Patent Application
20220169702
The present invention relates to TGF-β superfamily receptor ectodomain fusion molecules and uses thereof. More specifically, the present invention relates to TGF-β superfamily receptor ectodomain fusion molecules and their use in TGF-β superfamily ligand neutralization.
TGF-β is part of a superfamily of over 30 ligands that regulate several physiological processes, including cell proliferation, migration and differentiation. Perturbation of their levels and/or signaling gives rise to significant pathological effects. For instance, TGF-β and activin ligands play critical pathogenic roles in many diseases including cancer (Hawinkels & Ten Dijke, 2011; Massague et al, 2000; Rodgarkia-Dara et al, 2006). TGF-β, in particular, is considered as a critical regulator of tumor progression and is overexpressed by most tumor types. It favors tumorigenesis in part by inducing an epithelial-mesenchymal transition (EMT) in the epithelial tumor cells, leading to aggressive metastasis (Thiery et al, 2009). TGF-β also promotes tumorigenesis by acting as a powerful suppressor of the immune response in the tumor microenvironment (Li et al, 2006). In fact, TGF-β is recognized as one of the most potent immunosuppressive factors present in the tumor microenvironment. TGF-β interferes with the differentiation, proliferation and survival of many immune cell types, including dendritic cells, macrophages, NK cells, neutrophils, B-cells and T-cells; thus, it modulates both innate and adaptive immunity (Santarpia et al, 2015; Yang et al, 2010). The importance of TGF-beta in the tumor microenvironment is highlighted by evidence showing that, in several tumor types (including melanoma, lung, pancreatic, colorectal, hepatic and breast), elevated levels of TGF-β ligand are correlated with disease progression and recurrence, metastasis, and mortality. Hence, significant effort has been invested in devising anti-tumor therapeutic approaches that involve TGF-β inhibition (Arteaga, 2006; Mourskaia et al, 2007; Wojtowicz-Praga, 2003).
One approach to developing therapeutic agents that inhibit TGF-β function has been to use antibodies or soluble decoy receptors (also termed receptor ectodomain (ED)-based ligand traps) to bind and sequester ligand, thereby blocking access of ligand to its normal cell surface receptors (Zwaagstra et al, 2012). In general, receptor ED-based traps are a class of therapeutic agents that are able to sequester a wide range of ligands and that can be optimized using protein engineering approaches (Economides et al, 2003; Holash et al, 2002; Jin et al, 2009).
Previously, a novel protein engineering design strategy was used to generate single-chain, bivalent traps that are able to potently neutralize members of the TGF-β superfamily of ligands due to avidity effects (Zwaagstra et al, 2012) [WO 2008/113185; WO 2010/031168]. In this case, bivalency was achieved via covalent linkage of two TβRII ectodomains using portions of the intrinsically disordered regions (IDR) that flank the structured, ligand-binding domain of TβRII-ED. One example of these single-chain bivalent traps, T22d35, exhibited TGF-β neutralization potencies ˜100-fold higher than the monovalent non-engineered TβRII ectodomain, though it did not neutralize the TGF-β2 isoform and had a relatively short circulating half-life.
While research to date indicates that single-chain TGF-β traps have promising therapeutic potential, their circulating half-lives and manufacturability present challenges to the commercial application.
The present invention relates to TGF-β superfamily receptor ectodomain fusion molecules and uses thereof. More specifically, the present invention relates to TGF-β superfamily receptor ectodomain fusion molecules and their use in TGF-β superfamily ligand neutralization.
In some aspects, the invention relates to TGF-β superfamily receptor ectodomain-based polypeptides that are similar to typical Fc fusions in design, in that the ectodomain is fused to a dimeric antibody constant domain. In particular, with respect to the present polypeptides, the Fc portion occupies the N-terminal position. Fc fusions in the prior art typically provide the Fc portion at the C-terminal end of the fusion. As will be evident from the results presented herein, this difference in orientation provides a number of significant advantages.
In other aspects, the present polypeptides incorporate at least two TGF-β superfamily receptor ectodomains that are linked in tandem to the C-terminus of an antibody constant domain.
Thus, there is provided a polypeptide construct comprising: a first portion comprising the second constant domain (CH2) and/or third constant domain (CH3) of an antibody heavy chain, and a second portion comprising at least two TGF-β superfamily receptor ectodomains (TβSR-ED) linked in tandem; wherein the N-terminus of the second portion is linked to the C-terminus of the first portion.
There is also provided a polypeptide construct comprising: a first portion comprising the second constant domain (CH2) and/or third constant domain (CH3) of an antibody heavy chain, and a second portion comprising at least one TGF-β superfamily receptor ectodomains (TβSR-ED), wherein the N-terminus of the second portion is linked to the C-terminus of the first portion, and further wherein the first portion does not further comprise an antibody that binds to an antigen that is PD-L1, EGFR1, Her-2, CD4, CD6, CD20, CD25, MUC-1, IL-2, IL-6, or CTLA-4.
There is provided a polypeptide construct comprising: a first portion comprising the second constant domain (CH2) and/or third constant domain (CH3) of an antibody heavy chain, and a second portion comprising at least one TGF-β superfamily receptor ectodomain (TβSR-ED), wherein the N-terminus of the second portion is directly fused to the C-terminus of the first portion.
In an embodiment, there is provided a polypeptide construct comprising a first portion comprising the second constant domain (CH2) and/or third constant domain (CH3) of an antibody heavy chain, and a second portion comprising at least one TGF-β superfamily receptor ectodomain (TβSR-ED), wherein the N-terminus of the second portion is linked to the C-terminus of the first portion, and wherein the polypeptide construct neutralizes TGF-β with at least 100-fold more potency than the TβSR-ED alone.
In a preferred embodiment, the second portion comprises one, two, or multiple TGF-β superfamily receptor ectodomain (TβSR-ED). In a preferred embodiment, the TβSR-ED is a TGF-β receptor type II ectodomain (TβR-II-ED). In a preferred embodiment, the TβSR-ED comprises a sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:81, and a sequence substantially identical thereto.
The second portion may comprise a sequence selected from the group consisting of SEQ ID NO:43-SEQ ID NO:51, SEQ ID NO:61-SEQ ID NO:68, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, and a sequence substantially identical thereto.
In a preferred embodiment, the first portion of a polypeptide construct of the present invention further comprises a CH1, a CH1 and VH, or CH1 and scFv.
There is provided a polypeptide construct of the present invention wherein the antibody heavy chain is of human origin. In a preferred embodiment, the antibody heavy chain is selected from the group consisting of a human IgG1, IgG2, IgG3, or IgG4 heavy chain. More preferably, the antibody heavy chain is a human IgG1.
In accordance with the present invention, the polypeptide construct shows longer in vivo half-life compared to the half-life of the second portion alone.
There is provided a polypeptide construct of the present invention, wherein the polypeptide construct is a single chain polypeptide.
In an embodiment, the polypeptide construct forms a dimeric polypeptide. In another embodiment, the polypeptide construct is heterodimeric.
There is provided a polypeptide construct selected from the group consisting of any one of SEQ ID NO:91 to SEQ ID NO:120, and a sequence substantially identical thereto.
There is provided a polypeptide construct according to the present invention, wherein the construct comprises an antibody, antigen binding fragment thereof, or a targeting moiety. In a preferred embodiment, the antibody, the antigen binding fragment, or the targeting moiety is at the N-terminus of the first portion.
In a preferred embodiment, the antigen binding fragment may be selected from the group consisting of a Fv, scFv, Fab, or sdAb. In a preferred embodiment, the antigen binding fragment binds to any antigen, provided that it is not PD-L1, EGFR1, Her-2, CD4, CD6, CD20, CD25, MUC-1, IL-2, IL-6, or CTLA-4.
In a preferred embodiment, a polypeptide construct of the present invention comprises an antibody selected from the group consisting of Cetuximab, Avastin, Herceptin, Synagis, and FC5. In a preferred embodiment, the antibody is Cetuximab.
In a preferred embodiment, a polypeptide construct of the present invention comprises a targeting moiety, wherein the targeting moiety comprises a poly-aspartate sequence motif for bone targeting. In a preferred embodiment, the targeting moiety comprises D10.
There is provided a polypeptide construct according to the present invention wherein the construct is a dimeric polypeptide; wherein the dimeric polypeptide comprises: a first single chain polypeptide comprising a first portion comprising the second constant domain (CH2) and third constant domain (CH3) of an antibody heavy chain, and a heavy chain variable region of a given antibody; a second portion comprising one or more TGF-β superfamily receptor ectodomains (TβSR-ED), wherein the N-terminus of the second portion is linked to the C-terminus of the first portion, and a second single chain polypeptide comprising a first portion comprising the second constant domain (CH2) and third constant domain (CH3) of an antibody heavy chain, and a light chain variable region of said given antibody; a second portion comprising one or more TGF-β superfamily receptor ectodomain (TβSR-ED) which is the same or different from the ectodomain(s) in the first polypeptide, wherein the N-terminus of the second portion is linked to the C-terminus of the first portion.
There is also provided a nucleic acid molecule encoding the polypeptide construct of the present invention. There is also provided a vector comprising the nucleic acid molecule of claim the present invention.
There is also provided a composition comprising one or more than one independently selected polypeptide construct of the present invention and a pharmaceutically-acceptable carrier, diluent, or excipient.
There is also provided a transgenic cellular host comprising the nucleic acid molecule or a vector of the present invention. The transgenic cellular host further comprising a second nucleic acid molecule or a second vector encoding a second polypeptide construct different from the first polypeptide construct.
There is also provided the use of a polypeptide construct according to the present invention for treatment of a medical condition, disease or disorder; wherein the medical condition, disease or disorder comprises, but is not limited to, cancer, ocular diseases, fibrotic diseases, or genetic disorders of connective tissue.
In a preferred embodiment, there therefore provided a polypeptide construct comprising:
The antibody constant domain can further comprise, either linked thereto or formed integrally therewith, a binding agent such as a full size antibody, a ligand or any other protein of interest. In the alternative, the antibody constant domain comprises only the CH2 and/or CH3 regions, and not a full size antibody. In these and other types of constructs, the CH2 and/or CH3 region can be altered by deleting or substituting amino acids including one or more of the cysteines that provide cross-linking when the present constructs are provided as dimeric constructs.
In other aspects of the present invention, there is provided a polypeptide construct that incorporates one or more such ectodomains. When the constructs comprise only one ectodomain linked to the antibody constant domain, then the construct is further characterized by at least one of the following: (1) when the constant domain further comprises a full sized antibody, that antibody does not bind effectively to PD-L1 or to an immunoregulatory antigen selected, (2) the constant domain comprises only the CH2 and/or CH3 regions, (3) the constant domain comprises an amino acid alteration relative to a wild type counterpart, such as a cysteine residue alteration; and (4) the first portion is linked to the second portion directly and without intervening amino acids.
In another of its aspects, the present invention provides a polypeptide construct comprising
the N-terminus of the second portion is linked to the C-terminus of the first portion. These polypeptide constructs can neutralize TGF-β, and with at least 100-fold more potency than the TβSR-ED alone.
The second portion of the polypeptide construct of the present invention may comprise one or two or more TβSR-ED. In a preferred embodiment the construct comprises two or more independently selected ectodomains linked in tandem and to the C-terminus of the constant domain. The TβSR-ED may be selected from the group consisting of a TGF-β receptor type II ectodomain (TβRII-ED), a bone morphogenetic protein receptor type la ectodomain (BMPR-ED), an activin receptor type Ila ectodomain (ActRIIa-ED), and an activin receptor type IIb ectodomain (ActRII-ED). In another preferred embodiment, the ectodomain is a TβR-II ectodomain.
In the polypeptide construct described herein, the first portion further may comprise a CH1, a CH1 and VH, or a CH1 and scFv. It may constitute an Fc region, an antibody, or any ligand binding agent or moiety.
The polypeptide construct of the present invention may comprise a CH2 and CH3 from an antibody heavy chain that is of human or mouse origin. For example, and without wishing to be limiting, the antibody heavy chain may be selected from the group consisting of a human IgG1, IgG2, IgG3, or IgG4 heavy chain. In embodiments, the constant domain in the constructs is CH2 per se, or CH3 per se or CH2-CH3.
The polypeptide construct described herein may show longer in vivo half-life compared to the half-life of TβSR-ED alone.
In one example, the polypeptide construct of the present invention may be a single chain polypeptide. The polypeptide construct as described herein may also form a dimeric polypeptide. This dimeric polypeptide may be heterodimeric.
The present invention further provides a polypeptide construct comprising
wherein the N-terminus of the second portion is linked to the C-terminus of the first portion; additionally, in the construct as just described, the first portion is not derived from certain antibodies discussed infra.
The present invention also provides a polypeptide construct, comprising:
wherein the N-terminus of the second portion is linked to the C-terminus of the first portion, and
In alternative constructs of the present invention, the polypeptide construct comprises an antibody Fc fragment linked at the C-terminus of each heavy chain to at least one TGF-β superfamily receptor ectodomain (TβSR-ED), as described above. In embodiments the receptor ectodomain portion comprises two independently selected ectodomains that are linked in tandem, i.e., in a linear manner. In some embodiments, the ectodomains are the same in sequence, or least the same with respect to their target ligand. The construct may further comprise a binding fragment or moiety at the N-terminus of the Fc; the binding fragment may be selected from the group consisting of a Fv, scFv, Fab, or sdAb, or any other binding moiety such as a motif for bone targeting, also as described above. In the polypeptide constructs as described above, the TGF-β receptor ectodomain does not interfere in the native function or specificity of the binding fragment.
The present invention also provides a nucleic acid molecule encoding the polypeptide constructs as described herein. A vector comprising the nucleic acid molecule just described is also encompassed by the invention. The invention also includes a transgenic cellular host comprising the nucleic acid molecule or a vector as described herein; the cellular host may further include a second nucleic acid molecule or a second vector encoding a second polypeptide construct different from the first polypeptide construct. Systems used to produce the present polypeptides can be secretion systems, particularly in the case where dimerization through disulfide bridges is required, and the expression polynucleotides thus encode secretion signals that are cleaved by the host upon secretion into the culturing medium.
Compositions comprising one or more than one independently selected polypeptide construct described herein and a pharmaceutically-acceptable carrier, diluent, or excipient are also encompassed by the present invention.
Additional aspects and advantages of the present invention will be apparent in view of the following description. The detailed descriptions and examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the scope of the invention will become apparent to those skilled in the art in light of the teachings of this invention.
These and other features of the invention will now be described by way of example, with reference to the appended drawings, wherein:
The present invention relates to TGF-β superfamily receptor ectodomain fusion molecules and uses thereof. More specifically, the present invention relates to TGF-β superfamily receptor ectodomain fusion molecules and their use in TGF-β superfamily ligand neutralization.
The present invention provides polypeptide constructs, comprising
wherein the N-terminus of the second portion (ectodomain) is linked to the C-terminus of the first portion (Fc region), and wherein the polypeptide construct neutralizes TGF-β with at least 100-fold more potency than the TβSR-ED alone. The polypeptide construct referred to herein is a synthetic polypeptide produced via protein engineering. It comprises two protein “portions” (or “parts”) that are linked to form the chimeric polypeptide construct. When the polypeptide construct is expressed, two polypeptide chains dimerize, such that the CH2 and CHH domains form an antibody Fc region.
In specific embodiments of the present invention, descriptions of which are elaborated further herein, there are provided polypeptide constructs in which TGF-β superfamily receptor-ectodomains were fused to IgG Fc regions. Specifically, the T2m (single ectodomain) or T22d35 (double ectodomain) moieties were linked (fused) to the C-terminal end of the Fc region. It was observed that fusion constructs of this type have advantages relative to several other versions of receptor-ectodomain based molecules, including non-Fc fused monovalent or multivalent TGF-β receptor ectodomain constructs (such as T2m and T22d35) and constructs in which a receptor ectodomain is fused to the N-terminus of an Fc region. In particular, the constructs have improved manufacturability due to the presence of the Fc region (for example, purification can be accomplished using protein A chromatography). The Fc region also allows for improved circulating half-lives. Importantly, the present constructs have substantially higher TGF-β neutralization potencies compared to T2m and T22d35 alone or to constructs where a receptor ectodomain is fused to the N-terminus of an Fc region. Thus, an advantage of the present invention is unexpected high potency TGF-β superfamily ligand neutralization, including some degree of neutralization of TGF-β2, which was not observed for the non-Fc fused constructs T2m and T22d35. Finally, constructs in accordance with embodiments of the present invention, that is where the ectodomain(s) is/are fused to the C-terminus of an Fc region of an antibody, allows for preservation of the structure and function of the natural N-terminal regions/domains of an antibody; as such, antigen binding to the antibody CDR regions is not perturbed. This leads to the generation of a bifunctional construct able to interact with the target of the antibody while interacting with, and neutralizing, members of the TGF-β superfamily of ligands.
The invention relates not only to bifunctional constructs, but also to constructs that are monofunctional, and comprise an Fc region that consists only of the CH2 and/or CH3 regions of an antibody constant region. Preferably, the G1, G2 or G4 subclasses are used, and particularly G1 as well as G2. These constructs are monofunctional in the sense that the constant region itself has no particular activity, other than to act as a structure through which dimers of the polypeptide constructs can form. These minimal constant regions can also be altered to provide some benefit, by incorporating the corresponding hinge regions (SEQ ID NO:5-8) and optionally changing the cysteine residue composition. Thus, some or all of the cysteine residues involved in bridging the two Fc fragments or naturally used to bridge between the heavy and light chains of a full-length antibody can be replaced or deleted. These cysteine residues are seen in hinge sequences listed in SEQ ID NO:5-8. First, not all of the naturally-occurring inter-hinge disulfide bonds need to be formed for the Fc homodimerization to occur, while noting that the stability of the Fc homodimer may depend on the number of intermolecular disulphide bridges. Secondly and perhaps more importantly, the presence of hinge-region cysteine residues may become problematic when the Fc region lacks its N-terminal Fab fragment (i.e., is a headless Fc) as in the case of some polypeptide constructs of the present invention. This leads to untethering and exposure of these hinge-region cysteines, and in turn that may result in complex mixtures of high-order polymeric chains, which creates manufacturability issues in addition to potentially diminishing the intended biological activity and efficacy. Because it is practically impossible to predict the outcome of varying the number of inter-hinge disulphide bridges for the “headless” polypeptide constructs of the present invention, we generated a systematic array of N-terminal Fc variants for all four human IgG isotypes either by a deletion approach (in which hinge-region cysteine residues are progressively eliminated by N-terminal truncations) or by a mutagenesis approach (in which hinge-region cysteine residues are progressively mutated to serine from the N-terminus of the hinge region). Non-limiting examples of such N-terminal variants of headless Fc regions are listed in SEQ ID NO:9-34.
In the present disclosure, an “antibody”, also referred to in the art as “immunoglobulin” (Ig), refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM. The structure of an antibody and of each of the domains is well-established and familiar to those of skill in the art, though is summarized herein. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences; the immunoglobulin light chain folds into a variable (VL) and a constant (CL) domain, while the heavy chain folds into a variable (VH) and three constant (CH, CH2, CH3) domains. Once paired, interaction of the heavy and light chain variable domains (VH and VL) and first constant domain (CL and CH1) results in the formation of a Fab (Fragment, antigen-binding) containing the binding region (Fv); interaction of two heavy chains results in pairing of CH2 and CH3 domains, leading to the formation of a Fc (Fragment, crystallisable). Characteristics described herein for the CH2 and CH3 domains also apply to the Fc.
While the light and heavy chain variable regions show significant sequence diversity between antibodies, the constant regions show less sequence diversity and are responsible for binding a number of natural proteins to elicit important biochemical events. Specifically, and without wishing to be limiting, the Fc fragment binds to endogenous Fc receptors on the surface of lymphocytes.
The CH2 and CH3 domains of the first portion may be of any isotype, including one selected from the group consisting of IgA, IgD, IgE, and IgG. The CH2 and CH3 domains may also be from any suitable source. For example and without wishing to be limiting, the CH2 and CH3 domains may originate from a human, mouse and other rodents like rats and degu, rabbit, monkey, or other mammalian source. In one example, the CH2 and CH3 domains may be of the IgG isotype; in another example, the CH2 and CH3 domains are from human.
In a specific, non-limiting example, the CH2 and CH3 domains of the first portion may be of an isotype or comprise a sequence selected from the group consisting of:
and
In the protein constructs of the present invention, the first portion may further comprise a sequence corresponding to the hinge region at the N-terminus of the CH2 domain. For example, the first portion may further comprise a sequence selected from the group consisting of:
and a sequence substantially identical to any of the sequences listed above.
Thus, the first portion of the polypeptide construct of the present invention consists of naturally fused Fc and hinge regions for the various IgG isoforms and in embodiments is selected from the group consisting of SEQ ID NO:1-4 for the Fc region and SEQ ID NO:5-8 for the hinge region.
In specific embodiments, the first portion of the polypeptide construct of the present invention is selected from a group of sequences displaying variation in the N-terminal sequence as exemplified by SEQ ID NO:9-34. These differ in the number of cysteine residues retained from the hinge region as a means to modulating the degree of Fc-region dimerization and hence impacting on both efficacy and manufacturability. Thus, in embodiments, the polypeptide construct comprises a variation in the constant domain, wherein at least one cysteine residue involved in cross-linking is deleted or substituted. Suitable substitutions include serine or alanine, and preferably by serine. A substantially identical sequence may comprise one or more conservative amino acid mutations that still provide for proper folding upon secretion into the culturing medium. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physico-chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered “substantially identical” polypeptides. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). These conservative amino acid mutations may be made to the framework regions while maintaining the overall structure of the constant domains; thus the function of the Fc is maintained.
In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term “basic amino acid” it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term “neutral amino acid” (also “polar amino acid”), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term “hydrophobic amino acid” (also “non-polar amino acid”) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of (Eisenberg et al, 1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).
“Acidic amino acid” refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).
In another non-limiting example, a conservative mutation in the CH2 and/or CH3 domain may be a substitution that enhances a property selected from the group consisting of the stability, half-life, or Fc properties of CH2 and/or CH3 domains or alter glycosylation of the CH2 and/or CH3 domain. For example, and without wishing to be limiting in any manner, the mutation may be an alteration at position 228 (EU numbering, 241 according to Kabat) where the serine is substituted by a proline (S228P), which stabilizes the disulfide linkage within the Fc dimer. Another alteration is the mutation at position 409 (EU numbering, 440 according to Kabat) where an arginine is substituted to a lysine for further stabilization of the Fc homodimer at the CH3-domain level (Yang & Ambrogelly, 2014). Yet another alteration within the CH2 and/or CH3 domain may be a substitution of Asn297 (EU numbering, 314 according to Kabat) by glycine or alanine to alter glycosylation of the constant domain. In yet another example, the CH2 and/or CH3 domain may be altered by substitution of one or more threonine (T252L, T253S, and/or T256F; see [U.S. Pat. No. 6,277,7375]) to increase half-life. Particularly useful are those alterations that enhance Fc properties while remaining silent with respect to conformation, e.g., retaining Fc receptor binding.
In yet another non-limiting example, the conservative mutations in the CH2 and/or CH3 domain may be a substitution that is naturally-occurring. Such mutations may occur in nature as minor sequence differences between species or race.
Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), or any other appropriate software that is known in the art.
The substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may have an identity selected from the group consisting of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical, or any percentage there between, at the amino acid level to sequences described herein. Importantly, the substantially identical sequences retain the activity, specificity, and functionality of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s). In a non-limiting example, the first portion of the polypeptide construct of the present invention may comprise a Fc comprising a sequence selected from the group consisting of a sequence at least 95%, 98% or 99% identical to that of the Fc described herein.
Accordingly, it will be appreciated that the first portion of a construct will include at least an antibody region that preferably provides for cross-linking of the polypeptide constructs, thereby to provide a dimeric protein. This first portion comprises at least the minimal CH2 and/or CH3 domain. That portion can be altered (i) by substituting or deleting cysteine residues from the hinge regions (SEQ ID NO:5-8) involved in crosslinking between the antibody heavy chains or between the heavy and light chains in order to potentially improve preparation homogeneity and efficacy, and/or (ii) by deleting or suitably replacing (e.g., by mutation to alanine) the terminal lysine residue 447 (EU numbering, 478 according to Kabat) of an IgG heavy chain in order to improve chemical stability of C-terminal fusions to enzymatic proteolysis (e.g., by several serine proteases and typically by trypsin). These changes have a positive impact on potency and/or manufacturability, as revealed herein. The first portion can also be extended to become a full Fc region, by including the CH1 domain. As a full Fc, this portion will provide normal Fc effector functions that include involvement in immune cell recruitment, ADCC, CDC and other antibody functions. Moreover, and in embodiments of the present invention, the first portion can include a complete antibody or any equivalent thereof. In certain embodiments, such as when a construct comprises just one ectodomain that is a TGF-β receptor II ectodomain, there is the proviso that the second portion is not an antibody that binds to an immune checkpoint protein such as PD-L1 (programmed death ligand 1) and is not an antibody that binds to an immunomodulating agent that counteracts immune tolerance of cancer cells, the nature and identity of which is as described in [U.S. Pat. No. 8,815,247], and is not an antibody that binds one of EGFR1, her-2, CD4, CD6, CD20, CD25, MUC-1, IL-2, IL-6, and CTLA-4.
The second portion of the polypeptide construct of the present invention comprises at least one and preferably two TGF-β superfamily receptor ectodomain/s (TβSR-ED); for example, the second portion may comprise one or two TβSR-ED. The ectodomain of the Transforming Growth Factor-β superfamily receptor (TβSR) is the N-terminal extracellular, ligand-binding portion of the receptor. Without wishing to be limiting in any manner, the TβSR ectodomain may bind a molecule selected from the group consisting of TGF-β, bone morphogenetic protein (BMP) including BMP2, BMP3, BMP4, BMPS, BMP, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, an BMP15, activin including activins βA, βB and βC, growth differentiation factor (GDF-1) including GDF-3, GDF-8, GDF-9, and GDF-15, nodal, inhibin-α, anti-Mullerian hormone, Lefty-1, Lefty-2, arteman, persephin, neurturin, myostatin, or other known TGF-β superfamily ligands. For example, the TβR ectodomain may be selected from the group consisting of the human TGF-β receptor type II ectodomain (TβR-II-ED), the human TGF-β receptor type IIb (TβR-IIb) ectodomain, the human activin receptor type IIa (ActR-IIa) ectodomain, the human activin receptor type IIb (ActR-IIb) ectodomain, or the BMP type Ia (BMPR-Ia) ectodomain.
In a preferred embodiment the ectodomain binds TGF-β1 and/or TGF-β3. In another preferred embodiment, the ectodomain itself is a human TGF-β receptor type II ectodomain including particularly the TGF-β receptor type IIa (TβRIIa). In one specific, non-limiting example, the TβSR-ED is the TGF-β receptor type II ectodomain (TβRII-ED; SEQ ID NO:35).
In the second portion as described above, the TβSR ectodomain-based portion may further comprise natural linkers. Appropriate, naturally-derived linkers that can be used to fuse two ectodomains head-to-tail are known to those of skill in the art; for example, and without wishing to be limiting, suitable natural linkers are described in [WO2008/113185].
In this embodiment, the natural linker, if present, may be selected from the group consisting of
and
In a specific, non-limiting example, the second portion of the polypeptide construct of the present invention may comprise the sequence selected from the group consisting of:
and
In another specific, non-limiting example, the TGF-β receptor ectodomain is the bone morphogenetic protein receptor la (BMPRIa; SEQ ID NO:69). In this embodiment, the natural linker, if present, may be selected from the group consisting of
Thus, in a specific, non-limiting example, the second portion of the polypeptide construct of the present invention may comprise the sequence selected from the group consisting of:
and
In another specific, non-limiting example, the TβSR ectodomain is the activin receptor Ila (ActRIIa; SEQ ID NO:75). In this embodiment, the natural linker, if present, may be selected from the group consisting of
and
Thus, another specific non-limiting example of the second portion of the polypeptide construct of the present invention comprises the sequence selected from the group consisting of:
and
In another specific, non-limiting example, the TGF-β receptor ectodomain is the activin receptor IIb (ActRIIb; SEQ ID NO:81). In this embodiment, the natural linker, if present, may be selected from the group consisting of
and
Thus, another specific non-limiting example of the second portion of the polypeptide construct of the present invention comprises the sequence selected from the group consisting of:
and
Thus, in various embodiments of the present invention, the present constructs have an ectodomain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:69, SEQ ID NO:75, SEQ ID NO:81, and a sequence substantially identical thereto. In other embodiments, the second portion comprises the entire extracellular portion of a TβSR-ED consisting of a sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:73, SEQ ID NO:79, SEQ ID NO:85, and a sequence substantially identical thereto.
The at least two ectodomain portion can have the same or different ectodomains, all belonging to the superfamily. In embodiments, the ectodomains bind the same target. In other embodiments, the ectodomains originate from the same receptor species. In other embodiments, the ectodomains are identical and thus are homomeric. In other embodiments the ectodomains are different and thus are heteromeric. In these embodiments, the ectodomain can be for instance a TβRII-ED that is type a, and another ectodomain can be a TβRII-ED that is type b. A third ectodomain could be the same as either one of these, or different still. For example, when there is more than one ectodomain in the second portion of the polypeptide construct of the present invention, the ectodomains may be all the same (homomers) or all different (heteromers), or any combination of superfamily ectodomains.
Thus, in embodiments, the second portion of the polypeptide construct of the present invention comprises a repeat of a given TβSR-ED selected from the group consisting of SEQ ID NO:46, 47, 48, 74, 80, 86, and a sequence substantially identical thereto.
In specific embodiments, the second portion of the polypeptide construct of the present invention comprises heteromeric repeats of two distinct TβSR-EDs genetically fused and selected from the group consisting of SEQ ID NO:61, 62, 63, 88, and a sequence substantially identical thereto.
In yet other embodiments, the second portion of the polypeptide construct of the present invention comprises homo-multimeric and hetero-multimeric repeats of one or more TβSR-EDs selected for instance from the group consisting of SEQ ID NO:49, 50, 51, 64, 65, 66, 67, 68, and a sequence substantially identical thereto.
In the protein construct of the present invention, the first and second portions of the polypeptide construct of the present invention are linked. By the term “linked”, it is meant that the two portions are covalently bonded. The chemical bond may be achieved by chemical reaction, or may be the product of recombinant expression of the two portions in a single polypeptide chain. In one specific, non-limiting example, the C-terminus of the first portion is linked directly to the N-terminus of the second portion, that is, no additional “linker” amino acids are present between the two portions. In the case where no linker is present, that is to say direct fusion of the two portions, there will be a direct link between the N-terminus of the full ectodomain and the C-terminus of the antibody constant regions CH2-CH3. For example, in fusing the Fc variant SEQ ID NO:9 to the SEQ ID NO:43 via the intrinsically disordered linker with SEQ ID NO:36, which is part of the TβRII-ED (i.e., no additional “linker” amino acids added), one connects the glycine at the last position of SEQ ID NO:9 to the isoleucine at the first position of SEQ ID NO:43.
A common practice when producing fusion constructs is to introduce glycine or glycine-serine linkers (such as GGGGS, or [G4S]n) between the fused components. As taught in the above paragraph, the polypeptide fusions of the present invention can be produced by direct linkage without use of any additional amino-acid sequence except those present in the Fc portion and in the receptor ectodomain portion. One thus can refrain from utilizing foreign sequences as linkers, providing an advantage due to their potential for undesired immunogenicity and their added molecular weight. Entropic factors are also a potential liability for glycine and glycine-serine linkers, which are highly flexible and may become partially restricted upon target binding, hence causing a loss of entropy unfavourable to binding affinity. Therefore, only the flexible, intrinsically disordered N-terminal regions of the TβSR receptor ectodomains were employed as natural linkers in embodiments of the present invention. However, the particular amino acid compositions and lengths of these intrinsically disordered linkers (e.g., SEQ ID NO:36, 37, 70, 76, 82) precluded accurate prediction of whether the resulting direct-fusion constructs will have the required geometry and favourable molecular interactions for correct binding to their intended dimeric ligands.
The first and second portions of the polypeptide construct are, in embodiments, connected by natural intrinsically disordered polypeptide linkers selected from the group consisting of SEQ ID NO:36, 37, 38, 53, 70, 76, 82, and a sequence substantially identical thereto.
In embodiments, when multiple TβSR-ED structured regions are present, these can be fused directly or they can be connected by natural intrinsically disordered polypeptide linkers between the ectodomains, such as SEQ ID NO:40, 41, 42, 55, 58, 59, 60, 72, 78, 84, 87, and a sequence substantially identical thereto.
Non-limiting examples of full-length polypeptide constructs of the present invention that comprise the two aforementioned portions are selected from the group consisting of SEQ ID NO: 91-120, and a sequence substantially identical thereto.
It is particularly important to note that, in the polypeptide constructs of the present invention, the N-terminus of the second portion is linked to the C-terminus of the first portion (see for example
Some of the polypeptide constructs of the present invention display significantly greater potency of TGF-β neutralization compared to that of the TGF-β superfamily receptor ectodomain alone; for example, the polypeptide construct may be between at least 50-fold and 1×106-fold more potent. For example, the polypeptide constructs of the present invention may have a TGF-β neutralization potency selected from the group consisting of at least 50-, 75-, 100-, 150-, 200- 300-, 400-, 500-, 600, 1000- 1500-, 2000-, 3000-, 4000-, 5000-, 6000-, 7000-, 8000-, 9000, 10000-, 20000-, 30000-, 40000-, 50000-, 60000-, 70000-, 80000-, 90000-, 100000-, 150000-, 200000-, 250000-, 300000-, 350000-, 400000-, 450000-, 500000-, 550000-600000-, 650000-, 700000-, 750000-, 800000-, 850000-, 900000-, 950000, or 1000000-fold, more potent than the TβSR-ED alone, or any amount there between. In one example, the potency of the construct is at least 100-fold greater than the receptor ectodomain alone.
Additionally, when the polypeptide constructs of the present invention include a TβSR-ED that binds TGF-β, the polypeptide construct may neutralize, to varying extents, all three isotypes of TGF-β (that is, TGF-β1, TGF-β2, and TGF-β).
The polypeptide constructs of the present invention have, as assessed in cell-based assays, TGF-β neutralizing potencies that are significantly higher (100-fold or more) than those of bivalent comparator polypeptides, i.e. non-Fc-fused T22d35 and T2m-Fc. Within the series of polypeptide constructs of the present invention, those that contain two or more copies of the TβRII ectodomain fused to the C-terminus of the Fc constant region have potencies that are higher than those constructs that contain only one copy, as assessed in cell based assays. Additionally, within the series of polypeptide constructs of the present invention, if the first portion within the construct is “headless”, i.e. does not contain a Fab region, the potencies of the constructs are increased by engineering (optimizing) the number of cysteines in the hinge region at the “revealed” N-terminus. Engineering of the cysteine residues at the N-termini of “headless” constructs also markedly reduces the aggregation propensity of the constructs. Lastly, within the series of polypeptide constructs of the present invention, in vivo in tumor models, cysteine optimized “headless” constructs exhibit higher anti-tumor immuno-modulatory potencies than constructs in which the first portion is a full-sized antibody.
The polypeptide construct of the present invention is expressed as a single polypeptide chain. Once expressed, the polypeptide construct of the present invention forms a dimer wherein the CH2 and CH3 domains of the respective polypeptide constructs interact to form a properly assembled Fc region such as occurs when the expressed products are secreted into the culturing medium. For example, and without wishing to be limiting, examples of dimerized polypeptide constructs of the present invention are shown in
The first portion of the polypeptide construct described above may further comprise, at its N-terminus, any suitable antigen-binding antibody fragment known in the art. For example, and without wishing to be limiting in any manner, the first portion of the polypeptide construct may comprise CH2 and CH3 domains and one selected from the group consisting of a single-chain Fv (scFv; a molecule consisting of VL and VH connected with a peptide linker) and a single-domain antibody (sdAb, a fragment composed of a single VL or a single VH; see for example
The combination of constant domains and antigen-binding fragment may be naturally-occurring, or may be obtained by manipulation of a naturally-occurring antibody or by using recombinant methods. The polypeptide constructs such as those just described may require a sequence selected from the group consisting of linker sequences, disulfide bonds, hinge region sequences, and other type of covalent bond to link them to the CH2 and CH3 domains; those of skill in the art will be familiar with various suitable approaches.
In alternative constructs of the present invention, the polypeptide construct comprises an antibody Fc fragment linked at the C-terminus of each heavy chain to at least one TGF-β superfamily receptor ectodomain (TβSR-ED), as described above and as illustrated in
The antigen-binding antibody fragment described above, when present, may be directed to any suitable antigen. In certain limited embodiments, the antigen-binding antibody or fragment does not bind to an antigen that is PD-L1, EGFR1, her-2, CD4, CD6, CD20, CD25, MUC-1, IL-2, IL-6, or CTLA-4.
The present constructs can further comprise antibody or antibody fragments that target any antigen of interest. They can also comprise the antigen itself, or any other moiety of interest that is genetically encoded. Particular embodiments herein include the EGFR antibody cetuximab and its active fragments, Avastin, Herceptin, Synagis, FC5, or a poly-aspartate bone-localization motif, such a D10, or sequence substantially identical or equivalent thereto.
The present constructs can comprise a binding protein e.g., antibody and binding fragments thereof, that inhibits a checkpoint protein which may be CTLA-4, PD1, PDL1, PDL2, PDL3, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GALS, LAGS, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, CD28, CD86, or one of the B-7 family ligands or a combination thereof.
Illustrative immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-LI monoclonal Antibody (Anti-B7-HI; MED14736), MK-3475 (PD-1 blocker), Nivolumab (anti-PDI antibody), CT-011 (anti-PDI antibody), BY55 monoclonal antibody, AMP224 (anti-PDLI antibody), BMS-936559 (anti-PDLI antibody), MPLDL3280A (anti-PDLI antibody), MSB0010718C (anti-PDLI antibody) and Yervoy/ipilimumab (anti-CTLA-4 checkpoint inhibitor).
Other antibodies provided by the present constructs can include rituximab, muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, I-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101, volociximab, Anti-CD80 mAb, Anti-CD23 mAb, CAT-3888, CDP-791, eraptuzumab, MDX-010, MDX-060, MDX-070, matuzumab, CP-675,206, CAL, SGN-30, zanolimumab, adecatumumab, oregovomab, EGFR-binding antibodies cetuximab, nimotuzumab, necitumumab, panitumumab, matuzumab, and zalutumumab, as well as ABT-874, denosumab, AM 108, AMG 714, fontolizumab, daclizumab, golimumab, CNTO 1275, ocrelizumab, HuMax-CD20, belimumab, epratuzumab, MLN1202, visilizumab, tocilizumab, ocrerlizumab, certolizumab, eculizumab, pexelizumab, abciximab, ranibizimumab, mepolizumab, TNX-355, or MYO-029.
Still other antibodies that can be included in the present constructs are rituximab, zanolimumab, hA20, AME-133, HumaLYM, trastuzumab, pertuzumab, IMC-3G3, ch806, KSB-102, MR1-1, SC100, SC101, SC103, alemtuzumab, muromonab-CD3, OKT4A, ibritumomab, gemtuzumab, alefacept, abciximab, basiliximab, palivizumab, motavizumab, infliximab, adalimumab, CDP-571, etanercept, ABX-CBL, ABX-IL8, ABX-MA1 pemtumomab, Therex, AS1405, natalizumab, HuBC-1, natalizumab, IDEC-131, VLA-1; CAT-152; J695, CAT-192, CAT-213, BR3-Fc, LymphoStat-B, TRAIL-R1mAb, bevacizumab, ranibizumab, omalizumab, efalizumab, MLN-02, zanolimumab, HuMax-IL 15, HuMax-Inflam, HuMax-Cancer, HuMax-Lymphoma, HuMax-TAC, clenoliximab, lumiliximab, BEC2, IMC-1C11, DC101, labetuzumab, arcitumomab, epratuzumab, tacatuzumab, MyelomaCide, LkoCide, ProstaCide, ipilimumab, MDX-060, MDX-070, MDX-018, MDX-1106, MDX-1103, MDX-1333, MDX-214, MDX-1100, MDX-CD4, MDX-1388, MDX-066, MDX-1307, HGS-TR2J, FG-3019, BMS-66513, SGN-30, SGN-40, tocilizumab, CS-1008, IDM-1, golimumab, ONTO 1275, ONTO 95, ONTO 328, mepolizumab, MOR101, MOR102, MOR201, visilizumab, HuZAF, volocixmab, ING-1, MLN2201, daclizumab, HCD122, CDP860, PRO542, C14, oregovomab, edrecolomab, etaracizumab, siplizumab, lintuzumab, Hu1D10, Lym-1, efalizumab, ICM3, galiximab, eculizumab, pexelizumab, LDP-01, huA33, WX-G250, sibrotuzumab, Chimeric KW-2871, hu3S193, huLK26; bivatuzumab, ch14.18, 3F8, BC8, huHMFG1, MORAb-003, MORAb-004, MORAb-009, denosumab, PRO-140, 1D09C3, huMikbeta-1, NI-0401, NI-501, cantuzumab, HuN901, 8H9, chTNT-1/B, bavituximab, huJ591, HeFi-1, Pentacea, abagovomab, tositumomab, 105AD7, GMA161 and GMA321.
In other embodiments, the constant domain/first portion of the constructs can comprise a polypeptide having medicinal properties, such as agents that stimulate the immune system, in particular in relation to the ability of the immune system to attack tumor cells. These polypeptides can include cytokines (such as interleukin-2) or growth factors that stimulate immune cells directly or indirectly (i.e. act by providing gas to the immune system), as well as ectodomains or other binding agents that neutralize ligands which inhibit immune cells, either directly or indirectly (i.e. act by releasing a brake on the immune system).
In other embodiments, the constant domain/first portion of the constructs can comprise a polypeptide that does not have active medicinal properties pe se, but rather provides a localization signal. This localization motif will serve to focus the intrinsic TGF-β neutralization activity of the second portion of the construct to a particular region of the body. In one example, the first portion comprised a long poly-aspartate bone-localization motif, preferably D10 or an equivalent bone-localization moiety, which acts to enhance localisation of the construct to bone. By increasing the TGF-β neutralization activity of the construct within bone, more favourable dosing levels and schedules may be required for the treatment of bone-related diseases, such as osteogenesis imperfecta, relative to that required for a similar construct without the D10 motif.
Embodiments exemplifying polypeptide constructs of the present invention that include antigen-binding fragments at the N-terminus of the Fc region (first portion) are selected from the group consisting of SEQ ID NO: 121-131, and a sequence substantially identical thereto.
In other embodiments, polypeptide constructs of the present invention that include other targeting agents, e.g., a poly-aspartate bone-localization motif, at the N-terminus of the Fc region (first portion), are exemplified by SEQ ID NO: 136-150.
The polypeptide construct of the present invention may also comprise additional sequences to aid in expression, detection or purification of a recombinant antibody or fragment thereof. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or fragment thereof may comprise a targeting or signal sequence (for example, but not limited to ompA), a detection/purification tag (for example, but not limited to c-Myc, His5, His6, or His8G), or a combination thereof. In another example, the signal peptide may be MVLQTQVFISLLLWISGAYG (SEQ ID NO:89) or MDWTWRILFLVAAATGTHA (SEQ ID NO:90). In a further example, the additional sequence may be a biotin recognition site such as that described in [WO/1995/04069] or in [WO/2004/076670]. As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags, or may serve as a detection/purification tag.
The present invention also encompasses nucleic acid sequences encoding the molecules as described herein. Given the degeneracy of the genetic code, a number of nucleotide sequences would have the effect of encoding the desired polypeptide, as would be readily understood by a skilled artisan. The nucleic acid sequence may be codon-optimized for expression in various micro-organisms. The present invention also encompasses vectors comprising the nucleic acids as just described, wherein the vectors typically comprise a promoter and signal sequence that are operably linked to the construct-encoding polynucleotide for driving expression thereof in the selected cellular production host. The vectors can be the same or different provided both result in secretion of the dimeric polypeptide construct.
Furthermore, the invention encompasses cells, also referred to herein as transgenic cellular host, comprising the nucleic acid and/or vector as described, encoding a first polypeptide construct. The host cells may comprise a second nucleic acid and/or vector encoding a second polypeptide construct different from the first polypeptide construct. The co-expression of the first and second polypeptide constructs may lead to the formation of heterodimers.
The present invention also encompasses a composition comprising one or more than one polypeptide construct as described herein. The composition may comprise a single polypeptide construct as described above, or may be a mixture of polypeptide constructs. The composition may also comprise one or more than one polypeptide construct of the present invention linked to one or more than one cargo molecule. For example, and without wishing to be limiting in any manner, the composition may comprise one or more than one polypeptide construct of the present invention linked to a cytotoxic drug in order to generate an antibody-drug conjugate (ADC) in accordance with the present invention.
The composition may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with other ingredients in the composition, with the method of delivery of the composition, and is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form, powder form (for example, but limited to lyophilised or encapsulated), capsule or tablet form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, a suitable buffer, or additives to improve solubility and/or stability; reconstitution to produce the suspension is effected in a buffer at a suitable pH to ensure the viability of the antibody or fragment thereof. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be so formulated as to deliver the antibody or fragment thereof to the gastrointestinal tract of the subject. Thus, the composition may comprise encapsulation, time-release, or other suitable technologies for delivery of the antibody or fragment thereof. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present compounds.
The constructs of the present invention may be used to treat diseases or disorders associated with over-expression or over-activation of ligands of the TGF-β superfamily. The disease or disorder can be selected from, but not limited to, cancer, ocular diseases, fibrotic diseases, or genetic disorders of connective tissue.
In the field of cancer therapy, it has recently been demonstrated that TGF-β is a key factor inhibiting the antitumor response elicited by immunotherapies, such as immune checkpoint inhibitors (ICI's) (Hahn & Akporiaye, 2006). Specifically, therapeutic response to ICI antibodies results primarily from the re-activation of tumor-localized T-cells. Resistance to ICI antibodies is attributed to the presence of immunosuppressive mechanisms that result in a dearth of T-cells in the tumor microenvironment. Thus, it is now recognized that in order to elicit responses in resistant patients, ICI antibodies need to be combined with agents that can activate T-cells and induce their recruitment into the tumor, i.e. reversing of the “non-T-cell-inflamed” tumor phenotype. One publication noted that overcoming the non-T-cell-inflamed tumor microenvironment is the most significant next hurdle in immuno-oncology (Gajewski, 2015).
We have shown using a proof-of-principle TGF-β trap, T22d35, that blocking of TGF-β effectively reverses the “non-T cell inflamed” tumor phenotype (Zwaagstra et al, 2012). This positions anti-TGF-β molecules as potential synergistic combinations with ICI's and other immunotherapeutics. In support of this, a 2014 study (Holtzhausen et al., ASCO poster presentation) examined effects of a TGF-β blocker when combined an anti-CTLA-4 antibody in a physiologically-relevant transgenic melanoma model. The study demonstrated that while anti-CTLA-4 antibody monotherapy failed to suppress melanoma progression, the combination of the TGF-β antagonist and anti-CTLA-4 antibody significantly and synergistically suppressed both primary melanoma tumor growth as well as melanoma metastasis. These observations correlated with significant increases in effector T-cells in melanoma tissues.
Fibrotic diseases include those that affect any organ of the body, including, but not limited to kidney, lung, liver, heart, skin and eye. These diseases include, but are not limited to, chronic obstructive pulmonary disease (COPD), glomerulonephritis, liver fibrosis, post-infarction cardiac fibrosis, restenosis, systemic sclerosis, ocular surgery-induced fibrosis, and scarring.
Genetic disorders of connective tissue include, but are not limited to, Marfan syndrome (MFS) and Osteogenesis imperfecta (OI).
The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.
Several fusion molecules comprising full-size antibody (FSA), VHH-IgG Fc, D10-Fc or “headless” Fc C-terminally-fused to the T22d35 or T2m ectodomains were constructed (Table 1). All constructs comprising a heavy chain included the signal sequence MDWTWRILFLVAAATGTHA (SEQ ID NO:89) at the N-terminus, while constructs comprising a light chain included the signal sequence MVLQTQVFISLLLWISGAYG (SEQ ID NO:90) at the N-terminus. The DNA coding for constructs were prepared synthetically (Biobasic Inc. or Genescript USA Inc.). Constructs comprising FSA, D10-Fc and “headless” Fc were cloned into the EcoR1 (5′ end) and BamH1 (3′ end) sites and those comprising VHH-IgG Fc were cloned into the HindIII (5′ end) and BamH1 (3′ end) sites of the pTT5 mammalian expression plasmid vector (Durocher et al, 2002).
The Cet-T2m and Cet-T22d35 constructs were produced by transient co-transfection of Chinese Hamster Ovary (CHO) cells with the heavy chain (HC)-T2m or (HC)-T22d35 construct combined with the Cetuximab light chain (LC) construct which then assembled as the Cetuximab-T22d35 (Cet-T22d35) or Cetuximab-T2m (Cet-T2m) fusion molecules. Briefly, CetHC-T22d35 (SEQ ID NO:122) and CetLC (SEQ ID NO:121) plasmid DNAs (ratio=3:2) were co-transfected into a 10 L Wavebag culture of CHO-3E7 cells in FreeStyle F17 medium (Invitrogen) containing 4 mM glutamine and 0.1% Kolliphor p-188 (Sigma) in a Wavebag maintained at 37° C. Transfection conditions were: DNA (50% HC+LC plasmids, 30% ssDNA, 15% AKT plasmid, 5% GFP plasmid): PEI(polyethylenimine)pro (Polyplus) (ratio=1:2.5). At 24 hours post-transfection, 10% Tryptone N1feed (TekniScience Inc.) and 0.5 mM Vaporic acid (VPA, Sigma) were added and the temperature was shifted to 32° C. to promote the production and secretion of the fusion proteins and maintained for 15 days post transfection after which the cells were harvested. At final harvest the cell viability was 89.6%. The harvest supernatant (10.8 L) was filtered (0.2 μm) and loaded onto a 55 mL Protein A MabSelect Sure 55 mL column (GE Healthcare). The column was washed with 2 column volumes of PBS and protein was eluted with 3 column volumes of 0.1 M sodium citrate pH 3.6. To maximize the yield, the flow through was reloaded onto the Protein A column and eluted as described above. Eluted fractions were neutralized with 1 M Tris, evaluated by SDS-PAGE and those containing Cet-T22d35 were pooled (
Similar transfection, production and purification methods were performed for the other FSA-trap examples listed in Table 1. In the case of the VHH-Fc IgG-, D10 and “headless” Fc-fusion molecules the composition of the transfection mixture was modified as follows: DNA (80% plasmid construct, 15% AKT plasmid, 5% GFP plasmid): PEIpro (ratio 1:2.5).
The integrity of the pooled Prot-A and SEC fractions of Cet-T22d35 protein was analyzed by SDS-PAGE (4-15% polyacrylamide) under reducing and non-reducing conditions (
Similar methods were used to analyse expression levels, purifiability, aggregation levels, and dimeric assembly of several other Fc-ectodomain constructs. The results from these studies are shown in
The TGF-β neutralization potencies of purified Fc-ectodomain fusion constructs were determined and compared to those of non-Fc-fused T22d35. It should be noted that non-Fc-fused T2m does not neutralize any of TGF-β1, -β2, or -β3 (De Crescenzo et al, 2001).
TGF-β neutralization potencies for TGF-β1, -β2 and -β3 were determined for purified fusion constructs using two cell-based signaling assays: 1) the Mv1 Lu cell luciferase reporter assay with Mv1 Lu cells having a PAI-1-luciferase reporter (as described in (Zwaagstra et al, 2012)) and an A549 cell/IL-11 release assay adapted to the MSD (Meso Scale Discovery) platform.
Mv1Lu cell luciferase reporter assay: Briefly, cells were seeded onto 96-well plates (20,000 cells/well) and then treated with T2m, T22d35, or a fusion construct+25 pM TGF-β at 37° C. for 16 h in DM EM, 1% FBS, 0.1% BSA. Cells were then lysed and luciferase activity was measured (Promega Corp.) using a Synergy 2 plate reader (BioTek Instruments Inc.).
A549 cell IL-11 release assay: Human A549 lung cancer cells (ATCC-CCL-185, Cedarlane Burlington ON) were seeded in 96-well plates (5×103 cells/well). The following day 10 pM TGF-β in complete media in the absence or presence of a serial dilution of fusion protein was incubated for 30 min at RT prior to adding to the cells. After 21 h of incubation (37° C., 5% CO2, humidified atmosphere) conditioned medium was harvested and added to MSD Streptavidin Gold plates (Meso Scale Diagnostics, Gaithersburg, Md.) that were coated with 2 μg/mL biotinylated mouse anti-human IL-11 antibody (MAB618, R&D Systems, Minneapolis, Minn.). After 18 h (4° C.) plates were washed with PBS containing 0.02% Tween 20, a 2 μg/mL SULFO-tagged goat anti-human IL-11 antibody (AF-218-NA, R&D Systems Minneapolis, Minn.) was added and plates were incubated for 1 h at RT. After a final wash, plates were read in a MESO QuickPlex SQ120 machine (Meso Scale Diagnostics, Gaithersburg, Md.). IL-11 readouts were expressed as percent IL-11 release compared to control cells treated with TGF-β alone.
In one set of experiments, using the Mv1Lu cell reporter assay, the neutralization potency of Cet-T2m (construct Type F in
In another set of experiments using the Mv1 Lu cell reporter assay, similar extremely high potencies of TGF-β neutralization were observed for other C-terminus Fc fusion constructs (Table 2), e.g. for constructs in which T22d35 or T2m were fused with FSAs such as Herceptin (Her-T22d35), Avastin (Ava-T22d35) or Synagis (Syn-T22d35) [Type F and D constructs in
In order to confirm the relative potencies of Fc-ectodomain constructs, a second cell-based assay, an A549 cell IL-11 release assay, was used. This IL-11 release assay acts as a model of TGF-β-mediated biological responses that contribute to both tumor metastasis and fibrosis. In the set of experiments shown in
An additional set of experiments in which the A549 cell IL-11 release assay was used to compare TGF-β neutralization potencies is shown in
We have also compared the potencies of constructs that include three TβRII structured ectodomains with constructs carrying two ectodomains using the A549 cell IL-11 release assay. We observed that the triple-repeat based constructs (SEQ ID NO:111 and SEQ ID NO:116) are potent in neutralizing TGF-β1 in this assay, and typically have improved IC50 values relative to the corresponding double-repeat based constructs (SEQ ID NO:100 and
SEQ ID NO:106, respectively). All constructs involved in this comparative study had the same engineered N-terminus of the Fc portion, hIgG1FcΔK(C).
Binding to TGF-β: Binding of T22d35, Cet-T22d35, and Cet-T2m to TGF-β2 was measured using a competitive SPR binding experiment. In this assay, the molecule of interest was first allowed to bind to a fixed amount of TGF-β in solution. A 2-fold dilution series was prepared in PBS-0.05% Tween, starting with 1000 nM T22d35 trap or 20 nM Cet-T22d35 or Cet-T2m. Each diluted sample was pre-incubated with 1 nM TGF-β2 for 30 min at room temperature to allow binding. The mixture was then flowed over immobilized, pan-specific anti-TGF-β antibody 1D11 (2000 RU 1D11) in order to quantify the amount of ligand left unbound (TβRII ectodomain and 1D11 bind to a similar epitope on TGF-β) using a Biacore T200 instrument. The TGF-β2 binding EC50 values were determined by plotting the percent free TGF-β versus the protein concentration of the molecule of interest. Binding curves and EC50 values are shown in
The ability of an antibody alone or in a fusion construct of Example 1 to bind to its intended target antigen was evaluated using surface plasmon resonance (SPR).
Direct binding of Cet-T22d35 or Cetuximab to the EGF receptor extracellular domain (EGFR-ED) was quantified by SPR using a BIAcore T200 instrument, performed in the standard manner. Briefly, Cet-T22d35 or Cetuximab alone were captured on the SPR CM5 chip using immobilized anti-human IgG Fc-specific antibody (2000 RU). Variable concentrations of EGFR-ED in PBS-0.05% Tween were then flowed over the capture surface at 100 μl/min and 25° C. The resulting sensorgrams (data not shown) were analyzed using the Biacore T200 evaluation software. The KD values of Cet-T22d35 and Cetuximab were very similar (847 and 708 pM, respectively), indicating that fusion of T22d35 to the Fc portion of Cetuximab does not appreciably alter binding to EGFR-ED, compared to the non-fused FSA. Similar SPR methods and analyses were performed for other antibody-trap fusion examples, compared with their corresponding target antigens (see Table 4).
From Table 4 it is evident for each exemplified construct that fusion of a TGF-β superfamily receptor ectodomain(s) to the C-terminus of the Fc region of an antibody did not significantly alter antigen-binding affinities and KD values of the antibody. This indicates that the ectodomain(s) can be readily fused to any antibody without compromising the ability the antibody to bind its target antigen.
The ability of Cetuximab to maintain its therapeutic function (i.e. inhibition of EGF-induced EGFR phosphorylation and signaling) when fused to either T2m or T22d35 (Example 1) was evaluated.
Phosphorylation of EGFR: The ability of Cetuximab-comprising constructs to inhibit EGF-induced phosphorylation of EGFR in human lung cancer A549 cells was evaluated. A549 cells were seeded in 24-well plates (100,000 cells/well) and either mock treated (−) or pre-treated with Cetuximab, Cet-T2m, or Cet-T22d35 (all at 10, 1 or 0.1 nM) or T22d35 (10 nM) at 37° C. for 3 h, then treated with 50 ng/mL EGF at 37° C. for 10 min. Whole cell lysates were prepared and resolved by SDS-PAGE, western blotted and probed with anti-phosphoTyrosine antibody (Clone 4G10, Millipore 05-321). As shown in
Inhibition of EGFR signaling: Inhibition of autocrine EGFR signaling results in varying degrees of cytotoxicity in EGFR-expressing cells treated with Cetuximab. Cytotoxicity of Cet-T22d35 was compared to Cetuximab and T22d35 in human breast cancer cells (MDA-MB-468) and immortalized keratinocyte cells (HaCaT). These cells exhibited significant Cetuximab cytotoxicity, due to their intrinsic dependence on the EGF signaling pathway for basal growth.
The cells were seeded onto 96-well plates (MDA-MB-468, 2300 cells/well; HaCaT, 1500 cells/well) and then treated with different doses of inhibitor at 37° C. for 5 days. Cell viability was measured using sulforhodamine reagent to determine the percentage of viable cells relative to mock-treated controls. Results are shown in
The ability of FC5 VHH to maintain its function (i.e. transmigrate the blood-brain barrier) when fused with the T2m or T22d35 moieties (see description in Example 2 and activities in Table 2) was evaluated.
Briefly, SV40-immortalized Adult Rat Brain Endothelial Cells (Sv-ARBEC) were used to generate an in vitro blood-brain barrier (BBB) model as described (Garberg et al, 2005; Haqqani et al, 2013). Sv-ARBEC cells (80,000 cells/membrane) were seeded on 0.1 mg/mL rat tail collagen type I-coated tissue culture inserts (pore size-1 μm; surface area 0.9 cm2, Falcon) in 1 ml of growth medium. The bottom chamber of the insert assembly contained 2 ml of growth medium supplemented with the immortalized neonatal rat astrocytes-conditioned medium in a 1:1 (v/v) ratio. Equimolar amounts (5.6 μM) of positive (FC5-Fc) control; negative controls (A20.1); and T22d35, T2m, FC5-Fc-T22d35 or FC5-Fc-T2m were tested for their ability to cross the Sv-ARBEC cell monolayer. Following exposure of equimolar amounts of the proteins to the luminal side of the BBB, samples were taken after 15, 30, and 60 min from the abluminal side. The protein content of each sample was then quantified by mass spectrometry (multiple reaction monitoring-isotype labeled internal standards; MRM-ILIS) as described by (Haqqani et al, 2013) (see method description below).
Quantified values can be directly plotted or the Papp (apparent permeability coefficient) values can be determined using the following formula
The Papp value is commonly used to determine the specific permeability of a molecule, and is a measure of transport across the brain endothelial monolayer. [Qr/dt=cumulative amount in the receiver (bottom) compartment versus time; A=area of the cell monolayer; C0=initial concentration of the dosing solution (top chamber)].
Treatment of A549 cells with EGF plus TGF-β results in a strong epithelial to mesenchymal transition (EMT). The EMT is phenotypically characterized by changes in cell morphology (tight cellular junctions with “cobble-stone” appearance converts to elongated cells, see
Briefly, for the western blot analysis, A549 cells were seeded in 24-well plates (8000 cells/well) and then treated with EGF (50 ng/mL)+TGF-β1 (50 pM) at 37° C. for 3 days in the presence of Cet-T22d35, Cetuximab, or T22d35 (0, 0.05, 0.5, 5, 50, or 500 nM). Whole cell lysates were prepared and resolved by SDS-PAGE. The proteins were transferred to nitrocellulose and then probed with an E-cadherin antibody (BD Transduction laboratories Biosciences) (
The ability of Cet-T22d35, Cetuximab and T22d35 to block the EGF+TGF-β EMT response was further examined by flow cytometry using A549 cells treated with Cet-T22d35 or Cetuximab, (all at 50, 5, 0.5 nM) or the Cetuximab+T22d35 combination (50 nM+100 nM, 5 nM+10 nM, or 0.5 nM+1 nM, respectively) and evaluating the EMT associated changes in E-cadherin and N-cadherin cell surface expression levels (
Dissociation Buffer (Sigma)/well, centrifuged at 2000 rpm for 2 min and re-suspended in 100 μl RPMI-5 media at 4° C. AlexaFluor488-E-cadherin (Santa Cruz, SC21791) and AlexaFluor647-N-cadherin (BD Biosciences, 563434) antibodies ( 1/25 v/v dilutions) were added and samples were incubated at 4° C. for 1 h. Cells were then centrifuged, washed once in RPMI-5, and re-suspended in 400 μl RPMI-5 containing 15 μg/mL propidium iodide (Life Technologies) at 4° C. Mean fluorescent intensities (MFI) were measured by flow cytometry (BD LS RII flow cytometer, BD Biosciences) to quantify E-cadherin and N-cadherin levels. The results show that Cet-T22d35 was more effective in preventing down-regulation of E-cadherin (
PK studies were carried out in normal, healthy mice to determine whether fusion of T22d35 to an antibody increased its half-life in vivo, and whether removal of a lysine at the fusion site within constructs reduced the amount of cleavage occurring in vivo.
Results from Cet-T22d35 (construct Type D in
Results from several constructs with lysine deleted at the fusion site:
The same methods that were used in the PK study of Cet-T22d35 (
A FSA-T2m construct (Cet-T2m—Type F in
The protocols used for these syngeneic mouse model studies are described in (Zheng et al, 2013). Briefly, female BALB/c (H-2Kd) mice 6 weeks of age were purchased from The Jackson Laboratories and kept in filter-top cages. The 4T1 breast cancer cells and B16F10 cells were purchased from American type culture collection and cultured in RPMI-1640 supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 microgm/L streptomycin, 50 micromol/L 2-mercaptoethanol, and 10% fetal calf serum. Mice were inoculated subcutaneously in the left flank with 100 uL sterile saline containing 5×104 4T1 cells. Tumors were grown to -100 mm3, as measured by caliper, then mice were randomized and divided into the six treatment groups (8 animals/group) (Day 0). Treatments commenced on Day 1 and continued for 15 days with the animals being dosed at 5 mg/kg twice per week such that they received a total of 4 doses. Tumor growth was monitored by caliper measurements 3× per week. Animals were euthanized by exsanguination under anaesthesia on Day 15; T cells were isolated from draining lymph nodes and assessed for their capacity to kill mouse 4T1 and B16F10 tumour cells ex vivo. The capacity of T cells from mice treated with or without test agents to lyse target 4T1 tumor cells was measured using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega) according to the manufacturer's instructions. Briefly, naïve target 4T1 cells or melanoma cell line B16F10 cells are plated and incubated for 4 hr with CD8+ effector T cells isolated from 4T1 tumor-bearing mice using CD8 magnetic MicroBeads (BD Bioscience). The isolated CD8+ cells are confirmed, by flow cytometry, to be over 85% CD8+. A range of ratios of effectors to target cells is tested (100:1, 50:1, 25:1). Lactate dehydrogenase (LDH) release in response to effector T cells is measured in the buffer bathing target cells. Target cells incubated in the absence of effector cells are used as a comparator to control for spontaneous LDH release. Released LDH in culture supernatants is detected after a 30-min incubation using a coupled enzymatic assay. The intensity of the color formed is proportional to the number of lysed cells. Cytotoxic activity of CTL is calculated using the following formula:
Cytotoxic activity %=[(absorbance)−(spontaneous effector cell LDH release)−(spontaneous target cell LDH release)]/[(maximal LDH release)−(spontaneous target cell LDH release)]×100
The results presented in
To investigate whether these Fc-ectodomain fusions exhibit an immuno-modulatory effect in vivo on cytotoxic T lymphocyte cells (CTLs) present in tumor draining lymph nodes, lymph nodes were removed from mice treated with or without test agents; T-cells were then isolated and tested for their capacity to lyse target 4T1 tumor cells (and B16F10 cells as a test of tumor specificity) using the methods described above.
As shown in
Interestingly, with respect to the Fc-ectodomain fusions of this invention, the most potent constructs were the hIgG1FcΔK(CC)-T2m and hIgG2FcΔK(CC)-T2m constructs; both of these constructs containing two cysteines in the engineered hinge region. These constructs were more potent than the construct with one cysteine in the engineered hinge region, hIgG1FcΔK(C)-T2m, as well as being more potent than the full-size antibody-T2m construct, Cet-T2m. The difference in potency between constructs with one versus two hinge region cysteines may result from the construct with one cysteine having a lower relative stability. The lower potency of the full size antibody construct relative to the headless constructs with two hinge region cysteines may result from a difference in molecular weight, with the smaller constructs being able to penetrate the tumor microenvironment more effectively.
In vivo studies were carried out to investigate whether the addition of a 10 amino acid long poly-aspartate bone-localization motif (D10) to the N-terminus of the Fc region of constructs will promote their localization to bone. Optical imaging of D10-hIgG1 Fc-T2m fusions: Upon arrival, male Balb/c mice were housed 3 mice/cage. On the day of the experiment, animals were shaved dorsal and ventral and treated with the hair removal cream, NAIR®. Mice were injected with a single intravenous bolus of 10 mg/kg of two CF770 labeled constructs with a deca-aspartate motif for bone targeting (D10) at their N-termini (D10-hIgG1FcΔK(CC)-T2m, D10-GSL-hIgG1FcΔK(CC)-T2m) or with a control construct without the D10 motif (hIgG1FcΔK(CC)-T2m) and whole body bio-distribution followed using both in vivo and ex-vivo near infrared imaging. Imaging was conducted with a small-animal time-domain eXplore Optix pre-clinical imager MX3 (Advanced Research Technologies, ART) at various time points (prescan, 5 mins, 3 hr, 6 hr, 24 h, 48 h, 72 h, 96 h and 120 h).
The small animal time-domain eXplore Optix preclinical imagers consists of a 785-nm pulsed laser diode with a repetition frequency of 80 MHz and a time resolution of 12.5 ps light pulse was used for excitation. The fluorescence emission beyond 813 nm was collected by a highly sensitive time-correlated single photon counting system and detected through a fast photomultiplier tube. The data were recorded as temporal point-spread functions (TPSF) and the images were presented as fluorescence intensity maps using ART Optix Optiview analysis software 3.02.
For in vivo optical imaging, mice were first anesthetized using isofluorane (1.5-2%), positioned on the animal stage within a chamber which allows for gaseous anesthesia and maintenance of animal temperature at 36° C. The scanning of the mouse at each time point lasted up to 20 mins using a 2.5 mm step size and the mouse is placed back in its home cage between imaging time points.
At the end of the imaging protocol (120 hrs) animals were sacrificed by intracardiac perfusion using heparnized saline with deep anesthesia. The organs (brain, heart, lungs, liver, kidney, spleen and right and left leg bones) were imaged ex-vivo using a 1.0 mm step size using the eXplore Optix pre-clincal imager MX3.
Data analysis was done using eXplore Optix Optiview analysis software 3.02 (Advanced Research Technologies, Montreal, QC) to estimate the fluorescence total and average fluorescence intensity in region of interest containing the ex-vivo organs.
The results shown in
The results shown in
The embodiments and examples described herein are illustrative and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments, including alternatives, modifications and equivalents, are intended by the inventors to be encompassed by the claims. Furthermore, the discussed combination of features might not be necessary for the inventive solution.
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U.S. Pat. No. 8,815,247
U.S. 62/777,375
US2015/0225483
This application is a continuation of U.S. application Ser. No. 15/755,595, filed Feb. 27, 2018, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/IB2016/055204, filed Aug. 31, 2016, and claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application Ser. No. 62/212,058, filed Aug. 31, 2015, the entire contents of each of which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62212058 | Aug 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15755595 | Feb 2018 | US |
| Child | 17523602 | US |
