WO2025170888A1 - A combination of the axl inhibitor slc-391 and a pd-1 inhibitor for use in the treatment of blood cancer - Google Patents

Abstract

Provided herein are combination therapies for treating blood cancer, in particular, acute myeloid leukemia, by concurrently targeting AXL and PD-1.

Description

A COMBINATION OF THE AXL INHIBITOR SLC-391 AND A PD-1 INHIBITOR FOR USE IN THE TREATMENT OF BLOOD CANCER

BACKGROUND

Technical Field

[1] The present disclosure provides a combination therapy in the treatment of blood cancer, particularly acute myeloid leukemia (AML).

Description of the Related Art

[2] Acute myeloid leukemia (AML) is a rapidly progressing, often fatal hematopoietic malignancy characterized by clonal expansion of leukemic stem cells (LSCs) and differentiation block in the myeloid lineage, with accumulation of myeloid precursors (blasts) (Dohner et al., 2010; Dohner et al., 2015; Thomas and Majeti, 2017). An accumulation of rapid growth of abnormal cells severely disrupts normal hematopoiesis, and thus results in bone marrow failure. AML is a highly heterogeneous malignancy with complicated genetic mutations, and treatment of AML is one of most challenging hematopoietic malignancies. Although major progress has been made in identifying molecular and genetic subgroups, AML therapies and long-term patient outcomes have not improved significantly in the last 40 years. The 5-year survival rate thus remains less than 40% for patients under the age of 60, and only 10-20% for older patients (Dohner et al., 2010; Dohner et al., 2015; Khwaja et al., 2016). While standard induction chemotherapies, with anthracycline or cytocarabine, for example, lead to an initial reduction in myeloid blasts in most patients for a limited time period, resistance to frontline chemotherapy and relapses remain major causes for AML treatment failure. Accordingly, there is an urgent need for novel and more effective therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

[3] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [4] FIG. 1A-1C show SCL-391 treatment reduces the growth of AML cells and increases T-cell populations when co-cultured with activated PBMC. FIG. 1A - MV4-11- GFP⁺ cells were cultured with or without SLC-391 (0.05 pM, 0.1 pM, and 0.2 pM) or PBMCs at the effector: target ratio 5:1. Viable GFP⁺ cells were counted under fluorescent microscope at 3, 5 and 7 days after treatment, and the percentage of viable cells was normalized to untreated condition. The growth differences among various treatment conditions were measured after 7 days in culture. FIG. IB - Immunofluore scent photos were taken under a fluorescent microscope in MV4-11-GFP⁺ cells under the treatment condition indicated. FIG. 1C - The same cells were harvested after 7 days in culture and stained with antibodies to detect total CD3⁺ T cells, CD4⁺ and CD8⁺ T cells in each treatment condition by flow cytometry. The differences among each treatment condition were normalized to PBMC treatment only condition. Data shown are the mean ± SEM of measurements for at least three experimental replicates. P-values were calculated using 1- way ANOVA with Tukey’s correction for multiple comparisons using GraphPad Prism.

[5] FIG 2A-2F show pembrolizumab (PEM) treatment enhances the effects of SLC- 391 -mediated cell killing when co-cultured with PBMCs. FIG. 2A - In PBMC (effector: target ratio 5:1) co-culture conditions, MV4-11GFP⁺ cells were treated with SLC-391 (O.lpM, 0.2 pM) or PEM (lOpg/ml), either alone or in combination. Viable cells were counted under fluorescent microscope and normalized to PBMC treatment only condition. The growth differences among various treatment conditions were measured after 7 days in culture. FIG. 2B - Total CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were analyzed by flow cytometry and fold changes were shown as relative to PBMC treatment only condition. FIG. 2C - The percentage of CD3⁺/PD1⁺ cells was analyzed via flow cytometry under each treatment condition. FIG. 2D - PD-L1 expression in MV4-11-GFP⁺ cells was analyzed after 5 or 7 days of treatment by flow cytometry. The mean fluorescence intensity was then calculated under each treatment condition. FIG. 2E - CD3'CD56⁺ NK cells were analyzed under each treatment condition by flow cytometry and the fold change was normalized to PBMC treatment only condition. FIG. 2F - In PBMC co-culture conditions (effector: target ratio 5:1), MV411-GFP+ cells were treated with SLC-391 (0.05 pM, O.lpM, or 0.2pM) alone, PEM (O.lpg/ml, 1 pg/ml, or 10 pg/ml) alone, or with a combination of both PEM and SLC-391. Viable GFP+ cells were measured by counting beads via FACS analysis after 5 days in culture (bar graph). Synergistic effects among different treatment conditions were calculated based on various concentrations of either SLC-391 or PEM by SynergyFinder software. CI <1, CI =1, and CI >1 indicate synergistic, additive, and antagonistic effects, respectively. Other synergy scores, including the Loewe synergy score (left tensor) and HSA score (right tensor), were also determined. A score more than 10 or lower than -10 was classified as synergistic or antagonistic, respectively; a score between -10 and 10 was considered additive. Data shown are the mean ± SEM of measurements for at least three experimental replicates. P- values were calculated using 1-way ANOVA with Tukey’s correction for multiple comparisons using GraphPad Prism.

[6] FIG. 3A-3C show treatment of MV4-11-GFP⁺ cells with SLC-391 or in combination with PEM increases CD8⁺IFN-Y⁺ cells when co-cultured with PBMCs. FIG. 3A - After 7 days of treatment, cells were harvested and fixed for intracellular staining of IFN-y in the CD3⁺CD8⁺ T cell population. The percentage of CD8⁺IFN-y⁺ cells is shown, along with representative blots under each treatment condition by flow cytometry. FIG. 3B - Culture medium was harvested at the same time and absolute concentration of IFN-y (pg/mL) in the medium was measured using the Legendplex cytokine array kit from BioLegend. FIG. 3C - Using the LEGENDplex cytokine array kit, several other cytokines including TNF-a, Granzyme A, Galectin-9, sFasL, and Tim-3 were measured under each treatment condition.

[7] FIG. 4A-4G show combination treatment of SLC-391 and PEM inhibits the growth of AML stem/ progenitor cells and increases functional T-cells when co-cultured with PBMC. FIG. 4A - Pre- violet labeled CD34⁺ primary AML cells were cultured in the presence of SLC-391 (0.2 pM), anti-PDl(10pg/ml), alone or in combination, with or without PBMC for 7 days in six-growth factors supplementary medium. Cells were harvested and AccruCheck counting beads were added to each tube to calculate the remaining viable AML cells in each condition by flow cytometry analysis. Viable cells from each treatment were normalized to untreated condition. FIG. 4B - The fold change in CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ cells under PBMC co-culture condition was analyzed and normalized to PBMC treatment only condition. FIG. 4C - The PD1⁺CD3⁺ cells were compared among each group and the mean fluorescence intensity (MFI) was normalized to PBMC treatment only condition. Representative blots under each treatment condition are shown, as assessed by flow cytometry. FIG. 4D - The PD-L1 expression of primary AML cells was analyzed via flow cytometry analysis and the MFI under each treatment condition was normalized to the level of unstained control. FIG. 4E - The percentage of CD8⁺IFN-Y⁺ cells was determined via intracellular staining and representative blots under each treatment condition are shown. The concentration of IFN-y (pg/mL) in culture medium was measured using the LEGENDplex cytokine array kit. FIG. 4F - Heat map of each cytokine concentration with hierarchical clustering of the samples used for cytokine array assay. The row z-scores were used to display changes in cytokine concentration across all samples for different treatment conditions. FIG. 4G - Absolute concentrations of TNFa, sFasL and TIM3 (pg/mL) detected in co-cultured AML patient cells are shown.

[8] FIG. 5A-5G show combination treatment of SLC-391 and PEM decreases leukemia burden and enhances survival of mice in a humanized PDX model. FIG. 5A - Schematic of experimental design using a preclinical transplant model. CD34⁺ cord blood (CB) cells were intravenously (IV) injected into sub-lethal irradiated NSG mice at 4 weeks of age (0.4xl0⁵/mouse) and engraftment levels in peripheral blood (PB) were monitored via flow cytometry analysis. After 12 weeks transplantation, MV4-11 cells carrying a luciferase reporter and GFP marker were IV injected into the humanized immune mice (0.5xl0⁶/mouse). Two weeks after transplantation, treatment was administered for three weeks and the mice were monitored and analyzed as indicated. FIG. 5B - Engraftment levels of human CD45⁺, CD3⁺, CD3’CD56⁺(NK), HLR-DR⁺CD123⁺ (pDC) and other cell populations were determined at week 8 and week 12 after transplantation of CD34⁺ CB cells into mice. FIG. 5C - Bioluminescence images of mice after three weeks treatment and representative images from each group are presented. FIG. 5D - Three to four mice from each group were then sacrificed and images and weights of spleen from each mouse are shown. FIG. 5E - Engraftment levels of AML cells (CD45⁺GFP⁺) and CB-derived CD45⁺GFP" cells from hematological tissues including PB, spleen and BM are shown with representative flow cytometry plots. FIG. 5F - Overall survival of leukemic mice from each treatment group (n= 6 mice per group). FIG. 5G - Immunohistological staining was performed to evaluate the number of leukemic cells, CD3+ cells, and CD8+ cells for mice receiving combination treatment of SLC-391 and PEM. Spleens of treated mice were harvested and fixed in 10% formalin. The fixed tissues were further individually processed for Haemotoxylin and Eosin staining and IHC staining of anti-human CD3, CD8, and PDL1 antibodies. The representative 40X imaging of each treated mouse is shown. The percentage of CD3-positive, CD8-positive, and PDL1 -positive cells from each treatment condition are shown in the bar graph wherein the cell numbers were manually counted and calculated based on the total 4000 cells in each field. P-values were calculated using logrank test.

[9] FIG. 6A-6B show combination treatment of SLC-391 and PEM increased CD3⁺ T cells and other immune cell populations in a PDX model. Flow cytometry analysis of CD45⁺GFP" and CD45⁺CD3⁺ cells and other immune cell populations detected in PB (FIG. 6A) and BM (FIG. 6B) of mice under each treatment group.

DETAILED DESCRIPTION

[10] As disclosed herein, a combination treatment of AXE inhibitor with PD-1 inhibitor (e.g., an anti-PD-1 antibody) enhances anti-leukemic activity by reducing or eliminating the tumor-induced immunosuppression in AME. Accordingly, various embodiments of the present disclosure are directed to combination therapies utilizing an AXE inhibitor in combination with an PD- 1 inhibitor for treating blood cancer, in particular, acute myeloid leukemia.

[11] AXL is a receptor tyrosine kinase of the TAM family including TRYO3, AXL, and MER (Graham et al., 2014; Schoumacher and Burbridge, 2017; Zhu et al., 2019). TAM family members are overexpressed in many solid tumors, enhancing survival and resistance to apoptosis. Among four putative TAM ligands, growth arrest-specific gene 6 (GAS6) has subnanomolar affinity for AXL and is the most prominent and well-studied activating ligand for AXL (Ben-Batalla et al., 2013). AXL plays a critical role in mediating migration and invasiveness of cancer cells. Notably, most AML patient cells express increased levels of AXL protein as compared to normal individuals, and highly increased AXL activity has been reported in AML stem/progenitor cells, including patients harboring mixed-lineage leukemia (MLL) fusion, a poor prognostic group (Niu et al., 2021). The mechanistic studies indicated that AML cells can stimulate bone marrow- derived stoma cells to upregulate GAS6 activity and enhance the GAS6/AXL signaling that confers AML cells the resistance to chemotherapies (Ben-Batalla et al., 2013; Gay et al., 2017). Interestingly, suppression of AXL via shRNA or the use a newly developed AXL inhibitor, SLC-391, significantly increases apoptosis and decreases proliferation and survival of AML cell lines and patient cells in vitro and in vivo (Niu et al., 2021). Thus, targeting the GAS6/AXL activity is consequently a rational new treatment strategy in treating AML. Most interestingly, SLC-391 can also sensitize AML stem/progenitor cells to venetoclax, a BH3-mimetic and selective BCL-2 inhibitor (Delbridge et al., 2016; DiNardo et al., 2019), with strong synergistic effects in vitro and in patient-derived xenografts (PDX) models (Niu et al., 2021).

[12] Further, single-cell RNA- sequencing analysis of CD34⁺ AML stem/progenitor cells demonstrated that AXL inhibition is involved in T-cell-mediated cytotoxicity and T- cell immunity, suggesting a critical role of AXL in regulating tumor immune response in AML patients (Niu et al., 2021; Tirado-Gonzalez et al., 2021). Increasing evidence also indicates the specific role of AXL in tumor immune evasion by demonstrating increased AXL expression to be a component of anti-programmed cell death- l(PD-l) resistance in drug nonresponders (Hugo et al., 2016). Recent studies have shown that tumor immune escape is critical for tumor cell survival and progress. In particular, increased expression of inhibitory checkpoint molecules, such as programmed death-ligand 1 (PD-L1), has been demonstrated in AML patient cells as compared to healthy donors, which contribute to immune exhaustion and possibly AML disease relapse (Abaza and Zeidan, 2022; Williams et al., 2019). The PD-L1 blocking peptide can impede the binding between PD-1 and PD- Ll, disrupting the inhibitory signals on natural killer (NK) cells and T cells, leading to greater antitumor responses (Pardoll, 2012; Sharma and Allison, 2015). Similarly, the blockade of immune checkpoints, such as PD-1, can enhance antitumor immunity and the potential to produce enduring clinical responses. Clinical trials, such as anti-PD-1 therapy, Pembrolizumab (PEM), a humanized monoclonal IgG4 anti-PD-1 antibody, has shown promising clinical responses in melanoma and non-small cell lung cancer (Garon et al., 2015; Topalian et al., 2012). In AML, clinical trials of PEM monotherapy were tolerable and feasible in AML patients but its treatment effects were short-lived (Zeidner et al. 2021.; Guamera et al. 2023. Interestingly, the combination of hypomethylating agents and PD-1/PD-L1 inhibitors has shown promising results (Daver et al., 2019; Goswami et al., 2022). However, to date these therapies have not demonstrated a major and long-term benefit for these patients (Abaza and Zeidan, 2022; Gomez-Llobell et al., 2022; Krupka et al., 2016; Vaddepally et al., 2020).

Definitions

[13] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

[14] Unless otherwise defined herein, scientific and technical terms shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[15] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have," and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[16] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of this disclosure has "similar binding" or "similar activity" when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity).

[17] "Antigen" or "Ag", as used herein, refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells, activation of complement, antibody dependent cytotoxicity, or any combination thereof. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, stool samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen.

[18] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics. Where an antigen is or comprises a peptide or protein, the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding.

[19] The terms "antigen-binding site" or “antigen binding moiety” are used interchangeably herein and refer to the part of the antibody and/or immunoglobulin molecule that participates in binding to an antigen and/or epitope. In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. The "hypervariable regions" are three highly divergent stretches within the V regions of the heavy and light chains which are interposed between "framework regions," ("FR"), which are relatively conserved flanking stretches. The term "FR" refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In a human antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy ("H") and light ("L") chains are referred to as "complementarity-determining regions" or "CDRs." Antigen-binding sites can exist in an intact antibody, in an antigenbinding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv, using a peptide linker to connect the heavy chain variable domain to the light chain variable domain in a single polypeptide. An antigen binding site can comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[20] The term “antibody” as used herein encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies), antibody fusion proteins, antibodies that for heterodimers in engineered proteins, and antibody fragments so long as they exhibit the desired antigen-binding activity.

[21] An “antibody fragment” refers to a polypeptide or protein other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[22] Numbering of CDR and framework regions may be according to any known method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5^th ed.; Chothia and Lesk, J. Mol. Biol. 796:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and Pliickthun, J. Mol. Bio. 309:657-670 (2001)). Equivalent residue positions can be annotated and for different molecules to be compared using Antigen receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). The CDRs of an antigen-binding site can be determined according to known methods, such as the Kabat, Chothia, EU, IMGT, and AHo as described above. The CDRs determined under these definitions typically include overlapping or subsets of amino acid residues when compared against each other. The heavy chain CDRs and light chain CDRs of an antibody can be defined using different numbering conventions. For example, in certain embodiments, the heavy chain CDRs are defined according to Chothia, supra, and the light CDRs are defined according to Kabat, supra. CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 denote the light chain CDRs.

[23] As used herein, PD-L1 (also known as “programmed death-ligand 1” or CD274 in humans) refers to the protein of UniProt Accession No. Q0GN75 (human) and related isoforms and orthologs.

[24] As used herein, the term “inhibit” refers the reduction of a specified activity (e.g., immune suppression or tumor growth). Unless specified otherwise, an activity can be considered inhibited if the activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%, as measured by the methods disclosed herein or known in the art.

[25] ‘ ‘Mammal” or “mammalian subject” includes humans and domestic animals, such as cats, dogs, swine, cattle, sheep, goats, horses, rabbits, and the like.

[26] “Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted aryl” means that the aryl radical may or may not be substituted and that the description includes both substituted aryl radicals and aryl radicals having no substitution.

[27] “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[28] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene- 1,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4- amino salicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, -tolucncsul I'onic acid, trifluoroacetic acid, undecylenic acid, and the like.

[29] “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, A-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[30] Often crystallizations produce a solvate of the compound of the disclosure. As used herein, the term “solvate” refers to an aggregate that comprises one or more molecules of a compound of the disclosure with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compound of the disclosure may be true solvates, while in other cases, the compound of the disclosure may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

[31] A “pharmaceutical composition” refers to a formulation of a compound of the disclosure and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.

[32] “Therapeutically effective amount” refers to that amount of a compound of the disclosure which, when administered to a mammal, preferably a human, is sufficient to effect treatment, as defined below, of a disease or condition in the mammal, preferably a human. The amount of a compound of the disclosure which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[33] “Treating” or “treatment” as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or disorder of interest, and includes: (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting its development or reversing its progression; or

(iii) relieving the disease or condition, i.e., causing regression of the disease or condition.

Combination Therapy

[34] Described herein in more detail are therefore methods for treating blood cancer in a patient in need thereof, the method comprising concomitantly administering one or more AXL inhibitors with one or more PD- 1 inhibitor. The resulting therapeutic effects are surprisingly greater than the mere additive effects of monotherapies using each type of inhibitors alone. Such a synergistic combination is further accompanied by low toxicity.

[35] The AXL inhibitors suitable for the combination therapy disclosed are aminopyridine derivatives, known for being TAM family kinase inhibitors. See e.g., US Pat. No. 10,233,176, which is incorporated herein by reference in its entirety. In a preferred embodiment, the AXL inhibitor is 3-(5-(cyclopropylmethyl)-l,3,4-oxadiazol-2- yl)-5-(l-(piperidin-4-yl)-lH-pyrazol-4-yl)pyridin-2-amine (referred to herein as “Compound A” or “SLC-391”):

Compound A

[36] Programmed cell death protein 1 (PD-1, also known as CD279) is a cell surface receptor on T cells and B cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting selftolerance by suppressing T cell inflammatory activity. Unless otherwise indicated, PD-1 refers to the protein of UniProt Accession No. Q15116 (human) and related isoforms and orthologs. [37] PD-1 inhibitors are a class of drugs that block PD-1 and promote activation of the immune system to attack tumors and are used to treat certain types of cancer. In some embodiments a PD- 1 inhibitor is an anti-PD- 1 antibody that blocks the interaction of PD- 1 with a PD-1 ligand, such as PD-L1. Examples of anti-PD- 1 antibodies include pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012, AMP-224, AMP- 514 (MEDI0680), acrixolimab, and toripalimab. In some embodiments, a PD-1 inhibitor is anti-PD-Ll antibody that blocks the interaction of PD-L1 with PD-1. Examples of anti- PD-L1 antibodies include Atezolizumab, Avelumab, and Durvalumab.

[38] As used herein “combination therapy” refers to the administration of one or more AXL inhibitor, (e.g., Compound A), in combination with the administration of one or more PD-1 inhibitor (e.g., pembrolizumab). Unless stated otherwise, “combination therapy” may include simultaneous or sequential administration of the AXL inhibitor and the PD-1 inhibitor, in any order, in any dosage forms.

[39] The combination therapies of the invention are useful in preventing, treating or managing one or more blood cancers, in particular, leukemia. Examples of leukemia include acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemia and myelodysplastic syndrome, chronic leukemia such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia.

[40] The antiproliferative effect of a combination therapy of the invention may be assessed by administering the active ingredients of the combination therapy to a cultured tumor cell line. In the context of an in vitro assay, administration of an active ingredient may be simply achieved by contacting the cells in culture with the active ingredient in amounts effective to inhibit cell proliferation. Alternatively, the antiproliferative effect of a combination therapy of the invention may be assessed by administering the active ingredients of the combination therapy to an animal in an approved in vivo model for cell proliferation.

[41] The combination therapies of the invention can be tested for the treatment of AML by testing the combination therapy in a xenograft in SCID or nu/nu mouse model using human AXL-expressing AML leukemia cell lines. [42] Selection of the preferred prophylactically or therapeutically effective dose of an active ingredient used in the combination therapies of the invention can be determined (e.g., by clinical trials) by a skilled artisan based upon the consideration of several factors, including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; and the severity of the metastatic cancer.

[43] The precise dose of either the AXL inhibitor or the PD-1 inhibitor used in the combination therapies of the invention will also depend on the route of administration and the seriousness of the AML and should be decided according to the judgment of the medical practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[44] For example, a therapeutically effective daily dose for a AXL inhibitor may be, for a 70 kg mammal, from about 0.001 mg/kg (z.e., 0.07 mg) to about 300 mg/kg (z.e., 21.0 gm); preferably a therapeutically effective dose is from about 0.01 mg/kg (i.e., 0.7 mg) to about 100 mg/kg (i.e., 7.0 gm); more preferably a therapeutically effective dose is from about 0.1 mg/kg (i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 gm); and more preferably a therapeutically effective dose is from about 0.5 mg/kg (i.e., 35 mg) to about 25 mg/kg (i.e., 1.75 gm).

[45] Antibodies and antibody fragments can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses may be provided intravenously, subcutaneously, intraperitoneally, cutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose is generally at least 0.05 pg/kg body weight, at least 0.2 pg/kg, at least 0.5 pg/kg, at least 1 pg/kg, at least 10 pg/kg, at least 100 pg/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or at least 50 mg/kg (see, e.g., US20100266617, which is incorporated herein by reference).

[46] In the combination therapies of the invention, an AXL inhibitor is administered simultaneously with, prior to, or after administration of a PD-1 inhibitor, as described herein, by the same route of administration or by different routes. Such combination therapy includes administration of a single pharmaceutical dosage formulation which contains an AXL inhibitor and one or more additional chemotherapeutic agents, as well as administration of the AXL inhibitor and PD-1 in its own separate pharmaceutical dosage formulation. Where separate dosage formulations are used, the AXL inhibitor and the PD- 1 inhibitor can be administered to the patient at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially. All such combinations of administration are encompassed by the combination therapies of the invention.

[47] In certain embodiments of the combination therapies of the invention, the AXL inhibitor is administered to a patient concomitantly with a PD- 1 inhibitor useful for the treatment of cancer. The term “concomitantly” or “concurrently,” is not limited to the administration of the active ingredients i.e., the AXL inhibitor and the PD-1 inhibitor) at exactly the same time, but rather it is meant that the AXL inhibitor and the PD-1 inhibitor are administered to a patient in a sequence and within a time interval such that the AXL inhibitor can act together with PD-1 inhibitor to provide a synergistic benefit than if they were administered otherwise. For example, each active ingredient of the combination therapies of the invention may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. For example, the PD-1 inhibitor may be administered one time per week and the AXL inhibitor may be administered every day. In other words, the dosing regimens for the active ingredients are carried out concurrently even if the active ingredients are not administered simultaneously or within the same patient visit.

[48] In certain embodiments, the active ingredients of the invention are cyclically administered to a patient. Cycling therapy involves the administration of a first active ingredient, such as the AXL inhibitor, for a period of time, followed by the administration of the second and/or third active ingredient for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improves the efficacy of the treatment. [49] In yet other embodiments, the active ingredients of the combination therapies of the invention are administered in metronomic dosing regimens, either by continuous infusion or frequent administration without extended rest periods. Such metronomic administration can involve dosing at constant intervals without rest periods. Typically the chemotherapeutic agents, in particular cytotoxic agents, are used at lower doses. Such dosing regimens encompass the chronic daily administration of relatively low doses for extended periods of time. In one embodiment, the use of lower doses of the chemotherapeutic agent can minimize toxic side effects and eliminate rest periods. In certain embodiments, the active ingredients are administered by chronic low-dose or continuous infusion ranging from about 24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3 weeks to about 1 month to about 2 months, to about 3 months, to about 4 months, to about 5 months, to about 6 months. The scheduling of such dose regimens can be optimized by the skilled oncologist.

[50] In a preferred embodiment, the AXL inhibitor is administered every 24 hours to the patient and the PD-1 inhibitor is administered once weekly.

[51] Those skilled in the art are also familiar with determining administration methods (oral, intravenous, inhalation, sub-cutaneous, etc.), dosage forms, suitable pharmaceutical excipients and other matters relevant to the delivery of the compounds to a subject in need thereof.

[52] Administration of the compounds of the disclosure, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the disclosure can be prepared by combining a compound of the disclosure with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi- solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. Pharmaceutical compositions of the disclosure are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the disclosure in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the disclosure, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this disclosure.

A pharmaceutical composition of the disclosure may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet, capsule, or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.

EXAMPLES

EXAMPLE 1

SCL-391 TREATMENT SIGNIFICANTLY REDUCES THE GROWTH OF AML CELLS AND INCREASES FUNCTIONAL T-CELLS WHEN CO-CULTURED WITH ACTIVATED PBMCS

[53] To determine if the inhibitory effects of SLC-391 (Compound A) on AML cell growth can be enhanced by activated T-cells in vitro, the biological effects of SLC-391 on an AML cell line (MV4-11) was compared in the presence or absence of activated peripheral blood-derived mononuclear cells (PBMC). MV4-11 cells carrying a GBP marker (MV4-11-GLP⁺) were treated with various doses of SLC-391 (0.05 pM, 0.1 pM, and 0.2 pM) and viable cells were then counted at day 3, 5 and 7. As expected, a dosedependent reduction in cell viability was observed in MV4-11-GEP⁺ cells, particularly after 7 days in culture (P<0.0001, BIG. 1A) (Niu et al., 2021). Interestingly, this killing effect was significantly enhanced when MV4-11-GEP⁺ cells were co-cultured with CD3/CD28-stimulated human PBMCs, with an initial effector: target (PBMC: MV4-11- GFP⁺) ratio at 5:1, as compared to the same cells without co-culturing with PBMCs, (up to 80% inhibition, P<0.0004, FIG. 1A). This result was further supported by observation of a dose dependent reduction in GFP⁺ cells by SLC-391 treatment and co-culture with activated PBMCs as compared to controls, assessed by fluorescent microscope (FIG. IB). Flow cytometry analysis further demonstrated a dose-dependent increase in functional CD3⁺ T cells (up to 6-fold, P=0.0031), CD3⁺CD4⁺ (6-fold, P=0.0067) and CD3⁺CD8⁺ cells (7-fold, P=0.003) in SLC-391 -treated co-cultured cells, particularly under SLC 0.2 pM treatment, as compared to PBMC alone, without treatment (FIG. 1C), suggesting that SLC-391 treatment enhances functional CD3⁺CD4⁺ and CD3⁺CD8⁺ T-cells in a dosedependent manner under co-culture conditions.

EXAMPLE 2

PEMBROLIZUMAB TREATMENT ENHANCES THE EFFECTS OF SLC-391 -MEDIATED LEUKEMIC CELL KILLING WHEN CO-CULTURED WITH ACTIVATED PBMCS.

Next, it was determined whether the addition of Pembrolizumab (PEM), a clinically used immune checkpoint inhibitor, may further enhance the effect of SLC-391 in AML cells co-cultured with activated PBMC. The data show a combination of SLC-391 (0.1 pM and 0.2 pM) and PEM (lOpg/mL) and co-culture with activated PBMCs enhanced cell killing of MV4-11-GFP⁺ cells after 7 days in culture as compared to the same cells treated with SLC-391 or PEM alone (up to 95% inhibition, P<0.0001, FIG. 2A). It was noted that PEM treatment had limited effects compared to PBMC only condition (FIG. 2A). Most interestingly, flow cytometry analysis further demonstrated that the combination of SLC- 391 (0.05 pM, 0.1 pM and 0.2 pM) and PEM (lOpg/mL) co-cultured with activated PBMCs significantly increased total CD3⁺ T cells (up to 3-fold or 20-fold, P<0.0093), CD3⁺CD4⁺ (2.5-fold or 18-fold), and CD3⁺CD8⁺ T-cell populations (2.5-fold or 20-fold, P<0.035, respectively), as compared to SLC-391 or PEM treatment alone (FIG. 2B). As expected, PEM treatment reduced PD-1 expression in T-cells (FIG. 2C). A dosedependent increase in PD-L1 was also observed under the combination treatment conditions (up to 6-fold, FIG. 2D). Notably, CD3'CD56⁺ NK cells also increased in the presence of both SLC-391 and PEM as compared to PBMC or PBMC with SLC-391 or PEM (up to 20-fold, FIG. 2E), indicating that innate immunity is likely activated in these cells under a combination treatment co-cultured with activated PBMCs. Further studies were conducted to investigate if SLC-391 inhibits the growth of AML cells and shows superior synergistic effects in combination with PEM in vitro in a 5 day culture. In PBMC co-culture conditions (effector: target ratio 5:1), MV411-GFP+ cells were treated with SLC-391 (0.05 pM, O.lpM, or 0.2pM) alone, PEM (O.lpg/ml, 1 pg/ml, or 10 pg/ml) alone, or with a combination of both PEM and SLC-391. Viable GFP+ cells were measured by counting beads via FACS analysis after 5 days in culture (FIG. 2F, bar graph). The growth differences among different treatment conditions were normalized to PBMC-only treated conditions. Synergistic effects among different treatment conditions were calculated based on various concentrations of either SLC-391 or PEM by SynergyFinder software. CI <1, CI =1, and CI >1 indicate synergistic, additive, and antagonistic effects, respectively. Other synergy scores, including the Loewe synergy score (FIG. 2F, left tensor) and HSA score (FIG. 2F, right tensor), were also determined. A score more than 10 or lower than -10 was classified as synergistic or antagonistic, respectively; a score between -10 and 10 was considered additive. These results demonstrated that the combination treatment of SLC-391 and PEM in co-culture significantly reduced AML cell viability (95% cell killing), with a strong synergistic effect, particularly using SLC-391 at a concentration of 0.2pM (CKO.64) when compared to the PBMC-only condition, SLC-391 treatment alone, or PEM treatment alone.

Taken together, these results suggest that treatment of AML cells with a PD1 inhibitor can enhance immune response of these cells to further increase SLC-391 -induced antileukemic activity in vitro.

EXAMPLE 3

TREATMENT OF SCL-391 IN MV4-11-GFP⁺ CELLS INCREASES CD8⁺IFN-r⁺ CELLS WHEN COCULTURED WITH PBMC AND THIS EFFECT IS FURTHER ENHANCED BY PEM

[54] Activated cytotoxic T-cells are a major cell population that induces tumor cell killing and increased secretion of interferon gamma (IFN-y), a cytokine that plays an important role in inducing and modulating an array of immune responses; thus IFN-y activity is a critical indicator of activated status of cytotoxic T cells (Garris et al., 2018; Grasso et al., 2020). The activity of IFN-y was investigated under treatment of SLC-391 or PEM, alone and in combination, when co-cultured with activated PBMC and MV4-11- GFP⁺ cells. Intracellular staining analysis demonstrated that an increase in the CD8⁺IFN- y⁺ population was observed in SEC-391 -treated cells in comparison with PBMC-cultured cells (4-fold), although PEM treatment did not result in an obvious change (FIG. 3A). Interestingly, a combination of SLC-391 and PEM treatment significantly increased this specific cell population as compared to SLC-391 or PEM treatment alone (4-fold or 10- fold, P<0.0004, FIG. 3A). Furthermore, multiple cytokines were detected in co-cultured media obtained from MV4-11-GFP⁺ cells co-cultured with PBMCs and treated with either SLC-391 or PEM, alone or in combination, using a LEGENDplex multiplex assay; in particular, enhanced production of IFN-y after the combination treatment was observed (up to 15-fold, P<0.001, FIG. 3B). Other immune active cytokines, such as TNF-a, also displayed significantly increased concentrations with treatment of SLC-391 alone or SLC- 391 in combination with PEM when co-cultured with activated PBMCs in comparison to PBMC control or PEM treatment condition (up to 200-fold, P<0.008, FIG. 3C). These findings indicate that inhibition of AXL activity modulates activated human T-cell functions and synergizes with PEM in eradicating AML cells, suggesting its role as an enhancer of T-cell immunity and cytotoxicity.

EXAMPLE 4

DUAL INHIBITION OF AXL AND PD-1 SYNERGISTICALLY INHIBITS THE GROWTH OF AML STEM/ PROGENITOR CELLS AND INCREASES FUNCTIONAL T-CELLS

[55] To further investigate the efficacy of this combination treatment on primary AML patient cells, CD34⁺ cells from AML patients at diagnosis, with relatively high expression of AXL (n=6), were treated with SLC-391 or PEM, alone or combination, in the presence or absence activated PBMCs (at the effector: target ratio 5:1). Interestingly, SLC-391 or PEM treatment in the presence of activated PBMCs showed significantly reduced cell viability in AML patient cells, compared to the same cells without co-culturing with PBMC (50% or 60% reduction vs. 25% or 10%, P<0.014, FIG. 4A). Most interestingly, the combination treatment of SLC-391 and PEM in co-culture more effectively inhibited the growth of primitive AML cells when compared to PBMC only condition or SLC-391 or PEM treatment alone (up to 85% reduction, P<0.003, FIG. 4A). This combination treatment also demonstrated significantly increased CD3⁺CD4⁺ and CD3⁺CD8⁺ T-cell populations as compared to controls or single agents (2-fold, P<0.0001 and 4-fold, P<0.0001, respectively, FIG. 4B). As observed in the AML cell line model system, PEM treatment effectively blocked the PD-1 signal in T-cells as assessed by flow cytometry analysis (FIG. 4C). Increased PD-L1 was also observed under the combination treatment conditions when co-cultured with PBMC (up to 3-fold, P<0.0002, FIG. 4D). As expected, intracellular staining or cytokine array assays demonstrated increased IFN-y in T-cells or in co-culture media after the combination treatment, compared to control or single agents (P<0.0002, FIG. 4E), which correlated well with increased PD-L1 expression since IFN-y can induce upregulation of PD-L1. In addition, multiple cytokines involved in activation of innate and adaptive immune responses, such as TNF-a and sFasL, were detected at elevated levels in co-cultured media obtained from the combination treatment of AML stem/progenitor cells co-cultured with PBMCs as compared to controls or single agents, as assessed by a LEGENDplex multiplex assay (P<0.02, FIGS. 4F and 4G). Moreover, TIM3, an immune checkpoint receptor that is associated with regulation of T-cell and NK cell exhaustion, were reduced by the combination treatment, suggesting that its inhibition may boost innate and adaptive immune responses (up to 3-fold, P<0.03, FIG. 4G).

EXAMPLE 5

DUAL SLC-391 AND PEM TREATMENT DECREASES LEUKEMIA BURDEN AND ENHANCES SURVIVAL OF MICE IN A HUMANIZED PATIENT-DERIVED XENOTRANSPLANTATION (PDX) MODEL

[56] To create a pre-clinical PDX model to study the combination effect of AXL and PD-1 inhibitor therapy, a humanized PDX model was recently developed using an immunodeficient NSG mouse strain (NOD-scid IL2Rgnull), which allows mice to be humanized by engraftment of human healthy CD34⁺ cells (FIG. 5A). (Beyer and Muench, 2017; Miller et al., 2013) In vivo conditions for immune reconstitution were determined by transplanting CD34⁺ human cord blood (CB) cells into mice at 4 weeks old. Efficient engraftment of multiple-lineage circulating human CD45⁺ cells (-50%), T cells, NK cells and dendritic cells (DCs), etc. was demonstrated at 12 weeks, indicating successful immune reconstitution (FIG. 5B). MV4-11 cells carrying a luciferase reporter and GFP marker were intravenously (IV) injected into the mice (5xl0⁵/mouse). A strong and consistent bioluminescence signal was detected in all mice 16 days later. The mice were then treated with SLC-391 (50 mg/kg) daily by oral gavage or intraperitoneal (IP) injection of PEM (10 mg/kg) twice a week, or a combination of these or vehicle for three weeks, and then bioluminescent imaging were performed. SLC-391 treated mice have dramatically reduced bioluminescent signals compared to vehicle or PEM treated mice (FIG. 5C). Interestingly, the bioluminescent signal of the combination-treated mice was even lower than SLC-391 -treated mice or even below detection limits (FIG. 5C). There was no evidence of spleen enlargement in mice treated with SLC-391 or the combination, while there was clearly evidence of spleen enlargement in vehicle or PEM-treated mice (up to 4-fold, P<0.008, FIG. 5D). Flow cytometry analysis confirmed that the combination treatment significantly reduced leukemic cells and prevented the infiltration of leukemic cells in hematopoietic organs (up to 50-fold, P<0.03, FIG. 5E). Strikingly, 40% of mice with the combination treatment were still alive after 62 days compared to vehicle, PEM- treated mice and SLC-391 -treated mice (median survival 47, 47, and 56 days, respectively, FIG. 5F). In addition, the engraftment level of CB-derived CD45⁺GFP" cells was found to be higher in BM and spleen of the combination treated-mice as compared to vehicle or single drug-treated mice (P=0.03, FIG. 5E). Indeed, an increase in immune cells was also observed in the combination treated-mice, particularly CD3⁺ T cells, NK cells and pDC cells detected in peripheral blood or BM (up to 3-fold, FIG. 6A-B). Immunohistological staining was performed to evaluate the number of leukemic cells, CD3+ cells, and CD8+ cells for mice receiving combination treatment of SLC-391 and PEM. Spleens of treated mice were harvested and fixed in 10% formalin. The fixed tissues were further individually processed for Haemotoxylin and Eosin staining and IHC staining of antihuman CD3, CD8, and PDL1 antibodies. The representative 40X imaging of each treated mouse is shown in FIG. 5G. The percentage of CD3-positive, CD8-positive, and PDL1- positive cells from each treatment condition are shown in the bar graph (FIG. 5G) wherein the cell numbers were manually counted and calculated based on the total 4000 cells in each field. These results demonstrated that histological and IHC analyses revealed that the combination treatment reduced the engraftment of leukemic cells and prevented the infiltration of these cells in the spleen, and SLC-391 treatment alone or in combination with PEM increased CD3+ or CD8+ cells in this PDX model.

[57] Thus, disclosed herein a humanized PDX model that can potentiate anti-leukemia immunity and enhance cell killing of AML blast cells.

References

[58] Abaza, Y., and Zeidan, A. M. (2022). Immune Checkpoint Inhibition in Acute Myeloid Leukemia and Myelodysplastic Syndromes. Cells 11.

[59] Ben-Batalla, I., Schultze, A., Wroblewski, M., Erdmann, R., Heuser, M., Waizenegger, J. S., Riecken, K., Binder, M., Schewe, D., Sawall, S., et al. (2013). Axl, a prognostic and therapeutic target in acute myeloid leukemia mediates paracrine crosstalk of leukemia cells with bone marrow stroma. Blood 122, 2443-2452.

[60] Beyer, A. I., and Muench, M. O. (2017). Comparison of Human Hematopoietic Reconstitution in Different Strains of Immunodeficient Mice. Stem cells and development 26, 102-112.

[61] Daver, N., Garcia-Manero, G., Basu, S., Boddu, P. C., Alfayez, M., Cortes, J. E., Konopleva, M., Ravandi-Kashani, F., Jabbour, E., Kadia, T., et al. (2019). Efficacy, Safety, and Biomarkers of Response to Azacitidine and Nivolumab in Relap sed/Refractory Acute Myeloid Leukemia: A Nonrandomized, Open-Label, Phase II Study. Cancer discovery 9, 370-383.

[62] Delbridge, A. R., Grabow, S., Strasser, A., and Vaux, D. L. (2016). Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies. Nature reviews Cancer 16, 99-109.

[63] DiNardo, C. D., Pratz, K., Pullarkat, V., Jonas, B. A., Arellano, M., Becker, P. S., Frankfurt, O., Konopleva, M., Wei, A. H., Kantarjian, H. M., et al. (2019). Venetoclax combined with decitabine or azacitidine in treatment-naive, elderly patients with acute myeloid leukemia. Blood 133, 7-17.

[64] Dohner, H., Estey, E. H., Amadori, S., Appelbaum, F. R., Buchner, T., Burnett, A. K., Dombret, H., Fenaux, P., Grimwade, D., Larson, R. A., et al. (2010). Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 115, 453-474. [65] Dohner, H., Weisdorf, D. J., and Bloomfield, C. D. (2015). Acute Myeloid Leukemia. The New England journal of medicine 373, 1136-1152.

[66] Garon, E. B., Rizvi, N. A., Hui, R., Leighl, N., Balmanoukian, A. S., Eder, J. P., Patnaik, A., Aggarwal, C., Gubens, M., Hom, L., et al. (2015). Pembrolizumab for the treatment of non-small-cell lung cancer. The New England journal of medicine 372, 2018- 2028.

[67] Garris, C. S., Arlauckas, S. P., Kohler, R. H., Trefny, M. P., Garren, S., Piot, C., Engblom, C., Pfirschke, C., Siwicki, M., Gungabeesoon, J., et al. (2018). Successful Anti- PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-gamma and IL-12. Immunity 49, 1148-1161 el 147.

[68] Gay, C. M., Balaji, K., and Byers, L. A. (2017). Giving AXL the axe: targeting AXL in human malignancy. British journal of cancer 116, 415-423.

[69] Gomez-Llobell, M., Peleteiro Raindo, A., Climent Medina, J., Gomez Centurion, I., and Mosquera Orgueira, A. (2022). Immune Checkpoint Inhibitors in Acute Myeloid Leukemia: A Meta- Analysis. Frontiers in oncology 12, 882531.

[70] Goswami, M., Gui, G., Dillon, L. W., Lindblad, K. E., Thompson, J., Valdez, J., Kim, D. Y., Ghannam, J. Y., Oetjen, K. A., Destefano, C. B., et al. (2022).

Pembrolizumab and decitabine for refractory or relapsed acute myeloid leukemia. Journal for immunotherapy of cancer 10.

[71] Graham, D. K., DeRyckere, D., Davies, K. D., and Earp, H. S. (2014). The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer. Nature reviews Cancer 14, 769-785.

[72] Grasso, C. S., Tsoi, J., Onyshchenko, M., Abril-Rodriguez, G., Ross-Macdonald, P., Wind-Rotolo, M., Champhekar, A., Medina, E., Torrejon, D. Y., Shin, D. S., et al. (2020). Conserved Interferon-gamma Signaling Drives Clinical Response to Immune Checkpoint Blockade Therapy in Melanoma. Cancer cell 38, 500-515 e503.

[73] Hugo, W., Zaretsky, J. M., Sun, L., Song, C., Moreno, B. H., Hu-Lieskovan, S., Berent-Maoz, B., Pang, J., Chmielowski, B., Cherry, G., et al. (2016). Genomic and Transcriptomic Features of Response to Anti-PD- 1 Therapy in Metastatic Melanoma. Cell 165, 35-44. [74] Khwaja, A., Bjorkholm, M., Gale, R. E., Levine, R. L., Jordan, C. T., Ehninger, G., Bloomfield, C. D., Estey, E., Burnett, A., Cornelissen, J. J., et al. (2016). Acute myeloid leukaemia. Nat Rev Dis Primers 2, 16010.

[75] Krupka, C., Kufer, P., Kischel, R., Zugmaier, G., Lichtenegger, F. S., Kohnke, T., Vick, B., Jeremias, I., Metzeler, K. H., Altmann, T., et al. (2016). Blockade of the PD- 1/PD-L1 axis augments lysis of AML cells by the CD33/CD3 BiTE antibody construct AMG 330: reversing a T-cell-induced immune escape mechanism. Leukemia 30, 484-491.

[76] Miller, P. H., Cheung, A. M., Beer, P. A., Knapp, D. J., Dhillon, K., Rabu, G., Rostamirad, S., Humphries, R. K., and Eaves, C. J. (2013). Enhanced normal short-term human myelopoiesis in mice engineered to express human- specific myeloid growth factors. Blood 121, el-4.

[77] Niu, X., Rothe, K., Chen, M., Grasedieck, S., Li, R., Nam, S. E., Zhang, X., Novakovskiy, G. E., Ahn, Y. H., Maksakova, I., et al. (2021). Targeting AXL kinase sensitizes leukemic stem and progenitor cells to venetoclax treatment in acute myeloid leukemia. Blood 137, 3641-3655.

[78] Pardoll, D. M. (2012). The blockade of immune checkpoints in cancer immunotherapy. Nature reviews Cancer 12, 252-264.

[79] Schoumacher, M., and Burbridge, M. (2017). Key Roles of AXL and MER Receptor Tyrosine Kinases in Resistance to Multiple Anticancer Therapies. Current oncology reports 19, 19.

[80] Sharma, P., and Allison, J. P. (2015). Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell 161, 205-214.

[81] Thomas, D., and Majeti, R. (2017). Biology and relevance of human acute myeloid leukemia stem cells. Blood 129, 1577-1585.

[82] Tirado-Gonzalez, I., Descot, A., Soetopo, D., Nevmerzhitskaya, A., Schaffer, A., Kur, I. M., Czlonka, E., Wachtel, C., Tsoukala, I., Muller, L., et al. (2021). AXL Inhibition in Macrophages Stimulates Host-versus-Leukemia Immunity and Eradicates Naive and Treatment-Resistant Leukemia. Cancer discovery 11, 2924-2943.

[83] Topalian, S. L., Hodi, F. S., Brahmer, J. R., Gettinger, S. N., Smith, D. C., McDermott, D. F., Powderly, J. D., Carvajal, R. D., Sosman, J. A., Atkins, M. B., et al. (2012). Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. The New England journal of medicine 366, 2443-2454.

[84] Vaddepally, R. K., Kharel, P., Pandey, R., Garje, R., and Chandra, A. B. (2020). Review of Indications of FDA- Approved Immune Checkpoint Inhibitors per NCCN Guidelines with the Level of Evidence. Cancers 12.

[85] Williams, P., Basu, S., Garcia-Manero, G., Hourigan, C. S., Oetjen, K. A., Cortes, J. E., Ravandi, F., Jabbour, E. J., Al-Hamal, Z., Konopleva, M., et al. (2019). The distribution of T-cell subsets and the expression of immune checkpoint receptors and ligands in patients with newly diagnosed and relapsed acute myeloid leukemia. Cancer 125, 1470-1481.

[86] Zhu, C., Wei, Y., and Wei, X. (2019). AXL receptor tyrosine kinase as a promising anti-cancer approach: functions, molecular mechanisms and clinical applications.

Molecular cancer 18, 153.

[87] Zeidner JF, Vincent BG, et al. Phase II Trial of Pembrolizumab after High-Dose Cytarabine in Relapsed/Refractory Acute Myeloid Leukemia. Blood Cancer Discov. 2021 Sep 10;2(6):616-629.

[88] Guamera L, Bravo-Perez C, Visconte V. Immunotherapy in Acute Myeloid Leukemia: A Literature Review of Emerging Strategies. Bioengineering (Basel). 2023 Oct 20;10(10):1228

[89] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[90] These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full 1 scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1. A combination of an AXL inhibitor and a PD-1 inhibitor for use in a method for treating blood cancer, wherein the method comprises administering the AXL inhibitor concomitantly with the PD-1 inhibitor, wherein the AXL inhibitor is 3-(5- (cyclopropylmethyl)-l,3,4-oxadiazol-2-yl)-5-(l-(piperidin-4-yl)-lH-pyrazol-4-yl)pyridin- 2-amine (Compound A):

Compound A; and the PD-1 inhibitor is an anti-PD-1 antibody or antigen binding fragment thereof and/or a PD-L1 antibody antigen binding fragment thereof.

2. The combination of claim 1, wherein the anti-PDl antibody or an antigen binding fragment thereof is selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, vopratelimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, INCMGA00012, AMP-224, AMP-514 (MEDI0680), acrixolimab, and toripalimab, or an antigen binding fragment thereof.

3. The combination of claim 1 or 2, wherein the anti-PDl antibody or an antigen binding fragment thereof is pembrolizumab.

4. The combination of claim 1, wherein the anti-PD-Ll antibody or an antigen binding fragment thereof is selected from atezolizumab, avelumab, and durvalumab.

5. The combination of any one of the preceding claims wherein the blood cancer is acute myeloid leukemia (AML).

6. The combination of any one of the preceding claims wherein the AXL inhibitor is administered at a lower dose than a monotherapy using the same AXL inhibitor.

7. The combination of any one of the preceding claims wherein the PD- 1 inhibitor is administered at a lower dose than a monotherapy using the same PD-1 inhibitor.

8. A method of treating acute myeloid leukemia, comprising administering a therapeutically effective amount of an AXL inhibitor and a PD-1 inhibitor of any of the preceding claims to a patient.