Patent Application
20200079860
For the purpose of only those jurisdictions that permit incorporation by reference, all of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention.
Immune checkpoint blockade (ICB) is an approach to treating cancer that involves blocking inhibitory immune-cell receptors, such as PD-1, PD-L1, and/or CTLA-4, present on T-cells. Several such immune checkpoint inhibitors are currently in use clinically—including pembrolizumab, nivolumab, atezolizumab, and ipilimumab. While such methods can lead to durable and occasionally complete tumor regression in some patients, other patients remain insensitive to such treatments. For example, response rates to anti-PD-1 monotherapy range from approximately 44% in melanoma patients to markedly lower rates in breast and colorectal cancer patients. Accordingly, there is a need in the art for new and improved treatment regimens that can be used to treat tumors in that subset of patients for which immune checkpoint inhibitors are not effective.
The present invention is based, in part, on a series of important discoveries that are described in more detail in the Examples section of this patent specification. For example, it has now been discovered that certain combinations of agents, such as CD40 agonists and TLR agonists, can be used to treat tumors. Furthermore, it has been found that such combinations of agents can be used to sensitize tumor cells to treatment with immune checkpoint inhibitors, such as PD-1, PD-L1, and/or CTLA-4 inhibitors, leading to complete tumor regression, even in tumors that were previously resistant to such treatments. Building on these discoveries, and other discoveries presented herein, the present invention provides a variety of new and improved compositions and methods for the treatment of tumors. Some of the main aspects of the present invention are summarized below. Additional aspects of the invention are provided and described in the Detailed Description, Drawings, Examples, and Claims sections of this patent application.
In some embodiments the present invention provides a method of treating a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of: (a) a CD40 agonist (such as a CD40 agonist antibody) and (b) a TLR agonist. Similarly, in some embodiments the present invention provides a method of treating a tumor in a subject in need thereof, comprising administering to the subject an effective amount of: (a) a CD40 agonist (such as a CD40 agonist antibody), (b) a TLR agonist and (c) an immune checkpoint inhibitor (such as a PD-1, PD-L1, or anti-CTLA-4 inhibitor). Furthermore, each of the above embodiments may also comprise administering to the subject an effective amount of an IL10 receptor-blocking antibody or an IL10-blocking antibody. Similarly, each of the above embodiments may also comprise administering to the subject an effective amount of a vaccine adjuvant, or a vaccine antigen.
In each of the treatment methods of the present invention the various different active agents, or combinations thereof, can be administered either systemically or locally or a combination of both. Suitable routes of local administration include, but are not limited to, intratumoral, intrahepatic, intrapleural, intraocular, intraperitoneal, and intrathecal administration.
In some preferred embodiments the CD40 agonist (e.g. CD40 agonist antibody), the TLR agonist, and/or the IL10 receptor-blocking antibody or IL10 blocking antibody is administered locally, such as intratumorally. However, in other embodiments the CD40 agonist, the TLR agonist, and/or the IL10 receptor-blocking antibody/IL10-blocking antibody is administered systemically.
In some preferred embodiments the immune checkpoint inhibitor (such as an anti-PD-1, anti-PD-L1, or anti-CTLA-4 agent) is administered systemically. However, in other embodiments the immune checkpoint inhibitor is administered locally, such as intratumorally.
In some such embodiments the subject has a tumor that is resistant to treatment with an immune checkpoint inhibitor. In some such embodiments the subject has a PD-1, PD-L1, and/or CTLA-4 inhibitor resistant tumor. In some such embodiments the subject has previously been treated with an immune checkpoint inhibitor (such as a PD-1, PD-L1, or CTLA-4 inhibitor). In some such embodiments that patient has not previously been treated (with immunotherapy, checkpoint blockade, or otherwise). In some such embodiments the tumor is any solid tumor, including, but not limited to, a melanoma, a breast tumor, a lung tumor (such as a small cell lung cancer tumor), a prostate tumor, an ovarian tumor, a sarcoma, and a colon tumor.
In some embodiments the present invention provides various compositions, such as pharmaceutical compositions, that may be useful in the above methods. For example, in some embodiments the present invention provides compositions, such as pharmaceutical compositions, comprising: (a) a CD40 agonist (such as a CD40 agonist antibody), and (b) a TLR agonist, or compositions comprising any other combination of the active agents described (i.e. CD40 agonists, TLR agonists, IL10 receptor blocking antibodies/IL10 blocking antibodies, or immune checkpoint inhibitors (such as PD-1, PD-L1, and/or CTLA-4 inhibitors). In some such embodiments the compositions also comprise a vaccine adjuvant, or a vaccine antigen.
In some such embodiments the CD40 agonist (e.g. CD40 agonist antibody) and the TLR agonist, or any one or more of the active agents described above (i.e. CD40 agonists, TLR agonists, IL10 receptor or IL10 blocking antibodies, or immune checkpoint inhibitors), are connected via a linker moiety to form a single molecule, such as an antibody-drug conjugate molecule. In some such embodiments the agents may be connected using a lysine-bound linker or a cysteine-bound linker.
In some such embodiments any one or more of the active agents described above (i.e. CD40 agonists, TLR agonists, IL10 receptor blocking antibodies, or immune checkpoint inhibitors) may be provided together using a nanoparticle. For example, in some embodiments the CD40 agonist (e.g. CD40 agonist antibody) and the TLR agonist are provided together in a nanoparticle. Similarly in some embodiments the CD40 agonist (e.g. CD40 agonist antibody), the IL10 receptor blocking antibody, and the TLR agonist are provided together in a nanoparticle. In some such embodiments the CD40 agonist (e.g. CD40 agonist antibody) and/or the IL10 receptor-blocking antibody (or IL10-blocking antibody) is present on the surface of the nanoparticles. In particular it has been found that the nanoparticles of the invention are particularly effective when an IL10 receptor-blocking antibody is provided on the surface of the nanoparticles (e.g. in addition to a CD40 agonist antibody). However, in other embodiments these agents can be included inside nanoparticles—as cargo. In some such embodiments the TLR agonist and/or the immune checkpoint inhibitor (such as PD-1, PD-L1, and/or CTLA-4 inhibitor) is present inside the nanoparticles—i.e. as the “cargo” within the nanoparticle. In particular it has been found that the nanoparticles of the invention are particularly effective when the TLR3 agonist polyIC is provided as “cargo” within the nanoparticles. However, in other embodiments these agents can be used on the surface of the nanoparticles. The nanoparticles of the present invention can comprise the various active agents in any location—i.e. either coated on the surface of the nanoparticles or inside the nanoparticles.
In some such embodiments the nanoparticle is made using any suitable nanoparticle chemistry or technology known in the art. In some such embodiments the nanoparticle comprises one or more agents selected from the group consisting of mannose, chitosan, manosylated chitosan, protamine, chitosan with protamine, albumin, PLGA, and fucoidan. In some such embodiments the nanoparticles are formulated to release the active agent within them (i.e. their cargo) at endosomal pH, for example at the pH of early endosomes. The pH sensitivity of the nanoparticles can be adjusted (e.g., by adjusting their density) so the nanoparticles can be made to degrade within the acidic endosomes of APCs. In some the chemical features or physical properties (e.g., size, charge, etc) of the nanoparticles can be controlled such that systemic administration will lead to enrichment of the nanoparticles in certain organs of interest (e.g., the liver in the case of tumors within the liver or the lung in the case of tumors within the lungs). Means for altering the chemical or physical properties of nanoparticles to allow for tissue-specific enrichment are known in the art and can be used in connection with the present invention. For example, it is known that galactosamine-modified polymers can be used to target asiolaglycoprotein-receptor overexpressed by liver cells as a means for targeted delivery to the liver. See Seymour et al., “Hepatic drug targeting: phase I evaluation of polymer-bound doxorubicin,” J. Clin. Oncol. 2002, Vol. 20(6), pp. 1668-76, the contents of which are hereby incorporated by reference.
In those embodiments where nanoparticles are used to deliver the active agents of the invention, it has been found the nanoparticle compositions may be delivered using any suitable route of administration—whether local or systemic. However, in preferred embodiments intravenous administration is used. In particular, it has been found that the nanoparticle compositions of the invention are particularly potent when administered intravenously, such that the nanoparticles can be administered intravenously at approximately the same (low) dose with which they are administered intratumorally.
In some embodiments the CD40 agonist used in the methods and compositions described herein is selected from the group consisting of the following antibodies: FGK45, CP-870,984, APX005M, dacetuzumab, ChiLob 7/4, a CD40 agonist antibody as described in WO2005/063289, and a CD40 agonist antibody as described in WO2013/034904.
In some embodiments the TLR agonist used in the methods and compositions described herein is any TLR agonist known in the art that binds to a TLR expressed by an antigen presenting cell (APC), such as a dendritic cell (DC), macrophages, tissue-resident macrophages, monocytes, monocyte-derived cells, B-Cells, neutrophils, langerhans cells, histiocytes, or any so-called professional or non-professional APC. In some embodiments the TLR agonist is a TLR4 agonist, such as monophosphoryl lipid A (MPL). In some embodiments the TLR agonist is a TLR3 agonist, such as polyI:C.
In some embodiments the immune checkpoint inhibitor (including but not limited to PD-1, PD-L1, and/or CTLA-4 inhibitor) used in the methods and compositions described herein is an antibody. In some such embodiments the immune checkpoint inhibitor is an antibody selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, ipilimumab, and the PD-1 inhibitor antibody RMP1-14.
In some embodiments the IL10 receptor blocking antibody used in the methods and compositions described herein is the antibody 1B1.3A.
These and other embodiments are further described in other sections of this patent application. Furthermore, one of skill in the art will recognize that the various embodiments of the present invention described can be combined in various different ways, and that such combinations are within the scope of the present invention.
The two lower graphs provide averaged tumor growth curves for tumors treated as shown in the key. In each graph tumor size in mm2 is represented on the Y axis and time in days is indicated on the X axis. In all nanoparticle formulations MPL molecules are packaged inside the nanoparticles and anti-CD40 mAbs coated on the nanoparticle surface. As compared to the control group in which animals received systemic anti-PD-1 and intratumoral MPL and anti-CD40 (“non-formulated”), animals that received systemic anti-PD-1 and MPL with anti-CD40 co delivered as a nanoparticle (with MPL as the cargo and anti-CD40 on the surface of the nanoparticle) demonstrated improved control of injected and non-injected tumors.
While some of the main embodiments of the present invention are described in the above Summary of the Invention section of this patent application, as well as in the section of this application, this Detailed Description section provides certain additional description relating to the compositions and methods of the present invention, and is intended to be read in conjunction with all other sections of the present patent application.
Definitions and Abbreviations
As used herein the abbreviation “APC” refers to an Antigen Presenting Cell.
As used herein the abbreviation “CD40” refers to a cluster of differentiation 40—a receptor that may be found on APCs, where it is involved in stimulating APC activation.
As used herein the abbreviation “DC” refers to a Dendritic Cell
As used herein the abbreviation “IL10” refers to interleukin 10.
As used herein the abbreviation “IL10R” refers to an IL10 receptor, such as an IL10R present on APCs. The term “IL10R” include any and all subunits of the IL10 receptor, including, but not limited to, IL10RA, IL10RB, IL10R1, and IL10R2.
As used herein the abbreviation “IP” refers to intraperitoneal.
As used herein the abbreviation “IT: refers to intratumoral. For example a drug injected directly into a tumor is delivered intratumorally.
As used herein the abbreviation “IV” refers to intravenous. It is common to administer agents to mice via an IP route, which is considered to be analogous to administering an agent to a human subject by a IV route.
As used herein the abbreviation “MPL” refers to monophosphoryl lipid A. MPL is a TLR4 agonist.
As used herein the abbreviation PD-1” refers to Programmed Death 1, which is also known as Programmed Death Protein 1 or Programmed Cell Death Protein 1.
As used herein the abbreviation PD-L1 refers to a ligand for PD-1.
As used herein the abbreviation “TLR” refers to Toll-like receptor(s). TLRs on APCs are involved in stimulating APC activation.
As used herein the terms “inhibiting” and “blocking” are used interchangeably, as are the terms “inhibit” or “block” and the terms “inhibitor” or “blocker.”
As used herein, the terms “about” and “approximately,” when used in relation to numerical values, mean within + or −20% of the stated value. Other terms are defined elsewhere in this patent specification, or else are used in accordance with their usual meaning in the art.
Other abbreviations and definitions may be provided elsewhere in this patent specification, or may be well known in the art.
Active Agents for Use in the Compositions and Methods of the Invention
As described in the Summary of the Invention and other sections of this patent application, the methods and compositions provided by present invention involve various different active agents, including, but not limited to, CD40 agonist s (e.g. CD40 agonist antibodies), TLR agonists, immune checkpoint inhibitors (such as immune checkpoint inhibitor antibodies, PD-1 inhibitors (such as PD-1 inhibitor antibodies), PD-L1 inhibitors (such as PD-L1 inhibitor antibodies), CTLA-4 inhibitors (such as CTLA-4 inhibitor antibodies), and IL10 receptor blocking antibodies. Each of the embodiments described herein that involves one or more of such active agents, such as those known in the art (including, but not limited to the specific exemplary agents described herein), can, in some embodiments, be carried out using any suitable analogues, homologues, variants, or derivatives of such agents. Such analogues, homologues, variants, or derivatives should retain the key functional properties of the specific molecules described herein. For example, in the case of the CD40 agonist antibodies, any suitable analogue, homologue, variant, or derivative of such an antibody can be used provided that it retains CD40 agonist activity. In the case of the TLR agonists, any suitable analogue, homologue, variant, or derivative of such an agent can be used provided that it retains TLR agonist activity. In the case of PD-1 inhibitors, any suitable analogue, homologue, variant, or derivative of such an agent can be used provided that it retains PD-1 inhibitory activity. In the case of PD-L1 inhibitors, any suitable analogue, homologue, variant, or derivative of such an agent can be used provided that it retains PD-L1 inhibitory activity. In the case of CTLA-4 inhibitors, any suitable analogue, homologue, variant, or derivative of such an agent can be used provided that it retains CTLA-4 inhibitory activity.
Similarly, in the case of IL10 receptor blocking antibodies, any suitable analogue, homologue, variant, or derivative of such an agent can be used provided that it retains IL10 receptor blocking activity.
Several embodiments of the present invention involve antibodies. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibody, nanobody, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked, or conjugated to other molecules such as toxins, radioisotopes, or any of the other specific molecules recited herein.
The term “humanized antibody” refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region (FW) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.
Humanized antibodies can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, humanized antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. Nos. 5,225,539 or 5,639,641.
The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.
A “monoclonal antibody” (mAb) refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to “polyclonal antibodies” that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.
In particular, monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay (MA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated either in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid.
Alternatively, monoclonal antibodies can be made using recombinant DNA methods, as described in U.S. Pat. No. 4,816,567. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).
Polyclonal antibodies can be produced by various procedures well known in the art. For example, a host animal such as a rabbit, mouse, rat, etc. can be immunized by injection with an antigen to induce the production of sera containing polyclonal antibodies specific for the antigen. The antigen can include a natural, synthesized, or expressed protein, or a derivative (e.g., fragment) thereof. Various adjuvants may be used to increase the immunological response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art. Antibodies can be purified from the host's serum.
Conjugated Agents for Use in the Compositions and Methods of the Invention
Several embodiments of the present invention involve an antibody-drug conjugate molecule comprising a CD40 agonist (e.g. a CD40 agonist antibody) and a TLR agonist, linked together via a linker moiety. Any suitable CD40 agonist and TLR agonist known in the art or described herein can be used. Similarly, any suitable linker moiety can be used to connect the CD40 agonist to the TLR agonist. Several such linkers are known in the art, such as those that are conventionally used in the production of antibody-drug conjugates. In some embodiments the linker is a lysine-bound linker, such as, for example, the “SMCC” linker that is commercially available from ImmunoGen. In some embodiments the linker is a cysteine-bound linker, such as, for example, the “vc-pABC” linker that is commercially available from Seattle Genetics.
Compositions.
In certain embodiments, the present invention provides compositions, such as pharmaceutical compositions. The term “pharmaceutical composition,” as used herein, refers to a composition comprising at least one active agent as described herein, and one or more other components useful in formulating a composition for delivery to a subject, such as diluents, buffers, carriers, stabilizers, dispersing agents, suspending agents, thickening agents, excipients, preservatives, and the like.
Some of the compositions, such as pharmaceutical compositions, described herein comprise two or more of the active agents described herein. In some of such embodiments the two or more agents may, optionally, be provided: adsorbed to the surface of alum, or within an emulsion, or within a liposome, or within a micelle, or within a polymeric scaffold, or adsorbed to the surface of, or encapsulated within, a polymeric particle, or within an immunostimulating complex or “iscom,” or within charge-switching synthetic adjuvant particle (cSAP), or within PLGA: poly(lactic-co-glycolic acid) particles, or within other nanoparticles suitable for pharmaceutical administration.
In those embodiments of the present invention that involve nanoparticles, any suitable nanoparticle chemistry or nanoparticle technology known in the art may be used. In some embodiments the nanoparticles may comprise one or more agents selected from the group consisting of mannose, chitosan, manosylated chitosan, protamine, chitosan with protamine, albumin, PLGA, and fucoidan. In some embodiments the nanoparticles may comprise a CD40 agonist (e.g. CD40 agonist antibody) on the surface of the nanoparticle. In some embodiments the nanoparticles may comprise an IL10 receptor-blocking antibody on the surface of the nanoparticle. In some embodiments the nanoparticles may comprise a TLR agonist within the nanoparticle. In some embodiments the nanoparticles may comprise an immune checkpoint inhibitor (such as a PD-1 inhibitor or PD-L1 inhibitor or CTLA-4 inhibitor) within the nanoparticle. In some embodiments the nanoparticles may comprise any combination of the above agents on the surface on or within the nanoparticles.
Methods of Treatment
In certain embodiments the present invention provides methods of treatment. As used herein, the terms “treat,” “treating,” and “treatment” encompass a variety of activities aimed at achieving a detectable improvement in one or more clinical indicators or symptoms associated with a tumor. For example, such terms include, but are not limited to, reducing the rate of growth of a tumor (or of tumor cells), halting the growth of a tumor (or of tumor cells), causing regression of a tumor (or of tumor cells), reducing the size of a tumor (for example as measured in terms of tumor volume or tumor mass), reducing the grade of a tumor, eliminating a tumor (or tumor cells), preventing, delaying, or slowing recurrence (rebound) of a tumor, improving symptoms associated with tumor, improving survival from a tumor, inhibiting or reducing spreading of a tumor (e.g. metastases), and the like.
The term “tumor” is used herein in accordance with its normal usage in the art and includes a variety of different tumor types. It is expected that the present methods and compositions can be used to treat any solid tumor. Suitable tumors that can be treated using the methods and compositions of the present invention include, but are not limited to, melanomas, lung tumors, colon tumors, prostate tumors, ovarian tumors, sarcomas, and breast tumors, and the various other tumor types mentioned in the present patent specification.
In carrying out the treatment methods described herein, any suitable method or route of administration can be used to deliver the active agents or combinations thereof described herein. In some embodiments systemic administration may be employed, for example, oral or intravenous administration, or any other suitable method or route of systemic administration known in the art. In some embodiments intratumoral delivery may be employed. For example, the active agents described herein may be administered directly into a tumor by local injection, infusion through a catheter placed into the tumor, delivery using an implantable drug delivery device inserted into a tumor, or any other means known in the art for direct delivery of an agent to a tumor.
As used herein the terms “effective amount” or “therapeutically effective amount” refer to an amount of an active agent as described herein that is sufficient to achieve, or contribute towards achieving, one or more desirable clinical outcomes, such as those described in the “treatment” description above. An appropriate “effective” amount in any individual case may be determined using standard techniques known in the art, such as dose escalation studies, and may be determined taking into account such factors as the desired route of administration (e.g. systemic vs. intratumoral), desired frequency of dosing, etc. Furthermore, an “effective amount” may be determined in the context of any co-administration method to be used. One of skill in the art can readily perform such dosing studies (whether using single agents or combinations of agents) to determine appropriate doses to use, for example using assays such as those described in the Examples section of this patent application—which involve administration of the agents described herein to subjects (such as animal subjects routinely used in the pharmaceutical sciences for performing dosing studies).
For example, in some embodiments the dose of an active agent of the invention may be calculated based on studies in humans or other mammals carried out to determine efficacy and/or effective amounts of the active agent. The dose amount and frequency or timing of administration may be determined by methods known in the art and may depend on factors such as pharmaceutical form of the active agent, route of administration, whether only one active agent is used or multiple active agents (for example, the dosage of a first active agent required may be lower when such agent is used in combination with a second active agent), and patient characteristics including age, body weight or the presence of any medical conditions affecting drug metabolism.
In those embodiments described herein that refer to specific doses of agents to be administered based on mouse studies, one of skill in the art can readily determine comparable doses for human studies based on the mouse doses, for example using the types of dosing studies and calculations described herein.
In some embodiments suitable doses of the various active agents described herein can be determined by performing dosing studies of the type that are standard in the art, such as dose escalation studies, for example using the dosages shown to be effective in mice in the Examples section of this patent application as a starting point. Interestingly, and as illustrated in the Examples, it has been found that the methods and compositions of the present invention are, effective using much lower doses of the active agents than would normally be used in other applications and contexts. In some embodiments, where the active agents used are antibodies, the agents are administered at a dose of from about 1 mg/kg to about 10 mg/kg, or at a dose of from about 0.1 mg/kg to about 10 mg/kg.
Dosing regimens can also be adjusted and optimized by performing studies of the type that are standard in the art, for example using the dosing regimens shown to be effective in mice in the Examples section of this patent application as a starting point. In some embodiments the active agents are administered daily, or twice per week, or weekly, or every two weeks, or monthly.
In certain embodiments the compositions and methods of treatment provided herein may be employed together with other compositions and treatment methods known to be useful for tumor therapy, including, but not limited to, surgical methods (e.g. for tumor resection), radiation therapy methods, treatment with chemotherapeutic agents, treatment with antiangiogenic agents, or treatment with tyrosine kinase inhibitors. Similarly, in certain embodiments the methods of treatment provided herein may be employed together with procedures used to monitor disease status/progression, such as biopsy methods and diagnostic methods (e.g. MRI methods or other imaging methods).
For example, in some embodiments the agents and compositions described herein may be administered to a subject prior to performing surgical resection of a tumor, for example in order to shrink a tumor prior to surgical resection. In other embodiments the agents and compositions described herein may be administered both before and after performing surgical resection of a tumor. In other embodiments the subject has no tumor recurrence after the surgical resection.
Subjects
As used herein the term “subject” encompasses all mammalian species, including, but not limited to, humans, non-human primates, dogs, cats, rodents (such as rats, mice and guinea pigs), cows, pigs, sheep, goats, horses, and the like—including all mammalian animal species used in animal husbandry, as well as animals kept as pets and in zoos, etc. In preferred embodiments the subjects are human. Such subjects will typically have (or previously had) a tumor (or tumors) in need of treatment. In some embodiments the subject has previously been treated with an immune checkpoint inhibitor (such as a PD-1 inhibitor, PD-L1 inhibitor, or a CTLA-4 inhibitor). In some embodiments the subject has not previously been treated with an immune checkpoint inhibitor (such as a PD-1 inhibitor, PD-L1 inhibitor, or a CTLA-4 inhibitor). In some embodiments the subject has a tumor that is insensitive to, or resistant to, treatment with an immune checkpoint inhibitor (such as a PD-1 inhibitor, PD-L1 inhibitor, or a CTLA-4 inhibitor), or that is suspected of being insensitive to, or resistant to, treatment with an immune checkpoint inhibitor (such as a PD-1 inhibitor, PD-L1 inhibitor, or a CTLA-4 inhibitor). In some embodiments the subject has a tumor that has recurred following a prior treatment with an immune checkpoint inhibitor (such as a PD-1 inhibitor, PD-L1 inhibitor, or a CTLA-4 inhibitor) and/or with one or more other tumor treatment methods, including, but not limited to, chemotherapy, radiation therapy, or surgical resection, or any combination thereof. In some embodiments the subject has a tumor that has not previously been treated, whether with an immune checkpoint inhibitor (such as a PD-1 inhibitor, PD-L1 inhibitor, or a CTLA-4 inhibitor) or with one or more other tumor treatment methods, including, but not limited to, chemotherapy, radiation therapy, or surgical resection, or any combination thereof.
The invention is further described in the following non-limiting Examples, as well as the Figures referred to therein.
In each of these Examples, and unless stated otherwise, the indicated active agents were administered at the following doses: MPL—5 μg, anti-CD40—20 μg, anti-PD-1—250 μg, anti-IL10R—100 μg—regardless of the delivery route or formulation used. Also, unless indicated otherwise, controls were treated with isotype control antibodies and vehicle only. Furthermore, unless indicated otherwise, all experiments were performed using the mouse bilateral tumor model described below and summarized in
Immune checkpoint blockade (for example using anti-CTLA-4, PD-1, and PD-L1 monoclonal antibodies (mAbs)) offers the potential for durable remissions for patients across a broad range of cancers, including, but not limited to, lung, breast, colon and prostate cancer. However, despite this broad applicability, the majority (well over 80%) of cancer patients are, or become, resistant to it. The studies presented in this Example demonstrate a novel approach to overcome resistance to immune checkpoint blockade in manner applicable to most cancers, regardless of type or stage.
Cancers refractory to immune checkpoint blockade generally fail to mount significant antitumor T lymphocyte responses. Many cancers, including breast and colon cancer demonstrate defective antigen presenting cell (APC) activation. Since APCs prime T lymphocytes, this can explain the absence of a productive anti-tumor T lymphocyte response in these cancers.
We hypothesized that enforced activation of tumor-associated APCs, by directly injecting tumors, could potentially convert an individual tumor and/or lymphoid organs into ‘cellular factories’ of primed anti-tumor T lymphocytes that could then, potentially, recognize and kill cancer cells throughout the body, and induce direct tumor cell killing by activating innate immune cells (including APCs) at the tumor site.
A number of rationally-selected combinations of agents were chosen and tested in a murine model of aggressive melanoma, shown to be resistant to checkpoint blockade, with the aim of testing this hypothesis and identifying treatments with potent anti-tumor activity. The animals used had large tumors in two opposite flanks. One tumor was injected while the second remained non-injected (
Mice were treated with MPL (intratumoral) at 5 μg, anti-CD40 (FGK45/FGK4.5, intratumoral) at 20 μg, and anti-PD-1 (RMP1-14, systemically via intraperitoneal injection) at 250 μg, while control mice were treated with isotype control antibodies and vehicle only. It was found that the combination of low dose anti-CD40 and MPL in the setting of systemic anti-PD-1: (A) yielded no discernible toxicity, (B) consistently eradicated injected tumors, (C) controlled or eradicated large non-injected tumors (
Studies were next performed to determine whether conjugation of the anti-CD40 mAb and MPL, for example using nanoparticle technology (
Furthermore, additional studies demonstrated successful conversion of a living tumor into a ‘cellular factory’—which produces lymphocytes that, in turn, infiltrate and regress distant non-injected tumors. In particular, it was found that injected tumors were rapidly infiltrated with neutrophils—an important class of APCs (
Additional experiments were performed to determine whether the treatments outlined above might also be effective in other tumors and in other organs. Studies were performed using orthotopic lung cancer, sarcoma, and ovarian cancer models—as described in subsequent Examples. In each case potent treatment activity was observed.
Additional experiments were also performed to determine whether the agents could be effective systemically as well as intratumorally. Robust activity was also observed when the active agents were administered intravenously (IV) instead of intratumorally at the equivalent dose—as shown in other Examples.
Importantly, the compositions and methods described herein constitute an “off-the-shelf” method of priming and expanding tumor-specific T cells trained to recognize the patient's own tumor as it exists in the body and changes over time. This is in contrast to many other so-called “customized” approaches (e.g., vaccine, transgenic-T-cell, and CAR-T-cell therapy)—which instead often rely on directing lymphocytes to pre-defined targets associated with specific cancers. The treatment approaches described herein may therefore be less costly to produce, and more broadly applicable (for example across multiple cancer types and patients).
As shown in Example 1, injecting tumors with low-dose CD40 agonist mAb and MPL can synergize with intraperitoneal (IP) PD-1 mAb to treat cancer in an aggressive murine melanoma model, as well as other cancer models. The present Example extends upon the studies provided in Example 1 and provides data showing that intratumoral administration of a low-dose of CD40 agonist mAb and TLR4 agonist (MPL) can also synergize with intratumoral IL10R mAb—either alone or together with a PD-1 mAb—to treat cancer in the same B16 murine melanoma model. Experiments were performed to test the effects of intratumoral CD40 agonist mAb and intratumoral MPL in combination with either (A) IT IL10R mAb (
In experiments similar to those described in Example 1, C57BL/6 mice were initially implanted with 5×105 syngeneic B16F10 cells intradermally in bilateral flanks. 8 days post tumor cell implantation, when bilateral tumors measured ˜0.5 cm, intratumoral (IT) treatment with agents selected to activate APCs was initiated together with intraperitoneal (IP) anti-PD-1 mAb. Treatment was administered twice weekly for 4 weeks into one of the bilateral tumors. The contralateral tumor remained un-injected for the duration of the experiment (see
In other similar experiments the combination of intratumoral (IT) MPL, IT CD40 agonist mAb, and IT IL10R blocking mAb was tested using the same experimental methodology as described above and shown in
Experiments were also performed using a combination of intratumoral MPL, intratumoral CD40 agonist mAb, intratumoral IL10 receptor blocking mAb, and systemic anti-PD-1 delivered intraperitoneally—once again using the same experimental system described in
Although the data presented here uses a melanoma model, the treatments and mechanisms of action are not cancer-type specific and, as illustrated in Example 1, are expected to translate to all cancer types.
The present Example relates to experiments similar to those provided in the preceding Examples, but that were performed utilizing nanoparticle technology to deliver an anti-CD40 antibody and MPL in physical association with one another and to test several different nanoparticles—i.e. those containing chitosan, chitosan with protamine, albumin, mannose, PLGA, or fucoidan. The anti-CD40 antibody was coated onto the surface of the nanoparticles and the TLR agonist MPL was included as cargo inside the nanoparticles. The anti-CD40/MPL nanoparticles were tested in the same bilateral mouse tumor models described in the previous Examples.
Nanoparticles were produced using ionotropic gelation such that each intratumoral injection delivered 5 μg MPL (as nanoparticle cargo) and 20 μg anti-CD40 mAb FGK45 (on the nanoparticle surface). These nanoparticles were administered to animals receiving 250 μg intraperitoneal anti-PD-1 (RMP1-14) concurrently. Non-formulated control animals received mixtures of MPL (5 μg) and FGK45 (20μg) injected intratumorally with RMP1-14 delivered intraperitoneally. Anti-PD-1-only control animals received 250 μg of intraperitoneal RMP1-14 alone. Isotype/vehicle control animals received isotype control mAbs and vehicle corresponding to the nanoparticle-treated group.
Results from such experiments are shown in
Certain additional experiments were performed to expand upon the studies described above in the previous Examples, as follows:
Experiments were performed in which animals were treated with intratumoral MPL (5μg), the CD40 agonist mAb FGK45 (20μg), and intraperitoneal anti-PD-1 mAb RMP1-14 (250 μg) (see
Further experiments provided data showing that pigmented dendritic melanophages accumulate in the T-cell rich splenic peri-arterial lymphatic sheath 24 hours after a single treatment with intratumoral MPL (5μg), FGK45 (20μg), and anti-IL10R mAb 1B1.3A (100μg). See
Further experiments were performed using the bilateral tumor model, referred to elsewhere herein, with different tumor cell lines. C57BL/6 animals were challenged bilaterally with ovarian carcinoma ID8 syngeneic tumor cells. Established tumors were treated with intratumoral MPL (5μg), FGK45 (20μg), and anti-IL10R mAb 1B1.3A (100μg) and intraperitoneal anti-PD-1 mAb RMP1-14 (250 μg). Control animals received either intraperitoneal RMP1-14 alone or isotype mAb in vehicle. These data demonstrate that intratumoral treatment with MPL, anti-CD40, and anti-IL10R with IP anti-PD-1 provides superior local and distal tumor control compared with the two control groups. The results of these experiments are provided in
In additional experiments C57BL/6 animals were challenged intravenously (IV) with syngeneic HKP (krasG12D/+, p53f/f) lung carcinoma cells. Once bilateral lung tumors were established animals were treated once weekly for four weeks, and luminescence was assayed to monitor tumor growth. Animals received either isotype control mAbs, non-formulated mixtures of intravenous MPL (5 μg) and FGK45 (20 μg) together with 250 μg of RMP1-14 delivered intraperitoneally, or intravenous MPL (5 μg) and FGK45 (20 μg) formulated as a chitosan nanoparticle as described above together with 250 μg of RMP1-14 delivered intraperitoneally. The results of these experiments are shown in
Experiments were also performed to assess the effects of different surface antibodies and different cargo molecules in the context of the nanoparticles described herein.
Experiments were also performed to assess the contribution of each agent in the regimen consisting of intratumoral MPL (5 μg per injection), intratumoral anti-CD40 mAb FGK45 (20 micro-grams per injection) and intraperitoneal anti-PD-1 mAb (250 μg per injection). The results of these experiments are presented in
Experiments were performed to determine if anti-CD40 and/or anti-IL10R treatments could be effective systemically as well as intratumorally. As shown in
Experiments were also performed to test whether other TLR agonists could be used in the treatment methods and compositions of the invention. As shown in
Experiments were also performed to test the effects of using the treatment methods and compositions of the present invention together with chemotherapy. As shown in
Experiments were also performed to investigate resistance of previously-treated mice to later tumor re-challenge. It was found that mice treated with a regimen of intratumoral MPL, anti-CD40, and anti-IL10R (which eradicated injected tumors as shown in
Experiments were also performed to determine if the treatment regimens described herein could also be useful in the context of treatment with the immune checkpoint inhibitor anti-CTLA-4.
Experiments were also performed to determine if the anti-tumor activity observed at non-injected tumors using the methods and compositions of the invention is mediated by the adaptive immune system. If so, the antitumor activity should be lost in RAG1 knockout mice. Experiments were performed with control-treated animals, animals treated with systemic anti-PD-1, and intratumoral MPL and anti-CD40 (“440P”), animals treated with intratumoral MPL, anti-CD40, and anti-IL10R antibody (“41040”), and animals treated with systemic anti-PD-1, and intratumoral MPL, anti-CD40, and anti-IL10R (“41040P”). The anti-tumor activity manifested on non-injected tumors was virtually abolished in all treatment groups in the RAG knockout mice and survival was reduced—confirming that the effects are mediated by the adaptive immune system.
This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/202,163 filed on Aug. 6, 2015, and U.S. Provisional Patent Application No. 62/287,407 filed on Jan. 26, 2016, the contents of each of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/45970 | 8/8/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62202163 | Aug 2015 | US | |
| 62287407 | Jan 2016 | US |
