Patent Application
20230357344
Disclosed herein are interleukin 15 (IL15) constructs, as well as methods of use for the treatment of cancer.
IL15 is a cytokine originally described as a T cell growth factor. The cytokine belongs to the four α-helix bundle family, and its receptor consists of two subunits (the IL-2R/IL-15R p and γ chains) responsible for signal transduction. These receptors are expressed for example on activated T cells, and which can be activated with picomolar concentrations of IL15.
As a therapeutic, IL15 shows promise in the activation of T cells, especially CD8+ T cells, however, there are issues with dosing a patent due to the short half-life and rapid clearance of the molecule. Currently, there are no approved uses of recombinant IL-15, although several clinical trials are ongoing.
Thus, there is an unmet need in the art to provide for IL15 constructs that are able to deliver IL15 directly to the tumor microenvironment in a manner that provides for superior delivery of the molecule.
The present disclosure is directed to IL15 constructs. In one embodiment the IL15 construct is a bivalent, homodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A bivalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A bivalent, homodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A bivalent, homodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A bivalent homodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent, heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A monovalent heterodimeric interleukin 15 (IL15) construct comprising from N-terminus to C-terminus:
A pharmaceutical composition comprising the IL15 construct of in combination with at least one additional IL15 construct.
A method of treating cancer comprising administering to a patient in need an effective amount of the IL15 construct
The method, wherein the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma and sarcoma.
The method, wherein the IL15 construct is administered in combination with another therapeutic agent.
The method, wherein the therapeutic agent is an immune checkpoint agent.
The method, wherein the immune checkpoint agent is a PD-1, PD-L1, PD-L2, TIM3, LAG-3, OX40 or TIGIT antibody.
A method of increasing the survival of an immune cell, comprising administering an IL15 construct prior to, during or after administration of an effective amount of immune cells to a patient.
The method wherein the immune cell expresses a chimeric antigen receptor (CAR).
The method wherein the immune cell is an NK cell.
The method wherein the immune cell is a T-cell.
Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.
As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.
The term “or” is used to mean, and is used interchangeably with, the term “and/or” unless the context clearly dictates otherwise.
The term “anti-cancer agent” as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.
The term “Interleukin-15” or “IL15” is a cytokine that stimulates the proliferation of T-lymphocytes. The amino acid sequence of human IL15, (SEQ ID NO:1) can also be found at accession number X94223.
The term “Interleukin-15 receptor alpha” or “IL15Ra” is the high affinity receptor for IL15. The amino acid sequence of IL15Ra, (SEQ ID NO: 2) can also be found at accession number CR542023.
The term “Interleukin-2 receptor beta” or “IL2Rb” is a beta subunit receptor involved in receptor mediated endocytosis and transduces the mitogenic signals of IL2. It also associates with IL15Ra, involved in the stimulation of neutrophil phagocytosis by IL15. The amino acid sequence of human IL2Rb, (SEQ ID NO: 3) can also be found at accession number CR456506.
The terms “administration,” “administering,” “treating,” and “treatment” as used herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. The term “administration” and “treatment” also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term “subject” herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human. Treating any disease or disorder refer in one aspect, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another aspect, “treat,” “treating,” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another aspect, “treat,” “treating,” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another aspect, “treat,” “treating,” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.
The term “subject” in the context of the present disclosure is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having, or at risk of having, a disorder described herein).
The terms “cancer” or “tumor” herein has the broadest meaning as understood in the art and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of the present disclosure, the cancer is not limited to certain type or location.
The term “tumor associated antigen (TAA)” is an antigen expressed on a target tumor, wherein an antibody or antigen binding fragment of an antibody is directed to and specifically binds that TAA. For example, the TAA described herein is PD-L1 and the antibody that has been raised against this antigen is disclosed in WO 2016/000619.
In the context of the present disclosure, when reference is made to an amino acid sequence, the term “conservative substitution” means substitution of the original amino acid by anew amino acid that does not substantially alter the chemical, physical and/or functional properties of the IL15 construct, e.g. its ability to bind and activate the IL15 signaling pathway. Specifically, common conservative substations of amino acids are well known in the art and are shown below.
The term “knob-into-hole” technology as used herein refers to amino acids that direct the pairing of two polypeptides together either in vitro or in vivo by introducing a spatial protuberance (knob) into one polypeptide and a socket or cavity (hole) into the other polypeptide at an interface in which they interact. For example, knob-into-holes have been introduced in the Fc:Fc binding interfaces, CL:CHI interfaces or VH/VL interfaces of antibodies (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al, 1997, Protein Science 6:781-788). In some embodiments, knob-into-holes insure the correct pairing of two different heavy chains together during the expression of specific IL15 constructs. For example, IL15 constructs having knob-into-hole amino acids in their Fc regions can further comprise a first molecule of an IL15 construct and a second molecule of an IL15 construct, wherein these two molecules are assembled at least in part, through knob into hole interaction.
The term “knob” as used herein in the context of “knob-into-hole” technology refers to an amino acid change that introduces a protuberance (knob) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation.
The term “hole” as used herein in the context of “knob-into-hole” refers to an amino acid change that introduces a socket or cavity (hole) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation.
Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as values for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLAST program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), algorithm which has been incorporated into the GAP program in the GCG software package using either a BLOSUM62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
The term “operably linked” or in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
The terms “linker” “linked, “linked to” or “linkered” refer a polypeptide (protein) of at least two amino acids, that are inserted between two polypeptides thus joining them together. A linker can be non-cleavable or have a protease activatable (cleavable) moiety. Examples of linkers are shown below in Table 3 and Table 4.
In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, which include an IL15 construct described herein, formulated together with at least one pharmaceutically acceptable excipient. As used herein, the term “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The excipient can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g. by injection or infusion).
The compositions disclosed herein can be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, liposomes, and suppositories. A suitable form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusion solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the IL15 construct is administered by intravenous infusion or injection. In certain embodiments, the IL15 construct is administered by intramuscular or subcutaneous injection.
The term “therapeutically effective amount” as herein used, refers to the amount of an IL15 construct that, when administered to a subject for treating a disease, or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or symptom. The “therapeutically effective amount” can vary with the IL15 construct, the disease, disorder, and/or symptoms of the disease or disorder, severity of the disease, disorder, and/or symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated. An appropriate amount in any given instance can be apparent to those skilled in the art or can be determined by routine experiments. In the case of combination therapy, the “therapeutically effective amount” refers to the total amount of the combination objects for the effective treatment of a disease, a disorder or a condition.
The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also encompasses co-administration in multiple, or in separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Powders and/or liquids can be reconstituted or diluted to a desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
As used herein, the phrase “in combination with” means that an IL15 construct is administered to the subject at the same time as, just before, or just after administration of an additional therapeutic agent. In certain embodiments, an IL15 construct is administered as a co-formulation with an additional therapeutic agent.
The present disclosure provides for IL15 constructs that bind and activate the IL15 signaling pathway. Furthermore, the present disclosure provides IL15 constructs that have desirable pharmacokinetic characteristics and other desirable attributes, and thus can be used for reducing the likelihood of or treating cancer. The present disclosure further provides pharmaceutical compositions comprising IL15 constructs and methods of making and using such pharmaceutical compositions of IL15 constructs for the prevention and treatment of cancer and associated disorders.
Other IL15 constructs of the present disclosure include those where the amino acids or nucleic acids encoding the amino acids have been changed; yet have at least 60%, 70%, 80%, 90%, 95% or 99% percent identity to the sequences described in Table 2. In some aspects, it includes changes in the amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids have been changed when compared with sequences described in Table 2, while retaining substantially the same therapeutic activity.
It is also understood that the domains and/or regions of the polypeptide chains of the IL15 constructs can be contain linker regions of various lengths. In some embodiments, the IL15 construct domains are separated from each other by a linker region. For example, IL15Ra-(linker)-IL15. In some aspects, the linker can contain a protease activatable (cleavable) moiety.
In some embodiments, the amino acids glycine and serine comprise the amino acids of the linker (a “GS” linker). In another embodiment, the linker can be, without limitation the linkers in Table 3 or any combination thereof.
In other embodiments the linker contains a protease activatable (cleavable) moiety. In certain embodiment, the protease activatable moiety can be without limitation, the linkers in Table 4 or any combination thereof. Table 5 shows the placement of a protease activatable moieties in the context of representative constructs.
In yet other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions. For example, one or more amino acids can be replaced with a different amino acid residue such that the Fc region has an altered affinity for an effector ligand. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in, e.g., U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the Fc region has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in, e.g., U.S. Pat. No. 6,194,551 by Idusogie et al.
In yet another aspect, one or more amino acid residues are changed to thereby alter the ability of the Fc region to fix complement. This approach is described in, e.g., the publication WO 94/29351 by Bodmer et al. In a specific aspect, one or more amino acids of an IL15 construct of the present disclosure are replaced by one or more allotypic amino acid residues, for the IgG1 subclass and the kappa isotype. Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgG1, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).
In another aspect, if a reduction of ADCC is desired, the Fc region of IgG4 was shown in many previous reports to have only modest ADCC and almost no CDC effector function (Moore G L, et al. 2010 MAbs, 2:181-189). However, natural IgG4 was found less stable in stress conditions such as in acidic buffer or under increasing temperature (Angal, S. 1993 Mol Immunol, 30:105-108; Dall'Acqua, W. et al, 1998 Biochemistry, 37:9266-9273; Aalberse et al. 2002 Immunol, 105:9-19). Reduced ADCC can be achieved by operably linking the IL5 construct to an IgG4 Fc engineered with combinations of alterations that reduce FcγR binding or C1q binding activities, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of an IL15 construct as a biological therapeutic, one of the less desirable, intrinsic properties of IgG4 is dynamic separation of its two heavy chains in solution (Van der Neut Kolfschoten M, et al. 2007 Science, 317:1554-157). The mutation of serine to proline at position 228 (EU numbering system) appeared inhibitory to the IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al. 2002 Immunol, 105:9-19). Some of the amino acid residues in the hinge and γFc region were reported to have impact on Fc region interaction with Fcγ receptors (Chappel S M, et al. 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, K. L. et al. 1999 Eur J Immunol, 29:2613-2624; Clynes, R. A. et al, 2000 Nature Medicine, 6:443-446; Arnold J. N., 2007 Annu Rev immunol, 25:21-50). Furthermore, some rarely occurring IgG4 isoforms in human population can also elicit different physicochemical properties (Brusco, A. et al. 1998 Eur J Immunogenet, 25:349-55; Aalberse et al. 2002 Immunol, 105:9-19). To generate IL15 constructs with low ADCC and CDC but with good stability, it is possible to modify the hinge and Fc region of human IgG4 and introduce a number of alterations. These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88, U.S. Pat. No. 8,735,553 to Li et al.
The Fc domain can be modified via amino acid changes to provide “knob-into-hole” technology and to direct the pairing of two polypeptides together either in vitro or in vivo For example, “the knob-in-hole” mutations in the human IgG1 Fc were introduced to facilitate heterodimer formation (Ridgway et al., Prot. Eng. 1996 9:617-621). In addition, knob-into-holes were introduced in the Fc:Fc binding interfaces, CL:CHI interfaces or VH/VL interfaces of antibodies (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al, 1997, Protein Science 6:781-788). In some embodiments, knob-into-holes insure the correct pairing of two different heavy chains together to generate a specific IL15 construct.
IL15 Construct Production
The IL15 constructs can be produced by any means known in the art, including but not limited to, recombinant expression or chemical synthesis. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.
Also provided in the present disclosure are expression vectors and host cells for producing the IL15 constructs. The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding an IL15 construct. In some aspects, an inducible promoter is employed to prevent expression of inserted sequences except under the control of inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements can also be required or desired for efficient expression of an IL15 construct. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.
The host cells for harboring and expressing the an IL15 construct can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express IL15 constructs. Insect cells in combination with baculovirus vectors can also be used.
In other aspects, mammalian host cells are used to express and produce the IL15 constructs of the present disclosure. For example, they can be a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cells. For example, several suitable host cell lines capable of secreting intact polypeptides have been developed, including the CHO cell lines, various COS cell lines and HEK 293 cells. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.
Methods of Treatment
The IL15 constructs of the present disclosure are useful in a variety of applications including, but not limited to, methods for the treatment of cancer, infection or immune disorders.
In one aspect, the present disclosure provides a method of treating cancer. In certain aspects, the method comprises administering to a patient in need an effective amount of an IL15 construct. The cancer can include, without limitation, gastric cancer, colon cancer, pancreatic cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma and sarcoma.
The IL15 constructs as disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
IL15 constructs of the disclosure can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The IL15 construct need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of IL15 construct present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
For the prevention or treatment of disease, the appropriate dosage of an IL15 construct of the disclosure will depend on the type of disease to be treated, the severity and course of the disease, whether the IL15 construct is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the IL15 construct, and the discretion of the attending physician. The IL15 construct is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 100 mg/kg of IL15 construct can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. Such doses can be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the IL15 construct). An initial higher loading dose, followed by one or more lower doses can be administered. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
Combination Therapy
In one aspect, the IL15 constructs of the present disclosure can be used in combination with other therapeutic agents. Other therapeutic agents that can be used with the IL15 constructs of the present disclosure include: but are not limited to, a chemotherapeutic agent (e.g., paclitaxel or a paclitaxel agent; (e.g. Abraxane®), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitor (e.g., erlotinib), multikinase inhibitor (e.g., sitravatinib), CD-20 targeting agent (e.g., rituximab, ofatumumab), CD52 targeting agent (e.g., alemtuzumab), prednisolone, lenalidomide, Bcl-2 inhibitor (e.g., oblimersen sodium), aurora kinase inhibitor, proteasome inhibitor (e.g., bortezomib), MEK inhibitor (e.g., ABT-348), JAK-2 inhibitor (e.g., INCB018424), mTOR inhibitor (e.g., temsirolimus, everolimus), BCR/ABL inhibitor (e.g., imatinib).
In one aspect, the IL15 construct of the present disclosure is administered in combination with an immune checkpoint agent. Without limitation, immune checkpoint agents can be PD-1. PD-L1, PD-L2, TIM3, LAG-3, OX40 or TIGIT antibodies. In one aspect, the anti-PDI antibody can be Tislelizumab. In one aspect, the anti-PDI antibody can be Ociperlimab or a combination of Tislelizumab and Ocipcrlimab.
In another aspect, IL15 has been administered in cell therapy, providing a beneficial effect to immune cells such as T-cells or NK cells when administered prior, during or after administration of cell therapy to a patient. For NK cells containing an anti-CD19 chimeric antigen receptor (CAR), an IL15 fusion transgene was introduced in order to support NK cell function and persistence (Kaufman et al. Blood 2018 v. 32, supp. 1, 4541). An EGFR CAR introduced into NK cells was administered in combination with an IL15 construct to promote efficacy in a glioblastoma model (Ma et al., Cancer Res., 2021 81(13) 3635-48). NK92 cells transduced with a CD123 CAR were designed to target acute myeloid leukemia (AML). Retroviral vectors were used to introduce a transgene cassette for the constitutive expression of human IL-15 which allowed for increased NK cell persistence in vivo (Morgan et al., Viruses 2021 13(7): 1365).
Pharmaceutical Compositions and Formulations
Also provided are compositions, including pharmaceutical formulations, comprising an IL15 construct. In certain embodiments, compositions comprise one or more IL15 constructs, or one or more polynucleotides comprising sequences encoding one or more IL15 constructs. These compositions can further comprise suitable carriers, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.
Pharmaceutical formulations of an IL15 construct as described herein are prepared by mixing such IL15 construct having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Pat. No. 7,871,607 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
Exemplary lyophilized formulations are described in U.S. Pat. No. 6,267,958. Aqueous formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.
Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the IL15 construct, which matrices are in the form of shaped articles, e.g. films, or microcapsules. The formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
HH cells are a human T lymphocyte/leukemia cell line that were obtained from ATCC (CRL-2105). Cultures were maintained by the addition or replacement of fresh medium. Cell cultures were started at 2×105 cells/mL and maintain between 1×105 and 1×106 cells/mL, with culture medium be refreshed every 2-3 days. The IL15 used was unaltered IL15. P22339 is a published IL15 construct consisting of two molecules of IL15 linked to the sushi domain of IL15Ra and linked to an Fc (Hu et al. Sci. Rep. 2018 8:7675).
This assay provided insight as to the activity of specific constructs. In
M07e Cells
M07e cells are human megakaryocyte line. The M07e cells were obtained from Nanjing CoBioer Biosciences Co., Ltd (COBIOER #CBP60791). The cell cultures were maintained in RPMI 1640 with 10% FBS and GM-CSF (10 ng/ml) or IL-2 (10 ng/nl) by addition or replacement of fresh medium. Assay cultures were started at 2×105 cells/mL and maintained between 1×105 and 1×106 cells/mL. The M-07e cells proliferate in the presence of GM-CSF, IFN-alpha, IFN-beta, IFN-gamma, IL-2, IL-3, IL-4, IL-6, IL-15, NGF, SCF, TNT-alpha and TPO. IL15 reagents were obtained as described in Example 1. IL-2 was obtained from R&D Systems (#202-IL).
As shown in
ICR mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Female ICR mice were randomized into 13 groups according to body weight with 6 mice per group. Mice from 7 groups were intraperitoneally injected with single dose of vehicle (10 mM Histidine, 10 mM Acetic Acid, 240 mM sucrose, 0.02% Tween 20, pH5.5), or 0.3, 1 and 3 mg/kg P22339 or 10, 30 and 100 mg/kg MK137/MH7 for monitoring the general toxicological effect of each drug in ICR mice. Body weights were recorded every day and the mice were also monitored daily for clinical signs of toxicity throughout the study.
Mice from the other 6 groups were simultaneously intraperitoneally injected with single dose of 0.3, 1 and 3 mg/kg P22339 and 10, 30 and 100 mg/kg MK137/MH7 for evaluating the pharmacokinetics (PK) of each drug. A schematic of this is shown in
The survival time of all 78 mice was recorded to determine the maximal tolerated dose (MTD) of each drug. This result is shown in
Female C57BL/6 mice were subcutaneously inoculated with 1×107 GL261 cells. When the tumor volume reached around 100-200 mm3, animals were randomized according to the tumor volume with 7 animals per group. Mice were intraperitoneally injected with single dose of vehicle (10 mM Histidine, 10 mM Acetic Acid, 240 mM sucrose, 0.02% Tween 20, pH5.5) or 0.25 mg/kg P22339 or 10 and 30 mg/kg MK137/MH7. At 5 days post treatment, the mice were euthanized using carbon dioxide and 5 mice were selected for sample collection.
Blood samples were collected and used for further antibody staining directly. Tumor samples were minced using scissors into small pieces and followed by enzymatic digestion to isolate the tumor infiltrating lymphocytes (TILS). Blood cells and TILS were then stained with fluorescence conjugated antibody against certain biomarkers to identify NK cells, CD8+ T cells, CD4+ T cells and Treg cells for further analysis using flow cytometer.
MK137/MH7 at 10 mpk showed significant in vivo PD effect in tils but not in peripheral blood cells regarding to the increase of NK percentage in lymphocytes.
NCG mice are triple immunodeficient mice that lack functional T, B and NK cells and have reduced macrophage and dendritic cell function. Such characteristics makes these mice good models for research on immuno-oncology. Female NCG mice were obtained from Jiangsu GemPharmatech Co., Ltd., and were subcutaneously inoculated with a mix of 3×106 HT29 (human colon adenocarcinoma) cells which have high expression of matrix metalloproteases and 1×106 HH (human leukemia/lymphoma) cells which were chosen for their pSTAT5 response to IL15. Animals were randomized with 3 or 4 mice in each group when the average tumor size reached 400-600 mm3. A single dose of vehicle (10 mM Histidine, 10 mM Acetic Acid, 240 mM sucrose, 0.02% Tween 20, pH5.5), P22339, MK137/MH7 or MK138/MH7 (non-cleavable) were intraperitoneally administered to test the correlation of serum PK and IL15 induced tumor PD, in which pSTAT5 signaling from HH cells in HT29+HH tumor tissues was measured.
Serum intact MK137/MH7 and MK138/MH7 were detected by ELISA. Serum IL-15/IL-15Ra released from MK137/MH7 or MK138/MH7 were measured by MSD via their pSTAT5 induction level in HH cells which were spiked into serum obtained from NCG mice at different timepoints post treatment. Serum P22339 levels were examined by both ELISA and MSD as described. Tumor PD of MK137/MH7, MK138/MH7 and P22339 was evaluated by MSD at different timepoints post treatment by measuring pSTAT5 signaling from HH cells in HT29+HH tumor lysates.
Using the maximum tolerated dose (MTD) as determined above in Example 3 using IRC mice, the PK/PD correlation of P22339 and MK138/MH7 in the HT29+HH xenograft model was tested. As shown in
It is possible to calculate the equivalent amounts of molar IL15 between the MK137/MH7 and P22339 constructs, thus allowing these molecules to be dosed on that basis. When equivalent MK137/MH7 (5 mpk) and P22339 (2.5 mpk) doses were administered to the HT29+HH xenograft model, a similar PD was obtained in the tumor microenvironment as measured by ELISA (
Disclosed herein are a number of various IL15 constructs with cleavable linkers. It will be possible to create combinations of the IL15 constructs wherein one IL15 combined with at least one other IL15 construct as disclosed. This will account for differing amounts of protease expression in the tumor microenvironment. For example, a tumor expressing large amounts of a specific protease will cleave a protease activatable moiety in one IL15 construct very quickly, wherein a second protease that is not expressed as highly will cleave a second protease activatable moiety resulting in slower IL15 activation. If desired, this combination of IL15 constructs can allow for more construct delivered IL15 to remain in the tumor microenvironment longer. This can solve one of the issues associated with delivery of IL15, that of the very short half-life associated with systemic IL15 administration.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/115594 | Sep 2020 | WO | international |
| PCT/CN2021/106481 | Jul 2021 | WO | international |
| PCT/CN2021/116077 | Sep 2021 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/118679 | 9/16/2021 | WO |
