Patent Application
20220281971
This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:
Cancer immunotherapy broadly relates to directing immune responses to selectively attack tumor cells. The immunotherapeutic toolbox to treat cancer has been significantly enriched by the advent of chimeric antigen receptor (CAR)-directed T lymphocytes. The clinical experience with CAR-T cells demonstrates that T-lymphocytes, when adequately activated, can overcome resistance to chemotherapy, leading to major reduction in tumor burden, disease stabilization and, in some patients with B-cell leukemia and lymphoma, tumor eradication.18-26 In CAR-T cells, T cell stimulation occurs via the expression of chimeric molecules with antibody-like properties.
Current CAR T-cell methodologies do not harness the full potential of the immune system to target cancer cells.
Described herein is a peptide that includes a single-chain variable fragment (scFv) domain; a fragment crystallizable (Fc) domain; and a hinge domain joining the scFv and Fc domains. Also described are nucleic acids encoding the peptides described herein; vectors that include the nucleic acids, which encode the peptide described herein; immune cells (e.g., natural killer cells and T cells) that express the peptides described herein; and methods of making immune cells that express the peptides described herein.
The scFv domain can include an immunoglobulin variable light (VL) domain, an immunoglobulin variable heavy (VH) domain, and a linker domain joining the VL and VH domains. The linker domain can be (G4S)x, wherein x is an integer from 1 to 100. The linker domain can be (G4S)3.
The scFv domain can bind CD19, CD20, CD22, CD38, CD7, CD2, CD3, epidermal growth factor receptor (EGFR), CD123, CD33, B-cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD2), PD-L1 (CD274), CD80 or CD86.
The Fc domain can include an immunoglobulin constant heavy 2 (CH2) domain and an immunoglobulin constant heavy 3 (CH3) domain. The Fc domain can be human IgG1 Fc domain.
The peptide can further include a signal peptide that is N-terminal to the scFv domain.
The peptide can further include a self-cleaving peptide joining the Fc domain to a chimeric receptor, wherein the chimeric receptor includes: a receptor domain; a hinge and transmembrane domain; a co-stimulatory signaling domain; and a cytoplasmic signaling domain.
The self-cleaving peptide can be a 2A peptide. The receptor domain can be CD16. The hinge and transmembrane domain can be a CD8α hinge and transmembrane domain. The co-stimulatory domain can be 4-1BB co-stimulatory domain. The cytoplasmic signaling domain can be a CD3ζ cytoplasmic signaling. The chimeric receptor can be CD16V-4-1BB-CD3ζ.
In one particular embodiment, the scFv domain binds CD19 or CD20; the Fc domain is a human IgG1 Fc domain; and the hinge domain is an IgG1 hinge domain; the vector further includes a CD8α signal peptide that is N-terminal to the scFv domain; and the vector further includes a chimeric receptor that is CD16V-4-1BB-CD3ζ.
The vector can be a murine stem cell virus (MSCV).
The peptide can further include IL-15 joined to the Fc domain by a linker. The linker that joins IL-15 to the Fc domain is selected from the group consisting of SEQ ID NO: 51; A(EAAK)4ALEA(EAAAK)4A; (EAAAK)z; A(EAAAK)zA; and (XP)w, wherein z is an integer from 1 to 100; X is any amino acid, and w is an integer from 1 to 100.
Described herein is a peptide that includes a T-cell receptor (TCR) β domain; a first fragment crystallizable (Fc) domain joined to the TCR β domain; a TCR a domain; a self-cleaving peptide joining the Fc domain to the TCR α domain; and a second Fc domain joined to the TCR α domain. The peptide can further include a signal peptide joined to the T-cell receptor (TCR) β domain. The first Fc domain can be the same as the second Fc domain. The first Fc domain can be different from the second Fc domain. Also described are nucleic acids encoding the peptide and vectors that include the nucleic acid, which encodes the peptide.
Advantageously, the peptides described herein can be secreted by immune cells, such as T cells and NK cells. As a result, the immune cells can target and kill tumor cells without the need for exogenous administration of antibodies. NK cells can exert antibody-dependent cell cytotoxicity when the secreted peptides bind Fc receptors on the NK cell surface. T cells transduced with an Fc receptor can also exert antibody-dependent cell cytotoxicity. Moreover, the peptides can trigger phagocytosis of tumor cells by macrophages through interaction of Fc receptors on their cell surface. Finally, the peptides can kill tumor cells by inducing complement fixation.
The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.
A description of example embodiments follows.
Monoclonal antibodies are integral to the contemporary treatment of cancer. Antibodies exert anti-tumor activity via several mechanisms including direct induction of cell death, complement activation, and engagement of immune cells. Antibodies bound to tumor cells can trigger antibody-dependent cell cytotoxicity (ADCC).1-6 ADCC, which results from the engagement of Fc receptors (FcγR) expressed on the surface of natural killer (NK) cells,7 is central to the clinical efficacy of antibodies; polymorphisms of the gene coding FcγRIIIa (FCRG3A or CD16) leading to receptors with higher affinity for Fc have been associated with better tumor responses in patients.2,8-16 Other important mechanisms underlying the anti-tumor activity of antibodies include clearance of tumor cells by macrophages through antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).7,17
The immunotherapeutic toolbox to treat cancer has been significantly enriched by the advent of chimeric antigen receptor (CAR)-directed T lymphocytes. The clinical experience with CAR-T cells demonstrates that T-lymphocytes, when adequately activated, can overcome resistance to chemotherapy, leading to major reduction in tumor burden, disease stabilization and, in some patients with B-cell leukemia and lymphoma, tumor eradication.18-26 In CAR-T cells, T cell stimulation occurs via the expression of chimeric molecules with antibody-like properties.27-31 Another approach leading to tumor-specific T cell activation is through the expression of high-affinity CD16 as a component of a chimeric receptor including both stimulatory and co-stimulatory signals.32 Such receptor has the potential to significantly augment the anti-tumor effect of antibody therapy. Compared to CAR-T cells, it works in combination with other antibody-mediated mechanism, such as ADCP and CDC, resulting in a concerted anti-tumor effect. Moreover, by using multiple antibodies against weakly expressed antigens, vigorous T-cell responses can be elicited.
Described herein are methods that allow immune cells to produce binders with antibody-like function. These in vivo functional ligands (IFLs) are capable of triggering ADCC, ADCP and CDC, as well as cytokine stimulation. These can be expressed in NK cells and T cells, and in conjunction with CD16 chimeric receptors, to optimize effector functions.
While the particular examples described herein target CD20+ and CD19+ B-cells as a paradigm, the approach is applicable to targeting other antigens that are markers of cells in the pathogenesis of cancer and other diseases.
B-cell non-Hodgkin lymphoma (NHL) is a cancer of lymphoid blood cells. NHL inevitably progresses and is fatal if untreated. Standard treatment includes chemotherapy, antibody therapy, tyrosine kinase inhibitor therapy, and hematopoietic stem cell transplant. CD20 and CD19 are B-cell—specific antigens that are widely expressed in B-cell NHL (also referred to as B-NHL).
The vectors described herein can be used to generate modified T cells, which, in turn, can be used for targeted treatment of NHL. The processes described herein can be used to create transgenic T cells that can target CD20+ and CD19+ B-cells for destruction, thereby eradicating NHL and/or decreasing its severity.
Acute lymphoblastic leukemia (ALL) is also a cancer of lymphoid blood cells. ALL progresses rapidly and is fatal if untreated. Standard treatment includes chemotherapy and hematopoietic stem cell transplant. CD19 is a B-cell—specific antigen that is expressed on all leukemic cells in the majority of cases of ALL.
The vectors described herein can be used to generate modified T cells, which, in turn, can be used for targeted treatment of ALL. The processes described herein can be used to create transgenic T cells that can target CD19+ B-cells for destruction, thereby eradicating ALL and/or decreasing its severity.
As used herein, the term “nucleic acid” refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). “Nucleic acid” includes, for example, DNA (e.g., genomic DNA and cDNA), RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In certain embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, “nucleic acid” can refer to either or both strands of the molecule.
The terms “nucleotide” and “nucleotide monomer” refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.
As used herein, the term “sequence identity,” refers to the extent to which two nucleotide sequences, or two amino acid sequences, have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which a test sequences are compared. The sequence identity between reference and test sequences is expressed as the percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the entire length of the reference sequence.
Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, the alignment can include introduced gaps to provide for the maximal level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology).
When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul et al., J Mol Biol. 215(3):403-10 (1990)).
In various embodiments, two nucleotide sequences, or two amino acid sequences, can have at least, e.g., 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity. When ascertaining percent sequence identity to one or more sequences described herein, the sequences described herein are the reference sequences.
The terms “vector”, “vector construct” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter. Gene delivery vectors generally include a transgene (e.g., nucleic acid encoding an enzyme) operably linked to a promoter and other nucleic acid elements required for expression of the transgene in the host cells into which the vector is introduced. Suitable promoters for gene expression and delivery constructs are known in the art. Recombinant plasmids can also comprise inducible, or regulatable, promoters for expression of an enzyme in cells.
Various gene delivery vehicles are known in the art and include both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Such viral vectors include, e.g., vector derived from the herpes virus, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector (AAV), and murine stem cell virus (MSCV). The viral vector can be replicating or non-replicating. Such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
Non-viral vectors for gene delivery include naked DNA, plasmids, transposons, and mRNA, among others. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), pMAL plasmids (New England Biolabs, Beverly, Mass.). Such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
In certain embodiments, the vector comprises an internal ribosome entry site (IRES). In some embodiments, the vector includes a selection marker, such as an ampicillin resistance gene (Amp). In some embodiments, the nucleic acid encodes a fluorescent protein, such as green fluorescent protein (GFP) or mCherry. In some embodiments, the nucleic acid is suitable for subcloning into pMSCV-IRES-GFP between EcoRI and XhoI. In some embodiments, the vector contains a multiple cloning site (MCS) for the insertion of the desired gene.
Although the genetic code is degenerate in that most amino acids are represented by multiple codons (called “synonyms” or “synonymous” codons), it is understood in the art that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. Accordingly, in some embodiments, the vector includes a nucleotide sequence that has been optimized for expression in a particular type of host cell (e.g., through codon optimization). Codon optimization refers to a process in which a polynucleotide encoding a protein of interest is modified to replace particular codons in that polynucleotide with codons that encode the same amino acid(s), but are more commonly used/recognized in the host cell in which the nucleic acid is being expressed. In some aspects, the polynucleotides described herein are codon optimized for expression in T cells.
The scFv domain typically includes an immunoglobulin variable light (VL) domain, an immunoglobulin variable heavy (VH) domain, and a linker domain joining the VL and VH domains. The relative positions of the VL and VH domains can be reversed, but they are both N′ to the modified Fc domain, as illustrated in
The scFv domain targets an antigen of interest, such as an antigen of a tumor cell. One particular scFv described herein is an anti-CD19 single-chain variable fragment (anti-CD19 scFv). Another particular scFv described herein is an anti-CD20 single-chain variable fragment (anti-CD20 scFv).
While the embodiments described herein pertain to anti-CD19 construct and an anti-CD20 construct, a similar approach can be applied to generate constructs for other target antigens, such as CD22, CD123, CD33, B-cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD)-2, PD-L1 (CD274), CD80 or CD86. For example, based on the schema in
A hinge domain joins the scFv and modified Fc domains, though in some instances the hinge domain may be considered part of the Fc domain. An example of a hinge domain is the IgG hinge domain. The construct can also include an N-terminal signal peptide, such as a CD8α signal peptide (see SEQ ID NOS: 21 and 22).
A variety of linker domains between VL and VH domains are suitable. In some embodiments, the linker domain can be (G4S)x, wherein x is an integer from 1 to 100; preferably, x is an integer from 1 to 10; even more preferably, x is an integer from 2 to 5. In some embodiments, the linker domain can be (G4S)3. In other embodiments, the linker domain can be one or more glycine residues (e.g., (G)y, where y is an integer from 2 to 100. In other embodiments, the linker domain can be (EAAAK)3. (G4S)x, (G4S)3, and (G)y are examples of flexible linkers, while (EAAAK)3 is an example of a more rigid linker.
A variety of hinge domains are suitable. In some embodiments, the hinge domain can be a IgG hinge domain. In some embodiments, the hinge can be a plurality of amino acid residues. In some embodiments, the hinge domain can be a hinge domain from IgE, IgA, IgD, or CD8α.
In some embodiment, the construct is a bicistronic vector that also encodes a chimeric receptor, as illustrated in
The design of the IFL construct tested in this study can be further modified to enhance some its functions and/or widen the range of its specificities. For example, the modified Fc can be further altered to increase its affinity for Fc receptors in NK cells and macrophages, thus enhancing ADCC and ADCP, and/or to increase its capacity to fix complement.4 1-43
In one modification (
In another modification (
In another modification (
In another modification (
Described herein are methods of making a transgenic host cell, such as transgenic natural killer (NK) cells or transgenic T cells. The transgenic host cells can be made, for example, by introducing one or more of the vector embodiments described herein into the host cell.
In one embodiment, the method comprises introducing into a host cell a vector that includes a nucleic acid that encodes an IFL. In some embodiments, a nucleic acid, such as a bicistronic vector, expresses the IFL along with a chimeric receptor. In some embodiments, two separate vectors can be used to create a transgenic cell, such as a transgenic T cell, that expresses an IFL and a chimeric receptor.
In some embodiments, one or more of the nucleic acids are integrated into the genome of the host cell. In some embodiments, the nucleic acids to be integrated into a host genome can be introduced into the host cell using any of a variety of suitable methodologies known in the art, including, for example, homologous recombination, CRISPR-based systems (e.g., CRISPR/Cas9; CRISPR/Cpf1) and TALEN systems.
A variety of host cells are suitable for use in making transgenic host cells. Most commonly, the host cells are immune cells, such as natural killer (NK) cells or T lymphocyte cells.
As used herein, “natural killer cells” (“NK cells”) refer to a type of cytotoxic lymphocyte of the immune system. NK cells provide rapid responses to virally infected cells and respond to transformed cells. Typically, immune cells detect peptides from pathogens presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, triggering cytokine release, causing lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize stressed cells regardless of whether peptides from pathogens are present on MHC molecules. They were named “natural killers” because of the initial notion that they do not require prior activation in order to kill target. NK cells are large granular lymphocytes (LGL) and are known to differentiate and mature in the bone marrow from where they then enter into the circulation. NK cell can also kill tumor cells if antigens on the surface of tumor cells are bound by antibodies; the Fc portion of the antibody bind Fc receptors (CD16) on the surface of NK cells and triggers cytotoxicity, a process known as antibody-dependent cell cytotoxicity (ADCC).
As used herein, “T lymphocytes” or “T cells” refers to lymphocytes that mature in the thymus. T cells can be further characterized into subpopulations, including T helper (TH) cells, T cytotoxic (TC) cells, and T regulatory (Treg) cells. TH and TC cells can be characterized according to the presence or absence of membrane glycoproteins CD4 and CD8. Generally, TH cells express CD4 on their surface, while TC cells express CD8 on their surface. T helper cells can be further characterized as TH1 cells and TH2 cells. T cells can also exert ADCC if transduced with a receptor encoding CD16 and signaling molecules.32
In some aspects, the NK cell or the T cells are mammalian cells. Examples of “mammalian” or “mammals” include primates (e.g., human), canines, felines, rodents, porcine, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats and mice. In a particular aspect, the mammalian T or NK cell is a human T or NK cell.
Upon introducing into a host cell, a vector that includes a nucleic acid that encodes an IFL, the host cell becomes a transgenic host cell that expresses the IFL. Typically, the IFL is secreted by the transgenic host cell.
Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in various embodiments, unless the context clearly dictates otherwise. “About” in reference to a numerical value generally refers to a range of values that fall within ±8%, in some embodiments ±6%, in some embodiments ±4%, in some embodiments ±2%, in some embodiments ±1%, in some embodiments ±0.5% of the value unless otherwise stated or otherwise evident from the context.
EXEMPLIFICATION
Human cell lines RS4; 11 and Nalm-6 (B-cell leukemia), Ramos, Raji and Daudi (B-cell lymphoma) and Jurkat (T-cell leukemia) were obtained from the American Type Culture Collection (Rockville, Md.). The B-cell leukemia cell line OP-1 was established at our laboratory.33 We transduced Nalm-6 and Daudi with a murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-green fluorescent protein (GFP) retroviral vector (from the Vector Development and Production Shared Resource of St. Jude Children's Research Hospital, Memphis, Tenn.) containing firefly luciferase gene. We also transduced Ramos and Raji with the MSCV retroviral vector containing mCherry gene. Transduced cells were selected for their GFP or mCherry expression, respectively, using a MoFlo cell sorter (Beckman Coulter, Brea, Calif.). To have Nalm-6 express CD20 on the surface, we subcloned human CD20 gene in cytomegalovirus plasmid (pCMV6) vector (Origene, Rockville, Md.) into MSCV-IRES-GFP vector and transduced Nalm-6 with the CD20 gene. Nalm-6 cells expressing CD20 were selected using MoFlo sorter after staining with anti-CD20 antibody (BD Biosciences, San Jose, Calif.). Cell lines were cultured in RPMI-1640 (ThermoFisher Scientific, Waltham, Mass.) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin.
Peripheral blood was obtained from discarded products of platelet donations from healthy donors at the National University Hospital Blood Bank, Singapore. Mononucleated cells were isolated by a density gradient centrifugation with Lymphoprep (Axis-Shield, Oslo, Norway) and washed twice in RPMI-1640. For viral transduction, NK cells were expanded from the isolated mononucleated cells with the genetically modified K562-mb15-41BBL, previously established in our laboratory.34,35 T cells were activated by T cell TransAct (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in TexMACS medium (Miltenyi Biotec) with interleukin-2 (IL-2, Proleukin, Novartis, Basel, Switzerland, 100 IU/mL).
We designed IFLs composed of single-chain variable fragment (scFv) linking with a modified fragment crystallizable domain (Fc) of human immunoglobulin G1 (IgG1). The amino acid sequence of the signal peptide, scFv against CD20 and modified Fc of IgG1 was obtained from the sequence of rituximab described in DrugBank (http://www.drugbank.ca; Accession No. DB00073). The scFv sequence against CD19 was from anti-CD19-41BB-CD3ζ CAR previously developed in our laboratory.31 The variable domains of heavy and light chain were connected by a flexible linker sequence encoding (Gly4Ser)3. The linked scFv was joined to the signal peptide and the hinge followed by constant heavy domains 2 and 3 (CH2, CH3) of IgG1. The anti-CD20 IFL was fused with CD16V-4-1BB-CD3ζ, which could have T cells exert ADCC as previously described by our laboratory, through a self-cleaving 2A peptide (P2A).36 The gene was subcloned into the MSCV vector with or without GFP.
Gene transduction by retroviral vector was performed as previously described.37 Briefly, MSCV retroviral vector was added to RetroNectin-coated (Takara, Otsu, Japan) tubes and incubated at 4° C. for 16 hours. Then, activated NK cells or T lymphocytes were added to the tubes after removal of the supernatant and incubated at 37° C. in 5% CO2 for 24 hours. The transduction procedure was repeated one more time on the following day. Transduced cells were maintained in RPMI-1640, 10% FBS with IL-2.
To detect the IFL expression, transduced cells were stained with phycoerythrin (PE)-conjugated anti-human IgG antibody (SouthernBiotech, West Grove, Pa.) after permeabilizing by 8E reagent (a permeabilization reagent developed in our laboratory). CD16 and CD3 expression on the cell surface were determined by anti-CD16-PE (clone B73.1, BD Biosciences) and anti-CD3-APC (clone SK7, BD Biosciences), respectively.
For the specificity of IFLs, culture supernatant from transduced cells was added to Jurkat (CD20 negative, CD19 negative), Ramos (CD20 positive, CD19 positive), or RS4; 11 (CD20 negative, CD19 positive) at 1 μg/mL and incubated for 10 minutes. The IFLs bound on the cell surface were detected with PE-conjugated anti-human IgG antibody. Cell staining was analyzed using BD LSRFortessa (BD Biosciences).
The IFL concentration in culture supernatant from transduced cells was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, culture supernatant containing IFL or rituximab was incubated on plates coated with PE-conjugated anti-human IgG antibody for one hour and washed. Subsequently, horseradish peroxidase (HRP)-conjugated anti-Rituximab antibody (MB2A4, Bio-Rad, Hercules, Calif.) was added to the plates and incubated for one hour. Fluorescence was measured by Infinite 200 PRO (Tecan, Mannedorf, Switzerland) after adding QuantaBlu Fluorogenic Peroxidase Substrate (Thermo Fisher). The IFL concentration was determined by the standard curve prepared with rituximab.
Glycosylation analysis was performed by Proteodynamics (Riom, France). Briefly, IFLs in culture supernatant of transduced cells were concentrated by a dialysis membrane (Amicon Ultra-15 Centrifugal Filter Units, Merck Millipore, Burlington, Mass.) and purified using NAB Protein G Spin kit (Thermo Fisher). The purified IFLs were denatured in 0.5% sodium dodecyl sulfate (SDS) and 1% β—mercaptoethanol and deglycosylated by PNGase F (Promega, Fitchburg, Wis.). The PNGase released N-glycans were purified on Hypercarb Hypersep 200 mg (Thermo Fisher) and permethylated by sodium hydroxide, dimethyl sulfoxide (DMSO) and methyl iodide (ICH3), before MALDI-TOF MS analysis using an Autoflex speed mass spectrometer (Bruker, Billerica, Mass.).
Cytotoxicity Assays in vitro
For CDC assay, Ramos or SUDHL-4 in RPMI/10% FBS medium with or without 5% complement (Sigma-Aldrich, Saint Louis, Mo.) were plated, and Rituximab or anti-CD20 IFL was added at 0.05 μg/ml. Viable cells were counted by Accuri CD6 (BD Biosciences) after incubation at 37° C. in 5% CO2 for 2 hours.
To test ADCP, Ramos cells labelled with mCherry were cultured with or without THP-1 at a 1:1 ratio for 48 hours in the presence of anti-CD20 IFL or rituximab at 0.1 μg/ml. Ramos cells were counted by IncuCyte Zoom System (Essen BioScience, Ann Arbor, Mich.).
For ADCC assay, target cells stained with calcein AM (Thermo Fisher) were co-cultured with transduced NK cells or T lymphocytes at a 2:1 effector-to-target (E:T) ratio for 4 hours. Viable target cells were counted by flow cytometry. In other tests, target cells expressing mCherry were incubated with NK cells or T lymphocytes with IL-2 (200 IU/mL for NK cells, 100 IU/mL for T cells) at 37° C. in 5% CO2. As a control, rituximab was added to NK cells with GFP alone at 1.0 μg/ml. The target cells were counted using IncuCyte Zoom System every 8 hours for 3 days.
To measure plasma concentration of IFL secreted from T cells, NOD.Cg-Prkdcscid IL2rgtm1Wj1/SzJ (NOD/scid IL2RGnull) mice (The Jackson Laboratory, Bar Harbor, Me.) we injected intravenously (i.v.) 2×107 T cells transduced with anti-CD20 IFL-P2A-CD16V-4-1BB-CD3ζ, followed by 2×105 Nalm-6 expressing CD20 two days later. Mice also received 20,000 IU of IL-2 intraperitoneally every 2 days for three weeks. IFL in plasma was measured by ELISA.
To examine antitumor activity in vivo, luciferase-labelled Daudi was injected in NOD/scid IL2RGnull mice at 2×105 cells per mouse intraperitoneally (i.p.). Three and 6 days later, mice received T cells transduced with anti-CD20 IFL-P2A-CD16V-4-1BB-CD3ζ at 2×107 cells per mouse i.p. Other mice received 2×107 T cells transduced with GFP or 0.2 ml of RPMI 1640 only, instead of T cells. All mice received 20,000 IU of IL-2 every 2 days for one or three weeks. Growth of Daudi cells was measured using the Xenogen IVIS-200 System (Caliper Life Sciences, Waltham, Mass.) after injection of D-luciferin potassium salt (Perkin Elmer, Waltham, Mass.). Luminescence was analyzed with the Living Image 3.0 software (Perkin Elmer). Mice were euthanized when luminescence reached 1×1011 photons per second or physical signs warranting euthanasia appeared.
We first generated a scFv fragment from the public sequence of the anti-CD20 antibody rituximab. This scFv, as well as an anti-CD19 scFv previously developed in our laboratory,31 were linked to the hinge and heavy chain constant domain 2 (CH2) and 3 (CH3) of human IgG1 (
We determined whether the IFLs could bind their cognate target. As shown in
To measure the capacity of immune cells to produce IFLs, we collected the culture media from anti-CD20 IFL transduced cells and measured the concentration of antibody by ELISA, using an anti-idiotypic rituximab antibody. As shown in
To define the type of post-translational modification profile of the constructs produced by immune cells, we performed an analysis of the N-linked glycans bound to the modified Fc domain using MALDI-TOF. Twelve N-glycan structures were detected for the NK cell IFL, and 8 for the T cell IFL. For both, the dominant structure was a di-sialylated bi-antennary N-glycan ([M+Na]+2792) without core-fucose, containing 2 galactose and 2 terminal sialic acid named G2S2. Interestingly, 79% and 59% of Fc glycans were afucosylated when IFL were produced by NK and T cells, respectively. (
To test whether IFLs could mediate CDC, we incubated the CD20+ B-lymphoma cell lines Ramos, SUDHL-4 and Raji with different concentrations of anti-CD20 IFL (collected from supernatants of NK cells or T cells transduced with IFL) and 5% complement for 2 hours. In parallel tests, the anti-CD20 IFL was replaced by rituximab. As shown in
ADCP was tested by co-culturing Ramos with the monocytic cell line, THP-1, which can exert phagocytosis of tagged target cells.40 As shown in
To determine whether IFLs produced by NK cells and T cells could mediate ADCC, we co-cultured CD20+ lymphoma cell line Raji with NK cells transduced with GFP alone or anti-CD20 IFL at a E:T 1:1 ratio, using rituximab at 1 μg/mL with NK-GFP cells as a control. As shown in
We prepared a bicistronic construct containing anti-CD20 IFL and the CD16 (V158)-41BB-CD3ζ receptor, separated by P2A (
We next determined the levels of plasma IFL that can be measured in mouse plasma after intravenous injection of 2×107 T lymphocytes transduced with anti-CD20 IFL in NOD-SCID-IL2RGnull immunodeficient mice. As shown in
The IFL constructs were modified to enhance some its functions and/or widen the range of its specificities. For example, the modified Fc can be further altered to increase its affinity for Fc receptors in NK cells and macrophages, thus enhancing ADCC and ADCP, and/or to increase its capacity to fix complement. In particular, the modified IFLs of
The results of the experiments shown in
1. A peptide comprising:
a) a single-chain variable fragment (scFv) domain;
b) a fragment crystallizable (Fc) domain; and
c) a hinge domain joining the scFv and Fc domains.
2. The peptide of Embodiment 1, wherein the scFv domain comprises an immunoglobulin variable light (VL) domain, an immunoglobulin variable heavy (VH) domain, and a linker domain joining the VL and VH domains.
3. The peptide of Embodiment 2, wherein the linker domain is (G4S)x, wherein x is an integer from 1 to 100.
4. The peptide of Embodiment 3, wherein the linker domain is (G45)3.
5. The peptide of any one of Embodiments 1 through 4, wherein the scFv domain binds CD19.
6. The peptide of any one of Embodiments 1 through 4, wherein the scFv domain binds CD20.
7. The peptide of any one of Embodiments 1 through 4, wherein the scFv domain binds CD22, CD38, CD7, CD2, CD3, epidermal growth factor receptor (EGFR), CD123, CD33, B-cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disialoganglioside (GD2), PD-L1 (CD274), CD80 or CD86.
8. The peptide of any one of Embodiments 1 through 4, wherein the Fc domain comprises an immunoglobulin constant heavy 2 (CH2) domain and an immunoglobulin constant heavy 3 (CH3) domain.
9. The peptide of any one of Embodiments 1 through 4, wherein the Fc domain is human IgG1 Fc domain.
10. The peptide of any one of Embodiments 1 through 4, further comprising a signal peptide that is N-terminal to the scFv domain.
11. The peptide of any one of Embodiments 1 through 4, further comprising a self-cleaving peptide joining the Fc domain to a chimeric receptor, wherein the chimeric receptor comprises a receptor domain, a hinge and transmembrane domain, a co-stimulatory signaling domain, and a cytoplasmic signaling domain.
12. The peptide of Embodiment 11, wherein the self-cleaving peptide is a 2A peptide.
13. The peptide of Embodiment 11, wherein the receptor domain is CD16.
14. The peptide of Embodiment 11, wherein the hinge and transmembrane domain is a CD8α hinge and transmembrane domain.
15. The peptide of Embodiment 11, wherein the co-stimulatory domain is 4-1BB co-stimulatory domain.
16. The peptide of Embodiment 11, wherein the cytoplasmic signaling domain is a CD3ζ cytoplasmic signaling.
17. The peptide of Embodiment 11, wherein the chimeric receptor is CD16V-4-1BB-CD3ζ.
18. The peptide of any one of Embodiments 1 through 4, wherein the scFv domain binds CD19 or CD20, the Fc domain is a human IgG1 Fc domain, and the hinge domain is an IgG1 hinge domain; the peptide further comprising a CD8α signal peptide that is N-terminal to the scFv domain; the peptide further comprising a chimeric receptor that is CD16V-4-1BB-CD3ζ.
19. The peptide of any one of Embodiments 1 through 4, further comprising one or more of the following mutations: S239D; S267E; H268F; or 1332E.
20. The peptide of any one of Embodiments 1 through 4, further comprising one or more of the following mutations: E345K; E430G; or S440Y.
21. The peptide of any one of Embodiments 1 through 4, wherein the peptide further comprise IL-15 joined to the Fc domain by a linker.
22. The peptide of Embodiment 21, wherein the linker that joins IL-15 to the Fc domain is selected from the group consisting of SEQ ID NO: 51; A(EAAK)4ALEA(EAAAK)4A; (EAAAK)z; A(EAAAK)zA; and (XP)w, wherein z is an integer from 1 to 100; X is any amino acid, and w is an integer from 1 to 100.
23. The peptide of any one of Embodiments 1 through 4, wherein the peptide further comprises a ligand that binds 4-1BB (CD37), CD28, or OX40 (CD134) joined to the Fc domain by a linker.
24. The peptide of Embodiment 23, wherein the linker that joins IL-15 to the Fc domain is selected from the group consisting of SEQ ID NO: 51; A(EAAK)4ALEA(EAAAK)4A; (EAAAK)z; A(EAAAK)zA; and (XP)w, wherein z is an integer from 1 to 100; X is any amino acid, and w is an integer from 1 to 100.
25. A nucleic acid encoding the peptide of any of Embodiments 1 through 24.
26. A vector comprising a nucleic acid, the nucleic acid encoding the peptide of any of Embodiments 1 through 24.
27. The vector of Embodiment 26, wherein the vector is a murine stem cell virus (MSCV).
28. An immune cell that expresses a peptide, wherein the peptide comprises:
a) a T-cell receptor (TCR) β domain;
b) a first fragment crystallizable (Fc) domain joined to the TCR β domain;
c) a TCR a domain;
d) a self-cleaving peptide joining the Fc domain to the TCR a domain;
e) a second Fc domain joined to the TCR α domain.
29. The immune cell of Embodiment 28, further comprising a signal peptide joined to the T-cell receptor (TCR) β domain.
30. The immune cell of Embodiment 28, wherein the first Fc domain is the same as the second Fc domain.
31. A peptide comprising:
a) a T-cell receptor (TCR) β domain;
b) a first fragment crystallizable (Fc) domain joined to the TCR β domain;
c) a TCR α domain;
d) a self-cleaving peptide joining the Fc domain to the TCR α domain;
e) a second Fc domain joined to the TCR α domain.
32. The peptide of Embodiment 31, further comprising a signal peptide joined to the T-cell receptor (TCR) β domain.
33. The peptide of Embodiment 31, wherein the first Fc domain is the same as the second Fc domain.
34. A nucleic acid encoding the peptide of any one of Embodiments 31 through 33.
35. A vector comprising a nucleic acid, the nucleic acid encoding the peptide of any one of Embodiments 31 through 33.
36. A method of making a transgenic host cell, the method comprising introducing a vector into a host cell, the vector comprising a nucleic acid encoding the peptide of any of Embodiments 1 through 24 or Embodiments 31 through 33.
37. A method of enhancing antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the immune cells described herein.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.
1. Yu A L, Gilman A L, Ozkaynak M F, et al. Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J Med. 2010; 363(14):1324-1334.
2. Ferris R L, Jaffee E M, Ferrone S. Tumor antigen-targeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape. J Clin Oncol. 2010; 28(28):4390-4399.
3. Maloney D G. Anti-CD20 antibody therapy for B-cell lymphomas. N Engl J Med. 2012; 366(21):2008-2016.
4. Scott A M, Wolchok J D, Old L J. Antibody therapy of cancer. Nat Rev Cancer. 2012; 12(4):278-287.
5. Weiner L M, Murray J C, Shuptrine C W. Antibody-based immunotherapy of cancer. Cell. 2012; 148(6):1081-1084.
6. Galluzzi L, Vacchelli E, Fridman W H, et al. Trial Watch: Monoclonal antibodies in cancer therapy. Oncoimmunology. 2012; 1(1):28-37.
7. Nimmerjahn F, Ravetch J V. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008; 8(1):34-47.
8. Koene H R, Kleijer M, Algra J, Roos D, von dem Borne A E, de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood. 1997; 90(3):1109-1114.
9. Cartron G, Dacheux L, Salles G, et al. Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene. Blood. 2002; 99(3):754-758.
10. Weng W K, Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol. 2003; 21(21):3940-3947.
11. Dall'Ozzo S, Tartas S, Paintaud G, et al. Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship. Cancer Res. 2004; 64(13):4664-4669.
12. Hatjiharissi E, Xu L, Santos D D, et al. Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the Fc{gamma}RIIIa-158 V/V and V/F polymorphism. Blood. 2007; 110(7):2561-2564.
13. Musolino A, Naldi N, Bortesi B, et al. Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J Clin Oncol. 2008; 26(11):1789-1796.
14. Bibeau F, Lopez-Crapez E, Di Fiore F, et al. Impact of Fc{gamma}RIIa-Fc{gamma}RIIIa polymorphisms and KRAS mutations on the clinical outcome of patients with metastatic colorectal cancer treated with cetuximab plus irinotecan. J Clin Oncol. 2009; 27(7):1122-1129.
15. Ahlgrimm M, Pfreundschuh M, Kreuz M, Regitz E, Preuss K D, Bittenbring J. The impact of Fc-gamma receptor polymorphisms in elderly patients with diffuse large B-cell lymphoma treated with CHOP with or without rituximab. Blood. 2011; 118(17):4657-4662.
16. Veeramani S, Wang S Y, Dahle C, et al. Rituximab infusion induces NK activation in lymphoma patients with the high-affinity CD16 polymorphism. Blood. 2011; 118(12):3347-3349.
17. Weiskopf K, Weissman I L. Macrophages are critical effectors of antibody therapies for cancer. MAbs. 2015; 7(2):303-310.
18. Kochenderfer J N, Wilson W H, Janik J E, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood. 2010; 116(20):4099-4102.
19. Porter D L, Levine B L, Kalos M, Bagg A, June C H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011; 365(8):725-733.
20. Maude S L, Frey N, Shaw P A, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014; 371(16):1507-1517.
21. Davila M L, Riviere I, Wang X, et al. Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia. Sci Transl Med. 2014; 6(224):224ra225.
22. Kochenderfer J N, Dudley M E, Kassim S H, et al. Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma and Indolent B-Cell Malignancies Can Be Effectively Treated With Autologous T Cells Expressing an Anti-CD19 Chimeric Antigen Receptor. J Clin Oncol. 2014.
23. Lee D W, Kochenderfer J N, Stetler-Stevenson M, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet. 2014.
24. Turtle C J, Hanafi L A, Berger C, et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest. 2016; 126(6):2123-2138.
25. Sadelain M, Riviere I, Riddell S. Therapeutic T cell engineering. Nature. 2017; 545(7655):423-431.
26. Maude S L, Laetsch T W, Buechner J, et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med. 2018; 378(5):439-448.
27. Eshhar Z, Waks T, Gross G, Schindler D G. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. ProcNatlAcadSciUSA. 1993; 90(2):720-724.
28. Geiger T L, Leitenberg D, Flavell R A. The TCR zeta-chain immunoreceptor tyrosine-based activation motifs are sufficient for the activation and differentiation of primary T lymphocytes. J Immunol. 1999; 162(10):5931-5939.
29. Brentjens R J, Latouche J B, Santos E, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. NatMed. 2003; 9(3):279-286.
30. Cooper L J, Topp M S, Serrano L M, et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect. Blood. 2003; 101(4):1637-1644.
31. Imai C, Mihara K, Andreansky M, Nicholson I C, Pui C H, Campana D. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia. 2004; 18:676-684.
32. Kudo K, Imai C, Lorenzini P, et al. T lymphocytes expressing a CD16 signaling receptor exert antibody-dependent cancer cell killing. Cancer Res. 2014; 74(1):93-103.
33. Manabe A, Coustan-Smith E, Kumagai M, et al. Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia. Blood. 1994; 83(7): 1731-1737.
34. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. 2005; 106:376-383.
35. Fujisaki H, Kakuda H, Shimasaki N, et al. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res. 2009; 69(9):4010-4017.
36. Holst J, Szymczak-Workman A L, Vignali K M, Burton A R, Workman C J, Vignali D A. Generation of T-cell receptor retrogenic mice. NatProtoc. 2006; 1(1):406-417.
37. Shimasaki N, Campana D. Natural killer cell reprogramming with chimeric immune receptors. Methods Mol Biol. 2013; 969:203-220.
38. Chao M P, Alizadeh A A, Tang C, et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell. 2010; 142(5):699-713.
39. Hu W, Ge X, You T, et al. Human CD59 inhibitor sensitizes rituximab-resistant lymphoma cells to complement-mediated cytolysis. Cancer Res. 2011; 71(6):2298-2307.
40. Suzuki M, Yamanoi A, Machino Y, et al. Effect of trastuzumab interchain disulfide bond cleavage on Fcgamma receptor binding and antibody-dependent tumour cell phagocytosis. J Biochem. 2016; 159(1):67-76.
41. Lazar G A, Dang W, Karki S, et al. Engineered antibody Fc variants with enhanced effector function. Proc Natl Acad Sci USA. 2006; 103(11):4005-4010.
42. Richards J O, Karki S, Lazar G A, Chen H, Dang W, Desjarlais J R. Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther. 2008; 7(8):2517-2527.
43. Moore G L, Chen H, Karki S, Lazar G A. Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions. MAbs. 2010; 2(2):181-189.
44. Diebolder C A, Beurskens F J, de Jong R N, et al. Complement is activated by IgG hexamers assembled at the cell surface. Science. 2014; 343(6176):1260-1263.
45. Sneller M C, Kopp W C, Engelke K J, et al. IL-15 administered by continuous infusion to rhesus macaques induces massive expansion of CD8+ T effector memory population in peripheral blood. Blood. 2011; 118(26):6845-6848.
46. Imamura M, Shook D, Kamiya T, et al. Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15. Blood. 2014; 124(7):1081-1088.
47. Koh S, Shimasaki N, Bertoletti A. Redirecting T Cell Specificity Using T Cell Receptor Messenger RNA Electroporation. Methods Mol Biol. 2016; 1428:285-296.
48. de Jong R N, Beurskens F J, Verploegen S, et al. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface. PLoS Biol. 2016; 14(1):e1002344.
This application claims the benefit of U.S. Provisional Application No. 62/875,455, filed on Jul. 17, 2019. The entire teachings of the above application are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/056659 | 7/15/2020 | WO |
