Methods for targeted immunotherapy of acute myeloid leukemia (AML)

Abstract

This provides a method for targeted immunotherapy for acute myeloid leukemia (AML). [Solution] The disclosure of the present invention provides a method for treating an eligible subject having acute myeloid leukemia having high-risk disease features, comprising administering a targeted immunotherapy agent, which specifically targets B cell maturation antigens, to the intensified subject.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 460,255, filed on April 18, 2023, the content of which is hereby incorporated by reference in its entirety. The disclosure of the present invention relates to therapies for treating cancer, and more particularly to a targeted immunotherapy for treating cancers in which B - cell maturation antigen is expressed by blast cells.

Background Art

[0002] I. Immune Mechanisms Involved in the Body's Response to Cancer Immune Response The human immune system is a complex mechanism of cells and molecules that maintains immune homeostasis to preserve the integrity of an organism by eliminating all elements judged to be dangerous. Responses in the immune system can generally be divided into two sectors, referred to as "innate immunity" and "adaptive immunity". These two sectors of immunity do not operate independently of each other but function together to elicit an effective immune response.

[0003] Immune Synapse and Activation of Immune Cells The immune response is initiated by the interaction between antigen-presenting cells (APCs), such as dendritic cells (DCs), and responder cells, such as T cells, via tight cell contact boundaries called immunosynapses. Immunosynapses are highly organized intracellular structures that provide a platform for antigen presentation to receptors on the surface of responder cells, on the major histocompatibility class I and II complexes (MHC classes I and II) on the surface of APCs. In T cells, these contacts result in highly polarized membrane trafficking, thereby leading to the local release of lytic granules and the delivery and reuse of T cell receptors at the immunosynapse. Local trafficking also occurs on the APC side of the synapse, particularly in DCs where MHC class I and II-loaded antigens are presented and cytokines are specifically released at the synapse. Functional immunosynapses between DCs and naive T cells are essential for a functional T cell response. [Vergoogen, DRJ et al. Biomol Concepts (2016) 7(1): 17-28]

[0004] Not only DCs and T cells, but also other APCs, such as B cells or infected cells, and other effector cells, such as natural killer cells (NKs), form immunological synapses for the lethality of infected or abnormal target cells, in addition to intercellular communication. [Cited from Angus, KL and Griffiths, GM. Curr. Opin. Cell Biol. (2013) 25: 85-91; Friedl, P. et al. Nat. Rev. Immunol. (2005) 5: 53; Xie, J. et al. Immunol. Rev. (2013) 251: 65-79]. The structure of the synapse is highly dependent on the cell types involved, the presence and intensity of antigen recognition, and additional co-stimulatory interactions. [Ibid., Friedl, P. et al. Nat. Rev. Immunol. (2005) 5: 53; Thauland, TJ and Parker, DC. Immunology (2010) 131: 466-72; Azar, GA et al. Proc. Natl. Acad. Sci. USA (2010) 107: 3675-80 cited]. Immunological synapses can be functionally divided into two categories [citing Gerard, A. et al. Immunol. Rev. (2013) 251: 80-96]: (1) primary synapses, which are intercellular contacts that trigger the initial activation of immune cells, such as the synapse between DCs and T cells [citing Rodriguez-Fernandez, JL et al. Sci. Signal (2010) 3: re2], and (2) so-called secondary synapses resulting from interactions established after initial priming, such as activated T cells delivering stimulatory signals to B cells via CD40-CD40L interactions [citing Chaplin, DD. J. Allergy Clin. Immunol. (2010) 125: [Cited S3-23]; This category also includes synapses formed between NK or cytotoxic T cells that release lytic granules to kill APCs and their target APCs [Ibid., citing Stinchcombe, JC et al. Immunity (2001) 15: 751-61]. For both categories, immunological synapse formation can initiate intracellular signaling cascades in both APCs and T cells, resulting in cytoskeletal remodeling and re-routing of membrane trafficking pathways.

[0005] Membrane trafficking. Membrane trafficking plays a crucial role in T cell effector function by resulting in surface presentation of TCRs and other membrane proteins, reuse of depleted receptors, and release of cytokines and chemokines at immunological synapses. The best-understood form of exocytosis at immunological synapses is the release of cytolytic granules from CD8+ T cells and NK cells. Other types of cargo delivered and / or recycled on the T cell side of the immune synapse include cytokines (e.g., IFN-gamma) and membrane receptors (e.g., TCR, ICAM-1) [ibid., Griffiths, GM et al. J. Cell Biol. (2010) 189: 397-406; Angus, KL, Griffiths, GM. Curr. Opin. Cell Biol. (2013) 25: 85-91; Xie, J. et al. Immunol. Rev. (2013) 251: 65-79; Jo, JH et al. J. Cell Biochem. (2010) 111: 1125-37; Finetti, F. and Baldari, CT. Immunol. Rev. (2013) 251: 97-112; Das, V. et al. Immunity] [Cited from (2004) 20: 577-88; Soares H. et al. J. Exp. Med. (2013) 210: 2415-33]. Polarized delivery of these molecules to immune synapses enables more sensitive antigen presentation and / or enhances T cell effector function, while preventing unwanted activation of other nearby (immune) cells.

[0006] Polarized membrane trafficking also occurs on the APC side. In DCs, MHC classes I and II [ibid., Boes, M. et al. Nature (2002) 418: 983-8; Bertho, NJ Immunol. (2003) 171: 5689-96; Boes, M. et al. J. Immunol. (2003) 171: 4081-8; Compeer, EB et al. J. Biol. Chem. (2014) 289: 520-8] and the co-stimulatory molecule CD40 [ibid., Foster, N. et al. J. Immunol. (2012) 189: 5632-7] can be locally trafficked to and presented at immunosynapses. The local release of these molecules improves the efficiency of T cell activation and helps explain how T cells can detect small amounts of MHC ligands in the presence of large amounts of MHC bound to endogenous peptides [citing Xie, J. et al. Immunol. Rev. (2013) 251: 65-79]. In addition, IL-12 is also locally released by DCs at immunological synapses with T cells [citing Pulecio, J. et al. J. Exp. Med. (2010) 207: 2719-32]. IL-12 is T H It promotes the response, enhances the cytolytic activity of CD8+ T cells, and induces IFN-gamma production by T cells. Polarized release of IL-12 was also observed at the immunological synapse between DCs and NK cells [ibid., Borg, C. et al. Blood (2004) 104: 3267-75; Barreira da Silva, R. et al. Blood (2011) 118: 6487-98].

[0007] NK cells NK cells are innate immune cells that exhibit rapid and strong cytolytic activity in response to infected or abnormal cells [Gonzalez, H. et al. Genes & Development (2018) 32: 1267-84., citing Cerwenka A, Lanier LL. (2016). Nat Rev Immunol 16: 112-123]. NK cells possess a variety of inhibitory and stimulatory receptors on their cell surface used for immune surveillance. Inhibitory receptors target cancer cells lacking major histocompatibility class I (MHC-I) and mark them for programmed cell death [ibid., citing Marcus, A. et al. (2014). Adv Immunol 122: 91-128]. In contrast, in healthy cells, the binding of MHC-I molecules to their receptors on NK cells has a significant inhibitory effect on NK cell function [citing Bix, M. et al. (1991). Nature 349: 329-331; Liao, NS et al. (1991). Science 253: 199-202; Colonna, M. et al. (1992). Proc Natl Acad Sci 89: 7983-7985; Karlhofer, FM et al. (1992). Nature 358: 66-70; Wagtmann, N. et al. (1995). Immunity 2: 439-449; Lanier, LL (2005). J Immunol 174: 6565]. NK cells have well-established antitumor activity [citing Marcus, A. et al. (2014). Adv Immunol 122: 91-128; Iannello, A. et al. (2016). Curr Opin Immunol 38: 52-58].

[0008] The conventional NK (cNK) cell pool consists of a circulatory compartment and an intrinsic compartment in the inner epithelium and lamina propria of the gastrointestinal tract. [Jiao, Y. et al., Front. Immunol. (2020) 11: 282] cNK cells can sense pathogen, tumorigenetic, and tissue injury signals. Activation and conversion of cNK cells rely on an overall signaling input of activating, inhibitory, and exogenous cytokine signals, which further leads to changes in a group of specific transcription factors and pro-apoptotic proteins, ultimately determining the fate of the cNK cell. [Ibid., citing Viant C, et al. J Exp Med. (2017) 214:491-510]. When activated, cNK cells exert their cytotoxic function by releasing perforin, a cytolytic protein that forms pores, and granzymes, a cytotoxic protein. cNK cells also utilize the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) pathway and antibody-dependent cell-mediated cytotoxicity (ADCC) (ibid., citing Caligiuri MA. Blood. (2008) 112:461-9). At the same time, cNK cells have a strong capacity to produce cytokines such as TNF, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (ibid., citing Souza-Fonseca-Guimaraes F, et al. J Biol Chem. (2013) 288:10715-21).

[0009] cNK activation. The theory of cNK education emphasizes that the activation threshold of cNKs throughout their development is modulated by adjusting the expression levels of their activating and inhibitory receptors. The processes of cNK cell arming (meaning that downregulation of inhibitory receptors can upregulate the activation threshold) and cNK cell licensing (meaning a scenario in which activating receptors are downregulated to confer increased sensitivity to activation signals to cNK cells) ensure an appropriate activation strategy, i.e., that the self-response of cNK cells that do not recognize their own MHC class I molecules by inhibitory receptors is limited. Generally, educated cNK cells are characterized by elevated expression of the activating receptor DNAM-1, exhibit higher responsiveness to self-loss targets, and have increased degranulation and cytokine production capacity [ibid., citing Enqvist M, et al. J Immunol. (2015) 194:4518-27]. We hypothesized that the gastrointestinal tract may be one of the centers for cNK cells to acquire normal function and education. The acquisition of cytotoxic function by cNK cells depends on a dendritic cell-dependent priming process by symbiotic bacteria [citing Ganal SC, et al. Immunity. (2012) 37:171-86], and symbiotic lactic acid bacteria are major regulators of crosstalk between cNK cells. Lactic acid bacteria activate immature dendritic cells in the gastrointestinal tract to produce key cytokines such as IL-12 and IL-15, promoting the activation and proliferation of cNK cells [citing Rizzello V, et al. BioMed Res Int. (2011) 2011:473097].

[0010] T cells T cells are components of the adaptive immune system that act as orchestrators and effectors of the immune response. Depending on the immunological context, T cells can acquire functional and effector phenotypes in which their activity results in either direct inflammatory or anti-inflammatory consequences [Gonzalez, H. et al. Genes & Development (2018) 32: 1267-84, citing Speiser, DE et al. (2016) Regulatory circuits of T cell function in cancer. Nat Rev Immunol 16: 599-611]. T cells, after tumor-associated macrophages (TAMs), are the second most common immune cell type found in human tumors and have been extensively studied across various cancer types [citing Speiser, DE et al. (2016) Nat Rev Immunol 16: 599-611; Donadon, M. et al. (2017) J Gastrointest Surg 21: 1226-1236]. During the early stages of tumorigenesis, when sufficient immunogenic antigens are produced, naive T cells are expected to be primed in the influx region lymph nodes, subsequently activated simultaneously, and migrate to the tumor microenvironment (TME). From there, they initiate a protective effector immune response to eliminate immunogenic cancer cells. Histopathological analysis of human tumors shows that tumor-associated T cells extend beyond the edges of tumor infiltration and are dominant even in the hypoxic core [citing Halama, N. et al. (2011) Cancer Res 71: 5670-5677; Kirilovsky, A. et al. (2016) Int Immunol 28: 373-382].High levels of T-cell infiltration in tumors include melanoma [ibid., citing Clemente, CG et al. (1996) Cancer 77: 1303-1310], breast [ibid., citing Oldford, SA et al. (2006) Int Immunol 18: 1591-1602], lung [ibid., citing Dieu-Nosjean, MC et al. (2008) J Clin Oncol 26: 4410-4417], ovary [ibid., citing Kusuda, T. et al. (2005) Oncol Rep 13: 1153-1158], colorectal [ibid., citing Tosolini, M. et al. (2011) Cancer Res 71: 1263-1271], and kidney [ibid., citing Kondo, T. et al. (2006) Cancer It is associated with a favorable prognosis in cancers of the prostate [citing Sci 97: 780-786], prostate [citing the same, Vesalainen, S. et al. (1994) Eur J Cancer 30A: 1797-1803], and stomach [citing the same, Ubukata, H. et al. (2010) J Surg Oncol 102: 742-747; Fridman, WH et al. (2012) Nat Rev Cancer 12: 298-306; Kitamura, T. et al. (2015) Nat Rev Immunol 15: 73-86].

[0011] T cell activation. T cell activation depends on the interaction between the TCR / CD3 complex and peptides bound to the grooves of its homologous ligands, class I or class II MHC molecules. Full T cell responsiveness requires not only receptor engagement but also co-stimulatory activity delivered by accessory cells, such as engagement of CD28 on T cells by CD80 and / or CD86 on antigen-presenting cells (APCs). The soluble products of activated T lymphocytes are lymphokines (meaning cytokines produced by lymphocytes). Naive T cells must initially be activated by dendritic cells. [Yewdell, JW and BP Dolan. Nature (2011) 471 (73340): 581-82].

[0012] Interaction of MHC (pMHC) loaded with TCR peptide. TCR recognition by peptide-loaded MHC is unparalleled in terms of the diversity of interacting surfaces. All elements of the interaction are inherently extremely diverse: TCR generation by somatic recombination is approximately 10 15~18 This allows for the theoretically different TCR diversity of species, of which approximately 1.5 × 10 7 Species can be found within any individual [Zareie, P. et al. Viral Immunology 33 (3): 179-87 (2020), citing Davis, MM and Bjorkman, PJ. Nature (1988) 334: 395-402]; MHC genes are the most pleomorphic genes in the human genome, and the peptide cargoes to which MHC can bind are virtually limitless. Despite this diversity, key structural features have emerged that are highly conserved across virtually all interactions. The TCR contacts both the peptide cargo and the MHC (corecognition); the TCR binds to the top of the peptide, the peptide contacts at least one of the highly variable TCR CDR3 loops, and the TCR binds to the pMHC with highly conserved polarity. The TCR α chain is located across the MHCI α2 helix or the upper part of the MHCII β chain, while the TCR β chain is located on the MHCI α helix or the MHCII α chain. [Ibid., citing Rossjohn, J. et al. Annu. Rev. Immunol. (2015) 33: 169-200; Rudolph, MG et al. Annu. Rev. Immunol. (2006) 24: 419-66].

[0013] To understand MHC constraint, which addresses why T cells recognize only antigens presented by MHC molecules, there are two models currently under discussion: the germline model and the selection model. The germline model proposes that germline TCR sequences were selected during evolution to encode TCR structures that efficiently interact with MHC molecules. The opposing hypothesis stems from the selection model. Selection theory, which explains the bias of T cell receptors towards MHC, proposes that MHC constraint is ultimately driven by constraints imposed on the TCR during positive selection in the thymus and the nature of TCR signaling. [Zareie, P. et al. Viral Immunology 33 (3): 179-87 (2020), citing Rangarajan, S. and Mariuzza, RA. Cell Mol. Life Sci. (2014) 71: 3059-68; Van Laethem, F. et al. Trends Immunol. (2012) 33: 437-41]. TCRs themselves do not possess intrinsic signaling capabilities. Instead, TCR signaling relies on the delivery of lymphocyte-specific protein tyrosine kinase (Lck) to the CD3 complex upon association with the cytoplasmic tail of the CD4 or CD8 co-receptor [citing Turner, JM et al. Cell (1990) 60: 755-65]. Thus, CD4 and CD8 co-receptors act to concentrate TCRs on MHC molecules. This requirement for co-receptor facilitation of signaling prevents the development of T cells expressing non-MHC-specific TCRs and ensures MHC constraint.

[0014] TCR recognition by non-classical MHC molecules. Unlike classical MHC-I and MHC-II molecules, MHC I-like molecules such as CD1 and MR1 are monomorphic (meaning they have only one form) [Zareie, P. et al. Viral Immunology 33 (3): 179-87 (2020)., citing Mori, L. et al. Annu. Rev. Immunol. (2016) 34: 479-510], and present lipid or metabolite antigens, rather than peptides, to TCRs expressed by "non-conventional" T cells such as mucosa-associated invariant T (MAIT) cells, natural killer T (NKT) cells, and subsets of γδ T cells [reviewed in Godfrey DI, et al. Nat. Immunol. (2012) 13: 851-6].

[0015] The three main principles of conventional TCR-pMHC binding, namely TCR binding to peptides, TCR co-recognition of both MHC and peptide cargo; and the conserved docking polarity of TCRs, also apply to HLA-E [ibid., Gherardin, NA et al. Immunity (2016) 44: 32-45; Sullivan, LC et al. J. Biol. Chem. (2017) 292: 21149-58] and MR-1 [ibid., Corbet, AJ et al. Nature (2014) 509: 361-65; Eckle, SB et al. J. Exp. Med. (2014) 211: 1585-1600; Gherardin, NA et al. Immunity (2016) 44:] which present metabolites to MAIT cells and atypical MR1-restricted T cells. TCR recognition of non-classical MHC molecules, including the recognition of [32-45; Gherardin, NA et al. J. Immunol. (2018) 201: 2862; Patel, O. et al. Nat. Comun. (2013) 4: 2142], has also been observed.

[0016] CD4+ T cells. CD4+ T cells play a central role in organizing the host immune response against cancer and infectious diseases, as well as in autoimmunity [Wang, RF. Trends in Immunol. (2001) 22 (50): 269-76, citing Paradoll, DM and Topalian, SL. Curr. Opin. Immuno. (1998) 10: 588-94; O'Garra, A. et al. (1997) Curr. Opin. Immunol. 9: 872-83; Kalams, SA and Walker, BD. J. Exp. Med. (1998) 188: 2199-2204; Zajac, AJ et al. Curr. Opin. Immuno. (1998) 10: 444-49].

[0017] When DCs interact with CD4+ T cells, they interact with their different T helper cells (T H ) subset [Patente, TA, et al., Frontiers Immunol. (2019) doi.org / 10.3389 / fimmu.2018.03176., citing Iwasaki A, Medzhitov R. Nat Immunol. (2015) 16:343-353], for example T H 1[Amsen D, et al. Cell (2004) 117:515-26; Constant S, et al. J Exp Med (1995) 182:1591-6; Hosken NA, et al. J Exp Med. (1995) 182:1579-84; Kadowaki N. Allergol Int. (2007) 56:193-9; Maekawa Y, et al. Immunity (2003) 19:549-59; Pulendran B, et al. Proc Natl Acad Sci USA. (1999) 96:1036-41, T H2 [Ibid., Constant S, et al. J Exp Med (1995) 182:1591-6, Hosken NA, et al. J Exp Med. (1995) 182:1579-84, Jenkins SJ, P. et al. J Immunol. (2007) 179:3515-23, Soumelis V, et al. Nat Immunol. (2002) 3:673-680], T. H 17 [Cited from Bailey SL, Nat Immunol. (2007) 8:172-80; Iezzi G, et al. Proc Natl Acad Sci USA. (2009) 106:876-81; Huang G, et al. Cell Mol Immunol. (2012) 9:287-95], or other CD4+ T cell subtypes [Cited from Levings MK, et al. Blood (2005) 105:1162-9]. T cell differentiation in each subtype is a complex phenomenon that can be influenced by cytokines in the originating DC tissue [ibid., citing Rescigno M. Immunol Rev. (2014) 260:118-28], their maturation state [ibid., citing Reis e Sousa C. Nature Rev Immunol. (2006) 6:476-83], and the causes of tissue imbalance [ibid., citing Vega-Ramos J, et al. Curr Opin Pharmacol. (2014) 17:64-70].

[0018] Figure 1 schematically depicts the role of CD4+ T cells in modulating the immune response against cancer. As shown in Figure 1, CD4+ T cells play a central role in regulating the host immune response through the following mechanisms: (a) CD4+ T cells provide important help in the priming of CD8+ T cells through the activation of antigen-presenting cells (APCs). (b) CD4+ T cells secrete cytokines necessary to maintain the function and proliferation of CD8+ T cells. (c) CD4+ T cells can directly or indirectly inhibit tumor growth. (d) CD4+ T cells provide help for the activation of B cells. Therefore, both CD8+ T cells and antibody production require important help from CD4+ T cells. Cited from Wang, R-F. Trends in Immunol. (2001) 22 (50): 269-76.

[0019] CD4+ T cells can be divided into T helper 1 (T H 1) and T helper 2 (T H 2) cells based on their cytokine secretion profiles. [Wang, R-F. Trends in Immunol. (2001) 22 (50): 269-76, citing Morel, PA and Criss, TP. Crit. Rev. Immunol. (1998) 18: 275-303]. T H 2 cells activate B cells to become plasma cells that secrete antibodies. CD4+ T HOne cell helps prime the CD8+ T cell response [citing Toes, RE et al. J. Exp. Med. (1999) 189: 753-6]. Numerous studies have demonstrated that CD40-CD40 ligand (CD40L) interactions between DCs and CD4+ T cells activate DCs for effective priming and activation of CD8+ T cells [citing Schoenberger, SP et al. Nature (1998) 393: 473-4; Bennett, SR et al. Nature (1998) 393: 478-80; Ridge, JP et al. Nature (1998) 393: 474-78]. CD4+ T cells recognize antigens presented by professional APCs such as DCs, and subsequently activate the DCs to which the antigens are bound [citation from Banchereau, J. and Steinman, RM. Nature (1998) 392: 245-52]. Once activated, the DCs become capable of priming cytotoxic lymphocytes (CTLs) that recognize MHC class I restrictive determinants on the same APC. Therefore, antigen-specific recognition and activation of APCs by CD4+ T cells via CD40-CD40L engagement are essential for priming CD8+ T cells [citation from Schoenberger, SP et al. Nature (1998) 393: 473-4; Bennett, SR et al. Nature (1998) 393: 478-80; Ridge, JP et al. Nature (1998) 393: 474-78]. In addition, CD4+ T cells are essential for maintaining the effector function of CD8+ T cells by secreting cytokines such as IL-2, which are necessary for the growth and proliferation of CD8+ T cells. [Ibid., Rosenberg, SA et al. I Nature Med. (1998) 4: 321-7; Greenberg, PD. Adv. Immunol. (1991) 49: 281-355].

[0020] Most tumors express MHC class I but not MHC class II molecules. CD4+ T cells recognize peptides presented by MHC class II molecules on the cell surface of APCs. The formation of MHC class II peptide complexes on the cell surface is a complex multi-step process favorable for the presentation of exogenous protein antigens. The assembly of MHC class II α and β chains with the associated invariant chain (Ii) begins in the endoplasmic reticulum (ER). [Cited from Germain, RN. Cell (1994) 76: 287-99; Cresswell, P. Cell (1996) 84: 505-7]. The association of Ii prevents the antigenic peptide from binding to the αβ dimer and stabilizes the αβ complex. Ii contains an endosomal target sequence at its N-terminus and a class II-associated invariant-chain peptide (CLIP) between amino acids 81 and 104. The target sequence in the cytoplasmic tail of this II is involved in the transport of the nonumerate (αβIi)3 complex from the ER to an intracellular compartment with endosomal / lysosome characteristics, ultimately to an acidic endosome and lysosome-like structure called the MHC class II compartment (MIIC) [citations: Germain, RN. MHC-dependent antigen processing and peptide presentation providing ligands for T lymphocyte activation. Cell (1994) 76: 287-99; Cresswell, P. Cell (1996) 84: 505-7]. In this compartment, HLA-DM molecules facilitate the dissociation of residual II peptide (CLIP) from the peptide binding groove of mHC class II molecules and its replacement with antigenic peptides [citations: Cresswell, P. Cell (1996) 84: 505-7]. Therefore, the processing and presentation of MHC class II antigens require at least five genes, namely DRα, DRβ, Ii, DMA, and DMB, as well as specialized MIIC compartments.

[0021] CD8+ T cells. CD8+ T cells are the most prominent antitumor cells. When primed and activated by APC, CD8+ T cells differentiate into cytotoxic T lymphocytes (CTLs) and perform efficient antitumor attacks via exocytosis of granules containing perforin and granzyme, resulting in the direct destruction of target cells [Gonzalez, H. et al. Genes & Development (2018) 32: 1267-84, citing Hanson, HL et al. (2000) Immunity 13: 265-276; Matsushita, H. et al. (2012) Nature 482: 400-404]. On the other hand, CD4+ T helper 1 (T) cells mediated by the secretion of large amounts of pro-inflammatory cytokines such as IL-2, TNF-α, and IFN-γ. H 1) The mediated antitumor response promotes not only T cell priming and activation and CTL cytotoxicity, but also an overall increase in the antitumor activity of macrophages and NK cells, as well as tumor antigen presentation [Ibid., Kalams SA, Walker BD. (1998) J Exp Med 188: 2199-2204; Pardoll DM, Topalian SL. (1998). Curr Opin Immunol 10: 588-594; Shankaran, V. et al. (2001) Nature 410: 1107-1111]. Tumor-infiltrating CD8+ T cells and T in tumors H The presence of cytokine 1 correlates with a favorable prognosis in terms of disease-free survival and overall survival in many malignancies [Fridman, WH et al. (2012). Nat Rev Cancer 12: 298-306].

[0022] Preclinical studies in patients and mouse models suggest that cancer cells utilize the immunosuppressive properties of T cells, while simultaneously reducing the effector function of antitumor T cells, such as their ability to infiltrate tumors, as well as their survival, proliferation, and cytotoxicity [citing Grivennikov, SI et al. (2010) Cell 140: 883-899]. The antigen-dependent nature of effector T cells means that the effectiveness of the antitumor T cell immune response depends on both the ability of the tumor antigen to induce an immune response (immunogenicity) and the presence or absence of inhibitory signals that may impair T cell function [citing Speiser, DE et al. (2016) Nat Rev Immunol 16: 599-611]. Therefore, it is widely accepted that in T cell-dependent processes, most newly generated cells expressing highly immunogenic antigens are expected to be recognized and killed during the early stages of tumor development [citing Matsushita, H. et al. (2012) Nature 482: 400-404]. Cancer cells with lower immunogenicity evade T cell immune regulation and survive, a process called "cancer immunoediting" [citing Teng, MW et al. (2015) J Clin Invest 125: 3338-3346]. The ultimate outcome is that the surviving cancer cells adopt an immune resistance phenotype. In parallel, during tumor development, cancer cells develop mechanisms that mimic peripheral tolerance, preventing local cytotoxic responses from other cells such as tumor-associated macrophages (TAMs), NK cells, and tumor-associated neutrophils (TANs), in addition to the local cytotoxic response of effector T cells [citing Palucka AK, Coussens LM. (2016) The basis of oncoimmunology. Cell 164: 1233-47]. TANs are engaged in the tumor microenvironment by cytokines and chemokines and can be distinguished according to their activation and cytokine status, as well as their effects on tumor cell proliferation in N1 and N2 TANs. N1 TANs exert antitumor activity through direct or indirect cytotoxicity.[Masucci, MT et al. Front Oncol. 9: 1146]。

[0023] Immune checkpoints. During immune homeostasis, a key mechanism of peripheral tolerance is the regulation of effector T cell responses via immune checkpoints in CTLs and activated CD4+ T cells to protect tissues from inflammatory damage. Two well-described checkpoint molecules, CTLA-4 and PD-1, act as negative regulators of T cell function and are associated with immune evasion in cancer [Gonzalez, H. et al. Genes & Development (2018) 32: 1267-84., citing Pardoll, DM (2012) Nat Rev Cancer 12: 252-264]. The involvement of CTLA-4 signaling in cancer includes melanoma ([ibid., citing Bouwhuis, MG et al. (2010) Cancer Immunol Immunother 59: 303-312]), lung ([ibid., citing Khaghanzadeh, N. et al. (2010) Cancer Genet Cytogenet 196: 171-174]), breast ([ibid., citing Erfani N. et al. Cancer Genet. Cytogenet. (2006) 165 (2): 114-20]), stomach (ibid., citing Hadinia, A. et al. (2007) J Gastroenterol Hepatol 22: 2283-2287]), and colorectal cancer ([ibid., citing Hadinia, A. et al. (2007) J Gastroenterol Hepatol 22: [2283-2287; Cited Dilmec, F. et al. (2008). Int J Immunogenet 35: 317-321] This has been explained in cancer. Furthermore, the engagement of PD1 and its co-receptor PDL-1 (expressed by other immune cells, mesenchymal cells, vascular cells, and cancer cells) results in downregulation of T cell activity, which inhibits its antitumor activity, such as limiting T cell migration, proliferation, secretion of cytotoxic mediators, and cell lethality [Cited Topalian, SL et al. (2015) Cancer Cell 27: 450-461].The use of immune checkpoint inhibitors, such as anti-PD1 (e.g., lambrolizumab, pembrolizumab, and nivolumab), anti-PD-L1 (MPDL3280A), and anti-CTLA4 (ipilimumab), has successfully enhanced the effector antitumor response in various malignancies ([citing Gotwals, P. et al. (2017). Nat Rev Cancer 17: 286-301]), particularly in melanoma and lung cancer ([citing Hamid, O. et al. (2013) N Engl J Med 369: 134-144; Herbst, RS et al. (2014). Nature 515: 563-567; Topalian, SL et al. (2015) Cancer Cell 27: 450-461]).

[0024] As tumors grow and TMEs change, new antigens are produced, priming a new repertoire of T cells and altering the immune system's ability to direct them toward the tumor, thus altering the effectiveness of tumor containment. As the immune system functions to slow tumor growth, cancer cells and TMEs simultaneously suppress antitumor function by engaging immune checkpoints and the recruitment of regulatory CD4+ T cells (Tregs). Tregs engage other effector immune cells, such as T H1. Involved in suppressing the priming, activation, and cytotoxicity of CD4 T cells, CTLs, macrophages, NK cells, and neutrophils ([Ibid., Ward-Hartstonge KA, Kemp RA. (2017). Clin Transl Immunology 6: e154]). Treg-mediated immunosuppression is organized by contact-dependent mechanisms such as the expression of PDL-1, LAG-3, CD39 / 73, CTLA4, or PD1, the latter two of which further enhance the suppressive activity ([Ibid., Walker LS, Sansom DM. (2015) Trends Immunol 36: 63-70]), and is also organized by contact-independent mechanisms including IL-2 capture and the production of immunosuppressive molecules such as IL-10, TGF-β, prostaglandin E2, adenosine, and galectin-1 (Ibid., Francisco, LM et al. (2009). J [Cited from Exp Med 206: 3015-3029; Campbell, DJ (2015) Eur J Immunol 195: 2507-2513]. For example, in squamous cell carcinoma, inhibition of adhesion plaque kinase (FAK), a cell contact-independent mechanism, leads to CCL5 secretion by cancer cells, which in turn induces the recruitment of Tregs to the tumor site, where they suppress cytotoxic antitumor CD8+ T cells ([Cited from Serrels, A. et al. (2015) Cell 163: 160-173]. For example, in breast and lung adenocarcinoma, Tregs suppress T cell activation and antitumor immune responses in tumor-associated tertiary structures. Specific Treg depletion leads to tumor cell death and increased IFN-γ production ([Cited from Bos, PD et al. (2013) J Exp Med 210: 2435-2466; Joshi, [Cited NS et al. (2015) Immunity 43: 579-590]. Treg invasion in breast cancer was correlated with worsening patient outcomes [Ibid., Allaoui, R. et al. (2017) Cancer Biomark 20: 395-409].

[0025] In metastasis, cytotoxic T lymphocytes (CTLs) exert anti-metastatic effects in bone metastases [citing Bidwell, BN et al. (2012) Nat Med 18: 1224-1231], and predictive analyses of lung and breast cancer patients established an inverse correlation between the level of circulating cancer cells and the level of T cells in peripheral blood [citing Mego, M et al. (2016) Circulating tumor cells (CTC) are associated with defects in adaptive immunity in patients with inflammatory breast cancer. J Cancer 7: 1095-1104; Sun, WW et al. (2017) Onco Targets Ther 10: 2413-2424].

[0026] These data also extend to clinical trials reporting the therapeutic efficacy of immune checkpoint inhibitors in metastatic cancer [citing Di Giacomo, AM et al. (2012) Lancet Oncol 13: 879-886; Queirolo, P. et al. (2014) J Neurooncol 118: 109-116; Motzer, RJ et al. (2015) N Engl J Med 373: 1803-1813; Furudate, S. et al. (2016) Case Rep Oncol 9: 644-649; Goldberg, SB et al. (2016) Lancet Oncol 17: 976-983; Pai-Scherf, L. et al. (2017) Oncologist 22: 1392-1399]. Checkpoint inhibitors are remarkably effective in treating brain metastases from melanoma and lung cancer [cited from Queirolo, P. et al. (2014). J Neurooncol 118: 109-116; Goldberg, SB et al. (2016) Lancet Oncol 17: 976-983; Di Giacomo, AM et al. (2017) Cytokine Growth Factor Rev 36: 33-38]. Recent evidence suggests that the effectiveness of checkpoint inhibition in brain metastases from melanoma depends on extracranial disease and peripheral activation of CD8+ T cells [cited from Taggart, D. et al. (2018) Proc Natl Acad Sci 115: E1540-E1549]. On the other hand, high levels of circulating Treg cells have been associated with a higher risk of metastasis in patients with non-small cell lung cancer. [Ibid., citing Erfani, N. et al. (2012) Lung Cancer 77: 306-311].Similar associations have been described in breast cancer [ibid., citing Metelli, A. et al. (2016) Cancer Res 76: 7106-7117], metastatic colorectal cancer [ibid., citing Wang, Q. et al. (2014) Cell Immunol 287: 100-105], and hepatocellular carcinoma [ibid., citing Ye, LY et al. (2016) Cancer Res 76: 818-830].

[0027] B cells B cell activation. Mature B cells can be activated by encountering an antigen that expresses an epitope recognized by their cell surface immunoglobulin (Ig). The activation process may be direct, depending on the crosslinking of membrane Ig molecules by the antigen (crosslinking-dependent B cell activation), or indirect, occurring most efficiently in the context of close interaction with helper T cells ("cogeneic assistive process"). The soluble products of activated B lymphocytes are immunoglobulins (antibodies).

[0028] When activated in germinal centers of lymphoid organs, B cells expressing high-affinity antibodies differentiate into antibody-secreting plasma cells and memory B cells that mediate humoral immunity against pathogens [Gonzalez, H. et al. Genes & Development (2018) 32: 1267-84, citing De Silva NS, Klein U. (2015) Nat Rev Immunol 15: 137-148]. Although the presence of B cells in TMEs has been described in various cancers (particularly melanoma, as well as breast, ovarian, and prostate cancers) [citing Chin, Y. et al. (1992) Anticancer Res 12: 1463-1466; Yang, C. et al. (2013) PLoS One 8: e54029; Woo, JR et al. (2014) J Transl Med 12: 30; Pylayeva-Gupta, Y. et al. (2016) Cancer Discov 6: 247-255], the role of B cells in cancer progression is not as well understood as the role of T cells. Accumulated evidence has shown that B cells promote and support tumor growth; for example, using a transgenic mouse model of epithelial carcinogenesis, Coussens and collaborators [citing de Visser, KE et al. (2005) Cancer Cell 7: 411-423] demonstrated that the absence of mature B cells reduces tumor progression. Adoptive transfer of B cells restores chronic inflammation, angiogenesis, and tumor growth.From immunosuppression mediated by IL-10 secretion [cited from Schioppa, T. et al. (2011) Proc Natl Acad Sci 108: 10662-10667, ibid.] and TGFβ [cited from Olkhanud, PB et al. (2011) Cancer Res 71: 3505-3515, ibid.], various mechanisms ranging from direct stimulation of tumor cell proliferation by B cell-derived IL-35 in human pancreatic neoplasms and Kras-driven pancreatic neoplasms in mice have been described to explain the role of B cells in tumorigenesis [cited from Pylayeva-Gupta, Y. et al. (2016) Cancer Discov 6: 247-255, ibid.]. Furthermore, the accumulation of immunoglobulins in the TME also indirectly stimulates angiogenesis and chronic inflammation by B cells activating myeloid cells via FcRγ (Andreu, P. et al. (2010) Cancer Cell 17: 121-134).

[0029] Memory T cells The majority of human memory T cells are found in tissues such as lymphoid tissue, the intestines, lungs, and skin. By the end of puberty, lymphoid tissue, mucous membranes, and skin are predominantly composed of memory T cells, a state that persists into adulthood, and these cells represent the most abundant lymphocyte population in the body.

[0030] Memory T cells in humans are classically distinguished by the phenotype CD45RO+CD45RA- and comprise a heterogeneous population of memory T cell subsets. [Farber, DL, et al. Nat. Rev. Immunol. (2014) 14(1): 24-35] Naive T cells homogeneously express CCR7, which reflects their dominant presence in lymphoid tissue. Memory T cells are further trafficked into lymphoid tissues, and are CD45RA-CCR7+ central memory T(T) cells. CM )Cells and CD45RA-CCR7-Effector Memory T(T) which can move to multiple peripheral tissue sites EM ) can be divided into cells. Functionally, T CMand T EM Both cell subsets produce effector cytokines in response to viruses, antigens, and other stimuli [see, ibid., Wang A, et al. Sci Transl Med. (2012) 4:149ra12030-33; Pedron B, et al. Pediatr Res. (2011) 69:106-111; Champagne P, et al. Nature. (2001) 410:106-111; Ellefsen K, et al. Eur J Immunol. (2002) 32:3756-3764], T CM The cells exhibit higher proliferative capacity. (Ibid., citing Wang A, et al. Sci Transl Med. (2012) 4:149ra120, Fearon DT, et al. Immunol Rev. 2006;211:104-118). T memory stem (T SCM These cells are CD45RA+CD45RO- and resemble naive T cells in that they express high levels of costimulatory receptors CD27 and CD28, IL-7 receptor α chain (IL7Rα), CD62L and CCR7, and have high proliferative capacity, and these cells further develop into T cells. CM and T EM It possesses both self-renewal ability and pluripotency in that it can differentiate into other subsets, including cells [Ibid., citing Gattinoni L, et al. Nat Med. (2011) 17:1290-1297, Gattinoni L, et al. Clin Cancer Res. (2010) 16:4695-4701]. The progressive differentiation pathway, based on signal intensity and / or degree of activation, is naive (T N ), T SCM , T CM and T EMThe cells are positioned in the differentiation hierarchy and function as precursors to effector T cells [Ibid., citing Gattinoni L, et al. Nat Rev Cancer. (2012) 12:671-684; Klebanoff CA, et al. Immunol Rev. (2006) 211:214-224; Lanzavecchia A, Sallusto F. Nat Rev Immunol. (2002) 2:982-987].

[0031] In mice, tissue-resident memory T(T) RM ) cells are a non-circulating subset found in peripheral tissues and, in some cases, trigger a rapid in-situ defense response. Mouse CD4+T RM These cells may be generated in the lungs from adoptive transfer of activated (effector) T cells [citing Teijaro JR, et al. J Immunol. (2011) 187:5510-5514] or after respiratory virus infection [citing Turner, DL, et al. Mucosal Immunol. (2014) 7 (3): 501-510], exhibiting upregulation of their early activation marker CD69, tissue-specific retention of their cells in the lung niche [citing Turner, DL, et al. Mucosal Immunol. (2014) 7 (3): 501-510], and enhanced ability to mediate protection against influenza virus infection compared to circulating memory CD4+ T cells [citing Teijaro JR, et al. J Immunol. (2011)]. [Cited from 187:5510-5514] These are distinguished from splenic and circulating memory T cells. Similar non-circulating CD4+ T RM A cell subset has been identified in the bone marrow of mice after systemic viral infection exhibiting enhanced helper function. [Cited from Herndler-Brandstetter D, et al. J Immunol. (2011) 186:6965-6971]. Post-infection-generated CD8+T RMThe cells are found in the skin [citing Ibid., Clark RA, et al. Sci Transl Med. (2012) 4:117ra117; Liu L, et al. Nat Med. (2010) 16:224-227], vaginal mucosa [Ibid., Mackay LK, et al. Proc Natl Acad Sci US A. (2012) 109:7037-7042, Shin H, (2004) 20:551-562, Masopust D, et al. J Exp Med. (2010) 207:553-564, Masopust D, et al. J Immunol. (2006) It has been identified in multiple mouse tissues, including the lungs [citing 176:2079-2083, Turner, DL, et al. Mucosal Immunol. (2014) 7 (3): 501-510, Anderson KG, et al. J Immunol. (2012) 189:2702-2706] and the brain [citing Wakim LM, et al. Proc Natl Acad Sci US A. (2010) 107:17872-17879]. These cells are distinguished from splenic and circulating memory CD8+ T cells by increased expression of CD69 and further by the expression of integrin αEβ7 (also known as CD103) that binds to epithelial cells [Ibid., Mueller SN, et al. Annu Rev Immunol. (2013) 31:137-161, Mackay LK, et al. Proc Natl Acad Sci US A. (2012) 109:7037-7042, Casey KA, et al. J Immunol. (2012) 188:4866-4875; Masopust D, Picker LJ. J Immunol. (2012) 188:5811-5817; Gebhardt T, Mackay LK. Front Immunol. (2012) 3:340].

[0032] In humans, memory CD4+ T cells are dominant throughout the body, persisting as localized CCR7+ or CCR7- subsets in lymphoid tissue and mucosal sites, respectively. In contrast, memory CD8+ T cells persist primarily as CCR7- subsets in all sites, and in lymphoid tissue, CD8T cells are dominant. CM These cells are few in number, and these cells are very few in number in other sites [ibid., citing Sathaliyawala T, et al. Immunity (2013) 38:187-197]. Most memory T cells in peripheral tissue sites such as human mucosa, lymphocytes, and skin are putative T cells. RM While these cells express the cellular marker CD69 [see Goronzy JJ, Weyand CM. Nat Immunol. (2013) 14:428-436; Nikolich-Zugich J, Rudd BD. Curr Opin Immunol. (2010) 22:535-540; Clark RA, et al. J Immunol. (2006) 176:4431-4439, Mueller SN, et al. Annu Rev Immunol. (2013) 31:137-161, Casey KA, et al. J Immunol. (2012) 188:4866-4875], circulating blood memory T cells uniformly lack CD69 expression. [Ibid, citing Sathaliyawala T, et al. Immunity (2013) 38:187-197].

[0033] Human T RMCells also exhibit tissue-specific characteristics, suggesting in-situ influence. For example, memory T cells in the small and colon express the intestinal homing receptor CCR9 [cited from Kunkel EJ, et al. J Exp Med. (2000) 192:761-768] and integrin α4β7 [cited from Agace WW. Trends Immunol. (2008) 29:514-522], while memory T cells in the lung upregulate CCR6 expression [cited from Purwar R, et al. PLoS One. (2011) 6:e16245]. There is also evidence of crosstalk between mucosal sites, such as between the lungs and the intestines. For example, dendritic cells in the lungs induce the migration of protective T cells into the gastrointestinal tract. [Ibid., citing Ruane D, et al. J Exp Med. (2013) 210:1871-1888].

[0034] T RM There is evidence that it may be polyfunctional and also exhibits qualitative functional differences. Human lung T RM A significant proportion of cells produce multiple pro-inflammatory cytokines [ibid., citing Purwar R, et al. PLoS One. 2011;6:e16245], human gut T RM Cells are also polyfunctional [ibid., citing Sathaliyawala T, et al. Immunity. 2013;38:187-197]. Other functions are thought to be limited to specific subsets and / or tissue sites. For example, IL-17 in mucosal sites, specifically in the intestines of healthy individuals, is associated with CD4+T RMMemory T cells are produced by subsets of cells [ibid., citing Sathaliyawala T, et al. Immunity (2013) 38:187-197], by CCR6+ memory T cells in peripheral blood [ibid., citing Singh SP, et al. J Immunol. (2008) 180:214-221, Wan Q, et al. J Exp Med. (2011) 208:1875-1887], and by subsets of CD161+ T cells in inflamed tissues such as the skin of psoriasis patients [ibid., citing Cosmi L, et al. J Exp Med. 2008;205:1903-1916]. Therefore, the function of dominant memory T cells, such as IFNγ production, is widely distributed among multiple memory T cell subsets and tissues, but T cells in specific tissue locations... RM Cells can incorporate multiple or distinct functional properties, which may depend on tissue-specific inflammation.

[0035] Despite their specificity, human memory T cells exhibit cross-reactivity to previously unencountered antigenic epitopes, which may be due to the inherent characteristics of TCR recognition [citing Sewell AK. Nat Rev Immunol. 2012;12:669-677] as well as the range and breadth of human antigen experience. For example, memory CD4+ and CD8+ T cells specific to the unique epitope of avian influenza strain H5N1 were detected in healthy individuals unexposed to H5N1 infection, as assessed by serology [citing Lee LY, et al. J Clin Invest. (2008) 118 (10): 3478-90; Roti M, et al. J Immunol. (2008) 180:1758-1768]. In addition, HIV-specific memory T cells were identified in HIV-negative individuals [citing Su, LF et al. Immunity (2013) 38: 373-83, ibid.]. Virus-specific memory T cells also exhibit cross-reactivity to alloantigens, autoantigens, and unrelated pathogens [cited from D'Orsogna LJ, et al. Transpl Immunol. (2010) 23:149-155, Wucherpfennig KW. Mol Immunol. (2004) 40:1009-1017]; EBV-specific human memory T cells generated in HLA-B8 individuals exhibit allo-cross-reactivity to HLA-B44 [cited from Burrows SR, et al. J Exp Med. (1994) 179:1155-1161]; and influenza virus and HIV-specific memory CD4+ T cells recognize epitopes from unrelated bacterial pathogens [cited from Su LF, et al. Immunity. (2013) 38:373-383]. Furthermore, T cells specific to the autoantigen myelin basic protein (MBP) recognized multiple epitopes from viruses and pathogens [Ibid., citing Wucherpfennig KW. Mol Immunol. (2004) 40:1009-1017, Wucherpfennig KW, Strominger JL. Cell. (1995) 80:695-705].This cross-reactivity allows memory T cells to mediate defense even without the initial disease, a phenomenon known as "heteroimmunity" [citing Welsh RM, Selin LK. Nat Rev Immunol. (2002) 2:417-426]. Heteroimmunity has been demonstrated in humans in whom influenza virus-specific T cell clones have expanded and proliferated due to EBV infection [citing Clute SC, et al. J Clin Invest. 2005;115:3602-3612].

[0036] Analysis of human samples revealed influenza-specific T11. RM It has been revealed that a considerable number of these are found in lung tissue, highlighting their role in natural infection. Despite expressing low levels of granzyme B and CD107a, these CD8+T RM It possessed a diverse repertoire of T cell receptors (TCRs), high proliferative capacity, and was multifunctional [Muruganandah, V., et al. (2018). Front. Immunol., 9, 1574. doi:10.3389 / fimmu.2018.01574]. A history of influenza infection provides a greater level of protection against reinfection, particularly in the lungs where CD8+ T cells are present. RM This suggests that it may be due to the accumulation of [something]. Furthermore, the innate immune response to influenza A virus infection in rhesus monkey models shows that the majority of influenza-specific CD8+ T cells produced in the lungs are phenotypic CD69+CD103+T RM This was confirmed as the T of the lung parenchyma. RM Unlike the CD8+T of the airway RM It possesses insufficient cytolytic activity and participates in early control of viral replication by inducing a rapid and robust IFN-γ response. Bystander CD8+T RM It may also participate in the early immune response to infection through antigen-nonspecific NKG2D-mediated immunity. Functional T12 may protect against hetero-subtype influenza infection. RMThe generation of this substance is thought to be dependent on signals from CD4+ T cells. [Muruganandah, V., et al. (2018). Frontiers in Immunology, 9, 1574. doi:10.3389 / fimmu.2018.01574].

[0037] According to the typical paradigm of CD8+ T cell responses to acute viruses, CD8+ T cells are effectors in the presence of an antigen and become memory cells once the antigen is eliminated. However, in viral infections, it has become clear that memory T cell populations include multiple subtypes of cells distributed in various anatomical compartments, and in some cases recirculate among them. Memory CD8+ T cell responses to most viruses are phenotypic and functionally diverse and undergo dynamic changes during their development and maintenance in vivo. This heterogeneity is related to the nature of the infectious virus, its cytoafficity, the anatomical site of infection, and the placement of CD8+ T cells. In resolved acute infections, the presence of memory CD8+ T cells at the original viral entry and replication site is important for a rapid response to secondary infections. In latent infections, the presence of memory CD8+ T cells at sites where the virus persists is important for immune surveillance of viral reactivation. [Racanelli, V. et al., Rev. Med. Virol. (2011) 21 (6): 347-357].

[0038] II. Tumor Immunology The term "tumor" includes cancer cells and the stroma that supports them. Together, they are often referred to as a "neoplasm," which means an abnormal mass of cells that persists and proliferates after the stimulus that initiated its appearance has disappeared. Leukemia is a cancer caused by the neoplastic proliferation of blood cells, but it does not usually form a mass. There are two types of neoplasms: benign and malignant. The collective term for all malignant neoplasms is cancer. There is substantial evidence that malignant neoplasms / cancers are the result of multiple sequential mutations. As a result, certain molecules in cancer cells are mutants, which are upregulated or downregulated, or no longer expressed. Most, if not all, cancers exhibit epigenetic changes in gene expression. Cancer may require more than 10 mutations to develop into a sufficiently malignant tumor.

[0039] The first stage of cancer development is called tumor initiation, which is generally assumed to be irreversible due to various oncogenes, tumor suppressor genes, or somatic or germline mutations in DNA repair pathways. Initiated cells do not form a tumor. However, initiated cells clonally expand and proliferate, evolving into precancerous lesions over many years.

[0040] This second long-term stage is driven by tumor promotion (i.e., promotional conditions or exposure to chemicals). The most advanced stage of these precancerous lesions is called carcinoma in situ or neoplasm in situ. The precancerous process ends with invasion, the appearance of the first cancer cells. Invasion usually occurs before metastasis and is sufficient as a diagnostic criterion for cancer, although this criterion cannot be used for leukemia and mesenchymal tumors. There is molecular-level evidence of precancerous cells spreading to distal sites, to the effect that these cells remain precancerous unless malignant transformation is promoted. Following invasion, cancer cells may or may not invade lymphatic vessels, bloodstream, or body cavity fluids and implant themselves in sites discontinuous with the original tumor. Metastasis defines a tumor as malignant; benign tumors do not metastasize. [Paul, William E. Fundamental Immunology 7]th Ed. Wolters Kluwer Health / Lippincott Williams & Wilkins, Philadelphia (2013), Chapter 47, pp. 1200-1201; 1220-1222].

[0041] The term “tumor progression” describes the third phase of a multi-stage process. The invasive growth of the lesion typically culminates in a highly invasive, widespread metastatic cancer that ultimately leads to host death. There is compelling evidence that most cancers are inherently clonal, and compelling evidence that in cancer progression, new subpopulations of cells arise sequentially due to Darwinian selection of genetic variants with proliferative advantages, escape homeostatic control, or become resistant to destruction by defensive mechanisms or treatments. During this evolution, a series of mutations result in changes in growth rate, morphology, hormone dependence, enzyme and cytokine production, and surface antigen expression. [Paul, William E. Fundamental Immunology 7] th Ed. Wolters Kluwer Health / Lippincott Williams & Wilkins, Philadelphia (2013), Chapter 47, pp. 1200-1201; 1220-1222].

[0042] Factors that limit cancer immunity Various mechanisms allow cancer to evade innate or adaptive host immunity by (1) inducing a protective and supportive stromal microenvironment; (2) increasing resistance to direct immune attack; and (3) inducing T cell anergy. Cancer cells often appear to use a combination of these three.

[0043] The tumor microenvironment. Many escape variants maintain their antigens, but induce a stroma that is more effective than that of the parent cells in supporting proliferation and protecting against host destruction. Regulatory T cells (Tregs) play a role in tumor resistance to immune-mediated elimination, in addition to tolerance to tumor antigens [Mendelsohn, J., Gray, JW, Howley, PM, Israel, MA, and Thompson, CB. The Molecular Basis of Cancer, Chapter 52, pp., 715-17, citing Curiel, TJ et al. Nat. Med. (2004) 10: 942-9; Liyanage, UK et al. J. Immunol. (2002) 169: 2756-61]. Tregs are characterized by the expression of the central master regulatory transcription factor (FoxP3), and CD4+ Tregs selectively express numerous cell membrane molecules such as CD25, neuropilin (Treg, GITR, and LAG-3 surface markers) [ibid., citing Bruder, D. et al. Eur. J. Immunol. (2004) 34: 623-3078-80], but their overall suppressive capacity is entirely dependent on the sustained expression of FOXp3 [ibid., citing Williams LM et al. Nat. Immunol. (2007) 8: 277-84; Zheng, Y. and Rudensky, AY. Nat. Immunol. (2007) 8: 457-62]. The mechanisms of immunosuppression by Tregs are diverse and include the production of inhibitory cytokines, such as IL-10 and IL-35 [ibid., citing Vlovw, GC et al. N. [Cited from Engl. J. Med. (2000) 342: 1350-8].

[0044] The tumor microenvironment contains multiple inhibitory cells and molecules. Immature myeloid cells (iMCs) [Mendelsohn, J., Gray, JW, Howley, PM, Israel, MA, and Thompson, CB. The Molecular Basis of Cancer, Chapter 52, pp., 715-17, citing Kusmartsec, S. and Gabrilovich, DI. Cancer Immunol. Immunother. (2006) 55: 237-45; Yong, MR et al. Hum. Immunol. (2001) 62: 332-41] are often called myeloid suppressor cells (MDSCs) [ibid., Bronte, V. et al. Blood (2000) 96: 3838-46; Bronte, V. et al. J. Immunol. (2003) 170: 270-8; Mazzoni, A. et al. J. Immunol.]. (2002) 168: 689-95; citing Zea, AH et al. Cancer Res. (2005) 65: 3044-8), this represents a myeloid cell type, which overlaps somewhat with tumor-associated macrophages (TAMs) [see Zhang, Z. et al. Front. Cell & Devel. Biol. (2020) 8: 17; Bai, R. et al. Front. Oncology (2020) 10: 1290], and shares the common characteristic of inhibiting both the priming and effector functions of tumor-reactive T cells. It is unclear whether these myeloid cell types represent distinct lineages or different contexts of the same general immunosuppressive cell subset. [Pardoll, D. “Cancer Immunotherapy with vaccines and checkpoint blockade.” In The Molecular Basis of Cancer, Chapter 52, Mendelsohn, J., Gray, JW, Howley, PM, Israel, MA, and Thompson, CB. pp., 715-17].In mice, iMCs and MDSCs are characterized by co-expression of CD11b (considered a macrophage marker) and Gr1 (considered a granulocyte marker), but with low or absent expression of MHC class II or the co-stimulatory molecule CD86. In humans, they are defined as CD33+ and DR- due to the lack of mature macrophage, DC, or granulocyte markers. Numerous molecular species produced by tumors tend to drive iMC / MSC accumulation. Examples of these include IL-6, macrophage colony-stimulating factor (CSF-1), IL-10, and gangliosides. IL-6 and IL-10 are powerful inducers of STAT3 signaling and have been shown to be important for the persistence and activity of iMC / MDSCs.

[0045] In addition to inhibitory cytokine production, multiple types of myeloid cells in the tumor microenvironment express numerous enzymes whose metabolic activity ultimately leads to inhibition of T cell responses within the tumor microenvironment. These include the production of reactive oxygen species (ROS) and / or reactive nitrogen species (RNS). Nitric oxide (NO) production by iMC / MDSCs as a result of arginase and iNOS activity is well-established, and inhibition of this pathway with numerous drugs may mitigate the inhibitory effects of iMC / MDSCs. ROS, containing H2O2, has been reported to block T cell function related to the downmodulation of the zeta chain of the TCR signaling complex, a phenomenon associated with generalized T-cell unresponsiveness [citing Schmielau, J. and Finn, OJ. Cancer Res. (2001) 61: 4756-60].

[0046] Another mediator of cancer-related T cell unresponsiveness is the production of indoleamine 2,3-dioxygenase (IDO) [ibid., citing Munn, DH et al. Science (2002) 297: 18;67-70]. IDO appears to be produced by DCs either within the tumor or in tumor-associated lymph nodes. IDO in DCs has been reported to be induced via reverse signaling by B7-1 / 2, a homologous costimulatory ligand expressed on the surface of antigen-presenting cells (APCs) that binds to the CD28 family of receptors on lymphocytes and modulates the immune response via costimulatory or coinhibitory signaling upon ligation with CTLA-4 [Collins, M. et al. Genome Biol. 6 (6): 223]. [Ibid., citing Baban, B. et al. Int. Immunol. (2005) 1;7: 909-19; Mellor, AL et al. Int. Immunol. (2004) 16: 1391-1401]. The major IDO-producing DC subsets are either plasmacytoid DCs (PDCs) or pDC-associated cells that are B220+, an isoform of CD45 [Ibid., citing Munn, DH et al. J. Clin. Invest. (2004) 114: 280-90]. However, it has been shown that IDO is expressed by multiple cell types in the immunomicroenvironment, including tumor cells themselves [Ibid., citing Uyttenhove, C. et al. Nat. Med. (2003) 9: 1269-74]. IDO appears to inhibit the T cell response through tryptophan catabolism, and activated T cells are highly dependent on tryptophan and therefore sensitive to tryptophan depletion. A bystander mechanism has been proposed in which DCs in the local environment deplete tryptophan by upregulating IDO, thereby inducing metabolic apoptosis in locally activated T cells. [Ibid., citing Munn, DH et al. Science (2002) 297: 1867-70].The second enzyme that metabolizes tryptophan is tryptophan dioxygenase (TDO), which is generally upregulated in human cancers and may thereby inhibit the antitumor response in a microenvironment similar to that of IDO. [Ibid., citing Opitz, CA et al. Nature (2011) 478: 197-203]. Furthermore, the major product of tryptophan's IDO and TDO metabolism, namely kynurenine, has a potent effect on T cell differentiation. Under certain conditions, kynurenine can promote Treg development [Ibid., citing Mezrich, JD et al. J. Immunol. (2010) 185: 3190-8], and under other conditions, kynurenine is known to promote IL-17 production and its carcinogenic properties. H 17. It can promote cell development [Ibid., citing Favre, D. et al. Sci. Trans. Med. (2010) 2: 32ra36].

[0047] The primary inhibitory cytokine produced by iMC / MDSCs and other cell types involved in blunting the antitumor immune response is transforming growth factor beta (TGF-β), which has pleiotropic physiological effects. In most normal epithelial cells, TGF-β is a potent inhibitor of cell proliferation, causing cell cycle arrest in G1 phase [citation: Blobe, GC et al. N. Engl. J. Med. (2000) 342: 1350-8]. In many cancer cells, mutations in the TGFβ pathway confer resistance to cell cycle inhibition and enable uncontrolled proliferation.

[0048] To increase resistance to direct immune attacks. Primary or intrinsic resistance is a typical clinical manifestation of malignancies that do not respond to immunotherapy. Hyperprogression (HPD) is one form of primary resistance. For example, blocking immune checkpoints can functionally stimulate Tregs, potentially creating a locally immunosuppressive microenvironment, leading to the polarization of immunosuppressive cells and the production of immunosuppressive cytokines. H1 and T H By stimulating 17-mediated inflammatory responses, it may be possible to promote tumor growth and create conditions for resistance to immunotherapy. [Bai, R. et al. Front. Oncology (2020) 10: 1290, citing Champiat, S. et al. Clin. Cancer Res. (2017) 23: 1920-8].

[0049] Acquired resistance is a typical clinical manifestation in which a tumor initially responds effectively to immunotherapy, but recurs or progresses after the treatment period. Adaptive immune resistance is a mechanism by which a tumor can be recognized by the immune system, but allows it to evade immunity by changing itself to adapt to immune attack. [Ibid., citing Sharma, P. et al. Cell (2017) 168: 707-23].

[0050] The intrinsic mechanisms of tumor immune resistance include alterations in antitumor immune response pathways, changes in signaling pathways in tumor cells, and other changes in tumor cells that result in an inhibitory immunosuppressive microenvironment. For example, tumor cells often inhibit T cell activation by reducing or eliminating antigen expression [citing de Vries, TJ et al. Cancer Res. (1997) 57: 3223-9], and mediate immune evasion by modulating autoantigenicity through endocytosis antigens or antigen shedding. In addition, the host can selectively eliminate cells expressing tumor-specific antigens (TSAs) and, to some extent, promote the generation of variants that have lost tumor antigens [citing DuPage, M. et al. Nature (2012) 482: 405-9]. Tumor cells can also escape T cell-mediated attack by undergoing antigen drift, resulting in epitope mutations that alter the antigenicity of the tumor cell. [Id., citing Bai, XF et al. J. Clin. Invest. (2003) 111: 487-96].

[0051] Disruption of the antigen presentation signaling pathway can prevent T cells from being activated and lead to immune evasion, including mutations that interfere with changes in the structural composition of the proteasome, a multi-enzyme complex involved in regulating the ubiquitin-proteasome pathway of cell cycle progression, terminating signaling cascades, and removing mutated, damaged, and misfolded proteins; transporters involved in antigen processing; and the MHC itself. Abnormalities in MHC molecules can be broadly divided into structural defects resulting from gene mutations, such as mutations in the receptor-binding domain of MHC cells [citing Giannakis, M. et al. Cell Rep. (2016) 15: 857-65], and defects in regulatory mechanisms resulting from epigenetic changes, such as downregulation of transporters involved in antigen processing, downregulation of low molecular weight proteins and methomyl proteins, and inactivation of class I MHC gene transcription [citing Sokol, L. et al. J. Transl. Med. (2015) 13: 279]. For example, in certain malignancies such as progressive multiple myeloma, tumor cells can develop immune evasion by overexpressing non-classical MHC-I molecules (e.g., HLA-G) to escape lysis mediated by cytotoxic T lymphocytes (CTLs) and NK cells [citing Gao, M. et al. Acta Biochim. Biophys. Sinica (2014) 46: 597-604].

[0052] In patients undergoing immunotherapy, tumor cells may downmodulate or alter IFN-gamma signaling pathways, such as loss-of-function alleles of genes encoding changes in JAK1 / 2 and STAT1, to evade the effects of IFN-gamma, thereby leading to resistance. Epigenetic alterations related to IFN-gamma signaling pathways can influence immune resistance. For example, activating mutations in tyrosine-protein phosphatase nonreceptor type 2 (Ptpn2) negatively modulates JAK1 and STAT1 signaling, which is also associated with primary resistance to PD-1 blockade via resistance to IFN-gamma [citing Manguso, RT et al. Nature (2017) 547: 413-18].

[0053] Inducing cellular anergy T cell anergy is a tolerance mechanism in which lymphocytes are essentially functionally inactivated after encountering an antigen, but remain alive for extended periods in a low-response state. Models of T cell anergy affecting both CD4(+) and CD8(+) cells fall into two broad categories: one is clonal anergy, which is primarily a state of proliferation arrest, and the other is adaptive tolerance or in vivo anergy, which represents a more general inhibition of proliferation and effector function. Clonal anergy results from incomplete T cell activation, is mostly observed in previously activated T cells, is maintained by blocking in the Ras / MAP kinase pathway, and can be reversed by IL-2 or anti-OX40 signaling, usually without inhibiting effector function. [Schwartz, RH. Annu. Rev. Immunol. (2003) 21: 305-34].

[0054] In vivo anergy is, in most cases, initiated in naive T cells in vivo by stimulation in a co-stimulus-deficient or co-inhibitory environment. As the cells proliferate and differentiate to varying degrees, they then downregulate both functions upon persistent antigen exposure. This state involves an initial block in tyrosine kinase activation, predominantly inhibiting calcium mobilization, and independent mechanisms blocking signaling via the IL-2 receptor. Adaptive tolerance is reversed in the absence of the antigen. [Schwartz, RH. Annu. Rev. Immunol. (2003) 21: 305-34].

[0055] For example, anergy is a component of normal B cell behavior. [Packham, G. et al. Haematologica (2014) 99 (7): 1138-48, citing Cambier, JC et al. Nat. Rev. Immunol. (2007) 7 (8): 633-43]. This is a state of cellular inactivity resulting from antigen binding by B cells (signal 1) in the absence of significant CD4+ T cell support (signal 2). In fact, B cells remain in a state of unresponsiveness and are susceptible to apoptosis.

[0056] However, in chronic lymphocytic leukemia (CLL), the dominant B cell receptor response in vivo appears to be an anergy-like response, making autoreactive B cells unresponsive to activation via their cell surface BCR. Unlike normal B cells, CLL cells and their progeny are protected from death by the expression of the anti-IgM-induced pro-survival MCL1 protein detected in CLL blood cells, thus correlating with progressive disease like MYC. [Ibid., Petlickovski, A. et al. Blood (2005) 105 (12): 4820-7; Pepper, C. et al. Blood (2008) 112 (9): [Cited 3807-17]. In CLL, two major subsets arise at distinct points of differentiation, expressing U-CLL and M-CLL, respectively, which are either unmutated or mutated IGHV genes. The clinical behavior of the two subsets is substantially different, with U-CLL having a worse prognosis [Cited Hamblin, TJ et al. Blood (1999) 94 (6): 1848-54; Damle, RN et al. Blood (1999) 94 (6): 1840-7]. Extended survival also appears to occur in the anergenic fraction, primarily M-CLL, which is inconsistent with the known vulnerability of normal anergenic B cells, which have a short lifespan in vivo. [Cited Cambier, JC et al. Nat. Rev. Immunol. (2007) 7 (8): [Cited from 633-43]. Although anergy is observed in CLL cells, particularly M-CLL, a proliferative fraction exists in all CLL patients. [Messmer, BT et al. J. Clin. Invest. (2005) 115 (3): 755-64]. This positive response to antigen engagement occurs only in a small fraction of cells located in specific tissue sites, specifically within the proliferative centers of lymph nodes (LNs) [Ibid., Soma, LA et al. Hum. Pathol. (2006) 37 (2): 152-9].

[0057] III. Hematopoiesis, Crohn's hematopoiesis, and acute myeloid leukemia The term "hematopoiesis," as used herein, refers to the formation of new blood cells. The classical unidirectional model of hematopoietic differentiation involves differentiation into increasingly lineage-specific progenitor cells, although alternative pathways may exist that are used separately from or in combination with the classical pathway. In the classical pathway, hematopoietic stem cells that regrow over long periods (LTR-HSCs) are characterized by their ability to regenerate and differentiate into pluripotent cells. Pluripotent progenitor cells (MPPs) have reduced regenerative capacity and differentiate into lymphoid common progenitor cells (CLPs) or myeloid common progenitor cells (CMPs). CMPs differentiate into all blood lineages except lymphocytes. Increasing hematopoietic cells to lineage-restricted cells requires the stimulation and regulation of cytokines by transcription factors. This model has several drawbacks, including oversimplifying the complexity of hematopoietic stem and progenitor cells (HSPCs) and relying solely on transplantation using surface markers and bulk cells. Bulk cell analysis assumes that each cell with the same phenotype has the same function.

[0058] This classical model has had problems over the past few years, particularly in elucidating megakaryocyte formation. Advances in single-cell technology and genetic mouse models have broadened our understanding of hematopoiesis, leading to the identification of new types of HSPCs, which have been extensively studied due to their lineage bias. Figure 2 schematically depicts a harmonized model of hematopoietic stem cell differentiation, where HSCs first differentiate into MPP1 / short-term HSCs (ST-HSCs), followed by MPP2, MPP3, and MPP4 lymphocyte-priming MPPs (LMPPs). MPP2 can generate premegakaryocytes / erythrocyte progenitor cells (pre-MegE), which then either give rise to platelets via megakaryocyte progenitor cells (MkP) or produce erythrocytes via pre-CFU-E. MPP3 primarily gives rise to granulocyte and monocyte lineages, while MPP4 (LMPP) primarily contributes to lymphocytes. [Cheng, H. et al. New paradigms on hematopoietic stem cell differentiation. Protein Cell (2020) 11 (1): 34-44].

[0059] Clonal hematopoiesis in healthy individuals During each round of mitosis, DNA damage and inefficient repair occur, resulting in the accumulation of somatic mutations over time in aging hematopoietic stem and progenitor cells (HSPCs). The process of acquired mutations leads to clonal expansion of HSPCs, which is called clonal hematopoiesis (CH). CH is a risk factor for the development of hematological malignancies, most commonly myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloid neoplasms (MNs), including myeloproliferative neoplasms (MPNs). Mutations that drive CH confer a survival advantage over wild-type cells by resulting in clonal expansion of mutated cells. CH driven by single nucleotide variants (SNVs) or insertion-deletion mutants (indels) are the most common drivers of MNs. Examples include epigenetic modifiers (DNMT3A, TET2, ASXL1, IDH1, IDH2), splicing factors (SF3B1, SRSF2, U2AF1), and genes involved in the DNA injury response (TP53, PPM1D, CHEK2, and JAK2). Copy number events (amplification, deletion, or loss of copy-neutral heterozygosity) are also known to drive CH. Other classes of mutational events, such as moderate-sized (>50 bp) copy number events, structural events, and non-coding mutations, are also associated with hematological malignancies and may drive CH, but are not yet fully characterized. [Chan, ICC, et al. Front. Oncol. (2022) 12: 2022.]

[0060] Acute myeloid leukemia (AML) Acute myeloid leukemia (AML) is a malignant disease of the bone marrow in which hematopoietic precursors are inhibited in the early stages of development. Most AML subtypes are distinguished from other related hematological disorders by the presence of more than 20% blast cells in the bone marrow.

[0061] The underlying pathophysiology of AML consists of the inhibition of myeloid cell maturation in the earliest stages of development. The mechanisms of this inhibition are under investigation, but in many cases involve the activation or inactivation of genes via chromosomal translocations and other genetic and / or epigenetic abnormalities. [Seiter, K., medscape.com / article / 197802, updated Dec. 6, 2022, citing Arber, DA et al. Blood (2016) 127 (20): 2391-405; Smith MT et al. IARC Sci. Publ. (2004) 373-92; Ghiaur, G. et al. Semin. Hematol. (2015) 52 (3): 200-6]. This inhibition of development leads to two disease courses. First, the production of normal blood cells is significantly reduced, resulting in varying degrees of anemia, thrombocytopenia, and neutropenia. Secondly, the rapid proliferation of abnormal myeloblasts, along with a reduced ability to undergo programmed cell death (apoptosis), leads to their accumulation in the bone marrow, blood, and often the spleen and liver.

[0062] Cause theory. Numerous factors, including pre-existing hematological disorders, familial syndromes, environmental exposures, and drug exposures, are associated with the causes of AML. However, most patients who present with new-onset AML do not have any identifiable risk factors.

[0063] Pre-existing hematological disorders. The most common risk factor for AML is the presence of a pre-existing hematological disorder, the most common of which is myelodysplastic syndrome (MDS). Other pre-existing hematological disorders that increase the risk of AML include aplastic anemia and myeloproliferative disorders, particularly myelofibrosis. Congenital disorders. Some congenital disorders that increase the risk of developing AML include Bloom syndrome, Down syndrome, congenital neutropenia, Fanconi anemia, and neurofibromatosis. Typically, these patients develop AML in infancy, although it may rarely appear in adolescence. Even minor genetic disorders, such as polymorphisms in enzymes that metabolize carcinogens, can increase a patient's susceptibility to AML. For example, polymorphisms in NAD(P)H:quinone oxidoreductase (NQO1), an enzyme that metabolizes benzene derivatives, are associated with an increased risk of AML. [Ibid., citing Larson, RA et al. Blood (1999) 94 (2): 803-7] Specifically, an increased risk exists in cases of AML arising after chemotherapy for another disease, or in cases of new-onset AML due to abnormalities in chromosome 5, chromosome 7, or both. Similarly, polymorphisms in glutathione S-transferase are associated with secondary AML following chemotherapy for other malignancies. [Ibid., citing Allan, JM et al. Proc Natl Acad. Sci. USA (2001) 98 (20): 11592-7].

[0064] Familial syndromes. With the routine use of extended next-generation genetic panels in the bone marrow and confirmation in non-hematopoietic tissues, more patients are being diagnosed with germline mutations that predispose them to AML. Among these genes are DDX41 (which regulates the expression and alternative splicing of genes involved in tumorigenesis and immune responses), SRP72 (a component of signal-recognizing particles involved in blocking the translation of nascent proteins intended for secretion into the cell membrane or extracellularly, and their movement into the endoplasmic reticulum (ER) for precise trafficking); ANKRD26, which encodes a protein containing an N-terminal ankyrin repeat that functions in protein-protein interactions; and ETV6, which provides instructions for protein production and functions as a transcription factor. [Ibid., citing Guidugli, L. et al. Leukemia (2017) 31 (5): 1226-9].

[0065] Environmental exposure. Numerous studies have demonstrated a link between radiation exposure and leukemia. In several studies, smokers had a slightly increased risk of AML compared to non-smokers. Smokers had a slightly but statistically significant (odds ratio, 1.5) increased risk of developing AML. [Cited from Brownson, RC et al. Am. J. Epidemiol. (1991) 134 (9): 938-41]. Exposure to benzene is associated with aplastic anemia and pancytopenia; these patients often develop AML. Many of these patients have the erythroleukemia subtype of AML (AML-M6). Exposure to soot, creosote, ink, dyes, and sunscreen and charcoal dust has also been associated with AML. [Cited from Poynter, JN et al. Intl J. Cancer (2016) 140 (1): 23-33]. Previous exposure to chemotherapy agents. As more cancer patients survive their primary malignancies and more patients receive intensive chemotherapy (including bone marrow transplantation [BMT]), the number of patients with AML due to exposure to chemotherapy agents will increase.

[0066] prognosis The prognosis of AML depends on several factors. Aging is a detrimental factor because older patients are more likely to have pre-existing hematological impairments and / or poor-risk cytogenetic conditions and molecular markers that make leukemia resistant to chemotherapy. Older patients also more frequently have co-existing medical conditions that impair their ability to tolerate the full dose of chemotherapy. Pre-existing hematological impairments (most commonly MDS) are associated with poor treatment outcomes.

[0067] Findings from bone marrow cytogenetic analysis constitute one of the most important prognostic factors. Patients with t(8;21), t(15;17), or translocation 16 have the best prognosis, with a long-term survival rate of approximately 65%. Patients with normal cytogenetic findings have an intermediate prognosis, with a long-term survival rate of approximately 35%. Patients with poor-risk cytogenetic findings (especially -7, -5, or monosomal karyotype) have a poor prognosis, with a long-term survival rate of less than 10%. [Ibid., citing Valk, PJ et al. N. Engl. J. Med. (2004) 350 (16): 117-28]. Other cytogenetic abnormalities, such as +8, 11q23, and mixed types, have been reported to confer intermediate risk in some series and poor risk in others.

[0068] The presence of FLT3 mutations is associated with a poorer prognosis. There are two main types of FLT3 mutations: internal tandem duplication mutations (FLT3-ITD) in the near-membrane domain and point mutations or deletions (FLT3-TKD) in the tyrosine kinase domain. [Kiyoi, H. et al. Cancer Sci. (2020) 111 (2): 312-322]. Both mutant FLT3 molecules are activated via ligand-independent dimerization and transphosphorylation. Biallele mutations in the gene that provides instructions for producing a protein called CCAAT enhancer-binding protein alpha (CEBPA), a transcription factor involved in the maturation of certain blood cells, are associated with longer remission periods and longer overall survival. [Seiter, K., medscape.com / article / 197802, updated Dec. 6, 2022, citing Taskesen, E. et al. Blood (2011) 117 (8): 2469-75]. Mutations in the gene encoding nucleophosmin (NPM) are associated with an increased response to chemotherapy. Nucleophosmin, also known as B23, No38, or numatrin, is an abundant nucleolar protein found in the nuclei of proliferating cells. Patients with TP53 mutations have a particularly poor prognosis. [Ibid., citing Taylor, J. et al. Blood (2017) 130(4): 410-23].

[0069] Using the European LeukemiaNet (ELN) molecular risk classification of patients with primary cytogenetically normal AML, Metzeler et al. determined that mutations in the TET2 gene, which provides instructions for producing a protein of unknown function but thought to be involved in transcriptional regulation, had adverse prognostic effects in a subset of otherwise favorable-risk patients. [Cited from Metzeler, KH et al. J. Clin. Oncol. (2011) 29 (1): 1373-81]. In adults, treatment outcomes are generally analyzed separately for younger (18–60 years) patients with AML and older (>60 years) patients. Using current standard chemotherapy regimens, approximately 40–45% of adults younger than 60 years survive for more than 5 years and are considered cured. Outcomes in older patients are more unfortunate, with less than 10% surviving long-term. Overall, cure rates in younger patients have improved over the past few decades, but little progress has been made in improving survival rates in older patients. [Ibid., citing Bower, H. et al. Blood Cancer J. (2016) 6: e390]. The prognosis for therapy-related AML is particularly poor, with a 5-year survival rate of approximately 10%. The prognosis is better in a subset of patients with therapy-related AML who have favorable cytogenetic abnormalities. [Ibid., citing McNerney, ME et al. Nat. Rev. Cancer (2017) 17 (9): 513-27; Kayser, S. et al. Blood (2011) 117 (7): 2137-45].

[0070] Cytogenetic analysis Guidelines from an international expert panel representing European LeukemiaNet (ELN) recommend risk stratification of patients with AML based on genetic abnormalities. ELN identifies three levels of risk: favorable, intermediate, and adverse. [Cited from Dohner, H. et al. Blood (2017) 129 (4): 424-47]. The genetic abnormalities that convey a favorable risk are as follows: t(8;21)(q22;q22.1);RUNX1-RUNX1T1 inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 Mutant NPM1 lacking FLT3-ITD or possessing FLT3-ITD with a low allele ratio (<0.5) Bi-allele mutation CEBPA.

[0071] The genetic abnormalities that convey intermediate risk are as follows: Mutant NPM1 and FLT3-ITD with a high allele ratio (≧0.5) Wild-type NPM1 that lacks FLT3-ITD or has FLT3-ITD with a low allele ratio (no damage to the gene for adverse risk) t(9;11)(p21.3;q23.3);MLLT3-KMT2A.

[0072] The genetic abnormalities that convey harmful risks are as follows: t(6;9)(p23;q34.1);DEK-NUP214 t(v;11q23.3);KMT2A rearrangement t(9;22)(q34.1;q11.2);BCR-ABL1 inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2);GATA2, MECOM(EVI1) -5 or del(5q);-7;-17 / abn(17p) Complex karyotype or monosomal karyotype FLT3-ITD with wild-type NPM1 and high allele ratios Mutant RUNX1 Mutant ASXL1 A mutated TP53.

[0073] Numerous molecular abnormalities that are not detected by conventional cytogenetics have been shown to have prognostic importance in patients with AML. Next-generation sequencing identified multiple recurrent somatic mutations in >90% of patients with AML. [Kantarjiian, H. et al. Blood Cancer J. (2021) 11: 41, citing Papaemmanuil, E. et al. N. Engl. J. Med. (2016) 374: 2209-21; Angenendt, L. et al. J. Clin. Oncol. (2019) J. Clin. Oncol. 3;7: 2632-42]. Genes that were frequently mutated (frequency >5%) were FLT3, NPM1, DNMT3A, IDH1, IDH2, TET2, RUNX1, p53;NRAS;CEBPA, and WT1. [Ibid., citing Papaemmanuil, E. et al. N. Engl. J. Med. (2016) 374: 2209-21; Richard-Carpentier, G. and DiNardo, CD. Hematol. Am. Soc. Hematol. Educ. Program (2019) 548-56; Angenendt, L. et al. J. Clin. Oncol. (2019) J. Clin. Oncol. 3;7: 2632-42]. Based on functional analysis and known pathways, these are grouped into the following biological and functional categories: bone marrow transcription factor fusions or mutations; NPM1 mutations; tumor suppressor gene mutations; epigenetic alteration gene mutations; activation signaling pathway gene mutations; cohesion-complex gene mutations; and spliceosome complex gene mutations. [Kantarjian, H. et al. Blood Cancer J. (2021) 11: 41].

[0074] Translating this to clinical practice, key molecular subsets are based on the identification of FLT3 mutations (30% of AML), NPM1 mutations (40-50% of AML with a normal karyotype); isocitrate dehydrogenase 1 or 2 (IDH1 / 2) mutations (20% of AML); and TP53 mutations (2-20% of AML). [Ibid.]

[0075] Patients with AML that have NPM1 mutations have a better prognosis; however, among patients with a high FLT3 allele ratio (AR) and no NPM1 mutations, those with FLT3-ITD mutations have a poor prognosis. Patients with diploid karyotype AML (without harmful mutations such as TP53 or ASXL1) and patients with biallelic CEBPA mutations (less than 2% of AML cases) have a good prognosis. [Ibid., citing Dohner, H. et al. N. Engl. J. Med. (2015) 373: 1136-52]. FLT3 is the most commonly mutated gene in AML cases and is constitutively activated in one-third of AML cases. [Cited Griswold, IJ et al. Blood (2004) 104 (9): 2912-8] Internal tandem duplication (ITD) in the membrane-near domain of FLT3 is present in 25% of AML cases. In other cases, mutations are located in the activation loop of FLT3. Most studies have demonstrated that patients with AML and FLT3-ITD have a poor prognosis. Mutations in NPM1 are associated with an increased response to chemotherapy in patients with a normal karyotype. [Ibid., citing Falini, B. et al. N. Engl.J. Med. (2005) 352 (30: 254-66)]. A study by Thiede et al. of FLT3 and NPM1 in 1485 patients with AML analyzed the clinical impact in four groups (single mutations, double mutations, and wild-type [wt] of both NPM1 and FLT3-ITD) and found that patients with only NPM1 mutations (without FLT3-ITD) had significantly better disease-free survival and overall survival, as well as a lower cumulative incidence of relapse. [Ibid., citing Thiede, C. et al. Blood (2002) 99 (12): 4326-35].

[0076] Mutations in CEBPA are detected in 15% of patients with normal cytogenetic findings. Bi-allele mutations are associated with longer remission periods and longer overall survival. [Ibid., citing Frohling, S. et al. J. Clin. Oncol. (2004) 22 (4): 624-33]. ERG overexpression is a harmful predictor in cytogenetically normal AML. Studies in cancer and leukemia group B (CALGB) have found that high expression of the brain and acute leukemia cytoplasmic protein [BAALC] is associated with the absence of FLT3-ITD, wild-type NPM1, mutated CEBPA, mixed-phenotype leukemia-partial tandem duplication (MLL-PTD), FLT3 mutations in the tyrosine kinase domain (FLT3-TKD), and high ERG expression. In multivariate analysis, high BAALC expression independently predicted lower complete remission rates after adjusting for ERG expression and age, and shorter survival after adjusting for FLT3-ITD, NPM1, CEBPA, and WBC count. [Ibid., citing Schwind, S. et al. Blood (2010) 116 (25): 5660-9].

[0077] Staging of acute myeloid leukemia: The term "staging" generally describes or classifies cancer based on how much cancer is present in the body and where it is located at the time of initial diagnosis. This is often referred to as the extent of the cancer. Two staging systems are commonly used in AML. The French-American-British (FAB) classification defines specific immunotypes based on morphology. The World Health Organization (WHO) classification summarizes the evidence of chromosomal translocations and dysplasia. [Ibid., citing Arber, DA et al. Blood (2016) 127 (20): 2391-405]. Because acute myeloid leukemia (AML) begins in the bone marrow and is usually not detected until it has spread to other organs, conventional cancer staging is unnecessary. Instead of using the common methods for evaluating cancer, such as T (tumor size and any spread of cancer to nearby tissues); N (spread of cancer to nearby lymph nodes); and M (metastasis), subtypes of AML are classified using cytological (cellular) systems. The conventional French-American-British (FAB) classification of AML is as follows:

[0078] M0- undifferentiated leukemia In this stage of acute myeloid leukemia, bone marrow cells do not show significant signs of differentiation. M1- Myeloblastic, undifferentiated type Myeloid cells show some signs of granulocyte differentiation, with or without minimal cell maturation. M2- Myeloblastic, differentiated type Myelocyte maturation surpasses the promyelocyte (early granulocyte) stage. Various levels of granulocyte maturation can be observed. M3- Promyelocytic Most abnormal cells are early granulocytes, which are between myeloblasts and myelocytes in their developmental stage. These cells contain many small particles and have nuclei of varying sizes and shapes. M4- Myelomonocytic ;M4eo- Myelomonocytic with eosinophilia In this stage of AML, the bone marrow and circulating blood contain varying amounts of monocytes and differentiated granulocytes. The percentage of monocytes and promonocytes in the bone marrow is greater than 20 percent. There may also be an increased number of eosinophils, a type of granulocyte that often has a bilobed nucleus.

[0079] M5- monoblastic leukemia M5a - Monoblastic, undifferentiated type; M5b - Monocyte, differentiated type: This subset can be further divided into two distinct categories. The first subset is characterized by poorly differentiated monoblasts with lace-like genetic material. The second subset is characterized by numerous monoblasts, promonocytes, and monocytes. The proportion of monocytes in the bloodstream may be higher than that in the bone marrow. M6- Red blood leukemiaThis form of leukemia is characterized by cells that form abnormal red blood cells, which make up more than half of the nucleated cells in the bone marrow. M7- Megakaryoblastic leukemia In this form of leukemia, the blast cells resemble immature megakaryocytes (giant cells of the bone marrow) or lymphoblasts (cells that form lymphocytes). M7 leukemia can be distinguished by large deposits of fibrous tissue (fibrosis) in the bone marrow. The newer WHO classification is as follows [citing Arber, DA et al. Blood (2016) 127 (20): 2391-405]:

[0080] AML with recurrent genetic abnormalities: AML with t(8;21)(q22;q22), (AML1 / ETO); AML with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(p13)(q22), (CBFB / MYH11); APL with PML / RARa; AML with t(9;11)(p21.3;q23.3), (MLLT3-KMT2A); t(6;9)(p23;q3 4.1) AML with (DEK-NUP214); AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2), (GATA2, MECOM); AML (megakaryoblastic) with t(1;22)(p13.3q133), (RBM15-MKL1); AML with BCR-ABL1; AML with mutated NPM1; AML with diallele mutations in CEBPA; AML with mutated RUNX1; AML with spinal cord dysplasia-related changes (secondary to myelodysplastic syndrome (MDS) or MDS / myeloproliferative disorder (MPD); without prior MDS or MDS / MPD, but with dysplasia in at least 50% of cells of two or more lineages); Therapy-related myeloid neoplasms. AML and MDS, therapy-related - alkylating agent or radiation-related types; topoisomerase II inhibitor types; others; AML, unspecified AML, minimally differentiated AML; AML, unmaturated; AML, mature; acute myelomonocytic leukemia; acute monoblastic or monocytic leukemia; pure erythroleukemia; acute megakaryoblastic leukemia; acute basophilic leukemia; acute panmyelopathy and myelofibrosis; myelosarcoma.

[0081] Current barriers in AML treatment Biological characteristics of the AML niche Tumor evasion strategies in AML include the direct adaptation of AML cells to hide from immune recognition, as well as modifications mediated by tumor cells in the immune cell compartment, including effector T cells, natural killer cells (NKs), and dendritic cells (DCs). [Tettamanti, S. et al. Leukemia (2022) 36 (1): 13-22, citing Guo, R. et al. Biomark Res. (2021) 9: 1; Bailur, JK, et al. JCI Insight (2020); Bruck, O. et al. Blood Adv. (2020) 4: 274-86; Chretien, AS et al. Proc Ntl Acad. Sci. (2021) 118: e2020459118; Vadakekolathu, J. et al. Sci. Transl. Led. (2020) 12].

[0082] Crosstalk between AML blast cells and TME cellular components Figure 3 schematically illustrates the immune evasion strategies employed by AML blasts. AML blasts may interfere with the effector function of T cells and NK cells by abnormally overexpressing inhibitory T cell ligands (i.e., PD-L1, Gal-9, CD155, CD112, CD86) (1) or by releasing soluble forms of NKG2DL. By altering the cytokine environment, AML blasts further promote T cell depletion and apoptosis, driving the expansion and proliferation of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and inducing the switching of macrophages to tumor-associated macrophages (TAMs) (3). This is achieved through the release of other soluble factors within the BM niche, such as reactive oxygen species (ROS), indoleamine 2,3-dioxygenase-1 (IDO1), arginase II (ArgII), and extracellular vesicles (EVs) (4). Furthermore, AML blasts reduce the expression of their antigen-presenting molecules, thereby concealing themselves from immune cells such as dendritic cells (DCs) and macrophages (5). [Cited from Tettamanti, S. et al. Leukemia (2022) 36 (1): 13-22].

[0083] Gene expression profiling of AML blasts from patients who relapsed after HSCT revealed transcriptional signatures enriched by altered immune-related processes, including epigenetic downregulation of HLA class II genes, HLA genomic loss, epigenetic upregulation of T cell inhibitory ligands, and disregulated release of immunosuppressive molecules [citations: Toffalori, C. et al. Nat. Med. (2019) 25: 603-11; Jan, M. et al. Blood Adv. (2019) 3: 2199-204; Christopher, MJ et al. N. Engl. J. Med. (2018) 379: 2330-41; Stolzel, F. et al. Transplantation (2012) 93: 744-9].

[0084] Galectin-9 is a ligand for T cell immunoglobulin and mucin domain 3 (TIM-3), and is a co-inhibitory receptor expressed on IFN-γ-producing T cells, FoxP3+ Treg cells, and innate immune cells (macrophages and dendritic cells). It has been shown to suppress the response when it interacts with its ligand [Das, M. et al. Immunol. Rev. (2017) 276 (1): 97-111], and is highly involved in creating an autocrine loop that appears to be essential for the maintenance of leukemia stem cells (LSCs) [ibid., Kikusige, Y. et al. Stem Cell (2010) 7: 708-17; Kikushige, Y. et al. Oncology (2015) 89: 28-32]. In AML mouse models and patients, a strong association has been found between high frequencies of TIM-3+ and PD-1+ T cells and poor prognosis [Kong, Y. et al. Blood Cancer J. (2015) 5:330; Darwish, NH et al. Oncotarget (2016) 7: 57811; Kamal, AM et al. Oncol. Lett (2021) 21: 1-9; Jan, M. et al. Proc. Natl Acad. Sci. USA (2011) 108: 5009-14]. TIM-3 is a clear immune checkpoint in both effector T and NK cells. TIM-3 has been found to bind to galectin-9, which is highly expressed on AML blasts, and promote self-renewal via stimulative β-catenin and NFκB signaling, reducing the release of pro-inflammatory cytokines and ultimately leading to NK and T cell dysfunction. Another inhibitory receptor, TIGIT (T cell immunoglobulin and ITIM domain), binds to the same ligands as DNAM-1, CD155, and CD112, and has also been shown to be upregulated in AML blasts.In AML patients, low levels of DNAM-1 expression have been observed, but its ligands are highly expressed [ibid., citing Wang, M. et al. Clin. Immunol. (2018) 190: 64-73; Gao, J. et al. Cancer Sci. (2017) 108: 1934-8], suggesting that the binding of TIGIT to CD112 and CD155 ligands may represent a mechanism of tumor immunity evasion facilitated by immune cell inhibitory signaling. This idea is further supported by clinical observations showing that CD112 and CD155 expression is associated with poor prognosis in AML [ibid., citing Stamm, H. et al. Mamm Genome (2018) 29: 694-702]. Another recent study has shown that high mRNA levels of the inhibitory receptors cytotoxic T lymphocyte-associated protein 4 (CTLA4) and lymphocyte activation-3 (LAG-3) in AML blasts are also a predictor of an unfavorable prognosis [citing Radwan, S. et al. Clin. Lymphoma Myeloma Leuk (2020) 20: S198].

[0085] AML blasts alter the formation of T cell immunological synapses. Gene expression profiling has elucidated abnormal T cell activation signatures in AML patients. Identified differentially expressed genes involved in actin cytoskeleton formation, and correlated functional data demonstrate impaired T cell ability to form immunological synapses with AML blasts [citing LeDieu, R. et al. Blood (2009) 114: 39099-16]. Previous studies have shown that T cells isolated from AML patients are phenotypic effector cytotoxic T lymphocytes and express activation markers, but their cytotoxic potential, meaning their ability to express cytotoxic granules, is impaired [citing Lim, SH, et al. Leuk. Res. (1991) 15: 641-4].

[0086] Functional alterations of T cells in AML are also a consequence of dysregulation of the cytokine network, which is directly mediated by AML blasts. Numerous studies have reported a large number of Tregs in patients with AML [citations: Curti, A. et al. Blood (2007) 109: 2871-7; Ersvaer, E. et al. BMC Immunol. (2010) 11: 38]. Specifically, Shenghui et al. [Shenghui, Z. et al. Intl J. Cancer (2011) 129: 1373-81] showed that an increased frequency of CD4+CD25+CD127low / -Tregs in AML is associated with a poor prognosis. The enrichment of Treg cells in the AML niche has been shown to be associated with the ability of AML blast cells to secrete immunosuppressive factors such as IL-10, IL-35, transforming growth factor beta (TGF-β), and indoleamine 2,3-dioxygenase 1 (IDO1) [citations: Chen, W. et al. J. Exp Med. (2003) 198: 1875-86; Walker, RM, et al. J. Clin. Invest. (2003) 112: 1437-43; Cools, N. et al. Cell Mol. Med. (2008) 12: 690-700]. These soluble factors promote T cell polarization toward induced Treg cells, thereby facilitating T cell tolerance and leukemia progression. Specifically, IDO1 works to inhibit T cell proliferation by catabolizing tryptophan into N-formylkynurenine, leading to a reduction in local tryptophan concentrations and the accumulation of toxic tryptophan metabolites, and IDO1 has been shown to correlate with poor prognosis [citing Folgiero, V. et al. Oncotartet (2014) 5: 2052-64]. Furthermore, tryptophan-derived metabolites such as L-kynurenine inhibit antigen-specific T cell proliferation and induce T cell apoptosis.This cytokine imbalance reduces the production of pro-inflammatory cytokines such as IL-15 and interferon-gamma (IFN-γ), and further increases their negative effects on T cell effector function [citing Binder, S. et al. Cytokine Growth Factor Rev. (2018) 43: 8-15].

[0087] Other soluble factors associated with different metabolic pathways have also been reported to modulate TME in leukemia. For example, high levels of arginase II in the plasma of AML patients have been shown to reduce T cell proliferation and polarize monocytes into an immunosuppressive M2-like phenotype. In addition, increased arginine metabolism inhibits the proliferation of hematopoietic progenitor cells, which contributes to a broader suppressive TME [citing Mussai, F. et al. Blood (2013) 122: 749-58]. Upregulation of inducible nitric oxide synthase (iNOS) by AML blasts, along with arginine II, has been reported to correlate with inhibition of T cell proliferation, an increase in Tregs, and a decrease in NKT cell count [citing Jacamo, R. et al. Blood (2017) 130: 2443].

[0088] Furthermore, AML blasts can metabolize both glucose and fatty acids released by surrounding interstitial adipocytes to induce acetyl-CoA, driving the Krebs cycle and oxidative phosphorylation (OXPHOS) for ATP production. LSCs in the AML niche express the fatty acid transporter CD36, inducing lipolysis in BM adipocytes and promoting fatty acid oxidation (FAO) in leukemia cells [Ye, H. et al. Cell Stem Cell (2016) 19: 23-27].

[0089] AML blasts evade NK cell recognition. The mechanisms of NK cell evasion and escape by AML blasts include altered expression of NK cell ligands caused by epigenetic changes such as inaccurate hypermethylation of genes encoding ligands for the activating receptor NKG2D (NKG2DL), namely MICA, ULBP1, ULBP2, and ULBP3 genes [ibid., citing Baragano, RA, et al. Genes Immun. (2015) 16: 71-82]. NKG2DL-negative leukemia cells that evade immune recognition by NK cells have been shown to possess immature morphology and molecular and functional stem cell characteristics [Paczulla, AM et al. Nature (2019) 572: 254-9]. Furthermore, AML blasts have been shown to release a soluble form of NKG2DL (sNKG2DL) via metalloproteinase cleavage or exosome cleavage, leading to downregulation of the NKG2D receptor on NK cells and impaired cytotoxic activity. AML blasts also express high levels of ligands, such as CD112 and CD155, leading to a decrease in their activating receptor DNAM-1 on NK cells, ultimately altering NK degranulation and impairing cytotoxic activity [cited from Costello, RT et al. Blood (2002) 99: 3661-7; Sanchez-Correa, B. et al. Immunol. Cell Biol. (2012) 90: 109-15]. AML blasts can also escape NK cells by inducing co-inhibitory receptors on NK cells, including TIGIT, thereby inhibiting IFN-γ release [cited from Kong, Y. et al. Clin. Cancer Res. (2016) 22: 3057-66]. High TIGIT expression at engraftment has been shown to be associated with reduced NK cell counts in BM, a reduced incidence of acute graft-versus-host disease, and poor survival rates [cited from Hattori N. et al. Biol. Blood Marrow Transpl. (2019) 25: 861-7].

[0090] AML blasts increase bone marrow-derived suppressor cells and tumor-associated macrophages. Myeloid-derived suppressor cells (MDSCs) induce T cell tolerance through multiple mechanisms, including the expression of the V-domain Ig suppressor (VISTA), PD-L1, IDO1, and arginase for T cell activation, as well as the production of reactive oxygen species (ROS), peroxynitrates, and several cytokines (TGF-β and IL-10) [citing Yang, Y. et al. Front Immunol. (2020) 11: 1371]. AML blasts can promote MDSC proliferation by releasing extracellular vesicles (EVs) containing the oncogeneic protein MUC1, thereby increasing c-myc expression in EVs via the microRNA miR34a and leading to MDSC proliferation [citing Pyzer, AR et al. Blood (2017) 129: 1791-801]. The Akt / mTOR pathway has been shown to play a crucial role in the phenotypic and functional transition from monocytes to MDSCs induced by AML-EVs. Monocytes containing AML-derived EVs are CD14+HLA-DR low It acquires an inhibitory phenotype and upregulates the expression of MDSC-specific genes such as S100A8 / 9 and cEBPβ [citing Tohumeken, S. et al. Cancer Res. (2020) 80: 3663-76]. In AML patients, MDSCs have been reported to be more abundant in BM and peripheral blood (PB) compared to healthy controls [citing Pyzer, AR et al. Blood (2017) 129: 1791-801]. In myelodysplastic syndrome (MDS), there is also an association between Treg and MDSC number, which correlates with a higher risk of conversion to AML, suggesting that MDSCs may play a role in AML progression [citing Kittang, AO et al. Oncoimmunology (2015) 5: e1062208].

[0091] Macrophages are an important cellular component of immunosuppressive tumor-mediated mesenteric neuropathy (TMEs). The inherent plasticity of macrophages makes this cellular subset particularly susceptible to tissue-specific regulation. Within TMEs, tumor-associated macrophages (TAMs), generally defined as M2 macrophages, are characterized by anti-inflammatory activity through the secretion of arginase, metalloproteinase, TGF-β, IL-10, and other cytokines that induce immunosuppression, angiogenesis, and tissue repair [citing Mantovani, A. et al. Trends Immunol. (2002) 23: 549-55]. Al-Matary et al. reported elevated TAMs in tumor-mediated mesenteric tumors (BMs) of AML patients compared to healthy donors. Furthermore, AML blasts can directly drive TAM to an M2-like phenotype in the BM and spleen of tumor-bearing mice [citing Al-Matary, YS, et al. Haematologica (2016) 101: 1216-27, ibid.].

[0092] Interstitial and vascular niches promote resistance to immunotherapy. In AML, TMEs contribute to resistance to conventional chemotherapy and suppress the antitumor immune response. Leukemia-associated remodeling within the AML niche involves metabolic reprogramming in addition to changes associated with increased hypoxia and inflammation, facilitating immune evasion and activation of favorable survival pathways for AML progression [citing Mendez-Ferrer, S. et al. Nat. Rev. Cancer (2020) 20: 285-98].

[0093] The AML niche indicates changes in immune cell homing. CXCL12, expressed by BM stromal cells and its receptor CXC receptor 4 (CXCR4), plays a major role in the migration of LSCs into the BM niche. High expression of CXCR4 in AML blasts has been shown to predict poor prognosis [ibid., citing Spoo, AC et al. Blood (2007) 109: 786-91]. The CXCL12 / CXCR4 axis can also activate favorable pathways for AML blast survival, proliferation, and chemotherapy resistance [Cancilla, D. et al. Front. Oncol. (2020) 10: 1672]. CXCL12 expression appears to be reduced in MSCs in AML, promoting the migration of malignant LSCs that overexpress CXCR4 compared to normal hematopoietic stem cells (HSCs) [Hanoun, M. et al. Cell Stem Cell (2014) 15: 365-75]. CXCR4 is also involved in the trafficking of adoptive lymphocytes or CAR T cells into the BM niche. Suppression of the ability of stromal cells to produce CXCL12 in AML TME may reduce their migration and infiltration into BM, as has been reported for other hematological malignancies [citing Ponzetta, A et al. Cancer Res. (2015) 75: 4766-77].

[0094] Metabolic changes in the AML niche MSCs from AML patients have a higher likelihood of differentiating into adipocytes, and the interaction between AML blasts and adipocytes in the BM niche creates a unique microenvironment that supports the metabolic requirements of leukemia [citations: Ye, H. et al. Cell Stem Cell (2016) 19: 23-37; Azadniv, M. et al. Leukemia (2020) 34: 391-403]. AML blasts induce hormone-sensitive lipase in adipocytes, activating lipolysis and enabling FABP4-dependent transport of fatty acids to leukemia cells, thus enhancing fatty acid oxidation (FAO) [citations: Shafat, MS et al. Blood (2017) 129: 1320-32; Tabe, Y. et al. Cancer Res. (2017) 77: 1453-64]. Fatty acid richness may interfere with effector T cell function and promote Treg differentiation [citing Michalek, RD et al. J. Immunol. (2011) 186: 3299-303]. In fact, FAO can inhibit effector T cell activation by increasing PD-1 expression and inhibiting IFN-γ secretion, but promote Treg cell generation through activation of the MAPK signaling pathway. FAO also plays a major role in M2 macrophage polarization [citing O'Neill, LAJ et al. Nat. Rev. Immunol. (2016) 16: 553-65].

[0095] MSCs can transport mitochondria to AML cells via the endocytosis pathway or tunnel nanotube (TNT) pathway, a process further boosted by chemotherapy and associated with increased oxidative phosphorylation-derived ATP production in recipient cells [citing Moschoi, R. et al. Blood (2016) 128: 253-64]. AML-derived nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) drives mitochondrial migration via superoxide production [citing Marlein, CR et al. Blood (2017) 130: 1649-60]. Gap junction interactions between AML cells and MSCs in the leukemia niche have been shown to be involved in regulating leukemia cell metabolism [citing Kouzi, F. et al. Oncogene (2020) 39: 1198-212]. The constitutive activation of NOX and mitochondrial production leading to OXPHOS (the fundamental mitochondrial process that links the TCA cycle to ATP production) are major sources of large amounts of ROS, particularly abundant in M4 and M5 subtypes of AML [citations Hole, PS et al. Blood (2013) 122: 3322-30; Farge, T. et al. Cancer Disco. (2017) 7: 716-35]. AML blasts can use ROS to escape anti-leukemia effector lymphocytes because free radicals inactivate T and NK cells by initiating PARP-1-dependent apoptosis [citations Aurelius, J. et al. Blood (2012) 119: 5832-7].

[0096] AML progression has been shown to primarily cause significant endothelial remodeling mediated by nitric oxide (NO), increasing vascular permeability and reducing blood flow, leading to the formation of a hypoxic leukemia niche [citing Passaro, D. et al. Cancer Cell (2017) 32: 324-41.e6]. The BM region of the endosteum is a major site of this vascular loss [citing Duarte, D. et al. Cell Stem Cell (2018) 22: 64-77.e6]. As a result, numerous BM regions are in a state of hypoperfusion, leading to impaired drug distribution and immune cell trafficking [citing Carmeliet, P. and Jain, RK. Nat. Rev. Drug Disco. (2011) 10: 417-27; Rytelewski, M. et al. J. Immunother. Cancer (2019) 7: 1-13]. Ultimately, the adhesion characteristics of immune cells to the endothelium are also altered due to increased E-selectin levels induced by inflammation caused by AML blasts [citing Barbier, V. et al. Nat. Commun. (2020) 11: 2042].

[0097] By elucidating the heterogeneity of AML at the clinical, cytogenetic, and molecular levels, improvements in prognosis and predictive capabilities have been made, leading to the development of targeted therapies for each AML subset. The research efforts of the past decade have expanded the pathophysiological molecular subset of AML through the identification of prognostic, predictive, and targetable molecular abnormalities [Kantarjian, H. et al. Blood Cancer J. (2021) 11: 41, citing Patel, JP et al. N. Engl. J. Med. (2012) 366: 1079-89; Cancer Genome Atlas Research Network, et al. N. Engl. J. Med. (2013) 368: 2059-74; Ding, L. et al. Nature (2012) 481: 506-10; Papaemmanuil, E. et al. N. Engl. J. Med. (2016) 3;74: 2209-21; Grimwade, D. et al. Blood (2010) 116: 354-65; Pastore, F. et al. J. Clin. Oncol. (2014) 32: 1586-94; Richard-Carpentier, G. and Di Nardo, CD Hematol. Am. Soc. Hematol. Educ. Program (2019) 548-56; Short, NJ et al. Cancer Discov. (2020) 10: 506-25]. Particularly interesting ongoing research in AML and recently approved drugs include:

[0098] (1) Epigenetic therapy with hypomethylating agents (HMA; azacitidine, decitabine) in older patients (or patients unsuitable for intensive chemotherapy) in combination with venetoclax; and intensive chemotherapy in combination with venetoclax in younger / suitable patients. (2) Intensive chemotherapy with fms-like tyrosine kinase 3 (FLT3) inhibitors (such as gilteritinib, midostaurin, sorafenib, quizartinib, or clenolanib) or addition to HMA / low-intensity therapy in FLT3 mutant AML. (3) Addition of isocitrate dehydrogenase (IDH) inhibitors (ivosidenib, an IDH1 inhibitor; enasidenib, an IDH2 inhibitor) and / or venetoclax in IDH1 / 2 mutant AML. (4) Investigation of the roles of APR246 (TP53 regulator) and magrolimab (anti-CD47 monoclonal antibody that enhances macrophage-mediated phagocytosis) in TP53 mutant AML. (5) To explore the role of menin inhibitors in mixed-phenotype leukemia (MLL1)-reconstitution acute leukemia. Menin inhibitors inhibit proteins that are part of the menin KMT2A epigenetic complex. This complex is particularly active and important in leukemia development related to NPM1 mutations and KMT2A reconstitution AML.

[0099] (6) Investigate combination small molecule targeted therapies with or without standard intensive chemotherapy or hypomethylating agents (HMAs) to not only extend survival but also improve the potential rate of cure in previously untreatable subsets of AML (+ / - venetoclax; at the expense of worsening myelosuppression). (7) Establish maintenance therapy as an important strategy in AML (similar to in acute lymphoblastic leukemia [ALL]). (8) To develop oral anti-AML therapies (e.g., oral decitabine, oral azacitidine) to replace or improve the effects of parenteral therapies. (9) Approaches to enhance the T cell immune response to AML using T cell engagers (BiTEs), checkpoint inhibitors, and chimeric antigen receptor (CAR) T cell approaches (as performed in ALL). [Ibid.]

[0100] Measurable residual disease (MRD) in complete remission. Measuring residual disease in AML with complete remission (CR) is now part of the standard treatment for AML [ibid., Jongen-Lavrencic, M. et al. N. Engl. J. Med. (2018) 378: 1189-99; Grimwade, D. and Freeman, SD. Blood (2014) 124: 3345-55; Pastore, F. & Levine, RL. JAMA (2015) 314: 778-80; Kico, JM et al. JAMA (2015) 314: 811-22; Ravandi, F. et al. Cancer (2017) 123: 426-35; Short, NJ et al. JAMA Oncol. (2020) 6: 1890-99; Hourigan, CS et al. J. Clin. Oncol.] [Cited from (2020) 38: 1273-83]. Detection of MRD at the time of morphological complete remission is associated with higher relapse rates and worse survival rates in AML.

[0101] Younger patients with AML (and / or older patients who are suitable for intensive chemotherapy) The median age of patients with AML is 68 years [citing Sasaki, K. et al. Cancer (2021) 127 (12): 2049-61]. Most of the experience with 3+7 (daunorubicin 3 days + cytarabine 7 days) and other intensive chemotherapy regimens has been performed in younger patients, typically with an upper age limit of 60-65 years. The optimal state-of-the-art therapy for younger patients with AML is evolving. Increasing research suggests that there are induction-enhancing regimens that are superior to the 3+7 regimen. Modifications to state-of-the-art AML therapy include: (1) the use of high-dose cytarabine combinations during induction; (2) optimization of daunorubicin dose (60 mg / m2 three times daily, compared to 45 mg / m2 or 90 mg / m2 three times daily) and the use of other anthracyclines (e.g., idarubicin, mitoxantrone); (3) addition of adenosine nucleoside analogs (fludarabine, clofarabine, cladribine) to the cytarabine-anthracycline regimen; (4) addition of gemtuzumab ozogamicin (GO), a CD33-targeted monoclonal antibody; (5) addition of targeted therapies such as FLT3 and IDH inhibitors in appropriate patients; and (6) addition of venetoclax, a BCL-2 inhibitor, to the induction therapy currently under investigation. (7) Use of oral azacitidine as maintenance therapy. [Ibid.]

[0102] Older patients with AML (or younger patients who are not suitable for intensive chemotherapy). Patients with AML who are older tend to have poorer tolerance to intensive chemotherapy. A study by Lowenberg and his collaborators [citing Lowenberg, B. et al. N. Engl. J. Med. (2009) 361: 1235-48] evaluated 3+7 daily daunorubicin 45 mg / m2 versus 90 mg / m2 × 3 in 813 selected patients aged 60 years or older (median age 67 years), with a median survival time of 7–8 months and an estimated 3-year survival rate of 20%. This study reported an acceptable low early mortality rate of 11–12%.

[0103] The management of older patients with AML remains challenging. Acute myeloid leukemia in older patients has a distinctly different disease biology, associated with a high risk and often complex karyotype, a high incidence of cytogenetic abnormalities including monosomy 5 and 7 and chromosome 17 abnormalities, a high incidence of multiple mutations such as TP53 (20+%), and a high incidence of secondary / therapy-related AML (20-30%). Older patients have multiple comorbidities (hypertension; diabetes; organ dysfunction including cardiac, pulmonary, and renal abnormalities) that result in poor tolerance to intensive chemotherapy and a high early (4-8 week) mortality. In community implementations treating randomly selected older patients (SEER data; 2010-2017), the 4-week mortality rate was 24% among patients aged 60-69 years, and the 5-year survival rate was 18%. Among patients aged 70 years and older (45% of all AML), the 4-week mortality rate was 44%, and the 5-year survival rate was 4%. [Ibid.].

[0104] Faced with poor outcomes from intensive chemotherapy, researchers began evaluating lower-intensity strategies in the 1990s for patients unsuitable for intensive chemotherapy (those with a high probability of early death). These included low-dose cytarabine, HMA therapy, and targeted therapies (monoclonal antibodies; more recently, FLT3 inhibitors and IDH1 / 2 inhibitors). Numerous long-term clinical trials since 2000 have shown that lower-dose chemotherapy / HMA therapy combinations, since 2015, provide overall response rates as high as intensive chemotherapy, significantly reduced rates of early mortality and myelosuppression-related complications, and survival rates equivalent to or better than intensive chemotherapy [Quintas-Cardama, A. et al. Blood (2012) 120: 4840-45]; Takahashi, K. et al. Clin. Lymphoma Myeloma Leuk. (2016) 16: 163-8 e1-2]. The search for ligands and receptors involved in promoting or inhibiting cell proliferation, progression, and sensitivity to cytotoxic drugs in AML remains ongoing.

[0105] IV. B-cell maturation antigen (BCMA): A promising therapy for other hematological cancers such as multiple myeloma. Multiple myeloma (MM) is a hematological cancer characterized by the accumulation of neoplastic plasma cells in the bone marrow, associated with elevated serum and / or urinary monoclonal paraprotein levels. During the course of the disease, patients with MM typically suffer from debilitating clinical symptoms directly or indirectly associated with the accumulation of tumor plasma cells, such as lytic bone lesions, anemia, immunodeficiency, and renal dysfunction. [Tonon, G. and Anderson, KC. Chapter 30, Multiple Myeloma, in The Molecular Basis of Cancer, Mendelsohn, J., Gray, JW, Howley, PM, Israel, MA, and Thompson, CB. Elsevier Saunders; (2015), pp. 455-66]. Before MM, a condition called monoclonal immunoglobulinemia of uncertain significance (MGUS) almost always occurs [citing Landagren, O. et al. Blood (2009) 113: 5412-7; Weiss, BM et al. Blood (2009) 113: 5418-22], which is defined by the presence of less than 30 g / L of monoclonal paraproteins in the serum and less than 10% plasma cells in the bone marrow, in the absence of clinical symptoms. MGUS is present in 1% of adults over 25 years of age and progresses to malignant MM at a rate of 0.5–3% per year [citing Kyle, RA, et al. Leukemia (2010) 24: 1121-27; Zingone, A. and Kuehl, WM Semin. Hematol. (2011) 48: 4-12].

[0106] Unlike other blood cancers such as leukemia and lymphoma, which present a relatively intact karyotype, the MM genome is thoroughly reshuffled. The diverse genetic damage described in MM includes chromosome acquisition or loss, Ig-related chromosomal translocations, acquisition or loss of small chromosomal segments, and genetic and epigenetic alterations affecting single genes. Approximately half of patients present a high-diploid (HD) karyotype (where the number of chromosomes ranges from 48 to 74 compared to the 46 chromosomes of a normal human karyotype), often accompanied by an increased number of odd chromosomes (e.g., 3, 5, 7, 9, 11, 15, 19, or 21 in various combinations). The remaining patients belong to the non-hyperdiploid (NHD) group, which includes cases with hypodiploid, pseudodiploid, neardiploid, or tetraploid karyotypes [citing Steinman, RM and Dhodapkar, M. Int. J. Cancer (2001) 94: 459-73]. At the MGUS stage, patients present either an HD or NHD karyotype [citing Chng, WJ et al. Blood (1005) 106: 2156-61], which is maintained as the disease progresses [citing Chng, WJ et al. Leuk Res. (2006) 30: 266-71].

[0107] MM-specific chromosomal translocations, so-called primary translocations, are present from the MGUS stage [ibid., citing deJong, EC et al. J. Immunol. (2002) 168: 1704-9], including the immunoglobulin H (IgH) locus at 14q32.3 [ibid., citing van Duin, D. et al. Trends Immunol. (2006) 27: 49-55], and, at a lower frequency, the IgL locus (2p12, kappa or 22q11, lambda [ibid., citing van Duin, D. et al. Trends Immunol. (2006) 27: 49-55]), and include strong Ig enhancers paralleling various genes, resulting in increased expression of those genes. These primary translocations are primarily limited to the NHD group [ibid., Fonseca, R. et al. Blood (2003)]. [Citing 102: 2562-67].

[0108] Beyond this general framework, additional genetic or epigenetic modifications exist within the MM genome. For example, deletions affecting tumor suppressor genes such as TP53, UTX, FAM46C, and NFκB family members BIRC2, BIRC3, and CYLD, or localized amplifications of regions containing oncogenes such as MYC, HGF, MCL1, and IL6R have been reported. In MM cases, four pathways are remarkably enriched with somatic mutations; genes belonging to the NFκB pathway are frequently mutated; and high-frequency mutations affecting histone modification genes such as MLL, MLL2, MLL3, UTX, MMSET, and WHSC1L1 have been reported. Mutations affecting the same nucleotides were also found in the IRF4 transcription factor and its target PRMD1, confirming prior functional data that reported the crucial role of IRF4 in MM survival. [Ibid., citing Shaffer, AL et al. Nature (2008) 454: 226-31].

[0109] MM cells utilize the physiological mechanisms underlying the homing of healthy plasma cells to the bone marrow and the pathways that support long-lived plasma cells. MM plasma cells establish close interactions with virtually all BM components; the BM microenvironment includes the ECM (collagen, laminin, fibronectin, and osteopontin) and rich cellular components. The cellular BM compartment consists of hematopoietic and mesenchymal progenitor and precursor cells, such as hematopoietic stem cells (HSCs), bone marrow-derived circulating endothelial precursors (CEPs) and endothelial cells (BMECs); immune cells (dendritic cells; B and T lymphocytes, NKT and NK cells, monocytes, and macrophages); erythrocytes; megakaryocytes and platelets; and non-hematopoietic cells, including a group of undefined cells classified as fibroblasts / bone marrow stromal cells (BMSCs). It also includes cells involved in bone homeostasis, such as cartilage resorbers, osteoclasts (OCs), and osteoblasts (OBs). MM cells interact with BMSCs and ECM either directly, via adhesion molecules such as LFA1, VLA4, NCAM, ICAM1, and CD44, or indirectly, via chemokines, cytokines, and growth factors released by tumor cells and BMSCs, such as IL-6, IGF-1, TNF-α, TGFβ1, and VEGF. As a result of these multilayered interactions, numerous cancer-related pathways, including NFκB, JAK / STAT, P13K, and MAPK, are activated in both tumor and stromal cells, further increasing MM proliferation and survival. Homing of MM cells to BM also initiates a strong angiogenic response. [Ibid., Gabrilovich, DI et al. Clin. Cancer Res. (1999) 5: 2963-70, Vacca, A. and Ribatti, D. Leukemia (2006) 20: 193-9].

[0110] Smoldering / asymptomatic multiple myeloma (SMM) is an asymptomatic condition defined by the presence of ≥3 g / dL serum monoclonal (M) protein and / or 10%–60% clonal myeloid plasma cells (BMPCs) and the absence of evidence of terminal organ damage (i.e., CRAB criteria) or other events defining myeloma. [Rajkumar, SV et al. Blood (2015) 125 (20): 3069-757]. It is distinguished from MGUS based on serum M protein levels and percentage of clonal BMPCs. The disease definition of SMM has recently been updated to exclude patients with ≥60% BMPCs, a serum involved / uninvolved free light chain (FLC) ratio of ≥100, and two or more focal lesions on magnetic resonance imaging (MRI) (typically indicating focal myeloma). [Ibid., citing Rajkumar, SV et al. Lancet Oncol. (2014) 15 (12): e538-48]. Such patients are considered to have approximately a 40% annual risk of progression and currently have multiple myeloma (MM). [Ibid., Rajkumar, SV et al. N. Engl. J. Med. (2011) 365 (5): 474-5; Kastritis, E. et al. Leukemia (2013) 27 (4): 947-53; Larsen, JT, et al. Leukemia (2013) 27 (4): 941-6; Waxman, AJ et al. al. J. Clin. Oncol. (2014) 32 (5s) abstract 8607; Hillengass, J. et al. J. Clin. Oncol. (2010) 28 (9): 1606-10; citing Kastritis, E. et al. Leukemia (2014) 28 (12): 2402-3]. Light chain SMM is a subtype of SMM characterized by an excess of monoclonal FLC and the absence of immunoglobulin heavy chain expression [Kyle, RA et al. N. Engl. J. Med. (2006) 354 (13): 1362-9; this entity is characterized by excessive secretion of monoclonal FLC in the urine (Bence Jones proteinuria).

[0111] During the course of MM, patients may relapse or become resistant to proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib), immunomodulators (e.g., thalidomide, lenalidomide, pomalidomide [Kleber, M. et al. J. Clincal Med. (2021) 10: 4088, citing Siegel, RL et al. CA Cancer J. Clin. (2020) 70: 7-30]), and monoclonal antibodies, resulting in a very poor prognosis and a moderate overall survival (OS) of 5.6 months [ibid., Attal, M. et al. Lancet (201( 394: 2096-2017; Ghandi, UH et al. Leukemia (201() 33: [Citing 2266-75], this occurs particularly in patients with high-risk cytogenetic features or who fail to reach negative minimal residual disease [Ibid., Chim, CS et al. Leukemia (2018) 32: 252-62; Munshi, NC et al. JAMAA Oncol. (2017) 3: 28-35; Kastritis, E. et al. Clin. Lymphoma Myeloma Leuk. (2020) 20: 445-52]. Although a new generation of these drugs is becoming available, managing these patients remains challenging for clinicians [Ibid., Ntassnasis-Stathopoulos, I. et al. Clin. Lymphoma Myeloma Leuk. (2021) 21: 379-85].

[0112] B cell maturation antigen (BCMA) in MM BCMA is a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily [Yu et al. J. Hematol. & Oncology. (2020) 13: article 126. Doi.org / 10.1186 / s13045-020-00962-7, citing Madry, C. et al. (1998) 10 (11): 1693-702]. BCMA is encoded by the 2.92kb TNFRSF17 gene, located on the short arm of chromosome 16 (16p13.13) and consisting of three exons separated by two introns. The BCMA gene product is a 184-amino acid, 20.2 kDa type III transmembrane glycoprotein, with an extracellular N-terminus containing six conserved cysteine ​​motifs [Ibid., Kozlow, EJ et al. Blood (1993) 81 (2): 454-61; Laabi, Y. et al. Nucleic Acids Res. (1994) 22 (7): 1147-54; Laabi, Yl et al. EMO J. (1992) 11 (11): 3897-904; Zhou, LJ et al.].Immunol. / (1992) 149 (2): 735-42]. There are four native splice variants of human BCMA that exhibit different receptor binding affinities, membrane fixation capabilities, and intracellular domain signaling [citation: Laabi, Y. et al. Nucleic Acids Res. (1994) 22 (7): 1147-54, 23].

[0113] BCMA has binding sites for TNF-related factors (TRAF) 1, 2, and 3 in its cytoplasmic tail and can activate the NF-κB, Elk-1, p38MAPK, and JNK signaling pathways. [Gardam, S. and Brink, R. Front. Immunol. (2014) 4: article 509]. TRAF1 is a signaling adapter that plays a major role in pro-survivability signaling downstream of TNFR superfamily members such as TNFR1, LMP1, 4-1BB, and CD40. TRAF-2 typically signals cell survival via NF-κB and JNK activation and has been shown to be involved in the negative regulation of the activated and non-regulatory NF-κB pathway. [Shi, JH and Sun, SC, Front. Immunol. (2018) 9: 1849]. The adapter protein TRAF3 serves as a negative regulator in multiple aspects of B cell biology. [Bishop, GA et al. Front. Immunol. (2018) 9: 2161]. TRAF2-deficient B cells appear to have increased levels of TRAF3, indicating that TRAF2 helps target TRAF3 for ubiquitination and degradation.

[0114] BCMA, along with two other functionally related TNFR superfamily members, the B cell activator receptor (BAFF-R) and the transmembrane activator, calcium regulator, and cyclophylline ligand interactor (TACI), coordinates the regulation of B cell proliferation, maturation, survival, and differentiation into plasma cells (PCs) [Yu et al. J. Hematol. & Oncology. (2020) 13: article 126. Doi.org / 10.1186 / s13045-020-00962-7, citing Coquery, CM and Erickson, LD. Crit. Rev. Immunol. (2012) 32(4): 287-305; Marsters, SA, et al. Curr. Biol. (2000) 10 (13): 785-88; Gross, JA, et al. Nature (2000) 404]. (6781): 995-99; Thompson, JS et al. J. Exp. Med. (2000) 192 (1): 129-135; Sasaki, Y. et al. J. Immunol. (2004) 173 (4): 2245-52; Seshasayee, D. et al. Darce, JR et al. J. Immunol. (2007) 178 (9): 5612-22]. Unlike BAFF-R and TACI, BCMA is expressed almost without exception in plasmablasts [ibid., citing Avery, DT et al. J. Clin. Invest. (2003) 112 (2): 286-97] and PCs [O'Connor, BP et al. J. Exp. Med. (2004) 199 (1): 91-98]. It may also be weakly detected in some memory B cells and plasmacytoid dendritic cells involved in plasma cell differentiation [ibid., citing Tai, YT et al. Blood (2014) 123 (20): 3128-38].Due to the presence of plasma cells, BCMA is undetectable in naive B cells, hematopoietic stem cells, or normal non-hematological tissues, except in certain organs such as the testes, trachea, and parts of the gastrointestinal tract [citing Carpenter, RO et al. Clin. Cancer Res. (2013) 19 (8): 2048-60].

[0115] Upregulation of BCMA is induced by B lymphocyte-induced maturation protein 1 (Blimp-1), an essential transcription factor involved in plasma cell development and survival [citation: Deng, S. et al. Mol. Biol. Rep. (2010) 37 (8): 3747-55]. Long-term plasma cell survival is impaired in BCMA- / - mice. BCMA deficiency is associated with short-lived plasma cells, ineffective B cell development, or an early humoral immune response, while spleen structure and germinal centers appear intact in these BCMA-deficient mice [citation: O'Connor, BP et al. J. Exp. Med. (2004) 199 (1): 91-98; Xu, S. and Lam, KP. Mol. Cell Biol. (2001) 21 (12): 4067-74].

[0116] BCMA is identified on the surface of almost all MM cell lines (80-100%) and is more abundant in malignant PCs than in normal PCs [Lee, L. et al. Br. J. Haematol. (2016) 174 (6): 911-22; Eckhert, E. et al. Immunotherapy (2019) 11 (9): 801-11]. MM patients undergoing allogeneic transplantation often develop donor-derived anti-BCMA monoclonal antibodies (mAbs) after donor lymphocyte infusion, benefiting from the graft-versus-tumor response [Bellucci, R. et al. Blood (2005) 105 (10): 3945-50]. In contrast, TACI is expressed at significantly lower concentrations, and BAFF-R is barely detectable in MM cells [citing Claudio, JO et al. Blood (2002) 100 (6): 2175-86]. BCMA overexpression significantly promotes in vivo proliferation of xenografted MM cells in mouse models [citing Tai, YT, et al. Blood (2016) 127 (25): 3225-36]. Furthermore, BCMA expression is upregulated from normal to MGUS, SMM, and then to active MM during the pathogenesis and evolution of MM [Darce, JR et al. J. Immunol. (2007) 179 (11): 7276-86]. Since higher levels of BCMA are associated with a worse outcome [citing Lee, L. et al. Br. J. Haematol. (2016) 174 (6): 911-22], BCMA is shown to be a useful biomarker for disease activity and prognosis in MM.

[0117] BCMA has two agonist ligands: proliferation-inducing ligand (APRIL) and B-cell activator (BAFF; also known as BlyS), which are mainly secreted in the bone marrow (BM) by stromal cells, osteoclasts, and macrophages in a paracrine manner [Ibid., Tai, YT, et al. Blood (2016) 127 (25): 3225-36; Moreaux, J. et al. Blood (2004) 103 (8): 3148-57; Tai, YT et al. Cancer Res. (2006) 66 (13): 6675-82; Mulazzani, M. et al. J. Hematol. Oncol. (2019) 12 (1): 102] (Figure 4). APRIL exhibits a higher binding affinity to BCMA than BAFF [citing Day, ES et al. Biochemistry (2005) 44 (6): 1919-31], and APRIL also binds to TACI [citing Moreaux, J. et al. Eur. J. Haematol. (2009) 83 (2): 119-29], while BAFF further restricts selectivity to BAFF-R [citing Day, ES et al., Biochemistry (2005) 44 (6): 1919-31]. Therefore, APRIL is more specific to plasma cells and correlates with more downstream pathophysiological activity [citing Bolkun, L. et al. Ann. Hematol. (2014) 93 (4): 635-44].

[0118] When MM cell lines were xenotransplanted into APRIL- / - mice, their proliferation was significantly reduced [citing Matthes, T. et al. Leukemia (2015) 29 (9): 1901-8]. In MM patients, serum levels of APRIL and BAFF were elevated to approximately five times the serum levels of healthy controls [citing Moreaux, J. et al. Blood (2004) 103 (8): 3148-57], and the concentration of detected ligands increased as the stage of MM progressed [citing Pan, J. et al. Oncol. Lett (2017) 14 (3): 2657-62]. Studies have shown that MM cells can stimulate osteoclasts to produce more APRIL, contributing to an immunosuppressive BM microenvironment [Tai, YT et al. Blood (2016) 127 (25): 3225-36; Yaccoby, S. et al. Leukemia (2008) 22 (2): 406-13].

[0119] When ligands bind to BCMA, multiple proliferation and survival signaling cascades are activated in MM cells, most frequently activating the nuclear factor-κ light chain enhancer (NF-κβ) of activated B cells, but also including the rat sarcoma / mitogen-activated protein kinase (RAS / MAPK) and phosphoinositide-3-kinase-protein kinase B / Akt (PI3K-PKB / Akt) signaling pathways [citations: Eckhert, E. et al. Immunotherapy (2019) 11 (9): 801-11; Demchenko, YN, et al. Blood (2010) 115 (17): 3541-52; Hua, H. et al. J. Hematol. Oncol. (2019) 12 (1): 71]. These pathways lead to proliferation stimulation by modulating cell cycle checkpoints, increased viability by upregulating anti-apoptotic proteins (e.g., Mcl-1, BCL-2, BCL-XL), and production of cell adhesion molecules (e.g., ICAM-I), angiogenic factors (e.g., VEGF, IL-8), and immunosuppressive molecules (e.g., IL-10, PD-L1, TGF-β) [citations: Eckhert, E. et al. Immunotherapy (2019) 11 (9): 801-11; Tai, YT, et al. Blood (2016) 127 (25): 32225-3236; Tai, YT, and Anderson, DC. Expert Opin. Biol. Ther. (2019) 19 (11): 1143-56]. In vitro studies have shown that BCMA overexpression can even initiate activation of the NF-κβ and MAPK pathways in MM cells themselves, without the need for APRIL or BAFF stimulation [citing Hatzoglou, A. et al. J. Immunol. (2000) 165 (3): 1322-30]. In addition, there is a great deal of crosstalk between APRIL / BCMA signaling and other pathways.For example, APRIL interacts with CD138 / syndecane-1 and heparan sulfate proteoglycan (HSPG) to promote the proliferation and survival of MM cells [Hendriks, J. et al. Cell Death Differ. (2005) 12 (6): 637-48]. Blockade of coexisting FGF-R3 and JAK2 leads to downregulation of BCMA [Cassinelli, G. et al. Biochem. Pharmacol. (2009) 78 (9): 1139-47]. In vitro studies have shown that BCMA immunoprecipitates together with interferon regulator-4 (IRF-4), a master transcription factor that mediates MM cell survival, further highlighting its role in MM tumorigenesis [ibid., citing Shaffer, AL et al. Nature (2008) 454 (7201): 226-31].

[0120] Soluble BCMA (sBCMA) BCMA includes soluble sBCMA obtained from direct shedding of membrane BCMA via γ-secretase activity. sBCMA retains the extracellular domain and a portion of the molecular transmembrane region [citing Laurent, SA et al. Nature Commun. (2015) 6: 7333]. sBCMA is a representative example of a potential biomarker for B cell involvement in human autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis [citing Laurent, SA et al. Nat. Commun. (2015) 6: 7333; Gutierrez, et al. J. Immunol. Res. (2019) 2019: 3658215]. In MM patients, serum levels of sBCMA have been found to be significantly elevated compared to healthy individuals [citing Sanchez, E. et al. Br. J. Haematol. (212) 158 96]: 727-38]. Higher serum levels of sBCMA are independently correlated with a greater disease burden, a worse clinical and radiological response, and a worse prognosis [citing Ghermezi, M. et al. Haematologica (2017) 102 (4): 785-95]. The observation of a remarkable decrease in sBCMA levels in patients with an excellent response to BCMA-targeted immunotherapy suggests that sBCMA may be a novel biomarker for monitoring the response to MM therapy [citing Ali, SA et al. Blood (2016) 128 (13): 1688-700].

[0121] Unlike sBCMA, many studies have shown that levels of cell surface BCMA do not appear to affect the response to BCMA-targeted immunotherapy [citing Dettman, E. J et al., ibid.]. While the varying levels of surface BCMA observed in MM patients are simply a result of membrane BCMA shedding variations, it has been suggested that high levels of sBCMA may interfere with BCMA-targeted immunotherapy by reducing the total amount of cell surface BCMA and inhibiting efficient binding to MM cells by sequestering circulating ligands or anti-BCMA antibodies [citing Sanchez, E. et al. Clin. Cancer Res. (2016) 22 (13): 3383-97; Chen, H. et al. Leuk. Res. (2019) 81: 62-6, ibid.].

[0122] The γ-secretase inhibitor (GSI, LY3039478 / JSMD194) has been reported to reduce sBCMA concentration and simultaneously increase cell surface BCMA expression in MM cell lines and patient tumor cells in mouse models. This inhibitor significantly improved in vitro tumor recognition and in vivo antitumor efficacy of BCMA-specific chimeric antigen receptor (CAR)-T cells. Preclinical studies have also found that short-term GSI administration to MM patients significantly increased the percentage of BCMA-plus tumor cells [citing Pont, MJ et al. Blood (2019) 134 (19): 1585-97]. According to www.clinicaltrials.gov (accessed March 16, 2023), a Phase 1 clinical trial (NCT03502577) to evaluate the safety and efficacy of combining CAR-T therapy with GSI, plus cyclophosphamide (CTX) and fludarabine (FAMP) to treat patients with relapsed or persistent MM is pending further funding.

[0123] BCMA-targeted immunotherapy as a therapeutic strategy for MM During the course of MM, patients may relapse or become resistant to proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib), immunomodulators (e.g., thalidomide, lenalidomide, pomalidomide [see Kleber, M. et al. J. Clinical Med. (2021) 10: 4088, citing Siegel, RL et al. CA Cancer J. Clin. (2020) 70: 7-30] and monoclonal antibodies, resulting in a very poor prognosis and a moderate overall survival (OS) of 5.6 months [see Attal, M. et al. Lancet (201( 394: 2096-2017; Ghandi, UH et al. Leukemia (201() 33: ]). [Citing 2266-75], this occurs particularly in patients with high-risk cytogenetic features or who fail to reach negative minimal residual disease [Ibid., Chim, CS et al. Leukemia (2018) 32: 252-62; Munshi, NC et al. JAMAA Oncol. (2017) 3: 28-35; Kastritis, E. et al. Clin. Lymphoma Myeloma Leuk. (2020) 20: 445-52]. Although a new generation of these drugs is becoming available, managing these patients remains challenging for clinicians [Ibid., Ntassnasis-Stathopoulos, I. et al. Clin. Lymphoma Myeloma Leuk. (2021) 21: 379-85].

[0124] BCMA is a biomarker for the diagnosis of MM, and furthermore, a biomarker for monitoring disease progression and treatment response even in non-secretory MM patients where accurate monitoring of treatment efficacy is not available. [Ibid., citing Demel, I. et al. Br. J. Haematol. (2021) 193: 705-22]. Because BCMA is expressed at selectively high levels in both normal and malignant plasma cells, it is also significant as a target antigen for B-cell neoplasms, including MM therapy. [Yu, et al. J. Hematology & Oncology (2020) 13: 125, citing Lee, L. et al. Br. J. Haematol. (2016) 174 (6): 911-22; Wei, J. et al. J. Hematol. Oncol. (2019) 12 (1): 62]. Early studies of anti-BCMA antibodies have shown robust cytotoxic activity against MM cells in vitro [ibid., citing Ryan, MC et al. Mol. Cancer Ther. (2007) 6 (11): 3009-18].

[0125] Multiple BCMA-targeted treatment methods, including antibody-drug conjugates (ADCs), CAR-T cells, and bispecific T-cell engagers (BiTEs), are currently undergoing active clinical development for multiple myeloma [ibid., Wu, C. et al. J. Hematol. Oncol. (2019): 12 (1): 120; Liu, Q. et al. Mol. Cancer (2019) 18 (1): 154; Liu, D. et al. J. Hematol. Oncol. (2019) 12 (1): 15; Lonial, S. et al. Lancet Oncol. (2020) 21 (2): 207-21; Raje, N. et al. N. Engl. J. Med. (2019) 380 (18): 1726-37; Topp, M. et al. J. Clin. Oncol. (2019) 37 (no. 15_suppl): 8007-8007; Topp, M. et al. Blood (2018) 132 (Suppl. 1): 1010; Trudel, S. et al. Lancet Oncol. (2018) 19 (12): 1641-53; Zhao, WH et al. ,Blood (2018) 132 (Suppl. 1): 955; Zhao, WH et al. J. Hematol. Oncol. (2018) 11 (1): 141; citing Herrera, AF and Molina, A. Clinical Lymphoma, Myeloma and Leukemia (2018) 18 (7): 452-68.e454].

[0126] Antibody-drug conjugates (ADCs) ADCs are among the fastest-growing agents in plasma cell malignancies [Kleber, M. et al. J. Clincal Med. (2021) 10: 4088., citing Braunstein, M. et al. Expert Rev. Hematol. (2021) 14: 377-89; Demel, I. et al. Br. J. Haematol. (2021) 193: 705-22]. Due to the binding of their monoclonal antibodies (mAbs) to specific antigens on tumor cells, they can achieve conservation of normal cells and minimize systemic toxicity. This mode of action is limited by corneal toxicity.

[0127] Anti-BCMA / CD3 bispecific antibody Bispecific antibodies (BsAbs) are molecules that have affinity for two different epitopes, capable of monovalent or bivalent binding to CD3ε on MM cells and T cells [citing Suurs, FV; et al Pharmacol. Ther. (2019) 201: 103-19]. The therapeutic strategy is that BsAbs, which integrate a CD3 T cell receptor-binding domain and a tumor-binding domain, create an immune synapse by binding to tumor cells via T cells, leading to the release of granzymes and perforins and the induction of target cell lysis [citing Offner, S. et al. Mol. Immunol. (2006) 43: 763-71]. Activated T cells release interferon-y and additional cytokines, such as interleukin-6, -10, and tumor necrosis factor-α, which may induce cytokine release syndrome (CRS), causing flu-like side effects such as fever, fatigue, or headache in most patients [citing Demel, I. et al. Br. J. Haematol. (2021) 193: 705-22]. As of 2021, several bispecific T cell engagers (BITEs) targeting BCMA on MM cells and CD3 receptors on T cells were under investigation. This mode is limited due to its short lifespan and requires long IV infusion times via central venous access.

[0128] Anti-BCMA CAR-T cells in MM CAR strategies targeting BCMAs combine the characteristics of mAb target specificity with the cytotoxicity of T cells. A major advantage of CAR-T cells is that, in contrast to human leukocyte antigen (HLA)-restricted T cell receptors, they provide an HLA-type independent therapeutic strategy [citing Mikkilineni, L. and Kochenderfer, JN. Blood (2017) 130: 2594-2602]. Multiple clinical trials have demonstrated the promising therapeutic efficacy of CAR-T cells in patients with relapsed / refractory B-cell neoplasms [citing Maud, SL et al. N. Engl. J. Med. (2014) 371: 1507-17; Lee, DW et al. Lancet (2015) 385: 517-28; Maude, SL et al. N. Eng. J. Med. (2018) 378: 439-48; Kochendorfer, JN et al. J. Clin. Oncol. (2015) 33: 540-49]. The process involves producing CAR-T cells after transfusion of leukocyte-depleted peripheral leukocytes from the patient or a healthy donor to obtain autologous CAR-T cells and CD3+ T cells for allogeneic CAR-T cells. Subsequently, the collected leukocytes express CD3 and CD28 or 4-1-BB via mAb-coated beads after T cell stimulation [citing Nishida, H. Cancers (2021) 13: 2712]. In a subsequent step, these activated T cells can be transduced via a lentiviral vector encoding receptors for tumor-specific antigens on tumor cells, enabling them to express CAR genes [citing Nishida, H. Cancers (2021) 13: 2712].A key characteristic of CAR-T cells is their use of CARs against tumor cell antigens such as BCMA, CD19, CD138 (syndecan-1), orphan G protein-binding receptor class C group 5 member D (GPRC5D), and immunoglobulin kappa light chain [citations: Garfall, AL et al. N. Engl. J. Med. (2015) 373: 1040-7; Atamaniuk, J. et al. Eur. J. Clin. Investig. (2012) 42: 953-60; Ramos, CA et al. J. Clin. Investig. (2016) 126: 2588-96]. The role of BCMA in the pathogenesis of plasma cell malignancies offers novel and intriguing strategies for CAR-T cells [citing Carpenter, RO et al. Clin. Cancer Res. (2013) 19: 2048-60].

[0129] Table 1, quoted from Kleber, M. et al. J. Clinical Med. (2021) 10: 4088, summarizes some of the drugs that were in clinical development for MM at the time. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0130] BCMA as a treatment strategy for AML There is very little data linking BCMA as a therapeutic strategy for AML or myeloid cells. A systematic review of scientific literature reporting on the evaluation of BCMA expression (protein or mRNA) in hematological malignancies in patients of all ages with hematological malignancies, excluding animal studies, studies using non-human and / or non-hematological cell lines, studies investigating non-hematological conditions, and review articles, without mention of AML. Khattar [Khattar, P. et al. Blood (2017) 130 (Suppl. 1): 2755] reported that BCMA expression in normal lymphoid tissue is restrictive to plasma cells and germinal center B cells. BCMA expression was not detected in other tissues (epithelium, T cells, dendritic cells, and histiocytes / macrophages) using clinical-grade assays. BCMA expression was high in B-cell lineage malignancies and plasmacytomas, but was negative for BCMA immunohistochemistry in T-cell lymphomas, Hodgkin lymphomas, and myeloid and lymphoblastic lymphomas / leukemias. [Ibid.].

[0131] BCMA deficiency has been reported to lead to reduced viability of long-lived myeloid plasma cells [Bolkun, L. et al. J. Cancer (2016) 7: 1979-83, citing O'Connor, BP et al. J. Exp. Med. (2004) 199 (1): 91-8]. However, the soluble form of BCMA has been reported to inhibit the proliferative activity of APRIL in vitro and reduce the proliferation of HT29 colon cancer cells in nude mice [ibid., citing Rennert, P. et al. J. Exp. Med. (2000) 192 (11): 1677-84]. In addition, in xenograft models of lung and colon cancer, intratumoral delivery of soluble BCMA significantly reduced tumor growth in mice treated with soluble receptors. Ibid., citing Rennert, P. et al. J. Exp. Med. (2000) 192 (11): 1677-84. Although the anti-apoptotic activity of APRIL and BAFF has been demonstrated in B-lymphoma, MM, and chronic lymphocytic leukemia (B-CLL) [Bolkun, L. et al. J. Cancer (2016) 7: 1979-83, citing Chiu, A. et al. Blood (2007) 109 (2): 729-39; Kern, C. et al. Blood (2004) 103(2): 679-88; Moreaux, J. et al. Blood (2004) 103 (8): 3148-57], only limited studies have been reported on the role of APRIL and BAFF in regulating the apoptotic process in AML.

[0132] To determine whether baseline quantification of peripheral blood CD33+ blasts expressing BAFF, APRIL, and their receptors can improve the prognosis of treatment response, Bolkun et al. conducted a study of 24 patients with newly diagnosed AML ranging in age from 21 to 65 years, with a median age of 55 years, and reported their findings in 2016 [Bolkun, L. et al. J. Cancer (2016) 7: 1979-83]. All patients had a normal karyotype, and none had mutated CEBPA, NPM1, or FLT3-ITD. AML patients were treated with a 7-day induction chemotherapy regimen [ibid., citing Vardiman, JW. Et al. Blood (2009) 114 (5): 937-51]; after induction, morphological responses were assessed: 12 patients achieved complete remission after the first induction, and 12 were non-responders. Significant differences were found in the frequency of CD33+ blasts expressing APRIL and BAFF among groups of patients who showed varying clinical responses to the applied chemotherapy, but no significant differences were found in their mean fluorescence intensity (MFI). Non-responders (NR) showed elevated baseline frequencies of CD33+ blasts expressing APRIL and BAFF compared to patients who achieved complete remission (CR). There were no differences in plasma concentrations of APRIL and BAFF between CR patients and NF patients.

[0133] Regarding APRIL and BAFF receptors, AML patients who did not respond to induction chemotherapy showed very few detectable frequencies of CD33+BCMA+ cells (less than 1%). In CR patients, these cells were found at substantially elevated but still low frequencies. No difference was found in the frequency of AML blasts expressing TACI and BAFF-R. Patients who progressed to CR after induction chemotherapy showed significantly elevated MFI in AML cells with BAFF-R and TACI compared to NR, but not in BCMA. PCR analysis of APRIL expression levels. BAFF, BCMA, BAFF-R, and TACI did not show significant differences in expression levels in CD33+ AML cells (p>0.05). Compared to CR patients, a trend of higher APRIL expression was observed in NR patients at the statistical significance threshold. CR patients showed higher BCMA expression compared to NR patients.

[0134] Numerous studies have shown that abnormal DNA methylation may play a role in the progression of many neoplasms, including AML [citing Toyota, M. et al. Blood (2001) 97 (9): 2823-9], so we confirmed membrane BCMA expression in CD33+ AML blast cells by immunofluorescence staining. A significant negative correlation was observed between the level of BCMA promoter methylation and the frequency of CD33+ BCMA+ cells, suggesting that BCMA methylation may contribute to the therapy resistance of AML cells. No significant relationship was observed between BCMA methylation status and clinical features that may influence gene methylation (e.g., age, smoking). At baseline, BCMA expression on AML cells was detectable only in CR patients and not in NR patients. A significant negative correlation was observed between BAFF expression and BCMA expression in AML cells, rather than APRIL expression, suggesting a possible regulatory function of BAFF expression.

[0135] Next, we analyzed the DNA methylation status of the BCMA gene promoter sequence. No differences in methylation levels were observed regarding the type of clinical response to therapy; however, a significant negative correlation was found between the level of BCMA promoter methylation and the frequency of CD33+BCMA+ cells.

[0136] Finally, to directly evaluate the pro-viability effects of APRIL and BAFF on CD33+ cells, AML cells were treated with cytosine arabinoside (araC) in or without recombinant human APRIL and BAFF. As expected, AraC significantly reduced the frequency of viable CD33+ AML blasts. Stimulation with APRIL significantly reduced AraC-related induction of cell death in NR patients, but not in CR patients. A tendency to increase the frequency of viable cells (at the threshold of statistical significance) was observed after BAFF stimulation in the presence of AraC. These results were interpreted as confirming observations suggesting the possible involvement of BCMA in regulating apoptosis in AML cells and the potential usefulness of BCMA as a potential marker for predicting treatment response to induction chemotherapy. However, the exact function of BCMA and the regulation of its expression in AML blasts remain unclear.

[0137] The experiments by Matthes et al. concerned the development and differentiation of plasma cells in bone marrow progenitor cells from human bone marrow. [Matthes, T. et al. Blood 2011) 118 (7): 1838-44]. Matthes teaches that, after their generation in lymphoid organs, antibody-producing plasma cells (PCs) are highly dependent on environmental factors for survival [citing Sze, DM et al. J. Exp. Med. (2000) 192 (6): 813-21], and it is known that they enter the bloodstream and search for survival niches, such as those present in the bone marrow [citing Slifka, MK et al. J. Virol. (1995) 69 (3): 1895-1902; Manz, RA et al. Nature (1997) 388 (6638): 133-343]. The chemokine CXCL-12 mediates PC BM homing [citing Tokoyoda, K. et al. Immunity (2004) 20 (6): [Citing 707-18] Once PC enters the BM, it is retained in a specific survival niche via a VLA-4 / VCAM-1 adhesion mechanism. [Ibid., citing Tokoyoda, K. et al. Immunity (2004) 20 (6): 707-18]. These niches are thought to provide all the necessary PC survival factors. Since BM aspiration has revealed that PCs constitute less than 0.5% to 1% of the total BM cell density, it is widely recognized that the number of these niches is limited.

[0138] April has been shown to be involved in PC survival rates [citing Mackay, F. et al. Annu. Rev. Immunol (2003) 21: 231-64]. Recombinant APRIL has been reported to improve PC survival rates in vitro [cited from O'Connor, BP et al. J. Exp. Med. (2004) 199 (10: 91-98; Huard, B. et al. J. Clin. Invest. (2008) 118 (8): 2887-95; Bossen, C. et al. Blood (2008) 111 (3): 1004-12; Belnoue, E. et al. Blood (2008) 111 (5): 2755-64]. This function was confirmed in vivo in APRILnull mice [cited from Belnoue, E. et al. Blood (2008) 111 (5): 2755-64; Castigli, E. et al. Proc. Natl Acad. Sci. USA]. (2004) 101 (11): 3903-8; Benson, MJ et al. J. Immunol. (2008) 180 (6): 3655-9 cited). APRIL binds to two receptors, BCMA and TACI [ibid., Kalled, SL et al. Curr Dir. Autoimmun. (2005) 8: 206-42 cited]. In addition, APRIL uses heparin sulfate proteoglycan (HSPG, as a co-receptor) [ibid., Ingold, K. et al. J. Exp. Med. (2005) 201 (9): 1375-83; Hendriks, J. et al. Cell Death Differ. (2005) 12 (6): 637-48 cited].The co-expression of B cell maturation antigens and heparin sulfate proteoglycan (SSPG) CD138 by PC [ibid., O'Connor, BP et al. J. Exp Med. (2004) 199 (1): 91-98; Wijdenes, J. et al. Br. J. Haematol. (1996) 94 (2): 318-23] is consistent with the induction of survival signals by APRIL in these terminally differentiated, non-proliferating cells.

[0139] In situ studies have shown that megakaryocytes [ibid., Winter, O. et al. Blood (2010) 116 (11): 1867-75], eosinophils, and subsets of monocytes [ibid., Chu, VT et al. Nat. Immunol. (2011) 12 (2): 151-9] produce APRIL in mouse BM. In addition, osteoclasts differentiated in vitro also produce APRIL [ibid., Bond, D. et al. Blood (2004) 104 (1): 3169-72; Moreaux, J. et al. Blood (2005) 106 (3): 1021-30; Yacoby, S. et al. Leukemia (2008) 22 (2): 406-13]. All of these cell types constitute only a small fraction of all BM cells. Matthes et al. studied human BM and observed APRIL production mediated by cells from the myeloid lineage, the most abundant BM compartment, in both its immature and mature states, using two antibodies: Stalk-1 antibody to identify the cells that produce APRIL, and April-2 antibody to detect the secreted portion of APRIL. They successfully demonstrated that APRIL secretion by myeloid progenitor cells in BM plasma helps maintain PC survival.

[0140] (1) Bone marrow progenitor cells produce large amounts of APRIL before fully maturing and reaching the circulatory system, but the surrounding tissues do not retain the secreted APRIL. Up to 50% of human BM cells were APRIL-positive, but staining with Aprily-2 was negative. APRIL-producing cells readily secrete all of the APRIL they produce. The BM microenvironment surrounding these cells does not retain the secreted APRIL, which is likely due to the lack of HSPG expression. (2) Granulocyte-mediated APRIL production begins in the promyelocyte stage early in their differentiation; production peaks in the myelocyte stage and then declines with continued maturation. Both immature and mature cells are ready to secrete the APRIL they produce. (3) The absence of close contact between APRIL-producing myeloid cells and PCs in human BM indicates that APRIL-producing cells are not part of the PC survival niche. (4) Experiments to reproduce bone marrow hematopoiesis in vitro by incubating HSCs with early hematopoietic factors SCF, TPO, FLT-3L, which act on non-delegated cells capable of inducing multiple lineage pathways, and G-CSF, which selectively drives differentiation into granulocyte lineages [citing Barreda, DR et al. Dev. Comp. Immunol. (2004) 28 (5): 509-54] showed that cytokines acting in the early stages of hematopoiesis are involved in APRIL production by bone marrow precursor cells.

[0141] (5) Experiments to analyze the role of APRIL in BM PC survival in mice using APRIL blockade with the mouse antagonist mAb Apry-1-1 showed that a single shot treatment with the antagonist one day before cell migration was sufficient to reduce the number of recovered PCs by more than half. In mouse BM, immature myeloid cells are present in a cell population defined by low expression of the bone marrow marker Gr-1 [ibid., citing Ueda, Y. et al. J. Exp. Med. (2005) 201 (11): 1771-80]. This experiment showed that Gr-1 loThe study showed that BM cells, when present, included early stages of myeloid differentiation (myeloblasts, promyelocytes, and myelocytes), characterized by the presence of large cells with small nuclear openings. Eosinophils, which have been reported to produce APRIL in mouse BM cells, were not dominant in this population. Gr-1 hi The cells contain more mature cells (metamyelocytes, zonate nuclei, and neutrophils) characterized by the presence of cells containing nuclei with larger openings progressing toward complete segmentation. Q-PCR in purified cells sorted by FACS revealed immature Gr1 lo Cells mature Gr-1 hi This showed that more APRIL mRNA was expressed than in cells. In co-culture experiments, immature Gr-1 lo Cells maintain PC survival through mature Gr-1 hi It was more efficient than cells; this enhanced PC survival was April-dependent.

[0142] The disclosure of this invention provides BCMA as a novel target for enhanced immunotherapy for eligible patient populations of AML with high-risk disease. Immunotherapies include CAR-T for binding and subsequent receptor-mediated lethality; bispecific T cell engagers whose binding brings T cells closer to target cells; BCMA inhibitors that signal via NFκB suppression even without immune cell engagement; antibody-drug conjugates; and neutralizing antibodies. Data are presented demonstrating that BCMA is present on the cell surface of AML cell lines and in patient samples from young adults aged 26–45 years, and that the efficacy of BCMA in targeting bispecific T cell engagers in AML in vitro. [Overview of the project]

[0143] The disclosure of the present invention provides a method for treating an eligible subject having acute myeloid leukemia (AML) with high-risk disease features, comprising administering a targeted immunotherapy agent, which specifically targets B cell maturation antigen (BCMA), to the intensified subject. According to some embodiments of the method, an eligible subject with AML is a child; or an eligible subject with AML is a child under 2 years of age; or an eligible subject with AML is a teenager between 13 and 19 years of age, including upper and lower limits; or an eligible subject with AML is an adult; or an eligible subject with AML is an adult under 60 years of age; or an eligible subject with AML is a young adult between 26 and 50 years of age, including upper and lower limits; or an eligible subject with AML is an adult between 60 and 69 years of age, including upper and lower limits; or an eligible subject with AML is 70 years of age or older. According to some embodiments of the method, the characteristics of a high-risk disease include biological characteristics, clinical characteristics, or both.

[0144] According to some embodiments, high-risk biological features include pre-existing hematological disorders, the presence of more than 20% BM blasts in the bone marrow, 0, 1, 2, 3 or more clonal cytogenetic abnormalities, and molecular abnormalities. According to some embodiments, the preceding hematological disorder is myelodysplastic syndrome, refractory AML, AML in remission, or mixed phenotypic acute leukemia. According to some embodiments, clinical features include comorbidities, measurable residual disease at complete remission, resistance to induction chemotherapy; remission after induction therapy, relapse during remission, AML due to pre-existing hematological disorders; and AML in elderly patients. According to some embodiments, high-risk molecular features include FLT3 mutations, NPM1 mutations; isocitrate dehydrogenase 1 or 2 (IDH1 / 2) mutations, RUNX1 mutations, DNMT3A mutations, TET2 mutations, TP53 mutations, or combinations thereof. According to some embodiments, the FLT3 mutation is an FLT3-ILD mutation.

[0145] According to some embodiments, high-risk cytogenetic features include chromosomal translocations and monosomy of somatic cell chromosomes. According to some embodiments, the translocation is t(8;21)(q22;q22.1);RUNX1-RUNX1T1). According to some embodiments, the high-risk features in older adults include one or more of the following: comorbidities, a high incidence of cytogenetic abnormalities including monosomy 5 and 7 and chromosome 17 abnormalities, a high incidence of multiple mutations including TP53, and a high incidence of secondary / therapy-related AML. According to some embodiments, comorbidities include one or more of the following: hypertension; diabetes; and organ dysfunction including abnormalities of the heart, lungs, and kidneys.

[0146] According to some embodiments of the method, the immunotherapy agent comprises an immune cell engager ("ICE") selected from T cell engagers, natural killer (NK) cell engagers, and cytotoxic / phagocytic cell engagers; and / or an immune checkpoint therapy agent comprising an immune checkpoint inhibitor; and / or a gamma secretase inhibitor. According to some embodiments, the immune cell engager is a bispecific T cell engager (BiTe) comprising a BCMA-targeting molecule linked to a CD3-targeting molecule via a peptide linker (whose length is in the range of 3 to 20 amino acids, including upper and lower limits, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids), wherein the CD3-targeting molecule activates a specific chain of the CD3 complex associated with the T cell receptor (TCR) complex, mediating BCMA-specific cytotoxicity. The immune cell engager is either a CD16 or NKG2D receptor-targeted bispecific NK cell engager that participates in the assembly of epidemiotic synapses; or the immune engager is a chemically linked bispecific molecule that engages the non-ligand-binding site of a high-affinity receptor for immunoglobulin G (FcγRI, also known as CD64) that is selectively expressed by a population of cytotoxic / phagocytic immune cells that target BCMA antibody-dependent cell-mediated cytotoxicity in AML blasts.

[0147] According to some embodiments, the molecule that targets BCMA is an scFv fragment that targets BMCA, and the molecule that targets CD3 is an scFv fragment. According to some embodiments, the CD64-expressing population of cytotoxic / phagocytic immune cells is a population of monocytes, macrophages, dendritic cells, or cytokine-activated neutrophils. According to some embodiments, the T cell immune engager is a BiTE that targets CD3 on T cells and BCMA expressed on relapsed / refractory AML blasts. According to some embodiments, BiTE is tecristamab, erranatamab, AMG-701, AMG420 (formerly BI836909), REGN5458, or TNB-383B. According to some embodiments, the checkpoint inhibitor is lambrolizumab / pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), atezolizumab, TECENTRIQ®, or ipilimumab (YERVOY®). According to some embodiments, the gamma-secretase inhibitor is LY3039478 / JSMD194, dihydroergocristine (DHEC), RO4929097;LY900009;MK-0752;PF-03084014;BMS-986115;GSI-136;AL-101; or nilogacestat. According to some embodiments, the immunotherapy agent comprises a BCMA-targeted antibody drug conjugate or a BCMA-targeted CAR-T cell therapy agent as described in Table 1. [Brief explanation of the drawing]

[0148] [Figure 1]This diagram schematically illustrates the role of CD4+ T cells in modulating the immune response against cancer. As shown in Figure 1, CD4+ T cells play a central role in regulating the host immune response through the following mechanisms: (a) CD4+ T cells provide crucial assistance in priming CD8+ T cells via the activation of antigen-presenting cells (APCs). (b) CD4+ T cells secrete cytokines necessary to maintain the function and proliferation of CD8+ T cells. (c) CD4+ T cells can directly or indirectly inhibit tumor growth. (d) CD4+ T cells assist in the activation of B cells. Therefore, both CD8+ T cell activity and antibody production require the crucial assistance of CD4+ T cells. (Cited from Wang, RF. Trends in Immunol. (2001) 22 (50): 269-76.) [Figure 2] This diagram schematically depicts a harmonized model of hematopoietic stem cell differentiation. HSCs first differentiate into MPP1 / short-term HSCs (ST-HSCs), and then into MPP2, MPP3, and MPP4 lymphocyte-priming MPPs (LMPPs). MPP2 can generate premegakaryocye / erythrocyte progenitor cells (pre-MegE), which then either produce platelets via megakaryocyte progenitor cells (MkP) or erythrocytes via pre-CFU-E. MPP3 mainly produces granulocyte and monocyte lineages, while MPP4 (LMPP) mainly contributes to lymphocytes. [Cited from Cheng, H. et al. New paradigms on hematopoietic stem cell differentiation. Protein Cell (2020) 11 (1): 34-44] [Figure 3]This figure schematically illustrates the immune evasion strategies employed by AML blasts. AML blasts may interfere with the effector function of T cells and NK cells by abnormally overexpressing inhibitory T cell ligands (i.e., PD-L1, Gal-9, CD155, CD112, CD86) (1) or by releasing soluble forms of NKG2DL. By altering the cytokine environment, AML blasts further promote T cell depletion and apoptosis, driving the expansion and proliferation of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and inducing the switching of macrophages to tumor-associated macrophages (TAMs) (3). This is achieved through the release of other soluble factors such as reactive oxygen species (ROS), indoleamine 2,3-dioxygenase-1 (IDO1), arginase II (ArgII), and extracellular vesicles (EVs) within the BM niche (4). Furthermore, AML blasts reduce the expression of their antigen-presenting molecules, thereby concealing themselves from immune cells such as dendritic cells (DCs) and macrophages (5). (Cited from Tettamanti, S. et al. Leukemia (2022) 36 (1): 13-22.) [Figure 4]This diagram schematically illustrates the signaling pathway of B cell maturation antigen (BCMA) in multiple myeloma. BCMA has two agonist ligands: proliferation-inducing ligand (APRIL) and B cell activator (BAFF), which are primarily secreted in the bone marrow (BM) by stromal cells, osteoclasts, and macrophages in a paracrine manner. APRIL exhibits a higher binding affinity to BCMA than BAFF, and while APRIL also binds to TACI, BAFF ensures greater selectivity for BAFF-R. Subsequently, in multiple myeloma (MM) cells, multiple proliferation and survival signaling cascades are activated most frequently via NF-κβ, leading to upregulation of anti-apoptotic proteins and the production of cell adhesion molecules, angiogenic factors, and immunosuppressive molecules. These contribute to increased MM cell viability. Membrane BCMA can be cleaved by γ-secretase and released into the plasma as soluble BCMA (sBCMA). sBCMA can bind to APRIL and BAFF, which may interfere with the activation of the BCMA signaling pathway. (Cited from Yu et al. BCMA-targeted immunotherapy for multiple myeloma. J. Hematol. & Oncology. (2020) 13: article 126. Doi.org / 10.1186 / s13045-020-00962-7) [Figure 5] This diagram illustrates the pathway to antigen presentation [quoted from Yewdell, JW and Dolan, BP. Nature (2011) 471 (7340): 581-82]. [Figure 6]This diagram schematically illustrates the mechanism of action of immune cell engagers. Immune cell engagers can redirect immune effector cells to create artificial immune synapses between tumor cells (targeting tumor-associated antigens) and T cells (engaging CD3 or costimulatory molecules, e.g., CD28 or 4-1BB), NK cells (engaging CD16 or NKG2D), and cytotoxic / phagocytic cells (engaging CD64). [Cited from Fuca, G. et al. ESMO (2021) 6 (1): doi.org / 10.1016 / j.esmoop.2020.10046]. [Figure 7A] This figure provides mean fluorescence intensity data showing that BCMA is highly expressed in AML cell lines and patient cells. Figure 7A. Three human MM cell lines (MM1S, U266, NCI-H929) were analyzed for the expression of the MM-related antigen BCMA, as well as the AML-related antigens CD33, Flt3, and CD123. Mean median fluorescence intensity (MFI) and SEM are presented. [Figure 7B] This figure, Figure 7B, provides mean fluorescence intensity data showing that BCMA is highly expressed in AML cell lines and patient cells. Nine human AML cell lines (THP-1, OCI-AML-3, MOLM-13, K052, KASUMI-1, SET-2, NKM-1, HL-60, HEL) were analyzed for the expression of the MM-related antigen BCMA, as well as the AML-related antigens CD33, Flt3, and CD123. Mean MFI and SEM are presented. [Figure 7C] This figure provides mean fluorescence intensity data showing that BCMA is highly expressed in AML cell lines and patient cells. Figure 7C. Healthy human peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were analyzed for BCMA, Flt3, and CD123 expression. Mean MFI and SEM are presented. [Figure 7D] This figure provides mean fluorescence intensity data showing that BCMA is highly expressed in AML cell lines and patient cells. Figure 7D. AML patient cells were analyzed for the expression of BCMA, Flt3, and CD123. [Figure 7E]This figure, Figure 7E, provides mean fluorescence intensity data demonstrating the high expression of BCMA in AML cell lines and patient cells. BCMA expression was analyzed in healthy hematopoietic cells (CD34+HSPC, PBMC, BMMC, white column), as well as AML cell lines (gray column) and AML patient cells (black column). Mean MFI and SEM are presented. [Figure 7F] This figure provides mean fluorescence intensity data demonstrating the high expression of BCMA in AML cell lines and patient cells. Figure 7F. Summary of BCMA positivity rates (frequency of BCMA-positive cells) in AML cells from 33 patients. ND: newly diagnosed; R: relapsed. [Figure 8A] Figure 8A shows that U266 cells were more susceptible to BCMA BiTE-mediated lethality than AML cells, but BCMA BiTE treatment mediates lethality of AML cells in vitro. Figure 8A is a plot of percentage lethality over time for U266-luc human multiple myeloma cells (left) and MOLM-13-luc human AML cells (right) cultured in the presence of human peripheral blood mononuclear cells (PBMCs) at an effector:target ratio of 20:1. BCMA×CD3 BiTE (10 or 50 nM) was added to the culture medium, and relative luminescence units (RLU) were measured after 24 and 48 hours. Controls included: untreated target cells, target cells + PBMCs, and target cells + BiTE. Normalized values ​​(percentage of untreated target cells) are presented (mean and SEM). [Figure 8B]Figure 8B shows that U266 cells were more susceptible to BCMA BiTE-mediated lethality than AML cells, but BCMA BiTE treatment mediates lethality of AML cells in vitro. Figure 8B is a plot of percentage lethality over time for U266-luc human multiple myeloma cells (left) and OCI-AML3-luc human AML cells (right) cultured in the presence of human PBMCs at effector:target ratios of 10:1 and 20:1. BCMA×CD3 BiTE (0.4 nM) was added to the culture medium, and RLU was measured after 24, 48, and 72 hours. Controls included: untreated target cells, target cells + PBMC, and target cells + BiTE. Normalized values ​​(percentage of untreated target cells) are presented (mean and SEM). [Figure 8C] Figure 8C shows that U266 cells were more susceptible to BCMA BiTE-mediated lethality than AML cells, but BCMA BiTE treatment mediates lethality of AML cells in vitro. Figure 8C is a plot of percentage lethality for U266-luc cells (left), MOLM-13-luc cells (center), and OCI-AML3 cells (right) cultured in the presence of human PBMCs at effector:target ratios of 10:1 (U266), 20:1 (MOLM-13), and 30:1 (OCI-AML3). BCMA×CD3 BiTE was added to the culture medium at the specified concentrations, and RLU was measured after 24, 48, and 72 hours. Controls included untreated target cells and target cells + PBMCs. Normalized values ​​(percentage of untreated target cells) are presented (mean and SEM). [Figure 9] This figure shows that the BCMA gene (TNFRSF17) is expressed by all AML subsets. Normalized median expression (transcripts per million BCMA cells by AML cells) is shown based on the France-US-UK (FAB) classification. The figure was created using the TCGA bulk RNA sequencing gene expression dataset on the University of Alabama, Birmingham Cancer Data Analysis Portal (UALCAN) platform. [Figure 10]This study provides a Kaplan-Meier analysis of AML survival probabilities comparing patients with high (top 10%) and low (bottom 10%) normalized BCMA gene expression levels (transcripts per million, TPM). Differences between groups were analyzed using log-rank tests. The figures were generated using survival data from the Cancer Genome Atlast program (TCGA) on the OncoInc platform. The figures show that high BCMA expression levels are associated with poor prognosis in AML patients. [Figure 11A] This figure shows that BCMA×CD3 T cell engager (TCE) treatment mediates AML cell lethality in vitro. Figure 11A: OCI-AML3-luc human AML cells were cultured in the presence of human PBMCs in an effector:target ratio of 20:1. BCMA×CD3 TCE (0.4 nM) was added to the culture medium, and relative luminescence units (RLU) were measured after 24, 48, and 72 hours. Controls included: untreated target cells, target cells + PBMC, and target cells + TCE. Normalized values ​​(percentage of lethality based on untreated target cells) are presented (mean and SEM). [Figure 11B] This figure shows that BCMA×CD3 T cell engager (TCE) treatment mediates AML cell lethality in vitro. Figure 11B: THP-1-luc human AML cells were cultured in the presence of human PBMCs in an effector:target ratio of 10:1. BCMA×CD3 TCE (10 nM) was added to the culture medium, and RLU was measured after 24, 48, and 72 hours. Controls included the following conditions: untreated target cells, target cells + PBMC, and target cells + TCE. Normalized values ​​(percentage of lethality based on untreated target cells) are presented (mean and SEM). [Figure 11C]This figure shows that BCMA×CD3 T cell engager (TCE) treatment mediates AML cell lethality in vitro. Figure 11C: MOLM-13-luc human AML cells were cultured in the presence of human PBMCs in an effector:target ratio of 20:1. BCMA×CD3 TCE (10 or 50 nM) was added to the culture medium, and relative luminescence units (RLU) were measured after 24 and 48 hours. Controls included: untreated target cells, target cells + PBMC, and target cells + TCE. Normalized values ​​(percentage of lethality based on untreated target cells) are presented (mean and SEM). [Figure 12A] This figure shows that BCMA×CD3 TCE treatment exhibits strong anti-leukemic activity in an AML xenograft model. Figure 12A. NSG mice were intravenously administered THP-1 cells expressing 2.5×10⁶ luciferase cells. Engraftment was confirmed after 10 days, and the mice were assigned to the following groups: 1. Placebo (PBS) IV on day 0; 2. Human CD8 T cells (1×10⁶) IV on day 0; 3. Human CD8 T cells (1×10⁶) IV on day 0, followed by BCMA×CD3 bispecific T cell engager (TCE) 20 mg / kg IV on days 1, 4, 8, 11, 15, 18, and 22; 4. Human CD8 T cells (1×10⁶) IV on day 0, followed by TCE 80 mg / kg IV on days 1, 4, 8, 11, 15, 18, and 22. A: In vivo imaging of bioluminescence from day 4 to day 29 after treatment. Mean and SEM images are presented (n=3-4). [Figure 12B] This figure shows that BCMA × CD3 TCE treatment exhibits strong anti-leukemic activity in an AML xenograft model. Figure 12B: Kaplan-Meier analysis of survival. [Modes for carrying out the invention]

[0149] Glossary The term "4-1BB (CD137, tumor necrosis factor receptor superfamily 9)" is an inducible costimulatory receptor expressed on activated T and natural killer (NK) cells. Ligation of 4-1BB on T cells initiates a signaling cascade that leads to upregulation of anti-apoptotic molecules, cytokine secretion, and enhanced effector function. In NK cells, 4-1BB signaling can increase antibody-dependent cell-mediated cytotoxicity.

[0150] The terms “activate,” “stimulate,” “enhance,” “increase,” and / or “induce” (and similar terms) are generally used synonymously to refer to any action that directly or indirectly improves or increases the concentration, level, function, activity, or behavior compared to natural, expected, or mean conditions, or compared to control conditions. “Activate” refers to a primary response induced by the ligation of a cell surface region. For example, in the context of receptors, such stimulation is accompanied by receptor ligation and subsequent signaling events. Furthermore, the stimulating event can activate the cell and upregulate or downregulate the expression or secretion of molecules. Thus, ligation of a cell surface region can result in cytoskeletal reorganization or fusion of cell surface regions, even in the absence of direct signaling events, each of which may help enhance, modify, or alter subsequent cellular responses.

[0151] The term “administer” and its various grammatical forms mean, as used herein, but not limited to, that an exogenous ligand, reagent, placebo, small molecule, pharmaceutical substance, therapeutic agent, diagnostic agent, or composition come into contact with a subject, cell, tissue, organ, or biological fluid, when applied to a mammal, cell, tissue, organ, or biological fluid. This includes direct administration to tissue ex vivo, in addition to in vivo administration. Generally, a composition may be administered systemically by oral, buccal, parenteral, by inhalation or inhalation (i.e., through the mouth or nose), or rectally in the form of a dosage unit formulation containing a conventional non-toxic, pharmaceutically acceptable carrier, adjuvant, and vehicle as required, or topically by means such as injection, implantation, grafting, topical application, or parenteral administration. “Administration” may also refer, for example, to therapeutic, pharmacokinetic, diagnostic, investigational, placebo, and experimental methods. "Administration" also includes in vitro and ex vivo treatments, such as cell treatments, by reagents, diagnostic agents, conjugate compositions, or other cells. "Administration" should be understood to include administration via co-compounds (meaning those formulated together), as well as administration via one or more pharmaceutical compositions administered simultaneously (meaning at the same time, e.g., co-administration) or sequentially (meaning those administered later in time or order). In the case of cell therapy, administration may be intravenous (iv), for example, by infusion.

[0152] As used herein, the term "adapter protein" refers to a protein containing a set of protein-binding sites that link their respective interaction partners together, facilitating the formation of larger signaling complexes. The term "adoptive immunotherapy," also known as "cellular immunotherapy," is a type of immunotherapy in which T cells are given to a patient to help the body fight disease. Types of adoptive cell therapy include chimeric antigen receptor T cell (CAR T cell) therapy and tumor-infiltrating lymphocyte (TIL) therapy. The term “adverse event” or “AE,” as used herein, refers to an undesirable medical event that occurs in a subject to which a medicinal product has been administered, but which is not necessarily causally related to the treatment. This could be any adverse and unwanted sign (including abnormal experimental results), symptom, or transient illness associated with the use of the product, whether or not it is related to the product. The correlation between adverse events and the investigational drug therapy is as follows: positive, potentially related, possibly related, possibly unrelated, and not related at all. Table 2 below shows the severity of adverse events according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) scale.

[0153] [Table 2]

[0154] As used herein, the term "anergy" refers to a state of lymphocytes that are unresponsive to a specific antigen, induced by the encounter of lymphocytes with a congener antigen under suboptimal conditions, for example, in the absence of co-stimulation. As used herein, the term "antibody" includes, for example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal antibodies and monoclonal antibodies, as well as fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies and entirely synthetic antibodies, as well as fragments thereof. The term “antibody,” as used herein, is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, antibody fragments, chimeric antibodies, and synthetic antibodies as a whole, as long as they exhibit the desired antigen-binding activity. Originally, antibodies are serum proteins, and the molecules of serum proteins have small regions on their surface that are complementary to a small group of chemical substances on their target. These complementary regions (referred to as antibody combining sites or antigen binding sites) are present in at least two for the entire antibody molecule, and in some types of antibody molecules, as many as 10, 8, or even 12 in some species. Such regions react with their corresponding complementary regions (antigenic determinants or epitopes) on the antigen, allowing them to link together various molecules of a multivalent antigen to form a lattice. The basic structural unit of an antibody molecule consists of four polypeptide chains: two identical light (L) chains (each containing approximately 220 amino acids) and two identical heavy (H) chains (each typically containing approximately 440 amino acids). The two heavy chains and two light chains are held together by a combination of non-covalent and covalent (disulfide) bonds. Such a molecule consists of two identical halves, each of which has an identical antigen-binding site composed of the N-terminal region of the light chain and the N-terminal region of the heavy chain. Typically, both the light and heavy chains cooperate to form the antigen-binding surface.

[0155] The basic structural unit of an antibody molecule consists of four polypeptide chains: two identical light (L) chains (each containing approximately 220 amino acids) and two identical heavy (H) chains (each typically containing approximately 440 amino acids). The two heavy chains and two light chains are held together by a combination of non-covalent and covalent (disulfide) bonds. Such a molecule is composed of two identical halves, each of which has an identical antigen-binding site consisting of the N-terminal region of the light chain and the N-terminal region of the heavy chain. Typically, both the light and heavy chains cooperate to form the antigen-binding surface. Human antibodies exhibit two types of light chains, κ and λ, and individual immunoglobulin molecules generally possess only one or the other. In mammals, there are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each with its own heavy chain class. All five immunoglobulin classes differ from other serum proteins in that they exhibit a wide range of electrophoretic mobility and are not homogeneous. This heterogeneity, i.e., that individual IgG molecules differ from one another, for example, in net charge, is an inherent characteristic of immunoglobulins.

[0156] The principle of complementarity is often likened to putting a key into a lock, involving relatively weak binding forces (hydrophobic and hydrogen bonds, van der Waals forces, and ionic interactions) that can only act effectively when two reaction molecules are in extremely close proximity to each other, and when they are indeed that close, a protruding constituent atom or group of an atom of one molecule can complementarily fit into a recess or depression of the other. Antigen-antibody interactions exhibit a high degree of specificity, which manifests at many levels. At the molecular level, specificity means that the binding site of an antibody to an antigen has complementarity with the antigenic determinants of an unrelated antigen, not being entirely similar. Whenever the antigenic determinants of two different antigens have some structural similarity, some degree of fit of one determinant to the binding site of some antibody to the determinant of the other can occur, and this phenomenon causes cross-reactivity. Cross-reactivity is particularly important in understanding the complementarity or specificity of antigen-antibody reactions. Immunological specificity or complementarity allows for the detection of small amounts of impurities / contamination between antigens.

[0157] Monoclonal antibodies (mAbs) can be produced by fusing mouse spleen cells from an immunized donor with a mouse myeloma cell line and growing them in a selective medium to obtain an established mouse hybridoma clone. Hybridoma cells are immortalized hybrid cells resulting from the in vitro fusion of antibody-secreting B cells with myeloma cells. In vitro immunization refers to the primary activation of antigen-specific B cells in culture and is another well-established method for producing mouse monoclonal antibodies. Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ) chain variable genes from peripheral blood lymphocytes can also be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains (single-chain Fv or scFv) in which heavy and light chain variable domains are linked by polypeptide spacers can be constructed by randomly combining heavy and light chain V genes using PCR. The combinatorial library can then be cloned for presentation on the surface of filamentous bacteriophages by fusion to minor coat proteins at the phage tip.

[0158] The guided selection technique is based on shuffling the human immunoglobulin V gene with the rodent immunoglobulin V gene. This method involves (i) human V L The chain repertoire is the heavy chain variable region (V) of a mouse monoclonal antibody that has reactivity with the target antigen. H (ii) shuffling the domain; (ii) selecting a semi-human Fab on the antigen; (iii) selecting the V L (v) isolating a cloned Fab fragment containing a human light chain gene by using the gene as a "docking domain" for a human heavy chain library in a second shuffle; (v) transfecting mouse myeloma cells by electroporation with a mammalian cell expression vector containing the gene; and (vi) expressing the antigen-reactive Fab V gene as a complete IgG1 antibody molecule in mouse myeloma.

[0159] Antibodies may be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, anti-idiotype antibodies, and antibodies that can be labeled in soluble or conjugated forms, in addition to fragments, variants, or derivatives thereof, either alone or in combination with other amino acid sequences provided by known technologies. Antibodies may originate from any species. The term antibody also includes the antibody-binding fragments of the present invention. Antibody-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Exemplary fragments include Fv, Fab, Fab', single-chain antibodies (svFC), dimeric variable regions (diabodies), and disulfide-stabilized variable regions (dsFv). Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with publicly available or proprietary sequence databases. For example, computer-based comparison methods can be used to identify sequence motifs or predicted protein conformation domains present in other proteins with known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. For example, see Bowie et al. Science 253:164 (1991), which is incorporated in its entirety by reference. Antibodies other than “bispecific” or “bifunctional” antibodies are understood to have identical binding sites.

[0160] Design patterns for bispecific antibodies (BsAbs). Generally, bispecific antibodies can be divided into two main classes: those with an Fc region and those without. These are usually smaller than IgG and IgG-like bispecific molecules that contain an Fc region. The presence of an Fc region is important for Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement binding (CDC), and their larger size and longer half-lives resulting from Fc-Rn mediated reuse processes. [Kontermann, RE and Brinkmann (U., Drug Discovery Today (2015) 20 (7): doi.org / 10.1016 / j.drudis..2015.02.008.]

[0161] BsAbs lacking the Fc region rely entirely on their antigen-binding ability to exert their therapeutic activity. They either contain variable VH and VL domains or two antibodies, or are based on a Fab fragment. For example, one form used in BiTE technology is a genetic fusion of two scFV fragments, which results in a tandem scFv molecule in which the two scFv portions form independent folding units flexibly linked via a peptide linker. [Ibid., citing Hayden, MS et al. Ther. Immunol. (1994) 1: 3-15]. An alternative approach is based on a diabody form, in which variable domains from two antibodies A and B are expressed as two polypeptide chains VHA-VLB and VHB-VLA, and the domains are linked by a short peptide linker to result in heterodimerization of the two chains. {Ibid., citing Holliiger, P. et al. Proc. Natl Acad. Sci. USA (1994) 90: 6444-8}. This bivalent diabody configuration can be further improved by conversion to a single-chain version (scDb) [ibid., citing Brusselbach, S. et al. Tumor Target (1999) 4: 115-23] and its tetravalent dimeric derivative, the so-called "tandAb" molecule having two binding sites for each antigen [ibid., citing Kipriyanov, SM et al. J. Mol. Biol. (1999) 293: 41-56], as well as disulfide-stabilized variants, such as dual-affinity retargeting molecules [dual-affinity retargeting molecules: DART] [ibid., citing Johnson, S. et al. J. Mol. Biol. (2010) 399: 436-49]. BsAb can also be generated by fusing different antigen-binding sites (e.g., scFv or Fab) to other protein domains, thereby allowing for further functionalization.For example, two scFv fragments have been fused to albumin, thereby conferring a longer circulation time of serum albumin to the antibody fragment [citing Muller, D. et al. J. Biol. Chem. (2007) 282: 12650-60; McDonagh, CF et al. Mol. Cancer Ther. (2012) 11: 582-593]. Another example is the "dock-and-lock" approach based on heterodimerization of cAMP-dependent protein kinase A with A kinase-anchor proteins [citing Rossi, EA et al. Proc. Natl Acad. Sci. USA (2006) 103: 6841-46]. These domains can be linked to the Fab fragment and the entire antibody to form a polyvalent bsAb [Ibid., citing Rossi, EA et al. Bioconjug. Chem. (2012) 23: 309-23].

[0162] The term “antibody construct,” as used herein, refers to a polypeptide comprising one or more antigen-binding sites of the present invention linked to a linker polypeptide or immunoglobulin constant domain. A linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen-binding sites. Such linker polypeptides are well known in the art (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy chain and light chain constant domains are well known in the art. Antibody portions, e.g., Fab and F(ab')2 fragments, can be prepared from the whole antibody using conventional techniques such as papain or pepsin digestion of the whole antibody, respectively. Furthermore, antibodies, antibody moieties, and immunoadhesion molecules can be obtained using standard recombinant DNA technology.

[0163] The term "antibody-dependent cell-mediated cytotoxicity" (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is an immune mechanism in which effector cells possessing Fc receptors can recognize and kill target cells coated with antibodies expressing tumor or pathogen-derived antigens on their surface. This is mediated by the recruitment of cytotoxic effector cells, such as natural killer (NK) cells, macrophages, and polymorphonuclear leukocytes (PMNs), which express Fc gamma receptors (FcγRs) on their surface. The term "antibody-dependent phagocytosis" or ADCP refers to a powerful mechanism for eliminating antibody-coated foreign particles, such as microorganisms or tumor cells. Engagement of FcγRIIa and FcγRI expressed on macrophages initiates a signaling cascade, leading to the phagocytosis of IgG-opsonized particles.

[0164] The term “antibody-drug conjugate” or “ADC,” as used herein, refers to an antibody (e.g., monoclonal antibody, mAb) conjugated to a cytotoxic substance designed to induce target cell death in order to reduce systemic exposure and thus reduce the toxicity of the cytotoxic substance. The linker should be stable in circulation but release the cytotoxic substance when delivered to target cells. The specific antigenic target of the antibody component should be highly expressed in tumors but not expressed or low in healthy cells; it should be presented on the surface of tumor cells so that it is available to circulating antibodies; the ADS should have internalization properties to facilitate transport to cells, which in turn enhances the efficacy of the cytotoxic substance; or exerts a “bystander effect.”

[0165] The term “antigen,” as used herein, means a molecule containing one or more epitopes (linear, conformational, or both) that are expected to stimulate the host’s immune system to produce a humoral and / or cellular antigen-specific response. This term is used synonymously with the terms “immunogen” or “epitope.” Typically, B cell epitopes are expected to contain at least about five amino acids, but may contain as few as three to four. T cell epitopes, e.g., CTL epitopes, are expected to contain at least about seven to nine amino acids, and helper T cell epitopes are expected to contain at least about twelve to twenty amino acids. Typically, epitopes are expected to contain about seven to fifteen amino acids, e.g., 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. This term includes polypeptides that undergo modifications, such as deletions, additions, and substitutions (generally, essentially conserved) compared to their native sequence, as long as the protein maintains its ability to elicit an immune response as defined herein. These modifications may be planned, for example, through site-directed mutagenesis, or accidental, for example, through mutations in a host that produces an antigen.

[0166] As used herein, the term “antigen presentation” generally refers to the presentation of an antigen on the surface of a cell, for example, in the form of a peptide fragment bound to an MHC molecule. Figure 5 schematically illustrates the pathway to antigen presentation [quoted from Yewdell, JW and Dolan, BP. Nature (2011) 471 (7340): 581-82].

[0167] As used herein, the term “antigen-presenting cell (APC)” refers to a class of cells capable of displaying ("presenting") one or more antigens on their surface in the form of peptide-MHC complexes recognizable by specific effector cells of the immune system, thereby inducing an effective cellular immune response against the antigen or the antigen being presented. Examples of professional APCs include dendritic cells and macrophages, but any cell expressing MHC class I or II molecules may be capable of presenting peptide antigens. An APC may also be an “artificial APC,” which means a cell that has been engineered to present one or more antigens. Before T cells can recognize a foreign protein, the protein must be processed within the antigen-presenting cell or target cell so that it can be presented as a peptide-MHC complex on the cell surface. As used herein, the term "antigen processing" refers to the intracellular degradation of an exogenous protein into peptides that can bind to MHC molecules for presentation to T cells.

[0168] The term "apoptosis" or "programmed cell death" refers to a highly regulated and active process that contributes to biological homeostasis, consisting of a series of biochemical events that result in various morphological changes without damaging the organism, such as foam formation, changes in cell membranes (e.g., membrane asymmetry and loss of adhesion), cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Apoptotic cell death is induced by many different factors and involves a great many signaling pathways, some of which are caspase proteases (a class of cysteine ​​proteases) dependent, while others are caspase-independent. Apoptotic cell death can be initiated by many different cellular stimuli, such as cell surface receptors, mitochondrial responses to stress, and cytotoxic T cells, resulting in the activation of apoptotic signaling pathways. Caspases involved in apoptosis carry apoptotic signals in the proteolytic cascade. These caspases cleave and activate other caspases, which then degrade other cellular targets, leading to cell death. Caspases at the top of the cascade include caspase-8 and caspase-9. Caspase-8 is an early caspase involved in the response to receptors with a death domain (DD), such as Fas.

[0169] Receptors in the TNF receptor family are associated with inflammatory signaling in addition to inducing apoptosis. The Fas receptor (CD95) mediates apoptotic signaling via Fas ligand expressed on the surface of other cells. Fas-FasL interactions play a crucial role in the immune system, and a lack of this system leads to autoimmunity, indicating that Fas-mediated apoptosis eliminates autoreactive lymphocytes. Fas signaling is also involved in immune surveillance to eliminate transformed and virus-infected cells. Binding of Fas to oligomerized FasL on another cell activates apoptotic signaling via an intracellular domain called the death domain (DD), which interacts with signaling adapters such as FAF, FADD, and DAX to activate the caspase proteolysis cascade. First, caspases-8 and caspase-10 are activated, then downstream caspases cleave various cellular substrates, and are activated, leading to cell death.

[0170] Mitochondria participate in the apoptotic signaling pathway through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a major protein in electron transport, is released from mitochondria in response to apoptotic signals and activates Apaf-1, a protease released from mitochondria. Activated Apaf-1 activates caspase-9 and the rest of the caspase pathway. Smac / DIABLO inhibits IAP proteins, which are released from mitochondria and normally interact with caspase-9 to inhibit apoptosis. Apoptosis regulation by Bcl-2 family proteins occurs because family members enter the mitochondrial membrane and form complexes that regulate the release of cytochrome c and other proteins. While TNF family receptors that trigger apoptosis directly activate the caspase cascade, they can also activate Bid, a Bcl-2 family member that activates mitochondrial-mediated apoptosis. Another Bcl-2 family member, Bax, is activated by this pathway, localizes to the mitochondrial membrane, increases its permeability, and releases cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation and block apoptosis. Like cytochrome c, AIF (inducing apoptosis) is a protein found in mitochondria and released from them in response to apoptosis. While cytochrome c is linked to caspase-dependent apoptosis signaling, AIF release stimulates caspase-independent apoptosis, which then travels to the nucleus where AIF binds to DNA. DNA binding by AIF stimulates chromatin condensation and DNA fragmentation, likely via nuclease recruitment.

[0171] The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, leading to self-cleavage and activation of caspase-9. Caspases-3, -6, and -7 are downstream caspases activated by upstream proteases, and they themselves act to cleave cellular targets. Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, forming transmembrane pores and initiating apoptosis, possibly via caspase cleavage. However, a caspase-independent mechanism of granzyme B-mediated apoptosis has been suggested.

[0172] Fragmentation of the nuclear genome by multiple nucleases activated by apoptotic signaling pathways to create a nucleosome ladder is a characteristic cellular response of apoptosis. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase-activated DNA-degrading enzyme (CAD). DFF / CAD is activated during apoptosis via caspase protease cleavage of its associated inhibitor, ICAD. DFF / CAD interacts with chromatin components such as topoisomerase II and histone H1 to condense chromatin structures and possibly recruit CAD to chromatin. Another apoptosis-activated protease is endonuclease G (EndoG). EndoG is encoded in the nuclear genome but is localized to mitochondria in normal cells. EndoG may also play a role in apoptosis in addition to mitochondrial genome replication. Apoptotic signaling triggers the release of EndoG from mitochondria. The EndoG and DFF / CAD pathways are independent, as the EndoG pathway also occurs in cells lacking DFF.

[0173] Hypoxia, and subsequent hypoxia after reoxygenation, may initiate cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3), a serine-threonine kinase universally expressed in most cell types, appears to mediate or enhance apoptosis in response to numerous stimuli that activate mitochondrial cell death pathways. (Loberg, RD, et al., J. Biol. Chem. 277 (44): 41667-673 (2002)). It has been demonstrated that it induces caspase-3 activation and activates the pro-apoptotic tumor suppressor gene p53. GSK-3 has also been suggested to promote the activation and translocation of the pro-apoptotic Bcl-2 family member, Bax, and, upon aggregation and localization to mitochondria, induce cytochrome c release. Akt is an important regulator of GSK-3, and phosphorylation and inactivation of GSK-3 may mediate some of Akt's anti-apoptotic effects.

[0174] Proliferation-inducing ligand (APRIL) is a TNF-like ligand / cytokine synthesized as a type II transmembrane protein and cleaved by proteolysis at a polybasic motif. [Stein, JV et al. April modulates B and T cell immunity. J. Clin. Invest. (2002) 109 (12): 1587-98, citing Schneider, P. et al. J. Exp. Med. (1999) 189: 1747-56; Lopez-Fraga,, M. et al. EMBO Rep. (2001) 2: 945-51; Nardelli, B. et al. Blood (2001) 97: 198-204]. Because it is processed intracellularly by furincombatase before its secretion, it acts simply as a secreted factor. [Ibid., Schneider, P. et al. J. Exp. Med. (1999) 189: 1747-56; Lopez-Fraga,, M. et al. EMBO Rep. (2001) 2: 945-51; Nardelli, B. et al. Blood (2001) 97: 198-204] This can bind to the receptors TACI and BCMA [Ibid., Gross, JA et al. Nature (2000) 404: 995-99; Shu, HB et al. J. Leukoc. Biol. (1999) 65: 680-83; Thompson, JS et al. J. Exp. Med. (2000) 192: 129-35; Yan, M. et al. Nat. Immunol. (2000) [1: 37-41; citing Xia, XZ et al. J. Exp. Med. (2000) 192: 137-43].Despite the absence of a membrane-bound form of APRIL on the cell surface, the fusion protein known as TWE-PRIL, formed from the trans-splicing of apoptosis's weak TNF-related inducer (TWEAK, also known as TNFSF12) and APRIL, is membrane-bound and presents an APRIL receptor-binding domain on the cell surface [Gardam, S. and Brink, R. Frontiers Immunol. (2014) 4: article 509, citing Pradet-Balade, B. et al. EMBO J. (2002) 21: 2887-95]. TWE-PRIL is biologically active, but its physiological role has not yet been identified. [Ibid.] APRIL is expressed in hematopoietic cells and acts as an in vitro T cell stimulator, influencing the deletion phase of the CD4+ T cell response in vivo by signaling T cell survival. This is also involved in the B cell response.

[0175] The term "ARF" (Alternative Leading Frame), as used herein, refers to tumor suppressor proteins that accumulate in the nucleolus in response to abnormal tumorigenic / hyperproliferative signals and induce cell cycle arrest and apoptosis during G1 / S or G2 / M transitions. [Seite, P. (2011) in Schwab, M. (Eds). Encyclopedia of Cancer. Springer, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 978-3-642-16483-5_384]

[0176] As used herein, the term "B cell receptor" or "BCR" refers to an antigen-receptor complex of B lineage cells, which consists of a membrane-bound Ig(mIg) monomer and an Igα / Igβ complex required for intracellular signal transduction.

[0177] TNF family B cell activators (BAFFs) (also known as BlyS, THANK, TALL-1, and zTNF4) are TNF-like ligands / cytokines synthesized as type II transmembrane proteins that are cleaved by proteolysis at polybasic motifs. [Stein, JV et al. April modulates B and T cell immunity. J. Clin. Invest. (2002) 109 (12): 1587-98, citing Schneider, P. et al. J. Exp. Med. (1999) 189: 1747-56; Lopez-Fraga, M. et al. EMBO Rep. (2001) 2: 945-51; Nardelli, B. et al. Blood (2001) 97: 198-204]. This is released from the cell surface by the processing of membrane-bound BAFF [citations: Schneider, P. et al. J. Exp. Med. (1999) 189: 1747-56; Lopez-Fraga, M. et al. EMBO Rep. (2001) 2: 945-51; Nardelli, B. et al. Blood (2001) 97: 198-204]. It can bind to both TACI and BCMA, which are the third receptors (BAFF-R or BR-3) specific to BlysS [citations: Thompson, JS et al. Science (2001) 293: 2108-11; Yan, M. et al. Curr. Biol. (2001) 11: 1547-52]. BAFF is expressed in hematopoietic cells.Treatment of mice with soluble decoy forms of TACI or BCMA (TACI-Fc or BCMA-Fc) resulted in a reduction in B cell count and blockade of humoral responses [citations: Shu, HB et al. J. Leukoc. Biol. (1999) 65: 680-3; Yan, M. et al. Nat. Immunol. (2000) 1: 37-41; Xia, XZ et al. J. Exp. Med. (2000) 192: 137-43; Wang, H. et al. Nat. Immunol. (2001) 2: 632-7; Yu, G. et al. Nat. Immunol. (2000) 1: 252-6].These effects are thought to be due to BAFF sequesteration, which is because (a) BAFF acts as a co-stimulator of B cells in the presence of anti-IgM antibodies [see Moore, PA et al. Science (1999) 285: 260-3; Schneider, P. et al. J. Exp. Med. (1999) 189: 1747-56; Mukhopadhyay, A. et al. J. Biol. Chem. (1999) 274: 15978-81]; (b) in vivo administration of the soluble form of BAFF disrupts the splenic structure by increasing the number of B cells [see Moore, PA et al. Science (1999) 285: (c) Mice expressing BAFF as a transgene have hypertrophied spleens and lymph nodes and exhibit autoimmunity due to the enlargement and proliferation of B cells resulting from increased survival of B cells that are normally lost [ibid., Khare, SD et al. Proc. Natl Acad. Sci. USA (2000) 97: 3370-75; Gross, JA et al. Nature (2000) 404: 995-99; Mackay, F. et al. J. Exp. Med. (1999) 190: 1697-1710]; and (d) BAFF-deficient mice have a phenotype comparable to mice treated with TACI-Fc or BCMA-Fc, namely, a near-complete loss of mature B cells and a significant reduction in humoral responses [ibid., Schiemann, B. et al. Science (2001)]. [293: 2111-4; citing Gross, JA et al. Immunity (2001) 15: 289-302]. This last observation suggests that BAFF binding to TACI and / or BCMA is essential for B cell survival and function.Paradoxically, TACI-deficient mice exhibited B-cell proliferation rather than death, while BCMA knockout mice did not exhibit any clear phenotype [citing Schiemann, B. et al. Science (2001) 293: 2111-4; von Bulow, GU et al. Immunity (2001) 14: 573-82; Yan, M. et al. Nat. Immunol. (2001) 2: 638-43; Xu, S. and Lam, KP. Mol. Cell Biol. (2001) 21: 4067-74].

[0178] Given the limited expression of BAFF receptors in lymphocyte compartments, it is not surprising that most of the identified roles of BAFF are within lymphocytes. The greatest portion of the evidence regarding the role of BAFF-mediated NFκB2 signaling relates to peripheral B cell survival and maturation, antibody response generation, and plasma cell maintenance. [Gardam, S. and Brink R. Front. Immunol. (2014) 4: article 509]. BAFF / BAFF-R signaling is also essential for the complete maturation of B cells to the marginal zone phenotype in the spleen, in addition to providing a survival signal to peripheral B cells. Consistent with this finding, a mouse model with hyperactive NF-κB signaling presents an expansion and proliferation of MZ B cell populations. [Ibid., citing Mackay, F. et al. J. Exp. Med. (1999) 190: 1697-710].

[0179] One consequence of NF-κB signaling, which is thought to promote B cell survival, is the upregulation of anti-apoptotic molecules, such as Bcl-2 [Gardam, S. and Brink R. Front. Immunol. (2014) 4: article 509, citing Do, RK, et l. J. Exp. Med. (2000) 192: 953-64; Batten, M. et al. J. Exp. Med. (2000) 192: 1453-66]. Loss of either BAFF [Gardam, S. and Brink R. Front. Immunol. (2014) 4: article 509, citing Gardam, S. et al. Immunity (2008) 28: 391-401] or BAFF-R [ibid., citing Gardam, S. et al. Blood (2011) 117: 4041-51] can be fully compensated for B cell survival and maturation by the breakdown of the TRAF / cIAP ubiquitin ligase complex, and therefore by the constitutive hyperactivation of NF-κB2. While other evidence suggests that BAFF, BAFF-R, and NF-κB2 signaling can all individually contribute to B cell survival and maturation, these experiments definitively demonstrate that NF-κB2 activation more than compensates for the loss of BAFF or BAFF-R, meaning that the primary, perhaps even sole, purpose of BAFF / BAFF-R signaling in B cells is the activation of NF-κB2 signaling. This pathway promotes the transcriptional activity necessary for B cell survival and maturation in the periphery.

[0180] As used herein, the term "Bcl-2 family" refers to a family of intracellular proteins that include members that promote apoptosis (Bax, Bak, and Bok) and members that inhibit apoptosis (Bcl-2, Bcl-W, and Bcl-XL). The term "to bind" and its various grammatical forms refer to a persistent attraction between chemical substances. Binding specificity includes both binding to a specific partner and not binding to other molecules. Functionally important bindings can occur in a range from low to high affinity, and design elements can suppress undesirable cross-interactions. Post-translational modifications can also alter the chemistry and structure of interactions. "Promiscuous binding" may involve some degree of structural plasticity, which can result in different subsets of residues important for binding to different partners. "Relative binding specificity" is a characteristic in biochemical systems where a molecule interacts differentially with its target or partner, thereby differentially influencing them according to the identity of each individual target or partner.

[0181] The term “binding specificity,” as used herein, includes both binding to a specific partner and not binding to other molecules. Functionally important bindings can occur in a range from low to high affinity, and design elements may suppress undesirable cross-interactions. Post-translational modifications can also alter the chemistry and structure of interactions. “Promiscuous binding” may involve some degree of structural plasticity, which may result in different subsets of residues important for binding to different partners. “Relative binding specificity” is a feature in biochemical systems where a molecule interacts differentially with its target or partner, thereby differentially influencing them according to the identity of each individual target or partner.

[0182] As used herein, the term "biocompatible" means that a material does not cause irritation, injury, toxic reaction, or immune reaction to living tissues that are clinically relevant. As used herein, the term "biodegradable" refers to a material that is expected to degrade over time, either actively or passively, through simple chemical processes, the action of endogenous enzymes, or other similar biological activity mechanisms.

[0183] The term “biomarker” (or “biosignature”), as used herein, refers to any peptide, protein, nucleic acid, antibody, gene, metabolite, or other substance used as an indicator of a biological state. It is a feature that is objectively measured and evaluated as a cellular or molecular indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention. The term “indicator,” as used herein, refers to any substance, number, or ratio derived from a set of observed facts that may reveal relative changes as a function of time; or a visible signal, sign, mark, note, or symptom, or evidence of their existence or presence. Once a proposed biomarker is validated, it can be used to diagnose disease risk, the presence of disease, or to tailor treatment to disease in an individual (selection of drug treatment or administration regimen). In evaluating potential drug therapies, biomarkers may be used as a substitute for natural endpoints such as survival or irreversible morbidity. When a procedure alters a biomarker, that alteration is directly associated with the improved health status, and the biomarker may serve as a surrogate endpoint for evaluating clinical benefit. Clinical endpoints are variables that can be used to measure how a patient feels, functions, or survives. Surrogate endpoints are biomarkers intended to substitute for clinical endpoints, and these biomarkers have been demonstrated to predict clinical endpoints with an acceptable level of confidence for regulators and the clinical community.

[0184] As used herein, the term “bone marrow” refers to the tissue from which all cellular components of blood—namely red blood cells, white blood cells, and platelets—are first generated from hematopoietic stem cells, the site of further B cell development in mammals, and the source of stem cells that give rise to T cells upon migration to the thymus. The term "CD33," as used herein, refers to the sialoadhesin molecule and a member of the Ig supergene family. It is expressed by bone marrow stem cells (CFU-GEMM, CFU-GM, CFU-G, and E-BFU), myeloblasts and monoblasts, monocytes / macrophages, granulocyte precursors (expression decreases with maturation), and mast cells. Mature granulocytes may show very low levels of CD33 expression. CD33 may be abnormally expressed in some cases of plasma cell myeloma. This molecule is not expressed in erythrocytes, platelets, B cells, T cells, or NK cells. CD33 is a bone marrow marker and is commonly used for the diagnosis of AML. However, approximately 10–20% of B lymphoblastic or T lymphoblastic leukemia / lymphoma may abnormally express CD33. Faramarz, N. et al. Atlas of Hematopathology, 2d Ed., Chapter 2 Principles of Immunophenotyping (2018) Elsevier, Inc. pp. 29-56.

[0185] As used herein, the term "CD123" refers to the alpha chain of the interleukin-3 receptor (IL-3R). CD123 is a marker for the identification and targeting of LSCs in refractory or relapsed leukemia. [Shi, M. et al. Cardiovasc. Hematol. Disord. Drug Targets (2019) 19 (30: 195-204). As used herein, the term "CD135" refers to the receptor for the cytokine Flt3 ligand (FLT3L). Flt3 ligand is a transmembrane protein that binds to Flt3, an fms-like tyrosine kinase 3 receptor, initiating an intracellular signaling cascade that leads to the proliferation and development of hematopoietic stem cells and progenitor cells, as well as the development of the immune system. [Capitano, ML et al., Reference Module in Neuroscience and Biobehavioral Psychology (2017) doi.org / 10.1016 / B978-0-12-809324-5.03249-1]

[0186] The term "cancer stem cells," as used herein, refers to a small number of cells within a tumor that possess the ability to regenerate and drive tumorigenesis. The theory of cancer stem cells, which promotes the idea that cancer is primarily driven by a smaller population of stem cells, has significant implications. For example, if a therapy does not kill cancer stem cells within a tumor, these stem cells may allow the tumor to regrow and often give it resistance to the therapies previously used. The terms “carrier,” “excipient,” or “vehicle,” as used herein, refer to materials suitable for the formulation and administration of pharmaceutically acceptable compositions. The term “carrier,” as used herein, describes a material that does not cause significant irritation to the subject and does not impair the biological activity and properties of the immunotherapeutic agents of the compositions disclosed herein. Carriers must be sufficiently high in purity and sufficiently low in toxicity to be suitable for their administration to the mammal being treated. Carriers may be inert or may have pharmaceutically beneficial properties.

[0187] The term "CEBPA," as used herein, refers to the gene that provides instructions for the production of a protein called CCAAT enhancer-binding protein alpha. This protein is a transcription factor involved in the maturation of certain blood cells. It is also thought to act as a tumor suppressor. The terms "cell line" and "cultured cell line" are used synonymously to refer to a single type of cell adapted for continuous growth in a laboratory.

[0188] The term “cell proliferation,” as used herein, refers to the process by which cells accumulate mass and increase their physical size. Nature offers many diverse examples of how cells can proliferate. In some cases, cell size is proportional to DNA content. For example, continuous DNA replication in the absence of cell division (called intranuclear doubling) results in an increase in cell size. Megakaryoblasts, which mature into granular megakaryocytes—the cells that produce platelets in the bone marrow—typically proliferate in this manner. Through different strategies, adipocytes can proliferate to approximately 85–120 μm by the accumulation of intracellular lipids. In contrast to intranuclear doubling or lipid accumulation, some terminally differentiated cells, such as neurons and cardiomyocytes, proliferate without increasing their DNA content by ceasing division. These cells proportionally increase their macromolecular content (mostly proteins) to the point required to perform their specialized functions. This involves the coordination of extracellular signals from nutrients and growth factors with intracellular signaling networks involved in regulating cellular energy availability and macromolecular synthesis. Perhaps the most strictly regulated cell proliferation occurs in dividing cells, where cell proliferation and cell division are clearly separable processes. Dividing cells generally must increase in size through the cell division cycle in each passage to ensure that a consistent average cell size is maintained. In typical dividing mammalian cells, proliferation occurs in the G1 phase of the cell cycle and is closely linked to the S phase (DNA synthesis) and the M phase (mitosis). The combined effects of growth factors, hormones, and nutrient availability provide external signals to induce cell proliferation. [Guertin, DA, Sabatini, DM, “Cell Growth,” in The Molecular Basis of Cancer (4th Edn) Mendelsohn, J. et al Eds, Saunders (2015), 179-190].

[0189] As used herein, the term “cell proliferation” means the process that results in an increase in the number of cells, and is defined by the balance between cell division and cell loss through cell death or differentiation. As used herein, the term "chemokine" refers to a class of chemotactic cytokines that organize the migration and positioning of immune cells within tissues. Chemokines bind to seven-transmembrane G protein-coupled receptors that initiate intracellular signaling that promotes cell polarization, adhesion, and migration [Vilgelm, AE and Richmond, A. Front. Immunol. (2019) doi.org / 10.3389 / fimmu.2019.00333, citing Griffith, JW et al. Annu. Rev. Immunol. (2014) 32: 659-702; Nagarsheth, N. et al. Nat. Rev. Immunol. (2017) 17: 559-72]. Chemokines are classified into four families based on their structure: CXC, CC, CX3C, and C chemokines. These receptors follow a similar naming system based on the family of chemokines to which they bind. In addition, there is a family of atypical chemokine receptors, which do not directly couple to G proteins but have been reported to play various roles in development, homeostasis, inflammatory diseases, infections, and cancer [citing Nibbs, RJ, Graham, GJ. Nat. Rev. Immunol. (2013) 13: 815-29].

[0190] The terms “class switch,” “isotype switch,” or “class switch recombination,” as used herein, refer to a somatic recombination process in activated B cells that replaces one heavy chain constant region with one of different isotypes, thereby switching the antibody isotype from IgM to IgG, IgA, or IgE. This affects the effector function of the antibody but does not affect its antigen specificity. As used herein, the term "cognate help" refers to the process that occurs most efficiently under the circumstances of close interaction with helper T cells. As used herein, the term "compatibility" refers to the elements of a composition that can be combined with one another in such a manner that there are no interactions expected to substantially reduce the efficacy of the composition under normal use conditions.

[0191] The terms “complete response,” “complete remission,” or “CR,” as used herein, refer to the disappearance of all signs of cancer in response to treatment. This does not necessarily mean that the cancer is cured. As used herein, the term "element" means a component, part, or component that constitutes a part of something. As used herein, the term "composition" means a material formed from a mixture of two or more substances.

[0192] As used herein, the term “condition” means any health condition, including any disorder or disease caused by any underlying mechanism or disorder. The term “consolidation therapy,” also known as “remedial therapy” or “post-remission therapy,” as used herein, refers to treatment given after the initial therapy, after the cancer has disappeared. Consolidation therapy is used to kill any remaining cancer cells in the body. The term "contact" and its various grammatical forms, as used herein, mean a situation or state of touching, or a situation or state of direct or local proximity. Contact of a composition with a target destination can occur by any means of administration known to those skilled in the art.

[0193] As used herein, the term "costimulation" refers to a second signal required for completion of lymphocyte activation or prevention of anergy, which is provided by engagement of CD28 by CD80 and CD86 (T cells), and engagement of CD40 by CD40 ligand (B cells). As used herein, the term "costimulatory molecule" refers to a molecule presented on the cell surface that has a role in enhancing the activation of T cells that are already stimulated via their TCR. For example, HLA proteins present foreign antigens to the T cell receptor, which requires a costimulatory protein that binds to a complementary receptor on the surface of the T cell in order to result in enhanced T cell activation. As used herein, the term "costimulatory molecule" refers to highly active immunomodulatory proteins that play an important role in the development and maintenance of adaptive immune responses (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy, Chpt 5, 67-121 (2007)). The two-signal hypothesis of T cell responses involves the interaction of an antigen bound to an HLA molecule with its cognate T cell receptor (TCR), and the interaction of a costimulatory molecule with its ligand. Specialized APCs are carriers of the second signal for costimulation and can activate a T cell response after the HLA molecule has bound to the TCR. In contrast, somatic tissues do not express the second signal and thereby induce T cell non-responsiveness (ibid.). Many of the costimulatory molecules involved in the two-signal model can be blocked by coinhibitory molecules expressed by normal tissues (ibid.). In fact, the interactions of many types of immunomodulatory molecules expressed on a variety of tissues can exert both stimulatory and inhibitory functions depending on the immunological situation (ibid.). As used herein, the term "costimulatory receptor" means a cell surface receptor on naive lymphocytes that receives signals necessary for sufficient activation of lymphocytes in addition to those received via antigen receptors. Examples thereof are CD30 and CD40 on B cells, and CD27 and CD28 on T cells.

[0194] The term “cross-dressing,” as used herein, refers to a third pathway for cross-presentation. In cross-dressing, dendritic cells acquire pre-formed MHC class I molecules in the form of complexes with antigens from other cells, either through the process of trogocytosis (meaning the intercellular movement of cell membrane patches or individual proteins) [Yewdell, JW and Dolan, BP, “Cross-dressers turn on T cells”. Nature (2011) 471 (7340): 581-82, citing Joly,. E. and Hudrisier, D. Nature Immunol. (2003) 4: 815; Herrera OB et al. J. Imunol. (2004) 173: 4828-37] or via gap junctions. This allows antigen presentation by acceptor dendritic cells to occur immediately without any processing. Crossdressing is used to activate memory T cells in response to vial infection, but not to activate naive T cells [citing Wakins, LM and Bevan, MJ. Nature (2011) 471: 629-32, ibid.].

[0195] The term "cross-presentation," as used herein, refers to the process by which proteins absorbed by dendritic cells from the extracellular environment can produce peptides presented by MHC class I molecules. This allows antigens from extracellular sources to be presented by MHC class I molecules, thereby activating CD8 T cells. As used herein, the term "cross-priming" refers to the activation of CD8 T cells by dendritic cells in which an antigenic peptide presented by an MHC class I molecule is induced from an exogenous protein (i.e., by cross-presentation) rather than being directly produced within the dendritic cell (see direct presentation). The term "culture" and other grammatical forms, as used herein, mean the process of growing and multiplying a population of cells on a substrate in an artificial culture medium.

[0196] As used herein, the term "cytokine" refers to small soluble protein substances secreted by cells that have various effects on other cells. Cytokines mediate many important physiological functions such as proliferation, development, wound healing, and immune responses. Cytokines act by binding to their cell-specific receptors located on the cell membrane and initiating distinct signal transduction cascades within the cell, which ultimately are expected to result in biochemical and phenotypic changes in the target cells. Generally, cytokines act locally. Cytokines include type I cytokines, which include many interleukins, in addition to numerous hematopoietic growth factors; type II cytokines such as interferons and interleukin-10; tumor necrosis factor ("TNF")-related molecules such as TNFα and lymphotoxin; members of the immunoglobulin superfamily such as interleukin 1 ("IL-1"); and chemokines, a family of molecules that play important roles in a variety of immune and inflammatory functions. The same cytokine may have different effects on cells depending on the cellular context. Cytokines often regulate the expression of other cytokines and initiate their cascades. Non-limiting examples of cytokines include, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 / IL-23 P40, IL13, IL-15, IL-15 / IL15-RA, IL-17, IL-18, IL-21, IL-23, TGF-β, IFNγ, GM-CSF, Groα, MCP-1, and TNF-α.

[0197] As used herein, the term "cytotoxic T lymphocyte" (CTL) means referring to effector CD8+ T cells. Cytotoxic T cells kill their targets by inducing them to undergo apoptosis. Cytotoxic T cells induce target cells to undergo programmed cell death via extrinsic and intrinsic pathways. As used herein, the term "CXCR-4" refers to the G protein-bound chemokine receptor.

[0198] The term “decoy receptor,” as used herein, refers to a receptor that recognizes specific inflammatory cytokines with high affinity and specificity, but is structurally unable to signal to signaling receptor complexes or present agonists. Decoy receptors act as molecular traps for agonists and, furthermore, for signaling receptor elements. Interleukin-1 type II receptor (IL-1RII) was the first pure decoy identified. Subsequently, decoy receptors for tumor necrosis factor receptors and members of the IL-1R family were identified. [Mantovani, A. et al. Trends in Immunology (92001) 22 (6): 328-36]. The term "detectable marker" encompasses both selectable markers and assay markers. The term "selectable marker" refers to a variety of gene products that can be used to select or screen cells transformed with expression constructs. Examples include drug resistance markers, antigenic markers useful in fluorescence-activated cell sorting, and adhesion markers, such as receptors for adhesion ligands that enable selective adhesion.

[0199] The term “detectable response,” as used herein, means any signal or response that can be detected by an assay, whether or not it is performed with a detection reagent. Detectable responses include, but are not limited to, radiation decay and the emission, absorption, polarization, fluorescence, phosphorescence, conduction, reflection, or resonance transfer of energy (e.g., fluorescence, ultraviolet, infrared, visible light). Other detectable responses include chromatographic mobility, turbidity, electrophoretic mobility, mass spectra, ultraviolet spectra, infrared spectra, nuclear magnetic resonance spectra, and X-ray diffraction. Alternatively, a detectable response may be the result of an assay to measure one or more properties of a biological material, such as melting point, density, conductivity, surface acoustic waves, catalytic activity, or elemental composition. “Detection reagent” is any molecule that produces a detectable response indicating the presence or absence of a substance of interest. Detection reagents can be a variety of molecules, such as antibodies, nucleic acid sequences, and enzymes. For ease of detection, detection reagents may include markers.

[0200] As used herein, the term "derived" means all means of receiving, obtaining, or modifying something from its origin. The term "differentiate" and its various grammatical forms, as used herein, refer to a developmental process that increases the level of composition or complexity of a cell or tissue, often accompanied by a more specialized function. As used herein, the term “direct presentation” refers to the process by which a protein produced within a given cell gives rise to a peptide presented by an MHC class I molecule. This may refer to an APC (e.g., a dendritic cell) or to a non-immune cell that is expected to be a target of a CTL. As used herein, the terms “disease” or “disorder” refer to a state of impaired health or abnormal functioning. The terms “disease progression” or “progressive disease,” as used herein, refer to the continued growth or spread of cancer.

[0201] The term "DNAM-1" (also known as PTA1 or CD226) refers to a 65,000 molecular weight immunoglobulin-like transmembrane glycoprotein expressed on the surface of subsets of NK cells, T lymphocytes, platelets, monocytes, and B cells. [Xiong, P. et al. Immunology (2015) 146 (3): 369-78, citing Shibuya, A. et al. Immunity (1996) 4: 573-81] It is a co-activated receptor involved in NK cell adhesion, lymphocyte signaling, lymphokine secretion, and the regulation of cytotoxicity. DNAM-1 contains three domains: a 230-amino acid extracellular domain with two immunoglobulin-like domains and eight N-linked glycosylation sites; a 28-amino acid transmembrane domain; and a 60-amino acid cytosolic domain with four putative tyrosine residues and one serine residue for phosphorylation, which recruits signaling proteins. CD112 and CD155 are DNAM-1 ligands (DYNAM-1L) belonging to the nectin and nectin-like (Necl) protein family, containing nectin 1-4 and Necl 1-5, respectively. [Ibid., citing Bottino, C. et al. J. Exp. Med. (2003) 198: 557-67; Wang, L. et al. Vet Immunol. Immunopathol. (2009) 132: 257-63]. DNAM-1 receptor-ligand interactions mediate crosstalk between NK cells and other immune cells to maintain homeostasis. [Ibid., citing Pende, D. et al. Blood (2006) 107: 2030-6]. Currently, DNAM-1 has been shown to be involved in NK cell education and differentiation, immunological synapse formation, cytokine production, and crosstalk with DCs and T cells. DNAM-1 also modulates NK cell function by acting synergistically with CD96, TIGIT, and CRTAM.[Ibid., Hou, S. et al. J. Biol. Chem. (2014) 289: 6969-77; Nabekura, T. et al. Immunity (2014) 40: 225-34; Chan, CJ et al. Nat. Immunol (2014) 15: 431-8; Martinet, L. et al. Cell Rep. (2015) 11: 85-97; Martinet, L. and Smyth, MJ. Nat. Rev. Immunol. (2015) 15: 243-54; Pende, D. et al. Blood (2006) 107: 2030-6; Lozano, E. et al. J. Immunol. (2013) 191: 3673-80].

[0202] The term "DNMT3A," as used herein, refers to the gene that provides instructions for producing an enzyme called DNA methyltransferase 3-alpha. This enzyme is involved in DNA methylation, an epigenetic modification. DNMT3A mutations are an early event during leukemia induction and appear to confer a poor prognosis to patients with acute myeloid leukemia (AML). [Brunetti, L. et al. ColdSpring Harbor Perspect. Med. (2017) 7(2): a030320].

[0203] The term “dosage,” as used herein, means the amount of a therapeutic agent prescribed for single administration. The term “maximum tolerated dose,” as used herein, means the maximum dose of a drug or treatment that does not cause an unacceptable side effect. The term “dose escalation study,” as used herein, refers to a type of study in which enrolled patients receive different doses of a drug or investigational agent to determine a recommended phase 2 dose. As used herein, the term “dose-limiting toxicity” refers to a side effect of a procedure that is severe enough to stop increasing the dose of that procedure.

[0204] The term “ECOG Performance Status Scale,” as used herein, refers to a scale used to assess how a patient’s disease progresses, how the disease affects a patient’s ability to perform daily activities, and to determine appropriate treatment and prognosis. This scale is shown in Table 3 below and was developed by the Eastern Cooperative Oncology Group (ECOG), now part of the ECOG-ACRIN Cancer Research Group, Oken M, Creech R, Tormey D, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982;5:649-655.

[0205] [Table 3]

[0206] As used herein, the term “effective dose” generally refers to the amount of a therapeutic agent sufficient to induce a therapeutic effect. The effective dose may also refer to the amount of a therapeutic agent sufficient to delay or minimize the onset of symptoms. The effective dose may also refer to the amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease, disorder, or condition. Furthermore, the effective dose is the amount of a therapeutic agent, either alone or in combination with other therapeutic agents, that provides a therapeutic benefit in the treatment or management of a disease. The effective dose may also be the amount sufficient to enhance the immune response of the subject (e.g., human). The level of immunity can be monitored, for example, by measuring the amount of neutralizing antibodies, secretory antibodies, and / or serum antibodies, and can be monitored by, for example, plaque neutralization, complement fixation, enzyme immunoassay, or microneutralization assay. In the case of vaccines, the “effective dose” is the amount that prevents the disease and / or reduces the severity of symptoms.

[0207] For any therapeutic agent described herein, the effective dose may first be determined from preliminary in vitro studies and / or animal models. The therapeutically effective dose may also be determined from human data. The dose applied can be adjusted based on relative bioavailability and the potency of the administered compound. Adjusting the dose to achieve maximum efficacy based on the methods described above and other well-known methods is within the capabilities of those skilled in the art. The general principles for determining therapeutic efficacy can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference, and are summarized below. Pharmacokinetic principles provide criteria for modifying drug regimens to achieve the desired level of therapeutic efficacy while minimizing unacceptable adverse effects. Where plasma drug concentrations can be measured and are relevant to the therapeutic time range, additional guidance for dosage modifications can be obtained.

[0208] As used herein, the term “effector cell” refers to a cell that performs the final response or function. Major effector cells of the immune system include, for example, activated lymphocytes and phagocytic cells. As used herein, the term "effector function" refers to the actions taken by effector cells and antibodies to eliminate foreign substances, including, but not limited to, cytokine secretion, cytotoxicity, and antibody-mediated clearance.

[0209] As used herein, the term "eligible subject" refers to a subject who meets the requirements for treatment with immunotherapy under the professional judgment of the patient's physician. Eligibility criteria may include the subject's age, type and stage of cancer, current health status, medical history, and previous treatments. As used herein, the term "exclusion criteria" refers to characteristics that exclude subjects from a clinical study, and often includes factors such as comorbidities or concurrent treatments, or factors that may obscure the effect of a study treatment. As used herein, the term "expand" or "amplify", with respect to cells, refers to increasing the number of cells.

[0210] As used herein, the term "expression" and other grammatical forms refer to the production of an observable phenotype by a gene, usually by directing the synthesis of a protein. Expression includes mRNA biosynthesis, polypeptide biosynthesis, polypeptide activation, e.g., polypeptide activation by post-translational modification, or activation of expression by changing the intracellular location or by recruitment to chromatin. As used herein, the term "Fas" refers to a type 2 membrane protein found in lymphocytes that belongs to the TNF superfamily. Engagement of the cell death receptor Fas by Fas ligand (FasL) in cells expressing Fas results in apoptotic cell death mediated by caspase activation.

[0211] The term “Fc-gamma receptors (FcγRs),” as used herein, refers to receptors that recognize IgG-coated targets, such as opsonized pathogens or immune complexes (ICs). Crosslinking, along with the associated activation of downstream signaling cascades, leads to cargo internalization. FcγRs vary in their affinity for IgG and intracellular trafficking, and therefore have the opportunity to modulate antigen presentation by controlling the shuttleping and processing of their cargo. FcγRs bind to IgG molecules via their Fc (crystallizable fragment) moiety [Junker, F. et al. Front. Immunol. (2020) doi.org / 10.3389 / fimmu.2020.01393, citing Ravetch, JV, Bolland, S. Annu. Rev. Immunol. (2001) 19: 275-90]. In humans, three groups of FcγR—FcγRI, FcγRIIA / B, and FcγRIIIA / B—have been described across various cell types [citing Nimmerjahn, F., Ravetch, JV. Nat. Rev. Immunol. (2008) 8: 34-47]. These are expressed in various combinations on the surface membranes of various immune cells [citing Bruhns, P. Blood (2010) 119: 5640-9]. Examples of FcγRI include macrophages, neutrophils, eosinophils, and DCs. In the case of FcγRIIA, the cell types include macrophages, neutrophils, eosinophils, platelets, and Langerhans cells, as well as conventional, non-plasmacytoid dendritic cells (DCs) [citing Boruchov, AM et al. J. Clin. Invest. (2005) 115: 2914-23]. FcγRIIIIA has been found on natural killer (NK) cells and macrophages in other studies [citing Hayes, JM et al. J. Inflamm. Res. (2016) 9: 2009-19]. The inhibitory Fc gamma receptor FcγRIIB has been found on B cells, mast cells, as well as macrophages, neutrophils, and eosinophils.Importantly, it is also expressed in cDCs [citing Boruchov, AM et al. J. Clin. Invest. (2005) 115: 2914-23]. Flow cytometry experiments suggest that human pDCs are unlikely to express any FcγR, in contrast to mouse pDCs, which are claimed to express the inhibitory receptor FcγRIIB [citing Flores, M. et al. J. Immunol. (2009) 183: 129-39] [citing Boruchov, AM et al. J. Clin. Invest. (2005) 115: 2914-23; Patel, KR Front. Immunol. (2019) 10: 223]. Furthermore, some studies on pDCs may have included contamination from cDCs [citing Balan, S. Int. Rev. Cell Mol. Biol. (2019) 348: 1-68]. FcγRIIIB can be considered a decoy receptor because it lacks association with downstream signaling molecules (as discussed later in this paper), and it is primarily expressed in neutrophils, but may also be expressed in other immune cells such as basophils under certain conditions [citing Ravetch, JV, Bolland, S. Annu. Rev. Immunol. (2001) 19: 275-90; Bruhns, P. Blood (2012) 119: 5640-9].

[0212] The term "flow cytometry," as used herein, refers to a tool for matching the phenotype and characteristics of cells. Flow cytometry senses cells or particles as they move through a liquid stream using a beam of light passing through a laser (amplified light by stimulated emission of radiation) / sensing region. Relative light scattering of particles at the microscopic level and fluorescence, distinguished by color, are measured. Flow analysis and distinction of cells are based on size, particle size, and whether the cells possess fluorescent molecules in the form of antibodies or dyes. When a cell passes through a laser beam, the light is scattered in all directions, and light scattered forward at a low angle (0.5–10°) from the axis is proportional to the square of the radius of the sphere, and therefore proportional to the size of the cell or particle. Light can enter the cell; therefore, 90° (right-angle, lateral) scattering can be labeled with antibodies conjugated with fluorescent dyes, or stained with fluorescent membranes, intracytoplasmic, or nuclear dyes. Therefore, it is possible to easily distinguish between cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity, and DNA content. Flow cytometry is multi-parameterized and records numerous measurements in each cell; therefore, flow cytometry can identify homogeneous subpopulations within heterogeneous populations (Marion G. Macey, Flow cytometry: principles and applications, Humana Press, 2007). Fluorescence-activated cell sorting (FACS) allows for the isolation of distinct cell populations that are too similar in physical properties to be separated by size or density, and by using fluorescent tags to detect differentially expressed surface proteins, it enables high-precision identification among physically homogeneous cell populations.

[0213] The terms "FLT-3" or "Fms-like tyrosine kinase 3," as used herein, refer to type III receptor tyrosine kinases that play a vital role in the survival, proliferation, and differentiation of hematopoietic cells. Mutations in the FLT3 gene are the most frequent genetic alteration and are a poor prognostic factor in AML patients.

[0214] The term "FLT-3-ITD," as used herein, refers to FLT3 internal tandem duplication. The FLT3-ITD allele ratio is defined as the ratio of the area under the curve for FLT3-ITD to the area under the curve for FLT3-wildtype, using semi-quantitative DNA fragment analysis. Higher FLT2-ITD AR (generally defined as ≥0.5) is associated with worse survival than lower ratios. [Kantarjiian, H. et al. Blood Cancer J. (2021) 11: 41].

[0215] The term “germinal center” ("GC"), as used herein, refers to the site of vigorous B cell proliferation and differentiation that occurs in the lymphoid follicles of secondary lymphoid organs such as the spleen and lymph nodes during adaptive immune responses. Somatic hypermutation and class switching occur in germinal centers. Regulation of the GC response is crucial for ensuring high-affinity antibody production and pushing through self-tolerance by avoiding the emergence of autoimmune B cell clones. This regulation is not simply a result of the balance of follicular helper T cells / fT follicular regulatory cells, but also involves contributions from other cell types that modulate the GC microenvironment and evade autoimmunity. [Stebegg, M. et al. Front. Immunol. (2018) 9: 2469].

[0216] As used herein, the term "healthy subject" refers to a subject that shows no signs or symptoms of hematopoietic cancer. The term "helper T cell" or "T H When used herein, "effector CD4 T cells" refer to effector CD4 T cells that stimulate or "assist" B cells to produce antibodies in response to antigenic attack. The TH2, TH1, and THF subsets of effector CD4 T cells can exert this function. The term "IDH-1" or "isocitrate dehydrogenase-1," as used herein, refers to the primary source of NADPH reduction equivalents in the cytosol and peroxisomes. Both IDH1 and IDH2 are important in mitigating oxidative damage to cells induced by endogenous metabolism and extrinsic factors such as radiation. The term “intercellular adhesion molecules” or “ICAM,” as used herein, refers to the immunoglobulin superfamily cell adhesion molecules that bind to the leukocyte integrin CD11a:CD18(LFA-1). These are important for the binding of lymphocytes and other leukocytes to antigen-presenting cells and endothelial cells. As used herein, the term "IL-4Rα" refers to the cytokine-binding receptor chain for IL-4.

[0217] As used herein, the term “immune checkpoint” refers to a set of inhibitory pathways necessary for maintaining self-tolerance and modulating the duration and extent of the immune response to minimize damage to normal tissue. Immune checkpoint molecules, such as PD-1, PD-L1, and CTLA-4, are cell surface signaling receptors that play a role in modulating T cell responses in the tumor microenvironment. Tumor cells have been shown to exploit these checkpoints to their advantage by upmodulating their expression and activity. Given that tumor cells have the ability to utilize some immune checkpoint pathways as a mechanism for immune resistance, it has been hypothesized that checkpoint inhibitors, which bind to molecules on immune cells and activate or inactivate them, can mitigate the inhibition of the immune response. Immune checkpoint inhibitors have been reported to enable the persistence of the endogenous anti-cancer immune response by blocking another checkpoint in an active host immune response. Recent discoveries have identified immune checkpoints or targets such as PD-1, PD-L1, PD-L2, CTLA4, TIGIT, TIM-3, LAG-3, CCR4, OX40, OX40L, IDO, and A2AR as proteins involved in immune evasion.

[0218] The term “immune evasion” or “immune avoidance,” as used herein, refers to strategies for escaping the host’s immune response. This is characterized by the immune system’s inability to eliminate transformed cells before and after tumor development. Host contribution manifests as its inability to recognize antigens expressed by tumor cells, a phenomenon known as “host ignorance.” This results from defects in both the innate and adaptive arms of the immune system. Tumor contribution manifests as adapting tumor cells to evade the immune system or developing a microenvironment that suppresses the immune system. Qian J. et al. (2011) Immune Escape. In: Schwab M. (eds) Encyclopedia of Cancer. Springer, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 978-3-642-16483-5_2975. The term "immunohomeostasis" refers to the delicate and finely regulated balance of appropriate immune activation and suppression in tissues and organs, driven by countless cellular and chemical factors. [da Gama Duarte, J. et al. Immunology and Cell Biology (2018) 96: 497-506].

[0219] The terms “immune response” and “immune-mediated” are used synonymously herein and refer to any functional expression of the subject’s immune system to either external or autoantigens, regardless of whether the consequences of these reactions are beneficial or harmful to the subject. When used herein, the term “immune response” to an antigen or composition means that a humoral and / or cellular immune response to an antigen present in the composition of interest occurs in the subject. For the purposes of the disclosure of this invention, “humoral immune response” refers to an immune response mediated by antibody molecules, while “cellular immune response” refers to an immune response mediated by T lymphocytes and / or other leukocytes. One aspect of cellular immunity includes an antigen-specific response by cytolytic T cells (“CTLs”). CTLs have specificity for peptide antigens that are encoded by major histocompatibility complexes (MHCs) and presented in association with proteins expressed on the surface of cells. CTLs assist in the disruption of intracellular microorganisms or the induction and promotion of the lysis of cells infected with such microorganisms. Another aspect of cellular immunity includes an antigen-specific response by helper T cells. Helper T cells act to stimulate the function of nonspecific effector cells to cells that present peptide antigens associated with MHC molecules on their surface, thereby helping to concentrate their activity. “Cellular immune response” also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other leukocytes, such as those derived from CD4+ and CD8+ T cells. Therefore, an immune response may include one or more of the following actions: antibody production by B cells; and / or activation of suppressor T cells and / or γδ T cells specifically against antigens present in the composition or vaccine of interest. These responses may help neutralize infectivity and / or provide protection to an immunized host by mediating antibody-complement or antibody-dependent cell-mediated cytotoxicity (ADCC). Such responses can be determined using standard immunoassays and neutralization assays well known in the art.

[0220] The term “immunophenotype” or “immunotype,” as used herein, refers to the collective frequency of various immune cell populations and their functional responses (cellular signaling and antibody responses) to stimuli. [See Kaczorowski, KJ et al. Proc. Nat. Acad. Sci. USA (2017) doi / 10.1073 / pnas.1705065114]. The terms "immune surveillance" or "immunological surveillance" are used synonymously to refer to the process of monitoring by the immune system to detect and destroy virus-infected cells and tumor-transformed cells in the body. As used herein, the term “immune system” refers to the body’s defense system against disease, and includes the innate and adaptive immune systems. The innate immune system provides nonspecific first-line defense against pathogens. It includes both physical barriers (e.g., skin) and cellular (granulocytes, natural killer cells) and humoral (complementary system) defense mechanisms. The innate immune response is immediate but, unlike the adaptive immune system, does not provide permanent immunity against pathogens. Adaptive immune responses are typically the responses of the vertebrate immune system to specific antigens that produce immunological memory.

[0221] The term "immunely vulnerable," as used herein, refers to a state in which the immune system is weakened and its ability to fight infection and other diseases is reduced. Patients who are immunely vulnerable include those receiving long-term (>3 months) or high-dose (>0.5 mg / kg / day) steroids or other immunosuppressant drugs; organ or bone marrow transplant recipients; patients with solid tumors requiring chemotherapy within the past 5 years; or patients with hematological malignancies that have been treated, regardless of the time since diagnosis; patients with leukemia or lymphoma; patients with primary immunodeficiency; patients with HIV or AIDS; patients with autoimmune conditions; patients with asthma, which causes the immune system to overreact to harmless substances; elderly patients; and smokers.

[0222] The term “immunodeficiency” and other grammatical forms, as used herein, refer to the inability to produce a sufficient immune response due to a deficiency or absence of antibodies, immune cells, or both. Immunodeficiency disorders can be hereditary, such as severe combined immunodeficiency; they can be acquired, such as due to infection, such as HIV infection; or they can be caused by chemotherapy. The term "immunological repertoire" refers to the collection of transmembrane antigen-receptor proteins arranged on the surface of T and B cells. [Benichou, J. et al. Immunology (2011) 135: 183-191] The combinatorial mechanisms involved in receptor encoding do so by reshuffling the genetic code, and in humans, this has been observed for 10 years. 18 This can generate different T cell receptors (TCRs) across species [citing Venturi, Y. et al. Nat. Rev. Immunol. (2008) 8: 231-8] and a more diverse B cell repertoire. These sequences are then expected to be transcribed and translated into proteins to be presented on the cell surface. The recombination process that rearranges gene segments for receptor construction is crucial for the development of the immune response, and the precise formation of the rearranged receptor is important for its future antigen-binding affinity. For example, the diversity of TCR genes is generated by the rearrangement of the V and J gene segments during T cell development in the thymus (Makino, Y., et al (1993) J. Exptl Med. 177: 1399-1408). The V and J gene segments of the TCR, like the Ig gene, have a recombination signal, in which heptameric and nonameric sequences separated by a 12 / 23 bp spacer are flanked by germline V and J gene segments. Same as above.

[0223] The term "immunogen" and its various grammatical forms are used herein synonymously with the term "antigen." The terms “immunomodulatory,” “immune modulator,” “immunomodulatory,” and “immune modulatory” are used herein synonymously to refer to substances, drugs, or cells that can directly or indirectly increase or decrease the immune response, for example, by expressing chemokines, cytokines, and other mediators of the immune response.

[0224] The term “immunosynapse,” as used herein, refers to a highly structured entity that functions to enrich TCR signaling in a defined region. Immunosynapses are associated with the selective recruitment of signaling molecules and the exclusion of negative regulators. Synapses are stabilized by a ring of adhesion molecules, such as LFA1, which binds to ICAM1 on APCs. Co-stimulation via a second signaling pathway is required for T cells to become fully active. Many costimulators have been identified that share the common characteristic of being transmembrane receptors, often belonging to the TNFR superfamily, that bind to transmembrane ligands on APCs. The most important costimulator, CD28, binds to ligands CD80(B7.1) and CD86(B7.2), both expressed on activated APCs. Co-stimulation results in the clonal expansion and proliferation of CTLs with selected antigen specificity. CD80 / 86 expression is tightly regulated. High levels of expression occur only after APCs have received activating signals, such as inflammatory cytokines, or components of pathogens, such as lipopolysaccharides. CD28-mediated co-stimulation is crucial during the initiation of the immune response because it promotes IL-2 production and subsequently supports the development of effector T cells. Naive T cells stimulated in the TCR in the absence of co-stimulatory signals may become unresponsive to the antigen, a condition known as "anergy." Nutt, SL et al. Clinical Immunology (4th Ed.) chapter 17, Cytotoxic T lymphocytes and natural killer cells. (2013) 215-27.

[0225] The term “immunostimulant” and other grammatical forms, as used herein, refer to increasing an immune response, either directly or indirectly. The term “immunosuppression” and other grammatical forms, as used herein, refer to suppressing or reducing the immune response, either directly or indirectly.

[0226] The term “immunotherapy,” as used herein, refers to means employed using immunological methods and principles to target an overimmune or hypoimmune state in an organism, to artificially intervene in or modulate the immune function of an organism, or to enhance or weaken the immune response, so that a disease may be treated. This enhances the immune system’s ability to recognize, target, and eliminate cancer cells in the body. [Zhang, Z. et al. Front. Immunol. (2021) 12: Barbari, C. et al. Intl J. Mol. Sci. (2020) 21: 5009]. Some types of immunotherapy target only specific cells of the immune system. Others affect the immune system in a general way. For example, monoclonal antibodies can bind to specific proteins on the surface of cancer cells or immune cells to mark cancer as a target for the immune system or to boost the ability of immune cells to fight cancer. Another example, cytokine therapy, relies on proteins called interferons and interleukins to initiate an immune response. Interleukin-2 (IL-2) is used to treat kidney cancer and melanoma that have spread to other parts of the body. Interferon-alpha (IFN-alpha) is currently used to treat melanoma, kidney cancer, and certain leukemias and lymphomas. These cytokine treatments are also combined with other types of immunotherapy to increase their effectiveness.

[0227] The term "immunotherapy agent," as used herein, refers to a drug, molecule, nucleic acid, protein, composition, or cell that provides a therapeutic effect. The term "active substance," as used herein, refers to a component, element, or constituent of the composition of the present invention that is involved in the intended therapeutic effect. The terms "immunotherapy agent" and "active agent" are used synonymously herein. The term “immunotherapy element,” as used herein, refers to a therapeutically effective dosage (i.e., dose and frequency) that eliminates, reduces, or prevents the progression of a particular disease in a given percentage of a population. A commonly used example of a therapeutic element is ED50, which specifies a dosage that is therapeutically effective for the symptoms of a particular disease in 50% of a population. The term "induction therapy," also known as first-line therapy, primary therapy, or primary treatment, refers to the initial treatment given for a disease.

[0228] The term inhibitory receptor lymphocyte activator gene-3, or "LAG-3," as used herein, refers to a member of the immunoglobulin superfamily (IgSF) that binds to major histocompatibility complex (MHC) class II. LAG-3 expression in TILs is associated with tumor-mediated immunosuppression.

[0229] The term "lymphocyte" refers to small white blood cells formed in the lymphoid tissues throughout the body. In a normal adult, they make up approximately 22-28% of the total white blood cells in the circulating blood and play a significant role in defending the body from disease. Individual lymphocytes are specialized in that they are involved in responding to a limited group of structurally related antigens. This involvement is represented by the presence of receptors on the surface membrane of lymphocytes that are specific to the determinants (epitopes) on the antigen, and which exist before the immune system first comes into contact with a given antigen. Each lymphocyte has a population of receptors, all of which have identical binding sites. One set or clone of lymphocytes differs from another clone in the structure of its receptor binding region, and therefore in the epitopes it can recognize. Lymphocytes differ from one another not only in the specificity of their receptors but also in their function. Lymphocytes are more common in the lymphatic system, and examples include B cells, T cells, killer T cells, and natural killer (NK) cells. Lymphocytes are divided into two broad categories: T cells and B cells. T cells are involved in cell-mediated immunity, while B cells are involved in humoral immunity (related to antibodies). T cells are so named because these lymphocytes mature in the thymus, while B cells mature in the bone marrow. B cells produce antibodies that bind to pathogens and enable their destruction. CD4+ (helper) T cells regulate the immune response. CD8+ (cytotoxic) T cells and natural killer (NK) cells can kill, for example, cells in the body that are infected with a virus or present an antigenic sequence.

[0230] The term "lymphocyte activation" refers to the stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells, resulting in the synthesis of RNA, proteins, and DNA, as well as the production of lymphokines, which are soluble products of lymphocytes, followed by the proliferation and differentiation of various effector and memory cells. For example, mature B cells can be activated by encountering an antigen that expresses an epitope recognized by their cell surface immunoglobulin Ig. The activation process may be direct, depending on the crosslinking of membrane Ig molecules by the antigen (crosslinking-dependent B cell activation), or indirect, occurring most efficiently in the context of close interaction with helper T cells ("cogeneic assistive process"). T cell activation depends on the interaction between the TCR / CD3 complex and peptides bound to the grooves of its cogeneic ligands, class I or class II MHC molecules. The molecular events initiated by receptor engagement are complex. Full T-cell responsiveness requires not only receptor engagement but also co-stimulatory activity delivered by accessory cells, such as the engagement of CD28 on T cells by CD80 and / or CD86 on antigen-presenting cells (APCs).

[0231] The term "macrophage," as used herein, refers to mononuclear, active phagocytic cells arising from monocytic stem cells in the bone marrow. These cells are widely distributed throughout the body and vary in morphology and motility. Phagocytic activity is typically mediated by serum recognition factors such as specific immunoglobulins and complement system elements, but can also be nonspecific. Macrophages are also involved in both antibody production and cell-mediated immune responses, specifically antigen presentation to lymphocytes. They secrete a variety of immunomodulatory molecules. As used herein, the terms “maintenance therapy” or “continuous therapy” refer to the ongoing treatment of cancer to prevent recurrence after the cancer has responded to induction therapy.

[0232] The terms “Major Histocompatibility Complex (MHC),” “MHC-like molecule,” and “HLA” are used synonymously herein to refer to cell surface molecules that present molecular fragments known as epitopes or antigens and mediate interactions between leukocytes and other leukocytes or somatic cells. MHC is encoded by a large group of genes and can be systematically divided into three subgroups: Class I, Class II, and Class III. The MHC gene complex is called HLA (“Human Leukocyte Antigen”) in humans and H-2 (“Histocompatibility”) in mice. Both species have three major MHC class I genes, called HLA-A, HLA-B, and HLA-C in humans and H2-K, H2-D, and H2-L in mice. These encode the α chain of the respective MHC class I protein. Another subunit of the MHC class I molecule is β2-microglobulin. The Class II region includes genes (designated A and B) related to the α and β chains of the human MHC Class II molecules HLA-DR, HLA-DP, and HLA-DQ. The MHC Class II region also includes genes related to the TAP1:TAP2 peptide transporter, the PSMB (or LMP) gene encoding proteasome subunits, genes encoding the DMα and BMβ chains (DMA and DMB), genes encoding the α and β chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP). Class II genes encode various other proteins with immune functions. The DMA and DMB genes, which encode subunits of the HLA-DM molecule that catalyze peptide binding to MHC Class II molecules, are related to MHC Class II genes, as are the DOA and DOB genes, which encode subunits of the regulatory HLA-DO molecule. [Janeway's Immunobiology. 9th ed., GS, Garland Science, Taylor & Francis Group, 2017. pps. 232-233].In humans, there are three MHC class II isotypes encoded by the α and β chain genes within the human leukocyte antigen (HLA) locus on chromosome 6: HLA-DR, HLA-DP, and HLA-DQ [Wosen, JE et al. Front. Immunol. (2018) doi.10.3389 / fimmu.2018.02144].

[0233] As used herein, the term "MHC-restricted" refers to the requirement that an APC or target cell expresses an MHC molecule that the T cell recognizes as self in order for the T cell to respond to the antigen presented by that APC or target cell (the T cell is expected to recognize only the antigen presented by its own MHC molecule). For example, CD8 T cells bind to class I MHC, which is expressed on most cells in the body, and CD4 T cells bind to class II MHC, which is expressed only on specialized APCs. The terms “marker” or “cell surface marker,” as used herein, are synonymous to refer to antigenic determinants or epitopes found on the surface of a particular type of cell. Cell surface markers can facilitate the characterization, identification, and ultimately isolation of cell types. Cell sorting techniques are based on cellular biomarkers, and cell surface markers can be used for either positive or negative selection, i.e., inclusion or exclusion from a cell population. The term "mediated" and its various grammatical forms, as used herein, refer to relying on, acting by, or being connected through some kind of intervention.

[0234] The term "memory cells," as used herein, refers to B and T lymphocytes generated during the primary immune response that remain quiescent until fully activated (secondary immune response) by subsequent exposure to a specific antigen. Memory cells generally have higher sensitivity to antigens than naive lymphocytes and respond rapidly when re-exposed to the antigen that initially induced them. During the immune response, naive T cells (TNs) are primed by antigen-presenting cells (APCs). Depending on the intensity and quality of the stimulating signal, the proliferating T cells progress along a differentiation pathway that peaks in the generation of terminally differentiated, short-lived effector T (TEFF) cells. Once antigenic and inflammatory stimuli cease, the primed T cells become quiescent and, depending on the intensity of the signal received, enter the memory stem cell (TSCM), central memory (TCM) cell, or effector memory (TEM) cell pool. TSCM cells possess stem cell-like properties to a greater degree than any other memory lymphocyte population. Both TCM and TEM cells can undergo self-regeneration, but their ability to form diverse progeny is gradually limited. Therefore, only TCM cells are capable of generating all three memory subsets and TEFF cells, while TCM cells can produce TCM, TEM, and TEFF cells, and TEM cells can produce only themselves and TEFF cells. [Gattinoni, L. et al. Nature Revs. Cancer 12 (2012) 671-84].

[0235] The term “mixed phenotypic acute leukemia” or “MPAL,” as used herein, refers to a heterogeneous group of leukemias to which a single lineage origin cannot be assigned. This is currently defined by a limited set of lineage-specific markers proposed in a 2008 WHO study on the classification of tumors of hematopoietic and lymphoid tissues. [Wolach, O. and Stone, RM. Blood (2015) 125 (16): 2477-85].

[0236] The term “mutation,” as used herein, refers to a change in the DNA sequence within a gene or chromosome of an organism that produces a new feature or trait not found in the parental type, or to a process in a chromosome through either a change in the nucleotide sequence of the gene-coding DNA or a change in the physical arrangement of the chromosome. The three mechanisms of mutation include substitution (exchanging one base pair with another), addition (inserting one or more bases into a sequence), and deletion (losing one or more base pairs).

[0237] The terms "myeloblast" or "blast," as used herein, refer to a type of immature leukocyte. Myeloblasts have a diameter of 10–20 μm, are induced from HSCs, and are usually found in the bone marrow. Myeloblasts develop into mature granulocytes (neutrophils, basophils, and eosinophils). As used herein, the term "myeloid" refers to the lineage of blood cells that develop during bone marrow hematopoiesis, including granulocytes, monocytes, megakaryocytes, and dendritic cells. Circulating red blood cells and platelets also originate from myeloid progenitor cells. The term "bone marrow hematopoiesis," as used herein, refers to the development of non-lymphoid leukocytes. Bone marrow hematopoiesis begins with the differentiation of pluripotent stem cells into colony-forming units (CFU-GEMMs), small populations that produce myeloid cells (CFU-GMs), which then differentiate into granulocyte / macrophage colony-forming units (CFU-GMs), committed primitive bone marrow precursors. This process requires bone marrow hematopoietic cytokines (GM-CSF, SCF, IL-3, and IL-6). CFU-GMs give rise to more mature colony-forming units, CFU-G, CFU-M, CFU-Eo, and CFU-Baso, which differentiate into neutrophils, macrophages, eosinophils, and basophils, respectively. The term "myeloproliferative neoplasm" or "MPN," formerly known as myeloproliferative disorder, is characterized by the clonal proliferation of one or more hematopoietic cell lineages, predominantly in the bone marrow, but sometimes in the liver and spleen. In contrast to myelodysplastic syndromes (MDS), MPNs demonstrate the spread of peripheral myeloid cells into the peripheral blood. Examples of MPNs include chronic myeloid leukemia (CML), chronic neutrophilic leukemia, polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), chronic eosinophilic leukemia, mastocytosis, and unclassified MPNs.

[0238] As used herein, the term “naive T cell” refers to a T cell that has never been exposed to an antigen. Naive T cells are conventionally defined by the co-expression of the transmembrane phosphatase CD45 RA isoform, the lymph node homing molecule L-selectin (CD62L) and CCR7, and the costimulatory receptors CD27 and CD28. [De Rosa, SC et al. Nature Med. (2001) 7: 245-48]. Nectin and nectin-like molecules (Necl) have recently emerged as cell adhesion molecules that possess various cellular functions, including cell motility, proliferation, differentiation, polarization, and survival, as well as cell-to-cell adhesion. [Takai, Y. et al. Nature Rev. Molec. Cell Biol. (2008) 9: 603-15].

[0239] The term “natural killer (NK) cells,” as used herein, refers to lymphocytes in the same family as T and B cells, which are classified as innate lymphocytes of Group I. These cells possess the ability to kill tumor cells without any priming or prior activation, in contrast to cytotoxic T cells, which require priming by antigen-presenting cells. NK cells secrete cytokines such as IFNγ and TNFα, which act on other immune cells, such as macrophages and dendritic cells, to enhance the immune response. Activating receptors on the surface of NK cells recognize molecules expressed on the surface of cancer or infected cells, switching on the NK cells. Inhibitory receptors act as a check against NK cell lethality. Most normal, healthy cells express MHCI receptors, which mark them as “self.” Inhibitory receptors on the surface of NK cells recognize homologous MHCI, thereby switching off the NK cells and preventing their lethality. Once lethality is determined, NK cells cause lysis of target cells by releasing cytotoxic granules containing perforin and granzymes. Natural killer responsiveness, encompassing cytokine secretion and cytotoxicity, is regulated by a balance of inhibitory and activating receptors encoding numerous germline cells, including killer immunoglobulin-like receptors (KIRs) and innate cytotoxic receptors (NCRs). The presence of MHC class I molecules on target cells serves as one such inhibitory ligand for killer cell immunoglobulin-like receptors (KIRs) on NK cells, which are MHC class I-specific receptors. Engagement of the KIR receptor preserves its ability to respond to successive encounters by blocking NK activation and, paradoxically, initiating an inactivating signal. Therefore, if KIRs can adequately bind to MHC class I, this engagement may neutralize the signal for lethality, allowing target cells to survive. In contrast, if NK cells cannot adequately bind to MHC class I on target cells, lethality of the target cells may proceed. Consequently, tumors that express low levels of MHC class I and are thought to be able to evade T cell-mediated attack may instead be more susceptible to NK cell-mediated immune responses.

[0240] The abbreviation "NFκB," as used herein, refers to a pro-inflammatory transcription factor that switches on multiple inflammatory genes, such as cytokines, chemokines, proteases, and apoptosis inhibitors, resulting in an amplification of the inflammatory response [Barnes, PJ, (2016) Pharmacol. Rev. 68: 788-815]. Molecular pathways involved in NF-κB activation include numerous kinases. The classic (canonical) pathway for inflammatory stimuli and infections that activate NF-κB signaling involves the IKK (κB kinase inhibitor) complex, which consists of two catalytic subunits, IKK-α and IKK-β, and a regulatory subunit, IKK-γ (or a regulator essential for NFκB) [ibid., citing Hayden, MS and Ghosh, S (2012) Genes Dev. 26: 203-234]. The IKK complex phosphorylates IκB bound to Nf-κB, targeting them for degradation by the proteasome, thereby releasing NF-κB dimers composed of p65 and p50 subunits, which then transfer to the nucleus, where they bind to κB recognition sites in the promoter regions of inflammatory and immunogenes, leading to their transcriptional activation. This response relies primarily on the catalytic subunit IKK-β (also known as IKK2) that performs IκB phosphorylation. An atypical (alternative) pathway involves an upstream kinase, NF-κB-inducible kinase (NIK), which phosphorylates IKK-α homodimers, releases RelB in response to certain members of the TNF family, e.g., lymphotoxin-β, and processes p100 to p52 [ibid., citing Sun, SC. ​​(2012) Immunol. Rev. 246: 125-140]. This pathway can switch on different sets of genes and mediate different immune functions than the canonical pathway. Dominant-negative IKK-β inhibits most of the pro-inflammatory functions of NF-κB, while inhibiting IKK-α plays a role in certain cells, such as B lymphocytes, only in response to limited stimuli. Irregular pathways are involved in the development of the immune system and adaptive immune responses.The coactivator molecule CD40 is expressed on antigen-presenting cells such as dendritic cells and macrophages, and when it interacts with CD40L expressed on lymphocytes, it activates an irregular pathway [citing Lombardi, V et al. (2010) Int. Arch. Allergy Immunol. 151: 179-89]. Activation of irregular NF-κB signaling in response to TNF family B cell activator (BAFF) contributes to major events throughout B cell lifespan. Examples include activating transcriptional programs that make immature peripheral B cells more viable and allow them to mature into follicular and marginal zone (MZ) B cells; contributing to some T-independent immune responses; prolonging the duration of germinal center (GC) responses; and maintaining long-lived plasma cells in the germinal center (BM) [Gardam, S. and Brink, R. Front. Immunol. (2014) 4: article 509].

[0241] The term "NKG2D," as used herein, refers to the activating receptor expressed by all NK cells and T cell subsets (γδ T cells, CD8+ T cells, and CD4+ T cells) in humans. This is encoded by the KLRK1 gene (Killer cell lectin-like receptor subfamily K, member 1). The NKG2D receptor functions as an activating receptor through interaction with the signal transduction adapter dimer DAP10 in humans and with DAP10 and DAP12 in mice (Raulet, DH et al. Annu. Rev. Immunol. (2013) 31: 4123-41, citing Champsaur, M. and Lanier, LL. Immunol. REev. (2010) 235: 267-85; Wu, J. et al. Science (1999) 285: 730-32). When the receptor is ligated, DAP10 provides a signal that recruits the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and the GRB2-VAV1 complex. Engagement of NKG2D with NK cells induces degranulation and cytokine production.

[0242] NK cell activation resulting from NKG2D engagement can be modified or altered by engagement with other NK receptors. In naive human NK cells, synergistic activation occurs when NKG2D co-engages with 2B4, a SLAM family receptor whose ligand is widely expressed by hematopoietic cells, or with another activating receptor, NKp46 (citing Bryceson, YT et al. Blood (2006) 107: 159-6611)11). Conversely, if target cells express MHC class I molecules that engage with inhibitory receptors on NK cells, such as KIR (killer cell immunoglobulin-like receptor) in humans, NKG2D-induced NK activation may be inhibited (though not necessarily completely) [Ibid., Jamieson, AM et al. Immunity (2002) 17: 19-29l Rgunathan, J. et al.. Blood (2005) 105: 2133-40].

[0243] NKG2D binds to a number of different ligands, all of which are homologous to MHC class I molecules, but do not have a known role in antigen presentation [Ibid., Raulet, DH. Nat. Rev. Immunol. (2003) 3: 781-90; Champsaur, M. and Lanier, LL. Immunol. Rev. (2010) 235: 267-85; Eagle, RA and Trowsdale, J. Nat. Rev. Immunol. (2007) 7: 737-44; Machuldova, A. et al. Front. Immunol. (2021) 12: 651751, citing Stephens, HA. Trends Immunol. (2001) 22 (7): 378-85]. Like MHC proteins, NKG2D exhibits significant allele mutations. In humans, NKG2D ligands include MHC class I chain-related protein A (MICA) and MHC class I chain-related protein B (MICB), both encoded by genes in the MHC, as well as up to six different proteins known as unique long (UL)16-binding proteins (ULBPs), also known as retinoic acid early transcript 1 (RAET1) protein. Like MHC proteins, NKG2D ligands exhibit significant allele mutations.

[0244] All NKG2D ligands are encoded by distinct genes within the host's own genome; that is, the ligands are self-proteins. NKG2D ligands are poorly expressed or not expressed at all in most normal cells, but are upregulated in cancer cells and virus-infected cells. This type of recognition process, in which self-encoded ligands for activating receptors are induced in unhealthy cells, is called "induction of self-recognition" [citing Diefenbach, A. and Raulet, DH. Immunol. Rev. (2001) 181: 170-84], and is different from "loss of self-recognition" in which the loss of MHC ligands for NK inhibitory receptors makes cells more sensitive to elimination by NK cells. Various cellular pathways activated as a result of cellular stress, infection, or tumorigenesis regulate the expression of NKG2D ligands.

[0245] The structure of the NKG2D-ligand complex indicates that NKG2D binds diagonally to the α1 and α2 helices of the ligand, similar to how T cell receptors bind to MHC molecules. Despite the poor homology of different ligands, some of the key residues that interact with NKG2D are conserved, and the NKG2D residues involved in binding are similar in different structures. NKG2D ligands are generally underexpressed in normal cells but are upregulated in transformed cells, infected cells, and in some cases, stressed cells. NKG2D engagement is a sufficient stimulus to activate NK cell-mediated cell breakdown and cytokine production. However, it also provides an enhancing or co-stimulatory signal for the activation of CD8+ T cells and possibly other T cells.

[0246] As used herein, the term "novel antigen" refers to a tumor-specific antigen that is expressed only in tumor cells and is generated by mutations in tumor cells. As used herein, the term "neoepitope" refers to a tumor-specific MHCI-restricted epitope. The term “neutrophil” or “polymorphonuclear neutrophil (PMN),” as used herein, refers to the most abundant type of white blood cell in mammals, which forms an essential part of the innate immune system. It forms part of the polymorphonuclear cell family (PMN) along with basophils and eosinophils. Neutrophils are normally found in the bloodstream. During the onset of inflammation (acute phase), specifically as a result of bacterial infection or some cancers, neutrophils are one of the first responders of inflammatory cells that migrate toward the site of inflammation. Neutrophils migrate through blood vessels and then, in a process called chemotaxis, migrate through interstitial tissues in accordance with chemical signals such as interleukin-8 (IL-8) and C5a, which means the directed movement of a motile cell or part thereof along a chemical concentration gradient toward environmental conditions that it finds attractive and / or away from the surrounding environment that it finds unfavorable.

[0247] As used herein, the term "non-proliferative" refers to a population of cells that did not proliferate (in vitro) in a culture intended to increase the number of cells in a cell population. The abbreviation "NPM1," as used herein, refers to a mutant of nucleophosmin-1. NPM1 provides instructions for producing nucleophosmin found in the nucleolus, where it binds to ARF, thereby preventing cells from growing or dividing uncontrollably. When used herein, the term “Objective Response Rate” or “ORR” refers to the percentage of people in a study or treatment group who achieved a partial or complete response to a treatment within a specified period. As used herein, the term "overall survival" refers to the length of time a patient diagnosed with a disease, such as cancer, is still alive, from either the date of diagnosis or the start of treatment for that disease.

[0248] The term "paraprotein," as used herein, typically refers to monoclonal immunoglobulin fragments or intact immunoglobulins produced by malignant cone-like structures of plasma cells or B cells. These proteins are associated with a range of kidney disorders caused either by direct action on kidney cells or deposition in various kidney cells. Currently, these disorders are classified as monoclonal gamma globulinemia with kidney damage (MGRS). As used herein, the term "parenteral" refers to delivery to the body by injection (i.e., administration by injection), such as subcutaneous (i.e., injection just beneath the skin), intramuscular (i.e., injection into the muscle), intravenous (i.e., injection into a vein), subarachnoid (i.e., injection into the space around the spinal cord or the subarachnoid space of the brain), or by infusion techniques. The term “peptide” is used herein to refer to a series of amino acid residues typically linked to each other by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. A peptide is typically nine amino acids long, but may be eight short amino acids or fourteen long amino acids. A series of amino acids longer than approximately 14 amino acids, typically up to approximately 30–40 residues, is considered an “oligopeptide.” If the length of the amino acid residues exceeds 40, the series of amino acid residues is called a “polypeptide.”

[0249] As used herein, the term "perforin" refers to a molecule that can be inserted into the membrane of a target cell to promote the lysis of that target cell. Perforin-mediated lysis is enhanced by enzymes called granzymes. The term “peripheral blood mononuclear cell” or “PBMC” is used herein synonymously to refer to blood cells having a single round nucleus, such as lymphocytes or monocytes. PBMCs are an important component of the immune system’s response to infection. As used herein, the term "plasma cell" refers to a terminally differentiated B cell that secretes antibodies. These cells may be short-lived or long-lived, without undergoing isotype switching or somatic hypermutation, meaning they may have undergone isotype switching or somatic hypermutation.

[0250] Plasma cell differentiation. In bone marrow and fetal liver, precursor B lineage cells differentiate into immature B cells through a series of developmental steps characterized by the sequential rearrangement of immunoglobulin (Ig) heavy (H) and light (L) chain gene loci, expression of H and L chain proteins, their association to produce complete Ig molecules, and testing of the function of membrane-bound surface IgM (mIgM) that, together with Igα and Igβ, form the B cell receptor (BCR) for the antigen [Vale, AM et al. Development and function of B cell subsets. Chapter 7 in Molecular Biology of B cells, 2d Ed. Frederick W. Alt, Tasuku Honjo, Andreas Radbruch, Michael Reth, Eds., Elsevier / Academic Press, New York London (2015) pp. 99, citing Hardy, RR and Hayakawa, K. Annu. Rev. Immunol. (2001) 19: 595-621; Hardy, RR]. in. Chapter 8, B. Lymphocyte Development and Biology, Fundamental Immunology, Paul, W. Ed. 7 th[Ed. Wolters Kluwer (2013) Philadelphia 215-45]. Once these newly formed, immature B cells leave the tissue in which they were born, they undergo further selection steps as they enter the pool of mature B lymphocytes. [Ibid., citing Rolink, AG et al. Immunol. Rev. (2004) 197: 41-50]. These steps involve a series of developmental programs that include checkpoints to assess the composition, specificity, and reactivity of the BCRs carried by each individual lymphocyte. [Ibid., citing Nemazee, D. and Weigert, MJ Exp. Med. (2000) 191: 1813-17; Nemazee, D. Nat. Rev. Immunol. (2006) 6: 728-40]. These programs and checkpoints ultimately result in the generation of a set of mature B cells that present a diverse BCR repertoire capable of reacting with both a wide range of ancient and novel antigens [citing Goodnow, CC. Ann. NY Acad. Sci. (1997) 815: 55-66; Rolink, A. and Melchers, F. Immunol. Lett. (1996) 54: 157-61; Osmond, DC et al. Imunology Today (1998) 19: 65-8]. Cells within this B cell pool can be divided into subsets of mature IgM-bound B lymphocytes based on characteristic phenotypic differences in surface molecule expression, differences in anatomical arrangement, and differences in responses to immunological stimuli.

[0251] B cells are expected to be activated by antigens and, upon contact with the antigen, T cells, and / or cytokines, initiate proliferation, at which point the cells grow larger and resemble blast cells. Among antibody-secreting cells, plasmablasts are one distinct stage in the proliferation of antibody-secreting B cells [Tatlinton, D. Chapter 14b. “Plasma Cell Biology” in Molecular Biology of B cells, 2d Ed. Frederick W. Alt, Tasuku Honjo, Andreas Radbruch, Michael Reth, Eds., Elsevier / Academic Press, New York London (2015) pp. 232-35.

[0252] Plasmablasts (PBs) are B cells in lymph nodes that already exhibit some characteristics of plasma cells. They express BCRs, costimulatory molecules, and many markers of B cell lineages, such as B220 (an isoform specific to CD45 B cells) and CD80 / 86, and mechanisms related to antigen presentation mediated by PBs that secrete major histocompatibility complex II expression and Ig isotypes can be detected [citation from Oracki, SA et al. Immunol. Rev. (2010) 237: 140-59]. PBs are inherently short-lived, regardless of the properties of the antigen.

[0253] Plasma cells are found in the medulla of lymph nodes, red pulp, bone marrow, and mucosal tissues, and are antibody-secreting cells after mitosis. The conversion from B cells to plasma cells requires a dramatic change in the cell's transcriptional program [citing Nutt, SL, et al. Semin. Immunol. (2011) 23: 341-9], according to this model, in which B cells must silence the genes that define B cell identity and function, and express the corresponding genes related to plasma cell identity and function. Each state is relatively stable and maintained by master regulators, e.g., Pax5 and Bcl6 in the B cell state, and interferon regulator 4 (IRF4) and Blimp1 in the plasma cell state. Numerous models have been proposed, but none have identified a model that explains the key features of the molecular regulation of plasma cell differentiation. Blimp-1 primarily acts as a transcriptional repressor; its targets include Pax5 and Bcl6, genes that maintain B cell identity overall and within germinal centers, respectively.

[0254] Plasma cells are inherently short-lived, and most die within a day if, for example, they are removed from their in vivo location and placed in an in vitro culture. Factors that can maintain plasma cells include solubility and membrane-binding factors. IL6, TNF-alpha, CXCL12, APRIL, and BAFF all have the ability to support plasma cell survival, as do VEGF-1 and ICM-1 (VCAM-1, an α4β1 integrin that is a leukocyte ligand for fibronectin and osteopontin, and is involved in B cell acquisition of antigens and subsequent activation of them, lowering the activation threshold) and lymphocyte function-associated antigen-1 [LFA-1] (a member of the heterodimeric B2 integrin family), which bind to ultra-late antigen-4 (VLA-4), respectively, as well as hyaluronic acid and fibronectin which bind to CD44. In addition, there are survival factors that are thought to be active in inflammatory situations, specifically CXCL9, 10, and 11, which bind to CXCR3 [citing Winter, O. et al. J. Immunol. (2012) 189: 5105-11]. The T cell costimulatory molecule CD28 has been identified as providing a survival signal to plasma cells by binding to its ligand CD80 / 86 [citing Rozanski, CH et al. J. Exp. Med. (2011) 208: 1435-46]. Since CD28 is repressed by Pax5 in B cells, its early re-expression in plasma cells perfectly coincides with the loss of its Pax5 activity [citing Delogu, A. et al. Immunity (2006) 24: 269-81].

[0255] The term "plasmablast," as used herein, refers to the proliferative progeny of activated B cells. Plasmablasts develop into plasma cells. Antigen binding to the BCR initiates the activation of Src family kinases such as Lyn and Fyn, leading to phosphorylation of Igα (CD79a) and Igβ (CD79b), recruitment of Syk kinase, and subsequent recruitment and phosphorylation of BLNK, Btk, and PLCγ [Luo, W. et al. J. Immunol. (2014) 193(2): 909-20, citing Packard, TA & Cambier, JC. F1000 prime reports (2013) 5: 40]. These events activate the Ras pathway, PKC pathway, and calcium efflux, ultimately initiating the activation of NF-κB, Erk, and JNK. These positive signals are typically counteracted by negative signals that limit B cell activation and prevent spontaneous B cell proliferation and differentiation into plasma cells [citing Nitschke, L. Curr. Opin. Immunol. (2005) 17: 2990-97]. Negative signals are generated by a set of membrane receptors (CD22, CD72, FcγRIIb, PIR-B, Siglec-G, etc.) that are phosphorylated by Lyn. This reverses the phosphorylation of signaling molecules in the BCR pathway, allowing them to recruit phosphatases such as SHP1 and SHIP1, which reduce BCR signaling [citations: Poe, JC & Tedder, TF, Trends Immunol. (2012) 33: 413-20; Tsubata, T. Infectious disorders drug targets (2012) 12: 181-90; Vang, T. et al. Annu. Rev. Immunol. (2008) 26: 29-55].

[0256] The term "priming," as used herein, refers to the first encounter with a given antigen that generates a primary adaptive immune response. The term "unprimed cells" (also referred to as untreated, naive, or immature cells), as used herein, refers to T cells and B cells that have produced antigen receptors with specific specificity (TCRs in the case of T cells, and BCRs in the case of B cells) but have not yet encountered an antigen. For example, antigen-specific T cell precursors must be primed before helper T cells and B cells can interact to produce specific antibodies. Priming involves several steps: antigen uptake, processing, and cell surface expression bound to class II MHC molecules, recirculation and antigen-specific trapping of helper T cell precursors in lymphoid tissue, and T cell proliferation and differentiation. [Janeway, CA, Jr., “The priming of helper T cells, Semin. Immunol. (1989) 1(1): 13-20]. Helper T cells express CD4, but not all CD4 T cells are helper cells. Ibid. The signals required for the clonal expansion and proliferation of helper T cells differ from those required by other CD4 T cells. The antigen-presenting cell important for helper T cell priming appears to be macrophages, and the second important signal for helper T cell proliferation is the macrophage product interleukin-1 (IL-1). Ibid. When primed T cells and / or B cells receive a second co-stimulatory signal, they become activated T cells or B cells.

[0257] As used herein, the term "progression" refers to the course of a disease that begins to worsen or spreads within the body. The term “progression-free survival” or “PFS,” as used herein, refers to the length of time during and after treatment for a disease in which a patient is alive with the disease but whose disease does not progress. The term “proliferate” and its various grammatical forms, as used herein, mean the process that results in an increase in the number of cells, defined by the balance between cell division and cell loss through cell death or differentiation. As used herein, the term “proteasome” refers to a multicatalyzed proteinase complex that is crucial for the regulated degradation of cellular proteins into peptides by the ubiquitin-proteasome system (UPS).

[0258] The term "recurrent cancer" or "recurrence" usually refers to cancer that reappears after a period of time when the cancer was undetectable. The cancer may reappear in the same location as the primary tumor, or it may reappear in a different location within the body. The terms "refractory cancer" or "resistant cancer" refer to cancer that does not respond to treatment. Cancer may be resistant at the start of treatment, or it may become resistant during treatment. The term "relapse" refers to the return of a disease or its signs and symptoms after a period of improvement. The term “recurrence-free survival” (RFS) or “disease-free survival” (DFS) refers to the length of time a patient survives after the completion of primary treatment for cancer without any signs or symptoms of that cancer.

[0259] The term "RUNX1," as used herein, refers to the gene that provides instructions for the production of runt-related transcription factor 1 (RUNX1). The RUNX1 transcription factor helps regulate the activity of specific genes by binding to specific regions of DNA and interacting with core-binding factor beta (CBFβ), a protein produced from the CBFB gene, thereby helping RUNX1 bind to DNA and prevent its degradation. The RUNX1 transcription factor activates genes that help regulate the emergence and regulation of hematopoietic stem cells. [Swiers, G. et al. Int. J. Deve. PBiol. (2010) 54 (6-7): 1151-63]. The transcription factor RUNX1 is the fusion partner (ETO) of RUNX1T1 in relapsed t(8;21)(q22;q22) metastases, which are present in 8–13% of adult patients with novel acute myeloid leukemia (AML). [Greif, PA et al. Haematologica (2012) 97 (120): 1909-15].

[0260] The term “symptom” as used herein refers to evidence of disease provided by a healthcare provider. As used herein, the term "SLAM (signaling lymphocyte-activating molecule)" refers to a family of related cell surface receptors that mediate adhesion between lymphocytes, including, for example, SLAM, 2B4, CD84, LLy106, Ly9, and CRACC. The term “standard treatment,” as used herein, refers to a treatment for a disease that is approved by a physician and widely used. As used herein, the term "stimulate" means, in any of its grammatical forms, to induce activation or increase activation.

[0261] The terms “stimulate immune cells” or “to stimulate immune cells,” as used herein, mean a process that causes or results in a cellular response, such as the activation and / or proliferation of immune cells, such as CD8+ T cells (including, for example, signaling events or stimuli). The terms “subject,” “individual,” or “patient” are used synonymously and refer to members of animal species of mammalian origin, including, but not limited to, mice, rats, cats, goats, sheep, horses, hamsters, ferrets, pigs, dogs, guinea pigs, rabbits, and primates such as monkeys, apes, or humans. When used herein, the term "subject having acute myeloid leukemia" refers to a subject exhibiting diagnostic markers and / or symptoms associated with acute myeloid leukemia, or a subject diagnosed with acute myeloid leukemia. When used herein, the phrase “Persons requiring it” means (i) eligible patients who are expected to be administered an immunotherapy agent in accordance with the disclosure of the present invention; (ii) eligible patients who have received at least one immunotherapy in accordance with the disclosure of the present invention; or (iii) eligible patients who have previously received at least one immunotherapy in accordance with the disclosure of the present invention, unless the context and use of the phrase indicate otherwise.

[0262] As used herein, the term "symptoms" refers to a patient's subjective evidence of illness. The terms "T cell" or "T lymphocyte," used synonymously, refer to cells that mediate diverse immunological functions, such as the ability to help B cells develop into antibody-producing cells, the ability to enhance the bactericidal activity of monocytes / macrophages, the inhibition of certain types of immune responses, the direct killing of target cells, and the recruitment of inflammatory responses. These actions depend on the expression of their specific cell surface molecules and the secretion of cytokines. T cells mediate their function by recognizing antigens on the surface of antigen-presenting cells (APCs), interacting with these APCs, and altering their behavior. T cells can also be classified based on their function into helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells. T cell activation depends on the interaction between the TCR / CD3 complex and peptides bound to the grooves of its homologous ligands, class I or class II MHC molecules. The molecular events initiated by receptor engagement are complex. Among the earliest steps, the activation of tyrosine kinases appears to result in the tyrosine phosphorylation of a set of substrates that control numerous signaling pathways. These include adapter proteins that link the TCR to the ras pathway, phospholipase Cγ1 (whose tyrosine phosphorylation increases its catalytic activity, leading to increased intracellular free calcium concentration and activation of protein kinase C by engaging the inositol phospholipid metabolic pathway), and a set of other enzymes that regulate cell proliferation and differentiation. Full T cell responsiveness requires not only receptor engagement but also co-stimulatory activity delivered by accessory cells, such as the engagement of CD28 on T cells by CD80 and / or CD86 on antigen-presenting cells (APCs).

[0263] While the phylogenetic relationships between T cell subsets remain a subject of debate, T cell clusters within a population can be arranged as a progressive continuum based on phenotypic, functional, and transcriptional characteristics. T lymphocytes transition through progressive differentiation stages characterized by a stepwise loss of function and therapeutic potential, in the order of naive T (TN) cells to T memory stem cells (TSCMs) (T cells that have experienced the most immature antigens), T central memory (TCM) cells that patrol central lymphoid organs, and T effector memory (TEM) cells that patrol peripheral tissues. In contrast to TN cells, memory T cells are capa...

Claims

1. A method for treating an eligible subject having acute myeloid leukemia (AML) with high-risk disease characteristics, comprising administering a targeted immunotherapy agent, including an immunotherapy agent that specifically targets B cell maturation antigen (BCMA), to the intensified subject.

2. a. Is the eligible subject with AML a child? b. Whether the eligible subject with AML is a child under two years of age; c. Whether the eligible subject with AML is a teenager aged 13–19 years, including upper and lower limits; d. Whether the eligible subject with AML is an adult; e. Whether the eligible subject with AML is an adult under the age of 60; f. Whether the eligible subjects with AML are young adults aged 26 to 50 years, including the upper and lower limits; g. The eligible subject with AML is an adult aged 60–69 years, including upper and lower limits; or h. The method according to claim 1, wherein the eligible subject having AML is 70 years of age or older.

3. The method according to claim 1, wherein the characteristics of a high-risk disease include biological characteristics, clinical characteristics, or both.

4. The method according to claim 3, wherein the high-risk biological features include a pre-existing hematological disorder, the presence of 20% or more BM blasts in the bone marrow, 0, 1, 2, 3 or more clonal cytogenetic abnormalities, and molecular abnormalities.

5. The method according to claim 4, wherein the preceding hematological disorder is myelodysplastic syndrome, refractory AML, AML in remission, or mixed phenotypic acute leukemia.

6. The method according to claim 3, wherein the clinical features include comorbidities, measurable residual disease at complete remission, resistance to induction chemotherapy; remission after induction therapy, subjects who relapsed during remission, AML due to a prior hematological disorder; and AML in elderly patients.

7. The method according to claim 4, wherein the high-risk molecular features are FLT3 mutations, NPM1 mutations; isocitrate dehydrogenase 1 or 2 (IDH1 / 2) mutations, RUNX1 mutations, DNMT3A mutations, TET2 mutations, TP53 mutations, or combinations thereof.

8. The method according to claim 7, wherein the FLT3 mutation is an FLT3-ILD mutation.

9. The method according to claim 4, wherein high-risk cytogenetic features include chromosomal translocations and monosomy of somatic cell chromosomes.

10. The method according to claim 9, wherein the translocation is t(8;21)(q22;q22.1);RUNX1-RUNX1T1).

11. The method according to claim 6, wherein the high risk characteristics in elderly individuals include one or more of the following: a high incidence of cytogenetic abnormalities including comorbidities, monosomy 5 and 7 and chromosome 17 abnormalities, a high incidence of multiple mutations including TP53, and a high incidence of secondary / therapy-related AML.

12. The method according to claim 11, wherein the comorbidity includes one or more of the following: hypertension; diabetes; and organ dysfunction, including abnormalities of the heart, lungs, and kidneys.

13. Immunotherapy drugs, a. Immune cell engagers ("ICE") selected from T cell engagers, natural killer (NK) cell engagers and cytotoxic / phagocytic cell engagers; and / or b. Immunotherapy agents including immune checkpoint inhibitors, and / or c. Gamma secretase inhibitors The method according to claim 1, including the method described in claim 1.

14. a. The immune cell engager is a bispecific T cell engager (BiTE) containing a BCMA-targeting molecule linked to a CD3-targeting molecule via a peptide linker, wherein the CD3-targeting molecule activates a specific chain of the CD3 complex associated with the T cell receptor (TCR) complex and participates in the assembly of immune synapses that mediate BCMA-specific cytotoxicity; or b. The immune cell engager is a CD16 or NKG2D receptor-targeted bispecific NK cell engager that activates NK cells that target BCMA-specific cytotoxicity; or c. The method according to claim 13, wherein the immune engager is a chemically linked bispecific molecule that engages the non-ligand binding site of a high-affinity receptor for immunoglobulin G (also known as FcγRI or CD64), which is selectively expressed by a population of cytotoxic / phagocytic immune cells that target BCMA antibody-dependent cell-mediated cytotoxicity of AML blasts.

15. The method according to claim 14, wherein the molecule that targets BCMA is an scFv fragment that targets BCMA, and the molecule that targets CD3 is an scFv fragment that targets CD3.

16. The method according to claim 14, wherein the population of cytotoxic / phagocytic immune cells expressing CD64 is a population of monocytes, macrophages, dendritic cells, or cytokine-activated neutrophils.

17. The method according to claim 13, wherein the T cell immune engager is a BiTE that targets CD3 on T cells and BCMA expressed on relapsed / refractory AML blasts.

18. The method according to claim 17, wherein BiTE is tecristamatab, erranatamab, AMG-701, AMG420 (formerly BI836909), REGN5458, or TNB-383B.

19. The method according to claim 13, wherein the checkpoint inhibitor is lambrolizumab / pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), atezolizumab, TECONTRIQ®, or ipilimumab (YERVOY®).

20. The method according to claim 13, wherein the gamma-secretase inhibitor is LY3039478 / JSMD194, dihydroergocristine (DHEC), RO4929097; LY900009; MK-0752; PF-03084014; BMS-986115; GSI-136; AL-101; or nilogacestat.

21. The method according to claim 1, wherein the immunotherapy agent comprises a BCMA-targeted antibody drug conjugate or a BCMA-targeted CAR-T cell therapy agent as described in Table 1.

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