Method for regulating regulatory T cells, regulatory B cells, and immune responses using an APRIL-TACI interaction modulator.

Abstract

The present invention is based, in part, on a method for modulating regulatory T cells, regulatory B cells, and immune responses using modulators of APRIL-TACI interactions, including a method for selectively modifying the number and / or inhibitory immune activity of regulatory T cells (Tregs) and / or regulatory B cells (Bregs) in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one agent that modulates the interaction between a TACI receptor protein expressed by the Tregs and / or Bregs and an APRIL ligand, thereby selectively modifying the number and / or inhibitory immune activity of the Tregs and / or Bregs. [Selected Figure] Figure 1

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 522,167, filed Jun. 20, 2017; U.S. Provisional Application No. 62 / 573,264, filed Oct. 17, 2017; and U.S. Provisional Application No. 62 / 677,265, filed May 29, 2018, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0002] Statement Regarding Rights This invention was made with government support under Grant Nos. P50 CA100707 and R01 CA050947 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0003] The onset and progression of multiple myeloma (MM) are associated with genetic abnormalities and changes that occur in the bone marrow (BM) microenvironment, which promote the growth of malignant plasma cells (PCs) while suppressing host immunity. Indeed, MM is characterized by recurrent infections due to immunodeficiency and bone lesions due to enhanced osteoclast (OC) function. Moreover, the suppressive immune microenvironment underlies drug resistance and disease recurrence. However, to date, the regulatory mechanisms of MM - related immune cell dysfunction have not been fully characterized.

[0004] Regulatory T cells (Tregs) (conventionally defined as CD4+CD25+Foxp3+) are essential components of the immune surveillance system that maintains immune homeostasis and self-tolerance (Sakaguchi et al. (2008) Cell 133:775-787). Tregs are broadly classified into native Tregs (nTregs) derived from thymus-derived CD4+CD8+ T cells and peripheral Tregs (iTregs) induced from naive CD4+ T cells, depending on their lineage (Knutson et al. (2007) Cancer Immunol.Immunother.56:271-285). The latter are generated via intercellular contact and / or cytokine-dependent mechanisms, namely TGF-β and IL-10, to inhibit cellular and humoral immune responses (Campbell et al. (2001) J.Immunol.167:553-561). The functions of nTregs and iTregs are quite similar, making it difficult to distinguish between them. Recently, Tregs have been associated with long-surviving PCs in BM, further suggesting their role in regulating homeostasis in PC populations (Zaretsky et al. (2017) Cell Rep. 18: 1906-1916).

[0005] There is growing evidence that increased Treg counts contribute to dysfunction of the antitumor immune response, leading to immune evasion and progression in solid tumors and hematological malignancies, including MM (Fridman et al. (2012) Nat. Rev. Cancer 12:298-306, Tanaka et al. (2017) Cell Res. 27:109-118, Nishikawa et al. (2014) Curr. Opin. Immunol. 27:1-7, Kiniwa et al. (2007) Clin. Cancer Res. 13:6947-6958, Beyer et al. (2006) Blood 107:3940-3949, Feyler et al. (2009) Br. J. Haematol. 144:686-695, Raja et al. (2012) PloS One 7:e47077, Feng et al. (2017) Clin. Cancer Res. 23:4290-4300). Tumor cells can positively interact with Treg cells in the tumor microenvironment, inhibiting tumor-specific CD8+ and CD4+ T effector cell function and exhausting effector cells (Marabelle et al. (2013) J. Clin. Invest. 123:2447-2463, Bulliard et al. (2014) Immunol. Cell Biol. 92:475-480, Paiva et al. (2016) Blood 127:1151-1162, Arce Vargas et al. (2017) Immunity 46:577-586). In MM patients, the proportion of functionally circulating Tregs in T cells was increased, which correlated with disease burden and a higher risk of progression (Beyer et al. (2006) Blood 107:3940-3949, Feyler et al. (2009) Br.J. Haematol. 144:686-695, Raja et al. (2012) PloS One 7:e47077, Feng et al. (2017) Clin. Cancer Res. 23:4290-4300, Giannopoulos et al. (2012) Br.J. Cancer 106:546-552, Raja et al. (2012) PloS One 7:e49446).Elevated levels or numbers of Treg cells in MM patients may be derived from naive CD4 T cells stimulated by tumor cells and tumor bystander cells (Feng et al. (2017) Clin. Cancer Res. 23:4290-4300, Whiteside et al. (2012) Expert Opin. Biol. Ther. 12:1383-1397, Adeegbe et al. (2013) Front. Immunol. 4:190, Frassanito et al. (2015) Eur. J. Haematol. 95:65-74). As shown in ex vivo co-cultures, MM cells significantly induce the generation of iTregs from Tcones (Feng et al. (2017) Clin. Cancer Res. 23:4290-4300, Frassanito et al. (2015) Eur. J. Haematol. 95:65-74, Feyler et al. (2012) PloS One 7:e35981). CD38-expressing Tregs (both nTregs and iTregs) were identified and characterized as immunomodulators in MM patients (Feng et al. (2017) Clin. Cancer Res. 23:4290-4300, Krejcik et al. (2016) Blood 128:384-394, Tai et al. (2016) Blood 128:318-319). Importantly, therapeutic CD38-targeted monoclonal antibodies (mAbs) deplete CD38-expressing Tregs and stimulate effector T and NK cell function (Feng et al. (2017) Clin. Cancer Res. 23:4290-4300, Tai et al. (2017) Oncotarget 8:112166-112167, Krejcik et al. (2016) Blood 128:384-394). Overexpression of Foxp3 and CTLA-4 in BM samples further supports the local accumulation of immunosuppressive Tregs in the MM microenvironment (Braga et al. (2014) Cancer Immunol. Immunother. 63:1189-1197).Finally, MM cells directly drive Treg cells via a positive feedback loop in transplanted mouse models, promoting disease progression and poor outcomes (Kawano et al. (2018) J. Clin. Invest. DOI: 10.1172 / JCI88169).

[0006] The proliferation-inducing ligand (APRIL), an essential growth and survival factor for PCs, binds with high affinity to B-cell maturation antigen (BCMA), the most specific MM antigen expressed at high levels in malignant PCs in all MM patients (Carpenter et al. (2013) Clin. Cancer Res. 19:2048-2060, Tai et al. (2014) Blood 123:3128-3138). Most recently, targeting of BCMA with novel immunotherapies has achieved excellent clinical responses in relapsed and refractory MM (Carpenter et al. (2013) Clin. Cancer Res. 19:2048-2060, Tai et al. (2014) Blood 123:3128-3138, Tai et al. (2015) Immunotherapy 7:1187-1199, Ali et al. (2016) Blood 128:1688-1700, Mikkilineni et al. (2017) Blood 130:2594-2602). Constitutive activation of APRIL / BCMA signaling in vivo promotes MM cell progression and induction of immunosuppressants in MM cells (Tai et al. (2016) Blood 127:3225-3236). In addition, MM cell growth was significantly reduced in APRIL-deficient SCID mice, indicating that APRIL alone can induce MM progression in vivo (Matthes et al. (2015) Leukemia 29:1901-1908). OCs supporting myeloma produce APRIL (Moreaux et al. (2005) Blood 106:1021-1030, Tucci et al. (2011) Exp. Hematol. 39:773-783, Yaccoby et al. (2008) Leukemia 22:406-413, Abe et al. (2006) Leukemia 20:1313-1315) and PD-L1 in the melanoma (An et al. (2016) Blood 128:1590-1603), and OCs further block autologous T cell proliferation via immune checkpoint molecules including PD-L1 (An et al. (2016) Blood 128:1590-1603).However, it remains unknown whether Tregs mediate OC-induced immunosuppression, and whether APRIL controls these processes.

[0007] APRIL also binds to transmembrane activators, calcium modulators, and cyclophyllin ligand interactors (TACIs) (Marsters et al. (2000) Current Biol. 10:785-788), and these TACIs are expressed at lower levels and frequencies in patient MM cells compared to BCMA (Moreaux et al. (2005) Blood 106:1021-1030, Tai et al. (2006) Cancer Res. 66:6675-6682). Unlike BCMA, which is important only in long-term surviving and malignant PCs but not in normal B cells, TACI can negatively or positively regulate the B cell response (Yan et al. (2001) Nat.Immunol.2:638-643, Castigli et al. (2005) J.Exp.Med.201:35-39, Sakurai et al. (2007) Blood 109:2961-2967, Tsuji et al. (2011) Blood 118:5832-5839, Garcia-Carmona et al. (2015) Blood 125:1749-1758). Results from TACI and APRIL knockout mice demonstrate their roles in serum IgA production (Yan et al. (2001) Nat.Immunol.2:638-643, von Bulow et al. (2001) Immunity 14:573-582, Castigli et al. (2004) Proc.Natl.Acad.Sci.USA101:3903-3908, Planelles et al. (2004) Cancer Cell 6:399-408), and TACI requires heparan sulfate proteoglycan (i.e., CD138) for APRIL-induced IgA production (Sakurai et al. (2007) Blood 109:2961-2967, Guadagnoli et al. (2011) Blood 117:6856-6865). However, it is unclear whether APRIL directly acts on immunoregulatory T-lineage and B-lineage cells via TACI to downregulate effector T cells in MM.

[0008] Therefore, CD4 + CD25 高 FoxP3 高 Regulatory T cells (Tregs), such as T cells, are important regulators of the immune response because they inhibit immune effector cells (Feng et al. (2017) Clin. Cancer Res. DOI:10.1158 / 1078-0432. CCR-16-3192, Hori et al. (2003) Science 299:1057-1061, Fontenot et al. (2003) Nat. Immunol. 4:330-336, Vignali et al. (2008) Nat. Rev. Immunol. 8:523-532, Josefowicz et al. (2012) Annu. Rev. Immunol. 30:531-564, Shevach and Thornton (2014) Immunol. Rev. 259:88-102, Smigiel et al. al. (2014) Immunol. Rev. 259: 40-59). Similarly, CD19 + CD24 高 CD38 高Regulatory B cells (Bregs), such as B cells, are important regulators of the immune response because they also inhibit immune effector cells. In particular, Bregs suppress the immune response mainly through the production of the anti-inflammatory cytokine interleukin-10 (IL-10), and also regulate the activation and differentiation of CD4+ T cells (Zhang et al. (2017) Blood Cancer J.24:e547, Rosser et al. (2015) Immunity 42:607-612). Since Tregs and Bregs are involved in many diseases such as autoimmune states, cancer, and infections, it is desirable to regulate the number and / or inhibitory immune activity of Tregs and / or Bregs (Rosenblum et al. (2012) Science Transl. Med. 4:125sr121, Chapman and Chi (2014) Immunother. 6:1295-1311, Bluestone et al. (2015) J. Clin. Invest. 125:220-2260). However, selectively regulating the number and / or inhibitory immune activity of Tregs and / or Bregs is a challenge in this field because the genes and pathways expressed by these cells and related to cell growth, survival, and / or inhibitory immune activity are generally shared with the genes and pathways of other immunomodulatory cells such as effector T cells. Therefore, there is a great need in this field to identify and target genes and pathways that are selectively expressed by Treg and / or Breg, which control their cell growth, survival, and / or inhibitory immune activity, thereby enabling selective modification of these properties within Treg and / or Breg. Therefore, there is a great need in this field to understand the mechanisms of immune regulation in the tumor environment and to identify and target genes useful for cancer prevention and treatment in this pathway. In addition, there is a great need in this field to understand, identify, and target the pathways that are selectively expressed by Treg and / or Breg, which control their cell growth, survival, and / or inhibitory immune activity, thereby enabling selective modification of these properties in Treg and / or Breg. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Sakaguchi et al.(2008)Cell 133:775-787 [Non-Patent Document 2] Knutson et al.(2007)Cancer Immunol.Immunother.56:271-285 [Non-Patent Document 3] Campbell et al. (2001) J. Immunol. 167:553-561 [Non-Patent Document 4] Zaretsky et al.(2017)Cell Rep.18:1906-1916 [Non-Patent Document 5] Fridman et al.(2012)Nat.Rev.Cancer12:298-306 [Non-Patent Document 6] Tanaka et al.(2017)Cell Res.27:109-118 [Non-Patent Document 7] Nishikawa et al.(2014)Curr.Opin.Immunol.27:1-7 [Non-Patent Document 8] Kiniwa et al.(2007)Clin.Cancer Res.13:6947-6958 [Non-Patent Document 9] Beyer et al. (2006) Blood 107:3940-3949

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Summary of the Invention

Means for Solving the Problems

[0010] The present invention is at least partially based on the discovery that APRIL promotes immunosuppression in cancer cells through its interaction with TACI. For example, APRIL signaling via TACI significantly upregulates the proliferation, survival, and immunosuppressive functions of both Tregs and Bregs. Furthermore, targeting APRIL, alone and in combination with PD1 / PD-L1 blockade, reduces OC-induced immunosuppression in the tumor microenvironment. These findings provide a framework for targeting APRIL and / or the APRIL-TACI interaction to overcome immunosuppression, enhance the cytotoxic effects of cancer cells, and improve patient outcomes.

[0011] The present invention also is at least partially based on the fact that TACI, one of the two receptors for the APRIL ligand, is CD4 + CD25 高 FoxP3 高This is based on the finding that TACI is prominently expressed by regulatory T cells (Tregs), such as Tregs, while conventional T cells (Tcons), such as CD4+CD25- T cells, do not express TACI to a significant degree. The other receptor of the APRIL ligand, known as BCMA, is not expressed by either Tregs or Tcons. Similarly, regulatory B cells (Bregs) are also thought to express TACI. It is thought that the binding of APRIL to immune cells expressing TACI leads to the upregulation of growth and survival genes, and because TACI is selectively expressed by Tregs / Bregs, APRIL preferentially activates TACI in Tregs / Bregs rather than Tcons, selectively upregulating growth and survival genes in Tregs / Bregs, thereby increasing the number of Tregs / Bregs and / or inhibitory immune activity compared to Tcons, resulting in enhanced inhibitory immune function. Therefore, it is thought that modulating the APRIL / TACI interaction on Treg / Bregs enables selective modification (e.g., enhanced or reduced) or the number and / or inhibitory immune activity of Treg / Bregs, based on the direction of the APRIL / TACI interaction modulation (e.g., enhancement or reduction, respectively).

[0012] In one embodiment, a method is provided for selectively modifying the number and / or inhibitory immune activity of regulatory T cells (Treg) and / or regulatory B cells (Breg) in a subject, comprising administering a therapeutically effective amount of at least one agent to the subject, such that the number and / or inhibitory immune activity of Treg and / or Breg are selectively modified by modulating the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand.

[0013] Numerous embodiments are further provided, which may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the agent causes a reduction in the number of Treg and / or Breg and / or a reduction in the inhibitory immune activity of Treg and / or Breg by downregulating the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand, and optionally, the expression of IL10, PD-L1, and / or one or more growth or survival genes (e.g., MCL1, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3) is reduced. In another embodiment, the agent is a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interference agent, antisense oligonucleotide, peptide or peptide mimetic inhibitor, aptamer, or antibody. Still in another embodiment, the RNA interferant is a small interfering RNA (siRNA), CRISPR RNA (crRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA). Still in yet another embodiment, the RNA interferant is a CRISPR guide RNA (gRNA). Still in another embodiment, the agent comprises a blocking antibody or its antigen-binding fragment that specifically binds to a TACI receptor or APRIL ligand. Still in yet another embodiment, the antibody or its antigen-binding fragment is mouse, chimeric, humanized, composite, or human. Still in yet another embodiment, the antibody or its antigen-binding fragment is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. Still in another embodiment, the antibody or its antigen-binding fragment is conjugated to a cytotoxic agent. In yet another embodiment, the cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. In yet another embodiment, the method further includes administering an inhibitor of the STING pathway.In another embodiment, the agent increases the number of Treg and / or Breg and / or increases the inhibitory immune activity of Treg and / or Breg by upregulating the interaction between the TACI receptor protein expressed by Treg and / or Breg and the APRIL ligand, and optionally increases the expression of IL10, PD-L1, and / or one or more growth or survival genes (e.g., MCL1, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3). Still in yet another embodiment, the agent is a nucleic acid molecule encoding an APRIL ligand polypeptide or fragment thereof, an activated antibody or antigen-binding fragment thereof that specifically binds to the TACI receptor or APRIL ligand, or an antibody that specifically binds to both the TACI receptor and the APRIL ligand. Still in yet another embodiment, the antibody or antigen-binding fragment thereof is mouse, chimeric, humanized, complex, or human. In another embodiment, the antibody or its antigen-binding fragment is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. Still in yet another embodiment, the APRIL ligand polypeptide or fragment is a fusion protein. Still in yet another embodiment, the APRIL ligand polypeptide or fragment is fused to an Fc domain. In another embodiment, the method further comprises targeting an activator of the STING pathway (e.g., a STING agonist). Still in yet another embodiment, the method further comprises targeting at least one immunotherapy agent. Still in yet another embodiment, the immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines.In another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO1, IDO2, and A2aR. In yet another embodiment, the agent, either alone or in combination with a STING pathway inhibitor or activator and / or immunotherapeutic agent, i) does not significantly modulate the number of Tcones and / or immune activity, and / or ii) modulates immunomodulatory cytokine production in Tregs and / or Bregs. In yet another embodiment, the subject has cancer, and the agent, either alone or in combination with a STING pathway inhibitor or activator and / or immunotherapeutic agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing cancer cells, and optionally determines the responsiveness to the agent that modulates TACI receptor proteins expressed by Tregs and / or Bregs with APRIL ligands, measured by at least one criterion selected from the group consisting of clinical benefit rate, survival to death, pathological complete response, semi-quantitative measure of pathological response, clinical complete remission, clinical partial remission, clinical disease stabilization, recurrence-free survival, metastasis-free survival, disease-free survival, reduction of circulating tumor cells, circulating marker response, and RECIST criteria. In another embodiment, the method further comprises administering at least one additional therapeutic agent or regimen for treating cancer. Still in yet another embodiment, the agent, STING pathway inhibitor or activator, immunotherapy agent, and / or at least one additional therapeutic agent are administered non-systemically to a microenvironment containing Treg and / or Breg.

[0014] In another embodiment, a method is provided for selectively modifying the number and / or inhibitory immune activity of Treg and / or Bregs, comprising contacting Treg and / or Bregs with at least one agent such that the number and / or inhibitory immune activity of Treg and / or Bregs is selectively modified by modulating the interaction between TACI receptor proteins expressed by Treg and / or Bregs and APRIL ligands.

[0015] As described above, numerous embodiments are further provided, which may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the agent reduces the number of Treg and / or Breg and / or the inhibitory immune activity of Treg and / or Breg by downregulating the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand, and optionally reduces the expression of IL10, PD-L1, and / or one or more growth or survival genes (e.g., MCL1, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3). In another embodiment, the agent is a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interference agent, antisense oligonucleotide, peptide or peptide mimetic inhibitor, aptamer, or antibody. Still in another embodiment, the RNA interferant is a small interfering RNA (siRNA), CRISPR RNA (crRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA). Still in yet another embodiment, the RNA interferant is a CRISPR guide RNA (gRNA). Still in another embodiment, the agent comprises a blocking antibody or its antigen-binding fragment that specifically binds to a TACI receptor or APRIL ligand. Still in yet another embodiment, the antibody or its antigen-binding fragment is mouse, chimeric, humanized, complex, or human. Still in yet another embodiment, the antibody or its antigen-binding fragment is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. Still in another embodiment, the antibody or its antigen-binding fragment is conjugated to a cytotoxic agent. In yet another embodiment, the cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. In yet another embodiment, the method further includes administering an inhibitor of the STING pathway.In another embodiment, the agent increases the number of Treg and / or Breg and / or increases the inhibitory immune activity of Treg and / or Breg by upregulating the interaction between the TACI receptor protein expressed by Treg and / or Breg and the APRIL ligand, and optionally increases the expression of IL10, PD-L1, and / or one or more growth or survival genes (e.g., MCL1, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3). Still in yet another embodiment, the agent is a nucleic acid molecule encoding an APRIL ligand polypeptide or fragment thereof, an activated antibody or antigen-binding fragment thereof that specifically binds to the TACI receptor or APRIL ligand, or an antibody that specifically binds to both the TACI receptor and the APRIL ligand. Still in yet another embodiment, the antibody or antigen-binding fragment thereof is mouse, chimeric, humanized, complex, or human. In another embodiment, the antibody or its antigen-binding fragment is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. Still in yet another embodiment, the APRIL ligand polypeptide or fragment is a fusion protein. Still in yet another embodiment, the APRIL ligand polypeptide or fragment is fused to an Fc domain. In another embodiment, the method further comprises administering a STING pathway activator (e.g., a STING agonist) to the target. Still in yet another embodiment, the method further comprises contacting Treg and / or Breg with at least one immunotherapy agent. Still in yet another embodiment, the immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines.In another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO1, IDO2, and A2aR. Still in another embodiment, the agent, either alone or in combination with a STING pathway inhibitor or activator and / or immunotherapy agent, contacts Treg and / or Breg in the presence of Tcon to i) not significantly modulate the number and / or immune activity of Tcon, and / or ii) modulate immunomodulatory cytokine production in Treg and / or Breg. Still in another embodiment, the agent, either alone or in combination with a STING pathway inhibitor or activator and / or immunotherapy agent, contacts Treg and / or Breg in the presence of Tcon and cancer cells, and the agent, either alone or in combination with an immunotherapy agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing cancer cells. Still in another embodiment, the method further includes contacting cancer cells with at least one additional cancer treatment agent or regimen. Still in another embodiment, the agent, STING pathway inhibitor or activator, or immunotherapy agent, and / or at least one additional treatment agent contact Treg, Breg, Tcon, and / or cancer cells in vitro or ex vivo.

[0016] Still in another embodiment, a cell-based assay is provided for screening for agents that selectively modify the number and / or inhibitory immune activity of Treg and / or Breg, comprising contacting Treg and / or Breg with a test agent and determining the ability of the test agent to modulate the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand, wherein the test agent that modulates the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand selectively modifies the number and / or inhibitory immune activity of Treg and / or Breg.

[0017] As described above, numerous embodiments are further provided, which may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the contacting step occurs in vivo, ex vivo, or in vitro. In another embodiment, Treg and / or Breg are contacted with an inhibitor or activator of the STING pathway. Still in yet another embodiment, the activator of the STING pathway is a STING agonist. Still in yet another embodiment, Treg and / or Breg are contacted with at least one immunotherapy agent. In another embodiment, the immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines. In yet another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO1, IDO2, and A2aR. In yet another embodiment, Treg and / or Breg cells are contacted with the test agent, either alone or in combination with a STING pathway inhibitor or activator and / or immunotherapy agent, in the presence of Tcon cells, to determine i) a lack of significant modulation in the number and / or immune activity of Tcon cells, and / or ii) modulation of immunomodulatory cytokine production in Treg and / or Breg cells. In yet another embodiment, Treg and / or Breg cells are contacted with the test agent, either alone or in combination with a STING pathway inhibitor or activator, or immunotherapy agent, in the presence of Tcon cells and cancer cells, to determine a reduction in the number of proliferating cancer cells and / or a reduction in the volume or size of tumors containing cancer cells. Still in yet another embodiment, cancer cells are further contacted with at least one additional cancer treatment agent or regimen.

[0018] In another embodiment, the Treg is CD4+CD25+, CD4+FOXP3+, and / or CD4+CD25+FOXP3+ Treg, for example, CD4+CD25 高 In another embodiment, the Treg includes a FOXP3+ Treg. In yet another embodiment, the Breg is a CD19+CD24+CD38+ Breg, for example, a CD19+CD24 高 CD38 高 Contains Breg. In yet another embodiment, Tcon contains CD4+CD25-Tcon. In yet another embodiment, the subject has a condition in which upregulation of the immune response would be beneficial. Still in yet another embodiment, the subject has a condition selected from the group consisting of cancer, viral infection, bacterial infection, protozoan infection, helminthic infection, asthma associated with impaired airway tolerance, and immunosuppressive diseases. Still in yet another embodiment, the subject has cancer or the cell population contains cancer cells. In yet another embodiment, cancer is multiple myeloma. Still in yet another embodiment, cancer is an animal model of cancer, and optionally, the animal model is a mouse model. Still in yet another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a mouse or a human. Still in yet another embodiment, the mammal is a human. In certain embodiments, for example, the following are provided: (Item 1) A method for selectively modifying the number and / or inhibitory immune activity of regulatory T cells (Tregs) and / or regulatory B cells (Bregs) in a subject, comprising administering to the subject a therapeutically effective amount of at least one agent such that the number and / or inhibitory immune activity of the Tregs and / or Bregs are selectively modified by modulating the interaction between the TACI receptor protein expressed by the Tregs and / or Bregs and the APRIL ligand. (Item 2) The method according to item 1, wherein the agent downregulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, thereby reducing the number of the Treg and / or Breg and / or reducing the inhibitory immune activity of the Treg and / or Breg, and optionally reducing the expression of one or more growth or survival genes such as IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3. (Item 3) The method according to item 2, wherein the drug is a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interference agent, antisense oligonucleotide, peptide or peptide mimetic inhibitor, aptamer, or antibody. (Item 4) The method according to item 3, wherein the RNA interferant is a small interfering RNA (siRNA), CRISPR RNA (crRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA). (Item 5) The method according to item 3, wherein the RNA interferant is CRISPR guide RNA (gRNA). (Item 6) The method according to item 3, wherein the drug comprises a blocking antibody or an antigen-binding fragment thereof that specifically binds to the TACI receptor or the APRIL ligand. (Item 7) The method according to item 6, wherein the antibody or its antigen-binding fragment is mouse, chimeric, humanized, complex, or human. (Item 8) The method according to item 6 or 7, wherein the antibody or antigen-binding fragment is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and a diabody fragment. (Item 9) The method according to any one of items 6 to 8, wherein the antibody or its antigen-binding fragment is conjugated with a cytotoxic agent. (Item 10) The method according to item 9, wherein the cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. (Item 11) The method according to any one of items 1 to 10, further comprising administering an inhibitor of the STING pathway to the subject. (Item 12) The method according to item 1, wherein the agent upregulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, thereby increasing the number of the Treg and / or Breg and / or increasing the inhibitory immune activity of the Treg and / or Breg, and optionally increasing the expression of one or more growth or survival genes such as IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3. (Item 13) The method according to item 12, wherein the drug is a nucleic acid molecule encoding an APRIL ligand polypeptide or a fragment thereof, an APRIL polypeptide or a fragment thereof, an activated antibody or an antigen-binding fragment thereof that specifically binds to the TACI receptor or the APRIL ligand, or an antibody that specifically binds to both the TACI receptor and the APRIL ligand. (Item 14) The method according to item 12, wherein the antibody or its antigen-binding fragment is mouse, chimeric, humanized, complex, or human. (Item 15) The method according to item 13 or 14, wherein the antibody or its antigen-binding fragment is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and a diabody fragment. (Item 16) The method according to any one of items 1 to 15, wherein the APRIL ligand polypeptide or a fragment thereof is a fusion protein. (Item 17) The method according to item 16, wherein the APRIL ligand polypeptide or a fragment thereof is fused to the Fc domain. (Item 18) The method according to any one of items 12 to 17, further comprising administering a STING pathway activator to the subject. (Item 19) The method according to item 18, wherein the activator of the STING pathway is a STING agonist. (Item 20) The method according to any one of items 1 to 19, further comprising administering at least one immunotherapy agent to the subject. (Item 21) The method according to item 20, wherein the immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines. (Item 22) The method according to item 21, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO1, IDO2, and A2aR. (Item 23) The method according to any one of items 1 to 22, wherein the agent, either alone or in combination with the inhibitor or activator of the STING pathway and / or the immunotherapy agent, i) does not significantly modulate the number of Tcones and / or immune activity, and / or ii) modulates immunomodulatory cytokine production in the Tregs and / or Bregs. (Item 24) The method according to any one of items 1 to 23, wherein the subject has cancer, and the drug, either alone or in combination with the inhibitor or activator of the STING pathway and / or the immunotherapy agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing the cancer cells, and optionally determines the responsiveness to the drug that modulates the TACI receptor protein expressed by the Treg and / or Breg with APRIL ligand, as measured by at least one criterion selected from the group consisting of clinical benefit rate, survival to death, pathological complete response, semi-quantitative measure of pathological response, clinical complete remission, clinical partial remission, clinical disease stabilization, recurrence-free survival, metastasis-free survival, disease-free survival, reduction of circulating tumor cells, circulating marker response, and RECIST criteria. (Item 25) The method according to item 24, further comprising administering to the subject at least one additional therapeutic agent or regimen for treating the cancer. (Item 26) The method according to any one of items 1 to 25, wherein the drug, the inhibitor or activator of the STING pathway, an immunotherapy agent, and / or at least one additional therapeutic agent are administered non-systemically to a microenvironment containing Treg and / or Breg. (Item 27) A method for selectively modifying the number and / or inhibitory immune activity of Treg and / or Bregs, comprising contacting the Treg and / or Bregs with at least one agent such that the number and / or inhibitory immune activity of the Treg and / or Bregs is selectively modified by modulating the interaction between the TACI receptor protein expressed by the Treg and / or Bregs and the APRIL ligand. (Item 28) The method described in item 27, wherein the agent downregulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, thereby reducing the number of the Treg and / or Breg and / or reducing the inhibitory immune activity of the Treg and / or Breg, and optionally reducing the expression of one or more growth or survival genes such as IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3. (Item 29) The method according to item 28, wherein the drug is a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interferant, antisense oligonucleotide, peptide or peptide mimetic inhibitor, aptamer, or antibody. (Item 30) The method according to item 29, wherein the RNA interferant is a small interfering RNA (siRNA), CRISPR RNA (crRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA). (Item 31) The method according to item 29, wherein the RNA interferant is CRISPR guide RNA (gRNA). (Item 32) The method according to item 29, wherein the drug comprises a blocking antibody or an antigen-binding fragment thereof that specifically binds to the TACI receptor or the APRIL ligand. (Item 33) The method according to item 32, wherein the antibody or its antigen-binding fragment is mouse, chimeric, humanized, complex, or human. (Item 34) The method according to item 32 or 33, wherein the antibody or its antigen-binding fragment is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and a diabody fragment. (Item 35) The method according to any one of items 32 to 34, wherein the antibody or its antigen-binding fragment is conjugated with a cytotoxic agent. (Item 36) The method according to item 35, wherein the cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. (Item 37) The method according to any one of items 27 to 36, further comprising administering an inhibitor of the STING pathway to the subject. (Item 38) The method according to item 27, wherein the agent upregulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, thereby increasing the number of the Treg and / or Breg and / or increasing the inhibitory immune activity of the Treg and / or Breg, and optionally increasing the expression of one or more growth or survival genes such as IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and / or BIRC3. (Item 39) The method according to item 38, wherein the drug is a nucleic acid molecule encoding an APRIL ligand polypeptide or a fragment thereof, an APRIL polypeptide or a fragment thereof, an activated antibody or an antigen-binding fragment thereof that specifically binds to the TACI receptor or the APRIL ligand, or an antibody that specifically binds to both the TACI receptor and the APRIL ligand. (Item 40) The method according to item 38, wherein the antibody or its antigen-binding fragment is mouse, chimeric, humanized, complex, or human. (Item 41) The method according to item 39 or 40, wherein the antibody or its antigen-binding fragment is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and a diabody fragment. (Item 42) The method according to any one of items 27 to 41, wherein the APRIL ligand polypeptide or a fragment thereof is a fusion protein. (Item 43) The method according to item 42, wherein the APRIL ligand polypeptide or a fragment thereof is fused to the Fc domain. (Item 44) The method according to any one of items 38 to 43, further comprising administering a STING pathway activator to the subject. (Item 45) The method according to item 44, wherein the activator of the STING pathway is a STING agonist. (Item 46) The method according to any one of items 27 to 45, further comprising contacting the Treg and / or Breg with at least one immunotherapy agent. (Item 47) The method according to item 46, wherein the immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines. (Item 48) The method according to item 47, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO, IDO2, and A2aR. (Item 49) The method according to any one of items 27 to 48, wherein the agent, either alone or in combination with the inhibitor or activator of the STING pathway and / or the immunotherapy agent, contacts the Treg and / or Breg in the presence of Tcon, i) without significantly modulating the number and / or immune activity of the Tcon, and / or ii) modulates immunomodulatory cytokine production in the Treg and / or Breg. (Item 50) The method according to any one of items 27 to 49, wherein the agent, either alone or in combination with the inhibitor or activator of the STING pathway and / or the immunotherapy agent, contacts the Treg and / or Breg in the presence of Tcon and cancer cells, and the agent, either alone or in combination with the immunotherapy agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing the cancer cells. (Item 51) The method according to item 50, further comprising contacting the cancer cells with at least one additional cancer treatment agent or regimen. (Item 52) The method according to any one of items 27 to 51, wherein the drug, the inhibitor or activator of the STING pathway, or an immunotherapy agent and / or at least one additional therapeutic agent comes into contact with the Treg, Breg, Tcon, and / or cancer cells in vitro or ex vivo. (Item 53) A cell-based assay for screening for agents that selectively modify the number and / or inhibitory immune activity of Treg and / or Bregs, comprising contacting Treg and / or Bregs with a test agent and determining the ability of the test agent to modulate the interaction between the TACI receptor protein expressed by the Treg and / or Bregs and the APRIL ligand, wherein the test agent that modulates the interaction between the TACI receptor protein expressed by the Treg and / or Bregs and the APRIL ligand selectively modifies the number and / or inhibitory immune activity of the Treg and / or Bregs. (Item 54) A cell-based assay as described in item 53, wherein the contact step occurs in vivo, ex vivo, or in vitro. (Item 55) The cell-based assay according to item 53 or 54, further comprising contacting the Treg and / or Breg with an inhibitor or activator of the STING pathway. (Item 56) The cell-based assay described in item 55, wherein the activator of the STING pathway is a STING agonist. (Item 57) A cell-based assay according to any one of items 53 to 56, further comprising contacting the Treg and / or Breg with at least one immunotherapy agent. (Item 58) The cell-based assay described in item 57, wherein the immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines. (Item 59) The cell-based assay described in item 58, wherein the aforementioned immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophyllin, IDO1, IDO2, and A2aR. (Item 60) A cell-based assay according to any one of items 53 to 59, further comprising contacting the Treg and / or Breg with the test agent, either alone or in combination with the inhibitor or activator of the STING pathway and / or the immunotherapy agent, in the presence of Tcon, and determining i) a significant lack of regulation in the number and / or immune activity of Tcon, and / or ii) regulation of immunomodulatory cytokine production in the Treg and / or Breg. (Item 61) A cell-based assay according to any one of items 53 to 60, further comprising contacting the Treg and / or Breg with the test agent, either alone or in combination with the inhibitor or activator of the STING pathway and / or the immunotherapy agent, in the presence of Tcon and cancer cells, and determining a reduction in the number of proliferating cancer cells and / or a reduction in the volume or size of a tumor containing the cancer cells. (Item 62) A cell-based assay according to any one of items 59 to 61, further comprising contacting the cancer cells with at least one additional cancer treatment agent or regimen. (Item 63) The Treg is CD4+CD25+, CD4+FOXP3+, CD4+FoxP3+IL10+, CD4+FoxP3 高 IL10 高、 and / or CD4+CD25 高 A cell-based assay or method described in any one of items 1-62, including FOXP3+ Treg. (Item 64) The aforementioned Breg is CD19+CD24 高 CD38 高 A cell-based assay or method described in any one of items 1-63, including Breg. (Item 65) The method described in any one of items 1 to 64 or a cell-based assay, wherein the Tcon comprises CD4+CD25-Tcon. (Item 66) The method or cell-based assay described in any one of items 1 to 65, wherein the subject has a condition in which upregulation of the immune response would be beneficial. (Item 67) The method or cell-based assay described in item 66, wherein the subject has a condition selected from the group consisting of cancer, viral infection, bacterial infection, protozoan infection, helminthic infection, asthma associated with impaired airway tolerance, and immunosuppressive diseases. (Item 68) The method or cell-based assay described in any one of items 1 to 67, wherein the subject has cancer or the cell population contains cancer cells. (Item 69) The method or cell-based assay described in item 68, wherein the cancer is multiple myeloma. (Item 70) The method or cell-based assay described in item 68 or 69, wherein the cancer is an animal model of the cancer, and optionally, the animal model is a mouse model. (Item 71) The method or cell-based assay described in any one of items 1 to 69, wherein the subject is a mammal. (Item 72) The method according to item 71, wherein the mammal is a mouse or a human. (Item 73) The method described in item 72, wherein the mammal is a human. (Item 74) The method according to any one of items 1-10 and 27-45, further comprising administering a modulator of BCMA to the subject, or bringing the Treg and / or Breg into contact with the modulator of BCMA. [Brief explanation of the drawing]

[0019] [Figure 1] This study demonstrates that the anti-APRIL blocking antibody 01A, obtained from Aduro Biotech, blocks APRIL-induced and OC-induced multiple myeloma (MM) cell growth. [Figure 2] This study demonstrates that an anti-APRIL monoclonal antibody dose-dependently blocks APRIL-induced and OC-induced MM cell growth. [Figure 3]This study demonstrates that the anti-APRIL antibody 01A strongly inhibits the growth of APRIL-expressing MM cells compared to blocking APRIL-induced cell proliferation in parental RPMI8226 cells. Both APRIL and anti-APRIL are derived from Adipogen. [Figure 4] This study demonstrates that anti-APRIL mAbs strongly inhibit the growth of APRIL-expressing MM cells. [Figure 5] This study demonstrates that the anti-APRIL blocking antibody C4 more strongly blocks the proliferation of APRIL-expressing MM cells than O1A. [Figure 6] This study demonstrates that the anti-APRIL blocking antibody C4 selectively and potently inhibits APRIL-induced MM cell growth more strongly than O1A. [Figure 7] This study demonstrates that pre-incubation of APRIL in MM cells protects against daratumumab (Dara)-induced MM cell lysis, thereby indicating a therapeutic combination of anti-APRIL agents and Dara. [Figure 8] This study demonstrates that APRIL inhibits J6M0-induced MM1S cell lysis in a dose-dependent manner, thereby indicating a therapeutic combination of anti-APRIL agents with BCMA-related immunotherapies. J6M0 is a BCMA-specific anti-TNFRSF17 antibody. [Figure 9] This further demonstrates that APRIL inhibits J6M0-induced MM1S cell lysis in a dose-dependent manner, thereby indicating a therapeutic combination of anti-APRIL agents and BCMA-related immunotherapies. [Figure 10] This study demonstrates that C4(01A) overcomes J6M0-induced lysis, which is blocked by APRIL, in both MM cells sensitive to and resistant to current anti-MM therapies such as lenalidomide / pomalidomide. [Figure 11] This shows J6M0-induced ADCC using PBMC effector cells pretreated with C4 / 01A. [Figure 12] This study shows that when C4 was added during the ADCC assay, it did not alter anti-BCMA mAb-induced MM cell lysis. [Figure 13A]This study demonstrates that TACI is differentially expressed in Tregs compared to autologous Tcones from the same MM patient. For reference, the expression of other genes differentially expressed in Tregs compared to autologous Tcones (IL-10, CD38, Foxp3, CTLA-4, and TGFβ) is also shown (see also Feng et al. (2017) Clin. Cancer Res. 23:4290-4300 and Zhang et al. (2017) Blood Cancer J. 7:e547). The levels of the indicated Treg-related transcripts were examined in patient samples along with TACI. [Figure 13B] This study demonstrates that TACI is differentially expressed in Treg cells compared to self-Tcon cells. Treg cells were isolated from Tcon cells using CD3 T cells (T) derived from different donors (MM patients), followed by RNA extraction, and TACI transcripts were quantified by qRT-PCR. Foxp3, CTLA-4, and TGFβ served as control genes to identify Treg cells. After normalizing expression levels with the internal control GAPDH, relative expression levels between Treg and Tcon cells are shown. SLAMF7 is significantly and more highly expressed in Tcon cells compared to Treg cells in the self-controlled setting. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 14] The study shows that TACI protein levels are significantly higher on the surface of Tregs compared to Tcones from bone marrow and peripheral blood compartments derived from the same individual patient. TACI MFI for Tcones from paired Tregs derived from nine MM patients is presented. [Figure 15A] This study demonstrates that APRIL induces IL-10 expression in TACI-expressing Tregs compared to Tcon. [Figure 15B]This study demonstrates that APRIL induces Bcl2 and Bcl-xL expression in TACI-expressing Tregs compared to Tcon, and that this induction is suppressed by an antagonistic anti-APRIL antibody. Purified Tregs and paired Tcon (n=5) were incubated with APRIL over various timeframes. BCL2 and BCL2L1 expression levels were then determined by qRT-PCR and normalized by internal control GAPDH. Blocking anti-APRIL mAbs (A1, A2) were added to APRIL-containing medium and incubated for 6 hours and 1 day. Control (cnt), control medium; A2, clone Aprily-1-1. *p<0.02, **p<0.005, ***p<0.001, ****p<0.0001. [Figure 15C] This study demonstrates that APRIL induces CCND1 and CCND2 expression in TACI-expressing Tregs compared to Tcon, and that this induction is inhibited by an antagonistic anti-APRIL antibody. Purified Tregs and paired Tcon (n=5) were incubated with APRIL over various timeframes. CCND1 and CCND2 expression levels were then determined by qRT-PCR and normalized by internal control GAPDH. Blocking anti-APRIL mAbs (A1, A2) were added to APRIL-containing medium and incubated for 6 hours and 1 day. Control, control medium; A2, clone Aprily-1-1. *p<0.02, **p<0.005, ****p<0.0001. [Figure 15D] This study demonstrates that APRIL induces PD-L1 expression in TACI-expressing Tregs compared to Tcon. [Figure 16A] This study demonstrates that IL-10 is preferentially induced by APRIL in Tregs compared to Tcon, and is associated with higher TACI in Tregs compared to Tcon. [Figure 16B]This study demonstrates that APRIL selectively induces immunoregulatory and repressive genes in Treg cells but not in their paired Tcon cells. Specifically, APRIL induces the expression of Foxp3, IL-10, PD-L1, and TGFβ1, and this induction is suppressed by antagonistic anti-APRIL antibodies. Freshly purified Treg and Tcon cells from the same organism (n=5) were incubated with APRIL alone (left) or in the presence of antagonistic anti-APRIL mAbs (A1, A2; right) over the indicated timeframes. Control and control medium. Expression levels of the indicated genes by qRT-PCR were normalized by internal controls GAPDH and 18S. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 17-1]This study demonstrates that APRIL selectively enhances MM cell-inducible iTregs in CD4+ and CD8+ subsets in ex vivo co-cultures, and that this enhancement is blocked by anti-APRIL antibodies. U266 or RPMI8226 MM cells, pre-treated with mitomycin C, were washed and co-cultured with T cells in the presence of APRIL for 3 and 7 days. Neutralizing anti-APRIL mAbs (A1 or A2) were also added as shown. The percentage of gated CD4+CD25+Foxp3+ iTregs in CD4 T cells was determined by flow cytometry. Tcon cells were pre-stained with Cell Trace Violet (CTV) and co-cultured with U266 MM cells in APRIL-containing medium. Percentages of CTV-diluted iTregs (CTV-Foxp3+) (n=4) and dot plots of representative experiments are shown. The percentage of gated iTregs in CD8 T cells was also measured in the same co-cultures as above. Dot plots of additional representative experiments showed that proliferative iTregs (CTV-Foxp3+CD4+) were induced from 0 to 4.17% by U266 MM cells, which was further enhanced by APRIL from 4.17% to 8.02%. The percentage of CTV-diluted iTregs (CTV-Foxp3+) is shown. The percentage of resting iTregs versus proliferative iTregs and paired Tcon (n=3) in CD4+ T cells was determined under the conditions shown above. APRIL selectively increased the percentage of CTV-CD4+Foxp3+ iTregs induced by MM cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 17-2]This study demonstrates that APRIL selectively enhances MM cell-inducible iTregs in CD4+ and CD8+ subsets in ex vivo co-cultures, and that this enhancement is blocked by anti-APRIL antibodies. U266 or RPMI8226 MM cells, pre-treated with mitomycin C, were washed and co-cultured with T cells in the presence of APRIL for 3 and 7 days. Neutralizing anti-APRIL mAbs (A1 or A2) were also added as shown. The percentage of gated CD4+CD25+Foxp3+ iTregs in CD4 T cells was determined by flow cytometry. Tcon cells were pre-stained with Cell Trace Violet (CTV) and co-cultured with U266 MM cells in APRIL-containing medium. Percentages of CTV-diluted iTregs (CTV-Foxp3+) (n=4) and dot plots of representative experiments are shown. The percentage of gated iTregs in CD8 T cells was also measured in the same co-cultures as above. Dot plots of additional representative experiments showed that proliferative iTregs (CTV-Foxp3+CD4+) were induced from 0 to 4.17% by U266 MM cells, which was further enhanced by APRIL from 4.17% to 8.02%. The percentage of CTV-diluted iTregs (CTV-Foxp3+) is shown. The percentage of resting iTregs versus proliferative iTregs and paired Tcon (n=3) in CD4+ T cells was determined under the conditions shown above. APRIL selectively increased the percentage of CTV-CD4+Foxp3+ iTregs induced by MM cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 18] This study demonstrates that 01A blocks iTreg cells induced by MM cells, which are increased by APRIL in CD4+ and CD8+ subsets. [Figure 19]This study demonstrates that APRIL upregulates MM cell-induced iTregs, which are blocked by an obstructive anti-APRIL mAb. JJN3 and U266 MM cells were co-cultured with CD3 T cells for 4 days. The percentage of iTregs among CD4+ and CD8+ T cells was determined. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 20A] This study demonstrates that APRIL further promotes iTreg-mediated suppression of Tcon proliferation in ex vivo co-cultures, and that this suppression is inhibited by antagonist anti-APRIL antibodies (A1 or A2). MM cell-inducible iTregs were purified from the co-cultures and subjected to a CFSE dilution assay to determine the rate of autologous Tcon proliferation under the indicated conditions. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 20B] This study demonstrates that APRIL selectively induces immunosuppressive markers in MM cell-induced iTregs. Specifically, APRIL induces gene expression of IL-10, TGFβ, and CD15s in MM-induced iTregs (CD4+) and iTregs (CD8+). Three potential Treg suppression markers were assessed in CD4+ iTregs in the presence or absence of APRIL (upper panel). IL-10 and CD15s were also evaluated in CD8+ iTregs from the same culture (lower panel). TGFβ levels were determined by ELISA in the supernatant of the co-culture from the same co-culture. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 21A] This study demonstrates that OC further upregulates iTreg induction by MM cells in co-cultures. [Figure 21B]This study demonstrates that osteoclasts (OCs) further upregulate iTreg induction by MM cells in co-culture via intercellular contact and in an APRIL-dependent manner. iTreg induction is inhibited by antagonist anti-APRIL antibodies. Osteoclasts (OCs) were differentiated from CD14+ cells after 3 weeks of stimulation with M-CSF and RANKL, and then co-cultured with autologous T cells for 7 days in the presence or absence of anti-APRIL mAb (A1, 10 μg / ml). iTreg generation was determined by gateding CD25+Foxp3+ in CD4+ and CD8+ T cells. CD3 T cells were co-cultured with OCs from the same donor for 7 days. Flow cytometry analysis was also used to determine the percentage of CD25+Foxp3+ iTregs in CD4+ or CD8+ T cells in the same co-culture. A1 (50 μg / ml) was added where noted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 21C] This study demonstrates that OC culture supernatant upregulates iTreg induction by MM cells, which is specifically blocked by an antagonistic anti-APRIL antibody. Osteoclasts (OC) were differentiated from CD14+ cells after 3 weeks of stimulation with M-CSF and RANKL, and then co-cultured with autologous T cells for 7 days in or without anti-APRIL mAb (A1, 10 μg / ml). iTreg generation was determined by gateding CD25+Foxp3+ in CD4+ and CD8+ T cells. CD3 T cells were cultured for 7 days in supernatant (S) from 3-week OC cultures from the same donor. Flow cytometry analysis was also used to determine the percentage of CD25+Foxp3+ iTregs in CD4+ or CD8+ T cells in the same co-culture. A1 or A2 (50 μg / ml) was added where noted. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 21D]This study demonstrates that Tcon proliferation is inhibited by co-culture with autologous OCs. This inhibition is suppressed by antagonistic anti-APRIL antibodies, and to a higher degree by combinations of anti-APRIL, anti-PD1, and anti-PD-L1 antibodies. CD3 T cells pre-stained with CFSE were co-cultured with OCs from the same donor for 7 days under the indicated conditions, followed by flow cytometry analysis to determine the proportion of proliferative Tcon. Where noted, antagonistic anti-APRIL mAb, A1 or A2 (50 μg / ml) or anti-(α)-PD-1 / anti-(α)-PD-L1 mAb (10 μg / ml) was added. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 22] We demonstrate that APRIL ex vivo increases MM cell-induced iTregs, which are blocked by an obstructive anti-APRIL mAb. [Figure 23-1]Figure 23A shows that APRIL significantly protects Treg cells compared to matched Tcon cells via TACI. In the same individual, APRIL preferentially increases Treg growth and viability compared to Tcon cells and is associated with higher TACI in Treg cells compared to Tcon cells. The APRIL-dependent increase in Treg growth and viability is suppressed by an antagonist anti-APRIL antibody. Purified Treg and Tcon cells from the same patient were incubated with recombinant human APRIL in medium containing low-dose IL-2 (5 ng / ml) with or without neutralizing anti-APRIL mAb (A1, clone 01A), followed by luminescent cell viability assays CellTiter-Glo (CTG) and [3H]thymidine uptake assays. For time-course analysis (right panel), Tcon and Treg subsets were freshly isolated from normal donors. Purified Treg and paired Tcon cells were incubated with APRIL (200 ng / ml) for 4 and 7 days, followed by CTG-based survival assays and cpm-based proliferation assays. Neutralizing anti-APRIL mAbs (A1, A2) were added. *p<0.02, **p<0.005, ***p<0.001, ****p<0.0001. Figure 23B shows that APRIL inhibited caspase 3 / 7 and caspase 8 activity in Treg cells compared to autologous Tcon cells from MM patients, and that this inhibition was suppressed by antagonistic anti-APRIL antibodies. Purified Treg and Tcon cells from the same patients were incubated with recombinant human APRIL in medium containing low-dose IL-2 (5 ng / ml) with or without neutralizing anti-APRIL mAb (A1, clone 01A), followed by CTG-based caspase activity assays. *p<0.02, **p<0.005, ***p<0.001, ****p<0.0001. [Figure 23-2]Figure 23A shows that APRIL significantly protects Treg cells compared to matched Tcon cells via TACI. In the same individual, APRIL preferentially increases Treg growth and viability compared to Tcon cells and is associated with higher TACI in Treg cells compared to Tcon cells. The APRIL-dependent increase in Treg growth and viability is suppressed by an antagonist anti-APRIL antibody. Purified Treg and Tcon cells from the same patient were incubated with recombinant human APRIL in medium containing low-dose IL-2 (5 ng / ml) with or without neutralizing anti-APRIL mAb (A1, clone 01A), followed by luminescent cell viability assays CellTiter-Glo (CTG) and [3H]thymidine uptake assays. For time-course analysis (right panel), Tcon and Treg subsets were freshly isolated from normal donors. Purified Treg and paired Tcon cells were incubated with APRIL (200 ng / ml) for 4 and 7 days, followed by CTG-based survival assays and cpm-based proliferation assays. Neutralizing anti-APRIL mAbs (A1, A2) were added. *p<0.02, **p<0.005, ***p<0.001, ****p<0.0001. Figure 23B shows that APRIL inhibited caspase 3 / 7 and caspase 8 activity in Treg cells compared to autologous Tcon cells from MM patients, and that this inhibition was suppressed by antagonistic anti-APRIL antibodies. Purified Treg and Tcon cells from the same patients were incubated with recombinant human APRIL in medium containing low-dose IL-2 (5 ng / ml) with or without neutralizing anti-APRIL mAb (A1, clone 01A), followed by CTG-based caspase activity assays. *p<0.02, **p<0.005, ***p<0.001, ****p<0.0001. [Figure 24A]This study demonstrates that APRIL increases CD19+CD24-high CD38-high Breg cells, leading to further IL-10 secretion, which is inhibited by anti-APRIL mAbs. Regulatory B cells derived from MM patients express TACI, specifically mediating APRIL-induced IL-10 production. Bone marrow mononuclear cells (BMMCs) from MM patients were incubated with APRIL for 7 days in the presence of anti-APRIL mAbs. The percentages of Breg cells and IL-10+ Breg cells (CD19+CD24-high CD38-high) were determined by flow cytometry. The left panel shows dot blots of representative experiments. *p<0.02, **p<0.005, ***p<0.0005, ****p<0.0001. [Figure 24B] This study shows that TACI is highly expressed on the surface of BM-derived Breg cells (high CD19+CD24, high CD38) compared to naive B cells or memory B cells (high CD19+CD24, low CD38), and that this high expression of TACI on Breg cells is further enhanced by treatment with lipopolysaccharide (LPS), which induces IL-10 production from Breg cells. BM mononuclear cells isolated from MM patients were treated with LPS, and TACI levels were examined in the indicated B cell subsets: B regulatory cells (Breg) (defined as high CD19+CD24, high CD38), naive B cells (defined as medium CD19+CD38, medium CD24), and memory B cells (defined as low / - CD19+CD24, low / - CD38). (int, intermediate; LPS, lipopolysaccharide.) *p<0.02. [Figure 25] This study demonstrates that APRIL directly induces Treg proliferation based on an increase in the percentage of CFSE dilution. [Figure 26] This study demonstrates that APRIL induces myeloma cell-induced Tregs (iTregs) in CD4+ and CD4+ T cell subsets in ex vivo co-cultures of MM cells with T cells or Tcon cells. [Figure 27-1]This study demonstrates that APRIL further promotes Treg-mediated inhibition of autologous Tcon proliferation in a Treg / Tcon ratio, dose, and time-dependent manner, and that this inhibition of Tcon proliferation is suppressed by an antagonist anti-APRIL antibody. Purified Tcon were stained with 5 μM CFSE and then stimulated with CD3 / CD28 beads (beads) in the presence or absence of autologous Treg at the indicated Treg / Tcon ratios, with or without APRIL (200 ng / ml). Bead-stimulated Tcon were co-cultured with autologous Treg for 4 and 7 days in serial dilutions of APRIL (μg / ml) at two lower Treg / Tcon ratios. Tcon were co-cultured with Treg at lower Treg / Tcon ratios, with or without neutralizing anti-APRIL mAb (μg / ml), with or without APRIL (μg / ml). Chimeric homologs of C1, A1(01A). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 27-2] This study demonstrates that APRIL further promotes Treg-mediated inhibition of autologous Tcon proliferation in a Treg / Tcon ratio, dose, and time-dependent manner, and that this inhibition of Tcon proliferation is suppressed by an antagonist anti-APRIL antibody. Purified Tcon were stained with 5 μM CFSE and then stimulated with CD3 / CD28 beads (beads) in the presence or absence of autologous Treg at the indicated Treg / Tcon ratios, with or without APRIL (200 ng / ml). Bead-stimulated Tcon were co-cultured with autologous Treg for 4 and 7 days in serial dilutions of APRIL (μg / ml) at two lower Treg / Tcon ratios. Tcon were co-cultured with Treg at lower Treg / Tcon ratios, with or without neutralizing anti-APRIL mAb (μg / ml), with or without APRIL (μg / ml). Chimeric homologs of C1, A1(01A). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 28] This study demonstrates that 01A specifically inhibits APRIL-induced MM cell proliferation via BCMA. [Figure 29]This study demonstrates that anti-APRIL mAbs selectively block APRIL-induced MM cell proliferation. [Figure 30] This study demonstrates that anti-APRIL mAbs and O1A selectively block APRIL-induced MM cell proliferation via BCMA. [Figure 31] This study demonstrates that anti-APRIL mAbs and C4 / 01A selectively block APRIL-induced MM cell proliferation. [Figure 32] This study demonstrates that April further promotes Treg-mediated suppression of Tcon proliferation in a time-dependent manner. [Figure 33] TACI surface expression varied among T cell subsets, with the highest levels observed in CD4+(or CD8+)CD25-high cells from MM patient samples, followed by CD4+(or CD8+)CD25-low and CD4+(or CD8+)CD25- / negative cells. Flow cytometry analysis was used to measure TACI protein levels in the indicated subsets of CD4+ and CD8+ T cells from PB and BM compartments derived from MM patients (n=47). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 34A] This study shows that TACI protein levels are significantly elevated in CD4+(or CD8+)CD25-high FoxP3+ Tregs from MM patients compared to CD4+(or CD8+)CD25- Tcon cells. Flow cytometry analysis was used to determine the median fluorescence intensity (MFI) of TACI in a defined subset of CD4+ T cells from PB and BM compartments derived from MM patients (n=47). TACI protein levels were highest in the regulatory T subset (Treg, CD4+CD25+Foxp3+), followed by the CD4+CD25+Foxp3- subset. TACI MFI in normal T cells (Tcon, CD4+CD25-) was similar to that of isotype control Ab cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 34B]TACI levels were significantly higher in the CD4+FoxP3+IL10+ T cell subset compared to CD4+FoxP3-IL10- cells in the peripheral blood and bone marrow compartments of paired MM patients. Flow cytometry analysis was used to measure TACI protein levels in a specified subset of CD4+ T cells from the PB and BM compartments of MM patients (n=47). The percentage of the CD4+ T subset and TACI MFI were determined based on IL-10 and Foxp3 levels. TACI levels were highest in the CD4+IL-10+Foxp3+ subset in the PB and BM of MM patients. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 34C] TACI levels were significantly higher in the CD4+FoxP3-high IL10-high T cell subset compared to CD4+FoxP3-IL10- cells in the peripheral blood and bone marrow compartments of paired MM patients. IL-10 and TACI protein levels were measured in the CD4+CD25+Foxp3-high subset of CD4+CD25+Foxp3+ Treg cells from the PB and BM compartments of MM patients (n=47) using flow cytometry analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0020] For any figure, including histograms, note that the left-to-right bars for each discreet measurement correspond to the top-to-bottom boxes in the figure legend, as shown. [Modes for carrying out the invention]

[0021] Regulatory T and B cells negatively inhibit the immune response and are useful targets for modulating the immune response. However, it is difficult to identify genes and pathways selectively expressed by immune cell populations and to modify such genes and pathways to selectively regulate the number of immune cells and / or immune activity of subsets of immune cell populations. In this specification, TACI, the receptor for APRIL ligand, is expressed as CD4+ CD25 - Compared to normal T cells (Tcon), such as T cells, CD4+CD25+FoxP3+ Treg and CD4 + CD25 高 FoxP3 高 It has been determined that TACI is significantly expressed on Tregs such as Tregs. Furthermore, it has been determined in this specification that TACI is significantly expressed on CD8+CD25+FoxP3+ Tregs. It is also thought that Bregs selectively express TACI like Tregs. The binding of APRIL to immune cells expressing TACI is thought to lead to the upregulation of growth and survival genes, and since TACI is selectively expressed by Tregs / Bregs, APRIL preferentially activates TACI in Tregs / Bregs rather than Tcones, selectively upregulating growth and survival genes in Tregs / Bregs, thereby increasing the number of Tregs / Bregs and / or inhibitory immune activity compared to Tcones, resulting in enhanced inhibitory immune function. Therefore, modulating the APRIL / TACI interaction on Treg / Bregs is thought to enable selective modification of the number of Treg / Bregs and / or inhibitory immune activity (e.g., enhanced or reduced), based on the quality of the APRIL / TACI interaction modulation (e.g., enhanced or reduced, respectively).

[0022] Accordingly, the present invention relates in part to a method for selectively modifying the number and / or inhibitory immune activity of regulatory T cells (Treg) and / or regulatory B cells (Breg) in a subject, comprising administering a therapeutically effective amount of at least one agent to a subject, wherein the number and / or inhibitory immune activity of Treg and / or Breg are selectively modified by modulating the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand. In another embodiment, the present invention provides a method for selectively modifying the number and / or inhibitory immune activity of Treg and / or Breg, comprising contacting Treg and / or Breg with at least one agent, wherein the number and / or inhibitory immune activity of Treg and / or Breg are selectively modified by modulating the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand. Still in another aspect, the present invention provides a cell-based assay for screening for agents that selectively modify the number and / or inhibitory immune activity of Treg and / or Breg, comprising contacting Treg and / or Breg with a test agent and determining the ability of the test agent to modulate the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand, wherein the test agent that modulates the interaction between TACI receptor proteins expressed by Treg and / or Breg and APRIL ligand selectively modifies the number and / or inhibitory immune activity of Treg and / or Breg. Numerous other aspects and embodiments of the present invention are described below.

[0023] I. Definition The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, "(an) element" means one or more elements.

[0024] The term "administering" is intended to include the route of administration that enables the drug to perform its intended function. Examples of routes of administration that can be used for the treatment of the body include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. Injections may be bolus injections or continuous infusions. Depending on the route of administration, the drug may be coated with or placed in a material of choice to protect it from natural conditions that may adversely affect its ability to perform its intended function. The drug may be administered alone or in combination with a pharmaceutically acceptable carrier. The drug may also be administered as a prodrug, which is converted to its active form in vivo.

[0025] The terms “changed amount” or “changed level” refer to an increased or decreased copy number of a biomarker nucleic acid (e.g., germ cells and / or somatic cells), for example, an increased or decreased expression level in a cancer sample compared to the expression level or copy number of the biomarker nucleic acid in a control sample. The term “changed amount” of a biomarker also includes an increased or decreased protein level of the biomarker protein in a sample, for example, a cancer sample, compared to the corresponding protein level in a normal control sample. Furthermore, the changed amount of a biomarker protein may be determined by detecting post-translational modifications that may affect the expression or activity of the biomarker protein, such as the methylation status of the marker.

[0026] The amount of the biomarker in the subject is "significantly" higher or lower than the normal amount of the biomarker if it is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% higher or lower than the normal amount of the biomarker. Alternatively, the amount of the biomarker in the subject should be at least about 2, preferably at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%. Values ​​of %, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 2x, 3x, 4x, 5x, or more, or any range between them, e.g., 5% to 100%, respectively, if higher or lower, may be considered "significantly" higher or lower than normal and / or control levels. Such significant modulo values ​​may be applied to any measurement described herein, such as altered expression levels, altered activity, changes in cancer cell overgrowth, changes in cancer cell death, changes in biomarker inhibition, changes in test drug binding, etc.

[0027] The term "altered expression level" of a biomarker refers to the expression level or copy number of a biomarker in a test sample, for example, a sample from a patient with cancer, which is above or below the standard error of the assay used to assess the expression or copy number, and preferably at least twice, more preferably three times, four times, five times, or ten times, the expression level or copy number of the biomarker in a control sample (e.g., a sample from a healthy subject without the associated disease), and preferably at least twice, more preferably three times, four times, five times, or ten times, the average expression level or copy number of the biomarker in several control samples.

[0028] The term "altered activity" of a biomarker refers to increased or decreased activity of a biomarker in a diseased state, for example in a cancer sample, compared to the activity of the biomarker in a normal control sample. Altered activity of a biomarker may result from, for example, altered expression of the biomarker, altered protein levels of the biomarker, altered structure of the biomarker, or altered interactions with other proteins involved in the same or different pathways as the biomarker, or altered interactions with transcription activators or inhibitors.

[0029] The term "altered structure" of a biomarker refers to the presence of mutations or allele variants within the biomarker nucleic acid or protein, such as mutations that affect the expression or activity of the biomarker nucleic acid or protein compared to a normal or wild-type gene or protein. For example, mutations include, but are not limited to, substitutions, deletions, or additions. Mutations can be located in the coding or non-coding regions of the biomarker nucleic acid.

[0030] Unless otherwise specified herein, the terms “antibody” and “antibodies” broadly encompass native antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies and multispecific antibodies, as well as all fragments and derivatives thereof (such fragments and derivatives having at least an antigen-binding site). Antibody derivatives may include proteins or chemical moieties conjugated to the antibody.

[0031] In addition, intrabodies are well-known antigen-binding molecules that possess the characteristics of antibodies but can be expressed intracellularly to bind to and / or inhibit specific intracellular targets (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods for adapting antibodies to target (e.g., inhibit) intracellular parts are well known in the art, including the use of single-chain antibodies (scFvs), modification of the immunoglobulin VL domain for hyperstability, modification of antibodies to withstand reductive intracellular environments, and the creation of fusion proteins that increase intracellular stability and / or regulate intracellular localization. Intracellular antibodies can also be introduced into and expressed in one or more cells, tissues, or organs of a multicellular organism for prophylactic and / or therapeutic purposes (e.g., as gene therapy) (at least PCT publications WO08 / 020079, WO94 / 02610, WO95 / 22618, and WO03 / 014960, U.S. Patent No. 7,004,940, Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.), Kontermann (2004) Methods 34:163-170, Cohen et al. (1998) Oncogene 17:2445-2456, Auf der Maur et al. (2001) FEBS Lett. 508:407-412, Shaki-Loewenstein et al. See al. (2005) J.Immunol.Meth. 303:19-39.

[0032] As used herein, the term “antibody” also includes the “antigen-binding portion” (or simply “antibody portion”) of an antibody. As used herein, the term “antigen-binding portion” refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or a fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include: (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, i.e., bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of one arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using synthetic linkers that allow them to be constructed as a single protein chain (known as single-chain Fv (scFv)) in which the VL and VH regions pair up to form a monovalent polypeptide (see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of the antibody. To generate an expression vector encoding a complete IgG polypeptide or other isotype, either the VH and VL sequences of a specific scFv can be ligated to the cDNA or genomic sequence of the human immunoglobulin constant region. VH and VL can also be used in the generation of Fab, Fv, or other immunoglobulin fragments using either protein chemistry techniques or recombinant DNA techniques.Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between these two domains on the same chain forces them to pair with complementary domains on another chain, thereby creating two antigen-binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448 and Poljak et al. (1994) Structure 2:1121-1123).

[0033] Furthermore, the antibody or its antigen-binding moiety may also be part of a larger immunoadhesion polypeptide formed by covalent or noncovalent association with the antibody or antibody moiety of one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include the use of a streptavidin core region to produce tetrameric scFv polypeptides (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, biomarker peptides, and C-terminal polyhistidine tags to produce divalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol.Immunol.31:1047-1058). Antibody moieties such as Fab and F(ab')2 fragments can be prepared from intact antibodies using conventional techniques such as papain or pepsin digestion, respectively. Furthermore, antibodies, antibody moieties, and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques as described herein.

[0034] Antibodies may be polyclonal or monoclonal; heterogeneous, homogeneous, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, the antibodies of the present invention bind specifically or substantially specifically to a biomarker polypeptide or a fragment thereof. As used herein, the terms “monoclonal antibody” and “monoclonal antibody composition” refer to a group of antibody polypeptides containing only one species of antigen-binding site capable of initiating an immune response with a specific epitope of an antigen, while the terms “polyclonal antibody” and “polyclonal antibody composition” refer to a group of antibody polypeptides containing multiple species of antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity to the specific antigen with which they initiate an immune response.

[0035] Antibodies may also be “humanized,” meaning that humanization includes antibodies produced by non-human cells having variable and constant regions modified to closely resemble antibodies produced by human cells. For example, this is achieved by modifying the amino acid sequence of a non-human antibody to incorporate amino acids found in the immunoglobulin sequences of human germ cells. The humanized antibodies of the present invention may include, for example, amino acid residues in the CDR that are not encoded by the immunoglobulin sequences of human germ cells (e.g., mutations introduced in vitro by random or site-directed mutagenesis, or in vivo by somatic mutation). As used herein, the term “humanized antibody” also includes antibodies in which a CDR sequence derived from germ cells of another mammalian species, such as mouse, has been transplanted into a human framework sequence.

[0036] The term “assigned score” refers to a numerical value assigned to each biomarker after it has been measured in a patient sample. The assigned score correlates with the absence, presence, or estimation of the biomarker in the sample. The assigned score can be generated manually (e.g., by visual inspection) or with the assistance of imaging and analysis equipment. In certain embodiments, the assigned score is determined by qualitative assessment, e.g., detection of fluorescence readings on a graded scale, or by quantitative assessment. In one embodiment, a “total score” is determined, which refers to a combination of assigned scores from multiple measured biomarkers. In one embodiment, the total score is the sum of the assigned scores. In another embodiment, the combination of assigned scores involves performing mathematical operations on the assigned scores and then combining them into a total score. In certain embodiments, the total score is also referred to herein as the “prediction score.”

[0037] The term “biomarker” includes measurable entities of the present invention that are determined to be useful in modulating immune responses and / or predicting immunomodulatory responses. Biomarkers may include, but are not limited to, nucleic acids and proteins, including those shown in Table 1, Examples, and Figures, as well as interactions between such molecules (e.g., APRIL / TACI interactions). In addition, biomarkers may include immune cells that mediate immunomodulatory activity, such as the number and / or immune activity of Tregs, Bregs, and / or Tcones, their ratios, etc., as further described herein. Biomarkers include markers listed herein that are useful in diagnosing the sensitivity of cancer and / or its anti-cancer treatment, and also include, for example, tumor hyperactivity or hypoactivity, appearance, expression, growth, remission, relapse, or resistance before, during, or after treatment. The predictive function of a marker is, for example, (1) increased or decreased copy number (e.g., by FISH, FISH plus SKY, e.g. by single-molecule sequencing as described in at least J. Biotechnol., 86:289-301, or qPCR), overexpression or underexpression (e.g. by ISH, Northern blot, or qPCR), increased or decreased protein levels (e.g. by IHC), or increased or decreased activity (e.g., determined by modulation of the pathway in which the marker is involved) in approximately 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25% or more of human cancer types or cancer samples, or more than that, or (2) increased or decreased copy number (e.g. by FISH, FISH plus SKY, e.g. by single-molecule sequencing as described in at least J. Biotechnol., 86:289-301, or qPCR), increased or decreased protein levels (e.g. by IHC), or increased or decreased activity (e.g., determined by modulation of the pathway in which the marker is involved) in a human cancer sample, e.g., a subject with cancer, e.g., human-derived tissue, whole blood, serum, plasma, buccal scraping (buccal (3) Its presence or absence in a sample containing scrape, saliva, cerebrospinal fluid, urine, feces, or bone marrow; (4) Its presence or absence in a clinical subset of subjects with cancer (e.g., those who respond to or develop resistance to a particular therapy). Biomarkers also include “surrogate markers,” such as markers that are indirect markers of cancer progression.The term “biomarker” also includes markers listed herein that are useful in analyzing the effectiveness of anti-cancer treatments, such as tumor size, cancer cell proliferation and / or metastasis rate, number of cancer cells, and lifespan of subjects with cancer. Biomarkers also include markers listed herein in cellular signaling pathways, such as the number of Treg and / or other T cells, the number of Breg and / or other B cells, the number of either Treg or Breg (Treg / Breg) and / or inhibitory immune activity (acticity), differentiation rates and / or apoptosis / cytotoxicity rates of various T cells or other immune cells, expression of various proteins expressed on the cell surface of T cells or other immune cells, antigen presentation effectiveness, production of various signaling proteins (e.g., interferons) and their responsive genes, DNA methylation and transcriptional effectiveness, and aging / proliferative status.

[0038] The term "APRIL," also known as proliferation-inducing ligand, tumor necrosis factor ligand superfamily member 13 (TNFSF13), TALL-2, ZTNF2, and CD256, refers to a family of tumor necrosis factor (TNF) ligand proteins. APRIL is a ligand for TNFRSF17 / BCMA and TNFRSF13B / TACI. Both APRIL and its receptor are important for B cell development. In vitro experiments have shown that APRIL may be able to induce apoptosis in the long-term survival of plasma cells in the bone marrow through its interaction with other TNF receptor family proteins such as TNFRSF6 / FAS and TNFRSF14 / HVEM (Roth et al. (2001) Cell Death Diff. 8:403-410). Mice lacking APRIL have normal immune system development (Varfolomeev et al. (2004) Mol. Cell. Biol. 24:997-1006). However, APRIL-deficient mice have also been reported to have a reduced ability to support plasma cell survival (Belnoue et al. (2008) Blood 111:2755-2764). APRIL plays a role in regulating tumor cell growth and may be involved in monocyte / macrophage-mediated immune processes. APRIL also interacts with TNFRSF13B (Wu et al. (2000) J. Biol. Chem. 275:35478-35485) and B cell activators (Roschke et al. (2002) J. Immunol. 169:4314-4321).APRIL functions in multiple pathways, including at least PEDF-induced signaling (e.g., MIF-mediated glucocorticoid regulation, MIF-mediated regulation of innate immune cells, IL-6 pathway, STAT3 pathway, endothelin-1 signaling pathway, cytokine-cytokine receptor interactions, RAR-gamma-RXR-alpha degradation, and all-trans retinoic acid signaling in the brain), ERK signaling (e.g., Rho family GT passes), proteasome-mediated degradation of activated PAK-2p34 (e.g., TNFR2 non-classical NF-κB pathway, regulation of mRNA stability by proteins binding to AU-rich regions), TNF superfamily pathways (e.g., human ligand-receptor interactions and their related functions), and AKT signaling (e.g., p38 signaling). APRIL is considered a target in autoimmune diseases and B-cell malignancies (Ryan and Grewal (2009) Grewal IS, ed. Therapeutic Targets of the TNF Superfamily. Advances in Experimental Medicine and Biology. New York: Springer. pp. 52-63). APRIL is suggested to be associated with multiple diseases and disorders, including at least IgG4-related diseases, glioblastoma multiforme, opsoclonus-myoclonus syndrome, cryptococcal meningitis, and rheumatoid arthritis. It has been announced that at least one anti-APRIL monoclonal antibody, BION-1301, will enter a Phase I clinical trial for multiple myeloma (see Dulos et al.'s (2017) AACR Annual Meeting 2017 Online Proceedings, Session PO.IM02.10, No. 2645 / 4 at World Wide Web address: www.abstractsonline.com / pp8 / #! / 4292 / presentation / 6077).

[0039] The nucleic acid and amino acid sequences of representative human APRIL are publicly available in the GenBank database (gene ID 8741) and are shown in Table 1. The multiple transcript variants and protein isoforms of APRIL include, at a minimum, NM_003808.3 and NP_003799.1 representing the longest transcript variant alpha and the longest isoform alpha; NM_172087.2 and NP_742084.1 representing the transcript variant beta (lacking a selective in-frame exon in the central coding region compared to variant alpha) and coding isoform beta; NM_172088.2 and NP_742085.1 representing the transcript variant gamma (lacking a selective segment in the 3' coding region and 3'UTR compared to variant alpha) and coding isoform gamma (having a unique, shorter C-terminus compared to isoform alpha); and the transcript variant del This includes NM_001198622.1 and NP_001185551.1, representing the ta (lacking a selective in-frame segment in the 5' coding region compared to variant alpha) and coded isoform delta; NM_001198623.1 and NP_001185552.1, representing the transcript variant zeta (lacking a selective in-frame segment in the 5' coding region compared to variant alpha) and coded isoform zeta; and NM_001198624.1 and NP_001185553.1, representing the transcript variant eta (different in the 5'UTR compared to variant alpha, using a downstream start codon and lacking a selective in-frame segment in the 5' coding region) and coded isoform eta. The domain structure of the APRIL polypeptide is well known and is available in the UniProtKB database under accession number O75888, for example, including the TNF domain containing amino acids 117-248 of NP_003799.1.

[0040] Nucleic acid and polypeptide sequences of APRIL orthologs in non-human organisms are well known, including, for example, chimpanzees (Pan (Troglodytes) APRIL (NM_001205130.1 and NP_001192059.1), Canine APRIL (NM_001205169.1 and NP_001192098.1), Mouse APRIL (NM_023517.2 and NP_076006.2 representing longer transcript variant 1 and longer coding isoform 1, and NM_001159505.1 and NP_001152977.1 representing transcript variant 2 (using alternative in-frame splice sites in the central coding region compared to variant 1) and shorter coding isoform 2 (lacking one internal amino acid compared to isoform 1)), Bovine APRIL (NM_001034647.2 and NP_001029819.1), and Brown rat (Rattus) This includes norvegicus)APRIL(NM_001009623.1 and NP_001009623.1).

[0041] The term "APRIL activity" includes the ability of the APRIL polypeptide (as well as its fragments, domains, and / or motifs as considered herein) to bind to its substrate and / or biological activity. APRIL activity may also include one or more functions such as binding to its receptor and activation of downstream signaling pathways, and / or other functions disclosed herein. For example, APRIL may interact with TNFRSF17 / BCMA and / or TNFRSF13B / TACI to promote cell growth and survival, such as plasma cell and / or B cell survival. APRIL may also be modified proteolytically, such as by cleavage, ubiquitination, deubiquitination, or other modes disclosed herein, in order to function.

[0042] The term “APRIL substrates” refers to binding partners of APRIL polypeptides (and their fragments, domains, and / or motifs as considered herein), such as cellular receptors and / or other TNF superfamily members for various signaling pathways. Furthermore, APRIL substrates may also refer to downstream members in signaling pathways that are activated by APRIL binding to its receptor(s).

[0043] The term “APRIL-regulatory signaling pathways” includes signaling pathways through which APRIL (and any fragments, domains, and / or motifs thereof as discussed herein) binds to at least one of its substrates (e.g., its receptors), thereby altering at least one cellular function and / or activity and / or cellular protein profile. APRIL regulatory signaling pathways include, at a minimum, those described herein, such as PEDF-induced signaling (e.g., MIF-mediated glucocorticoid regulation, MIF-mediated regulation of innate immune cells, IL-6 pathway, STAT3 pathway, endothelin-1 signaling pathway, cytokine-cytokine receptor interactions, RAR-gamma-RXR-alpha degradation, and all-trans retinoic acid signaling in the brain), ERK signaling (e.g., Rho family GT passes), proteasome-mediated degradation of activated PAK-2p34 (e.g., TNFR2 non-classical NF-κB pathway, regulation of mRNA stability by proteins binding to AU-rich regions), TNF superfamily pathways (e.g., human ligand-receptor interactions and their related functions), and AKT signaling (e.g., p38 signaling).

[0044] The term "APRIL modulator" includes any natural or non-natural agent prepared, synthesized, manufactured, and / or purified by humans that is capable of modulating the ability of APRIL (and its fragments, domains, and / or motifs as considered herein) to be expressed, function, and / or bind to a binding partner. In one embodiment, the modulator promotes APRIL, and representative embodiments such as APRIL nucleic acid, polypeptide, multimer, and activating antibodies that multimerize APRIL are described herein. In another embodiment, the modulator inhibits APRIL. In one embodiment, such an inhibitor reduces or inhibits the binding / interaction between APRIL and its substrate or other binding partner. Still in yet another embodiment, such an inhibitor may increase or promote the turnover of APRIL, reduce or inhibit the expression and / or stability (e.g., half-life) of APRIL, and / or alter the cellular localization of APRIL, resulting in at least a decrease in APRIL levels and / or activity. Such inhibitors may include, but are not limited to, any molecule including small molecule compounds, antibodies or intrabodies, and RNA interference (RNAi) agents (including at least siRNA, shRNA, microRNA (miRNA), piwi, and other well-known agents). Such inhibitors may be specific to APRIL or may also inhibit at least one other TNF superfamily member. For example, the TGFβ2 inhibitor travedersen (AP12009) has been tested for its inhibition of APRIL (Tse (2013) Nat. Rev. Drug Dis. 12:179). Atacicept (TACI-Ig) is a recombinant fusion protein that combines the binding sites of B lymphocyte-stimulating factor (BLyS) and proliferation-inducing ligand (APRIL) with the constant region of an immunoglobulin (Hartung et al. (2010) Ther Adv Neurol Disord. 3:205-216). Atasicept (TACI-Ig) blocks the binding of Blys and APRIL to TNFSF13B / TACI, thereby inhibiting B cells and suppressing autoimmune diseases.Atacicept (TACI-Ig) is also being studied for the treatment of B-cell malignancies, including multiple myeloma, B-cell chronic lymphocytic leukemia, and non-Hodgkin lymphoma (Vasiliou (2008) Drugs Fut. 33:921). RNA interference methods for APRIL polypeptides are well-known and commercially available (e.g., human, mouse, or rat shRNA (catalog numbers TF300911, TF515490, and TF701276) and siRNA (catalog numbers SR406719, SR510783, and SR305759) products from Origene (Rockville, MD), as well as human or mouse gene knockout kits via CRISPR (catalog numbers KN203446 and KN317997), siRNA / shRNA products (catalog numbers sc-39822, sc-39823, and sc-141178) and CRISPR products (catalog numbers sc-403296, sc-427459, and sc-403150) from Santa Cruz Biotechnology (Dallas, Texas), and ready-to-package AAVs from Vigene Biosciences (Rockville, MD) shRNA clones (catalog numbers SH895874 and SH897133) are also known. Methods for the detection, purification, and / or inhibition of APRIL (e.g., by anti-APRIL antibodies) are also well known and commercially available (e.g., several anti-APRIL antibodies from Origene (catalog numbers TA306069, TA349496, TA351828, etc.), Novus Biologicals (Littleton, CO, catalog numbers NBP1-97587, MAB8843, NBP1-76767, etc.), abcam (Cambridge, MA, catalog numbers ab64967, ab16088, etc.), and Santa Cruz Biotechnology (catalog numbers sc-374673, sc-57035, etc.). Human APRIL knockout cell lines are also well known and commercially available from Horizon Discovery (Cambridge, UK, catalog number HZGHC8741).Selective APRIL blockade using monoclonal antibodies has been shown to delay systemic lupus erythematosus in mice (Huard et al. (2012) PLoS ONE 7:e31837).

[0045] The term "TACI," also known as transmembrane activator and CAML interactor, tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), CD267, and CVID2, refers to a transmembrane protein family member of the TNF receptor superfamily primarily found on the surface of B cells. TACI binds to B cell activator (BAFF) and APRIL, inducing the activation of several transcription factors such as NFAT, AP-1, and NF-κB, thereby regulating cellular activity. Functional defects in TACI can lead to immune system disorders and have been shown to cause lethal autoimmunity in mice (Seshasayee et al. (2003) Immunity. 18:279-288). TACI regulates T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis. TACI mediates the calcineurin-dependent activation of NF-AT, as well as the activation of NF-κB and AP-1. TACI is involved in stimulating B cell and T cell function and regulating humoral immunity. TACI is suggested to bind to multiple binding partners, including at least B cell activators, TRAF6, TRAF5, TNFSF13 / APRIL, TRAF2, and CAMLG (Xia et al. (2000) J.Exp.Med.192:137-143). TACI functions in multiple pathways, including at least the TNF superfamily pathway (human ligand-receptor interactions and their associated functions), AKT signaling (e.g., p38 signaling and Tec kinase signaling), RANK signaling in osteoclasts (e.g., APRIL pathway, B cell signaling, apoptosis, and BAFF in survival), PEDF-induced signaling (e.g., STAT3 pathway and cytokine-cytokine receptor interactions), TRAF pathway, and syndecan 2 or 4-mediated signaling events. TACI is suggested to be associated with several diseases and disorders, including at least unclassifiable immunodeficiency 2 (CVID2, also known as hypogammaglobulinemia due to TACI deficiency) and immunoglobulin A deficiency 2 (IGAD2).

[0046] Representative human TACI nucleic acid and amino acid sequences are publicly available in the GenBank database (gene ID 23495) and are shown in Table 1 (e.g., NM_012452.2 and NP_036584.1). The domain structure of TACI polypeptides is well known, and is available in the UniProtKB database under accession number Q4ACX1, including, for example, the three cysteine-rich domains (CRDs) containing amino acids 34-86, 89-170, and 172-230 of NP_036584.1, as well as the transmembrane region containing amino acids 166-186 of NP_036584.1.

[0047] Nucleic acid and polypeptide sequences of TACI orthologs in non-human organisms are well known, including, for example, chimpanzee (Pan troglodytes) TACI (XM_001161361.4 and XP_001161361.3, and XM_016932352.1 and XP_016787841.1), and rhesus macaque TACI (XM_015118722.1 and XP_014974208.1, and XM_015118723.1 and XP_014974209.1). This includes canine TACI (XM_005620177.2 and XP_005620234.1, as well as XM_005620179.2 and XP_005620236.1), mouse TACI (NM_021349.1 and NP_067324.1), and chicken TACI (NM_001097537.1 and NP_001091006.1 tumors).

[0048] The term "TACI activity" includes the ability of a TACI polypeptide (as well as its fragments, domains, and / or motifs as considered herein) to bind to its substrate and / or biological activity. TACI activity may also include one or more functions such as binding to its ligand and activation of downstream signaling pathways, and / or other functions disclosed herein. For example, TACI may interact with APRIL to promote B cell survival / proliferation. TACI may also be modified proteolytically, such as by cleavage, ubiquitination, deubiquitination, or other modes disclosed herein, in order to function.

[0049] The term “TACI substrates” refers to binding partners of TACI polypeptides (and their fragments, domains, and / or motifs as considered herein), such as ligands and / or other TNF superfamily members for various signaling pathways. Furthermore, TACI substrates may also refer to downstream members in signaling pathways that are activated by TACI binding to their receptors.

[0050] The term “TACI-regulatory signaling pathways” includes signaling pathways through which TACI (and its fragments, domains, and / or motifs as discussed herein) binds to at least one of its substrates (e.g., its ligands), thereby altering at least one cellular function and / or activity and / or cellular protein profile. TACI-regulatory signaling pathways include, at a minimum, those described herein, such as the TNF superfamily pathways (human ligand-receptor interactions and their associated functions), AKT signaling (e.g., p38 signaling and Tec kinase signaling), RANK signaling in osteoclasts (e.g., the APRIL pathway, B cell signaling, apoptosis, and BAFF in survival), PEDF-induced signaling (e.g., the STAT3 pathway and cytokine-cytokine receptor interactions), the TRAF pathway, syndecan 2 or 4-mediated signaling events, and others.

[0051] The term “TACI modulator” includes any natural or non-natural agent prepared, synthesized, manufactured, and / or purified by humans that is capable of modulating the ability of TACI (and its fragments, domains, and / or motifs as considered herein) to be expressed, function, and / or bind to a binding partner. In one embodiment, the modulator promotes TACI, and representative embodiments such as TACI nucleic acids, polypeptides, multimers, and activating antibodies that multimerize TACI are described herein. In another embodiment, the modulator inhibits TACI. In one embodiment, such inhibitors may reduce or inhibit the binding / interaction between TACI and its substrate or other binding partner. Still in yet another embodiment, such inhibitors may increase or promote the turnover of TACI, reduce or inhibit the expression and / or stability (e.g., half-life) of TACI, and / or alter the cellular localization of TACI, resulting in at least a decrease in TACI levels and / or activity. Such inhibitors may include, but are not limited to, any small molecule compounds, antibodies or intrabodies, or RNA interference (RNAi) agents (including at least siRNA, shRNA, microRNA (miRNA), piwi, and other well-known agents). Such inhibitors may be specific to TACI or may also inhibit at least one other TNF superfamily member (such as a cell receptor).RNA interference methods for TACI polypeptides are well-known and commercially available (e.g., human or mouse shRNA (catalog numbers TF308737 and TF503348) and siRNA (catalog numbers SR308311 and SR407026) products from Origene (Rockville, MD), as well as human or mouse gene knockout kits via CRISPR (catalog numbers KN211856 and KN317977), siRNA / shRNA products (catalog numbers sc-40243 and sc-40244) and CRISPR products (catalog numbers sc-406692 and sc-425465) from Santa Cruz Biotechnology (Dallas, Texas), and packaged AAV shRNA clones (catalog number SH860094) from Vigene Biosciences (Rockville, MD). Methods for detecting, purifying, and / or inhibiting TACI (e.g., using anti-TACI antibodies) are also well-known and commercially available (e.g., several anti-TACI antibodies from Origene (catalog numbers TA306064, TA352371, AM26557AF-N, etc.), Novus Biologicals (Littleton, CO, catalog numbers NBP2-11937, MAB174, NBP1-84596, etc.), abcam (Cambridge, MA, catalog numbers ab79023, ab89744, etc.), and Santa Cruz Biotechnology (catalog numbers sc-32775, sc-365253, etc.). Human TACI knockout cell lines are also well-known and commercially available from Horizon Discovery (Cambridge, UK, catalog number HZGHC23495).

[0052] The term "BCMA," also known as B cell maturation antigen, tumor necrosis factor receptor superfamily member 17 (TNFSF17), BCM, and CD269, refers to a family of transmembrane proteins of the TNF receptor superfamily found primarily on the surface of mature B cells. BCMA is important for B cell development and autoimmune responses. This receptor has been shown to specifically bind to tumor necrosis factor (ligand) superfamily member 13b (TNFSF13B / TALL-1 / BAFF), resulting in NF-κB and MAPK8 / JNK activation. BCMA also binds to various TRAF family members and can therefore transmit signals for cell survival and proliferation. In addition to BAFF, APRIL is also a ligand for BCMA. Other BCMA binding partners include at least TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 (Liu et al. (2003) Nature 423:49-56). BCMA functions in multiple pathways, including at least the TNF superfamily pathway (human ligand-receptor interactions and their associated functions), AKT signaling (e.g., p38 signaling and Tec kinase signaling), RANK signaling in osteoclasts (e.g., APRIL pathway, B cell signaling, apoptosis, and BAFF in survival), PEDF-induced signaling (e.g., STAT3 pathway and cytokine-cytokine receptor interactions), and the TGF-beta pathway (e.g., MAPK family pathway, JAK-STAT pathway, JNK pathway, regulation of eIF4 and p70S6K, SOCS pathway, etc.). TACI is suggested to be associated with multiple diseases and disorders, including at least unclassifiable immunodeficiency (e.g., acquired agammaglobulinemia), cryptococcal meningitis, chronic lymphocytic leukemia, blue cone monochoromacy, leukemia, lymphoma, and multiple myeloma. Representative human BCMA nucleic acid and amino acid sequences are publicly available in the GenBank database (gene ID 608) and are shown in Table 1 (e.g., NM_001192.2 and NP_001183.2).The domain structure of the BCMA polypeptide is well known and is available in the UniProtKB database under accession number Q02223, for example, including the TNFR-Cys domain containing amino acids 7-41 of NP_001183.2, and the transmembrane region containing amino acids 55-77 of NP_001183.2. Two cysteine-rich domains, for example, contain amino acids 4-21 and 24-126 of NP_001183.2.

[0053] Nucleic acid and polypeptide sequences of BCMA orthologs in non-human organisms are well known, including, for example, chimpanzee (Pan troglodytes) BCMA (XM_523298.5 and XP_523298.2), rhesus macaque BCMA (XM_001106892.3 and XP_001106892.1), canine BCMA (XM_005621530.2 and XP_005621587.1), bovine BCMA (XM_002697966.4 and XP_002698012.2), mouse BCMA (NM_011608.1 and NP_035738.1), and rat TACI (NM_011608.1 and NP_035738.1).

[0054] The term "BCMA activity" includes the ability of the BCMA polypeptide (as well as its fragments, domains, and / or motifs as considered herein) to bind to its substrate and / or biological activity. BCMA activity may also include one or more functions such as binding to its ligand and activation of downstream signaling pathways, and / or other functions disclosed herein. For example, BCMA may interact with APRIL to promote plasma cell survival / proliferation. BCMA may also be modified proteolytically, such as by cleavage, ubiquitination, deubiquitination, or other modes disclosed herein, in order to function.

[0055] The term “BCMA substrates” refers to binding partners of BCMA polypeptides (and their fragments, domains, and / or motifs as considered herein), such as ligands (e.g., APRIL and BAFF) and / or other TNF superfamily members for multiple signaling pathways. Furthermore, BCMA substrates may also refer to downstream members in signaling pathways that are activated by BCMA binding to its receptors.

[0056] The term “BCMA regulatory signaling pathways” includes signaling pathways through which BCMA (and its fragments, domains, and / or motifs as considered herein) binds to at least one of its substrates (e.g., its ligands), thereby altering at least one cellular function and / or activity and / or cellular protein profile. BCMA regulatory signaling pathways include, but are not limited to, those described herein, such as the TNF superfamily pathways (human ligand-receptor interactions and their associated functions), AKT signaling (e.g., p38 signaling and Tec kinase signaling), RANK signaling in osteoclasts (e.g., the APRIL pathway, B cell signaling, apoptosis, and BAFF in survival), PEDF-induced signaling (e.g., the STAT3 pathway and cytokine-cytokine receptor interactions), and TGF-beta pathways (e.g., the MAPK family pathways, JAK-STAT pathways, JNK pathways, regulation of eIF4 and p70S6K, SOCS pathways, etc.).

[0057] The term “BCMA modulator” includes any natural or non-natural agent prepared, synthesized, manufactured, and / or purified by humans that is capable of modulating the ability of BCMA (and its fragments, domains, and / or motifs as considered herein) to be expressed, function, and / or bind to a binding partner. In one embodiment, the modulator promotes BCMA, and representative embodiments such as BCMA nucleic acids, polypeptides, polymers, and activating antibodies that polymerize BCMA are described herein. In another embodiment, the modulator inhibits BCMA. In one embodiment, such an inhibitor may reduce or inhibit the binding / interaction between BCMA and its substrate or other binding partner. Still in yet another embodiment, such an inhibitor may increase or promote the turnover of BCMA, reduce or inhibit the expression and / or stability (e.g., half-life) of BCMA, and / or alter the cellular localization of BCMA, resulting in at least a decrease in BCMA levels and / or activity. Such inhibitors may include, but are not limited to, any small molecule compounds, antibodies or intrabodies, or RNA interference (RNAi) agents (including at least siRNA, shRNA, microRNA (miRNA), piwi, and other well-known agents). Such inhibitors may be specific to BCMA or may also inhibit at least one other TNF superfamily member (such as cell surface receptors).RNA interference methods for TACI polypeptides are well-known and commercially available (e.g., human or mouse shRNA (catalog numbers TL308735, TF514674, and TF704358) and siRNA (catalog numbers SR300419, SR404548, and SR502461) products from Origene (Rockville, MD), as well as human or mouse gene knockout kits via CRISPR (catalog numbers KN208851 and KN317980), siRNA / shRNA products (catalog numbers sc-40233 and sc-40234) and CRISPR products (catalog numbers sc-403058 and sc-423440) from Santa Cruz Biotechnology (Dallas, Texas), and packaged AAV shRNA clones (catalog number SH873263) from Vigene Biosciences (Rockville, MD). Methods for detecting, purifying, and / or inhibiting BCMA (e.g., by anti-BCMA antibodies) are also well known and commercially available (e.g., several anti-BCMA antibodies from Origene (catalog numbers TA306065, AP00250PU-N, TA311846, etc.), Novus Biologicals (Littleton, CO, catalog numbers NBP1-97637, AF593, NBP1-76774, etc.), abcam (Cambridge, MA, catalog numbers ab5972, ab17323, etc.), and Santa Cruz Biotechnology (catalog numbers sc-11746, sc-390147, etc.). Human BCMA knockout cell lines are also well known, and Horizon It is commercially available from Discovery (Cambridge, UK, catalog number HZGHC608). Another representative BCMA inhibitor is GSK2857916, which is an antibody-drug conjugate (ADC) consisting of a defucosylated humanized monoclonal antibody directed against B cell maturation antigen (BCMA), conjugated to an auristatin analog and the microtubule inhibitor monomethyl auristatin phenylalanine (MMAF), which has potential antineoplastic activity. The anti-BCMA antibody portion of the anti-BCMA ADC selectively binds to BCMA on the surface of tumor cells.Upon entering cells, the MMAF moiety binds to tubulin, inhibiting its polymerization and leading to G2 / M phase arrest, thereby inducing apoptosis in tumor cells. In addition, GSK2857916 induces antibody-dependent cell-mediated cytotoxicity (ADCC). Overall, this results in inhibition of cell proliferation in tumor cells overexpressing BCMA. Defucosylation of this antibody moiety increases ADCC.

[0058] The interactions between APRIL, BCMA, and TACI, as well as their functions, are well known in the art as described above (see, for example, Yu et al. (2000) Nat. Immunol. 1:252-256).

[0059] In addition, certain immune cells or their states may be biomarkers according to the present invention. The term “immune cells” refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. For example, antigen-reactive T cells are T cells that selectively bind to a target antigen based on antigen recognition and regulate the immunological response. Immune cells can be found in peripheral blood. The term “peripheral blood cell subtype” refers to cell types commonly found in peripheral blood, including but not limited to eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells. Some immune cells are “antigen-presenting cells,” which include professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes). Immune cells according to the present invention may be selected, determined, and / or modified to have the characteristics described herein. For example, Treg cells may be selected, determined, and / or modified to express TACI but not BCMA.

[0060] The term "B cell" refers to a type of leukocyte, a subtype of lymphocyte, that presents antigens, secretes cytokines, and, upon maturing into plasma cells, can secrete antibodies. "Immature B cells" are cells that can develop into mature B cells. Generally, pro-B cells (e.g., expressing CD45 or B220) undergo immunoglobulin heavy chain rearrangement to become pro-B / pre-B cells, and further undergo immunoglobulin light chain rearrangement to become immature B cells. Immature B cells include T1 and T2 B cells. Immature B cells can develop into mature B cells that can produce immunoglobulins (e.g., IgA, IgG, or IgM). Mature B cells express characteristic markers such as CD21 and CD23, but not AA41. B cells can be activated by drugs such as lipopolysaccharide (LPS), IL-4, and antibodies against IgM. B cells, their subtypes, and their developmental stages can be determined based on biomarkers well known in the art. For example, naive B cells are CD19+, CD24-, and CD38-, while memory B cells are CD19+, CD24-, CD38-, and -CD27+.

[0061] The term "Breg" refers to regulatory B cells, which are B cells that suppress resting and / or activated T cells. Bregs are well known in the art (see, for example, U.S. Patent Publication No. 2016 / 0375059, U.S. Patent Publication No. 2016 / 0152951, U.S. Patent Publication No. 2015 / 0110737, Zhang et al. (2017) Blood Cancer J.7:e547, and Blaire et al. (2010) Immunity 32:129-140). In one embodiment, Bregs are CD19 + CD24 高 CD38 高It expresses CD19+CD24. Generally, Breg produces IL-19, which has a potent anti-inflammatory effect and inhibits T cell-mediated inflammatory responses, such as Th1 immune responses. Breg can also produce TGF-β, another anti-inflammatory cytokine. In some embodiments, Breg can also produce cell surface molecules such as FasL and / or PD-L1 to induce targeted cell death. In some embodiments, Breg expresses CD19+CD24. 高 CD38 高 Bregs, which are a unique subset in bone marrow aspirates of MM patients compared to the same subset in peripheral blood compartments (Zhang et al. (2017) Blood Cancer J.24:e547). This unique Breg subset closely correlates with the loading of CD138+ myeloma cells in the bone marrow and peripheral blood compartments of MM patients. The interaction between Bregs and myeloma cells plays a vital role in Breg survival. These Bregs are functional because they induce the immunosuppressive cytokine IL-10 when stimulated by PMA. Furthermore, these Bregs reduce myeloma cell lysis induced by elotuzumab ex vivo. Therefore, this Breg subset is considered crucial in controlling the therapeutic response to anti-multiple myeloma therapies, including monoclonal antibody-based immunotherapies such as elotuzumab targeting SLAMF7 on multiple myeloma cells.

[0062] The term "T cell" is used, for example, in CD4 + T cells and CD8 + This includes T cells. The term T cell also includes both helper T1 type T cells and helper T2 type T cells. The term "antigen-presenting cells" includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0063] The term "Treg" refers to regulatory T cells, which constitute approximately 5-10% of the circulating CD4+ T cell population and are native CD4+CD25+FOXP3+ T lymphocytes that predominantly suppress autoreactive lymphocytes and act to regulate innate and adaptive immune responses (Piccirillo and Shevach (2004) Semin.Immunol.16:81-88, Fehervari and Sakaguchi (2004) Curr.Opin.Immunol.16:203-208, Azuma et al. (2003) Cancer Res.63:4516-4520, Cederbom et al. (2000) Eur.J.Immunol.30:1538-1543, Maloy et al. (2003) J.Exp.Med.197:111-119, Serra et al. (Also known as al. (2003) Immunity 19:877-889, Thornton and Shevach (1998) J. Exp. Med. 188:287-296, Janssens et al. (2003) J. Immunol. 171:4604-4612, Gasteiger et al. (2013) J. Exp. Med. 210:1167-1178, Sitrin et al. (2013) J. Exp. Med. 210:1153-1165). Tregs also include functionally inhibitory CD8+CD25+FOXP3+ T lymphocytes (Correale et al. (2010) Annu. Neurol. 67:625-638). Tregs achieve this inhibitory effect, at least partially, by inhibiting the proliferation, enlargement, and effector activity of normal T cells (Tcon). They also suppress effector T cells by destroying their (self) targets, either through intercellular contact by inhibiting T cell assistance and activation, or through the release of immunosuppressive cytokines such as IL-10 or TGF-β. reg It has been shown that IL-2-induced antitumor immunity is enhanced by cell depletion (Imai et al. (2007) Cancer Sci. 98:416-23).

[0064] Since both Tregs and Bregs inhibit the immune response, unless otherwise specified, any regulation of Tregs described herein applies to Bregs, and vice versa.

[0065] Conventional T cells, also known as Tcon or Teff, possess effector functions (e.g., cytokine secretion, cytotoxic activity, etc.) that enhance the immune response based on their expression of one or more T cell receptors. Tcon is defined as any T cell population that is not Treg, and includes, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated into, for example, Th1 or Th2 lineages. Therefore, increasing the number of Tregs, increasing Treg activity, and / or decreasing Treg cell death (e.g., apoptosis) are useful in suppressing unwanted immune responses associated with various immune disorders (e.g., cGVHD). For example, in a mouse model, adding a 1:1 mixture of CD4+CD25+ Tregs and CD25-effector T cells to donor bone marrow stem cells suppressed allogeneic immune activation and GVHD without increasing malignant relapse after transplantation (Edinger et al. (2003) Nat. Med. 9:1144-1150). In humans, active cGVHD is associated with impaired Treg rearrangement in HSCT recipients (Zorn et al. (2005) Blood 106:2903-2911). In participants with active cGVHD, impaired Treg rearrangement, low telomerase levels, and shortened telomeres are thought to contribute to reduced Treg survival (Zorn et al. (2005) Blood 106:2903-2911, Matsuoka et al. (2010) J. Clin. Invest. 120:1479-1493, Kawano et al. (2011) Blood 118:5021-5030). The role of IL-2 in Treg homeostasis and function is a major factor limiting its effectiveness as an anti-immunotoxic therapy, and it is thought to partially explain the finding that in vivo administration of syngeneic T-cell depleted donor bone marrow in addition to IL-2 prevents GVHD in MHC mismatch mice after allogeneic SCT without affecting the GVL response (Sykes et al. (1990) Proc. Natl. Acad. Sci. USA 87:5633-5647, Sykes et al. (1990) J. Exp. Med. 171:645-658).In mouse allogeneic HSCT models, co-infusion of ex vivo-enhanced Tregs with IL-2 also resulted in suppression of GVHD, along with improved immune rearrangement and preservation of the GVL response (Taylor et al. (2002) Blood 99:3493-3499, Trenado et al. (2003) J. Clin. Invest. 112:1688-1696). Tregs are also equally important in suppressing inflammation. In the context of ongoing inflammation, it is crucial that treatment preferentially enhances Tregs without activating conventional T cells (Tcon) or other effectors that could exacerbate GVHD. Effective in vivo enhancement of Tregs is also directly associated with other disorders due to impaired peripheral tolerance, such as SLE, T1D, MS, psoriasis, RA, IBD, and autoimmune diseases like vasculitis, where Treg dysfunction is increasingly believed to be involved (Grinberg-Bleyer et al. (2010) J.Exp.Med.207:1871-1878, Buckner (2010) Nat.Rev.Immunol.10:849-859, Humrich et al. (2010) Proc.Natl.Acad.Sci.USA107:204-209, Carbone et al. (2014) Nat.Med.20:69-74).

[0066] "Naive Tcon" refers to CD4 cells that have differentiated in the bone marrow and successfully undergone positive and negative central selection processes in the thymus, but have not yet been activated by exposure to an antigen. + These are T cells or CD8+ T cells. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), absence of activation markers such as CD25, CD44, or CD69, and absence of memory markers such as CD45RO. Therefore, naive T cells are thought to be quiescent and non-dividing, and require interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see at least WO2010 / 101870). The presence and activity of such cells are undesirable in relation to suppressing the immune response.

[0067] Unlike Tregs, "effector Tcon" are not anergistic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos.Trans.R.Soc.Lond.Biol.Sci.356:625-637). Effector Tcon can be CD4+ or CD8+ T cells. They recognize antigens associated with MHC class I or II molecules, respectively, and generally express activation markers such as CD25, CD44, or CD69, but generally do not express memory markers such as CD45RO. Generally, increasing the number of Tregs, increasing Treg activity, and / or decreasing Treg cell death (e.g., apoptosis) are useful in suppressing unwanted immune responses associated with various immunodeficiencies (e.g., cGVHD). Tregs are also equally important in suppressing inflammation. In the context of ongoing inflammation, treatment may preferentially enhance Tregs without the activation of Tcon or other effectors that could exacerbate GVHD. Effective in vivo enhancement of Tregs is also directly associated with other disorders due to impaired peripheral tolerance, such as SLE, T1D, MS, psoriasis, RA, IBD, and autoimmune diseases like vasculitis, where Treg dysfunction is increasingly believed to be involved (Grinberg-Bleyer et al. (2010) J.Exp.Med.207:1871-1878, Buckner (2010) Nat.Rev.Immunol.10:849-859, Humrich et al. (2010) Proc.Natl.Acad.Sci.USA107:204-209, Carbone et al. (2014) Nat.Med.20:69-74).

[0068] "Memory Tcon" refers to T cells that have experienced an antigen (i.e., T cells that have been previously exposed to and responded to an antigen), represented by at least three distinct subpopulations of T cells. Memory Tcon can rapidly replicate upon re-exposure to the antigen, eliciting a stronger immune response. Memory Tcon subpopulations can be identified based on the differential expression of the chemokine receptor CCR7 and L-selection (CD62L) (Sallusto et al. (2000) Curr. Top. Microbiol. Immunol. 251:167-171). For example, stem cell memory T cells (Tscm) are like naive cells, expressing CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting numerous functional characteristics specific to memory cells (Gattinoni et al. (2011) Nat. Med. 17:1290-1297). Central memory cells (Tcm) express L-selectin and CCR7 and secrete IL-2, but do not secrete IFNγ or IL-4. Effector memory cells (Tem) do not express L-selectin or CCR7, but produce effector cytokine-like IFNγ and IL-4.

[0069] A “fatigued Tcon” is a T cell that has gradually lost its function. “Fatigue” or “unresponsiveness” refers to a state in which a cell does not perform its normal function or activity in response to normal input signals, and includes the refractive activity of immune cells to stimuli such as stimulation via activating receptors or cytokines. Such functions or activities include, but are not limited to, proliferation or cell division, entry into the cell cycle, cytokine production, cytotoxicity, trafficking, phagocytic activity, or any combination thereof. Normal input signals may include, but are not limited to, stimulation via receptors (e.g., T cell receptors, B cell receptors, costimulatory receptors, etc.).

[0070] Exhausted immune cells may exhibit a reduction in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytic activity, or any combination thereof, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, compared to corresponding control immune cells of the same type. In one embodiment, the exhausted cell is a CD8+ T cell (e.g., an antigen-specific effector CD8+ T cell). CD8 cells normally proliferate in response to stimulation of T cell receptors and / or costimulatory receptors, as well as in response to cytokines such as IL-2 (e.g., colony growth). Therefore, an exhausted CD8 T cell is one that does not proliferate and / or produce cytokines in response to normal input signals. It is well known that the exhaustion of effector function can be described in several stages, eventually leading to terminal or complete exhaustion and ultimately to elimination (Yi et al. (2010) Immunol. 129:474-481, Wherry and Ahmed (2004) J. Virol. 78:5535-5545). In the first stage, functional T cells enter the "partial exhaustion I" stage, which is characterized by the loss of a subset of effector function, including loss of IL-2 production, reduced TNFα production, and reduced proliferative and / or ex vivo lytic capacity. In the second stage, partially exhausted T cells enter the "partial exhaustion II" stage, where both IL-2 and TNFα production following antigen stimulation cease, and IFNγ production is reduced. "Complete exhaustion" or "terminal exhaustion" occurs when CD8+ T cells lose all effector functions, including the absence of IL-2, TNFα, and IFNγ production following antigen stimulation, as well as loss of ex vivo lytic and proliferative capacity. Completely exhausted CD8+ T cells do not proliferate in response to normal input signals, do not lyse (cytotoxicize) target cells, and / or do not produce appropriate cytokines such as IL-2, TNFα, or IFNγ. Such a lack of effector function can occur when antigen loading is high and / or CD4 assistance is low.This hierarchical loss of function is also associated with the expression of co-inhibitor immune receptors such as PD-1, TIM-3, and LAG-3 (Day et al. (2006) Nature 443:350-4, Trautmann et al. (2006) Nat. Med. 12:1198-202, and Urbani et al. (2006) J. Virol. 80:1398-1403). Other molecular markers, such as high eomesodermin (EOMES) and low TBET expression, distinguish hierarchical stages of immune cell exhaustion (Paley et al. (2012) Science 338:1220-1225). Additional markers of exhausted T cells include decreased Bcl-b and increased BLIMP-1 (Pdrm1) production.

[0071] Immune cells can be obtained from a single source or multiple sources (e.g., a single subject or multiple subjects). Multiple means at least two (e.g., more than one). Still in another embodiment, the non-human mammal is a mouse. The animal from which the cell type of interest is obtained may be adult, neonatal (e.g., less than 48 hours postnatal), immature, or intrauterine. The cell type of interest may be primary cells, stem cells, established cancer cell lines, immortalized primary cells, etc.

[0072] Therefore, a decrease in the number of Treg / Bregs, a decrease in Treg / Breg activity, and / or an increase in Treg / Breg cell death (e.g., apoptosis) are generally useful in increasing the immune response associated with various immune disorders (e.g., cancer, infections, etc.). The reverse is also applicable to reducing the immune response by upregulating the number of Treg / Bregs and / or inhibitory immune activity. For example, effective in vivo enhancement of Tregs is also directly associated with other disorders due to peripheral tolerance dysfunction (e.g., autoimmune diseases such as SLE, T1D, MS, psoriasis, RA, IBD, and vasculitis), which are increasingly believed to involve Treg / Breg dysfunction (Grinberg-Bleyer et al. (2010) J.Exp.Med.207:1871-1878, Buckner (2010) Nat.Rev.Immunol.10:849-859, Humrich et al. (2010) Proc.Natl.Acad.Sci.USA107:204-209, Carbone et al. (2014) Nat.Med.20:69-74).

[0073] The number / activity of Treg / Bregs, the activity of Tcon, the regulation of Treg:Tcon interactions, and the regulation of Breg:Bcon interactions can be determined according to methods well known in the art and as illustrated in the examples. For example, the proliferation, activity, apoptosis, cytokine production repertoire of Treg / Bregs and / or Tcon, the activity of Treg / Bregs, the apoptosis of Treg / Bregs, the expression of cellular biomarkers (e.g., expression of CD4, CD19, CD24, CD25, CD38, CD25, FOXP3, etc.) can be analyzed. Furthermore, phenotypic analysis of lymphocyte subsets, functional assays of immunomodulation leading to reduced immune responses, and cytokines in plasma can be analyzed as further described herein.

[0074] The same well-known characteristics of immune cells can also be used to purify, concentrate, and / or isolate Treg / Breg, or alternatively, to modulate (e.g., reduce) or determine (e.g., confirm reduction) Treg / Breg. For example, the term "enriched Treg / Breg" refers to a composition containing Treg / Breg in a certain ratio in addition to other T cells, such that the composition has a Treg / Breg to Tcon (i.e., Treg to Tcon or Breg to Tcon), CD3+ cell, or Treg / Breg to another cell benchmark ratio of at least 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1, or greater, or any range or value between them. Such ratios can be achieved by purifying compositions containing T / B cells using various methods, such as CD8+ and CD19+ co-depletion combined with positive selection for CD25+ cells. Such enriched Treg / Breg cells can be further defined in terms of cell markers and / or viability. For example, an enriched Treg / Breg cell composition may have a total cell viability of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any range or value between them. It may contain cells expressing specific biomarkers of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any range or value between them. For example, it may contain FoxP3+ T cells that are greater than or equal to 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or any range or value between them.Similarly, the term "reduced Treg / Breg" refers to a reduction in Treg / Breg and can be quantified and qualified according to the reverse of the explanation provided above with respect to enriched Treg / Breg. The term "increased Treg / Breg" refers to the opposite of reduced Treg / Breg.

[0075] A “blocking” antibody or antibody “antagonist” is one that inhibits or reduces the biological activity of at least one of the antigens to which it binds. In certain embodiments, the blocking antibody or antagonist antibody or fragment thereof described herein substantially or completely inhibits the given biological activity of the antigen(s).

[0076] The term "body fluids" refers to fluids that are excreted or secreted from the body, as well as fluids that are not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, earwax and earwax, Cowper's fluid or bulbourethral gland fluid (pre-ejaculatory fluid), chyle, porridge, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menstrual fluid, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous fluid, and vomit).

[0077] The terms “cancer,” “tumor,” or “hyperproliferative” refer to the presence of cells that possess characteristics typical of oncogenic cells, such as uncontrolled growth, immortality, metastatic ability, rapid growth and proliferation rates, and certain characteristic morphological features. In some embodiments, such cells exhibit such characteristics partly or entirely due to the expression and activity of oncogenes, or defective expression and / or activity of tumor suppressor genes such as retinoblastoma protein (Rb). While cancer cells often take the form of tumors, such cells may exist alone in animals or may be non-tumorogenic cancer cells such as leukemia cells. As used herein, the term “cancer” includes pre-malignant and malignant cancers. Cancers include, but are not limited to, B-cell carcinomas such as multiple myeloma, Waldenström macroglobulinemia such as alpha-chain disease, gamma-chain disease, and mu-chain disease, benign monoclonal hypergammaglobulinemia, immunocellular amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and hematological cancers.Other non-limiting examples of cancer types to which the methods encompassed by the present invention are applicable include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminomas, fetal cancer, and viroma. Musculoskeletal tumors, cervical cancer, bone cancer, brain tumors, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, cancer is of an epithelial nature and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. Still in other embodiments, epithelial carcinoma is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial carcinoma may include, but is not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated, and may be characterized in a variety of other ways.

[0078] In certain embodiments, the cancer is multiple myeloma. Multiple myeloma, also known as plasma cell myeloma or Karel's disease, is a cancer of plasma cells, a type of white blood cell normally responsible for antibody production. In multiple myeloma, abnormal clusters of plasma cells accumulate in the bone marrow, where they interfere with the production of normal blood cells. Most cases of myeloma are also characterized by the production of paraproteins, abnormal antibodies that can cause kidney problems. Bone lesions and hypercalcemia (high blood calcium levels) are also frequently experienced. Diagnosing multiple myeloma is generally insufficient based on the results of any single test. Diagnosis is based on a combination of factors, including the patient's description of symptoms, the physician's physical examination of the patient, and the results of blood tests and optional X-rays. Diagnosis of multiple myeloma in a subject may occur through any established diagnostic procedure known in the art. Generally, multiple myeloma is diagnosed when a plasma cell tumor is confirmed by biopsy, or when at least 10% of the cells in the bone marrow are plasma cells, and in addition, there is evidence that either the blood or urinary level of M protein is above a certain level (e.g., 3 g / dL and 1 g / dL, respectively), or when imaging shows bone holes due to tumor growth or fragile bone (osteoporosis). In addition to the cancer therapies described herein, multiple myeloma and other cancers may, in some embodiments, respond to therapeutically effective doses of proteasome inhibitors, such as bortezomib. Bortezomib reversibly blocks the function of cellular proteasomes, affecting numerous biological pathways, including those related to cancer cell growth and survival. Numerous other effective proteasome inhibitors are known in the art, including, for example, carfilzomib, MLN9708, delanzomib, oprozomib, AM-114, marizomib, TMC-95A, curcusone-D, and PI-1840 (see, for example, U.S. Patent Publication 2017 / 0101684).Bortezomib is currently approved for use in patients with multiple myeloma who have previously received at least one treatment, whose disease has worsened since the last treatment, and who have either already undergone a bone marrow transplant or are not suitable for a bone marrow transplant. Bortezomib has significant activity in patients with relapsed multiple myeloma and in MM patients with renal failure. The efficacy of proteasome inhibitors like bortezomib is known to increase when used in combination with dexamethasone and other cancer drugs such as doxorubicin. Therefore, proteasome inhibitors may, in accordance with this disclosure, be used alone or in combination with other therapeutic agents described herein, such as melphalan, prednisone, doxorubicin, dexamethasone, immunomodulators, monoclonal antibody drugs including antibody fragment-based drugs, kinesin spindle protein (KSP) inhibitors, tyrosine kinase inhibitors, HDAC inhibitors, BCL2 inhibitors, cyclin-dependent kinase inhibitors, mTOR inhibitors, heat shock protein inhibitors, Bruton's kinase inhibitors, insulin-like growth factor inhibitors, RAS inhibitors, PARP inhibitors, and B-RAF inhibitors.

[0079] The term "coding region" refers to a region of a nucleotide sequence that contains codons, which are translated into amino acid residues, while the term "non-coding region" refers to a region of a nucleotide sequence that is not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0080] The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that an adenine residue in a first nucleic acid region can form a specific hydrogen bond ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region, if that residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid chain can base pair with a residue in a second nucleic acid chain that is antiparallel to the first chain, if that residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if at least one nucleotide residue in the first region can base pair with a residue in the second region when these two regions are arranged in an antiparallel configuration. Preferably, the first region comprises the first portion and the second region comprises the second portion such that at least about 50%, preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base-pairing with nucleotide residues in the second portion when the first and second portions are arranged in an antiparallel configuration. More preferably, all nucleotide residues of the first portion are capable of base-pairing with nucleotide residues in the second portion.

[0081] The term “control” refers to any reference standard suitable for providing a comparison with the expression product in the test sample. In one embodiment, the control comprises obtaining a “control sample,” from which the expression product level is detected and compared to the expression product level from the test sample. Such a control sample may include, but is not limited to, any suitable sample derived from a control patient with a known outcome (which may be a stored sample or a measurement from a previous sample); normal tissue or cells isolated from a subject such as a normal patient or a patient with the target condition (cancer is used below as a representative condition); cultured primary cells / tissues isolated from a subject such as a normal subject or a cancer patient; adjacent normal cells / tissues obtained from the same organ or body part of a cancer patient; tissue or cell samples isolated from a normal subject; or primary cells / tissues obtained from a depositary. In another preferred embodiment, the control may include, but is not limited to, expression product levels of a reference standard derived from any preferred source, housekeeping genes, expression product level ranges from normal tissue (or other previously analyzed control samples), a group of patients, or a previously determined expression product level range within a test sample derived from a pair of patients having a particular outcome (e.g., survival rates of 1, 2, 3, 4 years, etc.) or receiving a particular treatment (e.g., standard cancer therapy). Those skilled in the art will understand that such control samples and reference standard expression product levels may be used in combination as controls in the method of the present invention. In one embodiment, the control may include normal or non-cancerous cell / tissue samples. In another preferred embodiment, the control may include expression levels for a pair of patients, such as a pair of cancer patients, or for a pair of cancer patients receiving a particular treatment, or for a pair of patients having one outcome versus another. In the former case, the specific expression product levels of each patient may be assigned to percentile expression levels, or they may be expressed as being either higher or lower than the mean or average of the expression levels of a reference standard. In another preferred embodiment, the control may include normal cells, cells from patients treated with combination chemotherapy, and cells from patients with benign cancer.In another embodiment, the control may also include a measured value, for example, the mean expression level of a particular gene in a population compared to the expression level of a housekeeping gene in the same population. Such population may include normal subjects, cancer patients who have never received any treatment (i.e., treatment-naïve), cancer patients receiving standard treatment, or patients with benign cancer. In another preferred embodiment, the control includes, but is not limited to, a transformation of the expression product level ratio, which involves determining the ratio of the expression product levels of two genes in the test sample and comparing it to any preferred ratio of the same two genes in a reference standard; determining the expression product levels of two or more genes in the test sample and determining the difference in expression product levels in any preferred control; and determining the expression product levels of two or more genes in the test sample, normalizing their expression to the expression of a housekeeping gene in the test sample and comparing it to any preferred control. In a particularly preferred embodiment, the control includes a control sample belonging to the same series and / or type as the test sample. In another embodiment, the control may include expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment, a control expression product level is established, where, for example, a higher or lower expression product level compared to a specific percentile is used as a basis for predicting the outcome. In another preferred embodiment, a control expression product level is established using expression product levels from cancer control patients with known outcomes, and the expression product level from the test sample is compared to the control expression product level as a basis for predicting the outcome. As demonstrated by the data below, the method of the present invention is not limited to the use of specific cut points when comparing the expression product level in the test sample to the control.

[0082] The “copy number” of a biomarker nucleic acid refers to the number of DNA sequences encoding a particular gene product in a cell (e.g., germ cells and / or somatic cells). Generally, for a given gene, mammals have two copies of each gene. However, the copy number can be increased by gene amplification or duplication, or decreased by deletion. For example, changes in germ cell copy number include changes in one or more genomic loci, which are not explained by the copy number in the normal complement of germ cell copies in a control (e.g., the normal copy number in germ cell DNA for the same species as the specific germ cell DNA and the corresponding copy number determined). Changes in somatic cell copy number include changes in one or more genomic loci, which are not explained by the copy number in germ cell DNA in a control (e.g., the copy number in germ cell DNA for the same subject as the somatic cell DNA and the corresponding copy number determined).

[0083] The “normal” copy number of a biomarker nucleic acid (e.g., germ cells and / or somatic cells) or the “normal” expression level of a biomarker nucleic acid or protein is the activity / expression level or copy number in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, feces, and bone marrow, derived from a subject, e.g., a human without cancer, or from the corresponding non-cancerous tissue in the same subject with cancer.

[0084] The phrase “determining a suitable treatment regimen for a subject” is to be interpreted as meaning the determination of a treatment regimen for a subject (i.e., a single therapy or combination of different therapies used to prevent and / or treat cancer in the subject) that is initiated, modified, and / or terminated based on the results of the analysis according to the present invention, or essentially or at least partially. One example is deciding whether to provide targeted therapy for cancer, such as providing immunomodulatory therapy (e.g., APRIL / TACI interaction modulator therapy). Another example would be initiating adjuvant therapy after surgery aimed at reducing the risk of recurrence, and yet another would be modifying the dosage of a particular chemotherapy. The decision may be based on the results of the analysis according to the present invention, as well as the individual-specific characteristics of the subject to be treated. In most cases, the actual determination of a suitable treatment regimen for a subject will be made by the attending physician or doctor.

[0085] The term "expression signature" or "signature" refers to a group of two or more biomarkers that are expressed in coordination. For example, the genes, proteins, etc. that make up this signature may be expressed in a specific cell lineage, differentiation stage, or during a particular biological response. Biomarkers may reflect biological aspects of the tumor in which they are expressed, such as the origin cells of cancer, the nature of non-malignant cells in a biopsy sample, and the oncogenic mechanisms that cause cancer. Expression data and gene expression levels can be stored in computer-readable media, such as microarrays or computer-readable media used in conjunction with chip reading devices. Such expression data can be manipulated to generate expression signatures.

[0086] A molecule is “immobilized” or “attached” to a substrate if it associates with the substrate covalently or noncovalently, thereby allowing the substrate to be rinsed with a fluid (e.g., standard saline citrate, pH 7.4) without a substantial proportion of the molecules dissociating from the substrate.

[0087] The terms “high,” “low,” “intermediate,” and “negative” in relation to the expression of cellular biomarkers refer to the level of biomarker expression compared to the expression of the biomarker in one or more reference cells. Biomarker expression can be determined by any method described herein, including, but not limited to, the analysis of the cellular level, activity, structure, etc., of the genomic nucleic acid, ribonucleic acid, and / or polypeptide of one or more biomarkers. In one embodiment, these terms refer to defined percentages of cell populations that express the biomarker at the highest, intermediate, or lowest levels, respectively. Such percentages can be defined as the top 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15% or greater, or any range between them, including boundary values, of the cell population that either highly or weakly expresses the biomarker. The term "low" excludes cells that do not detectably express the biomarker, as they are "negative" in terms of biomarker expression. The term "intermediate" includes cells that express the biomarker, but at a lower level than the population that expresses it at a "high" level. In another embodiment, these terms may also refer to, or alternatively refer to, cell populations expressing a biomarker identified by a qualitative or statistical plot region. For example, cell populations selected using flow cytometry may be distinguished based on biomarker expression levels by identifying distinctly different plots based on an analysis of the detectable portion, for example, on average fluorescence intensity, according to methods well known in the art. Such plot regions may be refined with respect to the biomarker of interest according to number, shape, overlap, etc., according to methods well known in the art. Still in yet another embodiment, these terms may also be determined according to the presence or absence of expression of additional biomarkers.

[0088] The term "homologous" refers to the similarity of nucleotide sequences between two regions of the same nucleic acid chain or between regions of two different nucleic acid chains. Regions are homologous in a given position if the position of a nucleotide residue in both regions is occupied by the same nucleotide residue. A first region is homologous to a second region if at least one position of a nucleotide residue in each region is occupied by the same residue. The homology between two regions is expressed in terms of the ratio of the positions of nucleotide residues in the two regions that are occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first part and the second region comprises a second part such that at least about 50%, preferably at least about 75%, at least about 90%, or at least about 95% of the positions of nucleotide residues in each of the first and second parts are occupied by the same nucleotide residue. More preferably, all positions of nucleotide residues in each of these parts are occupied by the same nucleotide residue.

[0089] The term "STING," or "interferon gene stimulator," also known as transmembrane protein 173 (TMEM173), refers to a five-transmembrane protein that functions as a major regulator of the innate immune response to viral and bacterial infections. STING is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activated TBK1 / IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signaling and transcriptional activator 6) signaling pathways to induce a robust type I interferon and pro-inflammatory cytokine response. The term "STING" is intended to include its fragments, variants (e.g., allele variants), and derivatives. Representative human STING cDNA and human STING protein sequences are well known in the art and are generally available from the National Center for Biotechnology Information (NCBI). Human STING isoforms include the longer isoform 1 (NM_198282.3 and NP_938023.1) and the shorter isoform 2 (NM_001301738.1 and NP_001288667.1, which has a shorter 5'UTR and lacks an exon in the 3' coding region, thereby resulting in a shorter, distinctive C-terminus compared to variant 1).Nucleic acid and polypeptide sequences of STING orthologs in organisms other than humans are well-known, and these include, for example, chimpanzee CDH1 (XM_016953921.1 and XP_016809410.1, XM_009449784.2 and XP_009448059.1, XM_001135484.3 and XP_001135484.1), monkey CDH1 (XM_015141010.1 and XP_014996496.1), dog CDH1 (XM_022408269.1 and XP_022263977.1, XM_005617260.3 and XP_005617317.1, XM_022408249.1 and XP_022263957.1, XM_005617262.3 and XP_005617319.1, XM_005617258.3 and XP_005617315.1, XM_022408253.1 and XP_022263961.1, XM_005617257.3 and XP_005617314.1, XM_02240�240.1 and XP_022263948.1, XM_005617259.3 and XP_005617316.1, XM_022408259.1 and XP_022263967.1, XM_022408265.1 and XP_022263973.1), bovine CDH1 (NM_001046357.2 and NP_001039822.1), mouse CDH1 (NM_001289591.1 and NP_001276520.1, NM_001289592.1 and NP_001276521.1, NM_028261.1 and NP_082537.1), and rat CDH1 (NM_001109122.1 and NP_001102592.1).

[0090] STING agonists have been shown to be useful therapeutic agents for treating cancer. STING agonists are well known in the art and include, for example, MK-1454, STING agonist-1 (MedChem Express catalog number HY-19711), cyclic dinucleotides (CDNs), such as cyclic di-AMP (c-di-AMP), cyclic di-GMP (c-di-GMP), cGMP-AMP (2'3'cGAMP or 3'3'cGAMP), or 10-carboxymethyl-9-acridanone (CMA) (Ohkuri et al., Oncoimmunology. 2015;4(4):e999523), rationally designed synthetic CDN derivative molecules (Fu et al., Sci Transl Med. 2015:7(283):283ra52.doi:10.1126 / scitranslmed.aaa4306), and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) (Corrales et al. This includes (al., Cell Rep. 2015;11(7):1018-1030). These agonists bind to STING and activate it, resulting in a potent type I IFN response. On the other hand, targeting the cGAS-STING pathway with small molecule inhibitors may be beneficial in treating severe debilitating diseases such as inflammatory and autoimmune diseases, which are associated with excessive type I IFN production due to abnormal DNA sensing and signaling. STING inhibitors are also known, including, for example, CCCP (MedChem Express, catalog no. HY-100941) and 2-bromopalmitate (Tao, et al., IUBMB Life. 2016;68(11):858-870). It should be noted that this term may further be used to refer to any combination of the features described herein with respect to the STING molecule. For example, any combination of sequence composition, identity percentage, sequence length, domain structure, functional activity, etc., may be used to describe the STING molecule of the present invention.

[0091] The terms "STING pathway" or "cGAS-STING pathway" refer to the STING-regulated innate immune pathway that mediates cytosolic DNA-induced signaling events. Cytosolic DNA binds to cGAS and activates it, and cGAS catalyzes the synthesis of 2'3'-cGAMP from ATP and GTP. 2'3'-cGAMP binds to the ER adapter STING, causing STING to move to the ER-Golgi intermediate compartment (ERGIC) and Golgi apparatus. STING then activates IKK and TBK1. TBK1 phosphorylates STING, which in turn recruits IRF3 for phosphorylation by TBK1. The phosphorylated IRF3 dimerizes and then enters the nucleus, where it functions with NF-κB to turn on the expression of type I interferon and other immunomodulatory molecules. The cGAS-STING pathway not only mediates protective immune defense against infection by a wide range of DNA-containing pathogens but also detects tumor-derived DNA and generates endogenous anti-tumor immunity. However, abnormal activation of the cGAS-STING pathway by autologous DNA can also lead to autoimmune and inflammatory diseases.

[0092] The term "immunotherapy" refers to a form of targeted therapy that can include, for example, the use of cancer vaccines and / or sensitized antigen-presenting cells. For example, oncolytic viruses are viruses that can infect and lyse cancer cells while leaving normal cells intact, and thus they may be useful in immunomodulatory therapy. The replication of oncolytic viruses facilitates tumor cell destruction and also results in dose amplification at the tumor site. They can also act as vectors for anti-cancer genes and can be specifically delivered to the tumor site. Immunotherapy can involve passive immunity for short-term protection of the host, achieved by administration of pre-formed antibodies directed against cancer antigens or disease antigens (e.g., administration of monoclonal antibodies against tumor antigens, optionally conjugated to a chemotherapeutic agent or toxin). Immunotherapy can also focus on the use of epitopes of cancer cell lines recognized by cytotoxic lymphocytes. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides, etc. can be used to selectively modulate biomolecules associated with tumor or cancer initiation, progression, and / or pathology. As described above, immunotherapy against immune checkpoint targets such as PD-1, PD-L1, PD-L2, CTLA-4, etc. is useful.

[0093] The term “immune checkpoint” refers to a group of molecules on the cell surface of CD4+ and / or CD8+ T cells that fine-tune the immune response by downmodulating or inhibiting the antitumor immune response. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, IDO1, IDO2, and A2aR (see, for example, WO2012 / 177624). This term further encompasses fragments of bioactive proteins, as well as nucleic acids encoding full-length immune checkpoint proteins and fragments of those bioactive proteins. In some embodiments, this term further encompasses any fragments as described herein in relation to homology.

[0094] Immune checkpoints and their sequences are well known in the art, and representative embodiments are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that functions as a co-inhibitory receptor having PD-L1 and PD-L2 as known ligands. PD-1 was previously identified using a subtraction cloning-based technique to select a gene that is upregulated during TCR-induced activated T cell death. PD-1 is a member of the CD28 / CTLA-4 family of molecules based on its ability to bind to PD-L1. Similar to CTLA-4, PD-1 is rapidly induced on the surface of T cells in response to anti-CD3 (Agata et al. 25(1996) Int.Immunol. 8:765). However, in contrast to CTLA-4, PD-1 is also induced on the surface of B cells (in response to anti-IgM). PD-1 is also expressed on subsets of thymocytes and myeloid cells (Agata et al. (1996) (see above), Nishimura et al. (1996) Int.Immunol.8:773).

[0095] The term "IDO" refers to indoleamine 2,3-dioxygenase, a monomeric heme-containing cytosolic enzyme that catalyzes the first and rate-limiting step in tryptophan catabolism in the kynurenine pathway. IDO is encoded by the "IDO1" gene and can act on multiple tryptophan substrates, including, for example, D-tryptophan, L-tryptophan, 5-hydroxytryptophan, tryptamine, and serotonin. The term is intended to include its fragments, variants (e.g., allele variants), and derivatives. Representative human IDO1 cDNA and human IDO protein sequences are well known in the art and are generally available from the National Center for Biotechnology Information (NCBI) under accession numbers NM_002164.5 and NP_002155.1, respectively. Nucleic acid and polypeptide sequences of IDO1 / IDO orthologs in non-human organisms are well known, and include, for example, mouse IDO1 / IDO (NM_008324.1 and NP_032350.1), chimpanzee IDO1 / IDO (XM_001137531.2 and XP_0011373531.1), monkey IDO1 / IDO (NM_001077483.1 and NP_001070951.1), dog IDO1 / IDO (XM_532793.4 and XP_532793.1), bovine IDO1 / IDO (NM_001101866.2 and NP_001095336.1), and rat IDO1 / IDO (NM_023973.1 and NP_076463.1). Anti-IDO antibodies are well known in the art and include, for example, LS-C123833 (Lifespan Biosciences), AG-20A-0035 (Adipogen), MCA5433Z (AbD Serotec), HPA023149 (Atlas Antibodies), OAAB01406 (Aviva Systems Biology), and 210-301-E58 (Rockland).In addition, other inhibitors of IDO (e.g., small molecules) are known, including, for example, NSC-721782 (1-methyl-[D]-tryptophan, Muller et al. (2005) Nat. Med. 11:312-319), INCB024360 (Liu et al. (2010) Blood 115:3520-3530), and others (see, for example, Muller et al. (2005) Exp. Opin. Ther. Targ. 9:831-849). It should be noted that this term may also be used to refer to any combination of the features described herein with respect to the IDO1 / IDO molecule. For example, any combination of sequence composition, identity percentage, sequence length, domain structure, functional activity, etc., may be used to describe the IDO1 / IDO molecule of the present invention.

[0096] IDO is also encoded by the “IDO2” gene, which, like IDO1, can act similarly on multiple tryptophan substrates, including, for example, D-tryptophan, L-tryptophan, 5-hydroxytryptophan, tryptamine, and serotonin (Ball et al. (2007) Gene 396:203-213). Thus, references to the term “IDO” encompass both IDO and IDO2 proteins, as they have the same enzymatic activity, as desired by the embodiments described herein, unless each protein is specifically defined as either IDO or IDO2. The term is intended to include its fragments, variants (e.g., allele variants), and derivatives. Representative human IDO2 cDNA and human IDO2 protein sequences are well known in the art and are generally available from the National Center for Biotechnology Information (NCBI) under accession numbers NM_194294.2 and NP_919270.2, respectively. Nucleic acid and polypeptide sequences of IDO2 / IDO2 orthologs in non-human organisms are well known, including, for example, mouse IDO2 / IDO2 (NM_145949.2 and NP_666061.3), chimpanzee IDO2 / IDO2 (XM_528116.4 and XP_528116.4), monkey IDO2 / IDO2 (XM_001095833.2 and XP_001095833.2), and canine IDO2 / IDO2 (XM_005629824.1, XP_0056 This includes 29881.1, XM_005629827.1, XP_005629884.1, XM_005629826.1, XP_05629883.1, XM_005629825.1, XP_005629882.1, XM_005629828.1, and XP_005629885.1), and rat IDO2 / IDO2 (XM_001061228.2, XP_001061228.2, XM_003752920.1, and XP_003752968.1).Anti-IDO2 antibodies are well known in the art and include, for example, LS-C165098 (Lifespan Biosciences), 600-401-C69 and 210-301-E59 (Rockland), OAAB08672 and OAEBB02067 (Aviva Systems Biology), TA501378 (Origene), EB09548 (Everest Biotech), PA5-19180 (Thermo Fisher Scientific, Inc.), orb20285 and orb30411 (Biorbyt), and AP09441PU-N (Acris Antibodies). In addition, other inhibitors of IDO2 (e.g., small molecules) are known, including, for example, tenatoprazole (Bakmiwewa et al. (2012) Bioorg. Med. Chem. Lett. 22:7641-7646), 1-D-methyltryptophan (D-1MT) (Yuasa et al. (2010) Comp. Biochem. Physiol. B Biochem. Mol. Biol. 157:10-15), and others. It should be noted that this term may further be used to refer to any combination of the features described herein with respect to IDO2 / IDO2 molecules. For example, any combination of sequence composition, identity percentage, sequence length, domain structure, functional activity, etc., may be used to describe the IDO2 / IDO2 molecules of the present invention.

[0097] "Immune checkpoint" or "immune checkpoint inhibitor" or "immune checkpoint blockade" therapy refers to the use of drugs that inhibit immune checkpoint nucleic acids and / or proteins. Immune checkpoints share the common function of providing inhibitory signals that suppress the immune response, and inhibition of one or more immune checkpoints can block or otherwise neutralize inhibitory signaling, thereby upregulating the immune response to more effectively treat cancer. Exemplary drugs useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptide mimes, native ligands, and derivatives of native ligands that can either bind to and / or inactivate immune checkpoint proteins or fragments thereof, or inhibit immune checkpoint proteins or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc., that can downregulate the expression and / or activity of immune checkpoint nucleic acids or fragments thereof. Exemplary agents for upregulating the immune response include antibodies against one or more immune checkpoint proteins that block the interaction between a protein and its innate receptor(s); inactivated forms of one or more immune checkpoint proteins (e.g., dominant-negative polypeptides); small molecules or peptides that block the interaction between one or more immune checkpoint proteins and their innate receptor(s); fusion proteins that bind to their innate receptor(s) (e.g., extracellular components of immune checkpoint inhibitor proteins fused to the Fc portion of an antibody or immunoglobulin); and nucleic acid molecules that block the transcription or translation of immune checkpoint nucleic acids. Such agents can block inhibitory signaling and upregulate the immune response by directly blocking the interaction between one or more immune checkpoints and their innate receptor(s) (e.g., antibodies). Alternatively, agents can block inhibitory signaling and upregulate the immune response by indirectly blocking the interaction between one or more immune checkpoint proteins and their innate receptor(s).For example, soluble versions of immune checkpoint protein ligands, such as stabilized extracellular domains, can bind to their receptors and indirectly reduce the effective concentration of the receptor required to bind to the appropriate ligand. In one embodiment, anti-PD-1 antibodies, anti-PD-L1 antibodies, and / or anti-PD-L2 antibodies are used, either alone or in combination, to inhibit immune checkpoints. These embodiments are also applicable to specific therapies against particular immune checkpoints, such as the PD-1 pathway (e.g., anti-PD-1 pathway therapy, also known as PD-1 pathway inhibitor therapy). Numerous immune checkpoint inhibitors are known and generally available, including, for example, Keytruda® (pembrolizumab; anti-PD-1 antibody), Opdivo® (nivolumab; anti-PD-1 antibody), Tecentriq® (atezolizumab; anti-PD-L1 antibody), durvalumab (anti-PD-L1 antibody), and others.

[0098] The term "immunodeficiency" refers to a medical condition characterized by an unwanted immune response. In some embodiments, an immunodeficiency is one in which the desired response to the immunodeficiency is suppression of the immune response. Conditions in which downregulation of the immune response is desired are well known in the art and include, but are not limited to, situations of tissue, skin, and organ transplantation, graft-versus-host disease (GVHD), inflammation, or autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis, allergies, hypersensitivity reactions, and disorders requiring increased production or function of regulatory T cells, as further described herein. In other embodiments, an immunodeficiency is one in which the desired response is an increase in the immune response. Conditions in which upregulation of the immune response is desired are well known in the art and include, but are not limited to, disorders requiring increased production or function of CD4+ effector T cells, e.g., fighting cancer, infections (e.g., parasitic infections, bacterial infections, helminthic infections, or viral infections), disorders requiring improved vaccination efficiency, etc.

[0099] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term "immune response" includes immune responses indirectly affected by T cell activation, such as antibody production (humoral response), and cytokine-responsive cells, such as macrophage activation.

[0100] The term "immunotherapy agent" may include any molecule, peptide, antibody, or other agent that can stimulate the host's immune system to produce an immune response against a tumor or cancer in a target. Various immunotherapy agents are useful in the compositions and methods described herein.

[0101] The terms “inhibit” or “downregulate” include, for example, reducing, limiting, or blocking a particular action, function, or interaction. In some embodiments, cancer is “inhibited” if at least one symptom of cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if cancer recurrence or metastasis is reduced, slowed, delayed, or prevented. Similarly, a biological function, such as the function of a protein, is inhibited if it is reduced compared to a baseline state, such as a control, such as a wild-type state. For example, if a drug reduces the desired physical interaction between APRIL and TACI, for example, TACI expressed by Treg and / R Breg, then the binding of APRIL to TACI is inhibited by that drug. Such inhibition or impairment may be induced, such as by the application of the drug at a particular time and / or place, or constitutive, such as by a genetic mutation. Such inhibition or deficiency can also be partial or complete (e.g., essentially no measurable activity compared to a baseline state, such as a control like the wild-type state). Essentially complete inhibition or deficiency is referred to as blockade. The terms “promote” or “upregulate” have the opposite meaning.

[0102] When referring to interactions between two molecules, the term "interaction" refers to the physical contact (e.g., bonding) of these molecules with one another. Generally, such interactions result in the activation of one or both molecules (which produces biological effects).

[0103] "Isolated protein" means a protein that, if isolated from cells or produced by recombinant DNA techniques, substantially contains no other proteins, cellular material, isolation medium, or culture medium, or, if chemically synthesized, substantially contains no chemical precursors or other chemicals. "Isolated" or "purified" proteins or their bioactive portions substantially contain no cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide, or fusion protein originates, or, if chemically synthesized, substantially contains no chemical precursors or other chemicals. The phrase "substantially free of cellular material" includes preparations of biomarker polypeptides or fragments from which the protein is isolated or recombinantly produced from cellular components of the source cell. In one embodiment, the phrase “substantially free of cellular material” means that the preparation of a biomarker protein or fragment thereof contains less than about 30% (based on dry weight) of non-biomarker proteins (also referred to herein as “contaminating proteins”), more preferably less than about 20% of non-biomarker proteins, still more preferably less than about 10% of non-biomarker proteins, and most preferably less than about 5% of non-biomarker proteins. When an antibody, polypeptide, peptide, or fusion protein, or fragment thereof, for example, a bioactive fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., the culture medium accounts for less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%.

[0104] A “kit” is any product (e.g., package or container) comprising at least one reagent, e.g., a probe or small molecule, for specifically detecting and / or influencing the expression of the markers of the present invention. A kit may be marketed, distributed, or sold as a complete set for performing the methods of the present invention. A kit may contain one or more reagents necessary to express compositions useful in the methods of the present invention. In certain embodiments, a kit may further contain a reference standard, e.g., a nucleic acid encoding a protein that does not affect or control signaling pathways that control cell growth, division, migration, survival, or apoptosis. Those skilled in the art will know that many such control proteins can be envisioned, including, but not limited to, common molecular tags (e.g., green fluorescent protein and beta-galactosidase), proteins that do not fall into any of the pathways encompassing cell growth, division, migration, survival, or apoptosis according to GeneOntology criteria, or ubiquitous housekeeping proteins. The reagents in a kit may be supplied in individual containers or as a mixture of two or more reagents in a single container. In addition, the kit may include explanatory materials describing how to use the components included.

[0105] The term "neoadjuvant therapy" refers to treatment administered before primary treatment. Examples of neoadjuvant therapy include chemotherapy, radiation therapy, and hormone therapy.

[0106] A "normal" expression level of a biomarker refers to the expression level of the biomarker in cells of a subject that is not suffering from a disease such as cancer, for example, in the cells of a human patient. "Overexpression" or "significantly higher expression levels" of a biomarker refers to an expression level in a test sample that is at least 10%, more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than the standard error of the assay used to assess expression, preferably above the biomarker expression activity or level in a control sample (e.g., a sample from a healthy subject without a disease associated with the biomarker), and more preferably above the mean expression level of the biomarker in several control samples. A "significantly lower expression level" of a biomarker refers to an expression level in the test sample that is at least 10%, more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times lower than the expression level of the biomarker in a control sample (e.g., a sample from a healthy subject without a disease associated with the biomarker), preferably the average expression level of the biomarker in several control samples."Overexpression" or "significantly higher expression levels" of a biomarker refers to an expression level in a test sample that is at least 10%, more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than the standard error of the assay used to assess expression, preferably above the biomarker expression activity or level in a control sample (e.g., a sample from a healthy subject without a disease associated with the biomarker), and more preferably above the mean expression level of the biomarker in several control samples. A "significantly lower expression level" of a biomarker refers to an expression level in the test sample that is at least 10%, more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times lower than the expression level of the biomarker in a control sample (e.g., a sample from a healthy subject without a disease associated with the biomarker), preferably the average expression level of the biomarker in several control samples.

[0107] Such “significant” levels can also be applied to any other measured parameters described herein, such as expression, inhibition, cytotoxicity, and cell growth.

[0108] The term “default” biomarker quantity and / or activity measurement(s) may refer to biomarker quantities and / or activity measurement(s) used, for example, to evaluate subjects that may be selected for a particular treatment, to evaluate responses to therapeutic agents such as APRIL / TACI interaction modulators, either alone or in combination with one or more immunotherapeutic agents, and / or to evaluate disease status. Default biomarker quantities and / or activity measurement(s) may be determined in a population of patients with or without cancer. Default biomarker quantities and / or activity measurement(s) may be a single number equally applicable to all patients, or they may vary depending on a specific subpopulation of patients. Age, weight, height, and other factors of a subject may influence an individual’s default biomarker quantity and / or activity measurement(s). Furthermore, default biomarker quantities and / or activity may be determined individually for each subject. In one embodiment, the quantities determined and / or compared in the methods described herein are based on absolute measurements. In another embodiment, the amounts determined and / or compared in the methods described herein are based on relative measurements such as ratios (e.g., cell ratios, or serum biomarkers normalized to the expression of housekeeping biomarkers or otherwise generally constant biomarkers). The default biomarker amounts and / or activity measurements may be any preferred standard. For example, the default biomarker amounts and / or activity measurements may be obtained from the same or different individuals being assessed for patient selection. In one embodiment, the default biomarker amounts and / or activity measurements may be obtained from previous assessments of the same patient. In this way, the progress of patient selection can be monitored over time. In addition, if the subjects are humans, controls may be obtained from assessments of another or more individuals, for example, a group of selected individuals.In this way, the range of selection of individuals being evaluated can be compared with other suitable individuals, such as those suffering from similar or identical medical conditions and / or belonging to the same ethnic group, or other individuals in similar circumstances to the target individual.

[0109] The term “predictive” includes the use of biomarker nucleic acid and / or protein status, e.g., tumor hyperactivity or hypoactivity, appearance, expression, growth, remission, relapse, or resistance, before, during, or after treatment, to determine the likelihood of cancer response to immunomodulatory therapies such as APRIL / TACI interaction modulator therapy (e.g., APRIL / TACI interaction modulators alone or in combination with immunotherapeutic agents such as STING pathway modulators and / or immune checkpoint inhibitors). Such predictive use of biomarkers includes, for example, (1) increased or decreased copy numbers (e.g., by FISH, FISH plus SKY, monomolecular sequencing as described in at least J.Biotechnol., 86:289-301, or qPCR) in about 5%, 6%, 7%, 8%, 8%, 9%, 8%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of the human cancer type or cancer sample being assayed, overexpression or underexpression of biomarker nucleic acids (e.g., by ISH, Northern blot, or qPCR), increased or decreased biomarkers (2) the absolute or relatively regulated presence or absence of Karr proteins (e.g., by IHC) and / or biomarker targets, or increased or decreased activity thereof, in biological samples, e.g., samples containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, feces, or bone marrow, e.g., subjects, e.g., human-derived tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, feces, or bone marrow, e.g., subjects with cancer, e.g., those who respond to or develop resistance to certain immunomodulatory therapies (e.g., APRIL / TACI interaction modulators, either alone or in combination with STING pathway modulators and / or immunotherapeutic agents).

[0110] Terms such as "prevent," "preventing," "prevention," and "prophylactic measures" refer to reducing the probability of developing a disease, disability, or condition in individuals who do not currently have the disease, disability, or condition, but are at risk of developing it or are prone to developing it.

[0111] The term "probe" refers to any molecule that is selectively capable of binding to a specific target molecule, such as a biomarker nucleic acid encoded by or a corresponding nucleotide transcript or protein. Probes may be synthesized by those skilled in the art or derived from a suitable biological preparation. For the purpose of detecting a target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0112] The term "prognosis" includes predictions of the likely course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the prognosis of cancer in an individual is obtained by using statistical algorithms. For example, the prognosis may be surgical intervention, the development of a clinical subtype of cancer (e.g., solid tumors such as lung cancer, melanoma, and renal cell carcinoma), the development of one or more clinical factors, the development of colon cancer, or recovery from the disease.

[0113] The term “response to therapy” (e.g., APRIL / TACI interaction modulators, either alone or in combination with immunotherapeutic agents such as STING pathway modulators and / or immune checkpoint inhibitors) refers to any response to therapy (e.g., APRIL / TACI interaction modulators, either alone or in combination with immunotherapeutic agents such as STING pathway modulators and / or immune checkpoint inhibitors), and in the case of cancer, preferably relating to changes in cancer cell count, tumor burden, and / or volume after the initiation of neoadjuvant or adjuvant chemotherapy. The hyperproliferative impairment response may be assessed, for example, in terms of efficacy or in the neoadjuvant or adjuvant situation, in which case the size of the tumor after systemic intervention may be compared to the initial size and dimensions measured by CT, PET, mammography, ultrasound, or palpation. The response may also be assessed by calipas measurement or pathological examination of the tumor after biopsy or surgical resection. The response may be recorded in a quantitative manner, such as a percentage change in tumor volume, or in a qualitative manner, such as “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical disease progression” (cPD), or other qualitative criteria. Assessment of the hyperproliferative disorder response may be performed early after initiation of neoadjuvant or adjuvant therapy, for example, hours, days, weeks, or preferably months later. A typical endpoint for response assessment is after completion of neoadjuvant chemotherapy or surgical removal of residual tumor cells and / or tumor bed. This is typically 3 months after initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatment described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentages of patients in complete remission (CR), partial remission (PR), and stable disease (SD) at least six months remaining until the end of treatment. The abbreviated expression for this formula is CBR = CR + PR + SD for more than six months.In some embodiments, the CBR for a particular cancer therapy regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating response to cancer therapy are related to "survival time" and include all of the following: survival time to death, also known as overall survival time (the death may be due to any cause or may be tumor-related); "recurrence-free survival time" (the term recurrence is to include both local recurrence and distant recurrence); metastasis-free survival time; disease-free survival time (the term disease is to include cancer and related diseases). The length of the survival time can be calculated by reference to a defined starting point (e.g., at diagnosis or at the start of treatment) and an endpoint (e.g., death, recurrence, or metastasis). In addition, the criteria for treatment effectiveness can be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time frame, and probability of tumor recurrence. For example, to determine appropriate thresholds, a particular cancer therapy regimen can be administered to a population of subjects and the outcomes can be correlated to biomarker measurements determined prior to the administration of any immunomodulatory therapy. The measurement of the outcome may be a pathologic response to the therapy administered in a neoadjuvant setting. Alternatively, outcome measures such as overall survival time and disease-free survival time can be monitored over a period of time following immunomodulatory therapy for subjects for whom biomarker measurements are known. In certain embodiments, the dose administered is a standard dose known in the art for the cancer therapeutic agent. The period of time for which the subject is monitored can vary. For example, the subject may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.

[0114] The term "resistance" refers to acquired or innate resistance of a cancer sample or mammal to immunomodulatory therapy (i.e., being unresponsive to the therapeutic treatment or having a reduced or limited response to the therapeutic treatment), for example, having a response reduced by 5% or more, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, or 2x, 3x, 4x, 5x, 10x, 15x, or 20x or more. The reduction in response can be measured by comparing it to the same cancer sample or mammal before resistance was acquired, or by comparing it to a different cancer sample or mammal known not to be resistant to the therapeutic treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance can be mediated by P-glycoprotein or by other mechanisms, or it can occur when a mammal is infected with a multidrug-resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is customary in the art and can be measured within the skill of a clinical professional, for example, by cell proliferation assays and cell death assays, as described herein as “sensitization.” In some embodiments, the term “reversing resistance” means that, in situations where primary cancer therapy (e.g., chemotherapy or radiotherapy) alone cannot result in a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor, it is possible to achieve a statistically significant reduction in tumor volume (e.g., p<0.05) by combining a second agent with primary cancer therapy (e.g., chemotherapy or radiotherapy) compared to the tumor volume of an untreated tumor. This generally applies to tumor volume measurements performed when the untreated tumor is growing log-rhythmically.

[0115] The term “response” or “responsiveness” refers to a response to therapy. For example, an anti-cancer response may include a reduction in tumor size or inhibition of tumor growth. The term can also refer to an improvement in prognosis, such as an increase in time to recurrence, which is the time to the first recurrence with censoring a second primary cancer or death without evidence of recurrence as the first event, or an increase in overall survival, which is the time from treatment to death from any cause. To respond or have a response means to achieve a beneficial endpoint when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, sedated, or attenuated when exposed to a stimulus. It will be understood that assessing the likelihood that a tumor or subject will show a favorable response is equivalent to assessing the likelihood that a tumor or subject will not show a favorable response (i.e., show no response or be unresponsive).

[0116] As used herein, “RNA interferant” is defined as any agent that interferes with or inhibits the expression of a target biomarker gene by RNA interference (RNAi). Such RNA interferants include, but are not limited to, RNA molecules or fragments thereof homologous to the target biomarker gene of the present invention, short interfering RNA (siRNA), and nucleic acid molecules including small molecules that interfere with or inhibit the expression of a target biomarker nucleic acid by RNA interference (RNAi).

[0117] RNA interference (RNAi) is an evolutionarily conserved process in which the expression or introduction of RNA with a sequence identical or highly similar to that of a target biomarker nucleic acid results in sequence-specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from the target gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting the expression of the target biomarker nucleic acid. In one embodiment, the RNA is double-stranded RNA (dsRNA). This process has been described in plant, invertebrate, and mammalian cells. In nature, RNAi is initiated by Dicer, a dsRNA-specific endonuclease that facilitates the processive cleavage of long dsRNAs into double-stranded fragments called siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves the target mRNA. RNAi can also be initiated by introducing nucleic acid molecules, such as synthetic siRNA, shRNA, or other RNA interferants, to inhibit or silence the expression of a target biomarker nucleic acid. As used herein, “inhibition of target biomarker nucleic acid expression” or “inhibition of marker gene expression” includes any reduction in the expression or protein activity or level of the target biomarker nucleic acid or the protein encoded by the target biomarker nucleic acid. This reduction may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the expression or activity or level of the target biomarker nucleic acid or the protein encoded by the target biomarker nucleic acid that has not been targeted by the RNA interferant.

[0118] In addition to RNAi, genome editing can be used to modify the copy number or gene sequence of a target biomarker, such as constitutive or inducible knockout or mutation of a target biomarker like APRIL and / or TACI. For example, the CRISPR-Cas system can be used for precise editing of genomic nucleic acids (e.g., to create non-functional or null mutations). In such embodiments, CRISPR guide RNA and / or Cas enzymes may be expressed. For example, a vector containing only guide RNA may be administered to animals or cells genetically modified with the Cas9 enzyme. Similar strategies can be employed (e.g., designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases). Such systems are well known in the art (e.g., U.S. Patent No. 8,697,359, Sander and Joung (2014) Nat. Biotech. 32:347-355, Hale et al. (2009) Cell 139:945-956, Karginov and Hannon (2010) Mol. Cell 37:7, U.S. Patent Publications 2014 / 0087426 and 2012 / 0178169, Boch et al. (2011) Nat. Biotech. 29:135-136, Boch et al. (2009) Science 326:1509-1512, Moscow and Bogdanove (2009) Science 326:1501, Weber et al. (2011) PLoS One) See 6:e19722, Li et al. (2011) Nucl. Acids Res. 39:6315-6325, Zhang et al. (2011) Nat. Biotech. 29:149-153, Miller et al. (2011) Nat. Biotech. 29:143-148, Lin et al. (2014) Nucl. Acids Res. 42:e47). Such gene strategies may utilize constitutive or inducible expression systems according to methods well known in the art.

[0119] The term "small molecule" is a term used in the art and includes molecules with a molecular weight of less than approximately 1000 or less than approximately 500. In one embodiment, a small molecule is not exclusively composed of peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptide mimes, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compound is a small organic non-peptidic compound. In a further embodiment, the small molecule is not biosynthesized.

[0120] The term “sample” as used in relation to detecting or determining the presence or level of at least one biomarker typically refers to whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other body fluid (e.g., as set forth above under the definition of “body fluid”), or tissue samples such as small intestine, colon, or surgically resected tissue (e.g., biopsy specimens). In certain specific cases, the method of the present invention further comprises obtaining the sample from an individual before detecting or determining the presence or level of at least one marker in the sample.

[0121] The terms "selective modulator" or "selective modulator" applied to bioactive agents refer to the ability of a drug to modulate the signaling activity of a target cell population, etc., either directly or through interaction with the target, compared to the signaling activity of an off-target cell population, etc. For example, a drug that selectively inhibits the APRIL / TACI interaction more than another interaction between APRIL and another receptor such as BCMA, and / or selectively inhibits the APRIL / TACI interaction on a target cell population (e.g., soluble), may have at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 2x (times) or more higher activity against the APRIL / TACI interaction than the activity of the drug against at least one other APRIL receptor (e.g., at least Also about 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80 x, 85x, 90x, 95x, 100x, 105x, 110x, 120x, 125x, 150x, 200x, 250x, 300x, 350x, 400x, 450x, 500x, 60 (0x, 700x, 800x, 900x, 1000x, 1500x, 2000x, 2500x, 3000x, 3500x, 4000x, 4500x, 5000x, 5500x, 6000x, 6500x, 7000x, 7500x, 8000x, 8500x, 9000x, 9500x, 10000x, or above, or any range between them including boundary values). Such metrics are typically expressed in terms of the relative amount of drug required to reduce interaction / activity by half.

[0122] More generally, the term "selective" refers to a preferential action or function. The term "selective" can be quantified in terms of the preferential effect on a specific target compared to other targets. For example, a measured variable (e.g., regulation of Treg / Breg compared to other cells, e.g., other immune cells such as Tcon) is quantified as 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, The magnification can vary by 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 70x, 80x, 90x, 100x, or any range between them including those greater than or at the boundary (e.g., 50% to 16x). Using the same magnification analysis, the magnitude of the effect in a given tissue, cell population, measured variable, and measured effect can be confirmed, such as the Treg:Tcon ratio, Breg:Tcon ratio, growth rate or volume of hyperproliferating cells, or Treg / Breg proliferation rate or number.

[0123] In contrast, the term "specific" refers to an exclusive action or function. For example, specific modulation of the APRIL / TACI interaction refers to the exclusive modulation of the APRIL / TACI interaction, and does not refer to modulation of APRIL with other receptors such as BCMA. In another example, specific binding of an antibody to a given antigen refers to the ability of an antibody to bind to the target antigen without binding to other antigens. Typically, when an antibody is determined by surface plasmon resonance (SPR) technique in a BIACORE® assay instrument using the target antigen as the analyte and the antibody as the ligand, the binding rate is approximately 1 × 10⁻⁶. -7 Less than M, for example, approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 Affinity less than M, or even lower (KD ) binds to the predetermined antigen with an affinity at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, or 10.0 times or more than its affinity to nonspecific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). In addition, K D is, K A It is the reciprocal of [the specified value]. The terms "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used interchangeably in this specification with the term "antibody that specifically binds to an antigen."

[0124] The term "sensitize" means altering cells, such as cancer cells or tumor cells, to enable more effective treatment with the therapy (e.g., an APRIL / TACI interaction modulator either alone or in combination with an immunotherapy agent such as a STING pathway modulator and / or an immune checkpoint inhibitor). In some embodiments, normal cells are not affected to the extent that the therapy (e.g., an APRIL / TACI interaction modulator either alone or in combination with an immunotherapy agent such as a STING pathway modulator and / or an immune checkpoint inhibitor) causes excessive damage to normal cells. Increased or reduced sensitivity to therapeutic treatments is relevant to the specific treatments and methods described below in this specification, including cell proliferation assays (Tanigawa N, Kern DH, Kikasa Y, Morton DL, Cancer Res 1982;42:2159-2164), cell death assays (Weisenthal LM, Shoemaker RH, Marsden JA, Dill PL, Baker JA, Moran EM, Cancer Res 1984;94:161-173, Weisenthal LM, Lippman ME, Cancer Treat Rep 1985;69:615-632, Weisenthal LM, In:Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA:Harwood Academic). The susceptibility or resistance may also be measured in animals by measuring the reduction in tumor size over a period of time, for example, 6 months in the case of humans and 4-6 weeks in the case of mice.A composition or method sensitizes a response to a therapeutic treatment if the increase in therapeutic sensitivity or reduction in resistance is 5% or more, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2, 3, 4, 5, 10, 15, or 20 times, compared to therapeutic sensitivity or resistance in the absence of such composition or method. Determining sensitivity or resistance to a therapeutic treatment is customary in the art and within the scope of the skills of a clinical professional. It should be understood that any method described herein for enhancing the efficacy of immunomodulatory agents may be equally applicable to methods for sensitizing hyperproliferative or cancerous cells (e.g., resistant cells) to said therapy.

[0125] The term "synergistic effect" refers to the possibility that the combined effect of two or more therapeutic agents, such as two or more APRIL / TACI interaction modulators, an APRIL / TACI interaction modulator and an immunotherapy agent, or an APRIL / TACI interaction modulator alone or in combination with an immunotherapy agent such as a STING pathway modulator and / or an immune checkpoint inhibitor, may be greater than the sum of the individual effects of the anticancer agents alone.

[0126] Short interfering RNA (siRNA), also referred to herein as "small interfering RNA," is defined as a drug that functions to inhibit the expression of a target biomarker nucleic acid, for example, by RNAi. siRNA may be chemically synthesized, produced by in vitro transcription, or produced in a host cell. In one embodiment, the siRNA is a double-stranded RNA (dsRNA) molecule about 15 to about 40 nucleotides long, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides, and more preferably about 19, 20, 21, or 22 nucleotides long, and each strand may contain 3' and / or 5' overhangs having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhangs is independent between the two strands, i.e., the length of an overhang on one strand does not depend on the length of an overhang on the second strand. Preferably, the siRNA can promote RNA interference through the degradation of target messenger RNA (mRNA) or specific post-transcriptional gene silencing (PTGS).

[0127] In another embodiment, the siRNA is a small hairpin-type (also called stem-loop) RNA (shRNA). In one embodiment, these shRNAs consist of a short (e.g., 19-25 nucleotides) antisense strand, followed by a 5-9 nucleotide loop, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure, and the antisense strand may follow. These shRNAs are contained in plasmids, retroviruses, and lentiviruses and may be expressed from, for example, the pol III U6 promoter or another promoter (see, for example, Stewart, et al. (2003) RNA Apr;9(4):493-501, which is incorporated herein by reference).

[0128] RNA interfering agents, such as siRNA molecules, may be administered to patients who have cancer or are at risk of developing cancer to inhibit the expression of biomarker genes that are overexpressed in cancer, thereby treating, preventing, or inhibiting cancer in the subjects.

[0129] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from cancer, such as multiple myeloma, lung cancer, ovarian cancer, pancreatic cancer, liver cancer, breast cancer, prostate cancer, melanoma, and colon cancer. The term "subject" is interchangeable with "patient."

[0130] The term “survival” includes all of the following: survival to death, also known as overall survival (whether death is cause-independent or tumor-related); “recurrence-free survival” (the term recurrence includes both local and distant recurrence); metastasis-free survival; disease-free survival (the term disease includes cancer and related diseases). The length of such survival may be calculated by referring to a specified starting point (e.g., diagnosis or initiation of treatment) and endpoint (e.g., death, recurrence, or metastasis). In addition, criteria for the effectiveness of treatment may be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time frame, and probability of tumor recurrence.

[0131] The term "therapeutic effect" refers to a local or systemic effect caused by a pharmacologically active substance in animals, particularly mammals, and more specifically, humans. Therefore, this term means any substance intended for use in the diagnosis, cure, sedation, treatment, or prevention of disease in animals or humans, or in the enhancement of desirable physical or mental development and condition. The phrase "therapeutically effective dose" means the amount of a substance that produces some desired local or systemic effect with a reasonable benefit-risk ratio applicable to any treatment. In certain embodiments, the therapeutically effective dose of a compound will depend on its therapeutic index, solubility, etc. For example, a particular compound discovered by the method of the present invention may be administered in an amount sufficient to produce a reasonable benefit-risk ratio applicable to such treatment.

[0132] As used herein, the terms “therapeutically effective dose” and “effective dose” mean the amount of the compound, material, or composition containing the compound of the present invention that is effective in producing some desired therapeutic effect in at least a subpopulation of cells in an animal, in a reasonable benefit-to-risk ratio applicable to any medical treatment. The toxicity and therapeutic efficacy of the compound in question are expressed, for example, in terms of LD50. 50 and ED 50 The LD may be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Compositions exhibiting a large therapeutic index are preferred. In some embodiments, 50 The lethal dose (ED) can be measured and, for a drug, can be reduced by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to no administration of the drug. Similarly, ED 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured, and for a drug, it can be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to no administration of the drug. Similarly, IC 50(i.e., concentrations that achieve haploid cytopathy or cell division arrest in cancer cells) can be measured, and for a drug, this can increase, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to no administration of the drug. In some embodiments, cancer cell growth in the assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. Cancer cell death can be accelerated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, a reduction of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% in cancer cell number and / or solid malignancies can be achieved.

[0133] "Transcribed polynucleotide" or "nucleotide transcript" means a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analogues of such RNA or cDNA) that is complementary to or homologous to all or part of a mature mRNA, produced by the transcription of a biomarker nucleic acid and, where applicable, the normal post-transcriptional processing (e.g., splicing) of an RNA transcript, and the reverse transcription of an RNA transcript.

[0134] A known deterministic correspondence exists between the amino acid sequence of a particular protein and the nucleotide sequence that can encode that protein, as defined by the genetic code (shown below). Similarly, a known deterministic correspondence exists between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code. Genetic code Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp, D), GAC, GAT Cysteine ​​(Cys, C) TGC, TGT Glutamic acid (Glu, E), GAA, GAG Glutamine (Gln, Q), CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valin (V) GTA, GTC, GTG, GTT Termination signals (terminal): TAA, TAG, TGA

[0135] A well-known and important feature of the genetic code is its redundancy, which allows more than one coding nucleotide triplets to be used for most of the amino acids used to synthesize proteins (as illustrated above). Therefore, several different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than others). Furthermore, occasionally, purine or pyrimidine methylation variants may be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0136] In light of the foregoing, the amino acid sequence of a polypeptide can be derived by using the nucleotide sequence (or any portion thereof) of DNA or RNA encoding a biomarker nucleic acid, and translating the DNA or RNA into an amino acid sequence using the genetic code. Similarly, for the amino acid sequence of a polypeptide, the corresponding nucleotide sequence that can encode that polypeptide can be deduced from the genetic code (this will result in multiple nucleic acid sequences for any given amino acid sequence due to its redundancy). Therefore, in this specification, a description and / or disclosure of a nucleotide sequence encoding a polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by that nucleotide sequence. Similarly, in this specification, a description and / or disclosure of the amino acid sequence of a polypeptide should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode that amino acid sequence.

[0137] Finally, nucleic acid and amino acid sequence information relating to the loci and biomarkers of the present invention and related biomarkers (e.g., the biomarkers listed in Table 1) is well known in the art and readily available in publicly accessible databases such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences derived from publicly accessible sequence databases are provided below.

[0138] Representative sequences of the biomarkers described above are shown in Table 1 below. It should be noted that the above terms may be used to refer to any combination of the characteristics described herein with respect to the biomarkers. For example, any combination of sequence composition, identity percentage, sequence length, domain structure, functional activity, etc., may be used to describe the biomarkers of the present invention. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0139] * Table 1 includes RNA nucleic acid molecules (e.g., thymine replaced with uredine), nucleic acid molecules encoding orthologues of encoding proteins, and DNA or RNA nucleic acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity over the entire length of a DNA or RNA nucleic acid sequence with any sequence number listed in Table 1, or a portion thereof. Such nucleic acid molecules may have the functions of full-length nucleic acids as further described herein.

[0140] * Table 1 includes protein orthologues and polypeptide molecules having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across the entire length of the polypeptide molecule with the amino acid sequence of any SEQ ID NO listed in Table 1, or a portion thereof. Such polypeptides may have the functions of full-length polypeptides as further described herein.

[0141] * Table 1 includes interactions between APRIL and its receptor TACI; between APRIL and its receptor BCMA; and between APRIL and its receptors TACI and BCMA, as well as any known APRIL, TACI, and BCMA nucleic acids and polypeptide sequences and their variants as described herein.

[0142] II. Subject In one embodiment, the subject has a condition in which upregulation or downregulation of the immune response would be beneficial. The subject may be treated with at least one APRIL / TACI interaction modulator, either alone or in combination with an immunotherapy agent such as a STING pathway modulator and / or an immune checkpoint inhibitor. The subject may be a mammal (e.g., mouse, rat, primate, non-human mammal, domestic animal such as dog, cat, cattle, horse), and preferably a human. The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from an immune disorder. The term "subject" is interchangeable with "patient."

[0143] In another embodiment of the method of the present invention, the subject has never received treatment such as chemotherapy, radiotherapy, targeted therapy, and / or immunomodulatory therapy (e.g., at least one APRIL / TACI interaction modulator, either alone or in combination with immunotherapy agents such as STING pathway modulators and / or immune checkpoint inhibitors). Still in yet another embodiment, the subject has received treatment such as chemotherapy, radiotherapy, targeted therapy, and / or immunomodulatory therapy (e.g., at least one APRIL / TACI interaction modulator, either alone or in combination with immunotherapy agents such as STING pathway modulators and / or immune checkpoint inhibitors). Still in yet another embodiment, the subject is immune-responsive or immune-unresponsive. An "immune-responsive" subject is one that has the immune cells and immune functions necessary to establish a normal or desired immune response following exposure to an antigen. An "immune-unresponsive" subject is one that lacks one or more immune cell types or immune functions necessary to establish at least one normal or desired level of immune response following exposure to an antigen. Immunoresponsive subjects are susceptible to opportunistic infections, such as viral, fungal, protozoan, or bacterial infections, prion diseases, and certain neoplasms. "Immunodeficiency" subjects are those in which the innate host immune response may not be initiated at all, as seen in severe combined immunodeficiency (SCID) mice. "Immunopposed" subjects have a substantially reduced immune function compared to immune-responsive subjects. In all cases, the reduction or absence of immune function and / or immune cell types can occur in many different and well-known ways. For example, hematopoietic stem cells (HSCs), which give rise to all immune cells, are one such example and can be adversely affected in development, function, differentiation, and survival. Immunoresponsive subjects can be generated in many different ways known in the art. These can result from the modulation of the function and / or number of various parameters in numerous combinations.For example, to achieve a desired state of immune unresponsiveness, resting, mitotic, terminally differentiated, post-mitotic, inactivated, activated, and similar immune cell populations can be targeted for regulation. “Resting” cells refer to cells that are in a non-replicating state, not in the cell cycle (non-cycling). While resting cells may have the ability to replicate and divide when activated, they are quiescent because they are not in the cell cycle. Therefore, “resting” cells are not simply manipulated immune cells that have been stimulated to divide and then manipulated to return to a quiescent, non-mitotic state. Resting cells can be “naive,” meaning they are immune cells that have differentiated in the bone marrow, successfully undergone positive and negative selection in the thymus, matured, but are not activated and are not memory cells. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), absence of activation markers CD25, CD44, or CD69, and absence of the memory CD45RO isoform. These cells also express a functional IL-7 receptor, consisting of the IL-7 receptor-α subunit CD127 and the common gamma chain CD132. In the naive state, T cells are quiescent and non-dividing, and are thought to require the common gamma chain cytokines IL-7 and IL-15 for homeostatic survival mechanisms. In contrast, activated T cells have surface markers CD25, CD44, and CD62L. 低 , and express or upregulate the expression of CD69, which can further differentiate them into memory T cells. Naive B cells have never been exposed to an antigen in order to become either memory B cells or antibody-secreting plasma cells. In one embodiment, a resting cell becomes “activated” when it is induced to enter a replication or doubling state, which may include cells about to enter the cell cycle, cell division, or mitosis. In another embodiment, a resting cell may also become “activated” when it encounters an external signal such as an antigen or cytokine, which initiates activity by terminally differentiated mature immune cells to produce an immune response (e.g., T cell or B cell function).

[0144] In some embodiments, the subject requires an upregulated immune response to eliminate inhibition of the immune response, for example by reducing the number of Treg / Bregs and / or inhibitory immune activity. In some embodiments, the subject requires a downregulated immune response to promote inhibition of the immune response, for example by increasing the number of Treg / Bregs and / or inhibitory immune activity. Methods for upregulating and downregulating the immune response according to the present invention are described below.

[0145] The method of the present invention can be used to determine the responsiveness of many different disorders in the subjects described above to therapy (e.g., at least one APRIL / TACI interaction modulator, either alone or in combination with immunotherapeutic agents such as STING pathway modulators and / or immune checkpoint inhibitors).

[0146] The subjects and characteristics useful by the present invention also apply to cells used by the present invention, such as cancer cells, which are cells obtained from and / or cells derived from the subject that are brought into contact with at least one APRIL / TACI interaction modulator.

[0147] III. Sample Collection, Preparation, and Separation In some embodiments, the presence, absence, quantity, and / or activity measurements of a biomarker in a sample derived from the target, such as baseline Treg / Breg count, Treg ratio, Breg ratio, biomarker expression level, cytokine expression, etc., are compared to a predetermined control (standard) sample. The sample derived from the target is typically derived from affected tissue such as cancer cells or tissue, but can be any tissue of interest, such as serum or other body-derived samples described herein. The control sample may be derived from the same target or from a different target. The control sample is typically a normal, non-affected sample. However, in some embodiments, the control sample may be derived from affected tissue, for example, for disease staging or evaluation of treatment effectiveness. The control sample may be a combination of several samples derived from different targets. In some embodiments, the quantity and / or activity measurements of a biomarker derived from the target are compared to predetermined levels. This default level is typically obtained from a normal sample, such as the normal copy number, quantity, or activity of the biomarker in non-affected cells or tissues from the same species from which the test sample was obtained, or from the subject from which the test sample was obtained. When used herein, the “default” biomarker quantity and / or activity measurement(s) may be, for example, a biomarker quantity and / or activity measurement(s) used to evaluate a subject that may be selected for treatment, to evaluate the response to immunomodulatory therapy (e.g., at least one APRIL / TACI interaction modulator, either alone or in combination with an immunotherapeutic agent such as a STING pathway modulator and / or an immune checkpoint inhibitor), and / or to evaluate the response to combination immunomodulatory therapy (e.g., at least one APRIL / TACI interaction modulator, either alone or in combination with an immunotherapeutic agent such as a STING pathway modulator and / or an immune checkpoint inhibitor). The default biomarker quantity and / or activity measurement(s) may be determined in a population of patients with or without the target condition, such as cancer.The default biomarker amounts and / or activity measurements may be a single number equally applicable to all patients, or they may vary depending on a specific subpopulation of patients. Age, weight, height, and other factors may influence an individual's default biomarker amounts and / or activity measurements. Furthermore, the default biomarker amounts and / or activity can be determined individually for each subject. In one embodiment, the amounts determined and / or compared in the methods described herein are based on absolute measurements. In another embodiment, the amounts determined and / or compared in the methods described herein are based on relative measurements, such as ratios (e.g., biomarker expression normalized to housekeeping gene expression, or gene expression at various time points).

[0148] The default biomarker levels and / or activity measurements can be any suitable standard. For example, the default biomarker levels and / or activity measurements can be obtained from the same or different individuals being assessed for patient selection. In one embodiment, the default biomarker levels and / or activity measurements can be obtained from previous assessments of the same patient. This allows for monitoring the progress of patient selection over time. In addition, if the subjects are humans, controls can be obtained from assessments of another or more individuals, for example, a group of selected individuals. This allows for comparison of the range of selection of individuals being assessed with other suitable individuals, such as those with similar or the same disease(s) and / or belonging to the same ethnic group, or other individuals in similar circumstances to the target individuals.

[0149] In some embodiments of the present invention, the change from a predetermined level of the amount and / or activity measurement(s) of a biomarker is about 0.5 times, about 1.0 times, about 1.5 times, about 2.0 times, about 2.5 times, about 3.0 times, about 3.5 times, about 4.0 times, about 4.5 times, or about 5.0 times or more. In some embodiments, the magnification change is less than about 1, less than about 5, less than about 10, less than about 20, less than about 30, less than about 40, or less than about 50. In other embodiments, the magnification change in the amount and / or activity measurement(s) of a biomarker compared to a predetermined level is greater than about 1, greater than about 5, greater than about 10, greater than about 20, greater than about 30, greater than about 40, or greater than about 50.

[0150] Biological samples can be collected from a variety of patient-derived sources, including bodily fluid samples containing nucleic acids and / or proteins, cell samples, or tissue samples. “Body fluid” refers to fluids excreted or secreted from the body, as well as fluids that are not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, earwax and earwax, Cowper's fluid or bulbourethral gland fluid (pre-ejaculatory fluid), chyle, porridge, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menstrual fluid, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous fluid, vomit). In preferred embodiments, the subject and / or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin-embedded tissue, biopsy specimens, whole blood, papillary aspirate, serum, plasma, buccal scraping, saliva, cerebrospinal fluid, urine, feces, and bone marrow. In one embodiment, the sample is serum, plasma, or urine. In another embodiment, the sample is serum.

[0151] Samples can be repeatedly collected from an individual over a longitudinal period (e.g., approximately once every few days, weeks, months, yearly, or every six months). By obtaining a large number of samples from a given individual over a period of time, results from previous detections can be verified and / or changes in biological patterns can be identified, for example, as a result of disease progression or drug treatment. For example, a target sample can be collected and monitored monthly, every two months, or at intervals of one, two, or three months according to the present invention. In addition, the measured amounts and / or activity values ​​of the target biomarker obtained over time can be conveniently compared with each other and with those of a normal control during the monitoring period, thereby providing the value of the target itself as an internal control for long-term monitoring, i.e., an individual-specific control.

[0152] Sample preparation and separation may involve any of the following procedures, depending on the type of sample collected and / or the analysis of biomarker measurements. Such procedures may include, but are not limited to, concentration, dilution, pH adjustment, removal of large amounts of polypeptides (e.g., albumin, gamma globulin, and transferrin), addition of preservatives and calibration compounds, addition of protease inhibitors, addition of denaturants, desalting of the sample, concentration of sample proteins, and extraction and purification of lipids.

[0153] Sample preparation can also isolate molecules bound to other proteins (e.g., carrier proteins) within non-covalent complexes. This process may involve isolating molecules bound to specific carrier proteins (e.g., albumin), or it may involve a more general process such as releasing the bound molecules from all carrier proteins via protein denaturation (e.g., using acid), followed by removal of the carrier proteins.

[0154] Removal of unwanted proteins (e.g., proteins present in large quantities, of no informational value, or undetectable) from a sample can be achieved using high-affinity reagents, high-molecular-weight filters, ultracentrifugation, and / or electrodialysis. High-affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to proteins present in large quantities. Sample preparation may also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing, and related techniques. Molecular-weight filters include membranes that separate molecules based on size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration, and microfiltration.

[0155] Ultracentrifugation is a method for removing unwanted polypeptides from a sample. Ultracentrifugation involves centrifuging a sample at approximately 15,000–60,000 rpm while monitoring particle sedimentation (or lack thereof) using an optical system. Electrodialysis is a procedure using an electrical or semipermeable membrane in which ions are transported from one solution to another through a semipermeable membrane under the influence of a potential gradient. The membranes used in electrodialysis may have the ability to selectively transport positively or negatively charged ions and reject ions of the opposite charge, or to allow species to move through a semipermeable membrane based on size and charge, thus making electrodialysis useful for the concentration, removal, or separation of electrolytes.

[0156] The separation and purification in the present invention may include any procedure known in the art, such as capillary electrophoresis (e.g., in a capillary or on-chip) or chromatography (e.g., in a capillary, column, or on a chip). Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be performed in a gel, in a capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxide, agarose, or combinations thereof. Gels can be modified by crosslinking, addition of detergents or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography), and incorporation of pH gradients. Examples of capillaries used for electrophoresis include capillaries interfaced with electrospray.

[0157] Capillary electrophoresis (CE) is preferred for separating hydrophilic molecules and highly charged solutes of a complex. CE techniques can also be implemented on microfluidic chips. Depending on the type of capillary and buffer used, CE can be further divided into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isostatic electrophoresis (cITP), and capillary electrochromatography (CEC). Embodiments that combine CE techniques with electrospray ionization involve the use of aqueous mixtures containing volatile solutions, such as volatile acids and / or bases, and organic substances such as alcohols or acetonitrile.

[0158] Capillary isoelectric electrophoresis (cITP) is a technique in which analytes move through a capillary at a constant speed, but are still separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution electrophoresis (FSCE), is based on differences in the electrophoretic mobility of species, determined by the charge on the molecules and the frictional resistance encountered by the molecules during movement (which is often directly proportional to the size of the molecules). Capillary isoelectric focusing (CIEF) allows weakly ionic amphoteric molecules to be separated by electrophoresis under a pH gradient. CEC is a hybrid technique between conventional high-performance liquid chromatography (HPLC) and CE.

[0159] The separation and purification techniques used in the present invention include any chromatographic procedure known in the art. Chromatography may be based on the differential adsorption and elution of a particular analyte, i.e., the separation of the analyte between the mobile phase and the stationary phase. Various examples of chromatography include, but are not limited to, liquid chromatography (LC), gas chromatography (GC), and high-performance liquid chromatography (HPLC).

[0160] IV. Biomarker nucleic acids and polypeptides One aspect of the present invention relates to the use of isolated nucleic acid molecules corresponding to biomarker polypeptides such as APRIL, TACI, BCMA, cytokine-like IL-10, or biomarker nucleic acids encoding a portion of such polypeptides. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), as well as DNA or RNA analogs produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0161] An “isolated” nucleic acid molecule is one that has been separated from other nucleic acid molecules present in the natural source of that nucleic acid molecule. Preferably, an “isolated” nucleic acid molecule does not contain sequences that are naturally located on either side of the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (preferably protein-coding sequences) (i.e., sequences located at the 5' and 3' ends of the nucleic acid). For example, in various embodiments, an isolated nucleic acid molecule may contain nucleotide sequences that are naturally located on either side of the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates, at a concentration of about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB, or less than 0.1 kB. Furthermore, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture media if produced by recombinant techniques, or substantially free of chemical precursors or other chemical substances if chemically synthesized.

[0162] The biomarker nucleic acid molecules of the present invention can be isolated using standard molecular biology techniques and sequence information from database records described herein. Using all or part of such nucleic acid sequences, the nucleic acid molecules of the present invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0163] The nucleic acid molecules of the present invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The thus amplified nucleic acid molecules can be cloned into appropriate vectors and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or part of the nucleic acid molecules of the present invention can be prepared by standard synthesis techniques, for example, using an automated DNA synthesizer.

[0164] Furthermore, the nucleic acid molecule of the present invention may contain only a portion of a nucleic acid sequence, where the full-length nucleic acid sequence contains the marker of the present invention or encodes a polypeptide corresponding to the marker of the present invention. Such a nucleic acid molecule can be used, for example, as a probe or primer. The probe / primer is typically used as one or more substantially purified oligonucleotides. The oligonucleotide typically contains a region of a nucleotide sequence that, under stringent conditions, hybridizes to at least about seven, preferably about fifteen, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of the biomarker nucleic acid sequence. Using a probe based on the sequence of the biomarker nucleic acid molecule, a transcript or genomic sequence corresponding to one or more markers of the present invention can be detected. The probe contains a labeling group attached thereto, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

[0165] Due to the degeneracy of the genetic code, biomarker nucleic acid molecules are also conceivable that differ from the nucleotide sequence of the nucleic acid molecule encoding the protein corresponding to the biomarker, and therefore encode the same protein.

[0166] Furthermore, those skilled in the art will understand that DNA sequence polymorphisms leading to changes in amino acid sequences can exist within a population (e.g., a human population). Such genetic polymorphisms can exist among individuals within a population due to natural allele diversity. An allele is one of a group of genes that occur as substitutes at a given locus. In addition, it will be understood that there can also be DNA polymorphisms that affect RNA expression levels, which can affect the overall expression level of that gene (e.g., by affecting regulation or degradation).

[0167] As used herein interchangeably with "allele variant," the term "allele" refers to an alternative form of a gene or a portion thereof. Alleles occupy the same locus, or position, on homologous chromosomes. When an object has two identical alleles of a gene, it is said to be homozygous with respect to that gene or allele. When an object has two different alleles of a gene, it is said to be heterozygous with respect to that gene or allele. For example, alleles of a biomarker may differ from each other at a single nucleotide or at several nucleotides, and may include nucleotide substitutions, deletions, and insertions. Alleles of a gene may also be forms of the gene containing one or more mutations.

[0168] As used interchangeably herein, the terms “allelelicant of a polymorphic region of a gene” or “allelelicant” refer to an alternative form of a gene having one of several possible nucleotide sequences found in that region of the gene in a population. As used herein, allelelicant is intended to encompass functional allelelicants, non-functional allelelicants, SNPs, mutations, and polymorphisms.

[0169] The term "single nucleotide polymorphism" (SNP) refers to a polymorphic site that is occupied by a single nucleotide and is diverse between allele sequences. This site is usually preceded and followed by a highly conserved sequence of the allele (e.g., a sequence that differs in less than 1 / 100 or 1 / 1000 of a population). SNPs usually arise from the substitution of one nucleotide with another at the polymorphic site. SNPs can also arise from nucleotide deletions or nucleotide insertions compared to a reference allele. Typically, the polymorphic site is occupied by a base other than the reference base. For example, if the reference allele contains the base "T" (thymidine) at the polymorphic site, the altered allele may contain "C" (cytidine), "G" (guanine), or "A" (adenine) at the polymorphic site. SNPs can occur in protein-coding nucleic acid sequences, in which case they may result in defective or otherwise heteromorphic proteins, or genetic disorders. Such SNPs may alter the coding sequence of a gene and thus specify a different amino acid ("missense" SNPs), or they may introduce a stop codon ("nonsense" SNPs). If an SNP does not alter the amino acid sequence of a protein, it is called a "silent" SNP. SNPs can also occur in the non-coding region of a nucleotide sequence. This can result in defective protein expression, for example, as a result of alternative splicing, or it may have no effect on the protein's function.

[0170] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules containing an open reading frame encoding a polypeptide corresponding to a marker of the present invention. Such natural allele diversity can typically result in 1–5% differences in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in several different individuals. This can be easily carried out by identifying the same locus in various individuals using hybridization probes. Any such nucleotide diversity and resulting amino acid polymorphisms or variability that are a result of natural allele diversity and do not alter functional activity are intended to be within the scope of the present invention.

[0171] In another embodiment, the biomarker nucleic acid molecule is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, or 4500 nucleotides long and hybridizes under stringent conditions to a nucleic acid molecule corresponding to the marker of the present invention, or to a nucleic acid molecule encoding a protein corresponding to the marker of the present invention. As used herein, the term “hybridizes under stringent conditions” is intended to describe hybridization and washing conditions in which nucleotide sequences that are at least 60% (65%, 70%, 75%, 80%, preferably 85%) identical to each other typically remain hybridized. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1–6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989). A preferred non-limiting example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS, at 50–65°C.

[0172] In addition to the native allele variants of the nucleic acid molecules of the present invention that may exist in a population, those skilled in the art will further understand that sequence changes can be introduced by mutation, thereby altering the amino acid sequence of the encoding protein without altering the biological activity of the protein encoded by its amino acid sequence. For example, nucleotide substitutions can be made that result in amino acid substitutions at "non-essential" amino acid residues. "Non-essential" amino acid residues are those that can be modified from the wild-type sequence without altering biological activity, while "essential" amino acid residues are required for biological activity. For example, amino acid residues that are not conserved or are only semi-conserved among homologs of various species may be non-essential for activity and therefore would be suitable targets for modification. Alternatively, amino acid residues that are conserved among homologs of various species (e.g., mouse and human) may be essential for activity and therefore would not be suitable targets for modification.

[0173] Accordingly, another aspect of the present invention relates to nucleic acid molecules encoding the polypeptide of the present invention, which contains changes in amino acid residues that are not essential for activity. Such polypeptides have a different amino acid sequence from the native protein corresponding to the marker of the present invention, but still retain biological activity. In one embodiment, the biomarker protein has an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the amino acid sequence of the biomarker protein described herein.

[0174] Isolated nucleic acid molecules encoding variant proteins can be produced by introducing one or more nucleotide substitutions, additions, or deletions into the nucleic acid nucleotide sequence of the present invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoding protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the coding protein can be recombinantly expressed, and the protein's activity can be determined.

[0175] In some embodiments, the present invention further intends to use anti-biomarker antisense nucleic acid molecules, i.e., molecules complementary to the sense nucleic acid of the present invention, for example, complementary to the coding strand of a double-stranded cDNA molecule corresponding to the marker of the present invention, or complementary to the mRNA sequence corresponding to the marker of the present invention. Thus, the antisense nucleic acid molecule of the present invention can hydrogen bond to (i.e., anneal with) the sense nucleic acid of the present invention. The antisense nucleic acid may be complementary to the entire coding strand, or only to a portion thereof, for example, to all or part of the protein coding region (or open reading frame). The antisense nucleic acid molecule may also be antisense to all or part of the non-coding region of the coding strand of the nucleotide sequence encoding the polypeptide of the present invention. The non-coding region ("5' and 3' untranslated region") is the 5' and 3' sequence located on either side of the coding region and is not translated into amino acids.

[0176] Antisense oligonucleotides may be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides or longer. Antisense nucleic acids can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be chemically synthesized using native nucleotides, or a variety of modified nucleotides, such as phosphorothioate derivatives and acridine-substituted nucleotides, designed to increase the biological stability of the molecule or the physical stability of the double helix formed between the antisense nucleic acid and the sense nucleic acid, can be used. Examples of modified nucleotides that can be used to generate antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, and 5-methylam Examples include nomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxosine, pseudouracil, quosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.Alternatively, antisense nucleic acids can be biologically produced using subcloned expression vectors in which the nucleic acid is oriented in the antisense direction (i.e., the RNA transcribed from the inserted nucleic acid is oriented in the antisense direction relative to the target nucleic acid of interest (as further described in the following subsection)).

[0177] The antisense nucleic acid molecules of the present invention are typically administered to a target or generated in situ, and as a result, they hybridize with or bind to cellular mRNA and / or genomic DNA encoding a polypeptide corresponding to a selected marker of the present invention, thereby inhibiting the expression of the marker, for example, by inhibiting transcription and / or translation. Hybridization may occur by conventional nucleotide complementarity, such as forming a stable double helix, or, for example, in the case of antisense nucleic acid molecules that bind to a DNA double helix, through specific interactions in the major groove of the double helix. Examples of administration routes for the antisense nucleic acid molecules of the present invention include direct injection of antisense nucleic acids into a tissue site or injection of antisense nucleic acids into blood-related or bone marrow-related fluids. Alternatively, antisense nucleic acid molecules may be modified to target selected cells and then administered systemically. For example, in the case of systemic administration, antisense molecules may be modified to specifically bind to receptors or antigens expressed on the surface of selected cells, for example, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. Antisense nucleic acid molecules may also be delivered to cells using vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecules are placed under the control of a strong pol II or pol III promoter are preferred.

[0178] The antisense nucleic acid molecule of the present invention may be an α-anomeric nucleic acid molecule. In contrast to the usual α-unit, the α-anomeric nucleic acid molecule forms a specific double-stranded hybrid having complementary RNA strands that extend parallel to each other (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule may also include 2'-o-methylribonucleotides (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or chimeric RNA-DNA analogs (Inoue et al., 1987, FEBS Lett. 215:327-330).

[0179] The present invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules that possess ribonuclease activity capable of cleaving single-stranded nucleic acids, such as mRNA, in which they have complementary regions. Therefore, ribozymes (e.g., hammerhead ribozymes, as described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave mRNA transcripts, thereby inhibiting the translation of mRNA-encoded proteins. Ribozymes specific to nucleic acid molecules encoding polypeptides corresponding to the markers of the present invention can be designed based on the nucleotide sequence of the cDNA corresponding to the marker. For example, derivatives of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see U.S. Patent No. 4,987,071 and U.S. Patent No. 5,116,742 by Cech et al.). Alternatively, catalytic RNA having specific ribonuclease activity can be selected from a pool of RNA molecules using the mRNA encoding the polypeptide of the present invention (see, for example, Bartel and Szostak, 1993, Science 261:1411-1418).

[0180] The present invention also encompasses nucleic acid molecules that form a triple helix structure. For example, the expression of a biomarker protein may be inhibited by targeting a nucleotide sequence complementary to the regulatory region (e.g., promoter and / or enhancer) of the gene encoding the polypeptide, thereby forming a triple helix structure that blocks gene transcription in the target cell. For general information, see Helene (1991) Anticancer Drug Des. 6(6):569-84, Helene (1992) Ann. NYAcad. Sci. 660:27-36, and Maher (1992) Bioassays 14(12):807-15.

[0181] In various embodiments, the nucleic acid molecules of the present invention may be modified in the base moiety, sugar moiety, or phosphate backbone to improve, for example, molecular stability, hybridization, or solubility. For example, the deoxyribose phosphate backbone of a nucleic acid molecule may be modified to generate a peptide nucleic acid molecule (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1):5-23). ​​As used herein, the terms “peptide nucleic acid” or “PNA” refer to nucleic acid mimes, such as DNA mimes, in which the deoxyribose phosphate backbone is replaced by a pseudo-peptide backbone and only the four native nucleic acid bases are retained. The neutral PNA backbone has been shown to enable specific hybridization to DNA and RNA under low ionic strength conditions. The synthesis of PNA oligomers can be carried out using standard solid-phase peptide synthesis protocols such as those described in Hyrup et al. (1996) (see above) and Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

[0182] PNA can be used in therapeutic and diagnostic applications. For example, PNA can be used as an antisense or antigenic agent for sequence-specific regulation of gene expression, for example, by inducing transcription or translation arrest or inhibiting replication. PNA can also be used, for example, in the analysis of single base pair mutations in genes, for example, by PNA-directed PCR clamping, as an artificial restriction enzyme in combination with other enzymes, such as the S1 nuclease (Hyrup (1996) (see above)), or as a probe or primer for DNA sequencing and hybridization (Hyrup, 1996 (see above), Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93:14670-675).

[0183] In another embodiment, PNA may be modified to enhance, for example, their stability or cellular uptake, by attaching lipophilic groups or other helper groups to PNA, by forming a PNA-DNA chimera, or by using liposomes or other drug delivery techniques known in the art. For example, a PNA-DNA chimera may be generated that combines the advantageous properties of PNA and DNA. Such a chimera would allow DNA recognition enzymes, such as RNASE H and DNA polymerase, to interact with the DNA portion, while the PNA portion would provide high binding affinity and specificity. The PNA-DNA chimera may be linked using a linker of appropriate length selected in terms of base stacking, the number of bonds between nucleic acid bases, and orientation (Hyrup, 1996 (see above)). The synthesis of PNA-DNA chimeras can be carried out as described in Hyrup (1996) (see above) and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, DNA strands can be synthesized on a solid support using standard phosphoramidite coupling reactions and modified nucleoside analogs. Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxythymidine phosphoramidite can be used as a link between PNA and the 5' end of DNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). Then, PNA monomers are coupled stepwise to produce a chimeric molecule having a 5'PNA segment and a 3'DNA segment (Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63). Alternatively, a chimeric molecule having a 5'DNA segment and a 3'PNA segment can be synthesized (Peterser et al., 1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

[0184] In other embodiments, the oligonucleotide may include other appended groups, such as peptides (e.g., for targeting host cell receptors in vivo), or agents that facilitate transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556, Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652, PCT Publication WO88 / 09810) or the blood-brain barrier (see, e.g., PCT Publication WO89 / 10134). In addition, oligonucleotides may be modified with hybridization-triggered cleavage agents (e.g., Krol et al., 1988, Bio / Techniques 6:958-976) or insertors (e.g., Zon, 1988, Pharm.Res. 5:539-549). For this purpose, oligonucleotides may be conjugated with other molecules, such as peptides, hybridization-triggered crosslinking agents, transporters, or hybridization-triggered cleavage agents.

[0185] Another aspect of the present invention relates to the use of biomarker proteins and their bioactive moieties. In one embodiment, the native polypeptide corresponding to the marker can be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another embodiment, the polypeptide corresponding to the marker of the present invention is produced by recombinant DNA techniques. As an alternative to recombinant expression, the polypeptide corresponding to the marker of the present invention can be chemically synthesized using standard peptide synthesis techniques.

[0186] "Isolated" or "purified" proteins or their bioactive portions are substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein originates, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The phrase "substantially free of cellular material" includes protein preparations in which the protein has been separated from the cellular components of the cell from which it was isolated or recombinantly produced. Thus, proteins substantially free of cellular material include protein preparations having about 30%, 20%, 10%, or less than 5% (based on dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). If the protein or its bioactive portion is recombinantly produced, it is also preferably substantially free of culture medium, i.e., the culture medium accounts for about 20%, 10%, or less than 5% of the volume of the protein preparation. If the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it has been separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Therefore, such protein preparations contain approximately 30%, 20%, 10%, and less than 5% (based on dry weight) of chemical precursors or compounds in addition to the target polypeptide.

[0187] The bioactive portion of a biomarker polypeptide comprises a polypeptide that contains an amino acid sequence sufficiently identical to or derived from the amino acid sequence of the biomarker protein described herein, but contains fewer amino acids than the full-length protein, and exhibits at least one of the activities of the corresponding full-length protein. Typically, the bioactive portion comprises a domain or motif having at least one of the activities of the corresponding protein. The bioactive portion of the protein of the present invention may be a polypeptide having a length of, for example, 10, 25, 50, or 100 amino acids or more. Furthermore, other bioactive portions in which other regions of the protein are deleted can be prepared by recombinant techniques, and one or more of the innate functional activities of the polypeptide of the present invention can be evaluated.

[0188] Preferred polypeptides have the amino acid sequence of a biomarker protein encoded by a nucleic acid molecule described herein. Other useful proteins are substantially identical to one of these sequences (e.g., at least about 40%, preferably 50%, 60%, 70%, 75%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and have different amino acid sequences due to natural allele diversity or mutagenesis, while retaining the functional activity of the corresponding native protein.

[0189] To determine the identity percentage of two amino acid sequences or two nucleic acids, the sequences are aligned for the purpose of obtaining the best possible comparison (e.g., gaps may be introduced in the sequence of the first amino acid or nucleic acid sequence to obtain the best possible alignment with the second amino or nucleic acid sequence). Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The identity percentage between the two sequences is a function of the number of identical positions shared by those sequences (i.e., identity % = number of identical positions / total number of positions (e.g., duplicate positions) × 100). In one embodiment, the two sequences are of the same length.

[0190] The determination of the percentage of identity between two sequences can be performed using a mathematical algorithm. A preferred non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as shown in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. To obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention, a BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12. To obtain amino acid sequences homologous to the protein molecule of the present invention, a BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3. To obtain gap-inserted alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches to detect distant relationships between molecules. When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov). Another preferred non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm in Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When using the ALIGN program for amino acid sequence comparison, the PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used. Another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm, as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When using the FASTA algorithm for nucleotide or amino acid sequence comparison, the PAM120 weight residue table can be used, for example, with a k-tuple value of 2.

[0191] The percentage of identity between two sequences can be determined using the same techniques as described above, with or without allowing gaps. When calculating the percentage of identity, only exact matches are counted.

[0192] The present invention also provides chimeric proteins or fusion proteins corresponding to biomarker proteins. As used herein, “chimeric protein” or “fusion protein” comprises all or part (preferably a bioactive moiety) of the polypeptide corresponding to the marker of the present invention, operably linked to a heterogeneous polypeptide (i.e., a polypeptide other than the polypeptide corresponding to the marker). In fusion proteins, the term “operably linked” is intended to indicate that the polypeptide of the present invention and the heterogeneous polypeptide are fused to each other in frame. The heterogeneous polypeptide may be fused to the amino-terminus or carboxyl-terminus of the polypeptide of the present invention.

[0193] Useful fusion proteins include GST fusion proteins or Fc domain fusion proteins, in which a polypeptide corresponding to the marker of the present invention is fused to the carboxyl terminus of a GST sequence or an Fc domain, respectively. Such fusion proteins can facilitate the purification of the recombinant polypeptide of the present invention.

[0194] In another embodiment, the fusion protein contains a heterologous signal sequence, an immunoglobulin fusion protein, a toxin, or other useful protein sequence. The chimeric proteins and fusion proteins of the present invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of the gene fragments can be performed using anchor primers that create a complementary overhang between two consecutive gene fragments, and then annealed and re-amplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (see above)). Furthermore, many expression vectors that already encode the fusion portion (e.g., GST polypeptide) are commercially available. The nucleic acid encoding the polypeptide of the present invention can be cloned into such an expression vector such that the fusion portion is linked in-frame to the polypeptide of the present invention.

[0195] Signal sequences can be used to facilitate the secretion and isolation of secretory proteins or other proteins of interest. Signal sequences typically feature a hydrophobic amino acid core, which is generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that enable cleavage of the signal sequence from the mature protein as the mature protein passes through the secretory pathway. Thus, the present invention relates to the polypeptide having a signal sequence, and to polypeptides in which the signal sequence is proteolytically cleaved (i.e., cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence can be operably ligated in an expression vector to a protein of interest, such as a protein that is not normally secreted or, if not, difficult to isolate. The signal sequence directs the secretion of the protein from a eukaryotic host transformed by the expression vector, and the signal sequence is subsequently or simultaneously cleaved. The protein can then be readily purified from extracellular medium by methods recognized in the art. Alternatively, the signal sequence can be ligated to the protein of interest using a sequence that facilitates purification, such as using a GST domain.

[0196] The present invention also relates to variants of biomarker polypeptides described herein. Such variants have altered amino acid sequences that can function either as agonists (mimickings) or antagonists. Variants can be generated by mutagenesis, for example, by individual point mutations or shortenings. Agonists may retain substantially the same or a subset of the biological activity of the native protein. Protein antagonists may inhibit one or more of the activities of the native protein by competitively binding to, for example, downstream or upstream members of a cellular signaling cascade containing the protein of interest. Thus, specific biological effects may be elicited by treatment with variants with limited function. Treatment of a subject with a variant having a subset of the biological activity of the native protein may result in fewer side effects in the subject compared to treatment with the native protein.

[0197] Variants of a biomarker protein that function either as an agonist (mimicking) or an antagonist can be identified by screening a combinatorial library of variants of the protein of the present invention, such as truncated variants, for agonist or antagonist activity. In one embodiment, a diverse library of variants is generated at the nucleic acid level by combinatorial mutagenesis and encoded by a diverse gene library. A diverse library of variants can be produced, for example, by enzymatically ligating a mixture of synthetic oligonucleotides into a gene sequence such that a degenerate set of possible protein sequences can be expressed as individual polypeptides or, alternatively, as a set of larger fusion proteins (e.g., for phage display). There are various methods that can be used to produce a library of possible variants of the polypeptide of the present invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; and Ike et al., 1983 Nucleic Acid Res. 11:477).

[0198] In addition, a library of polypeptide coding sequence fragments corresponding to the markers of the present invention can be used to generate a diverse population of polypeptides for screening and subsequent variant selection. For example, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of the target coding sequence with a nuclease under conditions in which only about one cleavage occurs per molecule, thereby denaturing the double-stranded DNA, regenerating the DNA to form a double-stranded DNA that may contain sense / antisense pairs from different cleaved products, removing the single-stranded portion from the re-formed double helix by treatment with an S1 nuclease, and ligating the resulting library of fragments into an expression vector. This method can yield expression libraries encoding amino-terminus and internal fragments of various sizes of the target protein.

[0199] Several techniques are known in the art for screening gene products of combinatorial libraries constructed by point mutations or truncations, and for screening cDNA libraries for gene products possessing selected characteristics. The most widely used techniques for screening large gene libraries suitable for high-throughput analysis typically involve cloning the gene library into a replicable expression vector, transforming suitable cells with the resulting vector library, and expressing the combinatorial gene under conditions that facilitate isolation of the vector encoding the original gene from which the product was detected by detecting the desired activity. Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional variants in a library, can be used in combination with the screening assay for identifying variants of the protein of the present invention (Arkin and Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815, Delgrave et al., 1993, Protein Engineering 6(3):327-331).

[0200] The production and use of biomarker nucleic acids and / or biomarker polypeptide molecules described herein can be facilitated by using standard recombinant techniques. In some embodiments, such techniques use a vector, preferably an expression vector, containing a nucleic acid encoding a biomarker polypeptide or a portion of such polypeptide. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another ligated nucleic acid. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, in which an additional DNA segment can be ligated into a viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), when introduced into a host cell, are integrated into the host cell's genome and thereby replicate together with the host genome. Furthermore, certain vectors, namely expression vectors, can direct the expression of the gene to which they are operably ligated. Generally, expression vectors that are practical in recombinant DNA techniques are often in the form of plasmids (vectors). However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0201] The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for nucleic acid expression in host cells. This means that the recombinant expression vector comprises one or more regulatory sequences selected based on the host cell to be used for expression, which are operably ligated to the nucleic acid sequence to be expressed. In a recombinant expression vector, “operably ligated” means that the target nucleotide sequence is ligated to a regulatory sequence(s) such that the expression of the nucleotide sequence is possible (e.g., in an in vitro transcription / translation system or in the host cell if the vector is introduced into a host cell). The term “regulatory sequence” is intended to include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology: Gene Expression Technology vol. 185, Academic Press, San Diego, CA (1991). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of an expression vector may depend on factors such as the selection of host cells to be transformed and the desired expression level of the protein. The expression vector of the present invention can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein.

[0202] Recombinant expression vectors for use in the present invention can be designed to express polypeptides corresponding to the markers of the present invention in prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., insect cells {using baculovirus expression vectors}, yeast cells, or mammalian cells). Suitable host cells are further discussed in Goeddel (see above). Alternatively, recombinant expression vectors may be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and T7 polymerase.

[0203] Protein expression in prokaryotes is almost always carried out in E. coli using vectors containing constitutive or inductive promoters that direct the expression of either fusion or non-fusion proteins. Fusion vectors add several amino acids to the protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase the expression of the recombinant protein, 2) to increase the solubility of the recombinant protein, and 3) to assist in the purification of the recombinant protein by acting as a ligand in affinity purification. In most cases, proteolytic cleavage sites are introduced in fusion expression vectors at the junction between the fusion region and the recombinant protein, allowing for the separation of the recombinant protein from the fusion region following the purification of the fusion protein. Such enzymes, and their allorecognition sequences, include factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc, Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A to the target recombinant protein, respectively.

[0204] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studier et al., pp. 60-89, In ​​Gene Expression Technology: Methods in Enzymology vol. 185, Academic Press, San Diego, CA, 1991). Expression of target biomarker nucleic acids from the pTrc vector depends on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter. Expression of target biomarker nucleic acids from the pET 11d vector depends on transcription from a T7 gn10-lac fusion promoter mediated by co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from commensal prophages containing the T7 gn1 gene under transcriptional regulation by the lacUV 5 promoter.

[0205] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacterium that has impaired ability to proteolytically cleave the recombinant protein (Gottesman, pp. 119-128, In Gene Expression Technology: Methods in Enzymology vol. 185, Academic Press, San Diego, CA, 1990). Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that individual codons for each amino acid are preferentially utilized in E. coli (Wada et al., 1992, Nucleic Acids Res. 20: 2111-2118). Such modification of nucleic acid sequences in the present invention can be carried out by standard DNA synthesis techniques.

[0206] In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., 1987, EMBO J.6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).

[0207] Alternatively, expression vectors are baculovirus expression vectors. Baculovirus vectors available for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).

[0208] In another embodiment, the nucleic acids of the present invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO J.6:187-195). When used in mammalian cells, the regulatory function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and Simianvirus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (see above).

[0209] In another embodiment, recombinant mammalian expression vectors can be configured to preferentially direct nucleic acid expression in specific cell types (e.g., by using tissue-specific regulatory elements to express nucleic acids). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoters (liver-specific) (Pinkert et al., 1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235-275), particularly T cell receptor promoters (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Banerji et al., 1983, Cell 33:729-740, Queen and Baltimore, 1983, Cell 33:741-748), neuronal-specific promoters (e.g., nerve fibril promoters) (Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci. USA 86:5473-5477), and pancreas-specific promoters (Edlund et al.) Examples include mammary gland-specific promoters (e.g., whey promoter) (US Patent No. 4,873,316 and European Patent Publication No. 264,166). Developmentally regulated promoters, such as the mouse hox promoter (Kessel and Gruss, 1990, Science 249:374-379) and the α-fetal protein promoter (Camper and Tilghman, 1989, Genes Dev.3:537-546), are also included.

[0210] The present invention further provides a recombinant expression vector comprising a DNA molecule cloned into an expression vector in the antisense direction. That is, the DNA molecule is operably ligated to a regulatory sequence to enable the expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to the mRNA encoding the polypeptide of the present invention. A regulatory sequence operably ligated to the antisense-cloned nucleic acid can be selected to direct the continuous expression of the antisense RNA molecule in various cell types, such as a viral promoter and / or enhancer, or a regulatory sequence can be selected to direct the constitutive, tissue-specific, or cell-type-specific expression of the antisense RNA. The antisense expression vector may be in the form of a recombinant plasmid, phagemid, or attenuated virus, in which the antisense nucleic acid is produced under the control of a highly efficient regulatory region, and its activity may be determined by the cell type into which the vector is introduced. For a discussion of gene expression regulation using antisense genes, see (Weintraub et al., 1986, Trends in Genetics, Vol. 1(1)).

[0211] Another aspect of the present invention relates to host cells into which the recombinant expression vector of the present invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to specific target cells but also to the offspring or potential offspring of such cells. Such offspring may not be identical to the parent cells in practice, because certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they are still included within the scope of the terms used herein.

[0212] The host cell can be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., insect cell, yeast, or mammalian cell).

[0213] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of techniques recognized in the art for introducing exogenous nucleic acids into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (see above) and other laboratory manuals.

[0214] Regarding the stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells can incorporate exogenous DNA into their genomes. To identify and select these incorporates, genes encoding selectable markers (e.g., for antibiotic resistance) are generally introduced into host cells along with the target gene. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells incorporating the selectable marker gene survive while other cells die).

[0215] V. Analysis of biomarker nucleic acids, polypeptides, and cells Biomarker nucleic acids and / or biomarker polypeptides can be analyzed according to the methods described herein and techniques known to those skilled in the art for identifying such gene or expression changes useful for the present invention, including, but not limited to, 1) changes in the level of biomarker transcripts or polypeptides, 2) deletion or addition of one or more nucleotides from a biomarker gene, 4) substitution of one or more nucleotides in a biomarker gene, and 5) abnormal modifications of a biomarker gene, such as in an expression regulatory region.

[0216] a. Method for detecting copy number and / or genomic nucleic acid mutations Methods for evaluating the copy number of biomarker nucleic acids and / or the genomic nucleic acid state (e.g., mutations) are well known to those skilled in the art. The presence or absence of chromosome acquisition or loss can be easily assessed by determining the copy number of the regions or markers specified herein.

[0217] In one embodiment, a biological sample is tested for the presence of copy number variations at genomic loci containing a genomic marker.

[0218] Methods for evaluating the copy number of biomarker loci include, but are not limited to, hybridization-based assays. Hybridization-based assays include, but are not limited to, conventional "direct probe" methods such as Southern blotting and in-situ hybridization (e.g., FISH and FISH plus SKY), and "comparative probe" methods such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH. These methods can be used in a wide variety of forms, including, but are not limited to, substrate (e.g., membrane or glass) conjugation methods or array-based methods.

[0219] In one embodiment, the evaluation of the copy number of a biomarker gene in a sample is performed using Southern blotting. In Southern blotting, genomic DNA (typically fragmented and separated on an electrophoretic gel) is hybridized with a probe specific to the target region. The relative copy number of the target nucleic acid is estimated by comparing the intensity of the hybridization signal from the probe to the target region with a control probe signal obtained from the analysis of normal genomic DNA (e.g., non-amplified regions of the same or related cells, tissues, organs, etc.). Alternatively, Northern blotting may be used to evaluate the copy number of coding nucleic acids in a sample. In Northern blotting, mRNA is hybridized with a probe specific to the target region. The relative copy number of the target nucleic acid is estimated by comparing the intensity of the hybridization signal from the probe to the target region with a control probe signal obtained from the analysis of normal RNA (e.g., non-amplified regions of the same or related cells, tissues, organs, etc.). Alternatively, other methods well known in the art for detecting RNA can be used to estimate the relative copy number of the target nucleic acid from higher or lower expression levels compared to a suitable control (e.g., a non-amplified portion of the same or related cells, tissues, organs, etc.).

[0220] An alternative method for determining genome copy number is in-situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152:649). Generally, in-situ hybridization involves the following steps: (1) fixation of the tissue or biological structure to be analyzed, (2) pre-hybridization treatment of the biological structure to increase accessibility to target DNA and reduce nonspecific binding, (3) hybridization of a mixture of nucleic acids to the nucleic acids in the biological structure or tissue, (4) post-hybridization washing to remove nucleic acid fragments that were not bound during hybridization, and (5) detection of the hybridized nucleic acid fragments. The reagents and operating conditions used in each of these steps vary depending on the specific application. In a typical in-situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If nucleic acids are the target of probing, cells are typically denatured with heat or alkali. The cells are then brought into contact with a hybridization solution at a moderate temperature to allow annealing of a labeled probe specific to the nucleic acid sequence encoding the protein. The target (e.g., cells) is then typically washed at a predetermined stringency or with increasing stringency until a suitable signal-to-noise ratio is obtained. The probe is typically labeled, for example, with a radioisotope or a fluorescent reporter. In one embodiment, the probe is long enough to specifically hybridize with the target nucleic acid(s) under stringent conditions. Probes are generally in the range of approximately 200 to 1000 base pairs in length. In some applications, it is necessary to block the hybridization ability of repetitive sequences. Therefore, in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block nonspecific hybridization.

[0221] An alternative method for determining genome copy number is comparative genomic hybridization. Generally, genomic DNA is isolated not only from test cells (e.g., tumor cells) but also from normal reference cells and amplified if necessary. These two nucleic acids are distinctly labeled and then hybridized in situ to metaphase chromo...

Claims

1. A composition for use in a method for selectively reducing the number and / or inhibitory immune activity of regulatory T cells (Treg) and / or regulatory B cells (Breg) in a target, comprising: 1) at least one agent that modulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, the agent being a blocking antibody or an antigen-binding fragment that specifically binds to the TACI receptor; and 2) an antibody or an antigen-binding fragment that inhibits the PD-1 pathway.

2. The at least one of the agents causes the number of Treg and / or Breg to decrease and / or the inhibitory immune activity of Treg and / or Breg to decrease by downregulating the interaction between the TACI receptor protein expressed by Treg and / or Breg and the APRIL ligand, and optionally reduces the expression of one or more growth or survival genes selected from the group consisting of IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and BIRC3. The composition according to claim 1.

3. a) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is mouse, chimeric, humanized, complex, or human; b) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments; c) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof , conjugated with cytotoxic agents; and / or d) The cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. The composition according to claim 2.

4. The composition according to any one of claims 1 to 3, further comprising administering an inhibitor of the STING pathway to the subject.

5. a) The APRIL ligand is a fusion protein; and / or b) The APRIL ligand is fused to the Fc domain, The composition according to claim 1.

6. The method described above is a) Administering an activator of the STING pathway to the subject; b) administering at least one immunotherapy agent to the subject; and / or c) administering to the subject at least one additional therapeutic agent or regimen for the treatment of cancer; and / or d) Administering the BCMA modulator to the subject, or bringing the Treg and / or Breg into contact with the BCMA modulator. The composition according to claim 5, further comprising the following:

7. a) The activator of the STING pathway is a STING agonist; b) The immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines; c) The immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO1, IDO2, and A2aR; d) The agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, i) does not significantly modulate the number and / or immune activity of Tcon, and / or ii) modulates immunomodulatory cytokine production in the Treg and / or Breg; e) The subject has cancer, and the agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing the cancer cells, and optionally, the method includes determining the responsiveness to the agent that modulates the TACI receptor protein expressed by the Treg and / or Breg with APRIL ligand, as measured by at least one criterion selected from the group consisting of clinical efficacy rate, survival to death, pathological complete response, semi-quantitative measure of pathological response, clinical complete remission, clinical partial remission, clinical disease stabilization, recurrence-free survival, metastasis-free survival, disease-free survival, reduction of circulating tumor cells, circulating marker response, and RECIST criteria; f) The agent, the STING pathway activator, the immunotherapy agent, and / or at least one additional therapeutic agent are non-systemically administered to a microenvironment containing Treg and / or Breg; g) The Treg is CD4+CD25+, CD4+FOXP3+, CD4+FoxP3+IL10+, CD4+FoxP3 高 IL10 高、 and / or CD4 + CD25 高 Includes FOXP3+ Treg; h) The Breg is CD19 + CD24 高 CD38 高 Includes Breg; i) The Tcon includes CD4 + CD25 - Tcon; j) The subject has a condition in which upregulation of the immune response would be beneficial; k) The subject has a condition selected from the group consisting of cancer, viral infection, bacterial infection, protozoal infection, helminthic infection, asthma associated with impaired airway tolerance, and immunosuppressive diseases; l) The subject has cancer, or the cell population contains cancer cells; m) The cancer is multiple myeloma; n) The cancer is an animal model of the cancer, and optionally the animal model is a mouse model; and / or o) The subject is a mammal, mouse or human, The composition according to claim 6.

8. A composition for use in a method for selectively reducing the number and / or inhibitory immune activity of Treg and / or Breg, comprising: 1) at least one agent that modulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, the agent being a blocking antibody or an antigen-binding fragment that specifically binds to the TACI receptor; and 2) an antibody or an antigen-binding fragment that inhibits the PD-1 pathway. The method is characterized by comprising contacting the Treg and / or Breg with 1) at least one agent, which is a blocking antibody or an antigen-binding fragment thereof that specifically binds to the TACI receptor, and 2) an antibody or an antigen-binding fragment that inhibits the PD-1 pathway, so as to selectively reduce the number and / or inhibitory immune activity of the Treg and / or Breg.

9. The at least one of the agents causes the number of Treg and / or Breg to decrease and / or the inhibitory immune activity of Treg and / or Breg to decrease by downregulating the interaction between the TACI receptor protein expressed by Treg and / or Breg and the APRIL ligand, and optionally reduces the expression of one or more growth or survival genes selected from the group consisting of IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and BIRC3. The composition according to claim 8.

10. a) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is mouse, chimeric, humanized, complex, or human; b) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments; c) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or an antigen-binding fragment thereof, is conjugated to a cytotoxic agent; and / or d) The cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. The composition according to claim 9.

11. The composition according to any one of claims 8 to 10, further comprising contacting the Treg and / or Breg with an inhibitor of the STING pathway.

12. a) The APRIL ligand is a fusion protein; and / or b) The APRIL ligand is fused to the Fc domain, The composition according to claim 8.

13. The method described above is a) Contacting the Treg and / or Breg with the activator of the STING pathway; b) Contacting the Treg and / or Breg with at least one immunotherapy agent; c) Contacting the Treg and / or Breg in the context of cancer cells, and contacting the cancer cells with at least one additional cancer treatment agent or regimen; and / or d) The composition according to claim 12, further comprising bringing the Treg and / or Breg into contact with the modulator of the BCMA.

14. a) The activator of the STING pathway is a STING agonist; b) The immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines; c) The immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO, IDO2, and A2aR; d) The agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, contacts the Treg and / or Breg in the presence of Tcon, i) not significantly modulating the number and / or immune activity of the Tcon, and / or ii) modulating immunomodulatory cytokine production in the Treg and / or Breg; e) The agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, contacts the Treg and / or Breg in the presence of Tcon and cancer cells, and the agent, either alone or in combination with the immunotherapy agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing the cancer cells; f) The drug, the STING pathway activator, or the immunotherapy agent and / or at least one additional therapeutic agent comes into contact with the Treg, Breg, Tcon, and / or cancer cells in vitro or ex vivo; g) The Treg is CD4+CD25+, CD4+FOXP3+, CD4+FoxP3+IL10+, CD4+FoxP3 高 IL10 高、 and / or CD4 + CD25 高 Includes FOXP3+ Treg; h) said Breg is CD19+CD24 高 CD38 高 and includes Breg; i) The Tcon includes CD4 + CD25 - Tcon; j) The subject has a condition selected from the group consisting of cancer, viral infections, bacterial infections, protozoal infections, helminthic infections, asthma associated with impaired airway tolerance, and immunosuppressive diseases; k) The Treg and / or Breg are present in the context of cancer cells; l) The cancer is multiple myeloma; and / or m) The cancer is an animal model of the cancer, and optionally, the animal model is a mouse model. The composition according to claim 13.

15. In the manufacture of a pharmaceutical product for a method of selectively reducing the number and / or inhibitory immune activity of regulatory T cells (Treg) and / or regulatory B cells (Breg) in a target, the use of: 1) at least one agent which modulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, the agent being a blocking antibody or an antigen-binding fragment thereof that specifically binds to the TACI receptor; and 2) an antibody or an antigen-binding fragment thereof that inhibits the PD-1 pathway.

16. In the manufacture of a pharmaceutical product for a method of selectively reducing the number and / or inhibitory immune activity of Treg and / or Breg, the use of: 1) at least one agent which modulates the interaction between the TACI receptor protein expressed by the Treg and / or Breg and the APRIL ligand, the agent which is a blocking antibody or an antigen-binding fragment which specifically binds to the TACI receptor; and 2) an antibody or an antigen-binding fragment which inhibits the PD-1 pathway. The method is characterized by comprising contacting the Treg and / or Breg with 1) at least one agent, which is a blocking antibody or an antigen-binding fragment thereof that specifically binds to the TACI receptor, and 2) an antibody or an antigen-binding fragment that inhibits the PD-1 pathway, so as to selectively reduce the number and / or inhibitory immune activity of the Treg and / or Breg.

17. The at least one of the agents causes the number of Treg and / or Breg to decrease and / or the inhibitory immune activity of Treg and / or Breg to decrease by downregulating the interaction between the TACI receptor protein expressed by Treg and / or Breg and the APRIL ligand, and optionally reduces the expression of one or more growth or survival genes selected from the group consisting of IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and BIRC3. The use described in claim 15.

18. a) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is mouse, chimeric, humanized, complex, or human; b) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments; c) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or an antigen-binding fragment thereof, is conjugated to a cytotoxic agent; and / or d) The cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. The use described in claim 17.

19. The use according to any one of claims 15 and 17-18, further comprising administering an inhibitor of the STING pathway to the subject.

20. a) The APRIL ligand is a fusion protein; and / or b) The APRIL ligand is fused to the Fc domain, The use described in claim 15.

21. The method described above is a) Administering an activator of the STING pathway to the subject; b) administering at least one immunotherapy agent to the subject; and / or c) administering to the subject at least one additional therapeutic agent or regimen for the treatment of cancer; and / or d) Administering the BCMA modulator to the subject, or bringing the Treg and / or Breg into contact with the BCMA modulator. The use according to claim 20, further comprising:

22. a) The activator of the STING pathway is a STING agonist; b) The immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines; c) The immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO1, IDO2, and A2aR; d) The agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, i) does not significantly modulate the number and / or immune activity of Tcon, and / or ii) modulates immunomodulatory cytokine production in the Treg and / or Breg; e) The subject has cancer, and the agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing the cancer cells, and optionally, the method includes determining the responsiveness to the agent that modulates the TACI receptor protein expressed by the Treg and / or Breg with APRIL ligand, as measured by at least one criterion selected from the group consisting of clinical efficacy rate, survival to death, pathological complete response, semi-quantitative measure of pathological response, clinical complete remission, clinical partial remission, clinical disease stabilization, recurrence-free survival, metastasis-free survival, disease-free survival, reduction of circulating tumor cells, circulating marker response, and RECIST criteria; f) The agent, the STING pathway activator, the immunotherapy agent, and / or at least one additional therapeutic agent are non-systemically administered to a microenvironment containing Treg and / or Breg; g) The Treg is CD4+CD25+, CD4+FOXP3+, CD4+FoxP3+IL10+, CD4+FoxP3 高 IL10 高、 and / or CD4 + CD25 高 Includes FOXP3+ Treg; h) The Breg is CD19 + CD24 高 CD38 高 Includes Breg; i) The Tcon includes CD4 + CD25 - Tcon; j) The subject has a condition in which upregulation of the immune response would be beneficial; k) The subject has a condition selected from the group consisting of cancer, viral infection, bacterial infection, protozoal infection, helminthic infection, asthma associated with impaired airway tolerance, and immunosuppressive diseases; l) The subject has cancer, or the cell population contains cancer cells; m) The cancer is multiple myeloma; n) The cancer is an animal model of the cancer, and optionally the animal model is a mouse model; and / or o) The subject is a mammal, mouse or human, The use described in claim 21.

23. The at least one of the agents causes the number of Treg and / or Breg to decrease and / or the inhibitory immune activity of Treg and / or Breg to decrease by downregulating the interaction between the TACI receptor protein expressed by Treg and / or Breg and the APRIL ligand, and optionally reduces the expression of one or more growth or survival genes selected from the group consisting of IL10, PD-L1, and / or MCLA, Bcl-2, Bcl-xL, CCND1, CCND2, and BIRC3. The use described in claim 16.

24. a) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is mouse, chimeric, humanized, complex, or human; b) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or the antigen-binding fragment thereof, is detectably labeled and comprises an effector domain, an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments; c) The antibody that inhibits the PD-1 pathway and / or the blocking antibody that specifically binds to the TACI receptor, or an antigen-binding fragment thereof, is conjugated to a cytotoxic agent; and / or d) The cytotoxic agent is selected from the group consisting of chemotherapeutic agents, biological agents, toxins, and radioisotopes. The use described in claim 23.

25. The use according to any one of claims 16 and 23-24, further comprising contacting the Treg and / or Breg with an inhibitor of the STING pathway.

26. a) The APRIL ligand is a fusion protein; and / or b) The APRIL ligand is fused to the Fc domain, The use described in claim 16.

27. The aforementioned use, a) Contacting the Treg and / or Breg with the activator of the STING pathway; b) Contacting the Treg and / or Breg with at least one immunotherapy agent; c) Contacting the Treg and / or Breg in the context of cancer cells, and contacting the cancer cells with at least one additional cancer treatment agent or regimen; and / or d) The use according to claim 26, further comprising bringing the Treg and / or Breg into contact with the modulator of the BCMA.

28. a) The activator of the STING pathway is a STING agonist; b) The immunotherapy agent is selected from the group consisting of cell-based immunotherapy agents, cancer vaccines, viruses, immune checkpoint inhibitors, and immunomodulatory cytokines; c) The immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophylline, IDO, IDO2, and A2aR; d) The agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, contacts the Treg and / or Breg in the presence of Tcon, i) not significantly modulating the number and / or immune activity of the Tcon, and / or ii) modulating immunomodulatory cytokine production in the Treg and / or Breg; e) The agent, either alone or in combination with the STING pathway activator and / or the immunotherapy agent, contacts the Treg and / or Breg in the presence of Tcon and cancer cells, and the agent, either alone or in combination with the immunotherapy agent, reduces the number of proliferating cells in the cancer and / or reduces the volume or size of the tumor containing the cancer cells; f) The drug, the STING pathway activator, or the immunotherapy agent and / or at least one additional therapeutic agent comes into contact with the Treg, Breg, Tcon, and / or cancer cells in vitro or ex vivo; g) The Treg is CD4+CD25+, CD4+FOXP3+, CD4+FoxP3+IL10+, CD4+FoxP3 高 IL10 高、 and / or CD4 + CD25 高 Includes FOXP3+ Treg; h) The Breg is CD19 + CD24 高 CD38 高 Includes Breg; i) The Tcon includes CD4 + CD25 - Tcon; j) The subject has a condition selected from the group consisting of cancer, viral infections, bacterial infections, protozoal infections, helminthic infections, asthma associated with impaired airway tolerance, and immunosuppressive diseases; k) The Treg and / or Breg are present in the context of cancer cells; l) The cancer is multiple myeloma; and / or m) The cancer is an animal model of the cancer, and optionally, the animal model is a mouse model. The use described in claim 27.

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