Patent Application
20170260282
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 Feb. 21, 2017, is named JBI5082USNP_SL.txt and is 92,231 bytes in size.
The disclosure provided herein relates to monoclonal antibodies that specifically bind glucocorticoid-induced tumor necrosis factor receptor (GITR) and methods of producing and using the described antibodies.
Glucocorticoid-induced TNFR-related protein (GITR; also AITR, TNFRSF18, or CD357), a member of the TNFR superfamily, is expressed in many components of the innate and adaptive immune system and stimulates both acquired and innate immunity (Nocentini G et al., (1994) PNAS 94: 6216-6221; Hanabuchi S et al., (2006) Blood 107:3617-3623; Nocentini G & Riccardi C (2005) Eur J Immunol 35: 1016-1022; Nocentini G et al., (2007) Eur J Immunol 37: 1165-1169). It is expressed in several cells and tissues, including T, B, dendritic (DC) and Natural Killer (NK) cells and is activated by its ligand, GITR-L, mainly expressed on Antigen Presenting Cells (APCs), on endothelial cells, and also in tumor cells.
The GITR-GITRL system participates in the development of autoimmune/inflammatory responses and potentiates response to infection and tumors. For example, treating animals with GITR-Fc fusion protein ameliorates autoimmune/inflammatory diseases while GITR triggering is effective in treating viral, bacterial, and parasitic infections, as well in boosting immune response against tumors (Nocentini G et al., (2012) Br J Pharmacol 165: 2089-99). These effects are due to several concurrent mechanisms including: co-activation of effector T-cells, inhibition of regulatory T (Treg) cells, modulation of NK and dendritic cell function, activation of macrophages, and regulation of the extravasation process. The membrane expression of GITR is increased following T cell activation (Hanabuchi S et al, (2006) supra; Nocentini G & Riccardi C supra). Its triggering coactivates effector T lymphocytes (McHugh R S et al, (2002) Immunity 16: 311-323; Shimizu J et al, (2002) Nat Immunol 3: 135-142; Roncheti S et al, (2004) Eur J Immunol 34: 613-622; Tone M et al, (2003) PNAS 100: 15059-15064). GITR activation increases resistance to tumors and viral infections, is involved in autoimmune/inflammatory processes and regulates leukocyte extravasation (Nocentini G & Riccardi C (2005) supra; Cuzzocrea S et al, (2004) J Leukoc Biol 76: 933-940; Shevach E M & Stephens G L (2006) Nat Rev Immunol 6: 613-618; Cuzzocrea S et al, (2006) J Immunol 177: 631-641; Cuzzocrea S et al, (2007) FASEB J 21: 117-129).
Human GITR is expressed at very low levels in peripheral (non-activated) T cells. After T cell activation, GITR is strongly up-regulated for several days in both CD4+ and CD8+ cells (Kwon B et al, (1999) J Biol Chem 274: 6056-6061; Gurney A L et al, (1999) Curr Biol 9: 215-218; Ronchetti S et al, (2004) supra; Shimizu J et al, (2002) supra; Ji H B et al, (2004) supra; Ronchetti S et al, (2002) Blood 100: 350-352; Li Z et al, (2003) J Autoimmun 21: 83-92), with CD4+ cells having a higher GITR expression than CD8+ cells (Kober J et al, (2008) Eur J Immunol 38(10): 2678-88; Bianchini R et al, (2011) Eur J Immunol 41(8): 2269-78).
The role of human GITR in modulating immune responses indicates that it may be a suitable target for antibody-based therapy against diseases such as cancer. Antibodies against GITR are described (e.g. in WO200610502, WO2011028683, WO2015031667, WO20150353637, WO2015187835, WO2015184099, U.S. Pat. No. 9,255,151 and U.S. Pat. No. 9,255,152), but there is an ongoing need for novel agents and methods for modulating GITR activity against diseases, such as cancer.
Provided herein are antibodies that specifically bind to GITR and antigen-binding fragments thereof. Also described are related polynucleotides capable of encoding the provided GITR-specific antibodies and antigen-binding fragments, cells expressing the provided antibodies and antigen-binding fragments, as well as associated vectors and detectably labeled antibodies and antigen-binding fragments. In addition, methods of using the provided antibodies and antigen-binding fragments are described. For example, given the role GITR plays in modulating an immune response, the GITR-specific antibodies have utility in treating a variety of GITR-related diseases or disorders in which it is desirable to modulate an immune response. For example, the GITR specific antibodies can be used in a variety of immunotherapy applications, such as the treatment of a variety of cancers.
Described herein are isolated antibodies and antigen-binding fragments specific for GITR. In some embodiments, the GITR-specific antibodies and antigen-binding fragments bind human GITR. In some embodiments, the GITR-specific antibodies and antigen-binding fragments bind human GITR and cynomolgus monkey GITR. In some embodiments, the GITR-specific antibodies and antigen-binding fragments bind to an epitope including one or more residues from the GITR extracellular domain (ECD) as defined in SEQ ID NO:59. This GITR-specific antibody or antigen-binding fragment may bind to GITR with a binding affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less.
Table 1 provides a summary of examples of some GITR-specific antibodies described herein:
In some embodiments are provided a GITR-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 1 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 1.
The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans. They share more than 95% homology in the amino acid sequences of the Fc regions but show major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC and ADCP, the Fc region of an antibody binds to Fc receptors (FcgRs) on the surface of immune effector cells such as natural killers and macrophages, leading to the lysis or phagocytosis of the targeted cells. In CDC, the antibodies kill the targeted cells by triggering the complement cascade at the cell surface. The antibodies described herein include antibodies with the described features of the variable domains in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to effect different effector functions.
In some embodiments, the antibodies comprise the CDRs of the antibodies presented in Table 1 above. In some embodiments the described antibodies are capable of binding to GITR with a dissociation constant of 30 nM or less as measured by surface plasmon resonance (SPR). In some embodiments the described antibodies are capable of inducing an increase in luciferase expression in an NF-κB luciferase gene assay. In some embodiments the described antibodies are capable of inducing ADCC in vitro with an EC50 of 67 ng/mL or less.
In addition to the described GITR-specific antibodies and antigen-binding fragments, also provided are polynucleotide sequences capable of encoding the described antibodies and antigen-binding fragments. Vectors comprising the described polynucleotides are also provided, as are cells expressing the GITR-specific antibodies or antigen-binding fragments provided herein. Also described are cells capable of expressing the disclosed vectors. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacteria cells (such as E. coli). A process for the production of the described antibodies or antigen-binding fragments is also provided.
Methods of using the described GITR-specific antibodies or antigen-binding fragments are also disclosed. Particular antibodies for use in the methods discussed in this section include those with the set of CDRs described for antibodies in Table 1. For example, the key role that GITR plays in an immune response makes it an attractive target for immunotherapy, including inducing or enhancing an immune response against desired tumor antigens or pathogenic antigens (e.g., viruses and other pathogenic organisms). As such, the GITR-specific antibodies have utility in the treatment of various cancers and infectious disease.
As noted above, GITR activation sends a co-activating signal to CD4+ and CD8+ T cells and prevents suppression of an immune response by regulatory T cells. Thus, in one embodiment, a GITR-specific antibody is administered to inhibit the suppression of effector T cell activity by regulatory T cells. Such inhibition can be assayed by a variety of methods known in the art, including, for example, by monitoring T cell proliferation, expression of known markers of activation, or cytokine secretion. In another embodiment, a GITR-specific antibody is administered to a subject to decrease the level of regulatory T cells, for instance the level of tumor regulatory T cells. In yet another embodiment, the activity of effector T cells is induced or enhanced by administering a GITR-specific antibody as provided herein. Specific assays for each of these methods are provided in the EXAMPLES.
Described herein are kits including the disclosed GITR-specific antibodies or antigen-binding fragments thereof. The described kits may be used to carry out the methods of using the GITR-specific antibodies or antigen-binding fragments provided herein, or other methods known to those skilled in the art. In some embodiments the described kits may include the antibodies or antigen-binding fragments described herein and reagents for use in detecting the presence of GITR in a biological sample. Accordingly, the described kits may include one or more of the antibodies, or an antigen-binding fragment(s) thereof, described herein and a vessel for containing the antibody or fragment when not in use, instructions for use of the antibody or fragment, the antibody or fragment affixed to a solid support, and/or detectably labeled forms of the antibody or fragment, as described herein.
Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
“Isolated” means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if such composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An “isolated” antibody or antigen-binding fragment, as used herein, is intended to refer to an antibody or antigen-binding fragment which is substantially free of other antibodies or antigen-binding fragments having different antigenic specificities (for instance, an isolated antibody that specifically binds to GITR is substantially free of antibodies that specifically bind antigens other than GITR). An isolated antibody that specifically binds to an epitope, isoform or variant of GITR may, however, have cross-reactivity to other related antigens, for instance from other species (such as GITR species homologs).
As used herein, the terms “glucocorticoid-induced TNFR-related protein” and “GITR” specifically include the human GITR protein, for example as described in GenBank Accession No. AF241229, NCBI Reference Sequence: NP_004186.1 and UniProtKB/Swiss-Prot Accession No. Q9Y5U5 (see also Kwon et al. 1999, J. Biol. Chem. 274, 6056-6061). GITR is also known in the scientific literature as AITR, CD357, TNFRSF18, and GITR-D.
As used herein, the terms “GITR ligand”, “GITRL”, and “GITR-L” refer to glucocorticoid-induced TNFR-related protein ligand. GITRL is otherwise known as activation-induced TNF-related ligand (AITRL) and tumor necrosis factor ligand superfamily member 18 (TNFSF18). GenBank accession number AF125303 provides an exemplary human GITRL nucleic acid sequence. GenBank™ accession number NP 005083 and Swiss-Prot accession number Q9UNG2 provide exemplary human GITRL amino acid sequences. In a particular embodiment, the GITRL is a human GITRL of SEQ ID NO: 65.
“Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric, polymeric and chimeric forms, unless otherwise specified. Specifically encompassed by the term “antibody” are polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies.
“Antigen-binding fragments” are any proteinaceous structure that may exhibit binding affinity for a particular antigen. Antigen-binding fragments include those provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, antigen-binding fragments may comprise at least one variable region (either a heavy chain or light chain variable region) or one or more CDRs of an antibody known to bind a particular antigen. Examples of suitable antigen-binding fragments include, without limitation diabodies and single-chain molecules as well as Fab, F(ab′)2, Fc, Fabc, and Fv molecules, single chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, a Fd fragment consisting essentially of the V.sub.H and C.sub.H1 domains; a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 November; 21(11):484-90); camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 January; 5(1): 111-24); an isolated complementarity determining region (CDR), and the like. All antibody isotypes may be used to produce antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that may successfully incorporate polypeptide segments in an orientation that confers affinity for a given antigen of interest, such as protein scaffolds. Antigen-binding fragments may be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies. The phrase “an antibody or antigen-binding fragment thereof” may be used to denote that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.
The terms “CDR”, and its plural “CDRs”, refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDRL1, CDRL2 and CDRL3) and three make up the binding character of a heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, contact or any other boundary definitions. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called “hypervariable regions” within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography,” J. Mol. Biol. 262:732 (1996)), each of which is hereby incorporated by reference in its entirety.
Typically, CDRs form a loop structure that can be classified as a canonical structure. The term “canonical structure” refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); Chothia et al., “Conformations of Immunoglobulin Hypervariable Regions,” I 342:877 (1989); Martin and Thornton, “Structural Families in Loops of Homologous Proteins: Automatic Classification, Modelling and Application to Antibodies,” J. Mol. Biol. 263:800 (1996), each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework (i.e., outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues.
The term “polypeptide” is used interchangeably with the term “protein” and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein. “Specifically binds” or “binds specifically” or derivatives thereof when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1×10−8 M, as measured by a surface plasmon resonance assay or a cell-binding assay. Phrases such as “[antigen]-specific” antibody (e.g., GITR-specific antibody) are meant to convey that the recited antibody specifically binds the recited antigen.
“Polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
A “vector” is a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
As used herein, the term “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome. The terms “expression” and “production” are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications. The expression or production of an antibody or antigen-binding fragment thereof may be within the cytoplasm of the cell, or into the extracellular milieu such as the growth medium of a cell culture. The meaning of “substantially the same” can differ depending on the context in which the term is used. Because of the natural sequence variation likely to exist among heavy and light chains and the genes encoding them, one would expect to find some level of variation within the amino acid sequences or the genes encoding the antibodies or antigen-binding fragments described herein, with little or no impact on their unique binding properties (e.g., specificity and affinity). Such an expectation is due in part to the degeneracy of the genetic code, as well as to the evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the encoded protein. Accordingly, in the context of nucleic acid sequences, “substantially the same” means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or greater identity. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.
The degree of variation that may occur within the amino acid sequence of a protein without having a substantial effect on protein function is much lower than that of a nucleic acid sequence, since the same degeneracy principles do not apply to amino acid sequences. Accordingly, in the context of an antibody or antigen-binding fragment, “substantially the same” means antibodies or antigen-binding fragments having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibodies or antigen-binding fragments described. Other embodiments include GITR-specific antibodies, or antigen-binding fragments, that have framework, scaffold, or other non-binding regions that do not share significant identity with the antibodies and antigen-binding fragments described herein, but do incorporate one or more CDRs or other sequences needed to confer binding that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences described herein.
“Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff/kon, whereas KA is calculated from the quotient of kon/koff. kon refers to the association rate constant of, e.g., an antibody to an antigen, and koff refers to the dissociation of, e.g., an antibody to an antigen. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as surface plasmon resonance.
The term “subject” refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described methods, the subject is a human.
Described herein are isolated monoclonal antibodies or antigen-binding fragments that specifically bind GITR. The general structure of an antibody molecule comprises an antigen binding domain, which includes heavy and light chains, and the Fc domain, which serves a variety of functions, including complement fixation and binding antibody receptors.
The described GITR-specific antibodies or antigen-binding fragments include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin structure. The described antibodies or antigen-binding fragments also include the IgY isotype generally found in hen or turkey serum and hen or turkey egg yolk.
The GITR-specific antibodies and antigen-binding fragments may be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, non-human derived antibodies or antigen-binding fragments may be genetically or structurally altered to be less antigenic upon administration to a human patient.
In some embodiments, the antibodies or antigen-binding fragments are chimeric. As used herein, the term “chimeric” refers to an antibody, or antigen-binding fragment thereof, having at least some portion of at least one variable domain derived from the antibody amino acid sequence of a non-human mammal, a rodent, or a reptile, while the remaining portions of the antibody, or antigen-binding fragment thereof, are derived from a human.
In some embodiments, the antibodies are humanized antibodies. Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody may include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
The antibodies or antigen-binding fragments described herein can occur in a variety of forms, but will include one or more of the antibody CDRs shown in Table 1. Described herein are isolated antibodies and antigen-binding fragments that specifically bind to GITR. In some embodiments, the GITR-specific antibodies or antigen-binding fragments are human IgG, or derivatives thereof. While the GITR-specific antibodies or antigen-binding fragments exemplified herein are human, the antibodies or antigen-binding fragments exemplified may be chimerized.
In some embodiments are provided a GITR-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 1 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 1.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 12, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 39 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 2, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 13, a light chain CDR1 comprising SEQ ID NO: 29, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 36. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 40 and a light chain substantially the same as, or identical to, SEQ ID NO: 56 The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 6, a heavy chain CDR3 comprising SEQ ID NO: 14, a light chain CDR1 comprising SEQ ID NO: 30, a light chain CDR2 comprising SEQ ID NO: 33, and a light chain CDR3 comprising SEQ ID NO: 37. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 41 and a light chain substantially the same as, or identical to, SEQ ID NO: 57. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 15, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 42 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 16, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 43 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3 comprising SEQ ID NO: 17, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 44 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 9, a heavy chain CDR3 comprising SEQ ID NO: 18, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 45 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 10, a heavy chain CDR3 comprising SEQ ID NO: 19, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 46 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 20, a light chain CDR1 comprising SEQ ID NO: 31, a light chain CDR2 comprising SEQ ID NO: 34, and a light chain CDR3 comprising SEQ ID NO: 38. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 47 and a light chain substantially the same as, or identical to, SEQ ID NO: 58. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 11, a heavy chain CDR3 comprising SEQ ID NO: 21, a light chain CDR1 comprising SEQ ID NO: 31, a light chain CDR2 comprising SEQ ID NO: 34, and a light chain CDR3 comprising SEQ ID NO: 38. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 48 and a light chain substantially the same as, or identical to, SEQ ID NO: 58. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 22, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 49 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 23, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 50 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 24, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 51 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 25, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 52 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 26, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 53 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 11, a heavy chain CDR3 comprising SEQ ID NO: 21, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 54 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3 comprising SEQ ID NO: 16, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 63 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3 comprising SEQ ID NO: 26, a light chain CDR1 comprising SEQ ID NO: 28, a light chain CDR2 comprising SEQ ID NO: 32, and a light chain CDR3 comprising SEQ ID NO: 35. This GITR-specific antibody or antigen-binding fragment may comprise human framework sequences. This GITR-specific antibody or antigen-binding fragment may bind to GITR with an affinity of 30 nM or less, may induce an increase in luciferase expression in an NF-κB luciferase gene assay and may induce ADCC in vitro with an EC50 of 67 ng/mL or less. In some embodiments, the GITR-specific antibodies and antigen-binding fragments comprise a heavy chain substantially the same as, or identical to, SEQ ID NO: 64 and a light chain substantially the same as, or identical to, SEQ ID NO: 55. The heavy chain and light chain of antibodies discussed in this paragraph are suitable for inclusion in bispecific constructs in which one arm is an anti-GITR arm.
Also disclosed are isolated polynucleotides that encode the antibodies or antigen-binding fragments that specifically bind to GITR. The isolated polynucleotides capable of encoding the variable domain segments provided herein may be included on the same, or different, vectors to produce antibodies or antigen-binding fragments. Polynucleotides encoding recombinant antigen-binding proteins also are within the scope of the disclosure. In some embodiments, the polynucleotides described (and the peptides they encode) include a leader sequence. Any leader sequence known in the art may be employed. The leader sequence may include, but is not limited to, a restriction site or a translation start site.
The GITR-specific antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described GITR-specific antibodies or antigen-binding fragments. These variants may include: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Antibodies or antigen-binding fragments described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues. The techniques for obtaining these variants, including genetic (deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
The GITR-specific antibodies or antigen-binding fragments described herein may embody several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments the antibody isotype is IgG1, IgG2, IgG3, or IgG4 isotype, preferably IgG1 isotype. Antibody or antigen-binding fragment thereof specificity is largely determined by the amino acid sequence, and arrangement, of the CDRs. Therefore, the CDRs of one isotype may be transferred to another isotype without altering antigen specificity. Alternatively, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without altering antigen specificity. Accordingly, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.
The GITR-specific antibodies or antigen-binding fragments described herein have binding affinities for GITR that include a dissociation constant (KD) of less than about 30 nM. The affinity of the described GITR-specific antibodies, or antigen-binding fragments, may be determined by a variety of methods known in the art, such as surface plasmon resonance or ELISA-based methods. Assays for measuring affinity by SPR include assays performed using a BIAcore 3000 machine, where the assay is performed at room temperature (e.g. at or near 25° C.), wherein the antibody capable of binding to GITR is captured on the BIAcore sensor chip by an anti-Fc antibody (e.g. goat anti-human IgG Fc specific antibody Jackson ImmunoResearch laboratories Prod #109-005-098) to a level around 75 RUs, followed by the collection of association and dissociation data at a flow rate of 40 μl/min.
Also provided are vectors comprising the polynucleotides described herein. The vectors can be expression vectors. Recombinant expression vectors containing a sequence encoding a polypeptide of interest are thus contemplated as within the scope of this disclosure. The expression vector may contain one or more additional sequences such as but not limited to regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.
Recombinant expression vectors within the scope of the description include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements. Such regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5′ or 3′ flanking nontranscribed sequences, 5′ or 3′ nontranslated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host may also be incorporated.
The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. Exemplary vectors may be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).
In some embodiments, the antibody- or antigen-binding fragment-coding sequence is placed under control of a powerful constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. Such viral promoters include without limitation, Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus. In one embodiment, the GITR-specific antibody or antigen-binding fragment thereof coding sequence is placed under control of an inducible promoter such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon-stimulated response elements (ISRE) such as protein kinase R 2′,5′-oligoadenylate synthetases, Mx genes, ADAR1, and the like.
Vectors described herein may contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins. In some embodiments the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. Vector components may be contiguously linked, or arranged in a manner that provides optimal spacing for expressing the gene products (i.e., by the introduction of “spacer” nucleotides between the ORFs), or positioned in another way. Regulatory elements, such as the IRES motif, may also be arranged to provide optimal spacing for expression.
The vectors may comprise selection markers, which are well known in the art. Selection markers include positive and negative selection markers, for example, antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.
The vectors described herein may be used to transform various cells with the genes encoding the described antibodies or antigen-binding fragments. For example, the vectors may be used to generate GITR-specific antibody or antigen-binding fragment-producing cells. Thus, another aspect features host cells transformed with vectors comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds GITR, such as the antibodies or antigen-binding fragments described and exemplified herein.
Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells. Cells suitable for use in the expression of the GITR-specific antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin, for example but not limited to NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2/0 myeloma cells, and BHK cell lines, among others. In addition, expression of antibodies may be accomplished using hybridoma cells. Methods for producing hybridomas are well established in the art.
Cells transformed with expression vectors described herein may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant-positive cells are expanded and screened for subclones exhibiting a desired phenotype, such as high level expression, enhanced growth properties, or the ability to yield proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to inherent properties of a given subclone or to mutation. Mutations may be effected through the use of chemicals, UV-wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.
Provided herein are GITR-specific antibodies or antigen-binding fragments thereof for use in therapy. In particular, these antibodies or antigen-binding fragments may be useful in treating cancer. Accordingly, the invention provides a method of treating cancer comprising administering an antibody as described herein, such as GITR-specific antibodies or antigen-binding fragments. A couple aspects of GITR biology make it a potential target for the treatment of a variety of cancers. The first is that GITR activation, as described at length above, activates the immune system. Additionally, GITR-expressing effector T cells and regulatory T cells infiltrate multiple tumor types, yet there is little or no expression of GITR on non-hematopoetic cells. This distribution profile means that GITR-expressing cells can become concentrated at tumors. This combination of activities and distribution collectively makes GITR targeting an attractive approach for treating a variety of cancers. The antigen binding proteins can be used to treat both solid tumors, as well as hematological cancers, including leukemia.
The antibodies for use in these methods include those described herein above, for example a GITR-specific antibody or antigen-binding fragment with the features set out in Table 1, for example the CDRs or variable domain sequences, and in the further discussion of these antibodies.
In some embodiments described herein, immune effector properties of the GITR-specific antibodies may be modulated through Fc modifications by techniques known to those skilled in the art. For example, Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. may be provided and/or controlled by modifying residues in the Fc responsible for these activities. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated reaction in which non-specific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide component. Human IgG1 or IgG3 are N-glycosylated at Asn297 with the majority of the glycans in the well-known biantennary G0, G0F, G1, G1F, G2 or G2F forms. Antibodies produced by non-engineered CHO cells typically have a glycan fucose content of about at least 85%. The removal of the core fucose from the biantennary complex-type oligosaccharides attached to the Fc regions enhances the ADCC of antibodies via improved Fc.gamma.RIIIa binding without altering antigen binding or CDC activity. Such mAbs can be achieved using different methods reported to lead to the successful expression of relatively high defucosylated antibodies bearing the biantennary complex-type of Fc oligosaccharides such as control of culture osmolality (Konno et al., Cytotechnology 64:249-65, 2012), application of a variant CHO line Lec13 as the host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), application of a variant CHO line EB66 as the host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of print; PMID:20562582), application of a rat hybridoma cell line YB2/0 as the host cell line (Shinkawa et al., J Biol Chem 278:3466-3473, 2003), introduction of small interfering RNA specifically against the .alpha. 1,6-fucosyltrasferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or coexpression of .beta.-1,4-N-acetylglucosaminyltransferase III and Golgi .alpha.-mannosidase II or a potent alpha-mannosidase I inhibitor, kifunensine (Ferrara et al., J Biol Chem 281:5032-5036, 2006, Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008).
In some embodiments described herein, ADCC elicited by the GITR antibodies may also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are for example substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EU index) as described in U.S. Pat. No. 6,737,056.
The pharmaceutical compositions provided herein comprise: a) an effective amount of a GITR-specific antibody or antibody fragment of the present invention, and b) a pharmaceutically acceptable carrier, which may be inert or physiologically active. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described herein, or an antigen-binding fragment thereof. As used herein, the term “pharmaceutically acceptable carriers” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and/or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as any combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. In particular, relevant examples of suitable carrier include: (1) Dulbecco's phosphate buffered saline, pH.about.7.4, containing or not containing about 1 mg/mL to 25 mg/mL human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20®.
The compositions herein may also contain a further therapeutic agent, as necessary for the particular disorder being treated. Preferably, the GITR-specific antibody or antibody fragment and the supplementary active compound will have complementary activities that do not adversely affect each other. In a preferred embodiment, the further therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin 2. In a preferred embodiment, the further therapeutic agent is a chemotherapeutic agent.
The compositions of the invention may be in a variety of forms. These include for example liquid, semi-solid, and solid dosage forms, but the preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions. The preferred mode of administration is parenteral (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, the compositions of the invention are administered intravenously as a bolus or by continuous infusion over a period of time. In another preferred embodiment, they are injected by intramuscular, subcutaneous, intra-articular, intrasynovial, intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
Sterile compositions for parenteral administration can be prepared by incorporating the antibody, antibody fragment or antibody conjugate of the present invention in the required amount in the appropriate solvent, followed by sterilization by microfiltration. As solvent or vehicle, there may be used water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. These compositions may also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterile compositions for parenteral administration may also be prepared in the form of sterile solid compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium.
The GITR-specific antibody or antibody fragment may also be orally administered. As solid compositions for oral administration, tablets, pills, powders (gelatine capsules, sachets) or granules may be used. In these compositions, the active ingredient according to the invention is mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose or silica, under an argon stream. These compositions may also comprise substances other than diluents, for example one or more lubricants such as magnesium stearate or talc, a coloring, a coating (sugar-coated tablet) or a glaze.
As liquid compositions for oral administration, there may be used pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil. These compositions may comprise substances other than diluents, for example wetting, sweetening, thickening, flavoring or stabilizing products.
The doses depend on the desired effect, the duration of the treatment and the route of administration used; they are generally between 5 mg and 1000 mg per day orally for an adult with unit doses ranging from 1 mg to 250 mg of active substance. In general, the doctor will determine the appropriate dosage depending on the age, weight and any other factors specific to the subject to be treated.
Also provided herein are methods for killing a Treg cell by administering to a patient in need thereof a GITR specific antibody with ADCC activity and is able to recruit immune cells to kill the GITR-expressing Treg cell. Any of the GITR-specific antibodies or antibody fragments of the invention may be used therapeutically. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described herein or an antigen-binding fragment thereof.
In a preferred embodiment, GITR-specific antibodies or antibody fragments of the invention are used for the treatment of a hyperproliferative disorder in a mammal. In a more preferred embodiment, one of the pharmaceutical compositions disclosed above, and which contains a GITR-specific antibody or antibody fragment of the invention, is used for the treatment of a hyperproliferative disorder in a mammal. In one embodiment, the disorder is a cancer. A variety of different cancerous tumors have been demonstrated to contain GITR positive immune cells. Accordingly, these tumors are particularly attractive targets. Such tumors include, for instance, melanoma (including Stage III and Stage IV malignant melanoma), lung cancer (e.g., non-small cell lung cancer—NSCLC), head and neck cancer, prostate cancer, renal cell carcinoma and colorectal cancer. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described herein, or an antigen-binding fragment thereof.
Other cancers that can be treated with the antigen binding proteins include, but are not limited to, breast, prostate, endometrial, bladder, kidney, esophageal, testicular, ovarian, bladder, squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck-SCCHN), uveal melanoma, follicular lymphoma, cervical, brain, pancreatic, liver, lymphoma, Hodgkin's disease, multiple myeloma, gastric cancer, and astrocyctic cancer.
In treating any of the foregoing cancers, the treatment methods that are provided can be utilized to inhibit further tumor growth, induce tumor regression, increase progression-free survival and/or extend overall survival in an individual that has a tumor. In some embodiments, the GITR-specific antibody can also delay or prevent the onset of metastasis. Progress in treatment can be monitored using various methods. For instance, inhibition can result in reduced tumor size and/or a decrease in metabolic activity within the tumor. Both of these parameters can be measured by MRI or PET scans for example. Inhibition can also be monitored by biopsy to ascertain the level of necrosis, tumor cell death and the level of vascularity within the tumor. The extent of metastasis can be monitored using known methods. Accordingly, the pharmaceutical compositions of the invention are useful in the treatment or prevention of metastasis of a variety of cancers, including (but not limited to) the following: melanoma, lung, head and neck, renal cell, colorectal, breast, prostate, endometrial, bladder, kidney, esophageal, testicular, ovarian, squamous cell carcinoma (e.g., squamous cell carcinoma of the head and neck-SCCHN), uveal melanoma, follicular lymphoma, cervical, brain, pancreatic, liver, lymphoma, Hodgkin's disease, multiple myeloma, gastric, and astrocyctic.
Similarly, further provided herein is a method for inhibiting the growth of selected cell populations comprising contacting GITR-expressing immune cells with an effective amount of a GITR-specific antibody or antibody fragment of the present invention, either alone or in combination with other therapeutic agents. In preferred embodiments, the GITR-specific antibody is a GITR-specific antibody as described herein, or an antigen-binding fragment thereof. In a preferred embodiment, the further therapeutic agent is an immunotherapy i.e., an immunostimulatory agent that induces or enhances an immune response. Such agents can include, for example: 1) activators of dendritic cells, 2) vaccine adjuvants, 3) T cell stimulators, 4) inhibitors of immune checkpoints, and 5) inhibitors of suppressive cells, cytokines and/or enzymes.
In one embodiment, the immunostimulatory agent is a cancer vaccine. In addition to cancer vaccines comprised of cancer-associated antigens, vaccines useful in combination with the GITR-specific antibody include, without limitation, GM-CSF DNA and cell-based vaccines, dendritic cell vaccines, recombinant viral (e.g. vaccinia virus) vaccines, and heat shock protein (HSP) vaccines. Useful vaccines also include tumor vaccines, such as those formed of melanoma cells; and may be autologous or allogeneic. The vaccines may be, e.g., peptide, DNA or cell based. In one embodiment, the GITR-specific antibody is administered in combination with a CD8/CD4 Ag-specific peptide vaccine.
For clinical use, a therapeutically effective amount of the GITR-specific antibody or antigen-binding fragment is administered to a subject in need thereof. For example, the GITR-specific antibodies and antigen-binding fragments thereof may be useful in the treatment of cancerous tumors that contain GITR positive immune cells. In preferred embodiments, the GITR specific antibody is a GITR-specific antibody as described herein, or an antigen-binding fragment thereof. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the GITR-specific antibody or antigen-binding fragment will be administered as a solution that has been tested for sterility.
Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
The efficient dosages and the dosage regimens for the GITR-specific antibodies and fragments depend on the disease or condition to be treated and may be determined by one skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.001-10 mg/kg, such as about 0.001-5 mg/kg, for example about 0.001-2 mg/kg, such as about 0.001-1 mg/kg, for instance about 0.001, about 0.01, about 0.1, about 1 or about 10 mg/kg.
A physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the GITR-specific antibody or fragment employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a GITR-specific antibody of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Administration may e.g. be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, the GITR-specific antibody or fragment may be administered by infusion in a weekly dosage of calculated by mg/m2. Such dosages can, for example, be based on the mg/kg dosages provided above according to the following: dose (mg/kg)×70. Such administration may be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hr, such as of from 2 to 12 hr. In one embodiment, the GITR-specific antibody or fragment may be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.
In one embodiment, the GITR-specific antibody or fragment may be administered in a weekly dosage calculated as a fixed dose for up to eight times, such as from four to six times when given once a week. Such regimen may be repeated one or more times as necessary, for example, after six months or twelve months. Such fixed dosages can, for example, be based on the mg/kg dosages provided above, with a body weight estimate of 70 kg. The dosage may be determined or adjusted by measuring the amount of GITR-specific antibody of the present invention in the blood upon administration by for instance taking out a biological sample and using anti-idiotypic antibodies which target the GITR antigen binding region of the GITR-specific antibodies of the present invention.
In one embodiment, the GITR-specific antibody or fragment may be administered by maintenance therapy, such as, e.g., once a week for a period of six months or more.
A GITR-specific antibody or fragment may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission.
The GITR-specific antibodies and fragments thereof as described herein may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the antibody-containing medicament is for combination with one or more further therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the other therapeutic agents include, but are not limited to, anti-neoplastic agents including alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); Temodal™ (temozolamide), ethylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; pipodophylotoxins such as etoposide and teniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; Gemzar™ (gemcitabine), progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide. Therapies targeting epigenetic mechanism including, but not limited to, histone deacetylase inhibitors, demethylating agents (e.g., Vidaza) and release of transcriptional repression (ATRA) therapies can also be combined with the GITR antibodies.
Additional specific examples of chemotherapeutic agents include, taxol, taxenes (e.g., docetaxel and Taxotere), modified paclitaxel (e.g., Abraxane and Opaxio) doxorubicin, Avastin®, Sutent, Nexavar, and other multikinase inhibitors, cisplatin and carboplatin, etoposide, gemcitabine, and vinblastine. Specific inhibitors of other kinases can also be used in combination with the GITR antibodies, including but not limited to, MAPK pathway inhibitors (e.g., inhibitors of ERK, JNK and p38), PI3kinase/AKT inhibitors and Pim inhibitors. Other inhibitors include Hsp90 inhibitors, proteasome inhibitors (e.g., Velcade) and multiple mechanism of action inhibitors such as Trisenox.
Such combined administration may be simultaneous, separate or sequential, in any order. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate.
In one embodiment, a GITR-specific antibody or fragment thereof is administered in combination with another immunomodulatory agent, the immunomodulatory agent can be selected for example from the group consisting of a dendritic cell activator such as CD40 ligand and anti-CD40 agonist antibodies, as well as enhancers of antigen presentation, enhancers of T-cell tropism, inhibitors of tumor-related immunosuppressive factors, such as TGF-β (transforming growth factor beta), and IL-10.
Some methods involve administering a GITR-specific antibody or fragment thereof with a vaccine adjuvant. Such adjuvants include, for instance, IL-12, and various Toll Like Receptor (TLR) agonists, including CpG (a TLR 9 agonist), monophosphoryl lipid A (MPL—a TLR4 agonist), PolyI:C or PolyICLC (TLR3 agonist), and resiquimod and 852A (TLR 7/8 agonists).
In other therapeutic approaches, a GITR-specific antibody is administered in combination with T cell growth factors such as IL-15 and/or IL-17, or activators of these molecules. In related methods, a T cell stimulator is combined with a GITR antibody. Such stimulators include agonists of 4-1BB, such as agonist anti-4-1BB antibodies and 4-1BBL.
In one embodiment, a GITR-specific antibody or fragment thereof is administered with a T cell checkpoint inhibitor, e.g., molecules that send an inhibitory signal to the immune system. Examples of such agents include inhibitors of PD-1 or PD-L1 (B7-H1), such as anti-PD-1 antibodies, including nivolumab (Bristol-Myers Squibb) and pembrolizumab, also known as MK-3475 (Merck), pidilizumab (Curetech), AMP-224 (Amplimmune), and anti-PD-L1 antibodies, including MPDL3280A (Roche), MDX-1105 (Bristol Myer Squibb), MEDI-4736 (AstraZeneca) and MSB-0010718C (Merck). Other checkpoint inhibitors include antagonists of CTLA-4, such as anti-CTLA-4 antibodies. An exemplary anti-CTLA4 antibody is Yervoy® (ipilimumab) marketed by Bristol-Myers Squibb. Other exemplary CTLA-4 antibodies include tremelimumab (Pfizer), Ticilimumab (AstraZeneca) and AMGP-224 (Glaxo Smith Kline).
In yet other methods, a GITR specific antibody or fragment thereof is administered in combination with an inhibitor of an enzyme that has an immunosuppressive effect. An example is 1-methyl tryptophan (1MT), which is a small molecule inhibitor of indoleamine 2,3-dioxygenase.
The GITR specific antibody or fragment thereof can also be used in combination with T-VEC (talimogene laherparepvec) by Amgen.
In certain embodiments, the GITR specific antibody or fragment thereof is administered in combination with a bispecific antibody. The bispecific antibody can direct the immune system of a host, in particular the cytotoxic activity of T-cells, against cancer cells.
A GITR specific antibody or fragment thereof can also be administered in combination with a variety of targeted therapies. Examples of targeted therapies include, but are not limited to, use of therapeutic antibodies. Exemplary antibodies include, but are not limited to, those which bind to cell surface proteins Her2, CDC20, CDC33, mucin-like glycoprotein, and epidermal growth factor receptor (EGFR) present on tumor cells, OX40, PD-1, CD122, CD40, CTLA-4, and optionally induce a cytostatic and/or cytotoxic effect on tumor cells displaying these proteins. Exemplary antibodies also include HERCEPTIN® (trastuzumab), which may be used to treat breast cancer and other forms of cancer, and RITUXAN® (rituximab), ZEVALIN™ (ibritumomab tiuxetan), and LYMPHOCIDE™ (epratuzumab), which may be used to treat non-Hodgkin's lymphoma and other forms of cancer. Certain exemplary antibodies also include panitumumab (VECTIBIX®), ERBITUX® (IMC-C225); BEXXAR™ (iodine 131 tositumomab); KDR (kinase domain receptor) inhibitors; anti VEGF antibodies and antagonists (e.g., Avastin® and VEGAF-TRAP); anti VEGF receptor antibodies and antigen binding regions; anti-Ang-1 and Ang-2 antibodies and antigen binding regions; antibodies to Tie-2 and other Ang-1 and Ang-2 receptors; Tie-2 ligands; antibodies against Tie-2 kinase inhibitors; inhibitors of Hif-1a, and Campath™ (Alemtuzumab). In certain embodiments, cancer therapy agents are polypeptides which selectively induce apoptosis in tumor cells, including, but not limited to, the TNF-related polypeptide TRAIL.
In one embodiment, a GITR-specific antibody or fragment thereof, as provided herein is used in combination with one or more anti-angiogenic agents that decrease angiogenesis. Certain such agents include, but are not limited to, IL-8 antagonists; Campath, B-FGF; FGF antagonists; Tek antagonists (Cerretti et al., U.S. Publication No. 2003/0162712; Cerretti et al., U.S. Pat. No. 6,413,932, and Cerretti et al., U.S. Pat. No. 6,521,424); anti-TWEAK agents (which include, but are not limited to, antibodies and antigen binding regions); soluble TWEAK receptor antagonists (Wiley, U.S. Pat. No. 6,727,225); an ADAM distintegrin domain to antagonize the binding of integrin to its ligands (Fanslow et al., U.S. Publication No. 2002/0042368); anti-eph receptor and anti-ephrin antibodies; antigen binding regions, or antagonists (U.S. Pat. Nos. 5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447; 6,057,124); anti-VEGF agents (e.g., antibodies or antigen binding regions that specifically bind VEGF, or soluble VEGF receptors or a ligand binding regions thereof) such as Avastin® or VEGF-TRAP™, and anti-VEGF receptor agents (e.g., antibodies or antigen binding regions that specifically bind thereto), EGFR inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind thereto) such as panitumumab, IRESSA™ (gefitinib), TARCEVA™ (erlotinib), anti-Ang-1 and anti-Ang-2 agents (e.g., antibodies or antigen binding regions specifically binding thereto or to their receptors, e.g., Tie-2/TEK), and anti-Tie-2 kinase inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind and inhibit the activity of growth factors, such as antagonists of hepatocyte growth factor (HGF, also known as Scatter Factor), and antibodies or antigen binding regions that specifically bind its receptor “c-met” (e.g., rilotumumab and AMG 337, Amgen); anti-PDGF-BB antagonists; antibodies and antigen binding regions to PDGF-BB ligands; and PDGFR kinase inhibitors.
Other anti-angiogenic agents that can be used in combination with a GITR-specific antibody or fragment thereof include agents such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors. Examples of useful COX-II inhibitors include CELEBREX™ (celecoxib), valdecoxib, and rofecoxib.
A GITR-specific antibody or fragment thereof as provided herein can also be used in combination with a growth factor inhibitor. Examples of such agents, include, but are not limited to, agents that can inhibit EGF-R (epidermal growth factor receptor) responses, such as EGF-R antibodies (e.g., panitumumab (VECTIBIX®)), EGF antibodies, and molecules that are EGF-R inhibitors; VEGF (vascular endothelial growth factor) inhibitors, such as VEGF receptors and molecules that can inhibit VEGF; and erbB2 receptor inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, for example, HERCEPTIN® (Genentech, Inc.). EGF-R inhibitors are described in, for example in U.S. Pat. No. 5,747,498, WO 98/14451, WO 95/19970, and WO 98/02434.
In some treatment applications, particularly when the cancer has metastasized to the bone such that the bone is negatively impacted, it can be useful to administer a GITR-specific antibody or fragment thereof with a therapeutic agent that inhibits further bone loss or aids in restoring bone that has been lost. Accordingly, the GITR-specific antibody or fragment thereof can be administered with a therapeutically effective amount of a bone growth promoting (anabolic) agent or a bone anti-resorptive agent including but not limited to: bone morphogenic factors designated BMP-1 to BMP-12; transforming growth factor-3 and TGF-3 family members; fibroblast growth factors FGF-1 to FGF-10; interleukin-1 inhibitors (including IL-1ra, antibodies to IL-1 and antibodies to IL-1 receptors); TNFα inhibitors (including etanercept, adalibumab and infliximab); RANK ligand inhibitors (including soluble RANK, osteoprotegerin and antagonistic antibodies that specifically bind RANK or RANK ligand, such as denosumab (XGEVA®)), Dkk-1 inhibitors (e.g., anti-Dkk-1 antibodies), parathyroid hormone, E series prostaglandins, bisphosphonates and bone-enhancing minerals such as fluoride and calcium. Anabolic agents that can be used in combination with the GITR antibodies and functional fragments thereof include parathyroid hormone and insulin-like growth factor (IGF), wherein the latter agent is preferably complexed with an IGF binding protein. An IL-1 receptor antagonist suitable for such combination treatment is described in WO89/11540 and a suitable soluble TNF receptor-1 is described in WO98/01555. Exemplary RANK ligand antagonists are disclosed, for example, in WO 03/086289, WO 03/002713, U.S. Pat. Nos. 6,740,511 and 6,479,635. In one embodiment, a method for treating a cancer includes administration of a therapeutically effective amount of a GITR-specific antibody as described herein, along with radiotherapy to a subject in need thereof. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.
Provided herein are methods for detecting GITR in a biological sample by contacting the sample with an antibody, or antigen-binding fragment thereof, described herein. As described herein, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, cells that are not tissue associated (i.e., free cells), tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), histological preparations, and the like. In some embodiments the described methods include detecting GITR in a biological sample by contacting the sample with any of the GITR-specific antibodies or antigen-binding fragments thereof described herein.
In some embodiments the sample may be contacted with more than one of the GITR-specific antibodies or antigen-binding fragments described herein. For example, a sample may be contacted with a first GITR-specific antibody, or antigen-binding fragment thereof, and then contacted with a second GITR-specific antibody, or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are not the same antibody or antigen-binding fragment. In some embodiments, the first antibody, or antigen-binding fragment thereof, may be affixed to a surface, such as a multiwell plate, chip, or similar substrate prior to contacting the sample. In other embodiments the first antibody, or antigen-binding fragment thereof, may not be affixed, or attached, to anything at all prior to contacting the sample.
The described GITR-specific antibodies and antigen-binding fragments may be detectably labeled. In some embodiments labeled antibodies and antigen-binding fragments may facilitate the detection of GITR via the methods described herein. Many such labels are readily known to those skilled in the art. For example, suitable labels include, but should not be considered limited to, radiolabels, fluorescent labels, epitope tags, biotin, chromophore labels, ECL labels, or enzymes. More specifically, the described labels include ruthenium, 111In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes, rhodamine dyes, Alexafluor® dyes, and the like.
The described GITR-specific antibodies and antigen-binding fragments may be used in a variety of assays to detect GITR in a biological sample. Some suitable assays include, but should not be considered limited to, western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) or ELISA assay.
Provided herein are kits for detecting GITR in a biological sample. These kits include one or more of the GITR-specific antibodies described herein, or an antigen-binding fragment thereof, and instructions for use of the kit.
The provided GITR-specific antibody, or antigen-binding fragment, may be in solution; lyophilized; affixed to a substrate, carrier, or plate; or detectably labeled. The described kits may also include additional components useful for performing the methods described herein. By way of example, the kits may comprise means for obtaining a sample from a subject, a control or reference sample, e.g., a sample from a subject having slowly progressing cancer and/or a subject not having cancer, one or more sample compartments, and/or instructional material which describes performance of a method of the invention and tissue specific controls or standards.
The means for determining the level of GITR can further include, for example, buffers or other reagents for use in an assay for determining the level of GITR. The instructions can be, for example, printed instructions for performing the assay and/or instructions for evaluating the level of expression of GITR.
The described kits may also include means for isolating a sample from a subject. These means can comprise one or more items of equipment or reagents that can be used to obtain a fluid or tissue from a subject. The means for obtaining a sample from a subject may also comprise means for isolating blood components, such as serum, from a blood sample. Preferably, the kit is designed for use with a human subject.
To better and more fully describe the subject matter herein, this section provides enumerated exemplary embodiments of the subject matter presented.
1. An isolated antibody, or an antigen-binding fragment thereof, that specifically binds to human GITR comprising:
a. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
b. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 13, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 29, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 36;
c. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 6, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 14, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 30, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 33, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 37;
d. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 15, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
e. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 16, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
f. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 17, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
g. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 9, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 18, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
h. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 19, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
i. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 20, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 38;
j. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 21, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 34, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 38;
k. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 22, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
l. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 23, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
m. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 24, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
n. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 25, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35;
o. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 27, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 26, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35; or
p. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 21, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 28, a light chain CDR2 having the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 35.
2. An isolated antibody, or an antigen-binding fragment thereof, that specifically binds to human GITR comprising a heavy chain region selected from the group consisting of SEQ ID NOs: 39-54, 63 and 64.
3. The antibody of embodiment 2, wherein the antibody or antigen binding fragment thereof comprises a light chain region selected from the group consisting of SEQ ID NOs: 55-58.
4. The antibody of embodiment 2, wherein the antibody or antigen binding fragment thereof comprises a heavy chain region selected from the group consisting of SEQ ID NOs: 39-54, 63 and 64 and a light chain region selected from the group consisting of SEQ ID NOs: 55-58.
5. The antibody of embodiment 4, wherein
a. the heavy chain region comprises SEQ ID NO: 39 paired with a light chain region comprising SEQ ID NO: 55;
b. the heavy chain region comprises SEQ ID NO: 40 paired with a light chain region comprising SEQ ID NO: 56;
c. the heavy chain region comprises SEQ ID NO: 41 paired with a light chain region comprising SEQ ID NO: 57;
d. the heavy chain region comprises SEQ ID NO: 42 paired with a light chain region comprising SEQ ID NO: 55;
e. the heavy chain region comprises SEQ ID NO: 43 paired with a light chain region comprising SEQ ID NO: 55;
f. the heavy chain region comprises SEQ ID NO: 44 paired with a light chain region comprising SEQ ID NO: 55;
g. the heavy chain region comprises SEQ ID NO: 45 paired with a light chain region comprising SEQ ID NO: 55;
h. the heavy chain region comprises SEQ ID NO: 46 paired with a light chain region comprising SEQ ID NO: 55;
i. the heavy chain region comprises SEQ ID NO: 47 paired with a light chain region comprising SEQ ID NO: 58;
j. the heavy chain region comprises SEQ ID NO: 48 paired with a light chain region comprising SEQ ID NO: 58;
k. the heavy chain region comprises SEQ ID NO: 49 paired with a light chain region comprising SEQ ID NO: 55;
l. the heavy chain region comprises SEQ ID NO: 50 paired with a light chain region comprising SEQ ID NO: 55;
m. the heavy chain region comprises SEQ ID NO: 51 paired with a light chain region comprising SEQ ID NO: 55;
n. the heavy chain region comprises SEQ ID NO: 52 paired with a light chain region comprising SEQ ID NO: 55;
o. the heavy chain region comprises SEQ ID NO: 53 paired with a light chain region comprising SEQ ID NO: 55;
p. the heavy chain region comprises SEQ ID NO: 54 paired with a light chain region comprising SEQ ID NO: 55;
q. the heavy chain region comprises SEQ ID NO: 63 paired with a light chain region comprising SEQ ID NO: 55; or
r. the heavy chain region comprises SEQ ID NO: 64 paired with a light chain region comprising SEQ ID NO: 55.
6. The antibody or antigen-binding fragment of embodiment 5, wherein the antibody specifically binds to human GITR by interacting with GITR (SEQ ID NO: 62
amino acid residues:
a. 40-45; and
b. 75-79.
7. The antibody or antigen-binding fragment of embodiment 1 wherein the antibody or antigen-binding fragment thereof binds to a polypeptide having the amino acid sequence of SEQ ID NO: 59.
8. The antibody or antigen-binding fragment of embodiment 1 wherein the antibody or antigen-binding fragment thereof specifically binds human GITR with a binding affinity of at least 30 nM as measured by surface plasmon resonance using experimental design described in Example 9.
9. The antibody or antigen-binding fragment of embodiment 1 wherein the antibody or antigen-binding fragment induces an increase in luciferase expression in NF-κB luciferase gene assay.
10. The antibody or antigen-binding fragment of embodiment 1 wherein the antibody or antigen-binding fragment induces ADCC in vitro with an EC50 of less than about 67 ng/mL.
11. The antibody or antigen-binding fragment of embodiment 1 wherein the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment.
12. The antigen-binding fragment of embodiment 1 wherein the antigen binding fragment is a Fab fragment, a Fab2 fragment, or a single chain antibody.
13. The antibody or antigen-binding fragment of embodiment 1 wherein the antibody or antigen-binding fragment is recombinant.
14. The antibody or antigen-binding fragment of embodiment 1 wherein the antibody or antigen-binding fragment thereof are of IgG1, IgG2, IgG3, or IgG4 isotype.
15. The antibody or antigen-binding fragment of embodiment 1 is IgG1 isotype.
16. The antibody or antigen-binding fragment of any one of embodiments 1 wherein the antibody or antigen-binding fragment thereof specifically binds human GITR and cynomolgus monkey GITR.
17. A polynucleotide encoding the antibody or antigen binding fragment of any one of embodiment 1.
18. A vector comprising the polynucleotide of embodiment 17.
19. A host cell comprising the vector of embodiment 18.
20. A process for the production of an antibody or antigen-binding fragment, comprising:
culturing the host cell as defined in embodiment 19 under the conditions allowing the expression of the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding molecule from the culture.
21. A method of alleviating a symptom of a cancer or other neoplastic condition, the method comprising administering the antibody, or antigen binding fragment thereof, of embodiment 1 to a subject in need thereof in an amount sufficient to alleviate the symptom of the cancer or other neoplastic condition in the subject.
22. The method of embodiment 21, wherein the subject is a human.
23. The method of embodiment 21 further comprising one or more of the following:
a. administering chemotherapy
b. administering radiation therapy; or
c. administering one or more additional therapeutic agents.
24. The method of embodiment 23 wherein the additional therapeutic agent is an immunostimulatory agent.
25. The method of embodiment 24, wherein the immunostimulatory agent is selected from the group consisting of PD-1 antibody, CTLA-4 antibody, CD122 antibody, CD40 antibody, OX40 antibody, and a CD8 Ag-specific OVA peptide vaccine.
26. A pharmaceutical composition comprising the antibody, or antigen binding fragment thereof, of embodiment 1 and a pharmaceutically acceptable carrier.
27. A kit comprising the antibody, or antigen binding fragment thereof, of embodiment 1 and packaging for the same.
The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention.
Recombinant human (h) GITR-Fc fusion protein (R&D Systems catalog number 689-GR) corresponding to amino acid 26 to 161 of hGITR (SEQ ID NO:59). The protein was biotinylated for phage panning studies. This protein was also used for binding and affinity measurements.
GITR was expressed in HEK293F cells by transfection or lentiviral transduction for anti-GITR antibody reactivity confirmation, to test phage and Next Generation Sequencing panels, and to check cross-reactivity of GITR mAb hits against cyno-GITR.
The transfected cells presented the following GITR sequences:
The lentivirally-transduced cells presented the following GITR sequences:
Transient expression of HEK 293F cells was performed by placing cells in Freestyle™ 293 media (Gibco #12338) at a density of 1e6 cells/ml to a volume of 30 mls in a 125 ml vented cap shake flask with shaking at 130 RPM, 24 hours prior to transfection. Transfection was carried out using Freestyle max reagent (Invitrogen #16447). For a single 30 ml transfection, in one tube 37.5 μl of freestyle max reagent was diluted in 1 ml of OptiMEM media (Gibco #31985). In a separate tube, 37.5 μg of DNA (300 nanograms target and 37.2 μg of an unrelated carrier plasmid) was mixed into 1 ml OptiMEM. The two tubes were then mixed together, incubated in the biosafety cabinet for 1 minute and then the mixture added directly to the flask of HEK293F cells. After 48 hours of growth, the cells were ready for use in the indicated assays.
Lentiviral particles presenting full-length GITR generated by Genecopoeia (Genecopoeia catalog #LPP-U0202-LV105-200-S for human GITR and Genecopoeia catalog #LPP-U0202-LV105-200-S for cyno GITR) were transduced in cells using the manufacturer's protocol. Transduced cells were selected for stable plasmid integration and then single cell sorted using the BD Biosciences FACS Jazz cell sorter. The GITR surface expression was quantified by flow cytometry staining with R&D Systems FAB689P anti-huGITR antibody and analyzed on the BD Biosciences Accuri C6.
The de novo pIX Fab libraries (Shi, L., et al J Mol Biol, 2010. 397(2):p. 385-396. WO 2009/085462), consisting of VH1-69, 3-23 and 5-51 heavy chain libraries paired with Vκ VLK3-20, VLK4-1, VLK3-11, and VLK1-39 light chain libraries, were panned against biotinylated human GITR-ECD Fc fusion at a concentration of 100 nM (rounds 1-3) or 10 nM (round 4) in a selection process similar to that described in Rothe et al, J Mol Biol 376:1182-1200, 2008 and Steidl et al, Mol Immunol. 46: 135-144, 2008.
The pIX gene was excised from phagemid DNA following the fourth round of panning to generate soluble his-tagged Fab coding regions. Fabs were expressed in E. coli and screened for binding to GITR in an ELISA. Briefly, 96-well Nunc Maxisorp plates (Nunc #437111) were coated with either streptavidin (Promega) or sheep anti-human Fd (The Binding Site #PC075) in PBS at 5 μg/mL overnight at 4° C. Bacterial cultures containing the Fab expression vector were grown in 1 mL of 2×YT (Carbenecillin) in deep-well culture plates until turbid (OD600 Z 0.6). Fab expression was then induced by the addition of IPTG to a concentration of 1 mM. Cultures were grown overnight at 30° C. and then clarified by centrifugation the next day. Streptavidin-coated plates were washed thrice with TBS, 0.5% Tween-20 (Sigma #79039-10PAK), loaded with biotinylated GITR-Fc at 5 μg/mL, and held at room temperature for 30 minutes. Using the Biomek Liquid Handling Robot (Beckman Coulter) both anti-Fd coated Maxisorp plates and streptavidin-coated plates were washed three times with TBS, 0.5% Tween-20 (Sigma #79039-10PAK) and blocked with 200 μL PBS-Tween (0.5%)+nonfat dried milk (3%) per well for one hr at room temperature. At this step and all subsequent steps plates were washed three times with TBS, 0.5% Tween-20 (Sigma #79039-10PAK). Each well received 50 μL of Fab supernatant followed by one hr incubation at room temperature. After washing, 50 μL of goat anti-human kappa-HRP (Southern Biotech) was added at a 1:5000 dilution in TBST with 0.3% milk and plates were incubated for one hour at room temperature. Plates were washed and 50 uL chemiluminescent substrate, PoD (Roche #121-5829500001), was added according to manufacturer's instructions. Plates were then read for luminescence on an EnVision (Perkin Elmer) plate reader. Clones that were positive in both the fab expression ELISA and the GITR binding ELISA were selected for DNA sequencing. A total of 50 unique Fab sequences were discovered via this phage panning process. The unique heavy chain V-regions were cloned into human IgG1_G1m(17,1) expression vectors, the unique light chains were cloned into human kappa expression vectors, and the resultant antibodies were tested again for binding activity in an ELISA.
Binding of GITR antibodies to engineered cells was assessed using FACS. The object of the screening assay was to identify antibodies that bound to cells expressing hGITR.
Briefly, 300,000 cells per well were plated into a 96-well plate (Greiner bio one cat #651261) and the cells were pelleted. The cells were washed with 100 μL of FACS staining buffer (BD Pharmingen Stain Buffer (BSA) cat #554657), incubated at 4° C. for 30 minutes with a mixture of 50 μL FACS staining buffer and 20 μL per well of the unpurified antibody supernatants, and washed once with 200 μL of FACS staining buffer. For detection, the cells were subjected to a 30 minute incubation at 4° C. with 50 μL per well of Alexa Fluor 488 goat anti-human IgG (H+L) (Molecular Probes, cat #A11013) at 2 μg per mL in FACS staining buffer. The cells were washed once with 200 μL per well of FACS staining buffer, resuspended in 150 μL per well of FACS staining buffer, and then transferred to FACS tubes that contained 200 μL per well of FACS staining buffer. FACS analysis was then carried out. The assay was repeated for data consistency and top binders were selected for further development.
To assess agonist activity of the GITR antibodies, the panel was screened using an NF-κB-luciferase gene assay. Briefly, HEK293 cells were transiently transfected with a reporter plasmid encoding the luciferase gene under control of the NF-κB promoter together with human GITR expression plasmid. The cells were allowed to recover from the transfection and to express human GITR for four hours at 37° C., at which point the assay could be performed. To confirm that the assay worked as intended, recombinant human GITR ligand (R&D Systems 6987-GL/CF) was added to positive control wells at a final concentration of 2.5 micrograms per mL. Anti-GITR antibodies undergoing testing were added to experimental wells at a final concentration of 5 micrograms per mL. The plates were then incubated at 37° C. for four hours. Successful GITR signaling was expected to activate the NF-kB pathway, followed by luciferase expression, which could be detected by adding Steady Glo, as indicated by the manufacturer (Promega cat #E2550), and measuring the resultant luminescence in an Envision plate reader (Perkin Elmer).
Fifteen antibodies induced an increase in luciferase expression compared to treatment with PBS only (
Subsequent to the discovery of the first set of anti-GITR antibodies, DNA samples from the output of the final round of the phage selection were provided to Beckman Coulter Genomics for next generation sequencing of the heavy chain V-regions using the Roche 454 sequencing platform. Raw data from Beckman Coulter Genomics was subjected to an initial analysis at IMGT and then more closely examined internally using the proprietary Janssen software program 3DX. The idea of using next-generation sequencing to examine phage selection outputs more extensively has recently been developed as a method to increase the number and quality of antibodies discovered (Ravn et al, Methods 60 (2013) pg 99-110).
The sequences provided by IMGT were filtered for samples that were poor quality or that contained stop codons, and then the remaining sequences were sorted by heavy chain CDR3. This approach was chosen because CDR3 is expected to drive the majority of the binding energy for the antigen, and the majority of the diversity in the phage libraries is located in heavy chain CDR3. Eighty-seven V-regions were chosen for DNA synthesis and cloning into a human IgG1_G1m(17) vector based on both frequency of occurrence and lack of cysteines, methionines, or highly hydrophobic sequences.
After synthesis, the putative anti-GITR heavy chains were tested for binding to GITR as previously described. Because the next-generation sequencing dataset contained no information about the appropriate light chain partners for these heavy chain V-regions, the heavy chains were co-transfected with each of the 4 light chain germline genes found in the phage libraries: Vk3-20, Vk4-1, Vk3-11, and Vk1-39. Unpurified antibody supernatants from these four standard transfections were tested in an ELISA for the ability to bind to recombinant human GITR ECD-Fc fusion protein. The top binders from this assay were selected for further development.
After the anti-GITR mAbs from the next-generation sequencing dataset were shown to bind to a GITR ECD-Fc fusion protein in an ELISA, a subset was tested for binding to cell-surface GITR as described in EXAMPLE 3. Positive binders were tested for agonist activity using an NF-κB-luciferase gene assay as described in EXAMPLE 3. At 40 micrograms per mL, antibodies that induced an increase in luciferase expression equal to at least 20% of the increase over background observed upon treatment with the natural ligand were considered to have agonistic activity (
The purified antibodies from both phage display and next generation sequencing were tested for binding to the cynomolgus monkey GITR using flow cytomentry. Transiently transfected cells were incubated at 2-8° C. for 30 minutes with 0.1 mg/mL of test antibodies, washed, and incubated at 2-8° C. for 30 minutes with PE-labeled goat anti-human IgG. The cells were then washed and analyzed on a MACSQuant flow cytometer. Antibodies identified as positive binders exhibited a 1.5 to 2 log shift in the mean fluorescent intensity of cells transfected with human or cyno GITR compared to the mean fluorescent intensity of cells transfected with the empty vector. The binding results are compiled in Table 2.
Thus, in total a panel of 16 GITR antibodies—all depicted in Table 3—were found to bind to human and cyno GITR.
VH and VL of the 16 GITR mAbs are shown below in Table 4.
Antibodies that bound to both human and cyno GITR were tested for activity in ADCC and CDC assays. The huIgG1 isotype control antibody was included in these assays for comparison. ADCC assays were used to look at cell killing carried out by NK-92 cells genetically modified to express the high affinity FcγRIIIa 176V/V polymorphism. Three types of target cells were used: HuT102 cells, which endogenously express human GITR, pooled HT1080-huGITR stable transfectants, and HEK293 cells that were transiently transfected with either human GITR or cyno GITR. To carry out the ADCC assays, the target cells were labelled with calcein AM, washed, resuspended in assay medium, and seeded at 50,000 cells/50 microliters/well in V bottom 96 well plates. Anti-hGITR or control antibodies were added to the wells at various concentrations (100 microliters/well). NK-92 176V effector cells were washed, resuspended in assay medium, and seeded at 50,000 cells/50 microliters/well or 100,000 cells/50 microliters/well along with target cells and antibodies. Medium alone (background signal), target cells alone (spontaneous lysis signal), cells that would eventually be treated with Triton X-100 (Max lysis signal) and isotype control antibody at a final concentration of 1 microgram/mL were included as controls. After 1 hour incubation at 37° C., complete cell lysis was induced in the Max signal wells through the addition of 20 microliters of 2% Triton X-100 and the plates were centrifuged. 100 microliters of supernatants were removed and added to clear bottom black plates. Fluorescence intensity (FI) units were measured using a Molecular Devices SpectraMax5. Percent specific lysis was calculated after subtracting the average FI observed with medium alone from all wells. The formula to determine percent specific lysis was (Sample −Spontaneous lysis)/(Max lysis −Spontaneous lysis)*100. Analysis of the half maximal effective concentration (EC50) was carried out for each antibody in Prism.
Table 5 depicts the activity of the GITR antibodies in the different cell lines tested.
In preparation for cell line development, double gene construction was initiated for TRGB25, TRGB153, TRGB159, and TRGB160. During this process it was discovered that heavy chain of TRGB25 was in the human allotype IgG1_G1m(17,1) rather than the preferred human allotype IgG1_G1m(17). The heavy chain V-region from TRGB25 was switched into a human IgG1_G1m(17) allotype framework during double gene construction, thereby creating the new protein TRGB190. At this point it was also noted that TRGB160 had a framework mutation at the amino terminus of the heavy chain. During the construction of the double gene, the amino terminal residue of the TRGB160 heavy chain was switched from Q to E, thereby creating the new protein TRGB191. In addition, it was decided to produce a low-fucose version of TRGB191, ie TRGB191.CLF. Table 6 outlines the sequences of this modified anti-GITR antibodies.
The affinities of the GITR antibodies to recombinant human GITR ECD were measured by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 protein interaction array system (BioRad).
The rates of GITR ECD association and dissociation were measured for each variant. The biosensor surface was prepared by covalently coupling Goat anti-Human IgG (Fc) to the surface of a GLC chip (BioRad) using the manufacturer instructions for amine-coupling chemistry. Approximately 8800 RU (response units) of Goat anti-Human IgG (Fc) antibody (Jackson ImmunoResearch laboratories Prod #109-005-098) were immobilized. The RU immobilized also included a goat anti-mouse Fc antibody that was added to capture other antibodies not included in the ones reported here. Since the mixture was 1:1 about 50% of these RU immobilized are expected to be goat anti-human Fc. The binding kinetics experiments were performed at 25° C. in running buffer (PBS pH 7.4, 0.005% P20, 3 mM EDTA). Four-fold (1:3) serial dilutions of human GITR ECD or cyno GITR ECD starting at 100 nM were prepared in running buffer. An average of 300 RU of mAb (174-600) were captured on each channel of the sensor chip. The reference spots (Goat anti-Human IgG (Fc)-modified surface) containing no candidate captured were used as a reference surface. Capture of mAb was followed by 3 min injection (association phase) of antigen at 40 μL/min, followed by 10 min of buffer flow (dissociation phase). The chip surface was regenerated by injection of 0.85% phosphoric acid at 100 μL/min. Data were processed on the instrument software. Double reference subtraction of the data was performed by subtracting the curves generated by buffer injection from the reference-subtracted curves for analyte injections. Kinetic analysis of the data was performed using 1:1 Langmuir binding model with group fit. The result for each mAb was reported in the format of kon or on-rate, koff or off-rate, and KD (Equilibrium dissociation constant) (Table 7).
The results indicated that few of the antibodies met the objective of binding to the cyno GITR ECD with affinity within five-fold of binding to the human GITR ECD. This outcome seemed to conflict with the cell killing data discussed in EXAMPLE 7, in which most of the antibodies killed cells that expressed cyno GITR with a potency only slightly less than that shown against cells that expressed the human GITR protein. It is possible that the truncated GITR extracellular domains that had been overexpressed in insect cells may not have been properly folded, which resulted in the disagreement between the cell killing and affinity analysis experiments. It is further possible that full-length GITR expressed in human cells was more likely to fold properly, and that measurements of binding affinity to GITR-expressing cells would be more likely to align with the observed cell killing activity. To test these possibilities the affinities of these antibodies for cells that express either human or cyno GITR should be evaluated.
Cell-based affinity experiments were performed to assess the binding of anti-GITR antibodies with human and cyno GITR transfected HEK293 cell lines using MSD-Cell Affinity Technology (MSD-CAT). The MSD-CAT was developed in-house as a label-free method to determine affinity using intact cells in a high throughput format. The parental HEK293 cell line without any GITR expressed was used as a negative control.
In order to measure the affinity of an interaction by this technique, a series of solutions with a fixed concentration of anti-GITR antibody (300, 60, 12, 2.4 pM) and varying concentrations of human or cyno GITR expressing cells (2.0×107-1.0×103 cells/mL) were prepared and allowed to reach equilibrium by rotating the plates for 18 hr at 4° C. These samples were prepared in DMEM Glutamax medium (Invitrogen, Prod #10569-044) with 0.05% Azide, 1% BSA, 3 mM EDTA. After equilibration the plates were centrifuged for 5 min at 2000 rpm and free anti-GITR mAbs are detected in the supernatant. The free anti-GITR mAbs in the mixture were detected by electrochemiluminescence (ECL) using MSD reader instrument. For detection, MSD-Streptavidin plates (MesoScale Discovery, Prod #L11SA-1) were coated with 0.1 μg/mL of biotinylated human-GITR antigen in assay buffer at 50 μL/well and equilibrated overnight (˜16 hr at 4° C.). After equilibration, the plates were blocked by adding 150 μL/well of assay buffer without removing coating antigen, incubated for ˜1 hr at ambient temperature and washed 3 times with wash buffer. The supernatants from the centrifuged plates were transferred to antigen-coated plates (50 μL/well), incubated for 60 min, and then washed three times with wash buffer. After this, 50 μL/well of 0.7 μg/mL ruthenium-conjugated F(ab′)2 donkey anti-human IgG (H+L) (Jackson ImmunoResearch; Prod #709-006-149) was added and incubated for 1 hr. After 1 hr, the plates were washed three times with wash buffer and 150 μL of MSD Read Buffer (MesoScale Discovery Cat #R92TC-1; prepared by diluting 1:3 of stock into d. H2O) were added per well. The plates were read immediately on the MSD Sector Imager 6000 Reader for luminescence levels. ECL signal detected by MSD was expressed in term of % free antibody in the mixture and the data was analyzed to determine affinity using a user defined equation (derived from the law of mass action) introduced in Prism software. The free mAb concentration as a function of receptor concentration is subject to non-linear least squares analysis with a 1:1 binding model to determine binding affinities. Table 8 summarizes the cell binding affinities for all the tested molecules.
The affinities for cell-surface expressed GITR were measured by MSD-CAT for TRGB25, TRGB190, TRGB160, TRGB191.CLF and TRGB153. Four studies were performed where the first study was considered preliminary data and consisted of only one replicate. Further studies with larger number of repeats were performed (Studies 2 and 3). Studies 2 and 3 indicate that for TRGB190, the mAb affinity for cyno GITR is 1.5-1.6-fold weaker than for human GITR. Studies 2 and 3 also indicate that for TRGB191.CLF, the mAb affinity for cyno GITR is 1.5-3.2-fold weaker than for human GITR. Study 4 was carried out at a later date to confirm the TRGB191.CLF data from the earlier studies. The data in Study 4 indicates that for TRGB191.CLF, the mAb affinity for cyno GITR is 2.0-fold weaker than for human GITR.
Antigen primed T lymphocytes need to expand and persist to promote adaptive immunity. The growth and survival signals required are contributed in large part by the NF-κB pathway in activated T cells. Interferon gamma (IFNγ), a well-known target gene of the NF-kB transcription factor, is a critical cytokine for immunity against foreign pathogens, and is produced by Th1 CD4 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops (Schoenborn J R, Wilson C B. Adv Immunol. 2007; 96:41-101).
The tumor necrosis factor receptor (TNFR) superfamily members, of which GITR is a member, can provide a co-stimulatory signal to the T cells. This is initiated by binding to their respective ligand and via recruitment of adaptor proteins known as TRAFs (TNF receptor associated factor) which can signal through the NF-κB pathway. In addition, the strength of the co-stimulatory signaling is dependent on receptor oligomerization, which can be achieved with trimeric or hexameric soluble ligands, or via antibody-mediated cross-linking.
To detect the effect of anti-GITR antibody ligation on NF-κB activity, a modified version of the HEK-Blue NF-κB system (Invivogen) were utilized. These cells express the SEAP (Secreted Embroyonic Alkaline Phosphatase) reporter gene under the control of a minimal promoter fused to five NF-κB and AP-1 binding sites. They were stably transfected to express human GITR. GITR receptor crosslinking in this system drives NF-κB activity which can be detected by SEAP secretion in the supernatant. A trimeric soluble GITR ligand chimeric protein (R&D Systems) was used as a positive control.
For “agonist” testing 25,000 HEK-Blue-NF-κB-GITR cells were treated with serial 1:2 dilutions of soluble GITRL (starting at 100 ng/mL) or anti-GITR antibody (starting at 1 ug/mL) in the presence of 5× excess crosslinker antibody (anti-HA or anti-Fc, respectively) for 16-20 hours. Supernatant (40 uLs) was removed and mixed with 160 uLs of Quanti-Blue™ reagent. The colorimetric reaction was allowed to incubate at 37° C. for up to 1 hr before being read in a spectrophotometer at OD650 nm.
To test the antibodies in “antagonist” mode, cells were treated with serial 1:2 dilutions of anti-GITR antibody (starting at 2 ug/mL) in the presence of 25 ng/mL constant concentration of soluble GITRL. Antagonists were defined as antibodies that blocked binding and NF-κB activity of soluble GITRL by more than 50%. Representative graphs are provided below and serve to illustrate experiment variability.
In agonist mode, anti-GITR antibodies are capable of cross-linking GITR on the HEK-Blue-NF-κB-GITR cells, causing a dose-dependent increase in NF-κB activity, compared to an isotype control antibody, CNTO3930 (
A hallmark of immunity is the generation of memory T cells against foreign antigens such that an immune response can be mounted more rapidly upon subsequent exposure.
Cytomegalovirus (CMV) is a herpesvirus and is a common infection that is usually asymptomatic in healthy adults and children. It is estimated that 50-80% of adults are infected with CMV by the time they reach 40 years old. Tetanus toxin (TT) is produced bacteria called Clostridium tetani. Most adults in the US are vaccinated for tetanus 5 times before the age of 6, and receive boosters every 10 years thereafter.
By exposing sero-positive individuals to their respective antigens, memory T cells can be reactivated to mount a recall response. GITR expression has been shown to be upregulated on T cells and GITRL on the antigen presenting cells. An agonist GITR antibody could strengthen the T cell activation by signaling through GITR and further enhancing the antigen specific immune response.
Interferon gamma (IFNγ) is a critical cytokine for immunity against foreign pathogens, and is produced by Th1 CD4 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops (Schoenborn J R, Wilson C B. Adv Immunol. 2007; 96:41-101).
Here, we have developed a CMV and TT recall assay to characterize our anti-GITR antibodies for their ability to enhance T cell activation, as measured by IFNγ secretion. Briefly, 150,000 PBMCs obtained from sero-positive donors for CMV and TT were incubated in the presence of 0.1 ug/mL of CMV antigen or TT antigen (CMV whole antigen, Astarte #1004; TT antigen, Astarte #1002) in wells pre-coated with a 1:2 dilution series of test antibodies starting at 5 ug/mL to 156 ng/mL (left to right on x-axis). Supernatant was harvested 4-6 days afterwards and IFNγ levels were quantified by MSD. Antigen only controls were used to assess reactivity of the donor and CNTO3930 was an antibody isotype control. Each antibody concentration was run in replicates (n=6).
TRGB191.CLF augments CMV-dependent memory T cell activation in a dose dependent manner, as measured by IFNγ secretion, peaking at [Ab]=5 ug/mL (
The efficacy of anti-GITR on anti-tumor immunity can only be studied in tumor models where the host has an intact immune system. For this reason, the GITR mouse surrogate antibody, DTA-1, was studied in the established syngeneic colon carcinoma models, CT26 and MC38, in Balb/C or C57/BL6 mice, respectively
Mice were implanted subcutaneously (sc) with 5×105 CT26 or MC38 tumor cells on the right flank. On day 7 post-tumor cell implantation, mice were randomized into experimental groups with an average tumor size of approximately 85 mm3 or 120 mm3, respectively.
Mice were administered DTA-1 (BioXcell #BE0063) or rat IgG2b isotype control (clone LTF-2, BioXcell #BE0090) intraperitoneally at 200 μg/animal q3d-q4d for a total of 3 doses on days 7, 11 and 14 (n=10/group). Tumor caliper measurements were taken twice weekly until the end of the study. Tumor volume was calculated using the formula: Tumor Volume (mm3)=(1×w2/2); where ‘1’ represents the length, and ‘w’ the width of the tumor as determined by caliper measurements, and monitored twice weekly throughout the study. Percent tumor growth inhibition (% TGI) was defined as the difference between mean tumor volumes of the treated vs. control group, calculated as % TGI=[(TVc−TVt)/TVc)*100] where TVc is the mean tumor volume of a given control group and TVt is the mean tumor volume of the treatment group. As defined by NCI criteria, ≧60% TGI is considered biologically significant.
In the MC38 model, statistically significant tumor growth inhibition was achieved with DTA-1 treatment (80% TGI on day 21 vs. isotype control, p<0.0001) with tumor regressions observed as early as day 14 after DTA-1 treatment, and complete responses (CR) in 5/10 animals achieved by day 28. The CRs appear durable, with no re-growth observed up to 35 days post last treatment dose.
In the CT26 model, statistically significant tumor growth inhibition was achieved with DTA-1 treatment as compared to isotype treated control animals (>65% TGI on day 27, p<0.0001), with tumor regressions observed in half the group (5/10 animals) as early as day 14, and with complete responses (CRs) observed by day 31 (
The MC38 syngeneic colon carcinoma model was used to evaluate combination anti-GITR therapy in combination with anti-PD-1, anti-CTLA-4 checkpoint blockade or anti OX-40 antibody.
Mice were implanted subcutaneously (sc) with 5×105 MC38 tumor cells on the right flank. On day 14-21 post-tumor cell implantation, mice were randomized into experimental groups with an average tumor size of approximately 200 mm3. Mice were administered with surrogate anti-GITR (DTA-1, BioXcell #BE0063), anti-PD-1 (RMP1-14, BioXcell #BE0146), anti-CTLA-4 (9D9, BioXcell #BP0164), anti-OX40 (OX-86, BioXcell #BE0031) or rat IgG2b isotype control (LTF-2, BioXcell #BE0090) intraperitoneally at 100 μg/animal q4d for a total of 3 doses on days 1, 5 and 9 post randomization (n=10/group). Tumor caliper measurements were taken twice weekly until the end of the study. Tumor volume was calculated using the formula: Tumor Volume (mm3)=(1×w2/2); where ‘1’ represents the length, and ‘w’ the width of the tumor as determined by caliper measurements, and monitored twice weekly throughout the study. Percent tumor growth inhibition (% TGI) was defined as the difference between mean tumor volumes of the treated vs. control group, calculated as % TGI=[(TVc−TVt)/TVc)*100] where TVc is the mean tumor volume of a given control group and TVt is the mean tumor volume of the treatment group. As defined by NCI criteria, ≧60% TGI is considered biologically significant.
Statistically significant tumor growth inhibition was achieved with anti-GITR treatment compared to the isotype control cohort, even though treatment was initiated when tumors were larger, and the dosage was reduced from 200 μg/mouse to 100 ug/mouse. In the anti-GITR+anti-PD-1 combination group, tumor regressions in 5/10 animals were observed by day 26 after randomization (
The mechanisms by which combination therapy targeting GITR with PD-1 blockade augments Ag-specific CD8+ T cell responses in a vaccine setting were assessed. To address this, non-tumor bearing mice were immunized once with the OVA immunodominant CTL epitope OVA257-264 peptide vaccine (hereafter referred to as Vax) and treated with 200 μg anti-GITR on days 0, 3, and 6 and 200 μg and anti-PD-1 on days 3, 6, 9, and 12. Combination Vax/anti-GITR/anti-PD-1 therapy augmented CD8+ effector function over controls, as evidenced by increased levels of splenic Ag-specific IFNγ ELISpot responses, polyfunctional CD8 T cell responses, and increased levels of CD107a/IFNγ CD8+ T cells demonstrating cytolytic activity (
The extent to which combination therapy skewed Ag-specific CD8+ T cell differentiation toward an effector versus memory phenotype, by surface expression of CD44 and CD62L, 14 days after vaccine priming, was determined. The phenotypic profile for central memory (CM) is typically CD44+ and CD62L+, and effector memory (EM) cells are CD44+ and CD62L−. A significant increase was observed in the tetramer OVA-specific EM and CM CD8+ T cell population in mice given triple combination therapy, compared to other groups (
Given the increase of Ag-specific effector CD8+ T cell responses induced by the triple combination therapy in the non-tumor bearing setting, the next question was whether the combination could induce an antitumor response using the poorly immunogenic B16-OVA melanoma model. B16-OVA tumor cells were implanted into cohorts of naïve recipient B6 mice (n=10/group). Seven days after implantation when tumors reached an average size of ˜30-40 mm3, mice were randomized, and treated with the therapies as outlined in
To understand the mechanism of action of the combination therapy, the Ag-specific phenotype and functional response of CD8+ effector and CD4+ Tregs isolated from tumors following the various immunotherapies was characterized. Given the importance of multifunctional effector CD8+ T cell immunity in anti-tumor immunity (Villarreal D O, et al. Cancer Res 2014; 74:1789-800; Slaney C Y, et al. Cancer Res 2014; 74:7168-7174), the Ag-specific CD8+ T cell population and its expression of IFNγ and TNFα, in response to ex vivo OVA257-264 SIINFEKL peptide stimulation, was measured 15 days after tumor implantation (
Given that one mechanism of the anti-GITR mAb is to reduce CD4+ Tregs in the tumors (Cohen A D, et al. PloS one 2010; 5:e10436; Schaer D A, et al. Curr Opin Immunol 2012; 24:217-224; Schaer D A, et al. Immunother Cancer 2014:15:2-7), the effects of combined Vax/anti-GITR/anti-PD-1 immunotherapy on these cells in the tumors was evaluated. However, prior to assessing the Treg population in the tumors, the splenic Treg population at day 14 in non-tumor bearing mice was monitored, using the scheme in
Tumor-infiltrating CD8+ T cells showed a synergistic enhancement against an immunizing peptide in the Vax/anti-GITR/anti-PD-1 combination therapy, indicating that the superior induction of potent CTL responses was most likely critical for the efficacy of the combination therapy. Therefore, the relevance of the effector populations on tumor rejection induced by the combination therapy was investigated. In a therapeutic setting, CD8+ T cells, CD4+ T cells, and NK cells were depleted in tumor-bearing mice as illustrated in
The ultimate goal of both vaccination and active immunotherapy against cancer is the generation of long-lasting memory T cells, which can rapidly respond to subsequent Ag exposure. To assess memory responses, re-challenge experiments were carried out in tumor-free surviving animals, 6 months after completing treatment. All the mice that survived the first tumor challenge with Vax/anti-GITR/anti-PD-1 treatment survived a second tumor challenge against the same tumor 6 months later (
Extensive research in the field has demonstrated that CTLs play a major role in tumor rejection, and the numbers of tumor-infiltrating effector CD8+ T cells are often correlated with a good prognosis (Blohm, U., et al. Eur. J. Immunol. 2006; 36: 468-477; Boissonnas, A. et al. J. Exp. Med. 2007; 204:345-356: Steer, H. J., et al. Oncogene 2010; 29:6301-6313). More recently, several studies have begun to support the hypothesis that the subset of KLRG1+ effector memory CD8+ T cells may predict therapeutic efficacy against pathogens and tumors (Villarreal D O, et al. Molecular Therapy 2015; 10:1653-1662; Olson J A, et al. Immunity 2013; 38:1250-60; Cush S S, Flano E. J Immunol 2011; 186:4051-8; Ye F, et al. J Immunol 2012; 189:5206-11; van Duikeren S, et al. J Immunol 2012; 189:3397-403; Villarreal D O, et al. Cancer Res 2014; 74:1789-800; Slaney C Y, et al. Cancer Res 2014; 74:7168-7174; Brunner S M, et al. Hepatology 2015; 61:1957-67). The increase of KLRG1+CD8+ T cells in the peripheral blood of non-tumor bearing mice in
If the expansion of the KLRG1+CD8+ subset population is one mechanism that helped establish better tumor growth control/regression in the combination Vax/anti-GITR/anti-PD-1 therapy, it was important to determine whether depletion of the KLRG1+CD8+CD44+ effector T cell subpopulation would lead to a loss of tumor growth control. First, the ability of anti-KLRG1 (aKLRG1) antibody to deplete the target population was determined. To examine this, two groups of non-tumor bearing mice were vaccinated with the combination Vax/anti-GITR/anti-PD-1 therapy and one group was treated with 200 μg of anti-KLRG1 mAb (200 μg) at day 0, 2, 4, and 6 post-vaccination, and at day 7 after therapy initiation the expression of KLRG1 was monitored on CD8+ T cells from the blood and spleen (
A major challenge for developing effective cancer immunotherapies is driving potent antitumor responses against poorly immunogenic tumor associated antigens (TAAs), such as self-antigens. The results in
Although anti-CD122 as a monotherapy delayed tumor progression, it was not curative in a more stringent therapeutic intervention on 7-day tumors (with average tumor diameter ˜30 mm3) under the conditions tested (
A prime-boost vaccination strategy was applied on day 7, 10, and 14 for treating 7-day established tumors, and showed greater long-term survival (30%) than a single vaccine dose in this therapeutic setting (
Finally, given the improved efficacy by the additive benefit of reducing Tregs in the Vax/anti-CD122 combination and anti-CD4/anti-CD122 combination studies, we determined if anti-CD122 alone can synergize with anti-GITR mAb, an immunotherapy capable of reducing the number of CD4+ Tregs in the tumor (Schaer D A et al., Curr Opin Immunol 2012; 24:217-224). Therapeutic intervention on 4-day tumors using combinatorial therapy of anti-CD122 and anti-GITR targeting mAb demonstrated synergy, showing significant suppression of tumor growth that yielded ˜40% long-term survival, compared to anti-CD122 monotherapy (
To identify the binding epitopes for TRGB191 on human GITR extracellular domain, solution hydrogen/deuterium exchange-mass spectrometry (HDX-MS) was performed.
For pepsin/protease XIII digestion, 3.2 μg of human GITR in 133 μL control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured by adding 135 μL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH is 2.5) and incubating the mixture for 3 min at 25° C. Then, the mixture was subjected to online pepsin/protease XIII digestion and the resultant peptides was analyzed using an UPLC-MS system comprised of a Waters Acquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo). The peptides were separated on a 50 mm×1 mm C8 column with a 19 min gradient from 2-28% solvent B (0.2% formic acid in acetonitrile) for samples containing human GITR. Solvent A is 0.2% formic acid in water. The injection valve and pepsin/protease XIII column and their related connecting tubings are inside a cooling box maintained at 11° C. And the second switching valve, C8 column and their related connecting stainless steel tubings are inside another chilled circulating box maintained at 0° C. Peptide identification is done through searching MS/MS data against the human GITR sequence with Mascot. The mass tolerance for the precursor and product ions is 10 ppm and 0.05 Da, respectively.
10 μg human GITR was deglycosylated by incubation with 1 μL of PNGase F at 37□C for overnight. The sample was then dried down and glycan was reconstituted and incubated with 5 μL 400 mM procainamide (prepared in 3:7 ratio of acetic acid:DMSO (v/v) and 1M sodium cyanoborohydride) at 65□C for 3 h. To remove the excess labeling reagents, the sample was reconstituted in 90% ACN to a total of 500 μL solution. After conditioning the HILIC-SPE plate with 200 μL water and 200 μL 90% ACN, the sample was loaded to the HILIC-SPE plates, washed with 200 μL 90% ACN and eluted with 50 μL 20% ACN. The 75 μL ACN was added prior to further analysis. Glycan masses were measured using a UPLC-MS comprised of Waters ACQUITY UPLC and Bruker MicroTOF QII.
8 μL human GITR (3.2 μg) or 8 μL human GITR & mAb mixture (3.2 μg: 24 μg) was incubated with 125 μL deuterium oxide labeling buffer (50 mM sodium phosphate, 100 mM sodium chloride at pD 7.4) for 0 sec, 60 sec, 300 sec, 1800 sec, 7200 sec, and 14400 sec at 25□C. Hydrogen/deuterium exchange was quenched by adding 135 μL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH is 2.5). Subsequently, the quenched samples were subjected to on column pepsin/protease XIII digestion and LC-MS analysis as described above. The mass spectra were recorded in MS only mode.
Raw MS data was processed using HDX WorkBench, software for the analysis of H/D exchange MS data (J. Am. Soc. Mass Spectrom. 2012, 23 (9), 1512-1521). The deuterium levels were calculated using the average mass difference between the deuteriated peptide and its native form (to).
The deuterium levels at the identified peptides were monitored from the mass shift on LC-MS. Native human GITR-ECD show significant reduction in deuterium uptakes upon binding to mAb TRGB191.CLF at residues 28-50 and 70-79 of SEQ ID NO: 62. These regions with significant reduction in deuterium uptakes upon binding to mAb are thus assigned as the epitope peptides, which are highlighted in dark or light Grey in
HDX-MS measurements of mAb TRGB191 binding to human GITR ECD indicate that the binding epitope is discontinuous and located within two peptide regions of GITR:
region 1 (residues 28-50 of SEQ ID NO: 62)
region 2 (residues 70-79 of SEQ ID NO: 62):
The binding epitope of mAb TRGB191 was further refined by mapping the HDX data on the 3D model of GITR obtained from the crystal structure of GITR ECD in complex with TRGB159 Fab. According to the structure, the large portion of the two peptides is inaccessible for solvent. The exposed portions of the peptides are proximal in space and include residues 40-45 and 75-79 of SEQ ID NO:62 (highlighted in
The structure was determined as follows. The GITR:TRGB159 complex was prepared by mixing Fab with the 25% molar excess of GITR ECD in 20 mM Tris, pH 8.5, 250 mM NaCl, and incubated at 4° C. overnight. Formation of the complex was monitored on a Superdex 200 column. Crystallization of the complex was carried out by the vapor-diffusion method in sitting drops at 20° C. The crystals suitable for X-ray analysis were obtained from 14% PEG 3350 and 0.2 M Na formate in 0.1 M HEPES buffer, pH 7.5. For X-ray data collection, one crystal was soaked for a few seconds in a cryo-protectant solution containing mother liquor supplemented with 24% glycerol and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at the Advanced Photon Source (Argonne, Ill.) using a Mar225 detector. Diffraction intensities were detected to 2.8 Angstrom resolution and processed with the program XDS (Kabsch, W. (2010). XDS. Acta Cryst. D66, 125-132.). The structure was solved by molecular replacement with the program Phaser (McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658-674) using structure 5116 from the Protein Data Bank as a search model. When the Fab was positioned in the unit cell, the GITR molecule was manually built in the electron density using program Coot (Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501).
A polymorphism in the FcγRIIIA gene (rs396991) results in an amino acid substitution change from a valine to phenylalanine at position 158 (V158F), with the 158V allotype displaying a higher affinity for human IgG1 and increased ADCC; this polymorphism is occasionally denoted in the literature as V176F (Wu J, Edberg J C, Redecha P B, Bansal V, Guyre P M, Coleman K, Salmon J E, Kimberly R P. J Clin Invest; 1997; 100(5):1059-70) Cartron et al. confirmed the homozygous FcγRIIIA-158V genotype to be the single parameter associated with a positive clinical response to rituximab, an antibody whose mechanisms of action include ADCC of tumor cells (Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H. Blood; 2002; 99(3):754-8).
TRGB191.CLF is manufactured as a low fucose antibody and subsequently has a roughly 10-fold enhanced affinity to the FcγRIIIA, the Fc receptor present on NK cells, compared to a “regular” fucosylated version (RFV) of the same mAb (KD was ˜37 nM versus ˜370 nM to the high affinity FcγRIIIA-158V variant, and ˜180 nM versus 1,750 nM to the low affinity FcγRIIIA-158F variant, respectively).
Antibodies with enhanced affinity for FcγRIIIA have been demonstrated to possess increased ADCC activity (Strohl W, Strohl L. Therapeutic Antibody Engineering—Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry. 1st ed. Sawston: Woodhead Publishing; 2012). The ADCC activity of TRGB191.CLF was evaluated against several target cells or cell lines expressing varying levels of hGITR. For example, resting peripheral T cells express minimal levels of GITR, but GITR expression is upregulated on these cells when activated in vitro. The JJN-3 cell line is a human plasma cell leukemia line that expresses endogenous hGITR at more physiological levels compared to the HuT102 cells, and more similar to those seen on activated T cells and on in vitro differentiated Tregs.
The ADCC activity of TRGB191.CLF was characterized across a wide range of E:T cell ratios on primary resting or activated T cells (see
TRGB191.CLF had minimal ADCC activity on resting, unactivated CD4+ T cells (see
The JJN-3 cells were previously characterized to express lower levels of GITR than HuT102 cells, and within a range that was more comparable to activated primary T cells and in vitro generated TregS, although levels were several-fold higher. When JJN-3 cells were tested using the NK-92 158V high affinity effector cells across a wide range of E:T ratios (see
Recently published and internal data have demonstrated that GITR is expressed on tumor-infiltrating lymphocytes present within the tumor microenvironment in both mice and humans, with the highest level of expression being observed on CD4+ TregS in solid tumors.
Peripheral CD4+ T cells were differentiated and expanded to become functionally suppressive TregS that are defined as CD4+CD25+FOXP3+. These TregS express similar levels of GITR compared to activated primary CD4+ and CD8+ T cells. TRGB191.CLF induced antibody-dependent Treg cell killing comparable to the potency observed with JJN-3 cells (see
TRGB191.CLF has a roughly 10-fold enhanced affinity to both FcγRIIIA-158V/V and FcγRIIIA-158F/F compared to an RFV of the same mAb; its KD value to the low affinity FcγRIIIA-158F/F variant was equal to ˜180 nM, which is about 2-fold of the affinity of the RFV to the high affinity variant receptor (370 nM).
The ADCC activity of TRGB191.CLF to JJN-3 cells was comparable when using NK-92 effector cells expressing either the high affinity FcγRIIIA-158V/V or low affinity FcγRIIIA-158F/F variant, with the EC50 value varying ˜2-fold (40.01 ng/mL and 87.44 ng/mL, respectively) (see
Animals treated with isotype control antibodies with smaller starting tumor volumes (˜100 mm3) reached median time-to-endpoint (MTE) of roughly 19 days. DTA-1 dosed at a single of 10 mg/kg on day 1 led to 2 durable complete regressions (CR) and a delay of MTE to 29.3 days. FGK4.5, anti-CD40 dosed at 2 mg/kg on days 1, 5 and 9 resulted in 6 CRs and a measurable delay in MTE of 60 days. The combination of a single injection of 10 mg/kg of DTA-1 on day 1 along with FGK4.5 (q4dx3), resulted in 8 CRs up to day 40 at which point there appears to be 1 progression (
Animals treated with isotype control antibodies with larger starting tumor volumes (˜230 mm3) reached median time-to-endpoint (MTE) of roughly 10.5 days. FGK4.5 dosed at 10 mg/kg on days 1, 5 and 9 resulted in 1 complete response (CR), and a measurable delay in MTE of 33 days. The combination of a single injection of 10 mg/kg of DTA-1 on day 1 along with FGK4.5 (q4dx3), resulted in 4 durable CRs (
Concurrent combination of DTA-1 (10 mg/kg, q1, day 1) and OX86 (anti-OX40, 10 mg/kg, q4dx3 starting on day 1) antibodies also resulted in better anti-tumor growth responses with an increase from 2 CRs with DTA-1 to 5 CRs with DTA-1 plus OX86 combination. OX86 did not inhibit tumor progression as a single agent (
Concurrent combination of DTA-1 (10 mg/kg, q1, day 1) and RMP1-14 (anti-PD-1, 10 mg/kg, q4dx3) was also more efficacious than delaying one of the therapies by giving it two days after the first agent. Anti-PD-1 single agent led to 3 CRs, anti-GITR single agent led to 2 CRs and combination anti-GITR and anti-PD-1 therapies given concurrently resulted in 8 CRs. This efficacy was reduced to 4 or 5 CRs if anti-PD-1 or anti-GITR is dose sequenced first, respectively (
This application claims the benefit of U.S. Provisional Application Ser. No. 62/305,270, filed 8 Mar. 2016 and U.S. Provisional Application Ser. No. 62/407,106, filed 12 Oct. 2016. The entire content of the aforementioned applications is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62305279 | Mar 2016 | US | |
| 62407106 | Oct 2016 | US |
