WO2017085566A1 - Methods and compositions for increase/induction of immune responses - Google Patents

Abstract

Methods of decreasing immune tolerance in a subject in need thereof, e.g. immune tolerance of cancer cells, are provided. The methods involve administering to the subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, such as an anti- IL34 antibody.

Description

METHODS AND COMPOSITIONS FOR INCREASE/INDUCTION OF IMMUNE

RESPONSES

FIELD OF THE INVENTION:

The invention is in the field of immunotherapy to treat cancer. More particularly, the invention relates to a method of induction of an immune response. In particular, the invention provides methods of increasing/inducing/unleashing an immune response in a subject by administering an interleukin 34 inhibitor to said subject.

BACKGROUND OF THE INVENTION:

The immune system is a system of many biological structures and processes within an organism that protects against disease, detecting and destroying a wide variety of pathogens, including viruses, bacteria, and parasites. Another very important role of the immune system is immune surveillance, i.e. the identification and elimination of tumor cells. The transformed cells of tumors express antigens that are not found on normal cells, and to the immune system, these antigens appear foreign. This causes the immune cells to attack and destroy the transformed tumor cells, e.g. using killer T cells, sometimes with the assistance of helper T cells. In addition, sometimes antibodies are generated against tumor cells allowing for their destruction by the complement system.

Unfortunately, some tumors evade the immune system and go on to become cancers. Evasion occurs via a variety of mechanisms. For example, tumor cells often have a reduced number of MHC class I molecules on their surface, thus avoiding detection by killer T cells. Some tumor cells also release products that inhibit the immune response; for example by secreting the cytokine TGF-β, which suppresses the activity of macrophages and lymphocytes. In addition, immunological tolerance may develop against tumor antigens, so the immune system no longer attacks the tumor cells.

In addition, other conditions, and even some medical treatments, cause unwanted suppression of the immune system and leave the suppressed host vulnerable to infection and susceptible to disease.

It would be of great benefit to have an available agent which is able to safely and reliably stimulate the immune system of a subject in whom unwanted immune suppression has or is likely to occur. SUMMARY OF THE INVENTION

The invention relates to a method of unleashing immune responses in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, wherein said therapeutically effective amount is sufficient to decrease immune tolerance in the subject. In particular, the invention is defined by claims.

DETAILED DESCRIPTION

The present invention was developed in view of the surprising observation that inhibiting the activity of the cytokine IL34 results in an inhibition of the function of CD4⁺ and

CD8⁺ T regulatory cells (Tregs), thereby increasing/eliciting an immune response towards unwanted cells. One application of this finding is the use of an agent that inhibits or attenuates IL34 activity (e.g. an anti-IL34 antibody) to increase immune system functioning, especially cell-mediated immunity, in a subject in need thereof. For example, administration of at least one IL34-inhibiting agent to a subject increases primary T cell responses in the subject and results in increased T cell detection and destruction of unwanted cells such as cancer cells, cells infected by various pathogens, etc. In other exemplary aspects, administration of at least one IL34-inhibiting agent to a subject increases primary T cell responses in the subject and results in a more effective response to a vaccination.

As further provided herein, the use of an agent that inhibits or attenuates IL34 activity

(e.g. an anti-IL34 antibody) unexpectedly increases/augments the ratio of CD4⁺CD25^" T cells to regulatory T cells or Tregs (CD8+CD45RClow and CD4+CD25+), effector cell responses such as the one for B cells or T cells by inducing the expansion of CD4⁺CD25^" T cells or B cells, without reducing the number of Tregs, thereby decreasing the frequency of Tregs in a proportionate manner. As further provided herein, an agent that inhibits or attenuates IL34 activity (e.g. an anti-IL34 antibody) is administered with a tumor-associated or other antigen of interest, which enhances the immune system's response to the antigen.

Methods for increasing/inducing/eliciting an immune response The present disclosure describes an effective and safe immunotherapy and provides details regarding how the immunosuppressive effects of regulatory T cells can be overcome in order to trigger helpful immune responses. This immunotherapy employs the use of an agent that inhibits or inactivates IL34, such as an anti-IL34 antibody, thereby increasing an immune response toward an antigen of interest such as a tumor-associated antigen, an infectious disease antigen or an antigen in a vaccine preparation.

The present invention provides an effective and safe way to unleash regulated (i.e. suppressed or inhibited) T cell immune responses by Treg (e.g. CD8⁺ and CD4⁺ Tregs).

The present invention provides also a method of eliciting a T cell response in a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, wherein said T-cell response comprises increasing the expansion of CD4⁺CD25^" T cells in said subject.

The method of the present invention is suitable to use in a subject in need thereof. Typically, the invention provides methods of unleashing immune responses in a subject in need thereof. The methods comprise administering to said subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, wherein said therapeutically effective amount is sufficient to unleash immune responses in the subject.

In a particular embodiment, the invention relates to a method of unleashing immune responses in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, wherein said therapeutically effective amount is sufficient to decrease immune tolerance in the subject.

As used herein, the term "unleashing" refers to realizing or increasing an immune response against harmful substances (e.g. molecules on the surface of cancer cells, viruses, fungi, or bacteria). In the context of the invention, the unleashing refers to the induction of CD4⁺CD25^" T cells expansion in the tumor microenvironment for example.

As used herein, the term "subject" refers to any mammals, such as a rodent (e.g rat), a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with a cancer.

As used herein, the term "an agent that inhibits or inactivates IL34 activity" denotes a compound which blocks IL34 activity in the immune system. IL34 inhibition may be via direct inhibition of the IL34 molecule itself (e.g. by binding directly to IL34 and preventing its activity) or by other means described in detail below, e.g. by preventing or decreasing production of IL34, etc. Exemplary IL34 inhibiting compounds include but are not limited to antibodies, small organic molecules, polypeptides, and nucleic acids.

In one aspect, the compound that inhibits IL34 is an antibody. Antibodies directed against IL34 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against IL34 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-IL34 single chain antibodies. Compounds useful in practicing the present invention also include anti-IL34 antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity for IL34.

Humanized anti-IL34 antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397). Then, for this invention, neutralizing antibodies of IL34 are selected. In one aspect, the compound according to the invention is an anti-IL34 antibody as described for clones of MAbs: 2HCLC, E033E8 , 1D12, 578416 from R&D Systems, BioLegend, Abeam®, etc.

In a particular embodiment, the agent that inhibits or inactivates IL34 is an anti-IL34 antibody, wherein said anti-IL34 does not have the sequences: SEQ ID No: l; SEQ ID No:2; SEQ ID No:3; SEQ ID No:4; SEQ ID No:5; SEQ ID No:6; SEQ ID No:7; SEQ ID No:8; SEQ ID No:9; SEQ ID No: 10; SEQ ID No:l l; SEQ ID No: 12; SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15 and SEQ ID No: 16 of WO2016/097420.

In a particular embodiment, the agent that inhibits or inactivates IL34 is a bispecific antibody or trispecific antibody. Bispecific antibodies, also known as bifunctional antibodies, have at least one antigen recognition site for a first antigen (e.g IL34) and at least one antigen recognition site for a second antigen (e.g another cytokine). Such antibodies can be produced by recombinant DNA methods or chemically by methods known in the art. Chemically created bispecific antibodies include but are not limited to antibodies that have been reduced and reformed so as to retain their bivalent characteristics and antibodies that have been chemically coupled so that they have at least two antigen recognition sites for each antigen. Bispecific antibodies include all antibodies or conjugates of antibodies, or polymeric forms of antibodies which are capable of recognizing two different antigens. Antibodies can be immobilized on a polymer or particle. Typically, in the context of the invention, such antibody binds to IL34 and another cytokines which decrease immune tolerance. In a particular embodiment, the agent that inhibits or inactivates IL34 is selected from the group consisting of a bispecific antibody which binds to IL34 and IL 10, a bispecific antibody which binds to IL34 and IFNg, a bispecific antibody which binds to IL34 and TGFP and a bispecific antibody which binds to IL34 and MCSF.

Trispecific antibodies have at least one antigen recognition site for a first antigen, at least one antigen recognition site for a second antigen and at least one antigen recognition site for a third antigen. Trispecific antibodies include all antibodies or conjugates of antibodies, or polymeric forms of antibodies which are capable of recognizing three different antigens. Antibodies can be immobilized on a polymer or particle. In a particular embodiment, the agent that inhibits or inactivates IL34 is selected from the group consisting of a tripecific antibody which binds to IL34, IFNg and MCSF; a tripecific antibody which binds to IL34, IFNg and IL10; a tripecific antibody which binds to IL34, IL10 and MCSF and a tripecific antibody which binds to IL34, TGFb and MCSF.

In one embodiment, the compound that inhibits IL34 is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then, for this invention, neutralizing aptamers of IL34 are selected.

In one embodiment, the compound according to the invention is a polypeptide that is capable of binding to IL34, thereby preventing its interaction with its receptors, CD115 and PTPz or any receptor of IL34. The compound is thus a "functional equivalent" of the receptor and includes fragments, mutants, and muteins of CD115 or PTPz. The term "functionally equivalent" thus includes any equivalent of CD115 or PTPz obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to IL34. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence. Functional equivalents include molecules that bind IL34 and comprise all or a portion of CD115 or PTPz. Thus, an inhibitory IL34 polypeptide is able to improve immune surveillance of unwanted cells through its properties of "decoy receptor". In other words, the "decoy receptor" traps IL34 and prevents its physiological effects on the immune system (e.g. prevents suppression of T cell activity). Polypeptides are produced by any of the many methods known in the art, e.g. synthetically, by genetic engineering using recombinant technology, etc.

In one aspect, the compound that inhibits IL34 is a low molecular weight compound, 5 e. g. a small organic molecule. The term "small organic molecule" refers to a molecule (naturally occurring or synthetic) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Particular small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to i o about 1000 Da.

In other aspects, the compound that is an inhibitor of IL34 is an inhibitor of IL34 gene expression. For example, small inhibitory R As (siRNAs) can function as inhibitors of IL34 expression. IL34 gene expression can be reduced or eliminated by contacting a subject or cell

15 with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that IL34 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA- encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus,

20 MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of IL34 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific

25 hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of IL34 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for

30 ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of IL34 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense R A molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing IL34. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. Particular viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non- cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Particular viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In some embodiments, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

In one aspect, IL34 gene expression may be inhibited or inactivated by a genome editing technology based on the use of an artificial nuclease (ex: CRISPR/cas, ZFNs, TALENs).

Genome editing using targetable nucleases is an emerging technology for the precise genome modification of organisms ranging from bacteria to plants and animals, including humans. Recent approaches to targeted genome modification - zinc-finger nucleases (ZFNs) and transcription-activator like effector nucleases (TALENs) - have enabled researchers to generate permanent mutations by introducing double-stranded breaks. More recently, a new tool based on a totally distinct and specific system, namely bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable interest.

To achieve site-specific DNA recognition and cleavage, Cas9 must be complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. The simplicity of the type II CRISPR nuclease, with only three required components (Cas9 along with the crRNA and trRNA) made this system amenable to adaptation for genome editing. This potential was realized in 2012 by the Doudna and Charpentier laboratories (Jinek et al., 2012, Science, Vol.337 : 816-821). Based on the type II CRISPR system described previously, a simplified two-component system was developed by combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA). The sgRNA programmed Cas9 was shown to be as effective as Cas9 programmed with separate trRNA and crRNA in guiding targeted gene alterations. As it is well known in the art, a nucleic acid is introduced into a cell by any of a variety of techniques (e.g., calcium phosphate co-precipitation, lipofection, electroporation).

As used herein, the term "decrease immune tolerance" refers to reduce the state of unresponsiveness of the immune system to substances or tissue that have the capacity to elicit an immune response. In the context of the invention, the use of the agent that inhibits or inactivates IL34 results in the inhibition of T regulatory cells, thus leads to a decrease of immune tolerance.

Method of treating cancers

The present invention provides a method of eliciting an anti-tumor T cell response in a subject having said tumor, comprising the step of administering to said subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, wherein said T- cell response comprises increasing the expansion of CD4⁺CD25^" T cells. In another embodiment, eliciting an anti-tumor T cell response in a subject having a tumor or cancer allows treating said tumor or cancer in said subject. In another embodiment, eliciting an antitumor T cell response in a subject having a tumor or cancer prevents the establishment of metastases in said subject.

Provided herein are methods of increasing/inducing/eliciting an immune response in a subject in need thereof. For example, the subject may have developed or may be likely to develop immune tolerance to unwanted cells such as cancer (e.g. tumor) cells, and/or cells that are infected with a pathogen, etc. The methods involve administering to the subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, such as an anti- IL34 antibody.

It is a further object of this invention to provide methods of treating cancer in a subject in need thereof. The methods comprise unleashing or increasing immune responses to cancer cells in said subject by administering to said subject a therapeutically effective amount of an anti-IL34 antibody, wherein said therapeutically effective amount is sufficient to unleash or increase immune responses to the cancer cells in the subject. In another aspect, the invention relates to a method of treating cancer in a subject in need thereof, comprising increasing immune responses to cancer cells in said subject by administering to said subject a therapeutically effective amount of an anti-IL34 antibody wherein said therapeutically effective amount is sufficient to unleash immune responses to the cancer cells in the subject.

Thus, the invention relates to an anti-IL34 antibody for use in a method of treating cancer in a subject in need thereof.

As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). The methods may involve a step of identifying a suitable subject for treatment, e.g. a subject in which IL34 has inhibited the subject's immune response. For example, such a subject may have IL34 levels in the range of from about 10 pg/ml to about 10 000 pg/ml. In some aspects, the amount of inhibitor that is administered decreases the amount of detectable IL34 to below about 10 pg/ml, and IL34 may be undetectable after treatment. Alternatively, IL34 may still be detectable but the level of its activity (measured by methods that are known to those of skill in the art) is decreased by treatment sufficiently to allow the subject to mount an immune response to the unwanted cells, or to a vaccinogen. The subject to whom the anti- IL34 agent is administered may or may not have an immune response that is below "normal", i.e. below that that is observed in healthy, non-diseased control individuals who are not suffering from immune repression. In some aspects, the subject is not suffering from measurable or detectable immune suppression, but it may still be desired to further increase immune function by the practice of the present methods. For example, the subject may be a cancer patient who is recently diagnosed and whose immune system appears to function normally, but for whom it would be beneficial to prophylactically stimulate immune functioning to fight the disease, e.g. before or in addition to other cancer treatment(s). Alternatively, the subject may have received or be about to receive a vaccination, and an increase in an immune system response to antigens in the vaccine is desired.

The precise protocol of administration is determined by a skilled professional such as a physician. Generally, administration of an IL34 antibody is carried out, for example, from about 1-4 time per day, but may be less frequent, e.g. once per day, once every 2, 3, 4, 5, or 6 days, or even one per week, or once every 2 to 3 weeks, or even once per month. Further, administration may occur via "rounds" of treatment, e.g. over a day or a period of several days, followed by a rest period when no agent is administered for several days, and then followed by another round of treatment for at least one day, etc. The total duration of treatment may be for several days (e.g. 2-6 days or a week), or for several weeks, or for several months, or even for several years, e.g. while a patient is battling cancer long term. If the agent is an antibody, the amount that is administered is generally in the range of from about 1 mg/kg to about 100 mg/kg mg/kg of body weight, and is preferably in the range of from about 2 mg/kg to about 20 mg/kg.

As used herein, the terms "cancer", "tumor", "cancerous" or malignant" in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. The present invention provides methods for treating diseases or conditions characterized by the presence of unwanted cells in the body of a host subject. In particular, the immune system of the subject that is treated as described herein is ineffective with respect to detecting and removing one or more types of unwanted cells. In some aspects, one or more elements of a T cell response mediated by IL34 is inhibited in the subject. In some aspects, if it were not for the immune suppression, the body's natural defense system would detect and destroy the unwanted cells, and the practice of the present invention, e.g. administration of an IL34 inhibitor to a subject, restores the ability of the subject's immune cells to detect and kill unwanted cells. In other aspects, the practice of the present invention, e.g. administration of an IL34 inhibitor to a subject, increases the ability of the subject's immune cells to detect and kill unwanted cells, whether or not immune suppression per se has occurred. "Unwanted cells" include, for example, cancer (tumor) cells of any type; cells that are infected with a pathogenic agent such as a virus, bacterium, protozoan; etc.

Exemplary diseases and/or conditions that may be treated by the methods described herein include but are not limited to cancers such as: Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma, AIDS-Related Lymphoma, Primary CNS Lymphoma, Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, , Ewing Sarcoma Family of Tumors, and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumor (e.g. Astrocytomas, Brain and Spinal Cord Tumors, Brain Stem Glioma, Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma), Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor, Gastrointestinal, Cardiac (Heart) Tumors, Central Nervous System (e.g. Atypical Teratoid/Rhabdoid Tumors, Embryonal Tumors, Germ Cell Tumors, Lymphomas), Cervical Cancer, Childhood Cancers, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ (DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, , Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Malignant, and Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney (Renal Cell, Wilms Tumor and Other Childhood Kidney Tumors), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lung Cancer (Non-Small Cell, Small Cell), Lymphoma, Macroglobulinemia, Waldenstrom - see Non-Hodgkin Lymphoma, Melanoma, Intraocular (Eye), Merkel Cell Carcinoma, Mesothelioma, Malignant, Metastatic Squamous Neck Cancer with Occult Primary Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplasia Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma, Multiple, Myeloproliferative Neoplasms, Chronic, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cancer, Oral Cavity Cancer, Lip and Oropharyngeal Cancer, and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor, Pancreatic Cancer, Pancreatic

Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (, Kaposi, , Rhabdomyosarcoma, Soft Tissue, Uterine), Sezary Syndrome, Skin Cancer, Small Intestine Cancer, Squamous Cell Carcinoma, Squamous Neck Cancer with Occult Primary, Metastatic Stomach (Gastric) Cancer, T-Cell Lymphoma, Cutaneous, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Ureter and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Vaginal Cancer, Vulvar Cancer, and Wilms Tumor, CNS metastases etc. In a particular embodiment, the following cancers are not treated in the context of the invention: osteosarcoma, Ewing sarcoma and bone cancer.

The IL34 inhibitor may be administered with or in conjunction (coordinated) with administration of one or more other agents. For example, one or more cancer treatment modalities may be administered with the agent. Such modalities include but are not limited to: various chemotherapy agents such as methotrexate, Pt-based anti-cancer agents, radiation therapy, etc.

In one aspect, the IL34 inhibitor may be administered with an antigen such as a tumor- associated antigen or an infectious disease antigen, for example, in a vaccine preparation.

In one embodiment, the antigen is a tumor-associated antigen. For instance, an antigen for use in the compositions and methods provided herein is one of the following tumor antigens: a MAGE (Melanoma- Associated Antigen E) protein, e.g. MAGE 1, MAGE 2, MAGE 3, MAGE 4, a tyrosinase; a mutant ras protein; a mutant p53 protein; p97 melanoma antigen, a ras peptide or p53 peptide associated with advanced cancers; the HPV 16/18 antigens associated with cervical cancers, KLH antigen associated with breast carcinoma, CEA (carcinoembryonic antigen) associated with colorectal cancer, gplOO, a MARTI antigen associated with melanoma, or the PSA antigen associated with prostate cancer.

In one embodiment, the antigen is an infectious disease antigen. For instance, an antigen for use in the compositions and methods provided herein is one antigen derived from the following pathogens: BCG/Tuberculosis, Malaria, Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Rotavirus, Cholera, Diptheria-Tetanus, Pertussis, Haemophilus influenzae, Hepatitis B, Human papilloma virus, Influenza seasonal), Influenza A (H1N1) Pandemic, Measles and Rubella, Mumps, Meningococcus A+C, Oral Polio Vaccines, mono, bi and trivalent, Pneumococcal, Rabies, Tetanus Toxoid, Yellow Fever, Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox) and other related pox viruses, Francisella tularensis (tularemia), Viral hemorrhagic fevers, Arena viruses (LCM, Junin virus, Machupo virus, Guanarito virus, Lassa Fever), Bunyaviruses (Hantaviruses, Rift Valley Fever), Flaviruses (Dengue), Filo viruses (Ebola , Marburg), Burkholderia pseudomallei, Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), Chlamydia psittaci (Psittacosis), Ricin toxin (from Ricinus communis), Epsilon toxin of Clostridium perfringens, Staphylococcus enterotoxin B, Typhus fever (Rickettsia prowazekii), other Rickettsias, Food- and Waterborne Pathogens, Bacteria (Diarrheagenic E.coli, Pathogenic Vibrios, Shigella species, Salmonella BCG/, Campylobacter jejuni, Yersinia enterocolitica), Viruses (Caliciviruses, Hepatitis A, West Nile Virus, LaCrosse, California encephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus, Kyasanur Forest Virus, Nipah virus, hantaviruses, Tick borne hemorrhagic fever 5 viruses, Chikungunya virus, Crimean- Congo Hemorrhagic fever virus, Tick borne encephalitis viruses, Hepatitis B virus, Hepatitis C virus, Herpes Simplex virus (HSV), Human immunodeficiency virus (HIV), Human papillomavirus (HPV)), Protozoa (Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma), Fungi (Microsporidia), Yellow fever, Tuberculosis, including drug-resistant TB, Rabies, o Prions, Severe acute respiratory syndrome associated coronavirus (SARS-CoV), Coccidioides posadasii, Coccidioides immitis, Bacterial vaginosis, Chlamydia trachomatis, Cytomegalovirus, Granuloma inguinale, Hemophilus ducreyi, Neisseria gonorrhea, Treponema pallidum, Trichomonas vaginalis, or any other infectious disease known in the art that is not listed herein. In another embodiment, an infection occurs following a5 transplantation when a subject immune system may be compromised.

Pharmaceutical compositions

An agent such as an anti-IL34 antibody is generally administered in a composition, e.g. a composition that includes one or more substantially purified agents as described herein, o and a pharmacologically suitable carrier. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The liquids may be aqueous or oil-based suspensions or solutions. The active ingredients may be mixed5 with excipients which are pharmaceutically acceptable and compatible with the active ingredients, e.g. pharmaceutically acceptable salts. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. In addition, the composition may0 contain other adjuvants. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of agent in the formulations may vary. However, in general, the amount is from about 1-99%. Still other suitable formulations for use in the present invention can be found, for example in Remington's Pharmaceutical Sciences, Philadelphia, Pa., 19th ed. (1995).

Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene - polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

"Pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-.beta.-hydroxynaphthoates, gentisates, isethionates, di-p- toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, Ν,Ν'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like.

The agent may be administered in vivo by any suitable route including but not limited to: inoculation or injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like). In a preferred embodiment, the mode of administration is intravenous. In addition, the compositions may be administered in conjunction with other treatment modalities such as other substances that boost the immune system, various chemotherapeutic agents, antibiotic agents, and the like.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1A, B and C. Inhibition of IL34 in human and rat increases proliferation of CD4⁺ and CD8⁺ T effector cells. The relative proportion of CFSE-labelled dividing CD4 CD25 T cells stimulated with human allogeneic T cell-depleted PBMCs or rat sorted pDCs was analyzed after 5 days of culture, in the presence of human CD8⁺CD45RC^low (B) or human CD4⁺CD25^high Tregs (A) or rat CD8⁺CD45RC^low Tregs at 1 : 1 effector: suppressor ratios. The proliferation after addition of anti-IL34-blocking Ab was evaluated and compared to isotypic control (n=5-6 in triplicates). The proportion of dividing CD4⁺CD25 T effector cells in the control proliferation condition with allogeneic APCs only represented approximately 60% of the cells on day 5 and was given a value of 100 in each experiment. Results are expressed as mean ± SEM of the relative proportion of dividing CD4⁺CD25 T cells.

EXAMPLES

EXAMPLE 1. Inhibition of IL34 reverses immune suppression by CD4⁺ and CD8⁺ Tregs

To demonstrate the effect of IL34 inhibition on immune responses, either anti-human IL34 blocking Ab (578416 from R&D Systems) or a control isotype Ab was added to a Mixed Lymphocyte Reaction (MLR) assay where carboxyfluorescein succinimidyl ester (CFSE)- labeled CD4⁺CD25- effector T-cell proliferation in the presence of allogeneic T cell-depleted peripheral blood mononuclear cells (PBMCs) had been inhibited by Tregs. The results are presented in Figure 1A and B. Anti-human cross-reacting IL34 blocking Ab (578416 from R&D Systems) or a control isotype Ab was added to a Mixed Lymphocyte Reaction (MLR) assay where carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4⁺CD25- effector T- cell proliferation in the presence of allogeneic pDCs from the spleen had been inhibited by CD8⁺CD45RC^low Tregs. The results are presented in Figure 1C. As can be seen, blocking IL34 significantly reverted Treg-mediated suppression for both CD4⁺ and CD8⁺ Tregs compared with isotype control Ab. These results demonstrate that inhibition of IL34 increases CD4⁺ and CD8⁺ effector T cell proliferation. While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

1. A method of unleashing immune responses in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an agent that inhibits or inactivates IL34, wherein said therapeutically effective amount is sufficient to decrease immune tolerance in the subject.

2. The method of claim 1, wherein said agent that inhibits or inactivates IL34 is an anti-IL34 antibody, wherein said anti-IL34 does not have the sequences: SEQ ID No: l; SEQ ID No:2; SEQ ID No:3; SEQ ID No:4; SEQ ID No:5; SEQ ID No:6; SEQ ID No:7; SEQ ID No:8; SEQ ID No:9; SEQ ID No: 10; SEQ ID No:l l; SEQ ID No: 12; SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15 and SEQ ID No: 16 of WO2016/097420.

3. A method of treating cancer in a subject in need thereof, comprising increasing immune responses to cancer cells in said subject by administering to said subject a therapeutically effective amount of an anti-IL34 antibody, wherein said therapeutically effective amount is sufficient to unleash immune responses to the cancer cells in the subject.