WO2020006143A1 - Methods and compositions for regulating an immune response - Google Patents
Methods and compositions for regulating an immune response Download PDFInfo
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- WO2020006143A1 WO2020006143A1 PCT/US2019/039343 US2019039343W WO2020006143A1 WO 2020006143 A1 WO2020006143 A1 WO 2020006143A1 US 2019039343 W US2019039343 W US 2019039343W WO 2020006143 A1 WO2020006143 A1 WO 2020006143A1
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Definitions
- the present disclosure generally relates to a method for providing an immune suppressive therapy, in particular by inhibiting limb region 1 like (LMBR1L) in a subject in need thereof. Also, provided herein are compositions and kits that can be used in such methods.
- LBR1L limb region 1 like
- inflammatory and autoimmune diseases are among the most prevalent diseases in the United States, affecting more than 23.5 million people. Some inflammatory and autoimmune diseases are life-threatening, and most are debilitating and require a lifetime of treatment. Despite multiple therapeutic approaches, the proportion of the population living with an inflammatory or autoimmune related disease is predicted to increase by at least 37% before 2030.
- Excessive inflammation caused by abnormal recognition of host tissue as foreign, or prolongation of the inflammatory process may lead to autoimmune or inflammatory diseases as diverse as asthma, diabetes, arteriosclerosis, cataracts, reperfusion injury, and cancer, to post- infectious syndromes such as in infectious meningitis, and to rheumatic diseases such as systemic lupus erythematosus and rheumatoid arthritis.
- autoimmune or inflammatory diseases as diverse as asthma, diabetes, arteriosclerosis, cataracts, reperfusion injury, and cancer
- post- infectious syndromes such as in infectious meningitis
- rheumatic diseases such as systemic lupus erythematosus and rheumatoid arthritis.
- the centrality of the immune response in these varied diseases makes regulation of the immune system a critical component of disease treatment.
- an abnormal inflammatory response may be modulated by anti inflammatory agents such as corticosteroids, immunosuppressants, non-steroidal anti inflammatory drugs (NSAID), COX-2 inhibitors, and protease inhibitors
- corticosteroids may induce Cushingoid features, skin thinning, increased susceptibility to infection, and suppression of the hypothalamic-pituitary- adrenal axis.
- inflammatory and autoimmune diseases are often chronic, they generally require lifelong treatment and monitoring. Thus, a need exists for effective methods and compositions to treat inflammatory and autoimmune diseases.
- the methods include suppressing or reducing an immune response in a subject in need thereof, as well as methods related to decreasing the level of T cells, B cells, NK and/or NK T cells in a subject in need thereof.
- compositions and kits that can be used in such methods.
- a method of providing an immune suppressive therapy comprises inhibiting limb region 1 like (LMBR1L) in a subject in need thereof, thereby suppressing an immune response.
- LBR1L limb region 1 like
- said inhibiting comprises reducing the number of common lymphoid progenitors and/or lymphocytes in the subject.
- the lymphocytes can include, for example, one or more of T cells, B cells, NK and NK T cells.
- the subject can have an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection.
- the autoimmune disease can be systematic lupus erythematosus (SLE), Hashimoto’s thyroiditis, Grave’s disease, type I diabetes, multiple sclerosis and/or rheumatoid arthritis.
- the method can further include administering to the subject an effective amount of an LMBR1L inhibitor such as an anti-LMBRlL antibody or antigen binding fragment thereof, which binds to LMBR1L, such as an extracellular domain of LMBR1L.
- an LMBR1L inhibitor such as an anti-LMBRlL antibody or antigen binding fragment thereof, which binds to LMBR1L, such as an extracellular domain of LMBR1L.
- Another aspect relates to an LMBR1L inhibitor such as anti-LMBRlL antibody or antigen binding fragment thereof, which binds to limb region 1 like (LMBR1L), preferably an extracellular domain of LMBR1L.
- LMBR1L inhibitor such as anti-LMBRlL antibody or antigen binding fragment thereof, which binds to limb region 1 like (LMBR1L), preferably an extracellular domain of LMBR1L.
- a further aspect relates to a pharmaceutical composition for immunosuppression, comprising the LMBR1L inhibitor such as antibody or antigen binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.
- LMBR1L inhibitors such as anti-LMBRlL antibody or antigen binding fragment thereof disclosed herein, for the manufacture of a medicament for suppressing or reducing an immune response.
- FIGs. 1A-1R A heritable lymphopenia caused by LMBR1L deficiency in mice.
- T (C-J, M, N) Frequency and surface marker expression of T (C-F), B (H-J), NK (M), and NKl.l + T (N) cells in the peripheral blood from 12-week-old Lmbrll or Cers5 ; mice generated by the CRISPR/Cas9 system.
- K T cell-dependent b-gal-specific antibodies 14 days after immunization of 12-week-old Lmbrll or Cers5 mice with a recombinant SFV vector encoding the model antigen, b-gal (rSFV ⁇ Gal). Data presented as absorbance at 450 nm.
- mice were bled 48 h following adoptive transfer and killing of CFSE-labeled target cells was analyzed by flow cytometry.
- P Lmbrll mice generate reduced antigen-specific CD8 + T cell responses to aluminum hydroxide precipitated ovalbumin (OV A/alum). Lmbrll and wild-type littermates were immunized with OV A/alum at day 0.
- Total and memory K b /SIINFEKL (SEQ ID NO. 2) tetramer-positive CD8 + T cells were analyzed at day 14 by flow cytometry using CD44 and CD62L surface markers.
- Q NK cell cytotoxicity against MHC class I-deficient (B2m ' ) target cells in Lmbrll mice.
- Figs. 2A-2M A cell-intrinsic failure of lymphocyte development.
- A-D Repopulation of lymphocytes in spleen (A, B), thymus (C), and bone marrow (D) 12 weeks after reconstitution of irradiated wild-type (C57BL/6J; CD45.1) and strawberry (CD45.2) recipients with strawberry (CD45.2) or wild-type (C57BL/6J; CD45.1) bone marrow, or Rag2 recipients with a 1 : 1 mixture of Lmbrll ⁇ (CD45.2) and wild-type (C57BL/6J; CD45.1) bone marrow.
- A B and T cells
- B NK cells
- C thymocytes
- MR mature recirculating B cells
- Trans. transitional B cells
- Imm. immature B cells.
- Numbers adjacent to outlined areas or in quadrants (A-D) indicate percent cells in each.
- A, B, E-G Reconstitution of B (A, E), T (A, F), and NK (B, G) cells in the spleen of recipients with donor-derived cells 12 weeks after engraftment.
- C, D, H-K Repopulation of donor-derived T cell subsets in thymus (C, H, I) and B cell subsets in bone marrow (D, J, K) in recipients rescued from lethal irradiation.
- L, M The frequencies (L) and total numbers (M) of stem and progenitor cell subsets per femur in the LSK + and LK + compartments in the bone marrow of Lmbrll and wild-type littermates as determined by flow cytometry. Each symbol represents an individual mouse (E-M). P-values were determined by one-way ANOVA with Dunnett’s multiple comparisons (E-K) or Student’s /-test (L, M). Data are representative of two independent experiments with 6-7 mice per genotype. Error bars indicate S.D. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001.
- Figs. 3A-3M LMBRIL-deficient T cells die in response to expansion signals.
- LMBRlL-deficient peripheral T cells are activated.
- B Immunoblot analysis of TCF1/7, LEF1, Akt, phospho-Akt, S6, phospho-S6, phospho-p70S6K, phospho-p44/p42 MAPK, and GAPDH in total cell lysates (TCLs) of pooled CD8 + T cells from Lmbrli h or wild-type littermates.
- C Annexin V staining of CD4 + or CD8 + T cells in peripheral blood obtained from l4-week-old wild-type or strawberry mice.
- a 1 1 mixture of CellTrace Violet-labeled Lmbrll h (CD45.2) and Far Red-stained wild-type OT-I T cells (CD45.2) was adoptively transferred into wild-type hosts (C57BL/6J; CD45.1).
- H-M Impaired homeostatic expansion of LMBRlL-deficient T cells.
- Numbers adjacent to outlined areas indicate percent cells in each ⁇ SD. Each symbol represents an individual mouse (A, C, D, G, J, M). P-values were determined by Student’s /-test (A, C, D) or one-way ANOVA with Dunnett’s multiple comparisons (G, J, M). Data are representative of two independent experiments with 4-29 mice per genotype or group. Error bars indicate S.D. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001.
- LMBR1L negatively regulates Wnt signaling.
- A, B LMBR1L physically interacts with components of the Wnt signaling pathway.
- a Human protein microarray revealed binding between LMBR1L and GSK-3 proteins. A construct expressing both N-terminus FLAG-tagged and C-terminus V5-tagged human LMBR1L was transfected into HEK293T cells and the recombinant protein was purified using anti-FLAG M2 agarose beads. Binding between recombinant human LMBR1L and purified human proteins printed in duplicate on the microarray slide was probed with anti-V5-Alexa 647 antibody.
- HEK293T cells were transfected with either FLAG-tagged GSK-3 , b-catenin, ZNRF3, RNF43, FZD6, LRP6, DVL2, or empty vector (EV) and HA-tagged LMBR1L. Lysates were subsequently immunoprecipitated using anti-FLAG M2 agarose and immunoblotted with antibodies against HA or FLAG.
- LMBR1L-GP78-UBAC2 complex regulates maturation of Wnt receptors within the ER.
- A Immunoblots of the indicated proteins in membrane and TCLs of pooled CD8 + T cells isolated from the spleens of 12-week-old LmbrH or wild-type littermates. The upper band of FZD6 or LRP6 (red arrowhead) is the mature form; the lower band (blue arrowhead) is the ER form of FZD6 or LRP6 (also applies to B, C, E, F). Expression of GRP94 or BiP was determined with a KDEL antibody. GAPDH was used as loading control. *, an unknown KDEL-positive protein whose expression is unchanged.
- HEK293T cells were transfected with FLAG-tagged FZD6 and either HA-tagged LMBR1L, UBAC2, GP78, or empty vector. TCLs were immunoprecipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against FLAG, HA, and Ubiquitin (UB). GAPDH was used as a loading control.
- C HEK293T cells were transfected with FLAG-tagged LRP6 and HA-tagged LMBR1L, UBAC2, or empty vector. TCLs were immunoblotted using the indicated antibodies.
- D ER or plasma membrane proteins were isolated from LMBR1L-FLAG knock-in (KI) or parental HEK293T cells (WT).
- Endogenous LMBR1L expression was then analyzed by immunoblotting using a FLAG antibody. Expression of calnexin, E-cadherin, or a-tubulin were used as loading controls for ER, plasma membrane, or cytosol, respectively.
- E Immunoblots of indicated proteins in TCLs of pooled CD8 + T cells isolated from the spleens of 6-week-old Gp78 h or wild-type mice.
- F Constructs encoding FLAG-tagged LRP6 and HA-tagged LMBR1L were transfected into Gp78 h or parental HEK293T cells. TCLs were immunoblotted using the indicated antibodies.
- HEK293T cells were transfected with FLAG-tagged b-catenin and either HA-tagged LMBR1L, UBAC2, GP78, or empty vector.
- TCLs were immunoprecipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against FLAG, HA, and Ubiquitin (UB).
- UB Ubiquitin
- LMBR1L stabilizes GSK-3p.
- A HEK293T cells were transfected with FLAG-tagged GSK-3 and either HA-tagged LMBR1L or empty vector. TCLs were immuno precipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against p- GSK-3 , FLAG, and HA. GAPDH was used as a loading control.
- B HEK293T cells were transfected with FLAG-tagged GSK-3 and either HA-tagged LMBR1L or empty vector. The cells were treated with cyclohexamide (CHX) 14 h after transfection and harvested at various times post-treatment. TCLs were immunoblotted with the indicated antibodies. Two primary antibodies (anti-HA and GAPDH) were co-incubated to visualize LMBR1L (red arrowhead) and GAPDH (blue arrowhead, a loading control) on one membrane. Data are representative of three independent experiments.
- Figs. 7A-7C Deletion of b-catenin ( Ctnnbl ) attenuates apoptosis caused by LMBRIL-deficiency.
- Figs. 8A-8C Identification of a mutation in Lmbrll as causative for severe lymphopenia in mice.
- G3 third- generation descendants of a single ENU-mutagenized male mouse heterozygous for the mutations in Cers5 and Lmbrll were intercrossed to segregate the two ENU-induced point mutations.
- Peripheral blood lymphocytes from offspring with the indicated genotypes were analyzed by flow cytometry.
- A Representative flow cytometric analysis of CD3 + and B220 + cells in the peripheral blood of 12-week-old mice.
- B Activation marker (CD44 and CD62L) expression on the surface of CD3 + CD8 + T cells in the peripheral blood.
- C Quantification of the frequency in peripheral blood of B and T cells, and the frequency of naive, central memory (CM), and effector memory (EM) CD8 + T cells based on CD62L and CD44 expression. Each symbol represents an individual mouse. P-values were determined by one-way ANOVA with Dunnett’s multiple comparisons. Data are representative of three independent experiments with 4-9 mice per genotype. Error bars indicate S.D. * P ⁇ 0.05; *** P ⁇ 0.001.
- Figs. 9A-9H Whole-blood leukocyte counts in 12-week-old Lmbrlt and wild-type littermates. Each symbol represents an individual mouse. P-values were determined by Student’s /-test. Data are representative of three independent experiments with 12-14 mice per genotype. Error bars indicate S.D. * P ⁇ 0.05; *** P ⁇ 0.001; ns, not significant with P > 0.05.
- FIGs. 10A-10N Summary of the phenotypes observed in mice carrying an ENU- induced Lmbrll mutation.
- A-H, K, L Frequency and surface marker expression of T (A-D), B (F-H), NK (K), and NK T (L) cells in the peripheral blood from wild-type C57BL/6J (WT) mice, or a pedigree of G3 descendants of a single ENU-mutagenized male mouse with REF (+/+), HET ( strawberry/+ ), or VAR ⁇ strawberry/strawberry) genotypes for Lmbrll.
- F-O Splenocytes were analyzed by flow cytometry for surface markers encompassing the major immune lineages: B220, CD3s, CD4, CD5, CD8a, CDl lb, CDl lc, CD19, CD43, F4/80, and NK1.1. Each symbol represents an individual mouse. P-values were determined by Student’s I- test. Data are representative of two independent experiments with 8 mice per genotype. Error bars indicate S.D. * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0.001; NS, not significant with P > 0.05.
- Figs. 12A-12C Reduced antigen-specific CD8 + T cell responses in Lmbrlt h mice.
- Lmbrll 1 wild-type litermates, and OT-I mice were immunized with aluminum hydroxide precipitated ovalbumin (OV A/alum) at day 0.
- OV A/alum aluminum hydroxide precipitated ovalbumin
- Frequency of total (A, C) and memory (B, C) K b /SIINFEKL tetramer-positive CD8 + T cells were analyzed at day 14 by flow cytometry using CD44 and CD62L surface markers.
- CM central memory
- EM effector memory.
- P-values were determined by Student’s t- test. Data are representative of two independent experiments. Error bars indicate S.D. *** P ⁇ 0.001; NS, not significant with P > 0.05.
- Figs. 13A-13J Expression profile of Lmbrll and normal cytokine secretion by Lmbri peritoneal macrophages (PMs) in response to stimulation.
- HSPC hematopoietic stem/progenitor cells.
- C-J PMs from Lmbrir and wild-type litermates were stimulated with ParmCSlG (TLR2/1 ligand; C), poly(EC) (TLR3 ligand; D), bpopolysaccharide (LPS; TLR4 ligand; E), R848 (TLR7 ligand; F), CpG-obgodeoxynucleotide (CpG-ODN; TLR9 ligand; G), dsDNA (H), nigericin (inflammasome; I), and flagellin (TLR5 ligand; J) in vitro at the concentrations indicated in the materials and methods.
- IFN-a, IE-1b, and TNF-a in the culture medium were measured by ELISA 4 h later. Each symbol represents an individual mouse. P-values were determined by Student’s /-test. Data are representative of two independent experiments with 4-6 mice per genotype. Error bars indicate S.D. NS, not significant with P > 0.05.
- Figs. 14A-14B P/ii/;r/r -derived hematopoietic stem cells have a disadvantage in repopulating lymphoid-primed multipotent progenitors (LMPP) and common lymphoid progenitors (CLP) in competitive bone marrow chimeras.
- LMPP lymphoid-primed multipotent progenitors
- CLP common lymphoid progenitors
- A-B Repopulation of hematopoietic stem cell and progenitor populations in competitive bone marrow chimeras.
- a 1 1 mixture of Lmbrll +/+ BM (CD45.2) and congenic WT BM (C57BL/6J; CD45.1) competitor cells were injected into lethally -irradiated Rag2 recipients.
- Figs. 15A-15C Apoptosis of LmbrH or LmbrH sl/sl CD8 + T cells in response to antigen-specific or homeostatic expansion signals.
- A Annexin V staining of adoptively transferred wild-type OT-I or Lmbrlt OT-I T cells isolated from the spleens of wild-type (C57BL/6J; CD45.1) recipient mice, 48 h after injection of soluble OVA.
- FIGs. 16A-16B Lmbri CD4 + and CD8 + T cells can home to secondary lymphoid organs, but have proliferative defects in response to homeostatic expansion signals.
- a 10 1 mixture of CellTrace Violet-labeled ( Lmbrlt h ) or CellTrace Far Red-labeled ( Lmbrll +I+ ) pan T cells isolated from the spleen were adoptively transferred into sublethally irradiated (8.5 Gy) wild-type hosts (C57BL/6J; CD45.1).
- Figs. 17A-17E Enhanced intrinsic and extrinsic caspase activation in LmbrlP t,st T cells in response to stimulation.
- A, B Immunoblot analysis of caspase processing or cleavage of PARP in lysates of pooled splenic CD8 + T cells from Lmbrl st/st or WT littermates upon TNF- a (10 ng/ml; A) or FasL (25 ng/ml; B) stimulation for 0.5, 1, 2, 4 h or left untreated.
- C, D, E Representative flow cytometric analysis of CD3 + and B220 + cells in peripheral blood of 12- week-old Lmbr lV Tnf (C), Lmbr ir Fas lpr/lpr (D), Lmbrir Casp3 (E), or littermates with the indicated genotypes. Data are representative of three independent experiments with 3-7 mice per genotype. Figs. 18A-18C. LCN3 deficiency has no effect on lymphocyte development in mice.
- LMBR1L has been identified as a receptor for human lipocalin-l (13, 14).
- LCN3 the mouse orthologue of human lipocalin-l, using the CRISPR/Cas9 system.
- A Representative flow cytometric analysis of CD3 + and B220 + cells in the peripheral blood of l2-week-old Lcn3 or WT littermates.
- B Activation marker (CD44 and CD62L) expression on the surface of CD3 + CD8 + T cells in the peripheral blood.
- C Quantification of the frequency in peripheral blood of B and T cells, and the frequency of naive, central memory (CM), and effector memory (EM) CD8 + T cells based on CD44 and CD62L expression. Each symbol represents an individual mouse. P-values were determined by Student’s /-test. Data are representative of two independent experiments with 4-7 mice per genotype. Error bars indicate S.D. NS, not significant with P > 0.05.
- LMBR1L physically interacts with components of the endoplasmic reticulum-associated degradation (ERAD) system.
- A, B Constructs expressing either FLAG-tagged UBAC2, UBXD8, VCP, or empty vector were expressed with HA-tagged LMBR1L or UBAC2 in HEK293T cells. Cell lysates were subsequently immunoprecipitated using anti-FLAG M2 agarose and immunoblotted with antibodies against HA or FLAG.
- C, D Constructs expressing either the FLAG-tagged GP78 or empty vector were expressed with HA- tagged LMBR1L or UBAC2 in HEK293T cells. Immunoprecipitation and immunoblot were performed as described in (A, B). Data are representative of two independent experiments.
- LMBR1L deficiency results in nuclear accumulation of b-catenin.
- A Intracellular b-catenin in thymocyte subsets from Lmbr It ; or wild-type littermates.
- B Immunoblot analysis of b-catenin in total cell lysates of pooled mature single positive thymocytes, naive pan T cells, and pan T cells from the spleens of Lmbr It ; and WT littermates.
- C Immunoblot analysis of b-catenin in total cell lysates as well as cytosolic and nuclear extracts of pooled CD8 + T cells from Lmbr 11 ; or WT littermates.
- GAPDH and Histone H3 were used as markers for purity of cytosolic and nuclear fractions. Each symbol represents an individual mouse. P-values were determined by Student’s /-test. Data are representative of two independent experiments with 6 mice per genotype. Error bars indicate S.D. ** P ⁇ 0.01; *** P ⁇ 0.001.
- Figs. 21A-21B Immunoblot analysis of Wnt components in CD4 + T and B cells.
- Figs. 22A-22E Normal proliferation and b-catenin activation in the LmbrH small intestine and colon.
- C Ten fields of view per mouse were averaged to obtain the b-catenin mean fluorescence intensity (MFI) value.
- D Quantification of proliferating cells per crypt (Ki-67 + ) in Lmbrll and wild- type littermates.
- E Percentage of initial body weight on day 10 of 1.5% DSS treatment in drinking water for wild-type C57BL/6J mice (WT) or a pedigree of G3 descendants of a single ENU-mutagenized male mouse with REF (+/+), HET ( strawberry/+ ), or VAR ⁇ strawberry/strawberry) genotypes for Lmbrll.
- P-values were determined by Student’s /-test (C, D) or one-way ANOVA with Dunnett’s multiple comparisons (E). Error bars indicate S.D. NS, not significant with P > 0.05.
- LMBR1L induces retention of Frizzled-6 (FZD6) in the ER and inhibits its expression on the cell surface.
- Figs. 24A-24B Expression of Wnt components in resting Ubac2 or Gp78 cells.
- A Immunoblot analysis of FZD6, LRP6, b-catenin, UBAC2, GP78, GAPDH, and a-tubulin in total cell lysates of parental WT, l Jbac 2 ; . or Gp78 1 HEK293T (A) or EL4 cells (B). Red arrowheads indicate the mature form, and the blue arrowheads indicate the immature form. Data are representative of three independent experiments.
- Fig. 25 Effect of UBAC2 on LMBRIL-mediated FZD6 maturation. Constructs encoding the FLAG-tagged FZD6 and EGFP were co-expressed with increasing amounts of HA-tagged LMBR1L in parental HEK293T and ! Jbac 2 ; cells. Total cell lysates were immunoblotted using the indicated antibodies. Red arrowhead indicates the mature form and blue arrowhead indicates the ER form of FZD6. Data are representative of three independent experiments.
- (A) FLAG-tagged b-catenin constructs were expressed with either HA-tagged GP78 or empty -HA vector in HEK293T cells.
- Fig. 27 Effect of LMBR1L on expression of destruction complex proteins.
- HEK293T cells were co-transfected with constructs encoding the FLAG-tagged Axinl, DVL2, or ⁇ 8K-3b and HA-tagged LMBR1L or empty vector. Total cell lysates were immunoblotted using the indicated antibodies.
- LMBR1L is a transmembrane protein expressed on the plasma and ER membranes. It functions as a negative feedback regulator of Wnt signaling.
- GP78 is an ER membrane-anchored E3 ubiquitin ligase that prevents accumulation of misfolded proteins via ERAD.
- UBAC2 a central element in the GP78 complex, contains a functional poly -UB -binding domain at its C-teriminus (UBA).
- the LMBR1L-GP78-UBAC2 complex ubiquitinates and prevents maturation of FZD6 and Wnt co-receptor LRP6 within the ER of lymphocytes, and the complex may also regulate the ubiquitination and degradation of b-catenin. Absent this second destruction complex, FZD6 and LRP6 accumulate on the plasma membrane and enhanced Wnt signaling overwhelms the canonical destruction complex, causing b-catenin to flood the nucleus. Additionally, LMBR1L physically interacts with several components of the destruction complex including OdK-3b. These interactions help to stabilize OdK-3b, which is needed for tonic inactivation of b-catenin by phosphorylation to attenuate canonical Wnt signaling.
- Fig. 29 Amino acid sequence alignment of human (SEQ ID NO. 3) and mouse LMBR1L (SEQ ID NO. 4). Identical residues are highlighted in red and similar residues are highlighted in yellow. NCBI gene accession number for human LMBR1L is NP_060583.2, and mouse LMBR1L is NP 083374.1 .
- LMBR1L a mutation in LMBR1L, or a knockout of LMBR1L, causes a phenotype characterized by immunodeficiency, including decreased frequencies of CD3 + T cells in the peripheral blood, increased CD4 + to CD8 + ratio, increased surface glycoproteins CD44 and CD62L, impaired B cell development, diminished T cell- dependent and T cell-independent humoral immune responses, decreased cytotoxic T lymphocyte (CTL) killing activity, and reduced frequencies of natural killer (NK) and NK T cells.
- CTL cytotoxic T lymphocyte
- NK natural killer
- LMBR1L is essential for lymphopoiesis.
- An inhibitor of LMBR1L such as an antibody, can be used to reduce or suppress an immune response in a subject in need thereof.
- LMBR1L is a multi-spanning plasma membrane protein, previously of unknown function.
- all lymphocyte dependent immunity was strongly suppressed, and lymphoid cells were driven to apoptosis by stimuli that typically cause proliferation.
- the experiments of this disclosure demonstrate that LMBR1L is an essential component of the Wnt signaling pathway in lymphocytes of all lineages. Signaling via the Wnt pathway was abnormal in LMBR1L knockout mice, as b-catenin activity was constitutively high and the destruction complex could not engage (i.e., FRIZZLED-6 becomes highly upregulated and ZNRF3 was down-regulated at the cell membrane).
- LMBR1L can interact with several components of the Wnt signaling pathway in lymphocytes, including GSK3 , b-catenin, ZNRF3, RNF43, and FRIZZLED-6. Furthermore, it was unexpectedly discovered that LMBR1L deficiency inhibits autoimmune response such as the production of autoantibodies (dsDNA-specific IgG), which is a specific and sensitive indication for systemic lupus erythematosus and other autoimmune diseases.
- autoantibodies dsDNA-specific IgG
- LMBR1L inhibitors such as antibodies and small molecule antagonists, can be used for reducing or suppressing an immune response in a subject with conditions in which the immune system is excessive or overactive, e.g., inflammatory diseases, autoimmune diseases, graft versus host disease, or an allograft rejection.
- the articles“a” and“an” refer to one or more than one, e.g., to at least one, of the grammatical object of the article.
- the use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of "one or more,” “at least one,” and “one or more than one.”
- “about” and“approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.
- the term“substantially” means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.
- Lmbrir and“LMBR1L” also known as LIMR, are used interchangeably and refer to limb region 1 like, with“ LmbrlF generally referring to the gene or mRNA, and“LMBR1L” the protein product unless otherwise noted. It should be understood that the terms include the complete gene, the cDNA sequence, the complete amino acid sequence, or any fragment or variant thereof. In some embodiments, the LMBR1L is human LMBR1L.
- LMBR1L inhibitor is intended to include therapeutic agents that inhibit, down-modulate, suppress or down-regulate LMBR1L activity.
- the term is intended to include chemical compounds, such as small molecule inhibitors or antagonists and biologic agents (e.g., antibodies), interfering RNA (shRNA, siRNA), gene editing/silencing tools (CRISPR/Cas9, TALENs) and the like.
- An“anti-LMBRlL antibody” is an antibody that immuno-specifically binds to LMBR1L (e.g., its extracellular domain).
- the antibody may be an isolated antibody.
- Such binding to LMBR1L exhibits a K d with a value of, e.g., no greater than 1 mM, no greater than 100 nM, or no greater than 50 nM. Kd can be measured by any methods known to one skilled in the art, such as a surface plasmon resonance assay or a cell binding assay.
- An anti-LMBRlL antibody may be a monoclonal antibody or an antigen-binding fragment thereof.
- an“antibody” as used herein is a protein consisting of one or more polypeptides comprising binding domains that bind to a target epitope.
- the term antibody includes monoclonal antibodies comprising immunoglobulin heavy and light chain molecules, single heavy chain variable domain antibodies, and variants and derivatives thereof, including chimeric variants of monoclonal and single heavy chain variable domain antibodies.
- Binding domains are substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes, wherein the protein immuno-specifically binds to an antigen.
- the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
- Light chains are classified as either kappa or lambda.
- Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
- the typical immunoglobulin structural unit comprises a tetramer that is composed of two identical pairs of polypeptide chains, each pair having one“light” (about 25 kD) and one“heavy” chain (about 50-70 kD).
- VL” and VH refer to the variable domains of these light and heavy chains respectively.
- Loops of b-strands, three each on the VL and VH, are responsible for binding to the antigen and are referred to as the “complementarity determining regions” or“CDRs”.
- The“Fab” (fragment, antigen-binding) region includes one constant and one variable domain from each heavy and light chain of the antibody, i.e., VL, CL, VH, and CHL
- Antibodies include intact immunoglobulins as well as antigen-binding fragments thereof.
- antigen-binding fragment refers to a polypeptide fragment of an antibody which binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding).
- Antigen binding fragments can be produced by recombinant or biochemical methods that are well known in the art.
- antigen-binding fragments include Fv, Fab, Fab', (Fab')2, CDR, paratope, and single chain Fv antibodies (scFv) in which a VH and a VL chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
- Antibodies also include variants, chimeric antibodies, and humanized antibodies.
- antibody variant refers to an antibody with single or multiple mutations in the heavy chains and/or light chains.
- the mutations exist in the variable region.
- the mutations exist in the constant region.
- Chimeric antibodies refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chains is homologous to corresponding sequences in another.
- variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another.
- variable regions can conveniently be derived from presently known sources using readily available hybridomas or B cells from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation, and the specificity is not affected by its source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non-human source.
- “Humanized” antibodies refer to a molecule having an antigen-binding site that is substantially derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin.
- the antigen- binding site may comprise either complete variable domains fused onto constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domains.
- Antigen binding sites may be wild type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulin more closely.
- humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies).
- Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) which are altered with respect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs.
- amino acid residues of an antibody can be numbered according to the general numbering of Rabat (Rabat, et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, NIH, Bethesda, MD).
- binding refers to the process of a non-covalent interaction between molecules.
- said binding is specific.
- the specificity of an antibody can be determined based on affinity.
- a specific antibody can have a binding affinity or dissociation constant Rd for its epitope of less than 10 7 M, preferably less than 10 8 M.
- affinity refers to the strength of a binding reaction between a binding domain of an antibody and an epitope. It is the sum of the ahractive and repulsive forces operating between the binding domain and the epitope.
- affinity refers to the dissociation constant, Rd.
- antigen refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
- An antigen may have one or more epitopes.
- epitope includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor.
- epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
- An epitope is a region of an antigen that is bound by an antibody.
- an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
- Methods for epitope mapping are well known in the art, such as X-ray co-crystallography, array-based oligo-peptide scanning, site-directed mutagenesis, high throughput mutagenesis mapping, and hydrogen-deuterium exchange.
- paratope The site on the antibody that binds the epitope is referred to as“paratope,” which typically includes amino acid residues that are in close proximity to the epitope once bound. See Sela-Culang et al, Front Immunol. 2013; 4: 302.
- IHC Immunohistochemistry
- Immunospecific or“immunospecifically” refer to antibodies that bind via domains substantially encoded by
- an antibody binds immunospecifically to a cognate antigen with a Kd with a value of no greater than 50 nM, as measured by a surface plasmon resonance assay or a cell binding assay.
- the use of such assays is well known in the art.
- immune response includes T cell mediated responses, B cell mediated immune responses, and/or NK cell mediated responses, as well as changes in the number and/or development of T, B and/or NK cells (e.g., by regulating common lymphoid progenitors).
- immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
- the term“lymphopoiesis” has its general meaning in the art and refers to the generation of lymphocytes such as B, T and NK cells.
- lymphocytes such as B, T and NK cells.
- T-cell lymphopoiesis refers to the generation of T cells (i.e. T lymphocytes).
- autoimmune disease refers to a disease that arises from an overactive immune response in a subject, in which the subject’s immune system produces antibodies that attack the subject’s own cells, leading to the deterioration, and in some cases, the destruction of cells and/or tissue.
- autoimmune diseases include, without limitation, Type 1 diabetes, Multiple Sclerosis, coeliac disease, lupus erythematosus, systemic lupus erythematosus (SLE), Sjogren's syndrome, Churg-Strauss Syndrome, Hashimoto’s thyroiditis, Graves’ disease, idiopathic thrombocytopenic purpura, rheumatoid arthritis (RA), ankylosing spondylitis,
- inflammatory disease as used herein is defined as a disorder that results from an excessive inflammatory response (or inflammatory overresponse).
- An inflammatory disease is the result of an inappropriate and excessive response to an inappropriate antigen.
- inflammatory diseases include but are not limited to, allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, rheumatoid arthritis, lupus, preperfusion injury, transplant rejection, Addison’s disease, alopecia areata, dystrophic epidermolysis bullosa, epididymitis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis, among others.
- IBD Inflammatory Bowel Disease
- CD Crohn's Disease
- agent can include any molecule, peptide, antibody or other agent which can reduce or suppress an immune response in a subject with conditions in which the immune system is overactive, such as an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection.
- agents are useful in the compositions and methods described herein.
- cross-compete means the ability of an antibody or fragment thereof to interfere with the binding directly or indirectly through allosteric modulation of the anti-LMBRTL antibodies of the present disclosure to the target LMBR1L.
- the extent to which an antibody or fragment thereof is able to interfere with the binding of another to the target, and therefore whether it can be said to cross-block or cross-compete according to the present disclosure, can be determined using competition binding assays.
- One particularly suitable quantitative cross-competition assay uses a FACS- or an AlphaS creen-based approach to measure competition between the labelled (e.g.
- a cross-competing antibody or fragment thereof is, for example, one which can bind to the target in the cross-competition assay such that, during the assay and in the presence of a second antibody or fragment thereof, the recorded displacement of the immunoglobulin single variable domain or polypeptide according to the disclosure is up to 100% (e.g., in FACS based competition assay) of the maximum theoretical displacement (e.g., displacement by cold (e.g., unlabeled) antibody or fragment thereof that needs to be cross- blocked) by the to be tested potentially cross-blocking antibody or fragment thereof that is present in a given amount.
- cross-competing antibodies or fragments thereof have a recorded displacement that is between 10% and 100%, more preferred between 50% to 100%.
- inhibitor refers to any statistically significant decrease in biological activity (e.g., LMBR1L activity), including full blocking of the activity.
- inhibitor can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.
- subject or“patient” includes a human or other mammalian animal that receives either prophylactic or therapeutic treatment.
- the terms“treat,”“treating,” and“treatment” as used herein refer to therapeutic or preventative measures such as those described herein.
- the methods of“treatment” employ administration to a patient a LMBR1L inhibitor provided herein, for example, a patient with conditions in which the immune system is overactive, such as an inflammatory disease, graft- versus-host disease, allograft rejection, or an autoimmune disease (e.g., Hashimoto’s thyroiditis, Grave’s Disease, type I insulin-dependent diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS)), in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms in the patient with conditions in which the immune system is excessive or overactive, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
- an inflammatory disease e.g., Hashimoto’s thyroiditis, Grave’s Disease, type
- an agent such as a LMBR1L inhibitor, for example an anti-LMBRlL antibody, which is sufficient for reducing or suppressing an immune response in a patient with conditions in which the immune system is excessive or overactive, e.g., an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection, and/or effect treatment, prognosis, or diagnosis of an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection, when administered to a patient.
- an agent such as a LMBR1L inhibitor, for example an anti-LMBRlL antibody
- a therapeutically effective amount will vary depending upon the patient and disease condition being treated, the weight and age of the patient, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
- the dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 pg to about 3,500 mg, about 5 pg to about 3,000 mg, about 10 pg to about 2,600 mg, about 20 pg to about 2,575 mg, about 30 pg to about 2,550 mg, about 40 pg to about 2,500 mg,
- Dosing may be, e.g., every week, every 2 weeks, every three weeks, every 4 weeks, every 5 weeks or every 6 weeks. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (side effects) of the agent are minimized and/or outweighed by the beneficial effects.
- Administration may be intravenous at exactly or about 6 mg/kg or 12 mg/kg weekly, or 12 mg/kg or 24 mg/kg biweekly. Additional dosing regimens are described below.
- the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the disclosure.
- compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
- the limb region 1 like is a nine-transmembrane spanning cell surface protein, and as disclosed herein, is required for normal function of all lymphoid lineages, including T cells, B cells, NK and NK T cells.
- LMBR1L has been identified as a receptor for the small secretory protein, human Liopcalin-l (PMID: 23964685).
- the full gene sequence of human LMBR1L is 14,985 bp in length (GenBank ID No. NC_0000l2.l2).
- the LMBR1L protein was genetically linked to multiple congenital limb malformations.
- LMBR1L deficiency in T cells increased expression of the Wnt co-receptor frizzled-6 (FZD6) and low-density lipoprotein receptor-related protein 6 (LRP6), resulting in aberrant activation of the Wnt ⁇ -catenin pathway and apoptotic cell death upon stimulation.
- FZD6 Wnt co-receptor frizzled-6
- LRP6 low-density lipoprotein receptor-related protein 6
- LMBR1L Interaction of LMBR1L with ubiquitin-associated domain containing 2 (UBAC2) and glycoprotein 78 (GP78) causes downregulation of Wnt signaling in lymphocytes by preventing maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum.
- the present disclosure thus establishes an essential function for LMBR1L during lymphopoiesis and lymphoid activation, in which it acts as a negative regulator of the Wnt/b- catenin pathway.
- LMBR1L interacting proteins are essential components of the endoplasmic reticulum-associated degradation (ERAD) pathway, including ubiquitin associated domain containing 2 (UBAC2), transitional endoplasmic reticulum ATPase (TERA known as VCP), UBX domain-containing protein 8 (UBXD8, known as FAF2), and Glycoprotein 78 (GP78; known as AMFR).
- ESD endoplasmic reticulum-associated degradation
- UBAC2 ubiquitin associated domain containing 2
- TERA transitional endoplasmic reticulum ATPase
- UBXD8 UBX domain-containing protein 8
- GP78 Glycoprotein 78
- LMBR1L interactors including zinc and ring finger 3 (ZNRF3), low-density lipoprotein receptor-related protein 6 (LRP6), b-catenin, glycogen synthase kinase-3a (GSK3a), and OdK3b.
- ZNRF3 zinc and ring finger 3
- LRP6 low-density lipoprotein receptor-related protein 6
- b-catenin glycogen synthase kinase-3a
- OdK3b OdK3b
- LMBR1L has been identified herein as a novel negative regulator of Wnt/b- catenin signaling.
- LMBR1L exists in the GP78-UBAC2 complex to attenuate Wnt ⁇ -catenin signaling by inhibiting Wnt co-receptor maturation within the ER.
- the findings herein demonstrate the existence of a previously unrecognized pathway that regulates Wnt ⁇ -catenin signaling in lymphocytes.
- the exaggerated apoptosis of T cells that results in lymphopenia stems from aberrant activation of Wnt ⁇ -catenin signaling in LMBR1L- deficient mice.
- LMBR1L mature forms of Wnt co-receptors are highly upregulated and components of the destruction complex are downregulated.
- These alterations contribute to the accumulation of b-catenin, which enters the nucleus and promotes the transcription of target genes such as c-Myc, p53, and CD44.
- This signal transduction cascade favors apoptosis in an intrinsic and extrinsic caspase cascade-dependent manner.
- compositions for inhibiting LMBR1L and thus, reducing or suppressing an immune response in a subject with conditions in which the immune system is excessive or overactive are also provided.
- the composition can include one or more anti-LMBRlL antibodies disclosed herein, or an antigen binding fragment thereof.
- other LMBR1L inhibitors such as small molecule compounds, can also be used to inhibit one or more activities of LMBR1L.
- compositions and methods for treating an immunodeficiency disorder can include introducing a nucleic acid (DNA or mRNA) such as a transgene encoding LMBR1L into a subject in need thereof.
- a nucleic acid DNA or mRNA
- Inhibition of LMBR1L can reduce or suppress an immune response in a subject with conditions in which the immune system is excessive or overactive, such as an inflammatory disease, graft-versus-host disease, allograft rejection, or an autoimmune disease (e.g., Hashimoto’s thyroiditis, Grave’s Disease, type I insulin-dependent diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS)).
- autoimmune disease e.g., Hashimoto’s thyroiditis, Grave’s Disease, type I insulin-dependent diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS)
- LMBR1L inhibitors can be used as an effective agent in an immunosuppressive therapy.
- LMBR1L deficiency or inhibition can lead to apoptosis of lymphocytes such as T cells, as well as inhibition of autoimmune responses such as the production of autoantibodies (e.g., dsDNA-specific IgG).
- LMBR1L has an essential function during lymphopoiesis and lymphoid activation, acting as a negative regulator of the Wnt/b- catenin pathway.
- LMBR1L inhibitors are included in the present disclosure.
- examples include chemical compounds, such as small molecule inhibitors and biologic agents (e.g., antibodies) that can bind LMBR1L and inhibit or decrease its activity, e.g., measured in a Western Blot Analysis or ZNRF3, FRIZZLED-6, b-catenin, and/or c-Myc expression assay.
- Agents that regulate Lmbrll gene expression level are also included, such as interfering RNA (shRNA, siRNA) and gene editing/silencing tools (CRISPR/Cas9, TALENs, zinc finger nucleases) that are designed specifically to target the Lmbrll gene or a regulatory sequence thereto.
- a method for identifying an LMBR1L inhibitor can include contacting a cell with a test agent, wherein an increase in expression of FRIZZLED-6, b-catenin, and/or c-Myc, and/or a decrease in expression of ZNRF3, compared to a control cell that is not contacted with the test agent indicates that the test agent is an LMBR1L inhibitor.
- the LMBR1L inhibitor can be characterized by at least partial inhibition of proliferation (e.g., by at least 10% relative to control) of a cell expressing LMBR1L.
- the LMBR1L inhibitor is an anti-LMBRlL antibody, e.g., a monoclonal antibody, or an antigen-binding fragment thereof.
- the anti- LMBRlL antibody can be a modified antibody, e.g., chimeric or humanized antibody derived from a mouse anti-LMBRlL antibody. Methods for making modified antibodies are known in the art.
- the anti-LMBRlL antibody is an antibody or antigen binding fragment thereof which binds to an epitope present on the human LMBR1L protein, e.g., the extracellular ectodomain, or a portion thereof.
- the LMBR1L inhibitor such as anti-LMBRlL antibody can comprise a mixture, or cocktail, of two or more anti-LMBRlL antibodies, each of which binds to a different epitope on LMBR1L.
- the mixture, or cocktail comprises three anti-LMBRlL antibodies, each of which binds to a different epitope on LMBR1L.
- the LMBR1L inhibitor can include a nucleic acid molecule, such as an RNA molecule, that inhibits the expression or activity of LMBR1L.
- a nucleic acid molecule such as an RNA molecule
- Interfering RNAs specific for LMBR1L such as shRNAs or siRNAs that specifically inhibit the expression and/or activity of LMBR1L, can be designed in accordance with methods known in the art.
- an LMBR1L inhibitor for the manufacture of a medicament to reduce or suppress an immune response in a subject with conditions in which the immune system is excessive or overactive, such as an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection, is provided.
- a method of suppressing an immune response in a patient with conditions in which the immune system is excessive or overactive, such as an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection is provided, the method comprising administering to the patient an effective amount of an LMBR1L inhibitor.
- Anti-LMBRlL antibodies can be made using various methods generally known in the art. For example, phage display technology can be used to screen a human antibody library to produce a fully human monoclonal antibody for therapy. High affinity binders can be considered candidates for neutralization studies. Alternatively, a conventional monoclonal approach can be used, in which mice or rabbits can be immunized with the human protein, candidate binders identified and tested, and a humanized antibody ultimately produced by engrafting the combining sites of heavy and light chains into a human antibody encoding sequence.
- Antibodies typically comprise two identical pairs of polypeptide chains, each pair having one full-length“light” chain (typically having a molecular weight of about 25 kDa) and one full- length“heavy” chain (typically having a molecular weight of about 50-70 kDa).
- the amino- terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
- the carboxy-terminal portion of each chain typically defines a constant region responsible for effector function.
- the variable regions of each of the heavy chains and light chains typically exhibit the same general structure comprising four relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
- FR relatively conserved framework regions
- both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
- the assignment of amino acids to each domain is typically in accordance with the definitions of Rabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health, Bethesda, Md.), Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917, or Chothia et al, 1989, Nature 342:878-883).
- Monoclonal antibodies are produced using any method that produces antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma methods of Kohler et al. (1975, Nature 256:495-497) and the human B-cell hybridoma method (Kozbor, 1984, J. Immunol. 133:3001; and Brodeur et al, 1987 , Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, pp. 51-63).
- Monoclonal antibodies may be modified for use as therapeutics.
- One example is a “chimeric” antibody in which a portion of the heavy chain and/or light chain is identical with or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass.
- Other examples are fragments of such antibodies, so long as they exhibit the desired biological activity. See, U.S. Pat. No. 4,816,567; and Morrison et al. (1985), Proc. Natl. Acad. Sci. USA 81 :6851-6855.
- CDR-grafted antibody in which the antibody comprises one or more complementarity determining regions (CDRs) from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass.
- CDRs complementarity determining regions
- Humanization can be performed, for example, using methods described in the art (Jones et al, 1986, Nature 321:522-525; Riechmann et al, 1988, Nature 332:323-327; Verhoeyen et al, 1988, Science 239: 1534-1536), by substituting at least a portion of a rodent variable region for the corresponding regions of a human antibody.
- transgenic animals e.g., mice
- an antigen typically having at least 6 contiguous amino acids
- transgenic animals are produced by incapacitating the endogenous mouse immunoglobulin loci encoding the mouse heavy and light immunoglobulin chains therein, and inserting loci encoding human heavy and light chain proteins into the genome thereof. Partially modified animals, which have less than the full complement of modifications, are then cross-bred to obtain an animal having all of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific for these antigens having human (rather than murine) amino acid sequences, including variable regions. See PCT Publication Nos. W096/33735 and W094/02602, incorporated by reference. Additional methods are described in U.S. Pat. No. 5,545,807, PCT Publication Nos.
- Human antibodies may also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.
- phage display technology may be used to screen for therapeutic antibodies.
- antibody repertoires can be displayed on the surface of
- the constructed library may be screened for phages that bind to the immunogen.
- Antibody phage is based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection and phage propagation. This technique allows in vitro selection of LMBR1L monoclonal antibodies.
- the phage display process begins with antibody-library preparation followed by ligation of the variable heavy (VH) and variable light (VL) PCR products into a phage display vector, culminating in analysis of clones of monoclonal antibodies.
- VH variable heavy
- VL variable light
- VH and VL PCR products representing the antibody repertoire, are ligated into a phage display vector (e.g ., the phagemid pComb3X) that is engineered to express the VH and VL as an scFv fused to the pill minor capsid protein of a filamentous bacteriophage of
- a phage display vector e.g ., the phagemid pComb3X
- the phage display vector pComb3X does not have all the other genes necessary to encode a full
- a helper phage is added to the E. coli that are transformed with the phage display vector library.
- the result is a library of phages, each expressing on its surface a LMBR1L monoclonal antibody and harboring the vector with the respective nucleotide sequence within.
- the phage display can also be used to produce the LMBR1L monoclonal antibody itself (not attached to phage capsid proteins) in certain strains of E. Coli.
- Additional cDNA is engineered, in the phage display vector, after the VL and VH sequences to allow characterization and purification of the mAb produced.
- the recombinant antibody may have a hemagglutinin (HA) epitope tag and a polyhistidine to allow easy purification from solution.
- HA hemagglutinin
- Diverse antibody phage libraries are produced from -10 8 independent E. coli transformants infected with helper phage. Using bio-panning, a library can be screened for phage binding to the immunogen sequence listed above, or a fragment thereof, through the expressed surface of the monoclonal antibody. Cyclic panning allows for pulling out potentially very rare antigen-binding clones and consists of multiple rounds of phage binding to antigen (immobilized on ELISA plates or in solution on cell surfaces), washing, elution, and
- LMBR1L monoclonal antibody is characteristic for directed selection on the immobilized immunogen.
- Another method is to add a C-terminal His tag, suitable for purification by affinity chromatography, to the immunogen sequence listed above.
- Purified protein can be inoculated into mice together with a suitable adjuvant.
- Monoclonal antibodies produced in hybridomas can be tested for binding to the immunogen, and positive binders can be screened as described in the assays herein.
- Fully human antibodies can also be produced from phage-display libraries (as disclosed in Hoogenboom et al, 1991, J. Mol. Biol. 227:381; and Marks et al, 1991, J. Mol. Biol. 222:581). These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage and subsequent selection of phage by their binding to an antigen of choice.
- phage-display libraries as disclosed in Hoogenboom et al, 1991, J. Mol. Biol. 227:381; and Marks et al, 1991, J. Mol. Biol. 222:581).
- These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage and subsequent selection of phage by their binding to an antigen of choice.
- One such technique is described in PCT Publication No. WO99/10494, incorporated by reference, which describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk
- the extracellular domains of human LMBR1L can be used as the immunogen.
- the human LMBR1L has five extracellular domains (FIG. 1B).
- extracellular domains include:
- a fragment or portion of the human LMBR1L extracellular domain can also be used as the immunogen.
- Monoclonal antibodies can be raised using one or more immunogens. Potential therapeutic anti-LMBRlL antibodies can be generated.
- monoclonal antibodies that phenocopy the knockout mutation can be tested and identified as potential anti-LMBRlL antibody candidates.
- Monoclonal antibodies that phenocopy the knockout mutation can display phenotypes such as a reduced number of T cells (e.g., CD4+ and CD8+), B cells, NK and/or NK T cells.
- Such tests include screening endpoint(s), such as the augmentation of FRIZZLED-6, ZNRF3, b-catenin and/or c-Myc protein expression detected on, e.g., Western blot.
- screening endpoint(s) such as the augmentation of FRIZZLED-6, ZNRF3, b-catenin and/or c-Myc protein expression detected on, e.g., Western blot.
- Fully human monoclonal antibodies can be developed for preclinical testing and then tested in clinical human trials for safety and efficacy.
- Such antibodies can be clinical candidates that can reduce or suppress an immune response in a subject with conditions in which the immune system is excessive or overactive, such as an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection, and/or improve immune suppressive therapy (e.g., by decreasing the number of lymphocytes).
- Nucleotide sequences encoding the above antibodies can be determined. Thereafter, chimeric, CDR-grafted, humanized, and fully human antibodies also may be produced by recombinant methods. Nucleic acids encoding the antibodies can be introduced into host cells and expressed using materials and procedures generally known in the art.
- the disclosure provides one or more monoclonal antibodies against LMBR1L.
- the antibodies bind to one or more extracellular domains, or fragments thereof, of human LMBR1L.
- the disclosure provides nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to the variable regions thereof.
- sequences corresponding to CDRs specifically from CDR1 through CDR3, are provided.
- the disclosure provides hybridoma cell lines expressing such immunoglobulin molecules and monoclonal antibodies produced therefrom, preferably purified human monoclonal antibodies against human LMBR1L.
- the CDRs of the light and heavy chain variable regions of anti-LMBRlL antibodies of the disclosure can be grafted to framework regions (FRs) from the same, or another, species.
- the CDRs of the light and heavy chain variable regions of anti-LMBRlL antibody may be grafted to consensus human FRs.
- consensus human FRs FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence.
- the FRs of the anti-LMBRlL antibody heavy chain or light chain can be replaced with the FRs from a different heavy chain or light chain.
- Rare amino acids in the FRs of the heavy and light chains of anti-LMBRlL antibody typically are not replaced, while the rest of the FR amino acids can be replaced. Rare amino acids are specific amino acids that are in positions in which they are not usually found in FRs.
- the grafted variable regions from anti-LMBRlL antibodies of the disclosure can be used with a constant region that is different from the constant region of anti-LMBRlL antibody. Alternatively, the grafted variable regions are part of a single chain Fv antibody. CDR grafting is described, e.g., in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are hereby incorporated by reference for any purpose.
- antibodies of the disclosure can be produced by hybridoma lines.
- the antibodies of the disclosure bind to LMBR1L with a dissociation constant (Kd) of between approximately 4 pM and 1 mM.
- Kd dissociation constant
- the antibodies bind to LMBR1L with a Kd of less than about 100 nM, less than about 50 nM or less than about 10 nM.
- the antibodies of the disclosure are of the IgGl, IgG2, or IgG4 isotype, with the IgGl isotype most preferred.
- the antibodies comprise a human kappa light chain and a human IgGl, IgG2, or IgG4 heavy chain.
- the variable regions of the antibodies are ligated to a constant region other than the constant region for the IgGl, IgG2, or IgG4 isotype.
- the antibodies of the disclosure have been cloned for expression in mammalian cells.
- antibodies of the disclosure can be expressed in cell lines other than hybridoma cell lines.
- sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell.
- transformation can be achieved using any known method for introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art. Such procedures are exemplified by U.S. Pat. Nos.
- heterologous polynucleotides include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
- a nucleic acid molecule encoding the amino acid sequence of a heavy chain constant region, a heavy chain variable region, a light chain constant region, or a light chain variable region of a LMBR1L antibody of the disclosure is inserted into an appropriate expression vector using standard ligation techniques.
- the LMBR1L heavy or light chain constant region is appended to the C-terminus of the appropriate variable region and is ligated into an expression vector.
- the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
- expression vectors used in any of the host cells can contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
- sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
- sequences are well known in the art.
- Expression vectors of the disclosure may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.
- the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
- the transformation of an expression vector for an anti-LMBRlL antibody into a selected host cell may be accomplished by well- known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al, supra.
- the host cell when cultured under appropriate conditions, synthesizes an anti-LMBRTL antibody that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
- the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
- Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human
- ATCC American Type Culture Collection
- CHO Chinese hamster ovary
- HeLa HeLa
- BHK baby hamster kidney
- COS monkey kidney cells
- hepatocellular carcinoma cells e.g., Hep G2
- hepatocellular carcinoma cells e.g., Hep G2
- a heterologous antibody e.g., mouse myeloma cell lines NS0 and SP2/0
- compositions are provided that can be used in the methods disclosed herein, i.e., pharmaceutical compositions for reducing or suppressing an immune response in a subject with conditions in which the immune system is excessive or overactive, such as an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection, and/or improve immune suppressive therapy (e.g., by decreasing the number of lymphocytes).
- the pharmaceutical composition comprises an LMBR1L inhibitor and a pharmaceutically acceptable carrier.
- the LMBR1L inhibitor can be formulated with the pharmaceutically acceptable carrier into a pharmaceutical composition.
- the pharmaceutical composition can include, for example, instructions for use of the composition for the treatment of patients to reduce or suppress an immune response in a subject with conditions in which the immune system is overactive, and/or improve immune suppressive therapy.
- the LMBR1L inhibitor can be an anti-LMBRlL antibody or antigen binding fragment thereof.
- “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, and other excipients that are physiologically compatible.
- the carrier is suitable for parenteral, oral, or topical administration. Depending on the route of
- the active compound e.g., small molecule or biologic agent
- the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
- Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion, as well as conventional excipients for the preparation of tablets, pills, capsules and the like.
- sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion, as well as conventional excipients for the preparation of tablets, pills, capsules and the like.
- the use of such media and agents for the formulation of pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions provided herein is
- Supplementary active compounds can also be incorporated into the compositions.
- a pharmaceutically acceptable carrier can include a pharmaceutically acceptable antioxidant.
- pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium
- antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like
- metal chelating agents such as citric acid
- EDTA ethylenediamine tetraacetic acid
- sorbitol sorbitol
- tartaric acid tartaric acid
- phosphoric acid and the like.
- aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, and injectable organic esters, such as ethyl oleate.
- polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
- injectable organic esters such as ethyl oleate.
- proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
- isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
- agents that delays absorption for example, monostearate salts and gelatin.
- These compositions may also contain functional excipients such as preservatives, wetting agents, emulsifying agents and dispersing agents.
- compositions typically must be sterile, non-phylogenic, and stable under the conditions of manufacture and storage.
- the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
- Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization, e.g., by microfiltration.
- dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
- methods of preparation include vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- the active agent(s) may be mixed under sterile conditions with additional
- pharmaceutically acceptable carrier(s) and with any preservatives, buffers, or propellants which may be required.
- Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
- compositions comprising an LMBR1L inhibitor can be administered alone or in combination therapy.
- the combination therapy can include a composition provided herein comprising an LMBR1L inhibitor and at least one or more additional therapeutic agents, such as one or more chemotherapeutic agents known in the art, discussed in further detail below.
- Pharmaceutical compositions can also be administered in conjunction with radiation therapy and/or surgery.
- Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
- Exemplary dosage ranges for administration of an antibody include: 10-1000 mg (antibody )/kg (body weight of the patient), 10-800 mg/kg, 10-600 mg/kg, 10-400 mg/kg, 10-200 mg/kg, 30-1000 mg/kg, 30-800 mg/kg, 30-600 mg/kg, 30-400 mg/kg, 30-200 mg/kg, 50-1000 mg/kg, 50-800 mg/kg, 50-600 mg/kg, 50-400 mg/kg, 50-200 mg/kg, 100-1000 mg/kg, 100-900 mg/kg, 100-800 mg/kg, 100-700 mg/kg, 100-600 mg/kg, 100-500 mg/kg, 100-400 mg/kg, 100- 300 mg/kg, and 100-200 mg/kg.
- Exemplary dosage schedules include once every three days, once every five days, once every seven days (i.e., once a week), once every 10 days, once every 14 days (i.e., once every two weeks), once every 21 days (i.e., once every three weeks), once every 28 days (i.e., once every four weeks), and once a month.
- Unit dosage form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit contains a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with any required pharmaceutical carrier.
- the specification for unit dosage forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
- parenteral as used herein in the context of administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection, and infusion.
- parenteral administration and“administered parenterally” as used herein refer to modes of administration other than enteral (i.e., via the digestive tract) and topical administration, usually by injection or infusion, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection, and infusion. Intravenous injection and infusion are often (but not exclusively) used for antibody administration.
- agents provided herein are administered as pharmaceuticals, to humans or animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (e.g., 0.005 to 70%, e.g., 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
- the methods and uses provided herein for reducing or suppressing an immune response in a subject with conditions in which the immune system is excessive or overactive, and/or improve immune suppressive therapy can comprise administration of an LMBR1L inhibitor and at least one additional agent that is not an LMBR1L inhibitor.
- the improved effectiveness of a combination according to the disclosure can be demonstrated by achieving therapeutic synergy.
- therapeutic synergy is used when the combination of two products at given doses is more efficacious than the best of each of the two products alone at the same doses.
- therapeutic synergy can be evaluated by comparing a combination to the best single agent using estimates obtained from a two-way analysis of variance with repeated measurements (e.g., time factor) on parameter tumor volume.
- additive refers to when the combination of two or more products at given doses is equally efficacious than the sum of the efficacies obtained with of each of the two or more products, whilst the term“superadditive” refers to when the combination is more efficacious than the sum of the efficacies obtained with of each of the two or more products.
- compositions and methods for reducing or suppressing an immune response in a subject with conditions in which the immune system is overactive includes inhibiting LMBR1L in a subject in need thereof.
- inhibiting LMBR1L can reduce the number of T cells (e.g., CD4+ and CD8+), B cells, NK and/or NK T cells, thereby providing an immune suppressive therapy.
- LMBR1L inhibition e.g., an anti- LMBR1L antibody
- LMBR1L inhibition can be used in conjunction with other therapies.
- the methods disclosed herein can include administering to the subject an effective amount of LMBR1L inhibitor such as anti-LMBRlL antibody or antigen binding fragment thereof.
- the effective amount can be administered therapeutically and/or prophylactically.
- Treatment can be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk of developing such cancer. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
- a diagnostic test or opinion of a subject or health care provider e.g., genetic test, enzyme or protein marker, family history, and the like. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
- the formulations of the present disclosure are administered to a mammal in need of treatment with the LMBR1L inhibitors disclosed herein, preferably a human, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
- the formulations are administered to the mammal by subcutaneous (i.e., beneath the skin) administration.
- the formulation may be injected using a syringe.
- other devices for administration of the formulation are available such as injection devices (e.g., the INJECT-EASETM and GENJECTTM devices); injector pens (such as the GENPENTM); auto-injector devices, needleless devices (e.g.,
- kits for a single dose- administration unit comprise a container of an aqueous formulation of therapeutic protein or antibody, including both single or multi-chambered pre-filled syringes.
- exemplary pre-filled syringes are available from Verier GmbH, Ravensburg, Germany.
- the appropriate dosage (“therapeutically effective amount”) of the protein will depend, for example, on the condition to be treated, the severity and course of the condition, whether the protein is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to LMBR1L inhibitors, the format of the formulation used, and the discretion of the ahending physician.
- the LMBR1L inhibitor is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards.
- the LMBR1L inhibitor may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
- an initial candidate dosage can range from about 0.1-20 mg/kg for administration to the patient, which can take the form of one or more separate administrations.
- other dosage regimens may be useful. The progress of such therapy is easily monitored by conventional techniques.
- LMBR1L inhibitor (or a pharmaceutical composition comprising a combination of LMBR1L inhibitor and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course.
- the methods according to this aspect of the disclosure comprise sequentially administering to a subject multiple doses of an LMBR1L inhibitor such as anti-LMBRlL antibody of the disclosure.
- sequentially administering means that each dose of LMBR1L inhibitor is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
- the present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of an LMBR1L inhibitor, followed by one or more secondary doses of the LMBR1L inhibitor, and optionally followed by one or more tertiary doses of the LMBR1L inhibitor.
- the LMBR1L inhibitor may be administered at a dose of between 0.1 mg/kg to about 100 mg/kg.
- the terms “initial dose,” “secondary doses,” and “tertiary doses” refer to the temporal sequence of administration of the LMBR1L inhibitor of the disclosure.
- the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”);
- the “secondary doses” are the doses which are administered after the initial dose; and
- the “tertiary doses” are the doses which are administered after the secondary doses.
- the initial, secondary, and tertiary doses may all contain the same amount of LMBR1L inhibitor, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of LMBR1L inhibitor contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").
- each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1 1 ⁇ 2, 2, 21 ⁇ 2, 3, 3 1 ⁇ 2, 4, 41 ⁇ 2, 5, 51 ⁇ 2, 6, 61 ⁇ 2, 7, 71 ⁇ 2, 8, 81 ⁇ 2,
- the immediately preceding dose means, in a sequence of multiple administrations, the dose of LMBR1L inhibitor which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
- the methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of an LMBR1L inhibitor.
- any number of secondary and/or tertiary doses of an LMBR1L inhibitor may comprise administering to a patient any number of secondary and/or tertiary doses of an LMBR1L inhibitor.
- only a single secondary dose is administered to the patient.
- two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient.
- two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.
- each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose.
- the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
- the present disclosure includes administration regimens in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by administration of two or more maintenance doses to the patient on a less frequent basis.
- a first frequency e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.
- the maintenance doses may be administered to the patient once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, etc.).
- compositions disclosed herein have numerous therapeutic utilities, including, e.g., the treatment of conditions or diseases where the immune system displays an excessive or overactive response.
- the present disclosure provides, inter alia, methods for reducing or suppressing an immune response in a subject with conditions in which the immune system is excessive or overactive, such as an inflammatory disease, autoimmune disease, graft versus host disease, or an allograft rejection.
- Exemplary methods comprise administering to the subject a therapeutically effective amount of any of the LMBR1L inhibitors described herein to provide, e.g., an immunosuppressive therapy.
- immunosuppressive therapy examples include allo-immune diseases, auto-immune diseases, allergy, and other inflammatory diseases.
- Allo-immune diseases include organ transplant rejection, graft versus host disease (GVHD) (e.g., post allogeneic hematopoietic stem cell transplant, HSCT) and GVHD post allogeneic stem cell transplantation (SCT).
- GVHD graft versus host disease
- HSCT post allogeneic hematopoietic stem cell transplant
- SCT GVHD post allogeneic stem cell transplantation
- Autoimmune diseases are diseases in which the immune system attacks its own proteins, cells, and tissues.
- autoimmune diseases include Type 1 diabetes, Multiple Sclerosis, coeliac disease, lupus erythematosus, systemic lupus erythematosus (SLE), Sjogren's syndrome, Churg-Strauss Syndrome, Hashimoto’s thyroiditis, Graves’ disease, idiopathic thrombocytopenic purpura, rheumatoid arthritis (RA), ankylosing spondylitis, Crohn’s disease, dermatomyositis, Goodpasture's syndrome, Guillain-Barre syndrome (GBS), mixed Connective tissue disease, myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anaemia, psoriasis,
- Inflammatory diseases can be used to broadly define a vast array of disorders and conditions that are characterized by inflammation. Examples include allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, rheumatoid arthritis, lupus, preperfusion injury, transplant rejection, Addison’s disease, alopecia areata, dystrophic epidermolysis bullosa, epididymitis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis, among others.
- IBD Inflammatory Bowel Disease
- CD Crohn's Disease
- Example 1 LMBR1L regulates lymphopoiesis through Wnt/p-catenin signaling Abstract: Precise control of Wnt signaling is necessary for immune system development. Here we detected severely impaired development of all lymphoid lineages in mice resulting from a N- ethyl-iV-nitrosourea-induced mutation in limb region 1 like (l.mhrll). encoding a membrane- spanning protein with no previously described function in immunity.
- LMBR1L Interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain containing 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing destruction complex proteins.
- LMBRlL-deficient T cells exhibited hallmarks of Wnt/ -catenin activation and underwent apoptotic cell death in response to proliferative stimuli.
- LMBR1L has an essential function during lymphopoiesis and lymphoid activation, acting as a negative regulator of the Wnt/b- catenin pathway.
- the hematopoietic system consists of many cell types with specialized functions. Blood cells, derived from either the lymphoid or the myeloid lineage, are generated from hematopoietic stem cells (HSCs). HSCs continuously replenish all blood cell classes through a series of lineage-restricted steps and balance these mechanisms to maintain steady-state hematopoiesis throughout the lifetime of the organism. In the last two decades, canonical Wnt signaling (also known as Wnt/ b -catenin signaling) and non-canonical Wnt signaling (e.g.
- Wnt proteins function as growth-promoting factors but also affect cell-fate decisions including apoptosis and quiescence (5).
- Aberrant activation of the Wnt/ -catenin pathway in T cell lineages by deletion of adenomatous polyposis coli (Ape) causes T cell lymphopenia as a result of spontaneous activation and apoptosis of mature T cells in the periphery (6).
- Loss of ZNRF3 and RNF43 expression is predicted to result in hyper-responsiveness to Wnt stimulation, and mutations in ZNRF3 and RNF43 have been observed in a variety of cancers in humans (7, 9).
- negative feedback regulators that specifically control lymphopoiesis remain unknown.
- LMBR1L function and mechanism of action of LMBR1L in the negative regulation of Wnt signaling in lymphocytes.
- mice carrying A-ethyl-A-nitrosourea (ENU)-induced mutations We identified several mice descended from a common ENU-treated founder with low percentages of CD3 + T cells in the peripheral blood (inset in Fig. 1A).
- the phenotype which we called strawberry ( t ) was transmitted as a recessive trait.
- single-pedigree mapping a method that analyzes genotype versus phenotype associations from a pedigree (11), the strawberry phenotype correlated with mutations in Lmbrll and Cers5 (Fig. 1A).
- Lmbrll encodes limb region 1 like (LMBR1L), a transmembrane protein of unknown function in immunity, and Cers5 encodes ceramide synthase 5 (CERS5), an enzyme in ceramide synthesis.
- CERS5 ceramide synthase 5
- the Lmbrll mutation in strawberry mice results in the substitution of cysteine 212 with a premature stop (C212*) in the fifth transmembrane helix of LMBR1L (Fig. 1B). This mutation was considered a putative null allele.
- CRISPR/Cas9-targeted knockout mutations of both Cers5 and Lmbrll were generated, confirming that the mutation in Lmbrll was solely responsible for the observed phenotype (Fig. 1C).
- the CD4 + -to-CD8 + T cell ratio was increased in Lmbrll and strawberry mice (Figs. 1D, 10B).
- the B cell-to-T cell ratio was also increased (Figs. 1G, 10E).
- DN double-negative
- SP single-positive
- the antigen-specific cytotoxic T lymphocyte (CTL) killing activity in immunized Lmbrlt or strawberry mice was also decreased compared to wild-type littermates (Figs. 10, 10M).
- the antigen-specific CD8 + T cell response to immunization with aluminum hydroxide precipitated ovalbumin (OVA) was weaker in Lmbrlt mice compared to wild-type mice, as indicated by reduced total numbers (Fig.
- Figs. 12A-12C frequencies of K b /SIINFEKL-tetramer-positive CD8 + T cells
- Figs. 12A-12C frequencies of K b /SIINFEKL-tetramer-positive CD8 + T cells
- the frequencies and numbers of natural killer (NK; Figs. 1M, 10K, 111) and NKl.l + T cells (Figs. 1N, 10L, 11J) were reduced in Lmbrl or strawberry mice with a concomitant decrease in NK cell target killing (Figs. 1Q, 10N).
- the Lmbrl mice displayed susceptibility to mouse cytomegalovirus (MCMV) as determined by elevated viral titers in the liver (Fig. 1R) after challenge with a sublethal dose of MCMV.
- MCMV mouse cytomegalovirus
- Lmbrll mRNA was detected in a variety of mouse tissues and immune cells, with higher expression in the bone marrow, thymus, spleen, and lymphocytes (Figs. 13 A and 13B).
- LMBR1L deficiency had no effect on myeloid cell development (Figs. 11N and 110) or their function as determined by IFN-a, IE-1b, and TNF-a secretion in response to various stimuli (Fig. 13C-13J).
- LMBR1L is essential for lymphopoiesis.
- immature B cells were increased among repopulated B cells derived from strawberry donors compared to those from wild-type donors (B220 + IgM + IgD ; Figs. 2D, 2J), and very few of the B cells from strawberry donors progressed to the mature recirculating B cell stage (B220 + IgM + IgD + ; Figs. 2D, 2K).
- This developmental arrest occurred in both irradiated wild-type and Rag h recipients regardless of competition.
- Lmbrll mutations also impair B cell development.
- Lymphocytes including B, T, and NK cells originate from lymphoid-primed multipotent progenitors (LMPPs) and common lymphoid progenitors (CLPs), which are thought to develop from LMPPs. Therefore, we examined the hematopoietic stem and progenitor cell populations in the bone marrow. LMBR1L deficiency caused an increase in the proportion and numbers of LSK + cells compared to wild-type littermates (Figs. 2L, 2M). The composition of the LSK compartment was mildly altered in Lmbrll bone marrow, resulting in a reduction in the proportion of LMPPs and CLPs (Fig. 2L).
- LT-HSCs long-term-hematopoietic stem cells
- ST short-term-HSCs
- MPPs multipotent progenitors
- CMPs common myeloid progenitors
- MEPs megakaryocyte-erythrocyte progenitors
- GFPs granulocyte-macrophage progenitors
- Lmbrll -derived hematopoietic cells were at an advantage in repopulating LSKs, ST-HSCs, MPPs, CMPs, and MEPs, while showing a disadvantage in repopulating LMPPs, CLPs, and GMPs (Figs. 14A-14B).
- the observed HSC phenotype in Lmbrir mice corresponds to the HSC phenotype when Wnt signaling is modestly increased in mice carrying hypomorphic Ape mutations (12). This suggests a specific effect of LMBR1L deficiency on lymphoid lineage commitment that is cell-autonomous.
- T cell factor-l T cell factor-l
- LEF- 1 lymphoid enhancer-binding factor 1
- Akt mitogen-activated protein kinase
- p70S6K a mTORCl substrate
- ribosomal protein S6 a p70S6K substrate
- extrinsic death receptors and caspase-dependent apoptosis the balance between the expansion of activated (effector) T cells and their subsequent elimination during the termination of an immune response is controlled by extrinsic death receptors and caspase-dependent apoptosis, intrinsic mitochondria- and caspase- dependent apoptosis, or caspase-independent cell death.
- extrinsic death receptors such as tumor necrosis factor (TNF)-a or Fas ligand (FasL) enhanced proteolytic processing of caspases of the extrinsic apoptotic pathway (e.g., caspase-8, -3, -7 and PARP).
- LMBR1L was first identified as a receptor for human lipocalin-l (LCN1), an extracellular scavenger/carrier of lipophilic compounds that mediates ligand internalization and degradation (14-18). Later findings suggested that LMBR1L mediates internalization of bovine lipocalin b-lactoglobulin (BLG) (19), a major food-home allergen in humans, and that LMBR1L interacts with uteroglobin (UG), which has anti-chemotactic properties (20).
- BLG bovine lipocalin b-lactoglobulin
- UG uteroglobin
- mice carrying a targeted null allele of Lcn3 the mouse orthologue of human LCN1
- LCN3-deficient mice were overtly normal and did not exhibit lymphocyte development defects.
- the function of LMBR1L in lymphopoiesis is independent of its interaction with LCN3 (Figs. 18A-18C).
- LMBR1L interacting proteins identified by co-immunoprecipitation (IP) combined with mass spectrometry (MS) analysis which were increased more than 50 fold or Wnt components exclusively present in LMBR1L co-IP product relative to empty vector control.
- IP co-immunoprecipitation
- MS mass spectrometry
- ubiquitin associated domain containing 2 (UBAC2; elevated 297-fold), transitional endoplasmic reticulum ATPase (TERA known as VCP; elevated l20-fold), UBX domain-containing protein 8 (UBXD8, known as FAF2; elevated 7l-fold) (21), and glycoprotein 78 (GP78; known as
- LMBR1L showed binding affinity for casein kinase 1 (CK1) isoforms including CKla, g, d, and e, as well as for b-catenin.
- CK1L casein kinase 1
- HEK293T cells were co-transfected with HA-tagged LMBR1L and FLAG-tagged OdK-3b, b-catenin, ZNRF3, ring finger 43 (RNF43, a homologue of ZNRF3 with redundant function in Wnt receptor processing), FZD6, LRP6, or DVL2.
- LMBR1L co-immunoprecipitated with each of the FLAG- tagged proteins (Fig.
- LMBR1L may be a critical component of the Wnt ⁇ -catenin and ERAD signaling pathways.
- Wnt ⁇ -catenin signaling was examined in CD8+ T cells from Lmbrll -/- and wild-type mice.
- a key regulatory step in the Wnt ⁇ -catenin signaling pathway involves the phosphorylation, ubiquitination, and subsequent degradation of the Wnt downstream effector protein, b-catenin (22).
- LMBR1L deficiency resulted in b-catenin accumulation with concomitant decreased levels of phosphorylated ⁇ -catenin relative to those in wild-type cells (Fig. 4C).
- the b-catenin accumulation was observed in developing thymocytes (DN1-4, DP, SP4, SP8; Figs.
- Tonic b-catenin inactivation requires phosphorylation of b-catenin by GSK-Sa/b and CK1 within an intact destruction complex composed of scaffolding proteins Axinl and DVL2, followed by ubiquitination mediated by E3 ubiquitin ligase b-TrCP (5, 22).
- Lmbrll-/- CD8+ T cells showed decreased total GSK-301/b and CK1 levels with concomitant increased levels of the inactive form of ⁇ 8K-3b (phosphorylated-GSK ⁇ ; Fig. 4C). Additionally, Axinl, DVL2, and b-TrCP levels were reduced in Lmbrll-/- CD8+ T cells compared to wild-type cells (Fig. 4C).
- b-catenin nuclear accumulation of b-catenin upon Wnt activation facilitates upregulation of its target genes, including CD44 and c-Myc. Consistent with the increased b-catenin levels in the nuclear fraction of Lmbrll-/- CD8+ T cells, we found increased c-Myc expression in total cell lysates (Fig. 4C). c-Myc-induced apoptosis is p53-dependent. The anti-apoptotic cell cycle arrest protein p2l is a target of p53, and is transcriptionally repressed by c-Myc (23). LMBR1L deficiency increased p53 expression, suppressed p2l, and increased caspase-3 and -9 cleavage (Fig. 4C). LMBR1L deficiency produced similar effects in CD4+ T and B cells (Figs. 21A- 21B).
- the LMBR1L-GP78-UBAC2 complex regulates the maturation of Wnt receptors within the ER and stabilizes GSK-3P
- ZNRF3 and RNF43 are negative regulators of the Wnt pathway.
- ZNRF3 and RNF43 selectively ubiquitinate lysines in the cytoplasmic loops of FZD, which targets FZD for degradation at the plasma membrane (8).
- DVL proteins act as an intermediary for ZNRF3/RNF43-mediated ubiquitination and degradation of FZD (10).
- ZNRF3/RNF43 levels were altered in the membrane fraction of Lmbrl-/- CD8+ T cells compared to levels in wild-type cells. In the TCLs, ZNRF3 levels were unchanged, whereas RNF43 levels were slightly increased (Fig. 5A).
- UBAC2 is a core component of the GP78 ubiquitin ligase complex expressed on the ER membrane. UBAC2 physically interacts with and adds poly-UB chains to UBXD8, a protein involved in substrate extraction during ERAD (21, 25). We hypothesized that the interaction of LMBR1L with UBAC2, GP78, and UBXD8 might regulate the activity of the GP78 ubiquitin ligase complex towards FZD and/or LRP6. Transient co-transfection of HEK293T cells with FLAG-tagged FZD6 and HA-tagged LMBR1L or UBAC2 resulted in decreased total levels of the mature FZD6 (Fig. 5B).
- LMBR1L for which no functional domain has previously been reported, is known to localize at the plasma membrane (17, 18).
- LMBR1L may function as a core component of the GP78- UBAC2 ubiquitin ligase complex, and that LMBRlL-mediated maturation of Wnt co-receptors may be regulated within the ER.
- Lmbrll-/- T cells showed the decreased expression of phosphorylated-b- catenin and phosphorylated-LRP6 (Fig. 4C and Fig. 5A, respectively), increased phosphorylated-GSK ⁇ (Fig. 4C), and the activation of kinases such as Akt and p70S6K (Fig. 3B) which inactivates ⁇ 8K-3b by phosphorylation.
- the LMBR1L- GP78-UBAC2 complex may regulate the stability of destruction complex components such as GSK-3 , which has both inhibitory and stimulatory roles in Wnt ⁇ -catenin signaling by phosphorylating b-catenin and LRP6, respectively (26).
- Transient co-transfection of HEK293T cells with FLAG-tagged Axinl, DVL2, or GSK-3 and HA-tagged LMBR1L or empty vector resulted in decreased total levels of FLAG-tagged Axinl protein in the presence of HA- LMBR1L compared to empty vector control.
- LMBR1L had no effect on DVL2 or GSK-3 expression (Fig.
- HEK293T cells were transfected with FLAG- tagged GSK-3 and HA-tagged LMBR1L or empty vector. Fourteen hours after transfection, cells were treated with the translation inhibitor cycloheximide (CHX) and harvested at various times post-treatment. In the presence of LMBR1L, no detectable decrease of GSK-3 was observed up to 4 h after CHX treatment, suggesting that LMBR1L stabilizes GSK-3 (Fig. 6B).
- CHX translation inhibitor cycloheximide
- LMBR1L serves as a negative regulator of Wnt/b- catenin signaling.
- Ctnnbl Lmbrll, b-catenin
- EL4 cells Similar to the phenotype observed in primary Lmbrll-/- CD8+ T cells, Lmbrll-/- EL4 cells showed severe defects in proliferation even under normal culture conditions (Fig. 7A). Annexin V and PI staining showed that the majority of the Lmbrll-/- EL4 cells were apoptotic (Fig. 7B: top right, 7C).
- LMBR1L deficiency inhibits autoantibody production and B cell survival in mice
- LMBR1L LMBR1L
- GP78 GP78
- UBAC2 Wnt receptor availability independent of ligand binding
- LMBR1L supports the expression and/or stabilization of the canonical destruction complex including GSK-3 that is necessary for degradation of b-catenin and activation of LRP6.
- human and mouse LMBR1L orthologues share 96% identity (Fig. 29), we believe the same mechanism operates in human lymphoid cells and their progenitors.
- LMBR1L deficiency may be considered as a possible etiology in unexplained pan-lymphoid immunodeficiency disorders.
- Fas lpr , B2m tmlUnc (B2m ) and Tg(TcraTcrb) 1 lOOMjb (OT-I) transgenic mice were purchased from The Jackson Laboratory.
- mice were lethally irradiated with 13 Gy via gamma radiation (X-RAD 320, Precision X-ray Inc.). The mice were given an intravenous injection of 5 c 10 6 bone marrow cells derived from the tibia and femurs of the respective donors. For 4 weeks post-engraftment, mice were maintained on antibiotics. Twelve weeks after bone marrow engraftment, the chimeras were euthanized to assess immune cell development in bone marrow, thymus, and spleen by flow cytometry. Chimerism was assessed using congenic CD45 markers.
- Bone marrow cells, thymocytes, splenocytes, or peripheral blood cells were isolated, and red blood cell (RBC) lysis buffer was added to remove RBCs.
- RBC red blood cell
- Cells were stained at a 1:200 dilution with 15 mouse fluorochrome-conjugated monoclonal antibodies specific for the following murine cell surface markers encompassing the major immune lineages: B220, CD3s, CD4, CD5, CD 8 a, CDl lb, CDl lc, CD19, CD43, CD44, CD62L, F4/80, IgD, IgM, and NK1.1 in the presence of anti-mouse CD 16/32 antibody for 1 h at 4 °C. After staining, cells were washed twice in PBS and analyzed by flow cytometry.
- bone marrow was isolated and stained with Alexa Fluor 700-conjugated lineage markers (B220, CD3, CDl lb, Ly-6G/6C, and Ter-l l9), CD16/32, CD34, CD135, c-Kit, IL-7Ra, and Sca-l for 1 h at 4 °C. After staining, cells were washed twice in PBS and analyzed by flow cytometry.
- PE-conjugated K b /SIINFEKL tetramer a reagent specific for the ovalbumin epitope peptide SIINFEKL presented by H-2K b (MHC Tetramer Core at Baylor College of Medicine) was used to detect antigen-specific CD8 + T cell responses and memory CD8 + T cell formation in mice immunized with aluminum-hydroxide precipitated ovalbumin.
- thymi were homogenized to generate a single-cell suspension and surface stained for CD3, CD25, and CD44. Cells were then permeabilized using BD Cytofix/Cytoperm Kit followed by intracellular b-catenin staining. Data were acquired on an LSRFortessa cell analyzer (BD Bioscience) and analyzed with FlowJo software (Treestar).
- mice were immunized with another TD antigen, rSFV ⁇ Gal (2 c 10 6 IU; (28)) on day 0 and the T cell- independent antigen (TI) NPso-AECM-Ficoll (50 pg; Biosearch Technologies) on day 8 (i.p.) as previously described (29).
- TI T cell- independent antigen
- Cytolytic CD8 + T cell effector function was determined by a standard in vivo cytotoxic T lymphocyte (CTL) assay. Briefly, splenocytes were isolated from naive mice and divided in half. According to established methods (30), half were stained with 5 pM CFSE (CFSE hi ), and half were labeled with 0.5 pM CFSE (CFSE 10 ). CFSE 1 " cells were pulsed with 5 pM ICPMYARV peptide, which carries E. coli b-galactosidase MHC I epitope for mice with the H- 2 b haplotype (New England Peptide; (31). CFSE 10 cells were not stimulated.
- CTL cytotoxic T lymphocyte
- splenocytes from control C57BL/6J 0.5 pM Violet; Violet 10
- B2m mice 5 pM Violet; Violet hl
- Equal numbers of Violet 11 and Violet 10 cells were transferred by retro-orbital injection. Twenty- four hours after transfer, blood was collected and Violet intensity from each population was assessed by flow cytometry.
- % lysis [1 - (target cells/control cells) / (target cells/control cells 100.
- mice were infected with MCMV (Smith strain; 1.5 c 10 5 pfu/20 g of body weight) by intraperitoneal injection as described previously (32). Mice were euthanized 5 days after MCMV challenge to determine viral loads. Total DNA extracted from individual mouse spleen was used to measure copy numbers of MCMV immediate-early 1 (IE1) gene and control DNA sequence (b-actin). The viral titer is represented as the copy number ratio of MCMV IE1 to b-actin.
- IE1 immediate-early 1
- b-actin control DNA sequence
- Splenic CD45.2 + OT-I and Lm.br I ; ;OT-I T cells were purified using the EasySepTM Mouse CD8 + T Cell Isolation Kit (StemCell Technologies). Purities were over 95% in all experiments as tested by flow cytometry. Cells were labeled with 5 mM CellTrace Far Red (CD45.2 + OT-I) or 5 pM CellTrace Violet (l.mbrl 1 ;OT-I). and equal number of stained cells (2 x 10 6 ) cells were injected by retro-orbital route into wild-type CD45. l + mice.
- OVA Antigen-specific T cell activation was analyzed based on Far Red or Violet intensity of dividing OT-I cells after 48 h and 72 h.
- splenic pan T cells or mature SP thymocytes were isolated by using the EasySepTM Mouse Pan T Cell Isolation Kit (StemCell Technologies) or Dynal negative selection using biotinylated anti-CD24 mAh Ml/69 (eBioscience), respectively.
- Pan T or mature CD24 thymocytes isolated from Lmbrll , Lmbrll st/st or wild-type littermates were stained with 5 pM CellTrace Violet or CellTrace Far Red, respectively.
- a 1 : 1 or 10: 1 mix of labeled Lmbrll or wild-type cells was transferred into C45.
- mice that had been sublethally irradiated (8 Gy) 6 h earlier or into unirradiated controls.
- splenocytes were prepared, surface stained for CD45.1, CD45.2, together with CD3, CD4, and CD8 and then analyzed by flow cytometry for Far Red or Violet dye dilution.
- Annexin V/PI labeling and detection was performed with the FITC-Annexin V Apoptosis Detection Kit I (BD Bioscience) according to manufacturer’s instructions. Mass spectrometry analysis
- Co-immunoprecipitation and mass spectrometry were performed to identify Lmbrll- interacting proteins as described below.
- Transfection was performed in HEK293T cells (ATCC) using Lipofectamine 2000 reagent (Life technologies) with plasmid encoding Flag tagged- human Lmbrll or empty vector control. Forty-eight hours after transfection, cells were harvested in NP-40 lysis buffer for 45 min at 4 °C.
- Immunoprecipitation was performed using anti-FLAG M2 affinity gel (Sigma) for 2 h at 4 °C and beads were washed six times in NP-40 lysis buffer. The proteins were eluted with SDS sample buffer and heated at 95 °C for 10 min.
- Lysates were loaded onto 12% (wt/vol) SDS-PAGE gel and run ⁇ l cm into the separation gel. The gel was stained with Coomassie blue (Thermo Fisher) and whole stained lanes were subjected to mass spectrometry analysis (LC-MS/MS) as described previously (33).
- the Proto Array Human Protein Microarray V5.1 (Invitrogen) was used to identify human Lmbrll-interacting proteins according to manufacturer' s instructions. Briefly, Flag (N- terminus)- and V5 (C-terminus)-tagged recombinant human Lmbrll protein was expressed in HEK293T cells by transfection and purified with anti-FLAG M2 affinity gel (Sigma). The presence of the Flag and V5 tag on the protein was confirmed by standard immunoblot.
- Proteins from the plasma membrane or endoplasmic reticulum were isolated using the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher) or Endoplasmic Reticulum Enrichment Extraction Kit (Novus Biologicals), respectively, according to manufacturer’s instructions.
- Tumour suppressor RNF43 is a stem-cell E3 ligase that induces
- LMBR1L EST libraries determined that three (NP_083374.l, XP_011244060.1, and XP_0l 7172262.1) and fifteen (NP_060583.2, NP_00l287679. l, NP_00l287680. l, NP_00l 339090.1, NP_00l33909l.
- Human NP_060583.2 and murine NP 083374.1 as canonical LMBR1L proteins are 489-residue long with 9 transmembrane domains.
- Human LMBR1L protein has 97% identity to the murine LMBR1L protein (Fig. 20).
- LMBR1L The five extracellular domains of canonical human LMBR1L are marked in Fig. 1B. Based on this analysis, there are five targetable extracellular peptide sequences that are candidates for the anti-human LMBR1L inhibitors such as LMBR1L antibodies.
- a portion of any one or more of the five extracellular domains can also be used as an immunogen.
- Different methods known in the art, and those that have been disclosed herein, may be used to generate monoclonal, fully human or humanized anti-LMBRlL antibodies.
- fully human LMBR1L antibodies can also be produced from phage-display libraries.
- Humanized anti-LMBRlL antibodies can be prepared by humanizing monoclonal antibodies obtained from hybridomas.
- An exemplary approach can include:
- the LMBR1L protein is strongly conserved between humans and mice (Fig. 20). Use of phage display is likely to produce a reagent reactive with both species, useful in preclinical and clinical testing.
- chromatography can be added to the immunogen.
- Purified protein can be inoculated into mice together with a suitable adjuvant.
- Monoclonal antibodies produced in hybridomas can be tested for binding to the immunogen, and positive binders can be screened (e.g., decreased T cell- dependent and T-cell independent antibody responses, decreased T cells, B cells, NK cells and NK T cells) for ability to affect b-catenin, FRIZZLED-6, ZNRF3, and/or c-Myc expression in human lymphoid cells in the assays described above. Thereafter, antibodies can be humanized for preclinical and clinical studies.
- LMBR1L should be accessible to inhibition by an antibody. This would be reasonably expected to mimic the effects of the mutation.
- An antibody inhibitor of LMBR1L could be used to arrest, for example, graft-versus-host disease, allograft rejection, or autoimmune diseases, including (but not limited to) systemic lupus erythematosus,
- Hashimoto s thyroiditis
- Grave s disease
- type I diabetes a malignant neoplasm
- multiple sclerosis a malignant neoplasm
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AU2019295729A1 (en) | 2021-01-21 |
CN113226354A (en) | 2021-08-06 |
IL279687A (en) | 2021-03-01 |
KR20210038553A (en) | 2021-04-07 |
EP3814523A1 (en) | 2021-05-05 |
JP2021530457A (en) | 2021-11-11 |
US20210269519A1 (en) | 2021-09-02 |
CA3105595A1 (en) | 2020-01-02 |
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