DR5 agonists through combination treatment, (ii) developing biomarkers for better patient stratification, and (iii) the development of DR5-targeting agents that activate DR5 signaling and apoptosis-induction more effectively (reviewed in Lim et al., Expert Opin Ther Targets. 2015 May 25:1-15; Twomey et al., Drug Resist Updat. 2015 March; 19:13-21; Reddy et al., PLoS One. 2015 Sep. 17; 10(9)). Different therapeutic formats for increasing DR5 activation have been described and include oligomerization of synthetic DR5 binding peptides, linear fusions of DR5-specific scaffolds, nanoparticle-based delivery systems of rhTRAIL or conatumumab and multivalent DR5 antibody-based formats (reviewed in Holland et al., Cytokine Growth Factor Rev. 2014 April; 25(2):185-93). APG880 and derivatives exist of two single chain TRAIL receptor binding (scTRAIL-RBD) molecules (TRAIL mimics) fused to the Fc part of a human IgG. Each scTRAIL-RBD has three receptor binding sites resulting in a hexavalent binding mode in the fusion protein (WO 2010/003766 A2). A prototype scTRAIL-RBD (APG350) has been described to induce FcγR-independent antitumor efficacy in vivo (Gieffers et al., Mol Cancer Ther, 2013. 12(12): p. 2735-47). A tetravalent anti-DR5 antibody fragment-derived construct, assembled by fusion of an anti-DR5 scFv fragment, human serum albumin residues and the tetramerization domain of human p53, has been shown to induce apoptosis more potently than the monovalent construct (Liu et al., Biomed Pharmacother. 2015 March; 70:41-5). Nanobody molecules are single domain antibody fragments (VHH) derived from camelid heavy chain-only antibodies, which, similarly to scFvs, can be linked to form multivalent molecules. Preclinical in vitro studies showed that TAS266, a tetravalent anti-DR5 Nanobody® molecule, was more potent than TRAIL or crosslinked DR5 antibody LBY-135, which was attributed to more rapid caspase activation kinetics (Huet et al., MAbs. 2014; 6(6):1560-70). TAS266 was also more potent in vivo than the parental murine mAb of LBY-135. MultYbody™ molecules (MultYmab technology) are based on the fusion of a homomultimerizing peptide to the Fc of one heavy chains in an IgG heterodimer (knob into hole), making MultYbody molecules intrinsically multivalent in solution. An anti-DR5 MultYbody was shown to induce potent killing in vitro. Dual-affinity re-targeting (DART) molecules are covalently-linked Fv-based diabodies. DR5 targeting tetravalent Fc DARTs comprising either tetravalency for a single (mono-epitopic DARTs) or two DR5 epitopes (bi-epitopic DARTs) were shown to be more potent than TRAIL and a conatumumab variant in inducing in cytotoxicity in vitro and in vivo (Li et al., AACR Annual Meeting Apr. 20, 2015, Poster abstract #2464). Alternatively, FcγR-independent avidity-driven DR5 hyperclustering can be mediated by a bispecific DR5×FAP antibody (RG7386) through simultaneous binding to DR5 on the cancer cell and to fibroblast activation protein (FAP) that is expressed on fibroblasts in the tumor microenvironment (Friess et al., AACR Annual Meeting Apr. 19, 2015, Presentation abstract #952; Wartha et al., Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr. 5-9; San Diego, Calif. Philadelphia (Pa.): AACR; Cancer Res 2014; 74(19 Suppl):Abstract nr 4573. doi:10.1158/1538-7445.AM2014-4573). Finally, specific combinations of two anti-DR5 antibodies recognizing different epitopes have shown enhanced agonistic efficacy in vitro and in vivo compared to combinations of two anti-DR5 antibodies recognizing overlapping or similar epitopes (WO2014/009358).
Above described approaches show enhanced efficacy compared to the conventional anti-DR5 antibodies in preclinical studies, however clinical data indicate that there is still a need for improving the DR5 agonists. Moreover, it is desirable for antibody-based formats to preserve a pharmacokinetic (PK) as well as other Fc-mediated effector functions of regular IgG, which usually is not the case with antibody fragment-based constructs.
PCT/EP2016/079518, incorporated herein by reference, provides anti-DR5 antibodies comprising an Fc region of a human IgG and an antigen binding region binding to DR5, wherein the Fc region comprises a mutation at an amino acid position corresponding to position E430, E345 or S440. It was found that the introduction of a specific point mutation in the Fc region of an anti-DR5 antibody which facilitates hexamerization of the antibody on cell-surface antigen binding and conditional clustering of the antigen independent on secondary cross-linking, results in DR5 activation and significantly enhances the potency of the antibody in inducing apoptosis and cell death.
There is a need for providing stable formulations for the antibodies described in PCT/EP2016/079518, and more generally for antibodies that hexamerize more easily due to a mutation at an amino acid position corresponding to position E430, E345 or S440 in human IgG1 according to EU numbering, with the proviso that mutation in S440 is S440Y or S440W.
Surprisingly, the inventors of the present invention have found compositions that provide a stable formulation for variant antibodies that hexamerize more easily due to a mutation at an amino acid position corresponding to position E430, E345 or S440 in human IgG1, with the proviso that the mutation in S440 is S440Y or S440W. Two of such antibodies with entirely different sequences in their CDR domains were both found to be stable in the composition of the invention.
In a first main aspect, the invention relates to a pharmaceutical composition comprising:
In one embodiment of the invention the first and second Fc region comprises a mutation of an amino acid at a position corresponding to S440 in human IgG1, EU numbering, with the proviso that the mutation in S440 is S440Y or S440W.
Such formulations were found to provide excellent antibody solubility and stability under stress conditions, such as heating, freeze-thaw cycles and agitation. Minimal formation of macromolecular aggregates or other impurities such as degration products was observed.
In further aspects, the invention relates to the pharmaceutical composition of the invention for use as a medicament, to the use of a pharmaceutical composition of the invention for the manufacture of a medicament and to methods of treating individuals comprising administering to said individual an effective amount of a pharmaceutical composition of the invention.
In even further aspects, the invention relates to kits comprising two or more pharmaceutical compositions of the invention and to methods for preparing a pharmaceutical composition of the invention comprising the step of mixing two pharmaceutical compositions of the invention each comprising different antibodies.
In a preferred embodiment of the pharmaceutical composition of the invention, the antibody comprises an antigen-binding region which binds to human DR5, preferably wherein the antigen binding region comprises a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the amino acid sequences of:
e) the (VH) CDR1, CDR2, CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in any one of a) to d) above having one to five mutations or substitutions in total across said six CDR sequences.
Such antibodies binding to DR5 and comprising a hexamerizing-enhancing mutation in the Fc region corresponding to position E430, E345 or S440 of human IgG1 (according to EU numbering), with the proviso that the mutation in S440 is S440Y or S440 W, were found to be superior at inducing apoptosis in tumor cells expressing DR5 compared to antibodies binding DR5 without a mutation in one of the above mentioned positions.
In a further preferred embodiment, the pharmaceutical composition of the invention comprises at least two antibodies, comprising a first antibody and a second antibody, wherein
Such compositions comprising two anti-DR5 antibodies, which bind different epitopes on DR5, were found superior in in vitro and in vivo studies to compositions comprising the same anti-DR5 antibodies without the mutation. That is compositions with two antibodies of the present invention were superior at inducing apoptosis and/or inhibiting cell growth of tumor cells expressing DR5 compared to compositions comprising two DR5 antibodies without a mutation in the Fc region.
In describing the embodiments of the invention specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.
The term “immunoglobulin” as used herein, refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region of IgG antibodies typically is comprised of three domains, CH1, CH2, and CH3. The heavy chains are inter-connected via disulfide bonds in the so-called “hinge region”. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules (Brochet X., Nucl Acids Res. 2008; 36:W503-508 and Lefranc M P., Nucleic Acids Research 1999; 27:209-212; see also internet http address http://www.imgt.org/). Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present invention is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). The term “hinge region” as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering.
The term “CH2 region” or “CH2 domain” as used herein is intended to refer the CH2 region of an immunoglobulin heavy chain. Thus, for example the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering. However, the CH2 region may also be any of the other isotypes or allotypes as described herein.
The term “CH3 region” or “CH3 domain” as used herein is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering. However, the CH3 region may also be any of the other isotypes or allotypes as described herein.
The term “fragment crystallizable region”, “Fc region”, “Fc fragment” or “Fc domain”, which may be used interchangeably herein, refers to an antibody region comprising, arranged from amino-terminus to carboxy-terminus, at least a hinge region, a CH2 domain and a CH3 domain. An Fc region of an IgG1 antibody can, for example, be generated by digestion of an IgG1 antibody with papain. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation.
The term “Fab fragment” in the context of the present invention, refers to a fragment of an immunoglobulin molecule, which comprises the variable regions of the heavy chain and light chain as well as the constant region of the light chain and the CH1 region of the heavy chain of an immunoglobulin. The “CH1 region” refers e.g. to the region of a human IgG1 antibody corresponding to amino acids 118-215 according to the EU numbering. Thus, the Fab fragment comprises the binding region of an immunoglobulin.
The term “antibody” (Ab), as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof. The antibody of the present invention comprises an Fc-region of an immunoglobulin and an antigen-binding region. The Fc region generally contains two CH2-CH3 regions and a connecting region, e.g. a hinge region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The term “antibody” as used herein, also refers to, unless otherwise specified or contradicted by the context, polyclonal antibodies, oligoclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures, recombinant polyclonal antibodies, chimeric antibodies, humanized antibodies and human antibodies. An antibody as generated can potentially possess any class or isotype.
The term “human antibody”, as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another species, such as a mouse, have been grafted onto human framework sequences.
The term “chimeric antibody”, as used herein, refers to an antibody in which both chain types i.e. heavy chain and light chain are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (originating from a non-human species, or synthetic or engineered from any species including human) linked to a constant region of human origin.
The term “humanized antibody, as used herein, refers to an antibody in which both chain types are humanized as a result of antibody engineering. A humanized chain is typically a chain in which the complementarity determining regions (CDR) of the variable domains are foreign (originating from a species other than human, or synthetic) whereas the remainder of the chain is of human origin. Humanization assessment is based on the resulting amino acid sequence, and not on the methodology per se, which allows protocols other than grafting to be used.
The term “isotype”, as used herein, refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgA2, IgE, or IgM) that is encoded by heavy chain constant region genes. To produce a canonical antibody, each heavy chain isotype is to be combined with either a kappa (κ) or lambda (λ) light chain.
The term “allotype”, as used herein, refers to the amino acid variation within one isotype class in the same species. The predominant allotype of an antibody isotype varies between ethnicity individuals. The known allotype variations within the IgG1 isotype of the heavy chain result from 4 amino acid substitutions in the antibody frame as illustrated in
The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb”, or the like, as used herein refer to a preparation of Ab molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to Abs displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene repertoire and a human light chain transgene repertoire, rearranged to produce a functional human antibody and fused to an immortalized cell. Alternatively, the human mAbs may be generated recombinantly.
The term “antibody mimetics” as used herein, refers to compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides, proteins, nucleic acids or small molecules.
The term “bispecific antibody” refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets Examples of different classes of bispecific antibodies comprising an Fc region include but are not limited to: asymmetric bispecific molecules e.g. IgG-like molecules with complementary CH3 domains and symmetric bispecific molecules e.g. recombinant IgG-like dual targeting molecules wherein each antigen-binding region of the molecule binds at least two different epitopes.
Examples of bispecific molecules include but are not limited to Triomab® (Trion Pharma/Fresenius Biotech, WO/2002/020039), Knobs-into-Holes (Genentech, WO9850431), CrossMAbs (Roche, WO 2009/080251, WO 2009/080252, WO 2009/080253), electrostatically-matched Fc-heterodimeric molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y (Genentech), DIG-body, PIG-body and TIG-body (Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono, WO2007110205), Bispecific IgG1 and IgG2 (Pfizer/Rinat, WO11143545), Azymetric scaffold (Zymeworks/Merck, WO2012058768), mAb-Fv (Xencor, WO2011028952), XmAb (Xencor), Bivalent bispecific antibodies (Roche, WO2009/080254), Bispecific IgG (Eli Lilly), DuoBody® molecules (Genmab A/S, WO 2011/131746), DuetMab (Medimmune, US2014/0348839), Biclonics (Merus, WO 2013/157953), NovImmune (κλBodies, WO 2012/023053), FcΔAdp (Regeneron, WO 2010/151792), (DT)-Ig (GSK/Domantis), Two-in-one Antibody or Dual Action Fabs (Genentech, Adimab), mAb2 (F-Star, WO2008003116), Zybodies™ (Zyngenia), CovX-body (CovX/Pfizer), FynomAbs (Covagen/Janssen Cilag), DutaMab (Dutalys/Roche), iMab (MedImmune), Dual Variable Domain (DVD)-Ig™ (Abbott, U.S. Pat. No. 7,612,18), dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), Ts2Ab (Medlmmune/AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec, US007951918), scFv-fusions (Genentech/Roche, Novartis, Immunomedics, Changzhou Adam Biotech Inc, CN 102250246), TvAb (Roche, WO2012025525, WO2012025530), ScFv/Fc Fusions, SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Interceptor (Emergent), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics, WO2008/157379, WO2010/080538), BEAT (Glenmark), Di-Diabody (Imclone/Eli Lilly) and chemically crosslinked mAbs (Karmanos Cancer Center), and covalently fused mAbs (AIMM therapeutics).
The term “full-length antibody” when used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that class or isotype.
The term “oligomer” as used herein, refers to a molecule that consists of more than one but a limited number of monomer units (e.g. antibodies) in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers and hexamers. Greek prefixes are often used to designate the number of monomer units in the oligomer, for example a tetramer being composed of four units and a hexamer of six units. Likewise, the term “oligomerization”, as used herein, is intended to refer to a process that converts molecules to a finite degree of polymerization. Herein, it is observed, that antibodies and/or other dimeric proteins comprising target-binding regions according to the invention can form oligomers, such as hexamers, via non-covalent association of Fc-regions after target binding, e.g., at a cell surface.
The term “antigen-binding region”, “antigen binding region”, “binding region” or antigen binding domain, as used herein, refers to a region of an antibody which is capable of binding to the antigen. This binding region is typically defined by the VH and VL domains of the antibody which may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). The antigen can be any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion or in solution. The terms “antigen” and “target” may, unless contradicted by the context, be used interchangeably in the context of the present invention.
The term “target”, as used herein, refers to a molecule to which the antigen binding region of the antibody binds. The target includes any antigen towards which the raised antibody is directed. The term “antigen” and “target” may in relation to an antibody be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention.
The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of building blocks such as amino acids, sugar side chains or a combination thereof and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).
The term “binding” as used herein refers to the binding of an antibody to a predetermined antigen or target, typically with a binding affinity corresponding to a KD of about 10−6 M or less, e.g. 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte or visa versa, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and is obtained by dividing kd by ka.
The term “kd” (sec−1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value or off-rate.
The term “ka” (M−1×sec−1), as used herein, refers to the association rate constant of a particular antibody-antigen interaction. Said value is also referred to as the kon value or on-rate.
The term “KA” (M−1), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing ka by kd.
As used herein, the term “affinity” is the strength of binding of one molecule, e.g. an antibody, to another, e.g. a target or antigen, at a single site, such as the monovalent binding of an individual antigen binding site of an antibody to an antigen.
As used herein, the term “avidity” refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen binding sites of antibodies simultaneously interacting with a target. When more than one binding interactions are present, the two structures will only dissociate when all binding sites dissociate, and thus, the dissociation rate will be slower than for the individual binding sites, and thereby providing a greater effective total binding strength (avidity) compared to the strength of binding of the individual binding sites (affinity).
The term “hexamerization enhancing mutation”, as used herein, refers to a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1 according to EU numbering, with the proviso that the mutation in S440 is S440Y or S440W. The hexamerization enhancing mutation strengthens Fc-Fc interactions between neighbouring IgG antibodies that are bound to a cell surface target, resulting in enhanced hexamer formation of the target-bound antibodies, while the antibody molecules remain monomeric in solution as described in WO2013/004842; WO2014/108198.
The term “clustering” as used herein, is intended to refer to oligomerization of antibodies, polypeptides, antigens or other proteins through non-covalent interactions.
The term “repulsing mutation” or “self-repulsing mutation” or “hexamerization-inhibiting mutation”, as used herein, refers to a mutation of an amino acid position of human IgG1 that can result in charge repulsion between amino acids at the Fc-Fc interface, resulting in weakening of the Fc-Fc interaction between two adjacent Fc region containing polypeptides, and thus inhibiting hexamerization. Examples of such a repulsing mutation in human IgG1 are K439E and S440K. The repulsion in the Fc-Fc interaction between two adjacent Fc region containing polypeptides at the position of a repulsing mutation can be neutralized by introduction of a second mutation (complementary mutation) in the amino acid position that interacts with the position harboring the first mutation. This second mutation can be present either in the same antibody or in a second antibody. The combination of the first and second mutation results in neutralization of the repulsion and restoration of the Fc-Fc interactions and thus hexamerization. Examples of such first and second mutations are K439E (repulsing mutation) and S440K (neutralizing the repulsion by K439E), and vice versa S440K (repulsing mutation) and K439E (neutralizing the repulsion by S440K).
The term “complementary mutation”, as used herein, refers to a mutation of an amino acid position in an Fc region-containing polypeptide that relates to a first mutation in an adjacent Fc region containing polypeptide that preferably interacts with the Fc region-containing polypeptide containing the complementary mutation due to the combination of the two mutations in the two adjacent Fc region-containing polypeptides. The complementary mutation and the related first mutation can be present either in the same antibody (intramolecular) or in a second antibody (intermolecular). An example of intramolecular complementary mutations is the combination K409R and F405L that mediates preferential heterodimerization in a bispecific antibody according to WO 2011/131746. The combination of the K439E and S440K mutations that results in neutralization of repulsion and restoration of Fc-Fc interactions between two adjacent Fc region containing polypeptides and thus hexamerization is an example of complementary mutations that can be applied both inter- and intramolecularly.
The term “apoptosis”, as used herein refers to the process of programmed cell death (PCD) that may occur in a cell. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, phosphatidylserine exposure, loss of mitochondrial function, nuclear fragmentation, chromatin condensation, caspase activation, and chromosomal DNA fragmentation. In a particular embodiment, apoptosis by one or more agonistic anti-DR5 antibodies can be determined using methods such as, e.g., caspase-3/7 activation assays described in examples 19, 20, 25 and 45 or phosphatidylserine exposure described in examples 19 and 25. Anti-DR5 antibody at a fixed concentration of e.g. 1 μg/mL may be added to adhered cells and incubated for 1 to 24 hours. Caspase-3/7 activation can be determined by using special kits for this purpose, such as the PE Active Caspase-3 Apoptosis Kit of BD Pharmingen (Cat nr 550914) (example 19 and 25) or the Caspase-Glo 3/7 assay of Promega (Cat nr G8091) (examples 20 and 45). Phosphatidylserine exposure and cell death can be determined by using special kits for this purpose, such as the FITC Annexin V Apoptosis Detection Kit I from BD Pharmingen (Cat nr 556547) (examples 19 and 25).
The term “programmed cell-death” or “PCD”, as used herein refers to the death of a cell in any form mediated by an intracellular signaling, e.g. apoptosis, autophagy or necroptosis.
The term “Annexin V”, as used herein, refers to a protein of the annexin group that binds phosphatidylserine (PS) on the cell surface.
The term “caspase activation”, as used herein, refers to cleavage of inactive pro-forms of effector caspases by initiator caspases, leading to their conversion into effector caspases, which in turn cleave protein substrates within the cell to trigger apoptosis.
The term “caspase-dependent programmed cell death”, as used herein refers to any form of programmed cell death mediated by caspases. In a particular embodiment, caspase-dependent programmed cell death by one or more agonistic anti-DR5 antibodies can be determined by comparing the viability of a cell culture in the presence and absence of pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK) as described in examples 18 and 44. Pan-caspase inhibitor Z-VAD-FMK (5 μM end concentration) may be added to adhered cells in 96-well flat bottom plates and incubated for one hour at 37° C. Next, antibody concentration dilution series (e.g. starting from e.g. 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions) may be added and incubated for 3 days at 37° C. Cell viability can be quantified using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571).
The term “cell viability”, as used herein refers to the presence of metabolically active cells. In a particular embodiment, cell viability after incubation with one or more agonistic anti-DR5 antibodies can be determined by quantifying the ATP present in the cells as described in examples 8-18, 21-24, 38-44, 46 and 48. Antibody concentration dilution series (e.g. starting from e.g. 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions) may be added to cells in 96-well flat bottom plates, medium may be used as negative control and 5 μM staurosporine may be used as positive control for the induction of cell death. After 3 days incubation cell viability may be quantified using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571). The percentage viable cells can be calculated using the following formula: % viable cells=[(luminescence antibody sample−luminescence staurosporine sample)/(luminescence no antibody sample−luminescence staurosporine sample)]*100.
The term “DR5”, as used herein, refers to death receptor 5, also known as CD262 and TRAILR2, which is a single-pass type I membrane protein with three extracellular cysteine-rich domains (CRD's), a transmembrane domain (TM) and a cytoplasmic domain containing a death domain (DD). In humans, the DR5 protein is encoded by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO 46, (human DR5 protein: UniprotKB/Swissprot O14763).
The term “antibody binding DR5”, “anti-DR5 antibody” DR5-binding antibody”, “DR5-specific antibody”, “DR5 antibody” which may be used interchangeably herein, refers to any antibody binding an epitope on the extracellular part of DR5.”
The term “agonist” as used herein, refers to a molecule such as an anti-DR5 antibody that is able to trigger a response in a cell when bound to DR5, wherein the response may be programmed cell death. That the anti-DR5 antibody is agonistic is to be understood as that the antibody stimulates, activates or clusters DR5 as the result from anti-DR5 binding to DR5. That is an agonistic anti-DR5 antibody comprising an amino acid mutation in the Fc region according to the present invention bound to DR5 results in DR5 stimulation, clustering or activation of the same intracellular signaling pathways as TRAIL bound to DR5. In a particular embodiment, the agonistic activity of one or more antibodies can be determined by incubating target cells for 3 days with an antibody concentration dilution series (e.g. from 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions). The antibodies may be added directly when cells are seeded (described in examples 8, 9, 10, 39), or alternatively the cells are first allowed to adhere to 96-well flat-bottom plates before adding the antibody samples (described in examples 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 38, 40, 41, 42, 43, 44, 46, 48). The agonistic activity i.e. the agonistic effect can be quantified by measuring the amount of viable cells using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571).
The terms “DR5 positive” and “DR5 expressing” as used herein, refers to tissues or cell lines which show binding of a DR5-specific antibody which can be measured with e.g. flow cytometry or immunohistochemistry.
A “variant” or “antibody variant” of the present invention is an antibody molecule which comprises one or more mutations as compared to a “parent” antibody. Exemplary parent antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a bispecific antibody, a human antibody, humanized antibody, chimeric antibody or any combination thereof.
Exemplary mutations include amino acid deletions, insertions, and substitutions of amino acids in the parent amino acid sequence. Amino acid substitutions may exchange a native amino acid present in the wild-type protein for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:
In the context of the present invention, a substitution in a variant is indicated as:
The three letter code, or one letter code, are used, including the codes Xaa and X to indicate amino acid residue. Accordingly, the notation “E345R” or “Glu345Arg” means, that the variant comprises a substitution of Glutamic acid with Arginine in the variant amino acid position corresponding to the amino acid in position 345 in the parent antibody.
Where a position as such is not present in an antibody, but the variant comprises an insertion of an amino acid, for example: Position-substituted amino acid; the notation, e.g., “448E” is used. Such notation is particular relevant in connection with modification(s) in a series of homologous polypeptides or antibodies. Similarly when the identity of the substitution amino acid residues(s) is immaterial: Original amino acid-position; or “E345”. For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of Glutamic acid for Arginine, Lysine or Tryptophan in position 345: “Glu345Arg,Lys,Trp” or “E345R,K,W” or “E345R/K/W” or “E345 to R, K or W” may be used interchangeably in the context of the invention. Furthermore, the term “a substitution” embraces a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids. For example, a substitution of amino acid E in position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345Q 345R, 345S, 345T, 345V, 345W, and 345Y. This is, by the way, equivalent to the designation 345X, wherein the X designates any amino acid. These substitutions can also be designated E345A, E345C, etc, or E345A, C, ect, or E345A/C/ect. The same applies to analogy to each and every position mentioned herein, to specifically include herein any one of such substitutions.
For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment).
For the purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment).
The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative, physical or functional amino acids substitutions at most 5 mutations or substitutions selected from conservative, physical or functional amino acids in total across the six CDR sequences of the antibody binding region, such as at most 4 mutations or substitutions selected from conservative, physical or functional amino acids, such as at most 3 mutations or substitutions selected from conservative, physical or functional amino acids, such as at most 2 mutations selected from conservative, physical or functional amino acids or substitutions, such as at most 1 mutation or substitution selected from a conservative, physical or functional amino acid, in total across the six CDR sequences of the antibody binding region. The conservative, physical or functional amino acids are selected from the 20 natural amino acids found i.e, Arg (R), His (H), Lys (K), Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Cys (C), Gly (G), Pro (P), Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Trp (W), Tyr (Y) and Val (V).
The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative, physical or functional amino acids substitutions; for instance at least about 75%, about 80% or more, about 85% or more, about 90% or more, (e.g., about 75-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are mutations or substitutions selected from conservative, physical or functional amino acids residue replacements. The conservative, physical or functional amino acids are selected from the 20 natural amino acids found i.e, Arg (R), His (H), Lys (K), Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Cys (C), Gly (G), Pro (P), Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Trp (W), Tyr (Y) and Val (V).
An amino acid or segment in one sequence that “corresponds to” an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings. Hence a standard sequence alignment program can be used to identify which amino acid in an e.g. immunoglobulin sequence corresponds to a specific amino acid in e.g. human IgG1. Further a standard sequence alignment program can be used to identify sequence identity e.g. a sequence identity to SEQ ID NO:29 of at least 80%, or 85%, 90%, or at least 95%. For example, the sequence alignments shown in
The term “vector,” as used herein, refers to a nucleic acid molecule capable of inducing transcription of a nucleic acid segment ligated into the vector. One type of vector is a “plasmid”, which is in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, wherein the nucleic acid segment may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for instance bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas, such as CHO-S cells, CHO DG44 cells, HEK-293F cells, Expi293F cells, PER.C6, NS0 cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi, as well as prokaryotic cells such as E. coli.
As described above, in a first main aspect, the invention relates to a pharmaceutical composition comprising:
In one embodiment of the invention relates to a pharmaceutical composition comprising:
The pharmaceutical composition of the invention is typically a liquid aqueous solution.
In one embodiment of the pharmaceutical composition of the invention, the composition comprises from 5 mM to 100 mM histidine, e.g. from 5 mM to 75 mM, such as from 10 mM to 50 mM, e.g. from 15 mM to 45 mM, such as from 20 mM to 40 mM, e.g. from 25 to 35 mM, such as from 28 mM to 32 mM, e.g. 30 mM histidine.
In one embodiment, the pH is from 5.8 to 7.2, such as 5.5 to 6.5, e.g. 5.8 to 6.2, e.g. 5.9 to 6.1, such as 6.0.
In another embodiment, the pharmaceutical composition comprises from 25 mM to 500 mM sodium chloride, e.g. from 25 mM to 250 mM, such as from 50 mM to 250 mM, e.g. from 100 mM to 200 mM, such as from 125 mM to 175 mM, e.g. 150 mM sodium chloride.
In one embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 2 mg/ml to 40 mg/ml antibody at a pH between 5.5 and 6.5, preferably wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 20 mg/ml antibody at pH 6.
In one embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 15 mg/ml to 25 mg/ml antibody at a pH between 5.5 and 6.5, preferably wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 20 mg/ml antibody at pH 6.
In a further embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 2 mg/ml to 20 mg/ml antibody at a pH between 5.5 and 6.5, preferably wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 10 mg/ml antibody at pH 6.0.
In another embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 2 mg/ml to 20 mg/ml antibody at a pH between 5.5 and 6.5, preferably wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 20 mg/ml antibody at pH 6.0.
In a preferred embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 2 mg/ml to 40 mg/ml antibody at a pH between 5.5 and 6.5, preferably wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 20 mg/ml antibody at pH 6.0.
In another embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 2 mg/ml to 40 mg/ml antibody at a pH between 5.5 and 6.5, such as wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 30 mg/ml antibody at pH 6.0.
In a further embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 2 mg/ml to 40 mg/ml antibody at a pH between 5.5 and 6.5, such as wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 40 mg/ml antibody at pH 6.0.
The pharmaceutical compositions may be formulated with further pharmaceutically-acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in (Rowe et al., Handbook of Pharmaceutical Excipients, 2012 June, ISBN 9780857110275). Such optional further pharmaceutically-acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the antibody and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the chosen compound or pharmaceutical composition of the present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.) upon antigen binding).
Pharmaceutically-acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption-delaying agents, and the like that are physiologically compatible with the other components of the composition. Other examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof. A pharmaceutical composition of the present invention may further include fillers, salts, buffers, detergents (e. g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
In one embodiment, the pharmaceutical composition of the invention does not comprise a surfactant. In another embodiment, the pharmaceutical composition does not comprise a cryoprotectant. In a further embodiment, no other excepients than the histidine buffer and sodium chloride are added to the antibody preparation to prepare the composition.
The actual dosage levels of the antibody in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of antibody which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Pharmaceutical compositions for injection or infusion must typically be sterile and stable under the conditions of manufacture and storage.
In one embodiment, the antibody concentration in the pharmaceutical composition is from 0.5 mg/ml to 250 mg/ml, such as from 1 mg/ml to 100 mg/ml, e.g. from 1 mg/ml to 50 mg/ml, such as from 2 mg/ml to 20 mg/ml, e.g. from 5 ml/ml to 15 mg/ml, such as 10 mg/ml.
In a preferred embodiment of the invention the antibody concentration is the pharmaceutical composition is 20 mg/ml. In one embodiment of the invention the antibody concentration in the pharmaceutical composition is from 18-20 mg/ml. In one embodiment of the invention the antibody concentration in the pharmaceutical composition is from 19-21 mg/ml.
In one embodiment of the invention the antibody concentration is the pharmaceutical composition is 40 mg/ml.
In one embodiment of the invention the antibody concentration is the pharmaceutical composition is 60 mg/ml.
In one embodiment of the invention the antibody concentration is the pharmaceutical composition is 80 mg/ml.
In one embodiment of the invention the antibody concentration is the pharmaceutical composition is 100 mg/ml.
Antibodies Formulated in the Pharmaceutical Composition of the Invention
As described above, the antibody formulated in the pharmaceutical composition of the invention comprises an Fc region of a human immunoglobulin G and an antigen binding region, wherein the Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering, with the proviso that the mutation in S440 is S440Y or S440W. The positions corresponding to E430, E345 and S440 in human IgG1 according to EU numbering are located in the CH3 domain of the Fc region.
The antibody in the pharmaceutical composition of the invention comprises an Fc region comprising a first and a second heavy chain, wherein a mutation at a position corresponding to E430, E345 or S440 in human IgG1 according to EU numbering is present in both the first and the second heavy chain, or less preferred, is only present in one of the heavy chains. In the context of the present invention the term hexamerization enhancing mutation refers to an amino acid mutation at a position corresponding to E430, E345 or S440 in human IgG1 according to EU numbering, with the proviso that the mutation in S440 is S440Y or S440W. The hexamerixation enhancing mutation strengthens the Fc-Fc interactions between antibodies comprising the mutation when bound to the corresponding target on a cell surface (WO2013/004842; WO2014/108198).
In one embodiment, the Fc region of the antibody comprises a mutation corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y or S440W in human IgG1, EU numbering. Thus the antibody comprises a mutation selected from the group of: E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in human IgG1, EU numbering. Hereby are embodiments provided that allow for enhanced hexamerization of antibodies upon cell-surface antigen binding. The antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the above mentioned hexamerization enhancing mutations may be present in the first and/or the second heavy chain.
In a preferred embodiment, the Fc region comprises a mutation corresponding to E430G or E345K in human IgG1 EU numbering. Thus the Fc region comprises a mutation selected from E430G and E345K.
In one embodiment, the antibody comprises a mutation at an amino acid position corresponding to E430 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: E430G, E430S, E430F and E430T. In one embodiment the Fc region comprises a mutation corresponding to E430G. Thus in one embodiment the Fc region comprises an E430G mutation.
In one embodiment the antibody comprises a mutation at an amino acid position corresponding to E345 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: E345K, E345Q, E345R and E345Y. In one embodiment the Fc region comprises a mutation corresponding to E345K. Thus in one embodiment the Fc region comprises an E345K mutation.
In one embodiment the antibody comprises a mutation at an amino acid position corresponding to S440 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: S440W and S440Y. In one embodiment the Fc region comprises a mutation corresponding to S440Y. Thus in one embodiment the Fc region comprises an S440Y mutation.
In one embodiment the Fc region comprises a further hexamerization-inhibiting mutation such as K439E or S440K in human IgG1, EU numbering. The hexamerization-inhibiting mutation such as K439E or S440K prevent Fc-Fc interaction with antibodies comprising the same hexamerization inhibiting mutation, but by combining antibodies with a K439E mutation and antibodies with a S440K mutation the inhibiting effect is neutralized and Fc-Fc interactions is restored. In one embodiment the antibody comprises a further mutation at an amino acid position corresponding to one of the following positions S440 or K439 in human IgG1, EU numbering. In one embodiment the Fc region comprises a further mutation in a position corresponding to S440 or K439, with the proviso that the further mutation is not in position S440 if the hexamerization enhancing mutation is in S440. Antibodies comprising a mutation in a position corresponding to E430, E345 or S440 according to the present invention and a further mutation at an amino acid position corresponding to K439 such as a K439E mutation do not form oligomers with antibodies comprising a further mutation at an amino acid position corresponding to K439 such as a K439E mutation. However, antibodies comprising hexamerization enhancing mutation in E430, E345 or S440 and a further mutation in K439 such a K439E do form oligomers with antibodies comprising a hexamerization enhancing mutation in E430 or E345 and a further mutation in S440 such as S440K. Antibodies comprising a mutation in a position corresponding to E430 or E345 according to the present invention and a further mutation at an amino acid position corresponding to S440 such as a S440K mutation do not form oligomers with antibodies comprising a further mutation at an amino acid position corresponding to S440 such as a S440K mutation. However, antibodies comprising hexamerization enhancing mutation in E430 or E345 and a further mutation in S440 such a S440K do form oligomers with antibodies comprising a hexamerization enhancing mutation in E430 or E345 and a further mutation in K439 such as K439E. In one embodiment the Fc region comprises a hexamerization enhancing mutation such as E430G and a hexamerization inhibiting mutation such as K439E. In one embodiment the Fc region comprises a hexamerization enhancing mutation such as E345K and a hexamerization inhibiting mutation such as K439E. In another embodiment the Fc region comprises a hexamerization enhancing mutation such as E430G and a hexamerization inhibiting mutation such as S440K. In one embodiment the Fc region comprises a hexamerization enhancing mutation such as E345K and a hexamerization inhibiting mutation such as S440K. In one embodiment the Fc region comprises a hexamerization enhancing mutation such as S440Y and a hexamerization inhibiting mutation such as K439E Hereby embodiments are provided that allow for exclusive hexamerization between combinations of antibodies comprising a K439E mutation and antibodies comprising a S440K mutation.
In a preferred embodiment, the pharmaceutical composition of the invention comprises an anti-DR5 antibody, i.e. an antibody comprising an antigen binding region which binds to DR5.
In one embodiment, the pharmaceutical composition comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 2 mg/ml to 200 mg/ml anti-DR5 antibody at a pH between 5.5 and 6.5, preferably wherein the composition comprises 30 mM histidine, 150 mM sodium chloride and 20 mg/ml anti-DR5 antibody at pH 6.0. In one embodiment, the pharmaceutical composition comprise from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 10 mg/ml to 40 mg/ml anti-DR5 antibody at a pH between 5.5 and 6.5. In one embodiment, the pharmaceutical composition comprise from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 15 mg/ml to 30 mg/ml anti-DR5 antibody at a pH between 5.5 and 6.5.
In one embodiment, the pharmaceutical composition comprise from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and from 18 mg/ml to 25 mg/ml anti-DR5 antibody at a pH between 5.5 and 6.5 e.g. at a pH between 5.8 and 6.2.
In one embodiment of the invention the composition comprises 30 mM histidine, 150 mM sodium chloride and 10 mg/ml anti-DR5 antibody at pH 6.0. In one embodiment of the invention the composition comprises 30 mM histidine, 150 mM sodium chloride and 30 mg/ml anti-DR5 antibody at pH 6.0. In one embodiment of the invention the composition comprises 30 mM histidine, 150 mM sodium chloride and 40 mg/ml anti-DR5 antibody at pH 6.0. In one embodiment of the invention the composition comprises 30 mM histidine, 150 mM sodium chloride and 50 mg/ml anti-DR5 antibody at pH 6.0. In one embodiment of the invention the composition comprises 30 mM histidine, 150 mM sodium chloride and 100 mg/ml anti-DR5 antibody at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition from 2-200 mg/ml and said second anti-DR5 antibody is present in the composition from 2-200 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5, preferably wherein the composition comprises 10 mg/ml of said first anti-DR5 antibody, 10 mg/ml of said second anti-DR5 antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition from 10 mg/ml to 40 mg/ml and said second anti-DR5 antibody is present in the composition from 10 mg/ml to 40 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition from 10 mg/ml to 40 mg/ml and said second anti-DR5 antibody is present in the composition from 10 mg/ml to 40 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.8 and 6.2.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition from 15 mg/ml to 30 mg/ml and said second anti-DR5 antibody is present in the composition from 15 mg/ml to 30 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition from 15 mg/ml to 30 mg/ml and said second anti-DR5 antibody is present in the composition from 15 mg/ml to 30 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.8 and 6.2.
In one embodiment of the invention the pharmaceutical composition may also contain impurities, such as protein impurities e.g. antibody impurities. Protein impurities may be less than 0.1 mg/ml. In one embodiment the pharmaceutical composition comprises 0.1 mg/ml of protein impurities e.g antibody impurities. In one embodiment the pharmaceutical composition comprises less than 0.1 mg/ml of protein impurities e.g antibody impurities. In one embodiment the pharmaceutical composition comprises less than 0.09 mg/ml of protein impurities e.g antibody impurities. In one embodiment the pharmaceutical composition comprises less than 0.07 mg/ml of protein impurities e.g antibody impurities. In one embodiment the pharmaceutical composition comprises less than 0.05 mg/ml of protein impurities e.g antibody impurities. In one embodiment the pharmaceutical composition comprises less than 0.03 mg/ml of protein impurities e.g antibody impurities. In one embodiment the pharmaceutical composition comprises less than 0.001 mg/ml of protein impurities e.g antibody impurities.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition from 15 mg/ml to 30 mg/ml and said second anti-DR5 antibody is present in the composition from 15 mg/ml to 30 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride and less than 0.1 mg/ml of protein impurities e.g antibody impurities at a pH between 5.8 and 6.2.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition at 20 mg/ml and said second anti-DR5 antibody is present in the composition at 20 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 20 mg/ml of a first anti-DR5 antibody, 20 mg/ml of a second anti-DR5 antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody is present in the composition at 40 mg/ml and said second antibody is present in the composition at 40 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 40 mg/ml of a first anti-DR5 antibody, 40 mg/ml of a second anti-DR5 antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
The human DR5 molecule (Uniprot O14763) is comprised of 440 amino acids including a signaling peptide at the first 1-55 positions, followed by the extracellular domain at positions 56-210, a transmembrane domain at positions 211-231 and a cytoplasmic domain at positions 232-440. The extracellular domain is comprised of a 155 amino acid sequence. The short isoform of DR5 (Uniprot O14763-2) is missing 185-213 from the extracellular domain compared to the long version (Uniprot O14763) comprising the amino acids at position 56-210.
In one embodiment the anti-DR5 antibody comprises an antigen binding region binding to an epitope within the extracellular domain of DR5.
In one embodiment the antibody comprises an antigen binding region binding to the same binding site as TRAIL or a binding site overlapping with the binding site of TRAIL. The TRAIL binding motif is located in CRD2 and CRD3 based on a Crystal structure of TRAIL in complex with the DR5 ectodomain (Hymowitz et al., Mol Cell. 1999 October; 4(4):563-71). That is, in one embodiment the antibody comprises an antigen binding region binding to the same binding region on DR5 as TRAIL. Thus in one embodiment the DR5 antibody binds to CRD2 and/or CRD3 on DR5. In one embodiment the antibody comprises an antigen binding region that blocks TRAIL binding to DR5. In one embodiment the antibody comprises an antigen binding region that competes with TRAIL binding to DR5. In one embodiment the antibody blocks TRAIL induced mediated killing such as TRAIL induced apoptosis.
In another embodiment the antibody comprises an antigen binding region binding to an epitope on DR5 that is different from the binding site of TRAIL. In one embodiment the antibody comprises an antigen binding region binding to a different binding region on DR5 than TRAIL. In one embodiment the antibody does not block TRAIL induced mediated killing such as TRAIL induced apoptosis.
In an embodiment of the invention the antibody comprises an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 116-138 and one or more amino acid residues located within amino acid residues 139-166 of SEQ ID NO 46. That is the antigen binding region binds to or requires for binding to DR5 one or more amino acids located within positions 116-138 and one or more amino acids located within positions 139-166. That the antigen binding region binds to one or more amino acids comprised in a sequence is to be understood as the antigen binding region is in contact with or directly interacts with one or more amino acids within the sequence. That the antigen binding region requires one or more amino acids within a sequence means that no contact or direct interaction between antigen binding region and one or more amino acids in the sequence is needed, but that one or more amino acids are required for keeping the three-dimensional structure of the epitope.
The epitope or binding region on the extracellular domain on human DR5 of the antibodies of the present invention may be determined by use of the method of domain-swapped DR5 molecules as described in Example 6. In brief, domain-swapped DR5 molecules are transiently expressed in CHO cells, binding of antibodies to the domain-swapped human DR5 molecules are determined by a FACS assay. Loss of binding to the domain-swapped human DR5 molecules indicate that the swapped domain of human DR5 contains one or more amino acids that are involved in binding to the antibody.
In another preferred embodiment the antibody comprises an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 79-138 of SEQ ID NO 46.
In one embodiment the anti-DR5 antibody comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the amino acid sequences of:
In one embodiment the anti-DR5 antibody comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the amino acid sequences of:
In one embodiment the anti-DR5 antibody comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the amino acid sequences of:
That is in one embodiment up to five mutations such as substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains, wherein said VH region and said VL region has at least 75%, 80%, 85% 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the six CDR sequences selected from the group consisting of:
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains, wherein said VH region and said VL region has at least 75%, 80%, 85% 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the six CDR sequences selected from the group consisting of:
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains, wherein said VH region and said VL region has at least 75%, 80%, 85% 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the six CDR sequences selected from the group consisting of:
In one embodiment the anti-DR5 antibody comprises a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the CDR sequences selected from one of the groups consisting of:
c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in any one of (a) or (b) above having one to five mutations in total across said six CDR sequences. That is in one embodiment up to five mutations such as substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment the anti-DR5 antibody comprises a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the CDR sequences selected from one of the groups consisting of:
c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having up to five mutations in total across said six CDR sequences.
That is in one embodiment up to five mutations such as substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions, such as one, two, three, four or five mutations e.g. substitutions are made across the three CDRs of the VH region and no mutations are made across the three CDRs or the VL region. In other embodiments no mutations e.g. substitutions are made across the three CDRs of the VH region but up to five mutations e.g. substitutions are made across the six CDRs of the VL region, wherein the mutations e.g. substitutions are conservative or concern amino acids with similar physical or functional properties and preferably do not modify binding affinity to DR5.
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises an antigen binding region comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein said VH region and said VL region has at least 75%, 80%, 85% 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the VH and VL sequences selected from the group consisting of:
In one embodiment the antibody comprises an antigen binding region comprising a variable heavy chain (VH) region and a variable light chain (VL) region having the amino acid sequences of:
That is in one embodiment up to 10 mutations such as substitutions in total are allowed across the VH and VL regions defined by the VH and VL sequences. In some embodiments of the invention up to ten mutations e.g. substitutions, such as one, two, three, four, five, six, seven, eight, nine or ten mutations e.g. substitutions are made across the VH or VL sequences. In one embodiment of the invention up to 10 mutations or substitutions are made in the VH sequence and no mutations are made in the VL sequence. In one embodiment of the invention no mutations are made in the VH sequence and up to ten mutations e.g. substitutions are made in the VL sequence. Hereby are embodiments provided that allow for up to 10 mutations such as substitutions across the VH and VL sequences, wherein the mutations such as substitutions are conservative or concern amino acids with similar physical or functional properties, thereby allowing mutations e.g. substitutions within the VH and VL sequence without modifying binding affinity or function of the anti-DR5 antibody.
In one embodiment the antibody is a monoclonal antibody. In one embodiment of the present invention the antibody is of the IgG1, IgG2, IgG3 or IgG4 isotype. In a preferred embodiment of the invention the antibody is an IgG1 antibody.
In one embodiment the antibody is an IgG1m(f), IgG1m(z), IgG1m(a) or an IgG1m(x) allotype, or any allotype combination, such as IgG1m(z,a), IgG1m(z,a,x), IgG1m(f,a). In a preferred embodiment the antibody is an IgG1m(f).
In one embodiment the light chain is a kappa light chain. In one embodiment the light chain is a Km3 allotype. In one embodiment the antibody comprises an Fc region comprising an amino acid sequence selected from the group consisting of:
That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the Fc region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are allowed across the Fc region.
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:39 and wherein the HC has at least 75%, 80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:39 and wherein the HC has at least 75%, 80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in (HC) SEQ ID NO:38.
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC has at least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99% amino acid sequence identity set forth in SEQ ID NO:39 and wherein the HC has the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC has at least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99% amino acid sequence identity set forth in SEQ ID NO:39 and wherein the HC has the amino acid sequence as set forth in f) (HC) SEQ ID NO:38.
In one embodiment, the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:39 and wherein the HC comprises of one of the sequences selected from the group consisting of:
That is in one embodiment up to 10 mutations such as substitutions in total are allowed across the heavy chain defined by the heavy chain sequence. In some embodiments of the invention up to ten mutations e.g. substitutions, such as one, two, three, four, five, six, seven, eight, nine or ten mutations e.g. substitutions are made across the heavy chain sequence. Hereby are embodiments provided that allow for up to 10 mutations such as substitutions across the heavy chain sequence, wherein the mutations such as substitutions are conservative or concern amino acids with similar physical or functional properties, thereby allowing mutations or substitutions within the heavy chain sequence without modifying binding affinity or function of the anti-DR5 antibody.
In one embodiment, the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:39 and wherein the HC comprises the sequence of SEQ ID NO:38.
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:43 and wherein the HC has at least 75%, 80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:43 and wherein the HC has at least 75%, 80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in (HC) SEQ ID NO:42.
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC has at least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99% amino acid sequence identity set forth in SEQ ID NO:43 and wherein the HC has the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
In one embodiment, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC has at least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99% amino acid sequence identity set forth in SEQ ID NO:43 and wherein the HC has the amino acid sequence as set forth in the (HC) SEQ ID NO:42.
In one embodiment the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:43 and wherein the HC comprises of one of the sequences selected from the group consisting of:
That is in one embodiment up to 10 mutations such as substitutions in total are allowed across the heavy chain defined by the heavy chain sequence. In some embodiments of the invention up to ten mutations e.g. substitutions, such as one, two, three, four, five, six, seven, eight, nine or ten mutations e.g. substitutions are made across the heavy chain sequence. Hereby are embodiments provided that allow for up to 10 mutations such as substitutions across the heavy chain sequence, wherein the mutations such as substitutions are conservative or concern amino acids with similar physical or functional properties, thereby allowing mutations such as substitutions within the heavy chain sequence without modifying binding affinity or function of the anti-DR5 antibody.
In one embodiment the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:43 and wherein the HC comprises the sequence of SEQ ID NO:42.
In one embodiment the antibody is a human antibody, a chimeric antibody or a humanized antibody.
In one embodiment the antibody is an anti-DR5 antibody and said anti-DR5 antibody is agonistic. That the antibody is agonistic is to be understood as that the antibody clusters, stimulates or activates DR5. In one embodiment, an agonistic anti-DR5 antibody of the present invention bound to DR5 activates the same intracellular pathways as TRAIL bound to DR5. The agonistic activity of one or more antibodies can be determined by incubating target cells expressing DR5, such as COLO 205 cells (ATCC CCL-222) or HCT 116 cells (ATCC CCL-247), for 3 days with an antibody concentration dilution series (e.g. from 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions). The antibodies may be added directly when cells are seeded (described in examples 8, 9, 10, 39), or alternatively the cells are first allowed to adhere to 96-well flat-bottom plates before adding the antibody samples (described in examples 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 38, 40, 41, 42, 43, 44, 46, 48). The agonistic activity i.e. the agonistic effect can be quantified by measuring the amount of viable cells using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571).
In one embodiment the antibody is an anti-DR5 antibody and said anti-DR5 antibody has enhanced agonistic activity. That the anti-DR5 antibody has activity is to be understood as the antibody is able to cluster DR5 or activate at least the same intracellular pathways as TRAIL bound to DR5. That is anti-DR5 antibody with enhanced agonistic activity is able to induce increased level of apoptosis or programmed cell death in a cell or tissue expressing DR5 compared to TRAIL or a wild-type IgG1 antibody against DR5.
In one embodiment the antibody is an anti-DR5 antibody and said anti-DR5 antibody induces programmed cell death in a target cell. In one embodiment of the present invention the anti-DR5 antibody induces caspase-dependent cell death. Caspase-dependent cell death may be induced by activation of caspase-3 and/or caspase-7. In one embodiment of the invention the anti-DR5 antibody induces caspase-3 and/or caspase-7 dependent cell death. In one embodiment of the present invention the antibody induces apoptosis. Apoptosis by one or more agonistic anti-DR5 antibodies can be determined using methods such as, e.g., caspase-3/7 activation assays described in examples 19, 20, 25 and 45 or phosphatidylserine exposure described in examples 19 and 25. Anti-DR5 antibody at a fixed concentration of e.g. 1 μg/mL may be added to adhered cells and incubated for 1 to 24 hours. Caspase-3/7 activation can be determined by using special kits for this purpose, such as the PE Active Caspase-3 Apoptosis Kit of BD Pharmingen (Cat nr 550914) (example 19 and 25) or the Caspase-Glo 3/7 assay of Promega (Cat nr G8091) (examples 20 and 45). Phosphatidylserine exposure and cell death can be determined by using special kits for this purpose, such as the FITC Annexin V Apoptosis Detection Kit I from BD Pharmingen (Cat nr 556547) (examples 19 and 25).
In one embodiment the antibody is an anti-DR5 antibody and said anti-DR5 antibody induces phosphatidylserine (PS) exposure, which can be measured by Annexin-V binding. In one embodiment of the present invention anti-DR5 induces translocation of PS to the cell surface of the target cell. Therefore, Annexin-V binding correlates to programmed cell death and can be used to measure the anti-DR5 antibody's ability to induce cellular events leading to programmed cell death.
In a preferred embodiment the antibody is an anti-DR5 antibody which induces apoptosis in a target cell expressing DR5, such as a tumor cell.
In one embodiment the antibody is an anti-DR5 antibody which reduces cell viability.
In one embodiment the antibody is an anti-DR5 antibody which induces DR5 clustering. That the antibody can induce clustering and even enhance clustering leads to activation of at least the same intracellular signaling pathways as TRAIL bound to DR5.
In one embodiment, the compositions of the present invention comprise an anti-DR5 antibody and induce, trigger, increase or enhance apoptosis or cell death in cancer cells or cancer tissues expressing DR5. The increased or enhanced apoptosis or cell death can be measured by an increase or enhanced level of phosphatidylserine exposure on cells exposed to or treated with one or more anti-DR5 antibodies of the invention. Alternatively, the increase or enhanced apoptosis or cell death can be measured by measuring activation of caspase 3 or caspase 7 in cells that have been exposed to or treated with one or more anti-DR5 antibodies of the invention. Alternatively, the increase or enhanced apoptosis or cell death can be measured by a loss of viability in cell cultures that have been exposed to or treated with one or more anti-DR5 antibodies of the invention, compared to untreated cell cultures. Induction of caspase-mediated apoptosis can be assessed by demonstrating inhibition of the loss of viability after exposure to DR5 antibody by a caspase-inhibitor, for example ZVAD.
In one embodiment of the present invention, the antibody in a pharmaceutical composition of the invention is an anti-DR5 antibody which engages into oligomerization such as hexamerization of antibodies on target cells expressing DR5. Oligomerization such as hexamerization is mediated through Fc-Fc interactions. One method for determining this is by inhibiting Fc-Fc interactions which indicate that antibodies oligomerizies e.g. hexamerizies. The Fc-Fc interactions can be inhibited by a peptide binding to the hydrophobic patch involved in Fc-Fc interactions such as DCAWHLGELVWCT as described in example 15.
Antibodies to be formulated in a pharmaceutical composition of the invention may be produced recombinantly in a host cell by introducing an expression vector carrying sequences coding for the antibody chains. An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a humanized CD3 antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (as described in for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4−-precipitated construct (as described in for instance WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and 5,973,972).
A nucleic acid and/or vector may also comprise a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides, organelle-targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
In an expression vector of the invention, antibody-encoding nucleic acids and the first and the second polypeptides nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE (the skilled artisan will recognize that such terms are actually descriptors of a degree of gene expression under certain conditions).
Antibodies may be produced by use of recombinant eukaryotic or prokaryotic host cells. Examples of host cells include yeast, bacterial and mammalian cells, such as CHO or HEK-293 cells. For example, the host cell may comprise a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of an antibody described herein. The host cell may comprise a nucleic acid stably integrated into the cellular genome that comprise a sequence coding for expression of a first or a second polypeptide described herein. Alternatively, the host cell may comprise a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an antibody described herein.
Bispecific Antibodies Formulated in the Pharmaceutical Composition of the Invention
In another aspect, the pharmaceutical composition of the present invention comprises a bispecific antibody comprising at least one antigen binding region which binds to human DR5, as described herein.
In another aspect, the pharmaceutical composition of the present invention comprises a bispecific antibody comprising one or more antigen binding regions which binds to human DR5, as described herein.
In one embodiment hereof, the bispecific antibody comprises a first antigen binding region and a second antigen binding region which bind to human DR5, as defined herein.
In one such embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region and said second antigen binding region bind different epitopes on human DR5.
In another embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region binding to human DR5 does not block binding of said second antigen binding region binding to human DR5.
In one embodiment, the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the first and/or second Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering according to the invention. In one embodiment, the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the first and second Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering. In one embodiment, the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the first Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering. In one embodiment, the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the second Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region wherein a) said first antigen binding region comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antigen binding region comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NO:s 13, RTS, 14, or wherein the said first antigen binding region and said second antigen binding region comprises b) the six CDR sequences defined in (a having one to five mutations or substitutions in total across said six CDR sequences of each antigen binding region respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein,
That is the one or more mutations e.g. substitutions across the six CDR sequences of each antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following sequences (a) (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6, or b) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above having one to five mutations in total across said six CDR sequences and wherein said second antigen binding region comprises the following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (c) above having one to five mutations in total across said six CDR sequences.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein (a) said first antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen binding region or said second antigen binding region comprises one to five mutations in total across said six CDR sequences of each antigen binding region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following sequences (a) (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6, or (b) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above having one to five mutations in total across said six CDR sequences and wherein said second antigen binding region comprises the following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (c) above having one to five mutations in total across said six CDR sequences.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein (a) said first antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen binding region or said second antigen binding region comprises one to five mutations in total across said six CDR sequences of each antigen binding region.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following sequences (a) (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22, or (b) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above having one to five mutations in total across said six CDR sequences and wherein said second antigen binding region comprises the following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (c) above having one to five mutations in total across said six CDR sequences.
In one embodiment, the bispecific antibody comprises a first and a second antigen binding region, wherein (a) said first antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen binding region or said second antigen binding region comprises one to five mutations in total across said six CDR sequences of each antigen binding region.
If the antibody is a bispecific antibody that comprises an Fc region comprising a first and a second heavy chain, a mutation according to the present invention i.e. a mutation in a position corresponding to E430, E345 or S440 in IgG1, EU numbering, may in principle only be present in one of the heavy chains; i.e. in either the first or second heavy chain, although in a preferred embodiment according to the present invention, the mutation is present in both the first and second heavy chain of the bispecific antibody.
In a particular embodiment the antibody may be bispecific antibody such as the heterodimeric protein described in WO 11/131746, which is hereby incorporated herein by reference.
In one embodiment, the antibody is a bispecific antibody which comprises a first heavy chain comprising a first Fc region of an immunoglobulin and a first antigen-binding region, and a second heavy chain comprising a second Fc region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same antigen or on different antigens.
In a further embodiment said first heavy chain comprising a first Fc region comprises a further amino acid substitution at a position selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy chain; and wherein said second heavy chain comprising a second Fc region comprises a further amino acid substitution at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of a human IgG1 heavy chain, and wherein said further amino acid substitution in the first heavy chain comprising a first Fc region is different from the said further amino acid substitution in the second heavy chain comprising a second Fc region.
In a further embodiment said first heavy chain comprising a first Fc region comprises an amino acid substitution at a position corresponding to K409 in the Fc-region of a human IgG1 heavy chain; and said second heavy chain comprising a second Fc region comprises an amino acid substitution at a position corresponding to F405 in the Fc-region of a human IgG1 heavy chain.
In one embodiment, the bispecific antibody comprises introducing a first and second Fc region comprising a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In a further embodiment the mutation in the first and second Fc region in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, may be in the same amino acid residue position or a different position. In a further embodiment it may be the same or a different mutation in the same amino acid residue position in the first and second Fc region.
In another embodiment the bispecific antibody comprises a first or second CH2-CH3 region comprising a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment, the bispecific antibody comprises a first and a second heavy chain, wherein said first heavy chain comprises a mutation corresponding to F405L in human IgG1 according to EU numbering and said second heavy chain comprises a mutation corresponding to K409R in human IgG1 according EU numbering.
Compositions of the Invention Comprising Two or More Antibodies
In one aspect, the invention relates to a pharmaceutical composition comprising two or more antibodies, wherein at least one of the antibodies is an antibody comprising an Fc region of a human immunoglobulin G and an antigen binding region, wherein the Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering.
In one embodiment of the invention the pharmaceutical composition comprising two or more antibodies, wherein at least one of the antibodies is an antibody comprising an Fc region of a human immunoglobulin G and an antigen binding region, wherein the Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering, with the proviso that the mutation in S440 is S440Y or S440W.
In a further embodiment, the pharmaceutical composition of the invention comprises two different antibodies wherein both antibodies comprise an Fc region of a human immunoglobulin G and an antigen binding region wherein the Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering.
In a further embodiment, the pharmaceutical composition of the invention comprises two different antibodies wherein both antibodies comprise an Fc region of a human immunoglobulin G and an antigen binding region wherein the Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody as described herein. That is in one embodiment of the present invention the composition comprises a first antibody as described herein and a second antibody as described herein, wherein the first and the second antibody are not identical.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and comprising a mutation in the first Fc region at the position corresponding to E430 in human IgG1, EU numbering and a second anti-DR5 antibody having a second Fc region and comprising a mutation in the second Fc region at the position corresponding to E430 in human IgG1, EU numbering, wherein the first and second antibody bind different epitopes on DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and comprising a mutation in the first Fc region at the position corresponding to E430 in human IgG1, EU numbering and a second anti-DR5 antibody having a second Fc region and comprising a mutation in the second Fc region at the position corresponding to E430 in human IgG1, EU numbering, wherein the first antibody does not block binding of the second antibody to DR5. Blocking of one anti-DR5 antibody by another anti-DR5 antibody may be determined in a sandwich enzyme-linked immunoabsorbent assay (ELISA) as described in Example 7. In brief crossblocking by anti-DR5 antibodies may be determined by the following steps, a) 2 μg/ml of a first anti-DR5 antibody is coated on a 96-well flat bottom ELISA plate followed by b) blocking with PBSA and washing the plate in PBST followed by c) incubating the plate with 0.2 μg/ml DR5EDCD-FcHistag and 1 μg/ml of a second anti-DR5 antibody followed by d) washing in PBST and incubating the plate with an anti-His tag antibody followed by e) washing the plate and incubating the plate with poly-HRP followed by f) incubating the plate with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) followed by g) stopping the substrate reaction by adding 2% oxalic acid followed by h) measuring fluorescence at 405 nm on an ELISA reader. Whether one anti-DR5 antibody blocks binding to DR5 by another anti-DR5 antibody may be calculated by the following formula (% inhibition=100−[(binding in presence of competing antibody/binding in absence of competing antibody)]*100).
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody having a first and second Fc region comprising a mutation in the first and second Fc region at a position corresponding to E430 in human IgG1, EU numbering, such a mutation may be selected from the group consisting of: E430G, E430S and E430T.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and comprising an E430G mutation and a second anti-DR5 antibody having a second Fc region and comprising an E430G mutation, wherein the first and second antibody binds different epitopes on DR5. The epitope or binding region on the extracellular domain on human DR5 of the antibodies of the present invention may be determined by use of the method of domain-swapped DR5 molecules as described in Example 6. In brief, domain-swapped DR5 molecules are transiently expressed in CHO cells, binding of antibodies to the domain-swapped human DR5 molecules are determined by a FACS assay. Loss of binding to the domain-swapped human DR5 molecules indicate that the swapped domain of human DR5 contains one or more amino acids that are involved in binding to the antibody.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences:
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences:
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences:
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having an Fc region and comprising a mutation in the Fc region at a position corresponding to E345 in human IgG1, EU numbering and a second anti-DR5 antibody having an Fc region and comprising a mutation in the Fc region at a position corresponding to E345 in human IgG1, EU numbering, wherein the first and second antibody binds different epitopes on DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having an Fc region and comprising a mutation in the Fc region at a position corresponding to E345 in human IgG1, EU numbering and a second anti-DR5 antibody having an Fc region and comprising a mutation in the position corresponding to E345 in human IgG1, EU numbering, wherein the first antibody does not block binding of the second antibody to DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody having a first and second Fc region and comprising a mutation in the first and second Fc region at a position corresponding to E345, such a mutation may be selected from the group consisting of: E345K, E345Q, E345R and E345Y.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and comprising an E345K and a second anti-DR5 antibody having a second Fc region and comprising an E345K mutation, wherein the first and second antibody binds different epitopes on DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the first and second Fc region at a position corresponding to E345 in human IgG1, EU numbering.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E345K mutation in the first and second Fc region.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the first and second Fc region at a position corresponding to E345 in human IgG1, EU numbering.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E345K mutation in the first and second Fc region.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody having a first and second Fc region and comprising a mutation in the first and second Fc region at a position corresponding to S440 in human IgG1, EU numbering, such a mutation may be selected from the group consisting of: S440W and S440Y.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an S440Y mutation in the first and second Fc region.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an S440Y mutation in the first and second Fc region.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody having a first Fc region and a second anti-DR5 antibody having a second Fc region wherein the first and the second antibody comprises a further hexamerization-inhibiting mutation in the first and second Fc region corresponding to K439E or S440K in human IgG1 EU numbering. In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody having a first and second Fc region, wherein the first and second anti-DR5 antibody comprises a hexamerization enhancing mutation in the first and second Fc region at an amino acid position corresponding to E430, E345 or S440 in human IgG1, EU numbering and wherein the first antibody comprises a further mutation in an amino acid at a position corresponding to K439 and wherein the second antibody comprises a further mutation in an amino acid at a position corresponding to S440, with the proviso that the hexamerization enhancing mutation is not in S440 when the further mutation is in S440. That is in one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises a hexamerization enhancing mutation such as E430G and K439E, and wherein the second anti-DR5 antibody comprises a hexamerization enhancing mutation such as E430G and S440K. That is in one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises a hexamerization enhancing mutation such as E345K and K439E, and wherein the second anti-DR5 antibody comprises a hexamerization enhancing mutation such as E345K and S440K. Hereby are embodiments provided that allow compositions wherein hexamerization exclusively occur between combinations of antibodies comprising a K439E mutation and antibodies comprising a S440K mutation.
In one embodiment of the present invention the pharmaceutical composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody binding different epitopes on human DR5. In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 116-138 and one or more amino acid residues located within amino acid residues 139-166 of SEQ ID NO 46 and a second anti-DR5 antibody comprising an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 79-138 of SEQ ID NO 46.
In one embodiment of the present invention the pharmaceutical composition comprises said first anti-DR5 antibody binding to DR5, which does not block binding of said second anti-DR5 antibody to DR5. That is in one embodiment of the invention the composition comprises a first anti-DR5 antibody binding to DR5 and a second anti-DR5 antibody binding to DR5, wherein the first and the second anti-DR5 antibody does not compete for binding to DR5. Thus it is to be understood in the context of the present invention that a first anti-DR5 antibody that does not block binding of a second anti-DR5 antibody may be the same as a first anti-DR5 antibody that does not compete with a second anti-DR5 antibody.
In one embodiment of the invention, the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following: a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6; and said second antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14. In one embodiment thereof the sequence identity of the six CDR sequences in total of said first antibody and said second antibody is at least 85%, 90%, 95%, 97%, or 99%.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following six CDR sequences,
That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein
That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention, the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following: a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS; and said second antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14. In one embodiment thereof the sequence identity of the six CDR sequences in total of said first antibody and said second antibody is at least 85%, 90%, 95%, 97%, or 99%.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein
a) said first antibody comprises the following six CDR sequences, (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein
b) the said first antibody and said second antibody comprise the six CDR sequences defined in a) having one to five mutations or substitutions in total across said six CDR sequences respectively.
That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein
a) said first antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b) the said first antibody and said second antibody comprises the six CDR sequences of each antibody defined in (a) or comprising one to five mutations e.g. substitutions in total across said six CDR sequences respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention, the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following: a) (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6; and said second antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14.
In one embodiment thereof the sequence identity of the six CDR sequences in total of said first antibody and said second antibody is at least 85%, 90%, 95%, 97%, or 99%.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein
a) said first antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6 and said second antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b) the said first antibody and said second antibody comprise the six CDR sequences of each antibody defined in (a) or comprise one to five mutations e.g. substitutions in total across said six CDR sequences respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody as defined in any of the above embodiments wherein said first and second antibody further comprises a mutation in the Fc region corresponding to position K439 or S440 in human IgG1, EU numbering. In one embodiment of the invention the composition comprises a first antibody comprising a mutation corresponding to K439 such as K439E and a second antibody comprising a mutation corresponding to S440 such as S440K. In one embodiment o fthe invention the composition comprises a first antibody comprising a mutation corresponding to S440 such as S440K and a second antibody comprising a mutation corresponding to K439 such as K439E. Hereby embodiment are provided wherein the composition comprises a first antibody comprising at least two mutations such as E430G and K439E and a second antibody comprising at least two mutations such as E430G and S440K. In another embodiment of the present invention the composition comprises a first antibody comprising at least two mutations such as E345K and K439E and a second antibody comprising at least two mutations such as E345K and S440K. Hereby are embodiments provided that allow for hexamerization of antibodies with different specificities.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following sequences (a) (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody comprises the following sequences (b) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first and second antibody comprises the following CDR sequences (a) said first antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a) for each antibody comprising one to five mutations e.g. substitutions in total across said CDR sequences for each antibody. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following sequences (a) (VH) CDR1 SEQ ID NOs 1, CDR2 2, CDR3 3 and (VL) CDR1 SEQ ID NOs 5, CDR2 FAS, CDR3 6 and said second antibody comprises the following sequences (b) (VH) CDR1 SEQ ID NOs 10, CDR2 2, CDR3 11 and (VL) SEQ ID NOs CDR1 13, CDR2 RTS, CDR3 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first and second antibody comprises the following CDR sequences (a) said first antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a) for each antibody comprising one to five mutations e.g. substitutions in total across said CDR sequences for each antibody. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following sequences (a) (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antibody comprises the following sequences (b) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first and second antibody comprises the following CDR sequences (a) said first antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a) for each antibody comprising one to five mutations e.g. substitutions in total across said CDR sequences for each antibody. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the invention, the pharmaceutical composition comprises a first and a second antibody wherein both antibodies comprise an an Fc region of a human immunoglobulin G and an antigen binding region, wherein the Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering, wherein said first antibody and said second antibody are present in the composition at a 1:49 to 49:1 molar ratio, such as 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:35 molar ratio, a 1:40 molar ratio, a 1:45 molar ratio a 1:50 molar ratio, a 50:1 molar ratio, a 45:1 molar ratio, a 40:1 molar ratio, a 35:1 molar ratio, a 30:1 molar ratio a 25:1 molar ratio, a 20:1 molar ratio, a 15:1 molar ratio, a 10:1 molar ratio, a 9:1 molar ratio, a 8:1 molar ratio, a 7:1 molar ratio, a 6:1 molar ratio, a 5:1 molar ratio, a 4:1 molar ratio, a 3:1 molar ratio, a 2:1 molar ratio.
In one embodiment of the invention, the pharmaceutical composition comprises a first and a second antibody wherein both antibodies comprise an an Fc region of a human immunoglobulin G and an antigen binding region, wherein the Fc region comprises a mutation of an amino acid at a position corresponding to E430, E345 or S440 in human IgG1, EU numbering, with the proviso that the mutation in S440 is S440Y or S440W, wherein said first antibody and said second antibody are present in the composition at a 1:49 to 49:1 molar ratio, such as about a 1:1 molar ratio, about a 1:2 molar ratio, about a 1:3 molar ratio, about a 1:4 molar ratio, about a 1:5 molar ratio, about a 1:6 molar ratio, about a 1:7 molar ratio, about a 1:8 molar ratio, about a 1:9 molar ratio, about a 1:10 molar ratio, about a 1:15 molar ratio, about a 1:20 molar ratio, about a 1:25 molar ratio, about a 1:30 molar ratio, about a 1:35 molar ratio, about a 1:40 molar ratio, about a 1:45 molar ratio, about a 1:50 molar ratio, about a 50:1 molar ratio, about a 45:1 molar ratio, about a 40:1 molar ratio, about a 35:1 molar ratio, about a 30:1 molar ratio, about a 25:1 molar ratio, about a 20:1 molar ratio, about a 15:1 molar ratio, about a 10:1 molar ratio, about a 9:1 molar ratio, about a 8:1 molar ratio, about a 7:1 molar ratio, about a 6:1 molar ratio, about a 5:1 molar ratio, about a 4:1 molar ratio, about a 3:1 molar ratio, about a 2:1 molar ratio.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody and said second antibody are present in the composition at a 1:9 to 9:1 molar ratio.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody and said second antibody are present in the composition at about a 1:9 to 9:1 molar ratio.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody and said second antibody are present in the composition at about a 1:4 to 4:1 molar ratio, such as about a 1:3 to 3:1 molar ratio, such as about a 1:2 to 2:1 molar ratio.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody and said second antibody are present in the composition at approximately a 1:1 molar ratio.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody and said second antibody are present in the composition at a 1:1 molar ratio.
In a preferred embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody and second antibody and/or any additional antibodies are present in the composition at an equimolar ratio.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody is present in the composition at 5 mg/ml and said second antibody is present in the composition at 5 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 5 mg/ml of a first antibody, 5 mg/ml of a second antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody is present in the composition at 10 mg/ml and said second antibody is present in the composition at 10 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5, preferably wherein the composition comprises 10 mg/ml of said first antibody, 10 mg/ml of said second antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody is present in the composition at 15 mg/ml and said second antibody is present in the composition at 15 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 15 mg/ml of a first antibody, 15 mg/ml of a second antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody is present in the composition at 20 mg/ml and said second antibody is present in the composition at 20 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 20 mg/ml of a first antibody, 20 mg/ml of a second antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody is present in the composition at 30 mg/ml and said second antibody is present in the composition at 30 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 30 mg/ml of a first antibody, 30 mg/ml of a second antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody is present in the composition at 40 mg/ml and said second antibody is present in the composition at 40 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 40 mg/ml of a first antibody, 40 mg/ml of a second antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first antibody is present in the composition at 50 mg/ml and said second antibody is present in the composition at 50 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 50 mg/ml of a first antibody, 50 mg/ml of a second antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first and second antibody is present in the composition at a total antibody concentration of 10 mg/ml antibody and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises a first and a second antibody at a total antibody concentration of 10 mg/ml of antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first and second antibody is present in the composition at a total antibody concentration of 20 mg/ml antibody and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises a first and a second antibody at a total antibody concentration of 20 mg/ml of antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first and second antibody is present in the composition at a total antibody concentration of 30 mg/ml antibody and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises a first and a second antibody at a total antibody concentration of 30 mg/ml of antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first and second antibody is present in the composition at a total antibody concentration of 40 mg/ml antibody and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises a first and a second antibody at a total antibody concentration of 40 mg/ml of antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second antibody, wherein said first and second antibody is present in the composition at a total antibody concentration of 50 mg/ml antibody and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises a first and a second antibody at a total antibody concentration of 50 mg/ml of antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises an anti-DR5 antibody, the anti-DR5 antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:39 and wherein the HC comprises one of the sequences selected from the group consisting of:
In one embodiment of the invention the pharmaceutical composition comprises a an anti-DR5 antibody, the anti-DR5 antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:43 and wherein the HC comprises one of the sequences selected from the group consisting of:
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises a HC sequence selected from the group consisting of a) SEQ ID NO:33; b) SEQ ID NO:34; c) SEQ ID NO:35; d) SEQ ID NO:36; e) SEQ ID NO:37; or f) SEQ ID NO:38 and LC sequence ID NO: 39, said second anti-DR5 antibody comprises a HC sequence selected from the group consisting of g) SEQ ID NO:40; H) SEQ ID NO:41; or i) SEQ ID NO:42 and LC sequence NO:43, said first anti-DR5 antibody is present in the composition from 2 mg/ml to 200 mg/ml, and said second anti-DR5 antibody is present in the composition from 2 mg/ml to 200 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 10 mg/ml of a first anti-DR5 antibody, 10 mg/ml of a second anti-DR5 antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises HC sequence ID NO: 38 and LC sequence ID NO: 39, said second anti-DR5 antibody comprises HC sequence ID NO: 42 and LC sequence NO:43, said first anti-DR5 antibody is present in the composition from 2 mg/ml to 200 mg/ml, and said second anti-DR5 antibody is present in the composition from 2 mg/ml to 200 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5. In one embodiment of the invention the composition comprises 10 mg/ml of a first anti-DR5 antibody, 10 mg/ml of a second anti-DR5 antibody, 30 mM histidine, 150 mM sodium chloride at pH 6.0.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises HC SEQ ID NO: 38 and LC SEQ ID NO: 39, said second anti-DR5 antibody comprises HC SEQ ID NO: 42 and LC SEQ ID NO:43, said first anti-DR5 antibody is present in the composition from 10 mg/ml to 20 mg/ml, and said second anti-DR5 antibody is present in the composition from 10 mg/ml to 20 mg/ml and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises HC SEQ ID NO: 38 and LC SEQ ID NO: 39, said second anti-DR5 antibody comprises HC SEQ ID NO: 42 and LC SEQ ID NO:43, said first anti-DR5 antibody is present in the composition at 10 mg/ml, and said second anti-DR5 antibody is present in the composition at 10 mg/ml, and wherein the composition further comprises from 10 mM to 50 mM histidine, from 50 mM to 250 mM sodium chloride at a pH between 5.5 and 6.5.
In one embodiment of the invention the pharmaceutical composition comprises a first and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises HC SEQ ID NO: 38 and LC SEQ ID NO: 39, said second anti-DR5 antibody comprises HC SEQ ID NO: 42 and LC SEQ ID NO:43, said first anti-DR5 antibody is present in the composition at 10 mg/ml, and said second anti-DR5 antibody is present in the composition at 10 mg/ml, and wherein the composition further comprises 30 mM histidine, 150 mM sodium chloride at pH 6.
In a further aspect, the invention relates to a kit of parts comprising two or more pharmaceutical compositions according to any one of the preceding claims, wherein the compositions are for simultaneous, separate or sequential use in therapy. In one embodiment, the compositions are for simultaneous use in therapy, wherein the compositions are mixed immediately prior to use.
In a further aspect, the invention relates to a method for preparing a pharmaceutical composition according to the invention, said method comprising mixing a first pharmaceutical composition comprising a first antibody as defined herein with a second pharmaceutical composition comprising a second antibody as defined herein.
Therapeutic Applications
The pharmaceutical compositions according to any aspect or embodiment of the present invention may be used as a medicament, i.e. for medical, such as therapeutic applications.
Thus, in one aspect, the invention relates to a pharmaceutical composition according to the invention for use a medicament.
In another aspect, the present invention provides methods for treating or preventing a disorder, such as cancer, which method comprises administration of a therapeutically effective amount of pharmaceutical composition of the invention to a subject in need thereof.
The pharmaceutical composition may be administered by any suitable route and mode. Suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art.
In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. The terms “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-orbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.
In one embodiment, the pharmaceutical composition of the present invention is administered by intravenous or subcutaneous injection or infusion.
Pharmaceutical compositions according to the invention comprising one or more anti-DR5 antibodies can be used in the treatment or prevention of disorders involving cells expressing DR5. For example, the antibodies may be administered to human subjects, e.g., in vivo, to treat or prevent disorders involving DR5-expressing cells. As used herein, the term “subject” is typically a human to whom the anti-DR5 antibody or bispecific antibody is administered. Subjects may for instance include human patients having disorders that may be corrected or ameliorated by modulating DR5 function or by killing of the DR5-expressing cell, directly or indirectly.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in treatment of infectious disease, autoimmune disease or cardiovascular anomalies.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in treatment of cancer and/or tumors. The term “cancer” refers to or describes the physiological condition in mammals such as humans that is typically characterized by unregulated growth. Most cancers belong to one of two larger groups of cancers i.e., solid tumors and hematological tumors.
In a particular embodiment, the pharmaceutical composition is administered prophylactically in order to reduce the risk of developing cancer, delay the onset of an event in cancer progression or reduce the risk of recurrence when a cancer is in remission and/or a primary tumor has been surgically removed. In the latter case, the pharmaceutical composition could, for example, be administered in association with (i.e., before, during, or after) the surgery. Prophylactic administration may also be useful in patients where it is difficult to locate a tumor that is believed to be present due to other biological factors.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in treatment of solid tumors and/or hematological tumors
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in treatment of solid tumors such as, colorectal cancer, including colorectal carcinoma and colorectal adenocarcinoma, bladder cancer, osteosarcoma, chondrosarcoma, breast cancer, including triple-negative breast cancer, cancers of the central nervous system, including glioblastoma, astrocytoma, neuroblastoma, neural fibrosarcoma, neuroendocrine tumors, cervical cancer, endometrium cancer, gastric cancer, including gastric adenocarcinoma, head and neck cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), ovarian cancer, pancreatic cancer, including pancreatic ductal carcinoma and pancreatic adenocarcinoma, sarcoma or skin cancer, including malignant melanoma and non-melanoma skin cancers.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in treatment of hematological tumors such as, leukemia, including chronic lymphocytic leukemia and myeloid leukemia, including acute myeloid leukemia and chronic myeloid leukemia, lymphoma, including Non-Hodgkin lymphoma or multiple myeloma, including Hodgkin Lymphoma, and including myelodysplastic syndromes.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in treatment of a cancer selected from the following group of cancers; bladder cancer, bone cancer, colorectal cancer, sarcoma, endometrium cancer, fibroblast cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, muscle cancer, neural tissue cancer, ovary cancer, pancreas cancer and skin cancer.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in inhibiting growth of DR5 positive or DR5 expressing tumors or cancers.
In the present invention DR5 positive tumors or cancers are to be understood as tumor cells and/or cancer cells expressing DR5 on the cell surface. Such DR5 expression may be detected by immunohistochemistry, flow cytometry, imaging or other suitable diagnostic method. Tumors and cancer tissues that show heterogeneous expression of DR5 are also considered as DR5 positive tumors and cancers.
Tumors and/or cancers may express DR5 on some tumor and/or cancer cells and/or tissues showing DR5 expression, some tumor and/or cancers may show over-expression or aberrant expression of DR5, whereas other tumors and/or cancers show heterogeneous expression of DR5. Such tumors and/or cancers may all be suitable targets for treatment with anti-DR5 antibodies, bispecific antibodies and compositions comprising such antibodies according to the present invention.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention comprising one or more anti-DR5 antibodies for use in induction of apoptosis in DR5 expressing tumors.
In one embodiment of the invention, the use or the method of treating an individual having a cancer comprising administering to said individual an effective amount of pharmaceutical composition according to the invention, further comprises administering an additional therapeutic agent to the said individual.
In one embodiment of the invention the additional therapeutic agent is a single agent or a combination of agents comprising an agent or regimen selected from the group chemotherapeutics (including but not limited to paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), kinase inhibitors (including but not limited to sorafenib, sunitinib or everolimus), apoptosis-modulating agents (including but not limited to recombinant human TRAIL or birinapant), RAS inhibitors, proteasome inhibitors (including but not limited to bortezomib), histon deacetylase inhibitors (including but not limited to vorinostat), nutraceuticals, cytokines (including but not limited to IFN-γ), antibodies or antibody mimetics (including but not limited to anti-TF, anti-AXL, anti-EGFR, anti-IGF-1R, anti-VEGF, anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR, anti-VISTA (or other immunomodulatory targets) antibodies and antibody mimetics), and antibody-drug conjugates such as brentuximab vedotin, trastuzumab emtansine, HuMax-TF-ADC or HuMax-AXL-ADC.
When describing the embodiments of the present invention, the combinations and permutations of all possible embodiments have not been explicitly described. Nevertheless, the mere fact that certain measures are recited in mutually different dependent claims or described in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention envisages all possible combinations and permutations of the described embodiments.
LYTYYFDYWGQGTLVTVSS
LYTYYFDYWGQGTLVTVSS
WGTNVYFAYWGQGTLVTVSS
GDYYYGMDVWGQGTTVTVSS
FWSWIRQLPGKGLEWIGHIHNSGTTYYNPSLKS
GGDYYYGMDVWGQGTTVTVSS
LYTYYFDYWGQGTLVTVSSASTKGPSVFPLAPSS
LYTYYFDYWGQGTLVTVSSASTKGPSVFPLAPSS
LYTYYFDYWGQGTLVTVSSASTKGPSVFPLAPSS
LYTYYFDYWGQGTLVIVSSASTKGPSVFPLAPSS
LYTYYFDYWGQGTLVIVSSASTKGPSVFPLAPSS
LYTYYFDYWGQGTLVIVSSASTKGPSVFPLAPSS
WGTNVYFAYWGQGTLVTVSSASTKGPSVFPLA
WGTNVYFAYWGQGTLVTVSSASTKGPSVFPLA
WGTNVYFAYWGQGTLVTVSSASTKGPSVFPLA
SSYV
MSWVRQTPEKRLEWVATISSGGSYT
YYPDSVKG
RFTISRDNAKNTLYLQMSSLRS
GTA
VAWYQQKPGQSPKLLIYWASTRHTG
ESLSLSPGK
Expression Constructs for DR5
Codon-optimized constructs for expression of full-length DR5 proteins of human (SEQ ID NO 46), rhesus monkey (SEQ ID NO 25) and mouse (SEQ ID NO 26) were generated based on available sequences: human (Homo sapiens) DR5 (Genbank accession no. NP_003833, UniprotKB/Swiss-Prot O14763-1), Rhesus monkey (Macaca mulatta) DR5 (Genbank accession no. EHH28346), murine (Mus musculus) DR5 (UniprotKB/Swiss-Prot Q9QZM4). For mapping of the binding regions of the DR5 antibodies (as described in Example 6) the following chimeric human/mouse DR5 constructs were made; human DR5 in which, respectively, the following parts were replaced by the corresponding mouse DR5 sequence (numbers refer to human sequence), construct A aa 56-68, construct B aa 56-78, construct C aa 69-78, construct D aa 79-115, construct E 79-138, construct F aa 97-138, construct G aa 139-166, construct H aa 139-182, construct I aa 167-182, construct J 167-210, construct K aa 183-210. The loss-of-function mutation K415N was introduced in the human DR5 death domain (SEQ ID NO 44). In addition, codon-optimized construct for the extracellular domain (ECD) of human DR5 with a C-terminal His tag were generated: DR5ECD-FcHistag (SEQ ID NO 27) and DR5ECDdelHis (SEQ ID NO 28). All constructs contained suitable restriction sites for cloning and an optimal Kozak (GCCGCCACC) sequence. The constructs were cloned in the mammalian expression vector pcDNA3.3 (Invitrogen).
Expression Constructs for Antibodies
For antibody expression the VH and VL sequences, as earlier described, of the chimeric human/mouse DR5 antibodies DR5-01 and DR5-05 (based on EP2684896A1) and their humanized variants hDR5-01 and hDR5-05 (based on WO2014/009358) were cloned in expression vectors (pcDNA3.3) containing the relevant constant HC and LC regions. Desired mutations were introduced either by gene synthesis or site directed mutagenesis.
In some of the Examples, reference antibodies against DR5 were used that have been previously described. IgG1-CONA (based on U.S. Pat. No. 7,521,048 B2 and WO2010/138725) and IgG1-chTRA8 (based on EP1506285B1 and U.S. Pat. No. 7,244,429B2) were cloned in the relevant antibody expression vectors as supra.
In some of the examples the human IgG1 antibody IgG1-b12, a gp120-specific antibody was used as a negative control (Barbas et al., J Mol Biol. 1993 Apr. 5; 230(3):812-23).
Transient Expression
Antibodies were expressed as IgG1,κ. Plasmid DNA mixtures encoding both heavy and light chains of antibodies were transiently transfected in Expi293F cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by Vink et al. (Vink et al., Methods, 65 (1), 5-10 2014).
Membrane proteins were expressed in Freestyle CHO-S cells (Life technologies), using the freestyle Max reagent, as described by the manufacturer.
Purification and Analysis of Proteins
Antibodies were purified by immobilized protein G chromatography. His-tagged recombinant protein was purified by immobilized metal affinity chromatography. Protein batches were analyzed by a number of bioanalytical assays including SDS-PAGE, size exclusion chromatography and measurement of endotoxin levels.
Generation of Bispecific Antibodies
Bispecific IgG1 antibodies were generated by Fab-arm-exchange under controlled reducing conditions. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions as described in WO2011/131746. The F405L and K409R (EU numbering) mutations were introduced in anti-DR5 IgG1 antibodies to create antibody pairs with complementary CH3 domains. The F405L mutation was introduced in IgG1-DR5-05 and IgG1-DR5-05-E430G; the K409R mutation was introduced in IgG1-DR5-01 and IgG1-DR5-01-E430G. To generate bispecific antibodies, the two parental complementary antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL TE at 31° C. for 5 hours. The reduction reaction was stopped by removing the reducing agent 2-MEA using spin columns (Microcon centrifugal filters, 30 k, Millipore) according to the manufacturer's protocol. In this way the bispecific antibodies IgG1-DR5-01-K409R×IgG1-DR5-05-F405L (BsAb DR5-01-K409R×DR5-05-F405L) and IgG1-DR5-01-K409R-E430G×IgG1-DR5-05-F405L-E430G (BsAb DR5-01-K409R-E430G×DR5-05-F405L-E430G) were generated.
The K409R mutation and/or the F405L mutation have no effect on the antibody's binding to the corresponding antigen. That is the K409R mutation and/or the F405L mutation have no effect of the anti-DR5 antibody's binding to DR5.
DR5 density per cell was quantified for different human cancer cell lines by indirect immunofluorescence using QIFIKIT (DAKO, Cat nr K0078) with mouse monoclonal antibody B-K29 (Diaclone, Cat nr 854.860.000). Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm, washed with PBS and resuspended at a concentration of 2×106 cells/mL. The next steps were performed at 4° C. 50 μL of the single cell suspensions (100,000 cells per well) were seeded in polystyrene 96-well round-bottom plates (Greiner Bio-One, Cat nr 650101). Cells were pelleted by centrifugation for 3 minutes at 300×g and resuspended in 50 μL antibody sample or mouse IgG1 isotype control sample (BD/Pharmingen, Cat nr 555746) at 10 μg/mL saturating concentrations. After an incubation of 30 minutes at 4° C., cells were pelleted and resuspended in 150 μL FACS buffer (PBS+0.1% (w/v) bovine serum albumin (BSA)+0.02% (w/v) sodium azide). Set-up and calibration beads were added to the plate according to the manufacturer's instructions. Cells and beads in parallel were washed two more times with 150 μL FACS buffer and resuspended in 50 μL FITC-conjugated goat-anti-mouse IgG (1/50; DAKO, Cat nr F0479). Secondary antibody was incubated for 30 minutes at 4° C. protected from light. Cells and beads were washed twice with 150 μL FACS buffer and resuspended in 150 μL FACS buffer. Immunofluorescence was measured on a FACS Canto II (BD Biosciences) by recording 10,000 events within the population of viable cells. The Geometric mean of fluorescence intensity of the calibration beads was used to calculate the calibration curve that was forced to go through zero intensity and zero concentration using GraphPad Prism software (GraphPad Software, San Diego, Calif., USA). For each cell line, the antibody binding capacity (ABC), an estimate for the number of DR5 molecules expressed on the plasma membrane, was calculated using the Geometric mean fluorescence intensity of the DR5-antibody-stained cells, based on the equation of the calibration curve (interpolation of unknowns from the standard curve, using GraphPad Software). Generally, DR5 cell surface expression was low to moderate on the cell lines assessed here. Based on these data, cell lines were categorized according to low DR5 expression (ABC<10,000) and moderate DR5 expression (ABC>10,000). HCT-15 (ATCC, CCL-225), HT-29 (ATCC, HTB-38) and SW480 (ATCC, CCL-228) colon cancer, BxPC-3 (ATCC, CRL-1687), HPAF-11 (ATCC, CRL-1997) and PANC-1 (ATCC, CRL-1469) pancreatic cancer, and A549 (ATCC, CCL-185) and SK-MES-1 (ATCC, HTB-58) lung cancer cell lines were found to have low DR5 expression (QIFIKIT ABC range 3,081-8,411). COLO 205 (ATCC CCL-222™) and HCT 116 (ATCC CCL-247) colon cancer, A375 (ATCC, CRL-1619) skin cancer and SNU-5 (ATCC, CRL-5973) gastric cancer cell lines were found to have moderate DR5 expression (QIFIKIT ABC range 10,777-21,262).
The humanized antibodies hDR5-01 and hDR5-05 are described in patent application WO2014/009358. Binding of purified IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L to DR5-positive HCT 116 human colon cancer cells was analyzed and compared to binding of the chimeric antibodies IgG1-DR5-01-K409R and IgG1-DR5-05-F405L by FACS analysis. To prepare single cell suspensions, adherent HCT 116 cells were washed twice with PBS (B. Braun; Cat nr 3623140) before incubating with Trypsin 1×/EDTA 0.05% for 2 minutes at 37° C. 10 mL medium [McCoy's 5A medium with L-Glutamine and HEPES (Lonza; Cat nr BE12-168F)+10% Donor Bovine Serum with Iron (Life Technologies; Cat nr 10371-029)+100 Units Penicillin/100 Units Streptomycin (Lonza Cat nr DE17-603E)] was added before pelleting the cells by centrifugation for 5 minutes at 1200 rpm. Cells were resuspended in 10 mL medium, pelleted again by centrifugation for 5 minutes at 1200 rpm, and resuspended in FACS buffer at a concentration of 1.0×106 cells/mL. The next steps were performed at 4° C. 100 μL cell suspension samples (100,000 cells per well) were seeded in polystyrene 96-well round-bottom plates (Greiner Bio-One; Cat nr 650101) and pelleted by centrifugation at 300×g for 3 minutes at 4° C. Cells were resuspended in 100 μL samples of a serial dilution antibody preparation series (range 0 to 10 μg/mL in 5-fold dilutions) and incubated for 30 minutes at 4° C. Cells were pelleted by centrifugation at 300×g for 3 minutes at 4° C. and washed twice with 150 μL FACS buffer. Cells were incubated with 50 μL secondary antibody R-phycoerythrin (R-PE)-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C., protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 150 μL FACS buffer, and antibody binding was analyzed on a FACS Canto II (BD Biosciences) by recording 10,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
As can be seen from
Binding of purified antibody variants of IgG1-DR5-01-K409R, IgG1-DR5-05-F405L and bispecific antibody IgG1-DR5-01-K409R×IgG1-DR5-05-F405L (BsAb DR5-01-K409R×DR5-05-F405L) with and without a hexamerization-enhancing mutation (E430G or E345K) to human colon cancer cells COLO 205 was analyzed by FACS analysis. Cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent COLO 205 cells. Cells were centrifuged for 5 minutes at 1,200 rpm and resuspended in 10 mL culture medium [RPMI 1640 with 25 mM Hepes and L-Glutamine (Lonza Cat nr BE12-115F)+10% Donor Bovine Serum with Iron (Life Technologies Cat nr 10371-029)+50 Units Penicillin/50 Units Streptomycin (Lonza Cat nr DE17-603E)]. Cells were counted, centrifuged again and resuspended in FACS buffer at a concentration of 0.3×106 cells/mL. The next steps were performed at 4° C. 100 μL cell suspension samples (30,000 cells per well) were seeded in polystyrene 96-well round-bottom plates and pelleted by centrifugation at 300×g for 3 minutes at 4° C. Cells were resuspended in 50 μL samples of a serial dilution antibody preparation series (range 0 to 10 μg/mL final concentrations in 5-fold dilutions) and incubated for 30 minutes at 4° C. Plates were centrifuged at 300×g for 3 minutes at 4° C. and cells were washed twice with 150 μL FACS buffer. Cells were incubated with 50 μL secondary antibody R-PE-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C. protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a FACS Canto II (BD Biosciences) by recording 5,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
Binding of purified IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G to CHO cells expressing Rhesus macaque DR5 or human DR5 (described in Example 1) was analyzed by FACS analysis. One day before FACS analysis, CHO cells were transiently transfected with a vector encoding Rhesus macaque DR5, human DR5 or a non-coding vector (mock). To prepare single cell suspensions, cells were washed with PBS and resuspended in FACS buffer at a concentration of 1.0×106 cells/mL. The next steps were performed at 4° C. 75 μL cell suspension samples (75,000 cells per well) were seeded in polystyrene 96-well round-bottom plates and pelleted by centrifugation at 300×g for 3 minutes at 4° C. Cells were resuspended in 50 μL samples of a serial dilution antibody preparation series (range 10 to 0 μg/mL in 5-fold dilutions) and incubated for 30 minutes at 4° C. Plates were centrifuged at 300×g for 3 minutes at 4° C. and cells were washed twice with 150 μL FACS buffer. Cells were incubated with 50 μL secondary antibody R-PE-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C. protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a FACS Canto II (BD Biosciences) by recording 100,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
The amino acid sequences of the extracellular domains of human and murine DR5 show limited homology (
The competition between humanized DR5-01 and DR5-05 antibodies for binding to the extracellular domain of DR5 was measured by sandwich binding assays in a sandwich enzyme-linked immunosorbent assay (ELISA) as described in this example and by Bio-Layer interferometry (BLI) using a ForteBio Octet® HTX system (data not shown). For the ELISA, 96-well flat bottom ELISA plates (Greiner bio-one; Cat nr 655092) were coated overnight at 4° C. with 2 μg/mL DR5 antibody (IgG1-hDR5-01-E430G or IgG1-hDR5-05-E430G) in 100 μL. PBS. The wells were blocked by adding 200 μL PBSA [PBS/1% Bovine Serum Albumin (BSA; Roche Cat #10735086001)] and incubated for 1 hour at room temperature. The wells were washed three times with PBST [PBS/0.05% Tween-20 (Sigma-Aldrich; Cat nr 63158)]. Next, DR5ECD-FcHistag (SEQ ID 27) (0.2 μg/mL final concentration) and competing antibody (1 μg/mL final concentration) were added in a total volume of 100 μL PBSTA (PBST/0.2% BSA) and incubated for 1 hour at room temperature while shaking. After washing three times with PBST, wells were incubated on an ELISA shaker with 100 μL biotinylated anti-His tag antibody (R&D Systems; Cat nr BAM050; 1:2,000) in PBSTA for one hour at room temperature. After washing three times with PBST, wells were incubated with streptavidin-labelled Poly-HRP (Sanquin; Cat nr M2032; 1:10,000) in PBSTA for 20 minutes at room temperature on an ELISA shaker. After washing three times with PBST, the reaction was visualized through an incubation with 100 μL 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid [ABTS (Roche; Cat nr 11112597001)] for 30 minutes at RT protected from light. The substrate reaction was stopped by adding an equal volume of 2% oxalic acid. Fluorescence at 405 nm was measured on an ELISA reader (BioTek ELx808 Absorbance Microplate Reader).
A viability assay was performed to study the effect the hexamerization-enhancing mutation E430G in IgG1-DR5-01-K409R and IgG1-DR5-05-F405L on the capacity of the antibodies to kill human colon cancer cells COLO 205 and HCT 116. The antibodies were tested as single agent and as combinations of DR5-01 and DR5-05 antibodies. COLO 205 cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent cells. HCT 116 cells were harvested by trypsinization. Cells were passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspension (5,000 cells per well) was seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182). 50 μL of a serial dilution antibody preparation series (range 0.05 to 20,000 ng/mL final concentrations in 5-fold dilutions) was added and incubated for 3 days at 37° C. In samples that were treated with a combination of two antibodies, the total antibody concentration in the assay was the same as in the samples that were treated with single antibodies. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cultured cells was determined in a CellTiter-Glo luminescent cell viability assay (Promega, Cat nr G7571) that quantifies the ATP present, which is an indicator of metabolically active cells. From the kit, 20 μL luciferin solution reagent was added per well and mixed by shaking the plate for 2 minutes at 500 rpm. Next, plates were incubated for 1.5 hours at 37° C. 100 μL supernatant was transferred to a white OptiPlate-96 (Perkin Elmer, Cat nr 6005299) and luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism software.
In Example 8 it is shown that combining the two non-crossblocking anti-DR5 antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G with hexamerization enhancing mutations resulted in enhanced killing on cancer cell lines compared to the efficacy of the single antibodies. Here, we compare the efficacy of two non-crossblocking versus two crossblocking anti-DR5 antibodies. A viability assay was performed to study the capacity of the combination of antibodies IgG1-chTRA8-F405L-E430G with either non-crossblocking antibody IgG1-DR5-01-K409R-E430G or crossblocking antibody IgG1-DR5-05-F405L-E430G to induce killing of HCT 116 colon cancer cells in comparison to the single antibodies. A crossblock ELISA for antibodies IgG1-chTRA8-F405L and IgG1-DR5-05-F405L was performed as described in Example 7 and confirmed by a sandwich binding assay on an Octet® HTX system (data not shown). The viability assay on HCT 116 cells was performed as described in Example 8 with a serial diluted antibody series ranging from 0.00005 to 20 μg/mL final concentrations in 5-fold dilutions.
A viability assay was performed to study the capacity of another combination of two non-crossblocking antibodies (IgG1-CONA-K409R-E430G+IgG1-DR5-05-F405L-E345K) and its bispecific derivative BsAb IgG1-CONA-K409R-E430G×DR5-05-F405L-E345K to induce killing of HCT 116 colon cancer cells in comparison to the combination of antibodies and the bispecific antibody without hexamerization-enhancing mutation, respectively. A crossblock ELISA for antibodies IgG1-CONA-K409R and IgG1-DR5-05-F405L was performed as described in Example 7 and confirmed by a sandwich binding assay on an Octet® HTX system (data not shown). The viability assay on HCT 116 cells was performed as described in Example 8 with a serial diluted antibody series ranging from 0.01 to 20,000 ng/mL final concentrations in 5-fold dilutions.
A viability assay was performed to study the capacity of the combination of human-mouse chimeric antibodies IgG1-DR5-01-K409R+IgG1-DR5-05-F405L with and without the hexamerization-enhancing mutation E430G to induce killing of COLO 205, HCT-15, HCT 116, HT-29 and SW480 colon cancer, BxPC-3, HPAF-II and PANC-1 pancreatic cancer, SNU-5 gastric cancer, A549 and SK-MES-1 lung cancer, and A375 skin cancer cells. Adherent cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL [COLO 205, HCT-15, SW480 and BxPC-3: RPMI 1640 with 25 mM Hepes and L-Glutamine (Lonza Cat nr BE12-115F)+10% DBSI (Life Technologies Cat nr 10371-029)+Pen/Strep (Lonza Cat nr DE17-603E); HCT116 and HT-29: McCoy's5A Medium with L-Glutamine and Hepes (Lonza, Cat nr BE12-168F)+10% DBSI+Pen/Strep; HPAF-II and SK-MES-1: Eagle's Minimum Essential Medium (EMEM, ATCC Cat nr 30-2003)+10% DBSI+Pen/Strep; PANC-1 and A375: DMEM 4.5 g/L Glucose without L-Gln with HEPES (Lonza Cat nr LO BE12-709F)+10% DBSI+1 mM L-Glutamine (Lonza Cat nr 13E17-605E)+Pen/Strep; SNU-5: IMDM (Lonza Cat nr BE12-722F)+10% DBSI+Pen/Strep; A549: F-12K Medium (ATCC Cat nr 30-2004)+10% DBSI+1 mM L-Glutamine+Pen/Strep]. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. Supernatant of the adherent cells was replaced by 150 μL antibody sample (final concentration 10 μg/mL) and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. For all tested cell lines, the percentage viable cells was significant lower after incubation with 10 μg/mL of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G than after incubation with the non-target binding negative control antibody IgG1-b12 (
A viability assay was performed to compare the potency of the combination of chimeric antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G with the potency of the combination of humanized antibodies IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G to induce killing of BxPC-3 and PANC-1 pancreatic cancer cells in vitro. Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. Supernatant of the adherent cells was replaced by 150 μL antibody sample of a serial dilution antibody preparation series and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. The combination of the humanized antibodies with hexamerization-enhancing mutation IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G showed similar dose-response curves as the combination of the corresponding chimeric antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G (
Amino acid sequence N55-G56 was identified as a potential asparagine (Asn) deamidation motif in the CDR2 regions of the IgG1-hDR5-01 and IgG1-hDR5-05 heavy chains (SEQ ID NO:2). Deamidation at this position was mimicked by introduction of the N55D mutation in IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L to test the effect of deamidation on target binding. IgG1-hDR5-01-N55D-K409R and IgG1-hDR5-05-N55D-F405L were tested for binding to HCT 116 cells by FACS analysis as described in Example 3.
A viability assay was performed to compare the capacity of the combination of humanized antibodies IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with the capacity of the combination of humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G to induce killing of BxPC-3 pancreatic cancer cells. Viability was assessed as described in Example 11 with 1,000 cells per well and antibody concentrations series ranging from 0.0001 to 10,000 ng/mL final concentrations in 4-fold dilutions in a total volume of 200 μL.
To analyse the requirement of antibody hexamer formation by IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to induce cell death, we made use of the self-repulsing mutations K439E and S440K (Diebolder et al., Science. 2014 Mar. 14; 343(6176):1260-3). The Fc repulsion between antibodies that is introduced by the presence of either K439E or S440K in one IgG1 antibody or in a combination of antibodies results in inhibition of hexamerization, even in the presence of a hexamerization enhancing mutation such as E345K or E430G (WO2013/0044842). The repulsion by the K439E and S440K mutations is neutralized by combining both mutations in a mixture of two antibodies each harboring one or the other mutation, resulting in restoration of the Fc:Fc interactions and hexamerization. For both IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G, variants with either the K439E or S440K mutation were generated and tested in all different combinations. A viability assay was performed with serial dilution antibody preparation series ranging from 0.3 to 20,000 ng/mL total concentrations in 4-fold dilutions on BxPC-3 pancreatic and HCT-15 colon cancer cells as described in Example 11.
To test the involvement of Fc-Fc-mediated antibody hexamerization in the induction of cell death by the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G, we made use of the 13-residue peptide DCAWHLGELVWCT (DeLano et al., Science 2000 Feb. 18; 287(5456):1279-83) that binds the Fc in a region containing the core amino acids in the hydrophobic patch that are involved in Fc-Fc interactions (Diebolder et al., Science. 2014 Mar. 14; 343(6176):1260-3). A viability assay on BxPC-3 cells was performed as described in Example 11 for the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G in presence or absence of the DCAWHLGELVWCT peptide. Briefly, after overnight incubation of the cells at 37° C., culture medium was removed and replaced by 100 μL culture medium containing a dilution series (range 0-100 μg/mL) of the Fc-binding DCAWHLGELVWCT peptide, a non-specific control peptide GWTVFQKRLDGSV, or no peptide. Next, 50 μL antibody samples (833 ng/mL final concentration) were added and incubated for 3 days at 37° C. The capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of BxPC-3 cells was strongly inhibited by 100 μg/mL Fc-binding DCAWHLGELVWCT peptide (
A viability assay was performed to study the capacity of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G to induce killing of BxPC-3 pancreatic cancer cells, when combined at different ratios of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G. Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. 50 μL antibody sample with different ratios of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G (indicated as Ratio DR5-01:DR5-05 of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100 in serial dilution series ranging from 0.06 to 20 μg/mL final concentrations in 5-fold dilutions) was added and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. At 20 μg/mL and 4 μg/mL total antibody concentrations, killing was equally effective at all tested antibody ratios containing both antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G. At 0.8 μg/mL and 0.16 μg/mL total antibody concentrations, all tested antibody ratios containing both antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G induced killing (
A viability assay was performed to study the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of BxPC-3 pancreatic and HCT-15 colon cancer cells, when combined at different antibody ratios. Generally, the experiments were performed as described in Example 16. Briefly, pre-attached cells (5,000 cells per well) were incubated for 3 days at 37° C. in 150 μL in polystyrene 96-well flat-bottom plates with different ratios of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G (indicated in
A viability assay was performed to compare the cytotoxicity of the combination of humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G in the presence and absence of a caspase inhibitor. PANC-1 and BxPC3 pancreatic cancer cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. 25 μL pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK, 5 μM end concentration in 150 μL, Bachem, Cat nr 4026865.0005) was added to the cell cultures and incubated for one hour at 37° C. before adding 25 μL antibody sample of a serial dilution antibody preparation series (range 1 to 20 μg/mL final concentrations in 4-fold dilutions) and further incubation for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). Recombinant human TRAIL/APO-2L (eBioscience, Cat nr BMS356) was used at 6 μg/mL final concentration. The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. The combination of the humanized antibodies with hexamerization-enhancing mutation IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G was unable to reduce the viability of PANC-1 and BxPC3 pancreatic cancer cells in presence of the pan-caspase inhibitor Z-VAD-FMK, indicating that the combination of IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G induced caspase-dependent programmed cell death (
The kinetics of cell death induction was analyzed by Annexin V/Propidium Iodide (PI) double staining and active caspase-3 staining. Annexin-V binds phosphatidylserine that is exposed on the cell surface after initiation of programmed cell death, which is a reversible process. PI is a dye that intercalates into double-stranded DNA and RNA when it enters cells. Because PI cannot pass intact plasma and nuclear membranes, it will not stain living cells but only enter and stain dying cells that have decreased membrane integrity. Due to these characteristics, the Annexin V/PI double staining can be applied to discriminate between initiation (Annexin V-positive/PI-negative) and irreversible (Annexin V-positive/PI-positive) programmed cell death. Caspase-3 is activated by both the extrinsic death receptor-induced and intrinsic mitochondrial cell death pathways. Therefore, active caspase-3 is also a marker for initiation of the death cascade. The induction of cell death upon binding of the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was analyzed in the DR5-positive COLO 205 colon cancer cells. Cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent cells. Cells were passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.2×106 cells/mL. 500 μL of the single cell suspensions (100,000 cells per well) were seeded in 24-wells flat-bottom culture plates (Greiner Bio-One, Cat nr 662160) and incubated for 16 hours at 37° C. 500 μL antibody sample was added (1 μg antibody final concentration) and incubated for 5 hours or 24 hours at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). Cells were washed once with 250 μL 1×PBS. Adherent cells were harvested by incubating with 100 μL 0.05% trypsin for 10 minutes at 37° C. 200 μL medium was added to the trypsinized cells and cells were transferred to a 96-wells round-bottom FACS plate (Greiner Bio-One, Cat nr 650101) and pooled with the non-adherent cells. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm, resuspended in 200 μL ice cold PBS and divided into two samples of 100 μL in 96-Wells round-bottom FACS plates for the Annexin V/PI and active caspase-3 staining, respectively.
Annexin V/PI double staining was performed using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, Cat nr 556547). Cells were washed once with ice cold PBS and incubated in 50 μL Annexin V/PI Staining Solution (Annexin V-FITC 1:00 and PI 1:25) for 15 minutes at 4° C. Cells were washed with 100 μL Binding Buffer, resuspended in 20 μL Binding Buffer and fluorescence was measured on an iQue Screener (IntelliCyt) within 1 hour. Data were analyzed and plotted using GraphPad Prism software.
Active caspase-3 staining was performed using the PE Active Caspase-3 Apoptosis Kit (BD Pharmingen, Cat nr 550914). Cells were washed once with ice cold PBS, resuspended in 100 μL Cytofix/Cytoperm Fixation and Permeabilization Solution and incubated for 20 minutes on ice. Cells were pelleted at room temperature, washed twice with 100 μL 1× Perm/Wash Buffer and resuspended in 100 μL PE Rabbit Anti-Active Caspase-3 (1:10) for an incubation of 30 minutes at room temperature. Cells were pelleted at room temperature, washed once with 100 μL 1× Perm/Wash Buffer and resuspended in 20 μL 1× Perm/Wash Buffer. Fluorescence was measured on an iQue Screener. Data were analyzed and plotted using GraphPad Prism software.
After 24 hours incubation, the percentage of Annexin V/PI double-positive cells (D) was enhanced in samples treated with IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G, indicating that the cells had entered the irreversible stages of cell death. Also at this stage, the effect of the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was stronger (larger increase in the percentage of Annexin V/PI double-positive cells (E)) than in samples treated with a combination of DR5 antibodies without the E430G mutation (IgG1-DR5-01-K409R+IgG1-DR5-05-F405L) or any of the single antibodies. At the same time point, the percentage of Active Caspase 3 positive cells was highest in cells treated with IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G.
These data indicate that the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induces both the early and late stages of cell death in COLO 205 colon cancer cells, and does so more effectively than the combination of the antibodies without the E430G hexamerization enhancing mutation.
In example 19 it was described that incubation with the combination of chimeric DR5 antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced caspase-3 activation in COLO 205 colon cancer cells. The percentage of active caspase-3-positive cells was higher after 5 hours than after 24 hours of incubation with the antibody combination. In this example, Caspase-3/7 activation was measured in time using the Caspase-Glo 3/7 assay (Promega, Cat nr G8091), in which a substrate with the Caspase-3/7 recognition motif DEVD releases aminoluciferin, a substrate of luciferase, upon cleavage. Cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent COLO 205. Cells were passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.8×105 cells/mL. 25 μL of the single cell suspensions (2,000 cells per well) were seeded in 384-wells culture plates (Perkin Elmer, Cat nr 6007680) and incubated for 16 hours at 37° C. 25 μL antibody sample was added (1 μg antibody final concentration) and incubated for 1, 2, 5 and 24 hours at 37° C. Plates were removed from the incubator to let the temperature decrease till room temperature. Cells were pelleted by centrifugation for three minutes at 300 g. 25 μL supernatant was removed and replaced by 25 μL Caspase-Glo 3/7 Substrate. After mixing by shaking for one minute at 500 rpm, the plates were incubated for one hour at room temperature. Luminescence was measured on an EnVision Multilabel Reader (Perkin Elmer).
A viability assay was performed to compare the capacity of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in the absence and presence of secondary antibody crosslinker to induce killing of COLO 205 colorectal and BxPC-3 and PANC-1 pancreatic cancer cells. For comparison, two DR5 antibodies that are known to show enhanced killing in the presence of a secondary antibody crosslinker, IgG1-CONA and IgG1-chTRA8-F405L, were tested in the same settings. Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. Supernatant of the adherent cells was replaced by 150 μL antibody sample (final concentration 10 μg/mL) in the absence or presence of F(ab′)2 fragments of a goat-anti-human IgG antibody (1/150; Jackson ImmunoResearch; Cat nr 109-006-098) and incubated for 3 days at 37° C. As a positive control for cell killing, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. The antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced significant killing compared to the negative control of COLO 205, BxPC-3 and PANC-1 cancer cells, both in presence or absence of an Fc crosslinker (
In many of the experiments described in this application, the anti-DR5 antibodies IgG1-01 and IgG1-05 contain in the IgG Fc domain the K409R and F405L (EU numbering index) mutation, respectively. These mutations enable the generation of DR5 bispecific antibodies by Fab-arm-exchange reaction between IgG1-01-K409R and IgG1-05-F405L under controlled reducing conditions as described in WO2011/131746. Without Fab-arm exchange, human IgG1 antibodies bearing the K409R and F405L mutations are thought to show the same functional characteristics as wild type human IgG1 (Labrijn et al., Proc Natl Acad Sci USA. 2013 Mar. 26; 110(13):5145-50). Here we show that the presence of the K409R or F405L mutations has no effect on the capacity of the combination of the parental IgG1-01 and IgG1-05 antibodies to induce cell death in DR5-positive tumor cells in vitro. A viability assay was performed to compare the capacity of the combination of humanized antibodies IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G with the capacity of the combination of humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G to induce killing of BxPC-3 pancreatic cancer cells. The viability assay on The BxPC-3 was performed as described in Example 11 with a serial diluted antibody series ranging from 0.001 to 20,000 ng/mL final concentrations in 4-fold dilutions. The BxPC-3 pancreatic cancer cell line showed similar viability curves after incubation with the combination of the humanized antibodies IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G as with the combination of the humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G (
A bispecific antibody targeting two different DR5 epitopes was generated by Fab-arm exchange between the chimeric antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G as described in Example 1. A viability assay was performed as described in Example 11 to test the capacity of 10 μg/mL of the chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G to induce killing of cancer cells of different tissue origin (COLO 205 colorectal cancer, A375 skin cancer, SK-MES-1 lung cancer, BxPC-3 pancreatic cancer and SNU-5 gastric cancer cell lines). For all tested cell lines, the percentage viable cells was significantly lower when incubated with 10 μg/mL of the chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G antibody compared to the non-target binding negative control antibody IgG1-b12 (
A viability assay was performed to compare the potency of the chimeric BsAb IgG1-DR5-01-K409R-E430G×IgG1-DR5-05-F405L-E430G in the absence and presence of a secondary antibody crosslinker to induce killing of BxPC-3 pancreatic and COLO 205 colon cancer cells as described in Example 21. For comparison, two DR5 antibodies that are known to show enhanced killing in the presence of a secondary antibody crosslinker, IgG1-CONA and IgG1-chTRA8-F405L, were tested in the same setting. The chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G showed significant killing compared to the negative control of COLO 205 and BxPC-3 cancer cells, both in presence or absence of an Fc crosslinker (
The kinetics of cell death induction by 1 μg/mL BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G on COLO 205 cells was analyzed by Annexin V/Propidium Iodide (PI) double staining and active caspase-3 staining as described in Example 19.
These data indicate that BsAB IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G induces both the early and late stages of cell death in COLO 205 colon cancer cells, and does so more effectively than the bispecific antibody without the E430G hexamerization enhancing mutation.
The in vivo anti-tumor efficacy of different anti-DR5 antibodies and the combination of DR5-01+DR5-05 antibodies with hexamerization enhancing mutation was evaluated in a subcutaneous model with COLO 205 human colon cancer cells. At day 0, cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent cells. 3×106 cells were injected in a volume of 200 μL PBS into the flank of 6-11 weeks old female SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan). All experiments and animal handlings were approved by the local authorities, and were conducted according to all applicable international, national and local laws and guidelines. Tumor development was monitored at least twice per week by caliper (PLEXX) measurement as 0.52×(length)×(width)2. Tumors were measured until an endpoint tumor volume of 1,500 mm3, until tumors showed ulcerations, until serious clinical signs were observed, or until tumor growth blocked movements of the mouse. At day 6, the average tumor volume was ˜200 mm3 and the mice were sorted into groups with equal tumor size variance (Table 2 below). Mice were treated by intraperitoneal (i.p.) injection of 100 μg antibody in 200 μL PBS on day 6 and 13 (5 mg/kg per dose). To check for correct antibody administration, blood samples were obtained for IgG serum determination three days after the first dose. Three individual mice had no detectable human IgG plasma level and were excluded from statistical analysis (see Table 2 below). For the other mice, human antibody plasma concentrations were according to the expectations when assuming a 2-compartment model with Vcen=50 mL/kg, Vs=100 mL/kg and an elimination half-life of 11.6 days (data not shown). Tumors were measured until 16 weeks after tumor inoculation.
These data show that introduction of the E430G hexamerization-enhancing mutation in IgG1-DR5-05-F405L resulted in enhanced tumor inhibition in the subcutaneous COLO 205 colon cancer tumor model compared to IgG1-DR5-05-F405L without the hexamerization-enhancing mutation. Both DR5-01 and DR5-05 antibodies with hexamerization enhancing mutation (IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G), the bispecific antibodies with and without hexamerization enhancing mutation (BsAb DR5-01-K409R×DR5-05-F405L and BsAb DR5-01-K409R-E430G×DR5-05-F405L-E430G) and the combination of antibodies with hexamerization-enhancing mutation (IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G) showed better tumor inhibition as IgG1-CONA and IgG1-DR5-05-F405L without hexamerization-enhancing mutation.
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous COLO 205 human colon cancer xenograft model. Tumor cell inoculation, mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 26. At day 10, the average tumor volume was ˜400 mm3 and the mice were sorted into groups with equal tumor size variance (Table 3 below). Mice were treated by intravenous (i.v.) injection of 40 μg (2 mg/kg), 10 μg (0.5 mg/kg) or 2 μg (0.1 mg/kg) antibody in 100 μL PBS on day 10. Mice in the control group were treated with 40 μg (2 mg/kg) IgG1-b12. Tumors were measured until 17 weeks after tumor inoculation.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G had stronger anti-tumor efficacy compared to IgG1-CONA, since dosed at 2 mg/kg the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G significantly reduced tumor load at day 16 compared to IgG1-CONA, and at 0.5 mg/kg the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G combination significantly reduced tumor load at day 16 and prolonged progression free survival time (tumor size cut-off 500 mm3) compared to IgG1-CONA.
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA-F405L in the subcutaneous BxPC-3 human pancreatic cancer xenograft model. At day 0, adherent cells were harvested by trypsinization. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-11 weeks old female SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan).
Mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 26. At day 10, the average tumor volume was ˜250 mm3 and the mice were sorted into groups with equal tumor size variance (Table 4 below). Mice were treated by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 200 μL PBS on day 20 and 28. Mice in the control group were treated with 200 μg (10 mg/kg) IgG1-b12. To check for correct antibody administration, blood samples were obtained for IgG serum determination one week after dosing. Tumors were measured until 10 weeks after tumor inoculation.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA-F405L in an in vivo BxPC-3 human pancreatic cancer xenograft model.
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA-F405L in the subcutaneous A375 human skin cancer xenograft model. At day 0, adherent cells were harvested by trypsinization. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-11 weeks old female SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan). Mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 26. At day 19, the average tumor volume was ˜250 mm3 and the mice were sorted into groups with equal tumor size variance (Table 5 below). Mice were treated by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 200 μL PBS on day 19 and 26. Mice in the control group were treated with 200 μg (10 mg/kg) IgG1-b12. To check for correct antibody administration, blood samples were obtained for IgG serum determination one week after dosing. Tumor volumes were analyzed until 7 weeks after tumor inoculation.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA-F405L in an in vivo A375 human skin cancer xenograft model.
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous HCT-15 human colon cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. Adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-8 weeks old female BALB/c nude mice (Shanghai Laboratory Animal Center). The care and use of animals during the study were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V=0.5 a×b2 where a and b are the long and short diameters of the tumor, respectively. Eleven days after tumor inoculation, the mean tumor size reached 186 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA in an in vivo xenograft model with HCT-15 human colon cancer cells.
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous SW480 human colon cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in L-15 medium supplemented with 10% fetal bovine serum at 37° C. in 100% air. Adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 1×107 cells were injected in a volume of 200 μL PBS with Matrigel (1:1) into the flank of 6-8 weeks old female NOD/SCID mice (Beijing HFK Bioscience). Mouse handling and tumor volume measurements were performed as described in Example 30. Ten days after tumor inoculation, the mean tumor size reached 175 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy for doses of 10 mg/kg and 0.5 mg/kg was significantly better than for equivalent doses of IgG1-CONA in an in vivo SW480 human colon cancer xenograft model.
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G were evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous SNU-5 human gastric cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a suspension culture in IMDM medium supplemented with 20% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. Cells in an exponential growth phase were harvested and 1×107 cells were injected in a volume of 200 μL PBS with Matrigel (1:1) into the flank of 6-8 weeks old female CB17/SCID mice (Beijing HFK Bioscience). Mouse handling and tumor volume measurements were performed as described in Example 30. Eight days after tumor inoculation, the mean tumor size reached 169 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA in an in vivo SNU-5 human gastric cancer xenograft model.
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous SK-MES-1 human lung cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in MEM medium supplemented with 10% fetal bovine serum and 0.01 mM NEAA at 37° C. in an atmosphere of 5% CO2 in air. At day 0, adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-8 weeks old female BALB/c mice (Shanghai Laboratory Animal Center). Mouse handling and tumor volume measurements were performed as described in Example 30. Twenty-one days after tumor inoculation, the mean tumor size reached 161 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA at 0.5 mg/kg and 2 mg/kg in an in vivo SK-MES-1 human lung cancer xenograft model.
DR5 density per cell was quantified for different human cancer cell lines by indirect immunofluorescence using QIFIKIT with mouse monoclonal antibody B-K29 as described in Example 2. The cell lines were categorized according to low DR5 expression (ABC<10,000) and moderate DR5 expression (ABC>10,000). The human cancer cell lines SK-MEL-5 (ATCC, HTB-070) malignant melanoma, Jurkat (ATCC, TIB-152) acute T cell leukemia and Daudi (ATCC, CCL-231) Burkitt's lymphoma were found to have low DR5 expression (QIFIKIT ABC range 3,500-6,500). The human colorectal carcinoma cell lines SNU-C2B (ATCC, CCL-250), LS411N (ATCC, CRL-2159) and DLD-1 (ATCC, CCL-221) were found to have moderate DR5 expression (QIFIKIT ABC range 12,000-44,500).
Binding to human colon cancer cells HCT 116 was analyzed by flow cytometry for purified antibody variants of IgG1-hDR5-01-G56T and IgG1-hDR5-05 with and without the E430G mutation. Single cell suspensions were prepared and binding was analyzed for serial dilution antibody preparation series (range 0.0006 to 10 μg/mL final concentrations in 4-fold dilutions) as described in Example 3. After incubation with the secondary antibody, cells were washed twice, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences). Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
Antibody binding to HCT 116 human cancer cells with moderate DR5 expression was analyzed by flow cytometry for purified samples of Alexa 647-labeled IgG1-hDR5-01-G56T-E430G and Alexa 647-labeled IgG1-hDR5-05-E430G, both as single agents and as a combination of the two antibodies. 1 mg/mL IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G were labeled for 1 hour at room temperature with a 5 molar excess of Alexa Fluor® 647 carboxylic acid, succinimidyl ester (Molecular Probes; Cat # A-20006) in 0.1 M NaHCO3 conjugation buffer to reach a degree of labeling of three. Free excess Alexa 647 was removed on a PD 10 Column (Amersham Bioscience, Cat #17-0851-01). Single cell suspensions were prepared and binding was analyzed for serial dilution antibody preparation series (range 0.0019 to 30 μg/mL final concentrations in 5-fold dilutions) as described in Example 3. After antibody incubation, cells were washed twice, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences). Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
Binding of purified IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to CHO cells expressing the isoform short of human DR5 or cynomolgus monkey DR5 was analyzed by flow cytometry. Codon-optimized constructs for expression of the isoform short human DR5 protein with death domain loss-of-function mutation K386N (SEQ ID NO 47 based on Uniprot number 014763-2) and cynomolgus monkey DR5 protein with deletion of amino acids 185-213 and death domain loss-of-function mutation K420N (SEQ ID NO 50; based on NCBI accession number XP_005562887.1) were generated as described in Example 1. Binding to DR5-transfected CHO cells was analyzed, generally as described in Example 5. Transfected cells were stored in liquid nitrogen and quickly thawed at 37° C. and suspended in 10 mL medium. Cells were washed with PBS and resuspended in FACS buffer at a concentration of 1.0×106 cells/mL. 100 μL cell suspension samples (100,000 cells per well) were seeded in 96-well plates and pelleted by centrifugation at 300×g for 3 minutes at 4° C. 25 μL of serial dilution antibody preparation series (final concentrations 0 to 20 μg/mL in 6-fold dilutions) was added and incubated for 30 minutes at 4° C. Next, cells were washed and incubated with 50 μL secondary antibody R-PE-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C. protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 50 μL FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences) by recording 10,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
A viability assay was performed to study the effect the hexamerization-enhancing mutation E430G in IgG1-hDR5-01-G56T and IgG1-hDR5-05 on the capacity of the antibodies to kill human colon cancer cells COLO 205. The antibodies with and without the E430G mutation were tested as single agent and as combinations of the two non-crossblocking antibodies. COLO 205 cells were harvested as described in Example 8. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and allowed to adhere overnight at 37° C. Subsequently, 50 μL samples of antibody concentration series (range 0.3-20,000 ng/mL final concentration in 4-fold dilutions) were added and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8.
The effect of the hexamerization-enhancing mutation S440Y on the capacity of the single antibodies and the combination of IgG1-hDR5-01-G56T and IgG1-hDR5-05 to kill COLO 205 human colon cancer cells was studied in a viability assay. Cells were harvested and a CellTiter-Glo luminescent cell viability assay was performed as described in Example 8. Briefly, 100 μL single cell suspensions (5,000 cells per well) were seeded in 96-well plates and at the same time, 50 μL of serial dilution antibody preparation series (range 0.0003 to 20 μg/mL final concentrations in 4-fold dilutions) were added and incubated for 3 days at 37° C.
A crossblock ELISA for antibodies IgG1-DR5-CONA-K409R and IgG1-DR5-chTRA8-F405L was performed as described in Example 7. The K409R and F405L mutations are not relevant here and were previously shown to have no effect on the potency of antibodies with an E430G mutation (Example 22).
The efficacy of the combination of the non-crossblocking antibodies IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing was analyzed on different human cancer cell lines and compared to the parental antibody combination without the E430G mutation and TRAIL. A viability assay on HCT-15, HCT 116, HT-29 and SW480 colon cancer, BxPC-3, HPAF-II and PANC-1 pancreatic cancer, SNU-5 gastric cancer, A549 and SK-MES-1 lung cancer, and A375 skin cancer cells was performed, essentially as described in Example 11. Briefly, 100 μL single cell suspensions (5,000 cells per well) were seeded in 96-well plates and incubated at 37° C. overnight. 50 μL of antibody sample (133 nM final concentration) or human recombinant TRAIL/APO-2L (eBioscience, Cat nr BMS356; 133 nM final concentration) was added and incubated for 3 days at 37° C. Both TRAIL and the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G show killing of human cancer target cell lines originating from different indications (
The activity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was tested and compared to the activity of TRAIL in a panel of 235 cell lines representing 14 tumor lineages: kidney, neural tissue, colorectal, gastric, breast cancer (predominantly triple-negative breast cancer (TNBC)), non-small cell lung cancer (NSCLC), bladder, pancreatic, ovarian, melanoma, liver, endometrial, head and neck and small cell lung cancer (SCLC). A 72 hour ATPlite assay (except for DLD-1 and HCT116 cell lines, for which a 120 hour assay was performed) with growth inhibition analysis was performed in two parts at Horizon Discovery Ltd, UK. Samples were tested as four replicates in 384-well assay plates. Serial dilution series of antibody, starting from 0.072 μM final concentration was used for all tested cell lines. For TRAIL (Invitrogen; Cat # PHC1634) serial dilution series starting from 0.01 μM final concentration for the cell lines tested in the first part and 0.17 μM final concentration for the cell lines tested in the second part of the screening was used. Percentage inhibition was calculated using the formulas: If T≥V(0) than percentage inhibition=100*[1−(T−V(0))/(V−V(0))]; If T<V(0) than percentage inhibition=100%, with T=luminescence of the test sample, V(0)=luminescence of the medium control sample on day 0 and V=luminescence of the medium control sample on day 3. Responder and non-responder cell lines were grouped by a maximum response threshold value categorizing cell lines showing 70% inhibition as responders and cell lines showing ≤69% inhibition as non-responders (
A viability assay was performed to study the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of BxPC-3 pancreatic cancer cells and HCT-15 colon cancer cells, when combined at different ratios of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G. Antibody ratios of 1:0, 9:1, 3:1, 1:1, 1:3, 1:9 and 0:1 in serial dilution series (ranging from 0.006 to 20 μg/mL final concentrations in 5-fold dilutions) were tested in a CellTiter-Glo luminescent cell viability assay as described in Example 16.
At 20 μg/mL, 4 μg/mL and 0.8 μg/mL total antibody concentrations, killing of BxPC-3 (
A viability assay was performed to compare the cytotoxicity of the combination of antibody variants of IgG1-hDR5-01-G56T and IgG1-hDR5-05 with and without the hexamerization-enhancing mutation E430G in the presence and absence of a caspase inhibitor. A CellTiter-Glo luminescent cell viability assay with serial dilution series of antibody or TRAIL samples (range 0.002 to 133 nM final concentrations in 4-fold dilutions) was performed as described in Example 18.
The killing of BxPC-3 cells was inhibited in the presence of pan-caspase inhibitor Z-VAD-FMK for TRAIL and the antibody combinations IgG1-hDR5-01-G56T+IgG1-hDR5-05 and IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G (
Caspase-3/7 activation was measured in time using the Caspase-Glo 3/7 assay, essentially as described in Example 20. Briefly, cells were harvested by trypsinization, passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 1.6×105 cells/mL. 25 μL of the single cell suspensions (4,000 cells per well) were seeded in 384-wells culture plates (Perkin Elmer, Cat nr 6007680) and incubated overnight at 37° C. 25 μL sample was added (26.6 nM final concentrations) and incubated for 1, 2, 4 and 6 hours at 37° C. Plates were removed from the incubator to let the temperature decrease till room temperature. Cells were pelleted by centrifugation for three minutes at 300 g. 25 μL supernatant was removed and replaced by 25 μL Caspase-Glo 3/7 Substrate. After mixing by shaking for one minute at 500 rpm, the plates were incubated for one hour at room temperature. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer).
In the time course of 1, 2, 4 to 6 hours, both TRAIL and the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced more rapid and more potent caspase-3/7 activation on BxPC-3 cells compared to the WT antibody combination IgG1-hDR5-01-G56T+IgG1-hDR5-05 without the hexamerization enhancing mutation (
A viability assay was performed to compare the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of human HCT-15 colon cancer cells and BxPC-3 pancreatic cancer cells in the absence and presence of a secondary antibody crosslinker. IgG1-DR5-CONA, which is known to show enhanced killing in the presence of a secondary antibody crosslinker, was tested in the same assay for comparison. A viability assay in absence and presence of secondary crosslinker was performed, essentially as described in Example 21. Briefly, 100 μL of the single cell suspensions (5,000 cells per well) were seeded in 96-well plates and incubated overnight at 37° C. 50 μL antibody sample (final concentration 4 μg/mL) in the absence or presence of F(ab′)2 fragments of a goat-anti-human IgG antibody and incubated for 3 days at 37° C. As a positive control for cell killing, cells were incubated with 5 μM staurosporine. The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described Example 8.
The combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced potent killing in BxPC-3 and HCT15 cells, and cytotoxicity was not further enhanced in the presence of a secondary crosslinker (
To analyze the capacity of the antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to activate complement, an in vitro complement-dependent cytotoxicity (CDC) assay and deposition of complement component C3c was measured on CHO cells that were transiently transfected with the isoform short of either human or monkey DR5. The DR5 constructs harbored the K386N (human) or K420N (cynomolgus monkey) mutation in their death domain to prevent killing by the induction of apoptosis upon binding of the agonistic antibodies. Transient transfections of CHO cells with human or monkey (Macaca fascicularis) DR5 were performed as described in Example 1.
For the CDC assay, 0.1×106 cells were pre-incubated in polystyrene round-bottom 96-well plates (Greiner bio-one Cat #650101) with concentration series of purified antibodies in a total volume of 80 μL for 15 min on a shaker at RT. Next, 20 μL normal human serum (NHS; Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added as a source of complement and incubated in a 37° C. incubator for 45 min (20% final NHS concentration; 0.003-10.0 μg/mL final antibody concentrations in 3-fold dilutions). The reaction was stopped by putting the plates on ice before pelleting the cells by centrifugation and replacing the supernatant by 30 μL of 2 μg/mL propidium iodide solution (PI; Sigma Aldrich, Zwijnaarde, The Netherlands). The percentage of PI-positive cells was determined by flow cytrometry on an Intellicyt iQue™ screener (Westburg). The data were analyzed using best-fit values of a non-linear dose-response fit using log-transformed concentrations in GraphPad PRISM 5.
For the analysis of C3b deposition, 0.1×106 cells were pre-incubated in round-bottom 96-well plates with concentration series of purified antibodies (0.003-10.0 μg/mL final antibody concentrations in 3-fold dilutions) in a total volume of 80 μL for 15 min on a shaker at RT. Next, 20 μL C5-depleted serum (Quidel; Cat # A501) was added as a source of complement and incubated in a 37° C. incubator for 45 min (20% final NHS concentration). Cells were pelleted and subsequently incubated with 50 μL FITC-labeled polyclonal rabbit-anti-human C3c complement (Dako; Cat # F0201; 2 μg/mL) in FACS buffer for 30 minutes at 4° C. Cells were washed twice with FACS buffer and resuspended in 30 μL FACS buffer. The C3b-deposition on cells was determined by flow cytrometry on an Intellicyt iQue™ screener (Westburg). The data were analyzed using best-fit values of a non-linear dose-response fit using log-transformed concentrations in GraphPad PRISM 5.
Both complement-dependent killing (
In order to identify clinically relevant compounds that display synergistic inhibitory effects in combination with the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G, 100 compounds representing different therapeutic classes were screened for potential synergy in colon cancer cell lines. A 72 hour (for LS-411N, SNU-C2B and SW480) or 120 hour (for DLD-1 and HCT 116) ATPlite assay with growth inhibition analysis was performed in a 6×6 optimized combination matrix in 384-well assay plates at Horizon Discovery Ltd, UK. All samples were tested in four replicates. Percentage growth inhibition was calculated using the formulas: If T≥V(0) than percentage growth inhibition=100*[1−(T−V(0))/(V−V(0))]; If T<V(0) than percentage growth inhibition=100*[1−(T−V(0))/V(0)], with T=luminescence of the test sample, V(0)=luminescence of the medium control sample on day 0 and V=luminescence of the medium control sample on day 3. In order to identify synergistic effects, mean self-cross activity was determined for each therapeutic class using representative compounds. To measure combination effects in excess of Loewe additivity, Horizon Discovery Ltd has devised a scalar measure to characterize the strength of synergistic interaction termed the Synergy Score. The Synergy Score equation integrates the experimentally-observed activity volume at each point in the matrix in excess of a model surface numerically derived from the activity of the component agents using the Loewe model for additivity. Additional terms in the Synergy Score equation are used to normalize for various dilution factors used for individual agents and to allow for comparison of synergy scores across an entire experiment. The inclusion of positive inhibition gating or an Idata multiplier removes noise near the zero effect level, and biases results for synergistic interactions that occur at high activity levels. The Synergy Score (S) was calculated using the formula: S=log fX log fYΣ max(0,Idata)(Idata−ILoewe) with fx,y=dilution factors used for each single agent. Synergy Scores greater than the mean self-cross plus 3σ were considered candidate synergies at the 99% confidence levels.
Table 12 shows the Synergy Scores for all 100 tested compounds. Synergy with the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was observed for one or more cell lines with compounds from the different therapeutic classes, including chemotherapeutics (including cytoskeletal regulators and DNA/RNA damaging agents), kinase inhibitors, PI3K pathway inhibitors, RAS inhibitors, apoptosis-modulating agents, proteasome inhibitors, epigenetic modulators (including HDAC inhibitors) and others.
17.7
14.6
36
12.6
16.8
17.6
7.9
36.2
15.8
19.5
11.4
11.8
40.8
15.6
14.7
17.3
13.9
33
17.2
12.1
8.1
21.5
11
7.7
20.3
12.2
5.7
18.5
31.1
20.8
10.7
11.4
24.6
19.6
13.8
10.4
17.5
16.7
8.8
15.4
12.9
18.9
11.3
12
11.6
19.5
10
13.1
5.8
11.4
7.9
6
9
7.6
6.6
8.6
4.5
16.4
10.2
8.2
13.4
10.7
5.8
4.8
15.5
23.3
13.1
12.4
7.1
12.9
10
12.2
5.2
5.1
5.4
4.5
13.4
16.1
5.4
8.9
10.5
3.7
11.4
7.8
10.2
11.3
10.8
5.9
9.2
7.2
5.8
6
7.3
6.4
6
16.4
61.2
37
54.5
13.8
29.9
11.1
10.5
6.1
22.8
4.7
11.8
9
11.5
12.1
10.6
6
7.6
8
7.5
20.3
29
21.2
30.5
18.8
29.3
19.8
25.1
21.9
9.6
16.9
12.4
30.1
16.2
9.4
18
20.5
15
6.8
13.4
24.7
17
11.1
14.2
19.4
15.6
5.8
15.1
17.7
24.3
7.2
6.3
16.3
10.4
6.1
11
7.3
7.2
7.5
8.9
9.8
5.3
5.3
6.2
11.7
7.2
6
5.6
6.3
8
The in vivo anti-tumor efficacy of antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G was evaluated for the single antibodies and the combination of both antibodies and compared to the parental antibodies without the E430G mutation in the subcutaneous COLO 205 human colon cancer xenograft model. Tumor cell inoculation, mice handling, tumor outgrowth measurements and endpoint determination were performed, essentially as described in Example 26. 3×106 cells were injected in a volume of 100 μL PBS into the flank of 5-8 weeks old female SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan). At day 9, the average tumor volume was measured and the mice were sorted into groups with equal tumor size variance. Mice were treated by intravenous (i.v.) injection of 10 μg (0.5 mg/kg) antibody in 200 μL PBS on day 9. Mice in the control group were treated with 10 μg (0.5 mg/kg) IgG1-b12.
The in vivo anti-tumor efficacy of the anti-DR5 antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was compared to that of IgG1-hDR5-01-G56T+IgG1-hDR5-05 without the E430G hexamerization-enhancing mutation in the subcutaneous HCT15 human colon cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. Adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 7-9 weeks old female BALB/c nude mice. The care and use of animals during the study were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V=0.5 a×b2 where a and b are the long and short diameters of the tumor, respectively. Mice were assigned into groups using randomized block design and treatments were started when the mean tumor size reached 161 mm3 (8 mice per group). Mice were treated three times according to a Q7D regimen by i.v. injection of 0.5 mg/kg antibody (0.25 mg/kg of each antibody in the combination). Mice in the control group were treated in parallel with 0.5 mg/kg IgG1-b12.
These data indicate that introduction of the E430G hexamerization-enhancing mutation in the anti-DR5 antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G resulted in enhanced tumor growth inhibition in an in vivo xenograft model with HCT15 human colon cancer cells.
The in vivo anti-tumor efficacy of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was evaluated in combination with paclitaxel in the subcutaneous SK-MES-1 human lung cancer xenograft model at CrownBiosciences, Taicang, China. Cell culturing, tumor cell inoculation, mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 33. 21 days after tumor inoculation, the mean tumor size reached 167 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injections of 2 mg/kg antibody and 15 mg/kg paclitaxel both dosed in 10 μL PBS per g body weight as indicated in Table 14.
The clearance rate of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G was studied in a PK experiment in SCID mice for the single compounds and for the combination of the two antibodies in comparison to the parental antibodies without the E430G mutation.
7-10 weeks old female SCID (C.B-17/IcrHan@Hsd-Prkdc<scid, Harlan) mice (3 mice per group) were injected intravenously with 20 μg antibody (1 mg/kg) in a 200 μL injection volume. 50-100 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected into heparin-containing vials and centrifuged for 5 minutes at 10,000 g. Plasma samples were diluted 1:20 for the four first time points (15 μL sample in 285 μL PBSA (PBS supplemented with 0.2% bovine serum albumin (BSA)) and 1:10 for the last two time points (30 μL sample in 270 μL PBSA) and stored at −20° C. until determination of antibody concentrations.
Total human IgG concentrations were determined using a sandwich ELISA. Mouse anti-human IgG-kappa mAb clone MH16 (CLB Sanquin, Cat ## M1268) was used as capturing antibody and coated in 100 μL overnight at 4° C. to 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 μg/mL in PBS. Plates were blocked by incubating on a plate shaker for 1h at RT with PBSA. After washing, 100 μL of serial diluted plasma samples (range 0.037-1 μg/mL in 3-fold dilutions) were added and incubated on a plate shaker for 1h at RT. Plates were washed three times with 300 μL PBST (PBS supplemented with 0.05% Tween 20) and subsequently incubated on a plate shaker for 1h at RT with 100 μL peroxidase-labeled goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, Pa.; 1:10,000 in PBST supplemented with 0.2% BSA). Plates were washed again three times with 300 μL PBST before incubation for 15 minutes at RT with 100 μL substrate 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) [ABTS; Roche, Cat #11112 422001; 1 tablet in 50 mL ABTS buffer (Roche, Cat #11112 597001)] protected from light. The reaction was stopped by adding 100 μL 2% oxalic acid and incubation for 10 minutes at RT. Absorbance was measured in a microplate reader (Biotek, Winooski, Vt.) at 405 nm. Concentration was calculated by using the injected material as a reference curve. As a plate control, purified human IgG1 (The binding site, Cat # BP078) was included. Human IgG concentrations (in μg/mL) were plotted (
No difference in the plasma clearance rate was observed between IgG1-hDR5-01-G56T-E430G or IgG1-hDR5-05-E430G and their parental antibodies without the E430G mutation, both when injected as single agents or as the combinations of those (
The present study illustrate the ability of the anti-DR5 antibody IgG1-DR5-CONA with the hexamerization-enhancing mutation E430G to kill attached human colon cancer cells COLO 205. COLO 205 cells were harvested as described in Example 8. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in 96-well flat-bottom plates and incubated overnight at 37° C. 50 μL samples of antibody concentration series (range 0.04 to 10 μg/mL final concentrations in 4-fold dilutions) were added and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine. The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism software. The percentage viable cells was calculated using the following formula: % viable cells=[(luminescence antibody sample−luminescence staurosporine sample)/(luminescence no antibody sample−luminescence staurosporine sample)]*100.
Material:
Antibody IgG1-hDR5-05-E430G formulated at 20 mg/ml, unless stated otherwise.
Methods
Differential Scanning Calorimetry (DSC)
The melting temperature of the protein samples was determined using MicroCal Capillary DSC equipment.
Appearance
Appearance was determined by visual evaluation.
pH
pH was measured using a Mettler Toledo SevenMulti pH meter.
Protein Content by UV A280
Protein content was determined by UV/Vis Spectroscopy using an Agilent UV/Vis Spectrophotometer (Model 8453)
Size Exclusion Chromatography (SEC)
Size exclusion chromatography was performed on an Agilent 100 HPLC system, using a TOSOH, TSK-gel G-3000SWxL (7.8×300 mm) column (Sigma).
Imaging Capillary Isoelectric Focusing (icIEF)
Imaging capillary isoelectric focusing was performed using an iCE 3 Analyzer equipped with PrinCE Autosampler.
Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS)
Reduced and non-reduced capillary electrophoresis was performed using a Beckman Coulter PA800Plus Series Capillary Electrophoresis System. Beta Mercaptoethanol was used for reduced samples.
Dynamic Light Scattering (DLS)
Dynamic light scattering was performed using a Wyatt DynaPro Plate Reader.
Results
1. Baseline Biophysical Screening
Initial biophysical screening was performed to select buffer/pH combinations to move forward into the excipient screening. Table 15 displays the data obtained from the initial buffer screen, wherein glutamate, acetate, succinate, histidine, citrate and phosphate buffers were tested. DSC and DLS were used to assess thermal stability. DSC analysis provided the melting temperatures (Tm1 and Tm2) along with the Tonset. DLS analysis provided information on polydispersity and hydrodynamic radius of the protein.
Based on the DSC data, glutamate pH 5.0, acetate pH 5.5, and succinate pH 6.0 had higher Tonset values when compared to their counterparts at lower pH. Higher Tonset values are indicative of better thermal stability of the protein. All histidine formulations at pH 5.5, 6.0 and 6.5 displayed comparatively high Tonset values with these values increasing slightly with increasing pH. The Tonset for citrate buffer at pH 6.0 and 7.0 was 54° C. whereas the Tonset of phosphate at pH 7.5 was 54° C., respectively. Results from the DLS data from the initial biophysical screening did not correlate strongly with formulation results obtained from DSC. Specifically, high degrees of polydispersity were observed in formulations with higher pH that exhibited better thermal stability as observed in DSC. For example, histidine, pH 5.5 had a % Pd of 6.3, compared to pH 6.0 and 6.5 which displayed a % Pd of 10.8 and 15.6, respectively (Table 15). The phosphate and citrate formulations had highest % Pd compared to rest of the formulations. Based on the data obtained from DSC and DLS, glutamate pH 5.0, acetate pH 5.5, histidine pH 5.5 and succinate pH 6.0 formulations were further screened in the presence of various excipients. The phosphate and citrate formulations were not selected due to high % polydispersity and the potential possibility of destabilization of the protein in these buffers.
The formulations selected above were screened in the presence of 150 mM arginine, sodium chloride, sucrose and sorbitol. The data from this portion of the baseline biophysical screening is shown in Table 16. Glutamate pH 5.0 had lowest Tonset in the presence of excipients most likely due to the low pH, even in the presence of stabilizing excipients sorbitol and sucrose. Based on data shown in Table 16, it was observed that for the acetate formulations, the Tonset increased in the presence of sucrose. An increase in Tonset was observed for the histidine formulations in the presence of sucrose and sorbitol. Increased Tonset was only observed for the succinate formulation containing sucrose. For acetate samples, the formulations consisting of NaCl and arginine had Tonset values of 51° C. and 52° C., respectively. Histidine formulations containing NaCl displayed a higher onset value when compared to formulation in the presence of arginine. The Tonset value for histidine formulation with arginine was 47° C. whereas the Tonset value for the histidine formulation in the presence of NaCl was 51° C. The Tonset values for succinate formulations containing charged excipients were equivalent (54° C.). The onset values were overall the highest for succinate buffers containing charged excipients compared to the other three buffer types, while histidine and succinate buffers displayed higher onset values than glutamatate and acetate buffers in the presence of sorbitol and sucrose.
Based on DLS results (Table 16), all formulations consisting of sucrose and sorbitol displayed % Pd that were multimodal and high hydrodynamic radius which is indicative of formation of macromolecular aggregates. The DLS data also demonstrated that these formulations in the presence of charged excipients sodium chloride and arginine had low % Pd.
Overall, data obtain from both DSC and DLS suggested that 25 mM acetate at pH 5.5 in the presence of NaCl and arginine and Histidine formulations at pH 5.5 in the presence of sodium chloride were better candidates for further formulation development.
2. NaCl Screening
Antibody IgG1-hDR5-05-E430G was formulated at 40 mg/mL in 30 mM histidine, pH 5.5 in the presence of four different concentrations of NaCl (0, 25, 50, and 100 mM NaCl) to determine the effect on solubility and phase separation. Samples were stored at −5±3° C. for 24 h on a pre-cooled lyophilizer shelf. After 24 hours, the set of samples were tested by appearance. No phase separation was observed in any of the prepared samples.
3. Surfactant Screening
Antibody IgG1-hDR5-05-E430G was formulated in 30 mM histidine, pH 5.5 in the presence of 0, 0.03, or 0.06% w/v Tween-80 and stressed with three freeze-thaw cycles. Identical samples were agitated for period of 48 hours. After sample stress, the set of samples were tested by appearance, A280, SEC, reduced CE-SDS, and non-reduced CE-SDS.
3.1 Appearance
No visual differences were observed between samples any of the samples in the surfactant screening study. All samples were slightly yellow liquid, opalescent and free of visible particulates.
3.2 Protein Content by UV A280
The antibody concentrations obtained by UV analysis were not significantly different and ranged between 18.54 and 20.73 mg/mL (data not shown).
3.3 SEC
Monomer purity did not significantly differ, and no new peaks were not observed for any of the surfactant screening samples. The purity of all samples was between 98.8-99.0% (data not shown).
3.4 Reduced CE-SDS
Purity (LC and HC %) did not significantly differ, and no new peaks were observed for any of the surfactant screening samples. The purity of all samples was 95.6-96.1% (data not shown).
3.5 Non-Reduced CE-SDS
Main peak purity did not significantly differ, and no new peaks were observed for any of the surfactant screening samples. The purity of all samples was between 90.5% and 92.1% (data not shown).
3.6 Conclusions from Surfactant Screening
No changes in appearance, protein concentration, or purity were observed between unstressed and stressed samples containing concentrations of 0, 0.03, and 0.06% PS-80. These data indicate surfactants do not enhance the stability of the antibody in these formulations.
4. Cryoprotectant Screening
In the cryoprotectant screen, antibody IgG1-hDR5-05-E430G was formulated in 30 mM Histidine, pH 5.5 with three different concentrations (0, 5, or 10% w/v) of sucrose and stressed with three freeze-thaw cycles. Identical samples were agitated for a period of 48 hours. After sample stress, the set of samples were tested by appearance, A280, SEC, reduced CE-SDS, and non-reduced CE-SDS.
4.1 Appearance
No visual differences were observed between samples containing 0%, 5%, or 10% sucrose stressed with three freeze-thaw cycle and agitated for a period of 48 hours. All samples were slightly yellow liquid, opalescent, and free of visible particulates.
4.2 Protein Content by UV A280
The antibody concentrations obtained by UV analysis were not significantly different. Concentrations ranged between 19.06 and 24.86 mg/mL (data not shown).
4.3 SEC
Monomer purity did not significantly differ, and growth of new peaks was not observed for any of the cryoprotectant screening samples. The purity of all samples was between 98.9-99.1% (data not shown).
4.4 Reduced CE-SDS
Purity (LC and HC %) did not significantly differ, and growth of new peaks was not observed for any of the cryoprotectant screening samples. The purity of all samples was between 95.3-95.9% (data not shown).
4.5 Non-Reduced CE-SDS
Main peak purity did not significantly differ, and no growth of new peaks was observed for any of the cryoprotectant screening samples. The purity of all samples was between 91.5-92.0% (data not shown).
4.6 Conclusions from Cryoprotectant Screening
No changes in appearance, protein concentration, or purity were observed between unstressed and stressed samples containing concentrations of 0%, 5% and 10% sucrose. These data indicated that cryoprotectants do not enhance the stability of the antibody in these formulations.
5. DoE Stability Studies
Formulation design for DoE Study is shown in Table 17. Samples were stored for up to 4 weeks at 5±3° C. and 40±2° C./75±5% RH. The initial samples were tested by pH, UV, and DSC. After storage, the set of samples were tested by Appearance, pH, A280, DLS, SEC, icIEF, CE-SDS (reduced and non-reduced).
5.1 DSC (Initial)
The Initial DSC data is shown in Table 18. For the histidine formulations, it was observed that high Tonset values were obtained for formulations at higher pH. This trend correlated with data obtained in the initial baseline screen where higher Tonset values were observed with increasing pH. Histidine formulations pH 6.0 demonstrated highest Tonset values when compared to histidine formulations at pH 5.0 and pH 5.5. The Tonset values ranged from 50-53° C. for the histidine pH 6.0 formulations. Histidine pH 5.0 formulations ranged from 43-46° C. whereas Histidine pH 5.5 formulations ranged from 47-51° C. High Tonset values were observed for formulations consisting of sucrose and sorbitol. Formulations F13, F14, F28, and F29 displayed high Tonset values for formulations containing sucrose and sorbitol. For the acetate formulation, a similar trend was observed. The formulations at higher pH displayed higher onset values. Acetate formulations pH 6.0 and pH 5.5 displayed high Tonset values with and without the presence of NaCl and arginine. The acetate pH 6.0 formulations ranged from 52-54° C. whereas the acetate pH 5.5 formulations ranged from 51-54° C. Acetate pH 5.0 formulation displayed the lowest Tonset range of 45-49° C.
5.2 Protein Content by UV A280
Protein Content by UV A280 results showed for all sample protein concentrations between 18.47-21.95 mg/mL (data not shown). Samples F8, F24, and F22 had slightly lower protein concentrations which is likely due to experimental variability. Overall, there were no significant changes in protein concentration observed at the initial time point.
5.3 Appearance
All sample preparations at four week time point at 5±3° C. were clear and slightly yellow in color. Most sample preparations at 5±3° C. exhibited no particles that appeared to be non-product related. F5-3, F7, F8, F29, and F30 contained a few particles. Samples at 40±2° C./75±5% RH were slight yellow in color and clear except for samples F20-1 and F23 which were opalescent. Formulations at 40±2° C./75±5% RH ranged from having no particles to many particles. For the acetate formulations, the formulations F1 displayed no particles. Formulations F2, F5, F6, and F10 had few particles whereas formulations F3, F4, F7, F8, F9, F11, F12, F13, F14 and F15 had many particles. For the histidine formulations, F17 and F26 displayed many particles. F16, F18, F23, F25, F29, and F30 displayed few particles. The rest of the formulations F19, F20, F21, F22, F24, F27 and F28 had no particles.
5.4 pH
Target pH values for the formulations are shown in Table 19. For the acetate formulations, a significant shift was observed at the four week time point at 5±3° C. and 40±2° C./75±5% RH. The range for the difference in pH for the acetate formulations at initial time point and 5±3° C. was 0.12-0.30. The shift in pH observed in these samples stressed at 40±2° C./75±5% RH was 0.44-1.01. For the histidine formulations, a significant pH shift was not observed at the four week time point at 5±3° C. and 40±2° C./75±5% RH. Differences in pH of histidine formulations that were observed at the four week testing can be attributed to experimental variability. Overall, histidine pH 6.0 formulations did not undergo any changes in pH with and without excipients compared to the rest of the formulations. The acetate formulations were susceptible to shifts in pH over which makes acetate a less suitable component for the antibody. Significant pH shifts could also lead to accelerated degradation of the protein. Histidine pH 6.0 formulations on the other hand proved to be promising components. The stability results explained going forward will focus on histidine formulations (F16-F30) because of the observed pH shift for acetate formulations.
5.5 Protein Content by UV A280
The range of A280 readings for the 5±3° C. samples was 19.87-23.59 mg/mL whereas the range of A280 readings for the 40±2° C./75±5% RH was 19.81-26.38 mg/mL (data not shown). No significant shifts in A280 readings were observed. The observed ranges in UV content were likely due to experimental variability. The data did not show any trends with respect to buffer concentration, pH, and excipient concentration.
5.6 SEC
SEC results for four week time points are shown in Table 20. For the histidine formulations primarily at pH 6.0 it was observed that presence of charged excipients improved stability of the formulation. It was also observed that increase in pH in the presence of charged excipients improved the stability of the histidine formulations. A decrease in % total impurities for formulations in the presence of charged excipients was observed at 40±2° C./75±5% RH. Formulation F20-1 at 40±2° C./75±5% had the lowest purity of 84.2%. This is an unexpected and anomalous result since the other two replicates for this center point formulation are vastly more pure so this replicate is considered an outlier. For histidine pH 5.0 formulations F17, F18 and F19 at 40±2° C./75±5% RH, the % total impurities ranged from 5.0-5.8%. The % total impurities for the histidine pH 5.5 formulations F21, F22, and F23 ranged from 4.1-7.3% whereas histidine pH 6.0 formulations F25, F26, and F27 had % total impurities ranging from 3.5-3.8%. It was apparent that higher pH led to decrease in % total impurities and histidine pH 6.0 formulations in the presence of charged excipients showed better stability. An increase in % total impurities was observed for the histidine formulations containing sucrose or sorbitol. The % total impurities were 3.8% at 5±3° C. and 6.9% at 40±2° C./75±5% RH for pH 6.0 formulations without excipients. Overall, histidine pH 6.0 formulations in the presence of charged excipients NaCl and arginine (Formulations F25, F26, and F27) had better stability.
5.7 icIEF
Charge heterogeneity of samples was determined using icIEF at four weeks at 5±3° C. and 40±2° C./75±5% RH (Table 21). The icIEF results for samples at 5±3° C. and 40±2° C./75±5% RH showed that the percent acidic variants of histidine formulations at the four week time point at 5° C. ranged from 56.2-58.9% for formulations F16-F28 (data not shown). For formulations F29 and F30 that consisted of sucrose and sorbitol, the percent acidic variants were 60.4% and 63.8%, respectively. These differences appear to be significant as seen in a comparison of their profiles with F25. At 40±2° C./75±5% RH, F29 and F30 had percent acidic variants of 45.9% and 61.6%. For formulation F29 consisting of sucrose, there was a significant increase in basic variants to 32.4% at 40±2° C./75±5% RH. At the four week time point at 40±2° C./75±5% RH, all histidine formulations demonstrated increases in percent acidic variants ranging between 61.6%-71.6%. Formulation F29 showed percent acidic variants of 45.9% at 40±2° C./75±5% RH. The icIEF data showed that pH of the samples affected charge heterogeneity. Histidine formulations at pH 5.0 showed more significant increases in acidic variants when compared to histidine formulations pH 5.5 and 6.0. For histidine pH 5.0 formulations, the range of percent acidic variants at 40±2° C./75±5% RH was 71.3-71.6%. At 40±2° C./75±5% RH, The range of percent acidic variants for histidine pH 5.5 was 63.5-67.1% whereas the range of percent acidic variants for histidine pH 6.0 was 65.3-66.1%. This result is likely not due to deamidation because deamidation is known to be accelerated at higher pH values, and the opposite trend is observed here. Across all formulations, the results showed that histidine pH 5.5 and 6.0 were better formulations than histidine pH 5.0 formulations and significant degradation was observed in histidine formulations consisting of sucrose and sorbitol.
5.8 Reduced CE-SDS
Results for reduced CE-SDS are shown in Table 22. At the four week time point at 5±3° C., all histidine formulations regardless of pH showed comparable purity.
At the four week time point at 40±2° C./75±5% RH, the results showed an increase in impurities for all of the sample preparations. It was observed that formulations at lower pH displayed more degradation at 40±2° C./75±5% RH. Histidine pH 5.0 formulations showed a considerable decrease in percent purity. The range for the percent purity was 77.5-82.8%. For the histidine pH 5.5 formulations, the percent purity ranged from 80.1-91.2%. Significant degradation was not observed for the histidine pH 6.0 formulations. The percent purity for the histidine pH 6.0 formulations ranged from 89.7-91.1% at 40±2° C./75±5% RH four week time point. In addition, the % LMW for histidine samples was higher for histidine samples at lower pH and considerably lower for Histidine formulations at higher pH. The range of % LMW for histidine pH 5.0 formulations was 13.7-18.4%. Whereas the Histidine pH 5.5 and 6.0 formulations displayed % LMW range of 5.9-12.9% and 5.0-6.3%. For the Histidine pH 6.0 formulations, it was also observed that percent purity did not significantly decrease in the presence of charged excipients. Across all formulations, histidine pH 6.0 formulations in the presence of charged excipients showed better purity compared to the rest of the histidine formulations.
5.9 Non-Reduced CE-SDS
Results for non-reduced CE-SDS are shown in Table 23. Results obtained from acetate formulations (F1 to F15) will not be considered due to the pH shift observed for these formulations. Significant high % HMW impurities were observed for formulations consisting of arginine at 5±3° C., i.e. formulations F18, F19, F22, F23, F26 and F27. This increase in impurities was not observed for histidine pH 6.0 formulations in the presence of NaCl (F25), i.e. formulations F17, F21 and F25. Previous results suggested histidine pH 6.0 formulations in the presence of charged excipients (NaCl and arginine) to be optimal conditions. Results obtained from non-reduced CE-SDS data confirm that a histidine pH 6.0 formulation with NaCl is a better choice than histidine pH 6.0 with arginine.
5.10 DLS
Acetate formulations are not being considered due to the pH shift observed in these formulations. Based on the DLS data (not shown), it was observed that lower pH led to high polydispersity in the histidine formulations. Histidine pH 5.0 formulations F17, F18 and F19 had significant increases in polydispersity. For example, the % Pd of formulation F17 at 5±3° C. was 10.2 and 7.0 and increased to 20.8 and 18.7 after four weeks at 40±2° C./75±5% RH. Similarly histidine pH 5.5 formulations F21, F22, and F23 had increased % Pd at 40±2° C./75±5% RH. For example, formulation F23 had % Pd of 6.3 and 10.4 at 5±3° C. The % Pd increased to 17.1 and 21.7 at 40±2° C./75±5% RH. Most histidine pH 6.0 formulations in the presence of charged excipients (NaCl and arginine) were resistant to changes in polydispersity at both stress conditions. Formulations F25, F26, and F27 did not show significant increases in % Pd. For instance formulation F25 showed % Pd of 9.4 and 8.9 at 5±3° C. The % Pd at 40±2° C./75±5% RH was 8.3 and 10.2. Additionally, formulations in the presence of sucrose (F19) exhibited high polydispersity at both conditions. The % Pd for F29 was 23.7 and 23.4 at 5±3° C. whereas the % Pd at 40±2° C./75±5% RH was 23.2. The % Pd for the 5±3° C. condition was already fairly high for this method indicating, already, the presence of high order aggregates. The fact that the % Pd did not change at higher temperatures is thus not surprising. The high polydispersity was also observed in the initial baseline biophysical screen DLS data. Similarly high % Pd was observed for formulation with Sorbitol (F30). Interestingly, F28 which contains sorbitol and NaCl did not show a high % Pd which further supports the notion that NaCl is an ideal choice as a component for an optimal formulation. High % Pd was not observed for formulation F28 at the four week time point. The % Pd for formulation F30 at 40±2° C./75±5% RH was 15.4 and 14.2. Overall, histidine pH 6.0 formulations in the presence of charged excipients showed the least change in polydispersity. Both histidine formulations containing sucrose and sorbitol at pH 5.5 exhibited high % Pd at both stress conditions.
6. Conclusions
Based upon the results obtained from analytical testing of antibody IgG1-hDR5-05-E430G in the various formulations listed in Table 17, formulation F25 (30 mM histidine, 150 mM NaCl pH 6.0) was the optimal formulation for this molecule.
Initial baseline biophysical screening results suggested that acetate and histidine formulations at pH 5.5 were optimal buffer/pH conditions. Additionally, arginine and NaCl were better choice of excipients when compared to sorbitol and sucrose. The surfactant and cryoprotectant studies indicated that neither PS-80 nor sucrose was required to enhance the stability of the formulation. For the DoE stability study, the initial DSC results confirmed that 30 mM histidine pH 6.0 formulations had higher Tonset melting temperature values. Significant pH shifts were observed in all the 30 mM acetate formulations. The histidine pH 6.0 formulations did not exhibit any significant changes in pH over the four week stability at 5±3° C. and 40±2° C./75±5% RH. SEC data demonstrated that histidine pH 6.0 in the presence of charged excipients conferred the most stability for IgG1-hDR5-05-E430G. Results from icIEF showed that pH 5.5 and 6.0 samples were more resistant to changes in charge heterogeneity. It also showed that formulations in the presence of sucrose and sorbitol exhibited the most degradation. DLS data showed that histidine pH 6.0 formulations in the presence of charged excipients had the least change in polydispersity. The results for the reduced CE-SDS showed that histidine pH 6.0 formulations in the presence of charged excipients were the best formulations. Non-reduced CE-SDS data showed that the samples in the presence of arginine displayed high % HMW impurities that were not present in samples containing NaCl. Overall, the summation of the available data supports the choice of a formulation containing histidine and sodium chloride for this antibody.
Materials, Equipment and Methods
Materials, equipment and methods used were the same as in Example 54, except that the antibody was IgG1-hDR5-01-G56T-E430G rather than IgG1-hDR5-05-E430G.
Results
1. Initial Baseline Biophysical Screening
Initial biophysical screening was performed to select buffer/pH combinations to move forward into the excipient screening. DSC and DLS were used to assess thermal stability. DSC analysis provided the melting temperatures (Tm1 and Tm2) along with the Tonset. DLS analysis provided information on polydispersity and hydrodynamic radius of the protein.
A trend in DSC data was observed across the range of formulations. Tonset values ranged between 46° C. and 55° C. (data not shown). Higher Tonset values indicate greater thermal stability, thus the buffers of extreme low and high pH (glutamate, acetate, citrate, and phosphate), having the lowest Tonset values, are not optimal. The Tonset values of succinate and histidine buffers as well as acetate pH 5.5 indicated that these two buffers between pH 5.5 and 6.5 confer greater thermal stability.
Trends were observed across a range of buffers of increasing pH for DLS data (data not shown). In general, higher levels of polydispersity were observed for increasing pH across the range of buffers, indicating higher levels of aggregation for buffers of higher pH. Glutamate and Acetate buffers at both of their respective pH levels had the lowest levels of polydispersity (% Pd between 3.5%-7.6%). The remaining buffers, with the exception of 25 mM Histidine pH 5.5 (6.9% % Pd), had higher levels of polydispersity, ranging between 13.8%-23.3%.
Results from the DLS data from the initial biophysical screening did correlate strongly with formulation ranking results obtained from DSC for some formulations. Phosphate and citrate buffers exhibited high degrees of % Pd (14.1%-18.9%) as well as relatively lower Tonset values (48° C.-51° C.). Due to the evidence of aggregation and thermal instability, these two formulations were eliminated from further study. The other formulations (glutamate, acetate, succinate, histidine) did not have strong correlations between DSC and DLS data. For example, 25 mM histidine pH 6.0 and 6.5 had % Pd of 18.5% and 23.3%, respectively, however these same two buffers exhibited some of the highest Tonset values, 53° C. and 55° C., respectively. Both glutamate and acetate pH 4.5 buffers had somewhat lower Tonset values, between 46° C. and 50° C., but had the lowest % Pd values (between 3.5%-7.6%). Finally, succinate buffer at pH 5.5 and 6.0 exhibited higher Tonset values, 50° C. and 54° C., respectively, but were observed to have high levels of polydispersity (13.8% and 14.7%, respectively). Due to the inconclusive results in the aforementioned buffer formulations, all four buffers (glutamate, acetate, succinate, and histidine) were used in biophysical screening with excipients for further examination.
2. Biophysical Screening with Excipients
The formulations selected above were screened in the presence of 150 mM arginine, sodium chloride, sucrose or sorbitol. The data are shown in Table 24.
Trends were apparent in DSC and data for biophysical screening of antibody with excipients. Generally, the formulations with charged excipients had lower Tonset values than the formulations with sucrose or sorbitol. There was also a general increase in Tonset values (ranging between 46° C. and 55° C.) in DSC as pH increased across the range of buffers, indicating that increased buffer pH conferred greater thermal stability to the antibody.
A general trend was observed across DLS data as well. According to these data, the formulations containing charged excipients (arginine and NaCl) were found to have lower levels of polydispersity, thus lower levels of apparent aggregation, compared to formulations containing sugars (sorbitol and sucrose). These formulations containing sugars not only had higher levels of polydispersity, but in some cases contained two distinct proteinaceous populations, as indicated by the multimodal designation, for acetate buffer containing sorbitol and histidine buffer containing sorbitol and sucrose. The exception to this trend was found in all formulations of 25 mM succinate, which all showed high levels of polydispersity regardless of the presence of charged or sugar excipients.
Due to the significantly lower Tonset values for glutamate at lower pH, as well as the potential for low pH acid hydrolysis of the protein backbone, the glutamate buffers were excluded from further study. In addition, the succinate buffer was excluded from further examination due to the high levels of polydispersity among all of its formulations, including those with charged excipients. The biophysical screening data therefore suggest that 25 mM acetate and histidine formulations at pH 5.5 in the presence of sodium chloride and arginine were better candidates for further formulation development.
3. Solubility Study
The antibody was formulated at 40 mg/mL in its base formulation (30 mM histidine, pH 5.5) with four different concentrations of NaCl (0, 25, 50, and 100 mM NaCl) to determine the effect on solubility and phase separation. Samples were stored at −5±3° C. for 24 h on a pre-cooled lyophilizer shelf. After 24 hours, the set of samples were tested by appearance. No phase separation was observed in any of the prepared samples.
4. Surfactant Screening
The antibody was formulated in 30 mM histidine, pH 5.5 in the presence of 0, 0.03, or 0.06% w/v Tween-80 and stressed with three freeze-thaw cycles. Identical samples were agitated for period of 48 hours. After sample stress, the set of samples were tested by appearance, A280, SEC, reduced CE-SDS, and non-reduced CE-SDS.
4.1 Appearance
No visual differences were observed between samples any of the samples in the surfactant screening study. All samples were slightly yellow liquid, opalescent and free of visible particulates.
4.2 Protein Concentration by A280
The antibody concentrations obtained by UV analysis were not significantly different between agitated, freeze-thaw and control samples with different concentrations of PS-80, and ranged between 17.80 and 21.32 mg/mL (data not shown).
4.3 Size Exclusion Chromatography
Monomer purity did not significantly differ, and growth of new peaks was not observed for any of the surfactant screening samples. The purity of all samples was between 98.4-98.7% (data not shown).
4.4 Reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate
Purity (Light chain and heavy chain %) did not significantly differ, and no new peaks were observed for any of the surfactant screening samples. The purity of all samples was 95.4-95.8% (data not shown).
4.5 Non-Reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate
Main peak purity did not significantly differ, and no new peaks were observed for any of the surfactant screening samples. The purity of all samples was between 91.2% and 91.3% (data not shown).
4.6 Conclusions from Surfactant Screening
No changes in appearance, protein concentration, or purity were observed between unstressed and stressed samples containing concentrations of 0, 0.03, and 0.06% PS-80. These data indicate surfactants do not enhance the stability of the antibody.
5. Cryoprotectant Screening
In the cryoprotectant screen, the antibody was formulated in 30 mM histidine, pH 5.5 with three different concentrations (0, 5, or 10% w/v) of sucrose and stressed with three freeze-thaw cycles. Identical samples were agitated for a period of 48 hours. After sample stress, the set of samples were tested by appearance, A280, SEC, reduced CE-SDS, and non-reduced CE-SDS.
5.1 Appearance
No visual differences were observed between samples containing 0%, 5%, or 10% sucrose stressed with three freeze-thaw cycle and agitated for a period of 48 hours. All samples were slightly yellow liquid, opalescent, and free of visible particulates.
5.2 Protein Concentration by A280
The antibody concentrations obtained by UV analysis were not significantly different. Concentrations ranged between 19.03 and 22.92 mg/mL (data not shown).
5.3 Size Exclusion Chromatography
Monomer purity did not significantly differ, and growth of new peaks was not observed for any of the cryoprotectant screening samples. The purity of all samples was between 98.4-99.0% (data not shown).
5.4 Reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate
Purity (Light chain and heavy chain %) did not significantly differ, and growth of new peaks was not observed for any of the cryoprotectant screening samples. The purity of all samples was between 95.1-95.7% (data not shown).
5.5 Non Reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate
Main peak purity did not significantly differ, and no growth of new peaks was observed for any of the cryoprotectant screening samples. The purity of all samples was between 90.3-91.9% (data not shown).
5.6 Conclusions from Cyroprotectant Screening
No significant changes in appearance, protein concentration, or purity were observed between unstressed and stressed samples containing concentrations of 0%, 5% and 10% sucrose. These data indicated that cryoprotectants did not enhance the stability of the antibody.
6. DOE Stability Study
Study design and methodology was identical to that used in Example 54. See Table 17 above for listing of all formulations under examination.
6.1 DSC (Initial)
The Initial DSC data is shown in Table 25. For the histidine formulations, it was observed that high Tonset values were obtained for formulations at higher pH. This trend correlated with data obtained in the initial baseline screen where higher Tonset values were observed with increasing pH. Histidine formulations at pH 6.0 demonstrated higher Tonset values when compared to histidine formulations at pH 5.0 and pH 5.5. The Tonset values ranged from 47-52° C. for the histidine pH 6.0 formulations. Histidine pH 5.0 formulations ranged from 42-45° C. whereas histidine pH 5.5 formulations ranged from 46-50° C. High Tonset values were observed for formulations consisting of sucrose and sorbitol. Formulations F13, F14, F28, and F29 displayed high Tonset values for formulations consisting of sucrose and sorbitol. This is expected due to the effects osmolytes have on folded states of proteins. For the acetate formulations, a similar trend was observed. The formulations at higher pH displayed higher onset values. Acetate formulations pH 6.0 and pH 5.5 displayed high Tonset values with and without the presence of NaCl and arginine. The acetate pH 6.0 formulations ranged from 52-54° C. whereas the acetate pH 5.5 formulations ranged from 49-53° C. Acetate pH 5.0 formulation displayed the lowest Tonset range of 45-49° C.
6.2 UV (Initial)
The range of protein concentrations for the initial time point was between 18.52-21.86 mg/mL (data not shown). Overall, there were no significant changes in protein concentration observed at the initial time point.
6.3 Appearance
Most sample preparations at four week time point at 5±3° C. were clear and slightly yellow in color. Samples F28 and F29 (which contained either sorbitol or sucrose) were opalescent at 5±3° C. Most sample preparations in both acetate and histidine buffers at 5±3° C. and 40±2° C./75±5% RH exhibited few particles. Samples at both conditions were slightly yellow and clear with the exception of some samples prepared in histidine. For the 40±2° C./75±5% RH condition, the histidine formulations F20-1, F20-2, F20-3, F24, F28, F29, and F30, were opalescent. These formulations either do not have excipients or they have sucrose or sorbitol.
6.4 pH
For the acetate formulations, a significant pH shift was observed at the four week time point at 5±3° C. and 40±2° C./75±5% RH (data not shown). The range for the difference in pH for the acetate formulations at initial time point and 5±3° C. was 0.10-0.29. The shift in pH observed in these samples stressed at 40±2° C./75±5% RH was 0.07-1.30. For the histidine formulations, a pH shift was observed at the four week time point at 5±3° C. and 40±2° C./75±5% RH, but the changes were much less than that of the acetate samples. The range for the difference in pH for the histidine formulations at initial time point and 5±3° C. was 0.02-0.16. This type of change can be assigned to method variability for small volume samples. The shift in pH observed in these samples stressed at 40±2° C./75±5% RH was 0.02-0.94.
Significant pH shifts could also lead to accelerated degradation of the protein. The acetate formulations were susceptible to shifts in pH at a much higher level compared with histidine formulations, which makes acetate an unsuitable component for this antibody.
The stability results explained going forward will focus on histidine formulations (F16 to F30) because of the observed pH shift for acetate formulations.
6.5 Protein Content by UV A280
The range of protein content determination by A280 readings for the 5±3° C. samples was 14.66-21.70 mg/mL whereas the range of A280 readings for the 40±2° C./75±5% RH was 18.12-40.42 mg/mL (data not shown). Significant shifts in A280 readings were observed for F20-1, F20-2, F20-3, F24, and F28 at 40±2° C./75±5% RH. These same samples were observed to be opalescent in the appearance test. Due to the increase in observed protein concentration, it is likely that these results are due to a non-product related UV absorber inflating the apparent UV concentrations.
6.6 Size Exclusion Chromatography
SEC results for four week time points are shown in Table 26. Significant changes occurred in histidine formulations that were found to have been of high protein concentration and opalescent in appearance (F20-1, F20-2, F20-3, F24, and F28, F29, F30). For these samples, a significant UV absorbing peak was observed in the mobile phase flow through. These SEC data, as well as other supporting analyses already discussed, indicate that these formulations contained a UV absorbing non-product related component. At high temperature stress conditions, it is possible that the histidine formulation without charged excipients degraded and acted as this UV absorbing component. For the histidine formulations it was observed that presence of charged excipients improved stability of the formulation. It was also observed that an increase in pH improved the purity of the histidine formulations. A decrease in % total impurities for formulations in the presence of charged excipients was observed at 40±2° C./75±5% RH. For histidine pH 5.0 formulations F17, F18 and F19 at 40±2° C./75±5% RH, the % total impurities ranged from 4.2-4.9%. The % total impurities for the histidine pH 5.5 formulations F21, F22, and F23 ranged from 3.6-5.6% whereas histidine pH 6.0 formulations F25, F26, and F27 had % total impurities ranging from 3.2-3.4%. In general, higher pH led to decrease in % total impurities and histidine pH 6.0 formulations in the presence of charged excipients showed better stability. Overall, histidine pH 6.0 formulations in the presence of charged excipients NaCl and arginine had better stability.
6.7 Imaging Capillary Isoelectric Focusing
Charge heterogeneity of antibody samples was determined using icIEF (Table 27). Based on the data, the percent main peak of histidine formulations at the four week time point at 5±3° C. ranged from 45.6-47%. At 40±2° C./75±5% RH, formulations consisting of sorbitol or sucrose F28, F29 and F30 had significant increase in percent basic variants of 11.2%, 19.0%, and 11.5%, respectively. At the four week time point at 40±2° C./75±5% RH, all histidine formulations demonstrated increases in percent basic variants. The icIEF data showed that pH of the samples affected charge heterogeneity significantly. Histidine formulations at pH 5.0 showed more significant increases in basic variants when compared to histidine formulations pH 5.5 and 6.0. For histidine pH 5.0 formulations, the range of percent basic variants at 40±2° C./75±5% RH was 6.3-7.2%. At 40±2° C./75±5% RH, the range of percent basic variants for histidine pH 5.5 was 5.5-6.4% whereas the range of percent basic variants for histidine pH 6.0 formulation was 4.4-4.7%. This result is likely not due to deamidation because deamidation is known to be accelerated at higher pH values, and the opposite trend is observed here. The proliferation of basic variants may be due to other impurities like HMW or LMW species forming. Across all formulations, the results showed that histidine pH 6.0 were better formulations than histidine pH 5.0 and pH 5.5 formulations and significant degradation was observed in histidine formulations consisting of sucrose and sorbitol.
6.8 Reduced Capillary Electrophoresis—Sodium Dodecyl Sulfate
Results for reduced capillary electrophoresis are shown in Table 28. At the four week time point at 5±3° C., all histidine formulations regardless of pH showed comparable purity, however the formulations with sucrose and sorbitol were somewhat less pure.
At the four week time point at 40±2° C./75±5% RH, the results showed an increase in impurities for all of the sample preparations. It was observed that formulations at lower pH displayed more degradation at 40±2° C./75±5% RH. Histidine pH 5.0 formulations showed a considerable decrease in percent purity. The range for the percent purity was 86.3-88.9%. For the histidine pH 5.5 formulations, the percent purity ranged from 85.0-92.3%. Significantly less degradation was observed for the histidine pH 6.0 formulations. The percent purity for the histidine pH 6.0 formulations ranged from 90.1-93.1% at 40±2° C./75±5% RH four week time point. In addition, the % LMW for histidine samples was higher for histidine samples at lower pH and considerably lower for histidine formulations at higher pH. The range of % LMW for histidine pH 5.0 formulations was 8.3-11.0%, whereas the Histidine pH 5.5 and 6.0 formulations displayed % LMW range of 5.4-12.1% and 4.0-6.6%. For the Histidine pH 6.0 formulations, it was also observed that percent purity did not significantly decrease in the presence of charged excipients. Across all formulations, histidine pH 6.0 formulations in the presence of charged excipients NaCl and arginine showed better purity compared to the rest of the histidine formulations.
6.9 Non-Reduced Capillary Electrophoresis—Sodium Dodecyl Sulfate
Results for non-reduced capillary electrophoresis are shown in Table 29. For histidine formulations at 5±3° C., formulations F23 and F29 displayed high % HMW of 3.6% and 3.3% compared to rest of the histidine formulations. For the histidine formulations stressed at 40±2° C./75±5% RH, formulations F28, F29 and F30 displayed extremely high % total impurities of 36.8%, 49.3% and 37.4%, respectively. Additionally formulations F20 and F24 displayed high % impurities. These formulations either did not contain excipients or contained sucrose or sorbitol. Based on the data, it was observed that the % total impurities were lower at higher pH values for samples stressed at 40±2° C./75±5% RH. For histidine formulations pH 5.0 the % total impurities ranged from 11.8-15.0%. For histidine formulations at pH 5.5 (formulations F21, F22 and F23), the % total impurities ranged from 10.7-12.3% whereas the % total impurities for histidine formulations at pH 6.0 (F25, F26 and F27) ranged from 9.8-10.0%. Results obtained from non-reduced CE-SDS data confirm that histidine pH 6.0 formulations in the presence of charged excipients showed better purity compared to the rest of the histidine formulations.
6.10 Dynamic Light Scattering
Acetate formulations are not being considered due to the pH shift observed in these formulations. Based on the DLS data for histidine formulations, formulations F20-2, F24, F28 and F30 displayed high % Pd values at 5±3° C. (data not shown). Formulation F29 had multimodal designation for % Pd indicating presence of proteinaceous particles in solution. Previous data also indicated that the aforementioned formulations were not optimal conditions. The formulations consisting of sucrose and sorbitol exhibited high % Pd at both temperature conditions. This was also seen in the DLS data from the initial baseline screening study. For the histidine formulations at 40±2° C./75±5% RH, it was observed that at lower pH, there was increase in % Pd. For histidine formulations at pH 5.0, the % Pd at 40±2° C./75±5% RH ranged from 4.7-18.2%. For the histidine formulations at pH 5.5 (F21, F22, and F23), the % Pd ranged from 7.9-9.5%. Finally, the % Pd for the histidine pH 6.0 formulations (F25, F26, and F27) ranged from 7.8-9.8%. Histidine formulations at pH 5.5 (F21, F22, and F23) and 6.0 (F25, F26, and F27) displayed the lowest % Pd values compared at pH 5.0. For the histidine pH 6.0 formulations, F25, F26 and F27 were promising candidates as all formulations demonstrated low % Pd at both stress conditions and in the presence of charged excipients.
7. Conclusions
Based upon the results obtained from analytical testing of antibody IgG1-hDR5-01-G56T-E430G in the various formulations listed in Table 17, formulation F25 (30 mM histidine, 150 mM NaCl pH 6.0) was the optimal formulation for this molecule.
Initial baseline biophysical screening results suggested that acetate and histidine formulations at pH 5.5 in the presence of NaCl and arginine were optimal buffer/pH conditions. Additionally, arginine and NaCl were a better choice of excipients when compared to sorbitol and sucrose. The surfactant and cryoprotectant studies indicated that neither PS-80 nor sucrose was required to enhance the stability of the formulation. For the DoE stability study, the initial DSC results confirmed that 30 mM histidine pH 6.0 formulations had sufficiently high Tonset melting temperature values. Significant pH shifts were observed in all the 30 mM acetate formulations. The histidine pH 6.0 formulations did not exhibit any significant changes in pH over the four week stability at 5±3° C. and 40±2° C./75±5% RH. SEC data demonstrated that histidine pH 6.0 in the presence of charged excipients conferred the most stability for this antibody. Results from icIEF showed that histidine pH 6.0 samples were more resistant to changes in charge heterogeneity. It also showed that formulations in the presence of sucrose and sorbitol exhibited the most degradation. The results for the reduced and non reduced CE-SDS showed that histidine pH 6.0 formulations in the presence of charged excipients were best formulations. DLS data showed that histidine pH 5.5 and 6.0 formulations in the presence of charged excipients had the least change in polydispersity. Overall, the summation of the available data supports 30 mM histidine, 150 mM sodium chloride pH 6.0 as a formulation for antibody IgG1-hDR5-01-G56T-E430G.
A 1:1 mix of antibody IgG1-hDR5-01-G56T-E430G (20 mg/mL) and IgG1-hDR5-05-E430G (20 mg/mL), both formulated in 30 mM histidine, 150 mM sodium chloride pH 6.0 was stored at 5° in order to investigate the stability of the mix in the respective formulation. Samples were analysed after 2, 4, 8 and 12 weeks as well as after 6 months of storage, by Appearance, pH protein content, Size Exclusion Chromatography, Reduced and Non-Reduced Capillary Electrophoresis—Sodium Dodecyl Sulfate and Imaging Capillary Isoelectric Focusing, using the methods described in Example 54.
Results
No significant changes were observed in any of the tested properties. Thus, the antibody mixture was stable for at least 6 months at 5° C. storage temperature.
This application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP2018/065071, filed on Jun. 7, 2018, which claims priority to U.S. Provisional Application Nos. 62/614,801, filed on Jan. 8, 2018, and 62/516,489, filed on Jun. 7, 2017. The contents of the aforementioned applications are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/065071 | 6/7/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62614801 | Jan 2018 | US | |
| 62516489 | Jun 2017 | US |
