P-selectin inhibition for the treatment of cancer

Abstract

A method for treating brain metastasis cancer in a subject in need of such treatment is disclosed. The method comprises administering to the subject a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin and an immunomodulatory agent. Additional methods for treating cancer are also disclosed.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Application No. 63 / 320,310, filed March 16, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] FIELD OF THEINVENTION The present invention, in some embodiments thereof, relates to methods of treating brain metastatic cancer and glioblastoma by selectively reducing the activity or amount of P-selectin. [Background technology]

[0003] Primary and secondary brain malignancies affect many patients worldwide and in most cases lack effective treatments. Among primary brain tumors, glioblastoma (GB) is the most lethal and common type, exhibiting high heterogeneity, infiltration and invasiveness. Brain metastases affect 10-20% of all cancer patients and cause high mortality. Central nervous system tumors pose a major therapeutic challenge, in part due to the brain's native environment, which exhibits a strongly suppressive environment, low immune infiltration, and the lack of success of current targeted and immunotherapies. Among brain microenvironment cells, microglia promote the infiltration and immune suppression of central nervous system tumors. However, the reciprocal mechanisms by which tumor cells alter microglia / macrophage behavior are not fully understood.

[0004] It has previously been shown that P-selectin (SELP) mediates microglial proliferation and invasion of the GB by altering the activation state of microglia / macrophages (Yeini et al., Nature Communications 2021, 12 (1), 1912).

[0005] Further background art includes WO 2022 / 059008, U.S. Patent Publication No. 20200171064 (teaching isoquercetin or quercetin for the treatment of cancer, including glioblastoma), and U.S. Patent Publication No. 20190241665 (teaching P-selectin inhibitors for the treatment of metastatic cancer).

[0006] Shamay et al., Science Translational Medicine, Volume 8, issue 345, 29 June, 2016, teaches that P-selectin is expressed on cancer cells of several human tumor types.

[0007] Ferber et al., eLife 2017;6:e25281. DOI: www.doi.org / 10.7554 / eLife.25281 teach that P-selectin is expressed not only on tumor endothelium but also on glioblastoma cells and can be used as a target for selective delivery of anticancer drugs. Summary of the Invention

[0008] According to one aspect of the present invention, there is provided a method of treating brain metastasis cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin and an immunomodulatory agent to treat the brain metastasis cancer.

[0009] According to one aspect of the invention, there is provided a method of treating pancreatic or renal cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin, thereby treating the pancreatic or renal cancer.

[0010] According to one aspect of the present invention, there is provided a method of treating cancer in a subject in need of such treatment, the cancer being selected from the group consisting of pancreatic cancer, lung cancer, breast cancer, primary melanoma and renal cancer, comprising administering to the subject a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin and an immunomodulatory agent to treat the cancer.

[0011] According to an embodiment of the present invention, the brain metastasis cancer is brain metastasis melanoma, brain metastasis breast cancer, brain metastasis lung cancer, or brain metastasis colorectal cancer.

[0012] According to an embodiment of the invention, an agent that specifically reduces the amount and / or activity of P-selectin specifically binds to P-selectin or a polynucleotide encoding P-selectin.

[0013] According to an embodiment of the invention, an agent that specifically reduces the amount and / or activity of P-selectin binds to P-selectin glycoprotein ligand-1 (PSGL-1) or a polynucleotide encoding PSGL-1.

[0014] According to embodiments of the invention, the method further comprises administering to the subject an immunomodulatory drug.

[0015] According to an embodiment of the invention, the immunomodulatory agent comprises an immunomodulatory antibody.

[0016] According to an embodiment of the invention, the immunomodulatory antibody is selected from the group consisting of anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1, anti-PDL1, anti-LAG3, anti-IDO, and anti-TIGIT.

[0017] According to an embodiment of the invention, an agent that specifically reduces the amount and / or activity of P-selectin is an inhibitory antibody that binds to and inhibits P-selectin.

[0018] According to an embodiment of the invention, the inhibitory antibody is crizanlizumab or inlacumab.

[0019] According to an embodiment of the invention, the dosage of crizanlizumab is about 5 mg / kg once every two weeks or once every four weeks.

[0020] According to an embodiment of the invention, the inhibitory antibody is conjugated to a therapeutic agent.

[0021] According to an embodiment of the invention, the inhibitory antibody is not conjugated to a therapeutic agent.

[0022] According to an embodiment of the invention, the agent that specifically reduces the amount and / or activity of P-selectin is a small molecule drug.

[0023] According to an embodiment of the invention, the agent that specifically reduces the amount and / or activity of P-selectin is a polynucleotide drug.

[0024] According to an embodiment of the invention, an agent that specifically reduces the amount and / or activity of P-selectin is co-formulated with an immunomodulatory agent.

[0025] According to an embodiment of the invention, an agent that specifically reduces the amount and / or activity of P-selectin is included in the nanoparticles.

[0026] According to an embodiment of the invention, the nanoparticles are conjugated to targeting moieties that increase delivery across the blood-brain barrier.

[0027] According to an embodiment of the invention, an agent that specifically reduces the amount and / or activity of P-selectin is linked to a targeting moiety that increases delivery across the blood-brain barrier.

[0028] According to one aspect of the present invention, there is provided a method for treating glioblastoma or brain metastatic melanoma in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of crizanlizumab and an anti-PD1 antibody to treat the glioblastoma or brain metastatic melanoma.

[0029] According to an embodiment of the invention, crizanlizumab is delivered in a dose of about 5 mg / kg once every two weeks or once every four weeks.

[0030] According to an embodiment of the invention, the anti-PD1 antibody is selected from the group consisting of pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo) and dostarlimab (Jemperli).

[0031] According to an embodiment of the invention, the anti-PD1 antibody is nivolumab.

[0032] According to an embodiment of the invention, crizanlizumab and anti-PD1 antibody are initially administered once every two weeks for at least four weeks.

[0033] According to an embodiment of the invention, the dosage of anti-PD1 antibody is about 3 mg / kg once every two weeks.

[0034] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting.

[0035] Certain embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings, in which: Reference will now be made specifically in detail to the drawings, it being stressed that the particulars shown are by way of example and are for the purposes of illustrating embodiments of the invention, and in which the description with reference to the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. [Brief description of the drawings]

[0036] [Figure 1-1] Figure 1A-C. PD-1 / PD-L1 are highly expressed in glioblastoma patient tissues and their expression correlates with SELP / PSGL-1 expression. Analysis of the glioblastoma database showed high expression of PD-1 (PDCD1) and PD-L1 (CD207) in GB samples compared to healthy human brain tissue and low-grade gliomas (A-B), and positive correlation of PSGL-1 (SELPLG) expression with PD-1 and PD-L1 expression, and positive correlation of SELP expression with PD-1 expression. Analysis was performed using the GlioVis data portal. [Figure 1-2] Same as above [Figure 2-1] Figure 2A-B. SELP, PSGL-1, PD-1, and PD-L1 are co-expressed in BM and DIPG samples from multiple patients. Figure 2A. Immunostaining showing high expression of SELP in BM from melanoma, breast, lung, and CRC as well as DIPG patient samples. PSGL-1 was found to be highly expressed in BM from melanoma, breast, and lung as well as DIPG patient samples. Figure 2B. Immunostaining showing high expression of PD-1 in BM from melanoma and lung and high expression of PD-L1 in BM from melanoma, breast, lung, CRC as well as DIPG patient samples. All samples were compared to normal human brain tissue showing low expression of all staining markers. [Figure 2-2] Same as above [Figure 3-1] Figure 3A-Figure 3B. SELP, PSGL-1, PD-1, and PD-L1 are co-expressed in primary tumor samples from various patients. Figure 3A. Immunostaining shows high expression of SELP and PSGL-1 in primary melanoma, breast, and lung patient samples, and high expression of SELP in primary PDAC patient samples. Figure 3B. Immunostaining shows high expression of PD-1 and PD-L1 in primary melanoma, PDAC, and lung patient samples. [Figure 3-2] Same as above [Figure 4-1]Figure 4A-C. Microglia are activated in melanoma brain metastases in vivo and promote melanoma cell growth and invasion in vitro. Figure 4A. Iba-1 immunostaining showing positive staining of activated microglia at the tumor site using a patient-derived MBM mouse model and patient FFPE samples. Figure 4B. Co-culture proliferation assay showing concentration-dependent increased proliferation of mouse RET melanoma cells after addition of primary mouse microglial cells. Figure 4C. 3D spheroid invasion assay shows increased growth and invasion of mouse D4M melanoma cells when BV2 mouse microglia are incorporated into spheroids. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 5-1] Figure 5A-C. SELP is highly expressed by melanoma cells in 2D cultures and 3D spheroids. Figure 5A. Flow cytometry analysis shows that SELP is highly expressed in 2D cultured human A375, mouse B16-F10 and Ret melanoma cell lines. Figure 5B-C. Flow cytometry analysis shows that SELP is overexpressed in WM115 (B) and D4M.3A (C) 3D tumor spheroids compared to 2D cultures. [Figure 5-2] Same as above [Figure 6-1] Figure 6A-B. Combined treatment with SELP inhibitors and anti-PD-1 results in anti-cancer activity of splenocytes against D4M.3A melanoma spheroids in the presence of microglia. Figure 6A-B. D4M.3A spheroids composed of cancer cells alone (A) or cancer cells and primary mouse microglia (B) were co-cultured with freshly isolated mouse splenocytes. Spheroids were either untreated or treated with anti-PD-1 antibodies, SELP inhibitors (SELPi), or a combination of these. Combined treatment with SELPi and anti-PD-1 showed the highest inhibition of spheroid growth compared to treatment with SELPi or anti-PD-1 alone. [Figure 6-2] Same as above [Figure 7]Figure 7A-B. The SELP axis mediates invasion of breast cancer spheroids. Figure 7A. Flow cytometry analysis shows high expression of SELP in murine BRCA-mutated EMT6 breast cancer 3D spheroids. Figure 7B. Human MDA-BM-231 3D spheroids showed enhanced invasive ability when co-cultured with brain microenvironment cells and reduced invasion after treatment with a SELP inhibitor (SELPi). [Figure 8-1] Figure 8A-D. SELP has a role in lung cancer-microglia interactions and mediates the growth of lung cancer spheroids. Figure 8A. Growth of human A549 spheroids was increased in the presence of human microglia compared to spheroids composed of A549 cells alone. Figure 8B. As shown by flow cytometry analysis, A549 cells expressed high levels of SELP, whereas human microglia expressed high levels of PSGL-1 when co-cultured in 3D spheroids. Figure 8C. Treatment with a SELP inhibitor (SELPi) suppressed the growth of A549 spheroids in the presence of human microglia. Figure 8D. Treatment with SELPi dose-dependently reduced A549 spheroid growth and invasion into Matrigel. [Figure 8-2] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention, in some embodiments thereof, relates to methods of treating brain metastatic cancers and glioblastomas by selectively reducing the activity or amount of P-selectin.

[0038] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0039] Recently, SELP was found to be overexpressed in melanoma brain metastasis (MBM) cells and tissues in both patient-derived samples and mouse models. 40%-50% of stage IV melanoma patients will experience brain metastasis. Combinations of anti-CTLA-4 antibodies (ipilimumab) and anti-PD-1 antibodies (Keytruda (pembrolizumab), Opdivo (nivolumab), Libtayo (cemiplimab)) have proven effective in patients with melanoma brain metastasis, showing response rates of up to 50% in asymptomatic patients. However, heterogeneity of immune responses and treatment resistance are often observed due to the different infiltration of adaptive and innate immune cells into tumors.

[0040] We have now shown that the combination of anti-PD1 (or anti-PD-L1, e.g., Tecentriq (atezolizumab), Bavencio (avelumab) and Imfinzi (durvalumab), or small molecule inhibitors of PD-L1) with anti-SELP (antibody or small molecule) sensitizes tumors to immunotherapy (Figure 6). Thus, we propose that the combination of SELP inhibition with immunotherapy, such as checkpoint regulators, vaccines or CAR T therapy, has therapeutic potential for patients with primary and secondary brain malignancies and SELP-expressing primary tumors, such as PDAC / pancreas, RCC / kidney, melanoma / skin and lung (see Figures 2A-B and 3A-B).

[0041] Thus, according to one aspect of the present invention, there is provided a method of treating brain metastasis cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin and an immunomodulatory agent to treat the brain metastasis cancer.

[0042] According to another aspect of the invention, there is provided a method of treating pancreatic or renal cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin, thereby treating the pancreatic or renal cancer.

[0043] According to yet another aspect of the present invention, there is provided a method of treating cancer in a subject in need of such treatment, the cancer being selected from the group consisting of pancreatic cancer, lung cancer, breast cancer, primary melanoma and renal cancer, comprising administering to the subject a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin and an immunomodulatory agent to treat the cancer.

[0044] As used herein, the term "subject" refers to mammals, such as rodents, felines, canines, and primates. Preferably, the subject according to the present invention is a human. In one embodiment, the subject is a surgery- and radiation-ineligible subject. In one embodiment, the subject has a tumor that consists of two or more masses.

[0045] P-selectin is a member of the selectin family of adhesive glycoproteins, which also includes L-selectin and E-selectin. Selectins mediate recruitment, initial tethering, initial rolling, and adhesion of leukocytes to sites of inflammation. P-selectin is stored in the Weibel-Palade bodies of endothelial cells and in α-granules of platelets, and is rapidly recruited to the plasma membrane upon stimulation with vasoactive substances such as histamine and thrombin.

[0046] P-selectin is a transmembrane glycoprotein (SwissProt sequence P16109) consisting of an NH2-terminal lectin domain followed by an epidermal growth factor (EGF)-like domain and nine consensus repeat domains. P-selectin is anchored to the membrane by a single transmembrane domain and has a small cytoplasmic tail.

[0047] Human P-selectin (also called SELP) has the Uniprot number P16109 and the mRNA with REFSEQ:NM_003005.4.

[0048] P-selectin plays its central role in the recruitment of leukocytes to sites of inflammation and thrombosis by binding to its counter-receptor, P-selectin glycoprotein ligand-1 (PSGL-1) (or PSGL-1-like receptor on sickle cells), a mucin-like glycoprotein constitutively expressed on leukocytes including neutrophils and monocytes, platelets, and some endothelial cells.

[0049] The Uniprot number of human PSGL-1 is Q14242, and it has an mRNA with REFSEQ defined as NM_001206609.2 or NM_003006.4.

[0050] Thus, the present invention contemplates downregulating the function of P-selectin by using (1) antibodies against P-selectin, (2) antibodies against PSGL-1, (3) small molecules that mimic the binding domain of PSGL-1, and (4) other molecules that interfere with the binding of P-selectin to PSGL-1. Such agents are further described herein below.

[0051] In another embodiment, the agent downregulates the amount of P-selectin by decreasing the expression of P-selectin.

[0052] In yet another embodiment, the agent downregulates expression of PSGL-1.

[0053] As used herein, the phrase "downregulating expression" refers to downregulating the expression of P-selectin or PSGL-1 at the genomic level (e.g., homologous recombination and site-specific endonucleases) and / or transcript level using various molecules that interfere with transcription and / or translation (e.g., RNA silencing agents, CRISPR / Cas-9), or at the protein level (e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave polypeptides, antibodies, etc.).

[0054] Expression is generally expressed relative to expression in cells of the same type that have not been contacted with the agent or with a vehicle control (also referred to as a control) under the same culture conditions.

[0055] The downregulation of expression may be transient or permanent.

[0056] According to certain embodiments, downregulated expression refers to the absence of mRNA and / or protein as detected by RT-PCR or Western blot, respectively.

[0057] According to other specific embodiments, downregulating expression refers to a reduction in the levels of mRNA and / or protein as detected by RT-PCR or Western blot, respectively. The reduction may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

[0058] Non-limiting examples of agents capable of downregulating P-selectin or PSGL-1 expression are described in detail below.

[0059] Downregulation at the nucleic acid level Downregulation at the nucleic acid level is usually carried out using nucleic acid drugs with a nucleic acid backbone, DNA, RNA, their mimics or their combinations. The nucleic acid drug may be encoded by a DNA molecule or may be supplied to the cell itself.

[0060] According to certain embodiments, the down-regulating agent is a polynucleotide.

[0061] According to a particular embodiment, the downregulatory agent is a polynucleotide capable of hybridizing to the gene or mRNA encoding P-selectin.

[0062] According to certain embodiments, the down-regulating agent is a polynucleotide capable of hybridizing to the gene or mRNA encoding PSGL-1.

[0063] According to certain embodiments, the downregulatory agent interacts directly with P-selectin.

[0064] According to certain embodiments, the agent binds directly to P-selectin.

[0065] According to certain embodiments, the agent binds indirectly to P-selectin (eg, binds to an effector of P-selectin).

[0066] According to certain embodiments, the down-regulating agent is an RNA silencing agent or a genome editing agent.

[0067] Thus, downregulation of P-selectin or PSGL-1 can be achieved by RNA silencing.

[0068] As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules that result in the inhibition, or "silencing," of expression of corresponding protein-coding genes. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0069] As used herein, the term "RNA silencing agent" refers to an RNA that can specifically inhibit, or "silence", the expression of a target gene. In certain embodiments, the RNA silencing agent can prevent complete processing (e.g., complete translation and / or expression) of an mRNA molecule via a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules (e.g., double-stranded RNAs that include paired strands) and precursor RNAs that can generate such small non-coding RNAs. Exemplary RNA silencing agents include dsRNAs, such as siRNAs, miRNAs, and shRNAs. RNA silencing agents may be administered in naked oligonucleotides, may contain modified bases, or may be oligonucleotides stabilized by encapsulation or binding to nanoparticles or any nanocarrier (polymers, lipid-LNPs, liposomes, micelles, etc.). See, for example, Scomparin et al., Biotechnology Advances, Volume 33, Issue 6, Part 3, 1 November 2015, Pages 1294-1309.

[0070] In one embodiment, the RNA silencing agent is capable of inducing RNA interference.

[0071] In another embodiment, the RNA silencing agent is capable of mediating translational repression.

[0072] According to one embodiment of the invention, the RNA silencing agent is specific for the target RNA (e.g., P-selectin) and does not cross-inhibit or silence other targets or splice variants that exhibit 99% or less overall homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% overall homology to the target gene, as measured by PCR, Western blot, immunohistochemistry and / or flow cytometry.

[0073] RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNA (siRNA).

[0074] Below is a detailed description of RNA silencing agents that can be used in accordance with certain embodiments of the present invention.

[0075] DsRNA, siRNA and shRNA: The presence of long dsRNA in cells stimulates the activity of a ribonuclease III enzyme called Dicer. Dicer is involved in the processing of dsRNA into small fragments known as small interfering RNAs (siRNAs). Small interfering RNAs resulting from Dicer activity are usually about 21 to about 23 nucleotides long and consist of about 19 base pair duplexes. The RNAi response is also characterized by an endonuclease complex, commonly called the RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNAs with sequences complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex.

[0076] Thus, some embodiments of the present invention contemplate the use of dsRNA to downregulate protein expression from mRNA.

[0077] According to one embodiment, dsRNA longer than 30bp is used.Various studies have demonstrated that long dsRNA can be used to silence gene expression without inducing stress response or causing significant off-target effects.See, for example, [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810;Bhargava A et al., Brain Res. Protoc. 2004;13:115-125;Diallo M., et al., Oligonucleotides. 2003;13:381-392;Paddison PJ, et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448;Tran N., et al., FEBS Lett. 2004;573:127-134].

[0078] According to some embodiments of the present invention, dsRNA is provided to cells in which the interferon pathway is not activated.See, for example, Billy et al., PNAS 2001, Vol 98, pages 14428-14433 and Diallo et al., Oligonucleotides, October 1, 2003, 13(5): 381-392. doi:10.1089 / 154545703322617069.

[0079] According to one embodiment of the present invention, long dsRNA is specifically designed not to induce the interferon pathway and the PKR pathway to downregulate gene expression.For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] developed a vector named pDECAP to express long double-stranded RNA from an RNA polymerase II (Pol II) promoter.Since the transcript from pDECAP lacks both the 5'-cap structure and the 3'-poly(A) tail that promotes the transport of ds-RNA to the cytoplasm, the long ds-RNA from pDECAP does not induce the interferon response.

[0080] Another method to circumvent the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNA (siRNA) by transfection or endogenous expression.

[0081] The term "siRNA" refers to small interfering RNA duplexes (typically 18-30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21-mers with a central 19-bp duplex region and symmetric 2-base 3'-overhangs at the ends, but it has recently been shown that chemically synthesized 25-30 base-long duplexes can be up to 100-fold more potent than 21-mers of the same configuration. The observed improved potency obtained with longer RNAs in triggering RNAi suggests that this results from providing substrate (27-mer) to Dicer instead of product (21-mer), and that this improves the speed or efficiency of entry of the siRNA duplex into RISC.

[0082] The location of the 3'-overhang influences siRNA potency, with asymmetric duplexes with a 3'-overhang on the antisense strand generally being more potent than those with a 3'-overhang on the sense strand (Rose et al., 2005). This may be due to loading of the asymmetric strand into RISC, as the opposite efficacy pattern is observed when targeting antisense transcripts.

[0083] The strands of double-stranded interfering RNA (e.g., siRNA) may be linked to form a hairpin or stem-loop structure (e.g., shRNA). Thus, as mentioned above, the RNA silencing agent of some embodiments of the present invention may also be a short hairpin RNA (shRNA).

[0084] miRNAs and miRNA mimics: According to another embodiment, the RNA silencing agent may be a miRNA.

[0085] The terms "microRNA", "miRNA", and "miR" are synonymous and refer to a group of non-coding, single-stranded RNA molecules approximately 19-28 nucleotides long that regulate gene expression. miRNAs are found in a wide range of organisms and have been shown to be involved in development, homeostasis, and disease pathogenesis.

[0086] Antisense: Antisense is a single stranded RNA designed to block or inhibit the expression of a gene by specifically hybridizing to the mRNA of that gene. Downregulation of P-selectin can be achieved using antisense polynucleotides that can specifically hybridize to the mRNA transcripts encoding P-selectin. Downregulation of PSGL-1 can be achieved using antisense polynucleotides that can specifically hybridize to the mRNA transcripts encoding PSGL-1.

[0087] The design of antisense molecules that can be used to efficiently downregulate P-selectin or PSGL-1 must be undertaken while taking into account two aspects that are important for an antisense approach: the first is the delivery of the oligonucleotide to the cytoplasm of the appropriate cells, and the second is the design of an oligonucleotide that specifically binds to a designated mRNA within the cell so as to inhibit its translation.

[0088] Downregulation can be achieved by inactivating the gene (ie, the P-selectin gene or the PSGL-1 gene) by introducing targeted mutations involving loss-of-function mutations in the gene structure (eg, point mutations, deletions and insertions).

[0089] As used herein, the phrase "loss-of-function mutation" refers to any mutation in the DNA sequence of a gene that results in downregulation of the expression levels and / or activity of the expression product, i.e., the mRNA transcript and / or translated protein.Non-limiting examples of such loss-of-function mutations include missense mutations, i.e., mutations that change an amino acid residue in a protein to another amino acid residue, thereby causing the protein to lose enzymatic activity; nonsense mutations, i.e., mutations that introduce a stop codon (e.g., a premature stop codon) into a protein resulting in a shorter protein lacking enzymatic activity; frameshift mutations, i.e., mutations that change the reading frame of a protein, either resulting in a premature termination by introducing a stop codon into the reading frame (e.g., a truncated protein lacking enzymatic activity) or a nucleic acid mutation (usually a deletion or insertion) that can result in a longer amino acid sequence (e.g., a read-through protein) that affects the secondary or tertiary structure of the protein, resulting in a non-functional protein that lacks the enzymatic activity of the non-mutated polypeptide; read-through mutations with loss of enzymatic activity due to frameshift mutations or mutations due to change in the stop codon (i.e., where the stop codon is mutated to an amino acid codon); promoter mutations, i.e., These include mutations in promoter sequences, usually 5' to the transcription start site of a gene, that result in down-regulation of a particular gene product; regulatory mutations, i.e., mutations in upstream or downstream regions of a gene, or within a gene, that affect expression of the gene product; deletion mutations, i.e., mutations that delete coding nucleic acid within a gene sequence, which may result in a frameshift mutation or an in-frame mutation (deletion of one or more amino acid codons within the coding sequence); insertion mutations, i.e., mutations that insert coding or non-coding nucleic acid into a gene sequence, which may result in a frameshift mutation or an in-frame insertion of one or more amino acid codons; inversions, i.e., mutations that result in inverted coding or non-coding sequences; splice mutations, i.e., mutations that result in aberrant or defective splicing; and duplication mutations, i.e., mutations that result in duplicated coding or non-coding sequences, which may be in-frame or may cause a frameshift.

[0090] According to certain embodiments, the loss-of-function mutation of a gene may involve at least one allele of the gene.

[0091] As used herein, the term "allele" refers to any of one or more alternative forms of a genetic locus, both of which alleles are associated with a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0092] According to other specific embodiments, the loss-of-function mutation of the gene includes both alleles of the gene. In such cases, P-selectin or PSGL-1 may be homozygous or heterozygous.

[0093] Methods for introducing nucleic acid changes into a gene of interest are well known in the art [see, e.g., Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244: 1288-1292; Santiago et al., Proc Natl Acad Sci USA (2008) 105:5809-5814; International Publication No. 2014085593, No. 2009071334 and No. 2011146121; US ​​Patent No. 8771945, No. 8586526, No. 6774279 and US Patent Publication No. 20030232410, No. 20050026157, No. 20060014264; the contents of which are incorporated by reference in their entirety], including targeted homologous recombination, site-specific recombinase, PB transposase and engineered nuclease genome editing.The drug that introduces nucleic acid changes into gene of interest can be designed from publicly available sources or can be commercially obtained from Transposagen, Addgene and Sangamo Biosciences.

[0094] Below is a description of various exemplary methods used to introduce nucleic acid changes into a gene of interest, and agents for carrying same that can be used according to certain embodiments of the invention.

[0095] Genome editing using engineered endonucleases: This approach refers to a reverse genetic method using artificially engineered nucleases to cleave and create specific double-stranded breaks at desired locations in the genome, followed by repair by cell-intrinsic processes such as homology-directed repair (HDR) and non-homologous end joining (NHEJ). NHEJ directly joins DNA ends at double-stranded breaks, while HDR utilizes homologous sequences as templates to regenerate missing DNA sequences at the break points. In order to introduce specific nucleotide modifications into genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using conventional restriction endonucleases because most restriction enzymes recognize several base pairs on DNA as targets, and it is highly likely that the combination of recognized base pairs will be found at many locations on the genome, resulting in multiple breaks without being limited to the desired location. To overcome this challenge and create site-specific single-stranded or double-stranded breaks, several different classes of nucleases have been discovered and bioengineered to date. These include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

[0096] Meganucleases: Meganucleases are generally classified into four families (LAGLIDADG family, GIY-YIG family, His-Cys box family and HNH family). These families are characterized by structural motifs that affect catalytic activity and recognition sequence. For example, members of the LAGLIDADG family are characterized by having one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are significantly different from each other in terms of conserved structural elements and thus DNA recognition sequence specificity and catalytic activity. Meganucleases are commonly found in microbial species and have the unique property of having very long recognition sequences (>14bp), which naturally exerts very high specificity in cleaving at desired positions. This can be utilized to perform site-specific double-strand breaks in genome editing. Those skilled in the art can use these naturally occurring meganucleases, but the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to generate meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to generate hybrid enzymes that recognize new sequences. Alternatively, DNA-interacting amino acids of meganucleases can be altered to design sequence-specific meganucleases (see, for example, U.S. Pat. No. 8,021,867). Meganucleases can be designed using methods described, for example, in Certo, MT et al., Nature Methods (2012) 9:073-975, U.S. Pat. Nos. 8,304,222, 8,021,867, 8,119,381, 8,124,369, 8,129,134, 8,133,697, 8,143,015, 8,143,016, 8,148,098, or 8,163,514, the contents of each of which are incorporated herein by reference in their entirety.Alternatively, meganucleases with site-specific cleavage properties can be obtained using commercially available technologies, for example the Directed Nuclease Editor™ genome editing technology from Precision Biosciences.

[0097] ZFNs and TALENs: Two different classes of engineered nucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven effective in creating targeted double-stranded breaks ( Christian et al., 2010 , Kim et al., 1996 , Li et al., 2011 , Mahfouz et al., 2011 , Miller et al., 2010 ).

[0098] Essentially, ZFN and TALEN restriction endonuclease technologies utilize a non-specific DNA cleavage enzyme linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Usually, a restriction enzyme is selected in which the DNA recognition site and the cleavage site are separated from each other. The cleavage portion is separated and then linked to the DNA binding domain, thereby resulting in an endonuclease with very high specificity for the desired sequence. An exemplary restriction enzyme with such properties is Fokl. Furthermore, Fokl has the advantage that it requires dimerization to have nuclease activity, which means that specificity is dramatically improved since each nuclease partner recognizes a unique DNA sequence. To improve this effect, Fokl nucleases have been engineered to function only in heterodimers, increasing their catalytic activity. Nucleases that function in heterodimers avoid the possibility of unwanted homodimer activity, thus increasing the specificity of double-stranded cleavage.

[0099] Thus, for example, to target a specific site, ZFNs and TALENs are constructed as pairs of nucleases, with each member of the pair designed to bind to adjacent sequences of the target site. When transiently expressed in cells, these nucleases bind to the target site and the FokI domains heterodimerize to create double-stranded breaks. Repair of these double-stranded breaks via the non-homologous end joining (NHEJ) pathway most often results in small deletions or insertions of small sequences. Because each repair performed by NHEJ is unique, the use of a single nuclease pair can result in a series of alleles with a variety of different deletions at the target site. Deletions typically range in length from a few base pairs to a few hundred base pairs, although larger deletions have been successfully created in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). Furthermore, introduction of homologous DNA fragments into the target region along with nuclease pairs can repair double-strand breaks by homology-directed repair, resulting in specific modifications ( Li et al., 2011 , Miller et al., 2010 , Urnov et al., 2005 ).

[0100] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, whereas TALENs rely on TALEs. Both of these DNA recognition peptide domains have the characteristic of naturally occurring in combinations in their proteins. Cys2-His2 zinc fingers are usually found in repeats spaced at 3 bp intervals and are found in diverse combinations in various nucleic acid interacting proteins. On the other hand, TALEs are found in repeats with a one-to-one recognition ratio between the amino acid and the recognized nucleotide pair. As both zinc fingers and TALEs occur in a repeating pattern, a wide variety of sequence specificities can be created by trying different combinations. Approaches for producing site-specific zinc finger endonucleases include, for example, modular assembly (attaching zinc fingers associated with triplet sequences in a line to cover the required sequence), OPEN (low stringency selection of peptide domains versus triplet nucleotides in bacterial systems followed by high stringency selection of peptide combinations versus the final target), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs can also be designed and obtained commercially, for example, from Sangamo Biosciences™ (Richmond, Calif.).

[0101] The design and acquisition method of TALEN is described, for example, in Reyon et al. Nature Biotechnology 2012 May;30(5):460-5, Miller et al., Nat Biotechnol. (2011) 29: 143-148, Cermak et al., Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al., Nature Biotechnology (2011) 29 (2): 149-53. Recently developed, a web-based program named Mojo Hand has been introduced by Mayo Clinic to design TAL and TALEN constructs for genome editing applications (accessible at www.talendesign.org). TALEN can also be designed and acquired commercially, for example, by Sangamo Biosciences™ (Richmond, Calif.).

[0102] CRISPR-Cas systems: Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade the nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) genes that produce the RNA component, and CRISPR-associated (Cas) genes that code for the protein component. CRISPR RNAs (crRNAs) contain short stretches of homology to specific viruses and plasmids and act as guides to instruct Cas nucleases to degrade complementary nucleic acids of the corresponding pathogens. Studies of the type II CRISPR / Cas system of Streptococcus pyogenes have shown that the three components (Cas9 nuclease, crRNA containing 20 base pairs of homology to the target sequence, and trans-activating crRNA (tracrRNA)) form an RNA / protein complex that cooperates to exert sequence-specific nuclease activity (Jinek et al., Science (2012) 337: 816-821). Furthermore, it has been shown that a synthetic chimeric guide RNA (gRNA) composed of a fusion of crRNA and tracrRNA can direct Cas9 to cleave DNA targets complementary to the crRNA in vitro. It has also been shown that transient expression of Cas9 in combination with synthetic gRNAs can be used to generate targeted double-stranded breaks in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a,b; Jinek et al., 2013; Mali et al., 2013).

[0103] The CRIPSR / Cas system for genome editing comprises two distinct components, a gRNA and an endonuclease (e.g., Cas9).

[0104] gRNAs are typically 20 nucleotide sequences that code for the combination of a target homologous sequence (crRNA) and an endogenous bacterial RNA that tethers the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA / Cas9 complex is recruited to the target sequence by base pairing between the gRNA sequence and the complementary strand genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain an appropriate protospacer adjacent motif (PAM) sequence immediately following the target sequence. Binding of the gRNA / Cas9 complex localizes Cas9 to the genomic target sequence, allowing Cas9 to cleave both strands of DNA and create a double-stranded break. As with ZFNs and TALENs, the double-stranded break generated by CRISPR / Cas can perform homologous recombination or NHEJ.

[0105] The Cas9 nuclease has two functional domains, RuvC and HNH, which each cleave a different DNA strand. When both domains are active, Cas9 creates a double-stranded break in genomic DNA.

[0106] A major advantage of CRISPR / Cas is that the high efficiency of the system, combined with the ability to easily generate synthetic gRNAs, allows for the simultaneous targeting of multiple genes, and the majority of cells carrying mutations harbor biallelic mutations in the targeted genes.

[0107] However, the apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows for imperfect matching of the target sequence to be cleaved by Cas9.

[0108] Modified versions of the Cas9 enzyme that contain a single inactive catalytic domain, RuvC- or HNH-, are called "nickases." With only one active nuclease domain, Cas9 nickases cut only one strand of the target DNA, forming a single-stranded break or "nick." The single-stranded break or nick is usually immediately repaired by the HDR pathway, using the intact complementary DNA strand as a template. However, two adjacent, opposite-stranded nicks introduced by Cas9 nickases are often referred to as "double-nick" CRISPR systems and are treated as double-stranded breaks. The double nicks can be repaired by either NHEJ or HDR, depending on the desired effect on the gene target. Thus, when specificity and reduced off-target effects are critical, using Cas9 nickases to form double nicks with two gRNAs designed with adjacent target sequences and on opposite strands of genomic DNA would reduce off-target effects, since a nick generated by only one gRNA would not alter the genomic DNA.

[0109] A modified version of the Cas9 enzyme containing two inactive catalytic domains (deadCas9 or dCas9) does not have nuclease activity, but can still bind to DNA based on gRNA specificity. dCas9 can be utilized as a platform for DNA transcription regulators to activate or suppress gene expression by fusing the inactive enzyme to a known regulatory domain. For example, dCas9 alone can be bound to a target sequence in genomic DNA to prevent gene transcription.

[0110] For example, there are many publicly available tools that can help in the selection and / or design of target sequences and a list of bioinformatically determined unique gRNAs for different genes in different species, such as Target Finder from the Feng Zhang lab, Target Finder (E-CRISP) from the Michael Boutros lab, RGEN Tools:Cas-OFFinder, CasFinder (a flexible algorithm for identifying specific Cas9 targets in genomes) and CRISPR Optimal Target Finder.

[0111] Non-limiting examples of gRNA sequences that can be used in some embodiments of the invention are described in the literature (Sanjana NE, Shalem O., Zhang F. Nat Methods. 2014 Aug;11(8):783-4) and on the genscript website (see www.genscript.com / gRNA-detail / 6403 / SELP-CRISPR-guide-RNA).

[0112] According to certain embodiments, the gRNA sequence does not have significant off-target effects.Methods for measuring off-target effects are well known in the art, such as Human Whole Genome Sequencing by BGI (described in Nature; 491:65-56. 2012), next generation sequencing (NGS) using commercially available kits such as Alt-R-Genom Editing (IDT detection kit) or Sure select target enrichment <1% mutant allele frequency (Agilent).

[0113] To use the CRISPR system, both gRNA and Cas9 must be expressed in the target cell. The insertion vector can contain both cassettes on a single plasmid or can express the cassettes from two separate plasmids. CRISPR plasmids are commercially available, such as px330 plasmid from Addgene. Alternatively, target cells can be transfected with both gRNA and Cas9 without plasmids, for example using a transfection reagent such as CRISPRMAX [see, for example, Yu et al. (2016) JD1Biotechnol Lett. 38(6):919-29]. In some cells, electroporation can improve transfection of gRNA and Cas9 [see, for example, Liang et al. (2015) Journal of Biotechnology 208, 2015, Pages 44-53, and Liang et al. (2017) Journal of Biotechnology, Volume 241, 2017, pp. 136-146].

[0114] "Hit-and-run" or "in-out" involves a two-step recombination procedure. In the first step, the desired sequence change is introduced using an insertion vector containing a positive / negative dual selectable marker cassette. This insertion vector contains one continuous region of homology to the target locus and is modified to carry the desired mutation. This targeting construct is linearized with a restriction enzyme at one site within the homologous region and electroporated into cells, where positive selection is performed to isolate homologous recombinants. These homologous recombinants contain local duplications separated by intervening vector sequences containing the selection cassette. In the second step, the targeted clones are subjected to negative selection to identify cells that have lost the selection cassette through intrachromosomal recombination between the duplicated sequences. The duplication is removed by a local recombination event and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.

[0115] The "double replacement" or "tag and exchange" strategy involves a two-step selection procedure similar to the hit-and-run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a positive / negative double selectable cassette near the position where the mutation is to be introduced. After electroporation and positive selection, homologously targeted clones are identified. A second targeting vector containing the homologous region with the desired mutation is then electroporated into the targeted clone, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation, with the unwanted exogenous sequence excluded.

[0116] Site-specific recombinases: Cre recombinase from P1 bacteriophage and Flp recombinase from yeast Saccharomyces cerevisiae are site-specific DNA recombinases that recognize unique 34-base pair DNA sequences (termed "Lox" and "FRT", respectively), and sequences flanked by either Lox or FRT sites can be easily removed by site-specific recombination upon expression of Cre recombinase or Flp recombinase, respectively. For example, Lox sequences consist of an asymmetric 8-base pair spacer region flanked by 13-base pair inverted repeat sequences. Cre recombines the 34-base pair Lox DNA sequence by binding to the 13-base pair inverted repeat sequences and catalyzing strand cleavage and rejoining within the spacer region. The staggered DNA cleavage that Cre makes in the spacer region is separated by 6 base pairs to result in overlapping regions that function as homology sensors, ensuring that only recombination sites with identical overlapping regions recombine.

[0117] Essentially, site-specific recombinase systems provide a means to remove the selection cassette after homologous recombination. The system also allows for the creation of conditional mutant alleles that can be inactivated or activated in a transient or tissue-specific manner. Of note, Cre and Flp recombinases leave behind a 34-base pair Lox or FRT "scar". The remaining Lox or FRT sites usually remain within introns or the 3'UTR of the modified locus, although current evidence suggests that these sites usually do not significantly interfere with gene function.

[0118] Thus, Cre / Lox and Flp / FRT recombination involves the introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two Lox or FRT sequences, and a selectable cassette that is usually located between the two Lox or FRT sequences. Positive selection is applied to identify homologous recombinants that contain the targeted mutation. Transient expression of Cre or Flp is combined with negative selection to excise the selection cassette and select for cells in which the cassette has been lost. The final targeted allele contains a Lox or FRT scar of the exogenous sequence.

[0119] Transposase: As used herein, the term "transposase" refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome.

[0120] As used herein, the term "transposon" refers to a mobile genetic element that contains a nucleotide sequence that can move around to different locations in the genome of a single cell. In the process, transposons can cause mutations and / or changes in the amount of DNA in the genome of a cell.

[0121] Numerous transposon systems have been isolated or engineered that can transpose similarly in vertebrate cells, such as Sleeping Beauty [Izsvak and Ivics, Molecular Therapy (2004) 9, 147-156], piggyBac [Wilson et al. Molecular Therapy (2007) 15, 139-145], Tol2 [Kawakami et al., PNAS (2000) 97 (21): 11403-11408] or Frog Prince [Miskey et al., Nucleic Acids Res. Dec 1, (2003) 31(23):6873-6881]. In general, DNA transposons change position from one DNA site to another in a simple cut-and-paste manner. Each of these elements has its own advantages, for example, Sleeping Beauty is particularly useful for region-specific mutagenesis, whereas Tol2 has the greatest tendency to integrate into expressed genes. Sleeping Beauty and piggyBac allow the use of motility-enhancing systems. Most importantly, these transposons have different target site preferences, allowing sequence changes to be introduced into overlapping but distinct sets of genes. Therefore, the use of multiple elements is particularly preferred to cover genes as much as possible. As the basic mechanism is common between the different transposases, piggyBac (PB) is mentioned as an example.

[0122] PB is a 2.5 kb insect transposon originally isolated from Trichoplusia ni. The PB transposon consists of asymmetric terminal repeats flanking the transposase PBase. PBase recognizes the terminal repeats and directs transposition by a "cut-and-paste"-based mechanism, preferentially transposing into the host genome at a tetranucleotide sequence, TTAA. Upon insertion, the TTAA target site is duplicated such that the PB transposon is flanked by this tetranucleotide sequence. Upon mobilization, PB typically precisely excises itself and restores the single TTAA site, thereby restoring the host sequence to its pre-transposon state. After excision, PB can either transpose to a new location or disappear from the genome permanently.

[0123] Typically, transposase systems offer an alternative means to remove the selection cassette after completion of homologous recombination, similar to the use of Cre / Lox or Flp / FRT. Thus, for example, the PB transposase system involves the introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two PB terminal repeats at the site of the endogenous TTAA sequence, and a selection cassette placed between the PB terminal repeats. Positive selection is applied to identify homologous recombinants containing the target mutation. Transient expression of PBase is combined with negative selection to excise the selection cassette and select for cells in which the cassette has been lost. The final target allele contains no exogenous sequences and contains the introduced mutation.

[0124] For PB to be useful for introducing sequence changes, there must be a natural TTAA site relatively close to the position at which a particular mutation is to be introduced.

[0125] Genome editing using recombinant adeno-associated virus (rAAV) platform: This genome editing platform is based on rAAV vectors that allow the insertion, deletion or replacement of DNA sequences in the genome of living mammalian cells. The rAAV genome is a positive or negative sense single-stranded deoxyribonucleic acid (ssDNA) molecule, approximately 4.7 kb in length. These single-stranded DNA viral vectors have the unique property of having high transduction rates and stimulating endogenous homologous recombination in the absence of double-stranded DNA breaks in the genome. Those skilled in the art can design rAAV vectors to target desired genomic loci and perform both global and / or fine endogenous genetic changes in cells. rAAV genome editing has the advantage of targeting a single allele and not causing off-target genomic mutations. rAAV genome editing technology is commercially available, for example the rAAV GENESIS™ system from Horizon™ (Cambridge, UK).

[0126] It will be appreciated that the agent may be a mutagen that induces random mutations and may select for cells that exhibit downregulation of the expression level and / or activity of the target.

[0127] The mutagen may be, but is not limited to, a genetic agent, a chemical agent, or a radiation agent. For example, the mutagen may be, but is not limited to, ionizing radiation, such as ultraviolet light, gamma rays, or alpha particles. Other mutagens may include, but are not limited to, base analogs capable of inducing copy errors, deaminating agents such as nitrous acid, intercalating agents such as ethidium bromide, alkylating agents such as bromouracil, transposons, natural and synthetic alkaloids, bromine and its derivatives, sodium azide, psoralens (e.g., in conjunction with ultraviolet radiation). The mutagen may be a chemical mutagen, such as, but is not limited to, ICR191, 1,2,7,8-diepoxyoctane (DEO), 5-AzaC, N-methyl-N-nitrosoguanidine (MNNG), or ethyl methanesulfonate (EMS).

[0128] Methods of confirming validity and detecting sequence variations are well known in the art and include, but are not limited to, DNA sequencing, electrophoresis, enzyme-based mismatch detection assays, and hybridization assays such as PCR, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern blot, and dot blot analysis.

[0129] Sequence variations in specific genes can also be determined at the protein level using, for example, chromatography, electrophoresis, immunodetection assays (eg, ELISA and Western blot analysis), and immunohistochemistry.

[0130] Furthermore, one skilled in the art can easily design knock-in / knock-out constructs that contain positive and / or negative selection markers for efficient selection of transformed cells that have undergone a homologous recombination event with the construct. Positive selection provides a means to enrich the population of clones that have incorporated foreign DNA. Non-limiting examples of such positive markers include glutamine synthetase, dihydrofolate reductase (DHFR), markers that confer antibiotic resistance, such as neomycin, hygromycin, puromycin, and blasticidin S resistance cassettes. Negative selection markers are necessary for selection against random integration and / or removal of marker sequences (e.g., positive markers). Non-limiting examples of such negative markers include herpes simplex thymidine kinase (HSV-TK), which converts ganciclovir (GCV) into a cytotoxic nucleoside analog, hypoxanthine phosphoribosyltransferase (HPRT), and adenine phosphoribosyltransferase (ARPT).

[0131] Downregulation at the polypeptide level According to a particular embodiment, the agent capable of downregulating P-selectin is an antibody or antibody fragment capable of specifically binding to and inhibiting P-selectin.

[0132] Preferably, the antibody specifically binds to at least one epitope of P-selectin.

[0133] In another embodiment, the agent is an antibody or antibody fragment capable of specifically binding to and inhibiting PSGL-1.

[0134] Preferably, the antibody specifically binds to at least one epitope of PSGL-1.

[0135] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants usually consist of surface groupings of chemically active molecules such as amino acid side chains or carbohydrate side chains and usually have specific three-dimensional structural and charge characteristics.

[0136] Methods for producing polyclonal and monoclonal antibodies and fragments thereof are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0137] In the present invention, the term "antibody" includes not only intact molecules but also functional fragments thereof, which are capable of binding to an epitope of an antigen.

[0138] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitope determinants usually consist of surface groupings of chemically active molecules such as amino acid side chains or carbohydrate side chains and usually have specific three-dimensional structural and charge characteristics.

[0139] According to certain embodiments, antibody fragments include, but are not limited to, single chains, Fab, Fab' and F(ab')2 fragments, Fd, Fcab, Fv, dsFv, scFvs, diabodies, minibodies, nanobodies, Fab expression libraries, or single domain molecules such as VH and VL capable of binding to an epitope of an antigen in an HLA-restricted manner.

[0140] Antibody fragments suitable for practicing some embodiments of the present invention include the complementarity determining regions (CDRs) of an immunoglobulin light chain (referred to herein as the "light chain"), the complementarity determining regions of an immunoglobulin heavy chain (referred to herein as the "heavy chain"), the variable region of the light chain, the variable region of the heavy chain, the light chain, the heavy chain, Fd fragments, and antibody fragments containing essentially the entire variable regions of both the light and heavy chains, such as Fv, single chain Fv Fv (scFv), disulfide stabilized Fv (dsFv), Fab, Fab', and F(ab')2, or antibody fragments containing the Fc region of an antibody.

[0141] As used herein, the term "complementarity determining region" or "CDR" is used interchangeably to refer to the antigen-binding region found in the variable region of heavy and light chain polypeptides. Generally, an antibody comprises three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3), and three CDRs in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3).

[0142] The identity of the amino acid residues of a particular antibody that make up the variable regions or CDRs can be determined using methods well known in the art, including the sequence variability defined by Kabat et al. (see, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC), the location of the structural loop regions defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989), a compromise of Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and www.bioinf-org.uk / abs), available complex crystal structures defined with contact definitions (MacCallum et al., J. Mol. Biol. 262:732-745, 1996), and "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).

[0143] As used herein, the terms "variable region" and "CDR" may refer to variable regions and CDRs defined by any approach known in the art, including a combination of approaches.

[0144] A functional antibody fragment that contains all, or essentially all, of the variable regions of both the light and heavy chains is defined as follows: (i) Fv is defined as a genetically engineered fragment expressed as two chains, consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH); (ii) Single-chain Fv ("scFv") is a genetically engineered single-chain molecule that contains the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single-chain molecule; (iii) disulfide-stabilized Fv ("dsFv") is an engineered antibody containing a variable region of the light chain and a variable region of the heavy chain linked by an engineered disulfide bond; (iv) Fab is the fragment of an antibody molecule containing a monovalent antigen-binding portion that can be obtained by treating whole antibody with the enzyme papain to yield an intact light chain and an Fd fragment of the heavy chain consisting of its variable and CH1 domains; (v) Fab' is the fragment of an antibody molecule that contains a monovalent antigen-binding portion of the antibody molecule can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained per antibody molecule); (vi) F(ab')2 is the fragment of an antibody molecule containing a monovalent antigen-binding portion (i.e., a dimer of Fab' fragments held together by two disulfide bonds) obtained by treating whole antibody with the enzyme pepsin; (vii) single domain antibodies or nanobodies are composed of a single VH or VL domain that exhibits sufficient affinity for the antigen; (viii) Fcab is a fragment of an antibody molecule containing the Fc portion of an antibody that has been developed as an antigen-binding domain by introducing antigen-binding ability into the Fc region of the antibody.

[0145] Methods for producing polyclonal and monoclonal antibodies and fragments thereof are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0146] Exemplary methods for making antibodies include inducing in vivo production of antibody molecules, screening immunoglobulin libraries (Orlandi DR et al., 1989. Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter G. et al., 1991. Nature 349:293-299), or using continuous cell lines to make monoclonal antibody molecules. These include, but are not limited to, the hybridoma method, the human B cell hybridoma method and the Epstein-Barr virus (EBV) hybridoma method (Kohler G. et al., 1975. Nature 256:495-497; Kozbor D. et al., 1985. J. Immunol. Methods 81:31-42; Cote RJ. et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole SP. et al., 1984. Mol. Cell. Biol. 62:109-120).

[0147] When generating antibodies in vivo, if the target antigen is too small to elicit an adequate immunogenic response, such antigen (hapten) can be linked to an antigenically neutral carrier, such as keyhole limpet hemocyanin (KLH) or serum albumin (e.g., bovine serum albumin (BSA)) carrier (see, e.g., U.S. Pat. Nos. 5,189,178 and 5,239,078). The coupling of the hapten to the carrier can be performed using methods well known in the art. For example, direct coupling can be performed to an amino group, followed by optional reduction of the imino bond formed. Alternatively, the carrier can be coupled using a condensing agent such as dicyclohexylcarbodiimide or other carbodiimide dehydrating agents. Coupling can also be performed using a linker compound. Both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company (Rockford, Ill.). The resulting immunogenic complex can be injected into a suitable mammalian subject, such as a mouse or rabbit. A suitable protocol involves repeated injections of the immunogen in the presence of an adjuvant according to a schedule that enhances the production of antibodies in the serum. The titer of immune serum can be readily measured using immunoassay techniques well known in the art.

[0148] The antisera obtained can be used directly or monoclonal antibodies can be obtained as described above.

[0149] Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.

[0150] Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to obtain a 5S fragment designated F(ab')2. This fragment can be further cleaved with a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage with pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, in Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and the references contained therein, which are incorporated herein by reference in their entireties. See also Porter, RR [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies may also be used, such as separation of the heavy chain to form monovalent L-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical or genetic techniques, so long as the fragment binds to the antigen recognized by the intact antibody.

[0151] As mentioned above, an Fv fragment consists of an association of a VH chain and a VL chain. The linkage may be non-covalent, as described by Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720)]. Alternatively, the variable chains may be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragment comprises a VH chain and a VL chain linked by a peptide linker. These single-chain antigen-binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains linked by an oligonucleotide. This structural gene is inserted into an expression vector and subsequently introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, in Whitlow and Filpula, Methods 2: 97-105 (1991), Bird et al., Science 242:423-426. (1988), Pack et al., Bio / Technology 11:1271-77 (1993), and U.S. Pat. No. 4,946,778, which are incorporated herein by reference in their entireties.

[0152] Another form of antibody fragment is a peptide coding for a single complementarity determining region (CDR). A CDR peptide ("minimal recognition unit") can be obtained by constructing a gene coding for the CDR of a desired antibody. Such a gene is prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells using the polymerase chain reaction. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

[0153] As mentioned above, the antibody fragment may comprise the Fc region of the antibody, referred to as "Fcab". Such antibody fragments typically comprise the CH2-CH3 domains of the antibody. Fcabs are engineered to comprise at least one modification in the structural loop region of the antibody (i.e., the CH3 region of the heavy chain). Such antibody fragments can be generated, for example, by providing a nucleic acid encoding an antibody comprising at least one structural loop region (e.g., an Fc region), modifying at least one nucleotide residue in at least one structural loop region, transferring the modified nucleic acid into an expression system, expressing the modified antibody, contacting the expressed modified antibody with an epitope, and determining whether the modified antibody binds to the epitope. See, for example, U.S. Pat. Nos. 9,045,528 and 9,133,274, which are incorporated herein by reference in their entireties.

[0154] Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies comprise a human immunoglobulin (recipient antibody) in which residues forming the complementarity determining regions (CDRs) of the recipient are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Residues that are not found in the recipient antibody or in the imported CDR or framework sequences may also be included. In general, humanized antibodies comprise substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are consensus sequences of human immunoglobulins. The humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0155] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced from a source other than human. These non-human amino acid residues are often called imported residues and are usually taken from imported variable domains. Humanization can essentially be performed by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody according to the method of Winter and coworkers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)]. Such humanized antibodies are thus chimeric antibodies (US Pat. No. 4,816,567), in which significantly less than the intact human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0156] Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95). (1991)]. Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Human antibody production is observed by challenge testing and closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and in the following scientific publications: Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 856-859 (1995); 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0157] The antibodies described herein may be conjugated to a therapeutic moiety, which can be, for example, a cytotoxic moiety, a toxic moiety, a cytokine moiety, and a secondary antibody moiety that contains a different specificity than the antibody of the invention.

[0158] Non-limiting examples of therapeutic moieties that can be conjugated to the antibodies of the invention are provided in Table 1 below.

[0159] [Table 1]

[0160] Other therapeutic moieties which may be conjugated to the antibodies of the invention include anti-cancer drugs such as chemotherapeutic agents, including, but not limited to, tubulin inhibitors such as exatecan, belotecan, emtansine, etc.

[0161] Therapeutic moieties may be attached or conjugated to the antibodies of the invention in a variety of ways, depending on the context, use and purpose.

[0162] When the functional moiety is a polypeptide, the immunoconjugate may be produced by recombinant means. For example, a nucleic acid sequence encoding a toxin (e.g., PE38KDEL) or a fluorescent protein (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP) or yellow fluorescent protein (YFP)) may be ligated in frame with a nucleic acid sequence encoding an antibody of the invention and expressed in a host cell to produce a recombinant, labeled antibody. Alternatively, the functional moiety may be chemically synthesized by the stepwise addition of one or more amino acid residues in a defined order, e.g., by solid phase peptide synthesis techniques.

[0163] Also, functional moieties may be attached to the antibodies of the present invention using standard chemical synthesis techniques widely practiced in the art (see, for example, http: / / www.chemistry.org / portal / Chemistry) using any suitable chemical linkage, directly or indirectly, for example, via a peptide bond (if the functional moiety is a polypeptide) or via covalent attachment to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer. Chimeric peptides may be linked via the carboxy (C) or amino (N) terminus of the peptide, or via attachment to an internal chemical group, such as a straight-chain, branched-chain, cyclic side chain, internal carbon atom, or internal nitrogen atom. Detailed descriptions of fluorescent labeling of antibodies are found in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110.

[0164] Exemplary methods for conjugating peptide moieties (therapeutic or detectable) to the antibodies of the invention are described below.

[0165] SPDP conjugation: A non-limiting example of a method for SPDP conjugation is described in Cumber et al. (1985, Methods of Enzymology 112: 207-224). Briefly, a peptide such as a detectable or therapeutic moiety (e.g., 1.7 mg / ml) is mixed with a 10-fold excess of SPDP (50 mM in ethanol), an antibody is mixed with a 25-fold excess of SPDP in 20 mM sodium phosphate, 0.10 M NaCl (pH 7.2), and each reaction is incubated at room temperature for about 3 hours. The reactions are then dialyzed against PBS. The peptide is reduced, for example, with 50 mM DTT at room temperature for 1 hour. The reduced peptide is desalted by equilibrating with 50 mM KH2PO4 (pH 6.5) on a G-25 column (up to 5% sample / column volume). The reduced peptide is mixed with the SPDP antibody at a molar ratio of 1:10 antibody:peptide and incubated overnight at 4° C. to form the peptide-antibody conjugate.

[0166] Glutaraldehyde conjugation: A non-limiting example of a method for glutaraldehyde conjugation is described in GT Hermanson (1996, "Antibody Modification and Conjugation" in Bioconjugate Techniques, Academic Press, San Diego). Briefly, antibody and peptide (1.1 mg / ml) are mixed with a 10-fold excess of 0.05% glutaraldehyde in 0.1 M phosphate, 0.15 M NaCl, pH 6.8, and allowed to react for 2 hours at room temperature. 0.01 M lysine can be added to block excess sites. After reaction, excess glutaraldehyde is removed using a G-25 column (10% v / v sample / column volume) equilibrated with PBS.

[0167] Carbodiimide conjugation: Conjugation of peptides to antibodies can be carried out, for example, using a dehydrating agent such as carbodiimide in the presence of 4-dimethylaminopyridine. Carbodiimide conjugation can be used to form a covalent bond between a carboxyl group of a peptide and a hydroxyl group of an antibody (resulting in the formation of an ester bond), or an amino group of an antibody (resulting in the formation of an amide bond), or a sulfhydryl group of an antibody (resulting in the formation of a thioester bond). Similarly, carbodiimide coupling can be used to form a similar covalent bond between a carbon group of an antibody and a hydroxyl, amino or sulfhydryl group of a peptide [see J. March, Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985]. For example, peptides can be covalently conjugated to antibodies using carbodiimides such as dicyclohexylcarbodiimide [B. Neises et al. (1978), Angew Chem., Int. Ed. Engl. 17:522, A. Hassner et al. (1978, Tetrahedron Lett. 4475), EP Boden et al. (1986, J. Org. Chem. 50:2394) and LJ Mathias (1979, Synthesis 561)].

[0168] According to another embodiment, the antibodies described herein are not conjugated to a therapeutic or diagnostic moiety.

[0169] Another agent that can be used with some embodiments of the present invention to downregulate P-selectin is an aptamer. As used herein, the term "aptamer" refers to a double-stranded or single-stranded RNA molecule that binds to a specific molecular target, such as a protein. Various methods are known in the art that can be used to design protein-specific aptamers. Those skilled in the art can use SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection, as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).

[0170] Another agent capable of downregulating P-selectin is any molecule that binds to and / or cleaves P-selectin. Such molecules are small molecules, P-selectin antagonists, or P-selectin inhibitory peptides.

[0171] Another agent that may be used to downregulate P-selectin includes proteolysis-targeting chimaera (PROTAC). Such agents are heterobifunctional, including a ligand that binds to ubiquitin ligase (such as E3 ubiquitin ligase) and a ligand that binds to P-selectin, and optionally a linker that connects the two ligands. Binding of PROTAC to target protein results in ubiquitination of exposed lysines on the target protein, followed by protein degradation by the ubiquitin proteasome system (UPS).

[0172] In one embodiment, the P-selectin inhibitor is a monoclonal antibody against P-selectin, such as crizanlizumab or inlacumab. These antibodies against P-selectin were developed to treat sickle cell anemia and myocardial damage after myocardial infarction, respectively. Both antibodies were well tolerated by patients when administered systemically.

[0173] According to certain embodiments, the P-selectin inhibitor is a monoclonal antibody against PSLGL-1. An example of such an antibody is VTX-0811, which is being developed by Verseau therapeutics (www.verseautx.com / pipeline).

[0174] In another embodiment, the P-selectin inhibitor is a small molecule such as rivipancel or tinzaparin, which were developed to treat sickle cell anemia and as an anticoagulant, respectively. Rivipancel is not specific for P-selectin, but inhibits several members of the selectin family. Tinzaparin is a heparinoid.

[0175] In another embodiment, the P-selectin inhibitor is KF 38789 from Tocris (i.e., 3-[7-(2,4-dimethoxyphenyl)-2,3,6,7-tetrahydro-1,4-thiazepin-5-yl]-4-hydroxy-6-methyl-2H-pyran-2-one).

[0176] Further exemplary P-selectin inhibitors are summarized in Table 2 below.

[0177] [Table 2] TIFF2025509755000004.tif248160

[0178] Agents used to downregulate the amount and / or activity of P-selectin may be formulated to cross the blood-brain barrier.

[0179] Exemplary methods of formulating the above drugs for increased permeability through the blood-brain barrier are described in Yeini et al., Advanced Therapeutics, DOI: 10.1002 / adtp.202000124.

[0180] Thus, for example, the agents described above can be formulated into nanoparticles, such as liposome-based nanoparticles, amphiphilic micelles, dendrimers, inorganic nanoparticles and polymeric nanoparticles.

[0181] Specifically for the delivery of oligonucleotides, the use of cationic nanoemulsified biodegradable poly(β-amino esters) (PBAEs), cell-derived extracellular vesicles, and spherical nucleic acid nanoparticles may be considered to improve delivery to the brain.

[0182] Because the BBB restricts the passage of most therapeutic agents from blood to the brain, receptor-mediated transcytosis can provide a non-invasive transport system for delivering targeted carriers to the brain parenchyma. Furthermore, this approach allows for selective targeting of tumor cells in brain tissue, thus reducing toxicity in other tissues and non-tumor cells in the brain. Examples of receptor-mediated approaches include manipulation of apolipoprotein receptors, targeting of epidermal growth factor receptors, targeting of transferrin receptors, targeting of insulin receptors, and targeting of adhesion molecules, all of which are contemplated.

[0183] It will be appreciated that the agent which inhibits P-selectin may be directly or indirectly bound to a moiety which targets the agent to the blood-brain barrier (e.g., the agent which inhibits P-selectin may be contained in a carrier which is capable of binding to the targeting moiety).

[0184] According to one aspect of the invention, agents that inhibit P-selectin are used (in combination with immunomodulatory agents) to treat brain metastatic cancer.

[0185] Metastatic brain tumors (also called secondary brain tumors) are caused by cancer cells that spread (metastasize) to the brain from different parts of the body.

[0186] According to certain embodiments, the brain metastasis cancer is selected from the group consisting of:

[0187] As mentioned above, the P-selectin inhibitor may be administered / co-formulated with an immunomodulatory agent. In one embodiment, the immunomodulatory agent is a nanoparticle containing a glioblastoma neoantigen peptide (e.g., GL261). Methods for formulating such nanoparticles are disclosed in WO2020 / 136657, the contents of which are incorporated herein by reference.

[0188] In one embodiment, the immunomodulatory agent is a checkpoint inhibitor.

[0189] The phrase "checkpoint blockade" or "checkpoint inhibitor" refers to a type of immunotherapy that aims to help the patient's own immune system fight cancer. This can be done using substances such as monoclonal antibodies or binding fragments thereof, which can be designed to target very specific molecules on the cell surface. For example, antibodies unblock responses that block the immune system's natural attack on invading cancer cells. In another example, a ligand-receptor interaction that has been studied as a target for cancer therapy is the interaction between the transmembrane programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1, CD274). In normal physiology, PD-L1 on the surface of cells binds to PD1 on the surface of immune cells, thereby inhibiting their activity. It appears that upregulation of PD-L1 on the surface of cancer cells can enable cancer cells to evade the host immune system by inhibiting T cells that might otherwise attack tumor cells. Thus, antibodies that bind to either PD-1 or PD-L1, thereby blocking the interaction, can enable T cells to attack tumors. In some alternatives, the checkpoint blockade therapeutic comprises an anti-PD-1 antibody or a binding fragment thereof (e.g., a monoclonal antibody or a humanized version thereof or a binding fragment thereof). In some alternatives, the checkpoint blockade therapeutic comprises PD-L1.

[0190] Examples of immunomodulatory agents include immunomodulatory cytokines, including but not limited to, IL-2, IL-15, IL-7, IL-21, GM-CSF, and any other cytokine that can further enhance an immune response; immunomodulatory antibodies, including but not limited to, anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1, and anti-PDL1; and immunomodulatory agents, including but not limited to, lenalidomide (Revlimid).

[0191] Exemplary anti-PD1 antibodies include pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), and dostarlimab (Jemperli).

[0192] According to a particular embodiment, the anti-PD1 antibody is nivolumab.

[0193] Further anti-PD1 antibodies include JTX-4014, Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBI308), Tislelizumab (BGB-A317), Toripalimab (JS 001) INCMGA00012 (MGA012), AMP-224 AMP-514 (MEDI0680).

[0194] Other contemplated anti-cancer drugs that may be administered to a subject in combination with the P-selectin inhibitors described herein include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adriamycin, adzelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bimesilate, bisulfite, bisulfite hydrochloride ... Snafide, bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carbicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, desaguanine, desaguanine mesylate, diazicon, docetaxel , doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine rabin phosphate, fluorouracil, flurocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride,lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospel, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, noga ramycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, rogletimide, safingol, safingol hydrochloride, sems tin, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, taxol, tecogalan sodium, tegafur, teroxantrone hydrochloride, temoporfin, teniposide, teroxylon, testolactone, thiamiprine, thioguanine, thiotepa, thiazophylline, tirapazamine, topotecan hydrochloride, toremifene citrate, trestro vintrexate acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tuburozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride. Additional antineoplastic agents include those described in Chapter 52 and its preface in Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner),Goodman and Gilman, "The Pharmacological Basis of Therapeutics", pp. 1202-1263, Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).

[0195] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Specific examples of cancer diseases include, but are not limited to, myeloid leukemias such as chronic myeloid leukemia, acute myeloid leukemia with maturation, acute promyelocytic leukemia, acute nonlymphocytic leukemia with basophilia, acute monocytic leukemia, acute myelomonocytic leukemia with eosinophilia; malignant lymphomas such as Burkitt's lymphoma, non-Hodgkin's lymphoma; lymphocytic leukemias such as acute lymphoblastic leukemia, chronic lymphocytic leukemia; solid tumors. myeloproliferative disorders such as ovarian tumors, benign meningiomas, mixed tumors of the salivary glands, and colon adenomas; adenocarcinomas such as small cell lung cancer, kidney, uterine, prostate, bladder, ovarian, colon, sarcoma, liposarcoma, myxoid, synovial sarcoma, rhabdomyosarcoma (alveolar), extraskeletal myxoid chondrosarcoma, and Ewing's tumor; and others include dysgerminoma of the testis and ovary, retinoblastoma, Wilms' tumor, neuroblastoma, malignant melanoma, mesothelioma, breast, skin, prostate, and ovary.

[0196] According to certain embodiments, the cancer is a solid tumor.

[0197] Exemplary cancers that can be treated with combination therapy (ie, immunomodulatory agent plus P-selectin inhibitor) include pancreatic cancer, lung cancer, breast cancer, primary melanoma and renal cancer.

[0198] The P-selectin inhibitor may be co-formulated with an immunomodulatory agent described herein or may be provided to the subject as a separate composition.

[0199] Thus, each drug in a combination may be formulated separately for use in combination. Drugs are said to be used in "combination" if the effect of one drug enhances or at least affects the effect of the other drug in the recipient of both drugs.

[0200] The two drugs in the combination drug work together to provide an effect on target cells that exceeds the effect of either drug alone. This advantage is manifested as a statistically significant improvement in a given parameter of target cell effect. In an embodiment, the improvement obtained by the treatment with the combination of drugs can be manifested as at least an additive effect, preferably a synergistic effect, compared with the result obtained by using only a single drug.

[0201] When used, each drug in the combination can be formulated similarly to the monotherapy in terms of dosage, formulation, and administration regimen. In this regard, synergistic effects achieved by the combination may allow for the use of smaller doses or less frequent administration, as would be demonstrated in appropriately controlled clinical trials.

[0202] According to one embodiment, the P-selectin inhibitor and the immunomodulatory agent are administered simultaneously.

[0203] According to another embodiment, the P-selectin inhibitor and the immunomodulator are administered sequentially, and the first agent is used, for example, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 1 month or more after the second agent. Such determination is within the ability of a person skilled in the art. In another embodiment, the P-selectin inhibitor and the immunomodulator are administered sequentially, and the second agent is used, for example, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 1 month or more after the first agent.

[0204] The P-selectin inhibitors (and immunomodulators) of some embodiments of the present invention can be administered to an organism per se or in a pharmaceutical composition mixed with a suitable carrier or excipient.

[0205] As used herein, the term "pharmaceutical composition" refers to a formulation of one or more active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0206] As used herein, "active ingredient" refers to the P-selectin inhibitor that is responsible for the biological effect.

[0207] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not significantly irritate an organism and does not impair the biological activity and properties of the compound being administered. Adjuvants are included in these terms.

[0208] Here, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0209] Techniques for drug formulation and administration are described in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA (latest edition), which is incorporated herein by reference.

[0210] Suitable routes of administration may include, for example, oral, rectal, transmucosal (particularly nasal), intestinal or parenteral delivery (including intramuscular, subcutaneous, intramedullary, as well as intrathecal, direct intraventricular, intracardiac (e.g., intraright or left ventricular), intracoronary, intravenous, intraperitoneal, intranasal, or intraocular injections).

[0211] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular engineering of drugs in an attempt to exploit one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins that include a combination of a transport peptide with affinity for an endothelial cell surface molecule and a drug that cannot cross the BBB by itself); pharmacological strategies designed to increase the lipid solubility of the drug (e.g., conjugation of a water-soluble drug to a lipid or cholesterol carrier); and temporary disruption of the integrity of the BBB by hyperosmotic disruption (injection of a mannitol solution into the carotid artery or use of biologically active agents such as angiotensin peptides). However, each of these strategies has limitations, such as the inherent risks associated with invasive surgical procedures, size limitations imposed by the limitations inherent in endogenous transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that can be activated outside the CNS, and the potential risk of brain damage that may occur within regions of the brain where the BBB is disrupted, making them suboptimal delivery methods.

[0212] Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example, by injecting the pharmaceutical composition directly into the patient's brain.

[0213] The pharmaceutical compositions of some embodiments of the present invention may be produced by processes well known in the art, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0214] Thus, pharmaceutical compositions for use according to some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate processing of the active ingredient into a medicament that can be used pharma- ceutically. Appropriate formulations depend on the selected route of administration.

[0215] For injection, the active ingredient of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0216] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by patients. Pharmaceutical preparations for oral administration can be prepared with solid excipients by optionally grinding the resulting mixture, optionally adding suitable auxiliary agents, and then processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

[0217] Dragee cores are provided with suitable coatings.For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and concentrated sugar solutions, optionally containing suitable organic solvents or solvent mixtures, may be used.Dyes or pigments may be added to the tablet or dragee coating for identification or to characterize different combinations of active compound doses.

[0218] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally, a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All formulations for oral administration should be in a dosage suitable for the chosen route of administration.

[0219] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0220] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0221] The pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Injections may be provided in unit dosage form, for example, in ampoules or multi-dose containers, optionally with the addition of preservatives. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0222] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active formulation in water-soluble form. In addition, suspensions of the active ingredient may be prepared as oily or aqueous injection suspensions as needed. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow the preparation of highly concentrated solutions.

[0223] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile, pyrogen-free water-based solution, before use.

[0224] Pharmaceutical compositions of some embodiments of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0225] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient (e.g., a P-selectin inhibitor) effective to prevent, alleviate or ameliorate symptoms of disease or to prolong the survival of the treated subject.

[0226] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0227] For any formulation used in the method of the present invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to obtain a desired concentration or titer. Such information can be used to more accurately determine effective doses in humans.

[0228] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures, or in experimental animals. The data obtained from these in vitro assays, cell culture assays, and animal studies can be used in formulating a range of dosages for use in humans. Dosages will depend on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition (see, for example, Fingl, et al. in "The Pharmacological Basis of Therapeutics", 1975, Ch. 1, p.1).

[0229] Dosage and intervals may be adjusted individually to provide levels of active ingredient (e.g., brain levels) sufficient to induce or inhibit a biological effect (minimal effective concentration, MEC). The MEC varies from formulation to formulation but can be estimated from in vitro data. The dose required to achieve the MEC depends on individual characteristics and route of administration. Detection assays can be used to measure plasma concentrations.

[0230] For any formulation used in the method of the present invention, the dose or therapeutically effective amount can be estimated initially from in vitro and cell culture assays. For example, doses can be formulated in animal models to obtain a desired concentration or potency. Such information can be used to more accurately determine effective doses in humans. Since administration of the disclosed combinations is expected to provide improved results over administration of a single agent, the therapeutically effective amount of each agent in the combination treatment can be, for example, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the FDA approved dose.

[0231] For example, the therapeutically effective amount of an immunomodulatory agent (e.g., an immunomodulatory antibody) in a combination treatment may be, for example, less than 50%, 40%, 30%, 20%, or 10% of the FDA approved dose. Conversely, the therapeutically effective amount of a P-selectin inhibitor in a combination treatment may be, for example, less than 50%, 40%, 30%, 20%, or 10% of the FDA approved dose.

[0232] Depending on the severity and responsiveness of the condition being treated, administration may be a single dose or multiple doses and the duration of treatment may last from several days to several weeks, or until a cure or abatement of the disease is observed.

[0233] According to another aspect of the present invention, there is provided a method of treating glioblastoma or brain metastatic melanoma in a subject in need thereof, comprising administering to the subject therapeutically effective amounts of crizanlizumab and an anti-PD1 antibody, thereby treating the glioblastoma or brain metastatic melanoma.

[0234] As used herein, the term "glioblastoma" (GBM), also known as glioblastoma multiforme or "grade IV astrocytoma" according to the WHO classification, refers to a primary tumor of the central nervous system derived from glial cells. GBM is one of the most lethal human cancers in the world, with an incidence rate of approximately 3.5 / 100,000 per year (Cloughesy, TF, WK Cavenee, and PS Mischel, Glioblastoma: from molecular pathology to targeted treatment. Annu Rev Pathol, 2014. 9: p. 1-25). Currently, the prognosis remains very poor, with an overall survival of approximately 15 months despite aggressive standard treatments including surgery, chemotherapy, and radiation therapy.

[0235] According to certain embodiments, the glioblastoma is in an early stage (eg, when the tumor diameter is less than 14 mm).

[0236] An exemplary therapeutic regimen for the treatment of cancer (eg, glioblastoma) is as follows. 1. The subject is administered crizanlizumab (e.g., 1-20 mg / kg, more specifically, about 5 mg / kg) plus an anti-PD1 antibody (e.g., nivolumab) (e.g., 1-20 mg / kg, more specifically, about 3 mg / kg) once every two weeks. 2. After 2-3 rounds of (1), subjects are administered crizanlizumab (e.g., 1-20 mg / kg, more specifically, about 5 mg / kg) once every 4 weeks. In stage 2, anti-PD1 antibodies (e.g., nivolumab) (1-20 mg / kg, more specifically, about 3 mg / kg) may be administered once every 2 weeks or once every 4 weeks.

[0237] According to certain embodiments, the active agent is administered following resection of the glioblastoma tumor.

[0238] The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0239] The compositions of some embodiments of the present invention may be provided in a pack or dispenser device, such as an FDA approved kit, if desired, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be packaged in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, in accordance with a notice on the container, which notice reflects the approval by the governmental agency of the composition or form of administration to humans or animals. Such notice may, for example, be the notice on the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the notice on the approved package insert. The compositions comprising the formulated formulations of the present invention in a compatible pharmaceutical carrier may also be prepared, packaged in an appropriate container, and labeled for the treatment of an indicated condition, as further detailed above.

[0240] Treatment method The term "treating" refers to inhibiting, preventing or arresting the onset of a pathology (disease, disorder or condition) and / or reducing, ameliorating or regressing a pathology. Those skilled in the art will appreciate that a variety of methods and assays can be used to assess the onset of a pathology, and may also be used to assess the reduction, amelioration or regression of a pathology.

[0241] As used herein, the term "preventing" refers to preventing the development of a disease, disorder, or condition in a subject who is at risk for the disease, but has not yet been diagnosed as having the disease.

[0242] Treatment regimen As used herein, the phrase "therapeutic regimen" refers to a treatment plan that specifies the type, dose, schedule and / or duration of treatment provided to a subject in need of treatment (e.g., a subject diagnosed with a medical condition). The selected treatment regimen can be aggressive, which is expected to provide the best clinical outcome (e.g., complete cure of the medical condition), or it can be more moderate, which may alleviate symptoms of the medical condition but not completely cure the medical condition. It will be understood that in some cases, a more aggressive treatment regimen may involve some discomfort to the subject or side effects (e.g., damage to healthy cells or tissues). Types of treatment can include surgical intervention (e.g., removal of lesions, diseased cells, diseased tissues or diseased organs), cell replacement therapy, administration of therapeutic agents (e.g., receptor agonists, antagonists, hormones, chemotherapy drugs) in local or systemic modes, exposure to radiation therapy using external sources (e.g., external beam radiation) and / or internal sources (e.g., brachytherapy), and / or any combination thereof. The dosage, schedule and duration of treatment may vary depending on the severity of the condition and the type of treatment selected, but one of skill in the art would be able to adjust the dosage, schedule and duration of treatment to suit the type of treatment.

[0243] As used herein, the term "about" refers to ±10%.

[0244] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0245] The term "consisting of" means "including and limited to."

[0246] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if they do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0247] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0248] Throughout this application, various embodiments of the invention may be described in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation of the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0249] In this specification, whenever a range of numerical values ​​is given, it is meant to include any cited numbers (decimals or integers) within the range given. In this specification, the phrases "range / range between" a first and a second numerical value and "range to / range to" a first numerical value "from" a second numerical value are used interchangeably and are meant to include the first and second numerical values ​​and all decimals and integers therebetween.

[0250] As used herein, the term "method" refers to methods, means, techniques and procedures for accomplishing a given task, including but not limited to those that are known, or that are readily developed from, methods, means, techniques and procedures known to practitioners in the chemical, pharmacological, biological, biochemical and medical fields.

[0251] As used herein, the term "treatment" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, or substantially ameliorating the clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0252] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination or as appropriate with any other described embodiment of the invention. Certain features described in the context of various embodiments are not considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0253] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed below, find experimental support in the following examples. EXAMPLES

[0254] Reference is now made to the following examples which, together with the above descriptions, illustrate certain non-limiting embodiments of the present invention.

[0255] material DMEM, fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, mycoplasma detection kit, EZ-RNA II total RNA isolation kit, and fibronectin (1 mg / ml, dilution: 1:100) were purchased from Biological Industries Ltd. (Beit Haemek Kibbutz, Israel). Percoll medium (catalog no. p4937) and all other chemical reagents, including salts and solvents, were purchased from Sigma-Aldrich (Rehovot, Israel). Milli-Q water was prepared using a Millipore water purification system. Amicon ultracentrifugal filters (molecular weight cut-off (MWCO) 5 or 3 kDa) and poly-L-lysine (PLL) (catalog no. A-005-C; 0.1 mg / ml) were purchased from Merck Millipore (Burlington, MA, USA). qScript™ cDNA synthesis kit was purchased from Quantabio (Beverly, MA, USA). Fast SYBR™ green Master Mix was purchased from Applied Biosystems (CA, USA). Collagenase IV, Dispase II (neutral protease) and DNase I were purchased from Worthington Biochemical Corporation (NJ, USA). RBC Lysis Solution (Cat. No. 420301) was purchased from BioLegend (San Diego, CA, USA). MACS MS Magnetic Column for Cell Isolation (Cat. No. 130-042-201), CD11b MicroBeads for Cell Isolation (Cat. No. 130-093-634) and CD45 (TIL) MicroBeads for Cell Isolation (Cat. No. 130-110-618) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The SELP inhibitor (SELPi) KF38789 (cat. no. 2748) was purchased from Tocris BioScience (Bristol, UK).Recombinant human SELP (catalog no. ADP3; lot no. ARL6019071), recombinant mouse SELP (rSELP) (catalog no. 10094-PS; lot no. DKLJ0118111), human SELP ELISA kit (catalog no. DPSE00), Total NO / Nitrite / Nitrate Immunoassay (catalog no. KGE001), Mouse XL Cytokine Array Kit (catalog no. ARY028), Human Cytokine Array Kit (catalog no. ARY005B), anti-human PSGL-1 neutralizing antibody (catalog no. MAB3345; lot no. CLYK0120111; Clone 688102), and anti-human SELP neutralizing antibody (catalog no. AF137; lot no. FBX0518051) were purchased from R&D Systems (Minneapolis, MN, USA). Human L-507 Cytokine Array Kit (Cat. No. AAH-BLM-1A-4; Lot No. 102920 009) was purchased from RayBiotech (Norcross, GA, USA). Anti-human / mouse CD44 neutralizing antibody (Cat. No. NBP2-2530; Lot No. VC289186) was purchased from Novus (CO, USA). Anti-mouse PSGL-1 neutralizing antibody (Cat. No. BE0188; Lot No. 676818M2) was purchased from Bio X Cell (MA, USA). MEBCYTO Apoptosis Kit was purchased from MBL International (UK) and recombinant mouse GM-CSF (Cat. No. 315-03-50ug; Lot No. 091855) was purchased from PeproTech (Rehovot, Israel). Latex beads for phagocytosis assay (cat. no. L4655) were purchased from Sigma-Aldrich (Rehovot, Israel). ProLong® Gold with DAPI (cat. no. p36935) and Hoechst 33342 (cat. no. H3570) were purchased from Invitrogen (Carlsbad, CA, USA).Mayer's hematoxylin solution (cat. no. 05-06002) and eosin Y solution (cat. no. 05-10002) were purchased from Bio-Optica (Milan, Italy). Anti-PD-1 antibody was purchased from ichorbio Ltd (Wantage, USA, cat. no. ICH1132; lot no. 1220L520). Immunostaining antibodies: Mouse anti-human SELP (cat. no. BBA1; lot no. APB081704; clone BBIG-E; dilution 1:30) was purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-mouse SELP (cat. no. 148302; lot no. B186735; clone RMP-1; dilution 1:50) was purchased from BioLegend (San Diego, CA, USA). Goat anti-mouse Alexa Fluor® 647 (cat. no. ab15115; lot no. GR309891-3; dilution 1:300), flow cytometry antibody: mouse anti-human SELP (cat. no. BBA1; lot no. APB081704; clone BBIG-E; dilution 1:20), mouse IgG1 isotype control (cat. no. mab002; dilution 1:20) were purchased from R&D Systems (Minneapolis, MN, USA).

[0256] method cell culture Metastatic melanoma 131 / 4-5B1 cells

[92] were cultured in RPMI medium supplemented with 10% FBS, 100 IU / mL penicillin, 100 μg / mL streptomycin, 12.5 IU / mL nystatin, and 2 mM L-glutamine. Primary melanoma A375 cells (ATCC, USA) were cultured in RPMI medium supplemented with 10% FBS, 100 IU / mL penicillin, 100 μg / mL streptomycin, 12.5 IU / mL nystatin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 25 mM HEPES. Primary melanoma WM115 cells (ECACC, Porton Down, Salisbury, UK) were cultured in MEM medium supplemented with 10% FBS, 100 IU / mL penicillin, 100 μg / mL streptomycin, 12.5 IU / mL nystatin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1× MEM NAA. Primary melanoma B16-F10 cells (ATCC, USA) were cultured in DMEM supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 12.5 IU / mL nystatin, and 2 mM L-glutamine. Primary melanoma B2905 was grown in RPMI supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 12.5 IU / mL nystatin, 25 mM HEPES, and 2 mM L-glutamine. Primary melanoma Mel-ret cells (kindly provided by Neta Erez) were grown in RPMI supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 12.5 IU / mL nystatin, 1 mM sodium pyruvate, and 2 mM L-glutamine. Primary melanoma D4M.3A cells (kindly provided by David W. Mullis) were grown in Advanced DMEM supplemented with 5% FBS, 100 IU / mL penicillin, 100 μg / mL streptomycin, 12.5 IU / mL nystatin, and 2 mM Glutamax. All melanoma cell lines were labeled with pQC-mCherry retroviral particles as previously described.

[0257] Isolation of primary mouse microglial cells Brains dissected from healthy 5-8 week old C57BL / 6 mice were minced and incubated with 1mg / ml collagenase IV, 2mg / ml dispase II (neutral protease), and 0.02U / ml DNase I for 50 min at 37°C. Red blood cells (RBCs) were lysed with RBC lysis solution, followed by myelin separation on a Percoll gradient. The resulting cell suspension was incubated with CD11b microbeads, and the desired populations were isolated with a MACS MS magnetic column. Mouse microglia were then seeded onto poly-L-lysine (PLL)-coated plates in microglia medium (ScienCell, CA, USA).

[0258] Isolation of splenocytes Splenocytes were freshly isolated from the spleens of healthy 7-11 week old C57BL / 6 mice. Spleens were crushed and passed through a 70 μm nylon strainer prior to RCB lysis. Flasks were coated with anti-CD3ε antibody (catalog 100340; lot B302116; clone 145-2C11) and splenocytes (30 × 106 cells / 75 cm2 flask) were incubated with 2 μg / ml anti-CD28 (catalog 102116; lot B331922; clone 3751) and 10 U / ml rhIL-2 for 6 days.

[0259] Flow cytometry In the flow cytometry assay, cells were harvested using a cell scraper, then washed with PBS, followed by further washing with PBS supplemented with 1% BSA and 5 mM EDTA. Tumor spheroids were recovered from Matrigel using Cell Recovery Solution (Corning) and washed with FACS buffer. To evaluate the expression of P-selectin, cells were then incubated with anti-human or anti-mouse P-selectin antibodies on ice for 1 hour, then washed and incubated with Alexa-488-labeled anti-mouse IgG binding protein on ice for 1 hour.

[0260] Co-culture proliferation assay Primary mouse microglia were seeded at different concentrations in 24-well plates (Corning), and the same amount of mCherry-labeled mouse Mel-ret (at ratios of 1:4, 1.5:1, 3:1, and 6:1) was added 24–72 h after seeding. The plates were then incubated in microglia medium for 96 h (37°C; 5% CO2), and the proliferation of fluorescently labeled cells was measured with an IncuCyte Zoom Live cell analysis system (Essen Bioscience). Melanoma cells in microglia medium were used as a control.

[0261] Co-culture of splenocytes with melanoma spheroids Tumor spheroids were prepared from D4M m-cherry-labeled tumors. D4M spheroids were prepared by seeding 500 cells per well in U-bottom low-attachment 96-well plates. Splenocytes were harvested from C57BL / 6J mice and seeded at a 1:50 ratio into the 96-well plates containing D4M spheroids and incubated in RPMI complete medium supplemented with 10% (v / v) FBS, 1% (v / v) PEST, 1% (v / v) HEPES, 1% (v / v) sodium pyruvate, and 0.1% (v / v) 2-mercaptoethanol. Co-cultured cells were treated with the SELP inhibitor KF38789 (Tocris, 0.5 μM), or PSI-697 (10 μM), with or without anti-PD-1 antibody (0.5 mg / ml). The growth of fluorescently labeled melanoma spheroids was measured using the IncuCyte Zoom Live cell analysis system (Essen Bioscience).

[0262] RNA isolation Total RNA was isolated using the EZ-RNA II total RNA isolation kit (Biological Industries Ltd., Israel) according to the manufacturer's protocol. Briefly, samples were immersed in 0.5 ml of denaturing solution / 10 cm. 2The RNA was dissolved in 100% culture plates. Water-saturated phenol was then added and the samples were centrifuged. Isopropanol was added to precipitate the RNA, and the centrifuged RNA pellet was washed with 75% ethanol, centrifuged, and resuspended in double-distilled ultrapure water. RNA concentration was assessed using a NanoDrop® ND-1000 spectrophotometer according to the manufacturer's V3.5 user manual (Nano-Drop Technologies, Wilmington, DE).

[0263] Synthesis of cDNA cDNA was synthesized using the qScript™ cDNA Synthesis Kit for RT-PCR according to the manufacturer's protocol. Briefly, 1 μg of total RNA sample was mixed with qScript reverse transcriptase, dNTPs, and nuclease-free water. The reaction tube was then incubated at 42°C for 30 min and heated at 85°C for 5 min to stop cDNA synthesis.

[0264] Real-time PCR Expression levels of target genes were assessed by SYBR green real-time PCR (StepOne plus, Life Technologies) and normalized with the GAPDH housekeeping gene.

[0265] Animal and ethical statement Animals were housed in the animal facility at Tel Aviv University. All experiments received ethical approval from the Institutional Animal Care and Use Committee (IACUC) of Tel Aviv University (protocol numbers 01-16-054 and 01-21-006) and were performed in accordance with NIH guidelines.

[0266] Animal models To generate primary melanoma tumors, mouse or human cells (0.5 × 10 6 1.5 × 10 cells / 100 μl) were inoculated intradermally (id) into immunocompetent C57BL / 6 mice or 6-8 week old male immunodeficient SCID mice, respectively. To generate intracranial melanoma tumors, mouse or human cells (1.5 × 10 41000 cells / 2μl) were stereotactically inoculated intracranially into the striatum of 8-10 week-old immunocompetent C57BL / 6 mice or 6-8 week-old male immunodeficient SCID mice, respectively. The mice were monitored twice weekly for weight changes and tumor growth was measured using a 4.7T MRI (MR Solutions, UK). Mice were euthanized and then the brains were removed for further immunostaining and flow cytometry analysis.

[0267] Tissue fixation with cryo-OCT Tumor-bearing mice were anesthetized with IP injection of ketamine (150 mg / kg) and xylazine (12 mg / kg) and perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were removed and incubated in 4% PFA for 4 hours, then incubated in 0.5 M sucrose (BioLab) for 1 hour and 1 M sucrose overnight (ON). Brains were then embedded in optimal cutting temperature (OCT) compound (Scigen) on dry ice and stored at -80°C.

[0268] immunostaining OCT-embedded tumor samples were cut into 5 μm thick sections. Staining was performed using a BOND RX automated stainer (Leica). Sections were stained with hematoxylin and eosin (H&E) and immunostained for P-selectin. Prior to antibody incubation, slides were incubated with 10% goat serum in 1x PBS + 0.02% Tween-20 for 30 min to block nonspecific binding sites. Slides were incubated with primary antibodies for 1 h, washed, and incubated with secondary antibodies for an additional 1 h. The slides were then washed and treated with ProLong® Gold with DAPI before being cover slipped. Stained samples were imaged using an EVOS FL Auto cell imaging system (ThermoFisher Scientific). At least three areas of each sample were imaged and quantified using ImageJ 1.52v software. Quantitation of positive staining was performed by measuring the total area stained in each image after background subtraction using monochromatic images representing the correlating marker.

[0269] statistical analysis Data are presented as mean ± standard deviation (sd) for in vitro assays and mean ± standard error (sem) for in vivo assays. Statistical significance was determined using unpaired two-tailed t-tests for comparisons between two groups and multiple comparison ANOVA test for comparisons between three or more groups. P < 0.05 was considered statistically significant. For Kaplan-Meier survival curves, p-values ​​were determined using the log-rank test. For in vivo tumor growth curves, p-values ​​were determined using one-way ANOVA, Dunn's test, or Holm-Sidak's test. Statistical analysis was performed using GraphPad Prism 8.

[0270] result GB tumors are characterized by a highly suppressive tumor microenvironment. To investigate whether PD-1 and its ligand PD-L1 are highly expressed in GB tumors, we investigated the GB patient database using the GlioVis data portal (Bowman, R. L et al., Neuro-oncology 2017, 19 (1), 139-141). Analysis showed that both PD-1 (PDCD1) and PD-L1 (CD274) were highly expressed in GB samples compared to healthy brain and low-grade glioma samples. Furthermore, their expression was negatively correlated with patient survival, confirming the role of the PD-1 / PDL-1 axis in GB progression (Figure 1A-B). Furthermore, in GB patient tissues, PSGL-1 (SELPLG) expression was found to be positively correlated with both PD-1 and PDL-1 expression, and SELP expression was found to be positively correlated with PD-L1 expression. This may suggest that immunosuppressive microglia / macrophage cells express both PSGL-1 and PD-L1 and promote PD-1 expression by T cells, and that GB cells express SELP and PD-L1. These results may explain why anti-PD-1 shows no therapeutic benefit as monotherapy in GB patients.

[0271] To evaluate whether the SELP and PD-1 axis is relevant in other brain tumors, FFPE samples of harvested normal human brain were collected along with patient samples of melanoma, breast, lung and CRC brain metastasis (BM) and patient diffuse pontine glioma (DIPG) samples. Immunostaining revealed high expression of SELP and PD-L1 in all samples, high expression of PSGL-1 in DIPG and BM of melanoma, breast and lung, and high expression of PD-1 in BM of melanoma and lung (Figure 2A-B). In addition, high expression of SELP was found in primary melanoma, breast, lung and pancreatic ductal adenocarcinoma (PDAC) patient samples. PSGL-1 was found to be highly expressed in primary melanoma, breast and lung samples, and PD-1 and PD-L1 in primary melanoma, PDAC and lung samples (Figure 3A-B).

[0272] To investigate the role of microglia in cancer brain metastasis, several melanoma brain metastasis (MBM) mouse models were established. By using Iba-1 immunostaining, activated microglia were identified in the tumor sites of all the different models and in formalin-fixed paraffin-embedded (FFPE) samples of MBM patients (Figure 4A). To evaluate the impact of microglia on MBM progression, primary mouse microglia were co-cultured with mouse RET MBM cell lines and cancer cell proliferation was observed. A direct correlation was observed between the increased proliferation rate of mouse RET MBM cells and the concentration of mouse microglia in the culture (Figure 4B).

[0273] Furthermore, we established a 3D in vitro model of melanoma using human WM115 and mouse D4M.3A melanoma cell lines. In the presence of microglia within the spheroids, we observed increased invasion of D4M.3A spheroids (Figure 4C) and higher expression of SELP compared to melanoma cells grown on 2D plastic culture dishes (Figure 5A-C). This expression increased over time after Matrigel seeding of D4M.3A spheroids (Figure 5C).

[0274] To evaluate the therapeutic potential of combined treatment with SELP inhibitors (SELPi) and anti-PD-1 antibodies, we co-cultured D4M.3A tumor spheroids with primary mouse microglia. After spheroid formation, freshly isolated mouse splenocytes were added with different treatments and cancer cell killing by splenocytes was monitored. The results showed a synergistic effect of SELPi treatment and anti-PD-1 antibodies in spheroids composed of both melanoma cells and microglia, whereas only a minor effect was observed in D4M.3A spheroids without microglia (Figure 6A-B). This indicates that microglia are required to induce cancer cell killing by splenocytes after inhibition with SELP and PD-1.

[0275] We validated these results in further cancer models, finding high expression of SELP in the EMT-6 mouse breast cancer cell line (Figure 7A). Furthermore, using human astrocyte-MDA-MB-231 breast cancer multicellular 3D spheroids, we were able to reduce cancer cell invasion after SELP inhibition (Figure 7B). This may suggest that SELP is also important for the interaction between breast cancer cells and astrocytes.

[0276] Focusing on lung cancer cells, we observed increased invasion of human A549 spheroids when co-cultured with human microglia (Figure 8A). Flow cytometry analysis of these spheroids showed high expression of SELP and PSGL-1 in A549 cells and human microglia, respectively (Figure 8B). Indeed, inhibition of SELP reduced spheroid growth and invasion in the A549-microglia 3D model (Figure 8C-D).

[0277] Taken together, these results demonstrated the potential therapeutic benefit of combining SELP inhibition with other immunomodulatory agents (e.g., anti-PD-1) for both primary and secondary brain malignancies.

[0278] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0279] It is the intention of the applicants to incorporate by reference all publications, patents and patent applications referenced herein in their entireties as if each individual article, patent or patent application was individually and specifically set forth when referenced. Furthermore, citation or identification of a reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. Additionally, the priority documents of this application are incorporated herein by reference in their entireties.

Claims

1. A combination agent for treating a cancer selected from the group consisting of brain metastatic cancer, pancreatic cancer, lung cancer, breast cancer, primary melanoma, and kidney cancer, comprising a therapeutically effective amount of an agent that specifically reduces the amount and / or activity of P-selectin, and an immunomodulator.

2. The combination drug according to claim 1, wherein the brain metastasis cancer is brain metastasis melanoma, brain metastasis breast cancer, brain metastasis lung cancer, or brain metastasis colorectal cancer.

3. The combination agent according to claim 1 or 2, wherein the agent that specifically reduces the amount and / or activity of P-selectin specifically binds to P-selectin or the polynucleotide encoding P-selectin.

4. The combination agent according to claim 1, wherein the agent that specifically reduces the amount and / or activity of P-selectin is bound to P-selectin glycoprotein ligand-1 (PSGL-1) or a polynucleotide encoding PSGL-1.

5. The combination drug according to claim 1, wherein the immunomodulatory agent comprises an immunomodulatory antibody.

6. The combination drug according to claim 5, wherein the immunomodulatory antibody is selected from the group consisting of anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1, anti-PDL1, anti-LAG3, anti-IDO, and anti-TIGIT.

7. The combination drug according to claim 1, wherein the agent that specifically reduces the amount and / or activity of P-selectin is an inhibitory antibody that binds to and inhibits P-selectin.

8. The combination drug according to claim 7, wherein the inhibitory antibody is chryzanlizumab or incrumab.

9. The combination drug according to claim 1, wherein the agent that specifically reduces the amount and / or activity of P-selectin is a low-molecule drug.

10. The combination drug according to claim 1, wherein the agent that specifically reduces the amount and / or activity of P-selectin is a polynucleotide drug.

11. The combination agent according to claim 1, wherein the agent that specifically reduces the amount and / or activity of P-selectin is contained in nanoparticles.

12. The combination drug according to claim 11, wherein the nanoparticles are bound to a targeting portion that increases delivery across the blood-brain barrier.

13. The combination agent according to claim 1, wherein the agent that specifically reduces the amount and / or activity of P-selectin is bound to a targeting portion that increases delivery across the blood-brain barrier.

14. A combination drug for treating glioblastoma or melanoma metastasis in subjects requiring treatment for glioblastoma or melanoma metastasis, comprising a therapeutically effective dose of crizanlizumab and an anti-PD1 antibody.

15. The combination drug according to claim 14, wherein the crizanlizumab is delivered at a dose of 5 mg / kg once every two weeks or once every four weeks.

16. The combination agent according to claim 14 or 15, wherein the chryzanlizumab and the anti-PD1 antibody are initially administered once every two weeks for at least four weeks.

17. A drug for treating pancreatic cancer or kidney cancer that specifically reduces the amount and / or activity of P-selectin.

18. The agent according to claim 17, for use in combination with an immunomodulator.

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