阅读说明：本技术 Cd24用于预防和治疗移植物抗宿主病和粘膜炎的使用方法 (Method of use of CD24 for the prevention and treatment of graft versus host disease and mucositis ) 是由 Y.刘 P.郑 M.德文波特 于 2019-06-03 设计创作，主要内容包括：本发明涉及CD24蛋白在预防或治疗移植物抗宿主病和粘膜炎中的用途。(The invention relates to application of CD24 protein in preventing or treating graft-versus-host disease and mucositis.)

1. A method of treating or preventing Graft Versus Host Disease (GVHD) in a subject in need thereof comprising administering to the subject a CD24 protein.

2. The method of claim 1, wherein said method reduces the risk of grade III-IV acute GVHD in said subject.

3. A method of treating or preventing mucositis in a subject in need thereof comprising administering to said subject a CD24 protein.

4. The method of any one of claims 1-3, wherein the subject has cancer.

5. The method of claim 4, wherein the cancer is Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Myelogenous Leukemia (CML), myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML).

6. The method of any one of claims 1-5, wherein the CD24 protein is administered at a dose of 240mg or 480 mg.

7. The method of any one of claims 1-6, wherein the subject will undergo or has undergone hematopoietic stem cell transplantation (HCT).

8. The method of claim 7, wherein said CD24 protein is administered before or after said HCT.

9. The method of claim 8, wherein said CD24 protein is administered the day before said HCT.

10. The method of claim 8 or 9, wherein the CD24 protein is administered more than once.

11. The method of claim 10, wherein said CD24 protein is administered in a once-three-two-week dose comprising a dose one day before said HCT, a dose 14 days after said HCT, and a dose 28 days after said HCT.

12. The method of claim 11, wherein the dose of CD24 protein is 480mg, 240mg, and 240mg, respectively.

13. The method of any one of claims 1-12, wherein said CD24 protein comprises a mature human CD24 polypeptide fused at its N-or C-terminus to the Fc region of a mammalian immunoglobulin (Ig) protein.

14. The method of claim 13, wherein the mature human CD24 polypeptide comprises the sequence set forth in SEQ ID No. 1 or 2.

15. The method of claim 14, wherein the Ig protein is human, and wherein the Fc region comprises a hinge region and CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, or IgA.

16. The method of claim 14, wherein the Ig protein is human, and wherein the Fc region comprises a hinge region and CH2, CH3, and CH4 domains of IgM.

17. The method of claim 15, wherein said CD24 protein comprises the sequence set forth in SEQ ID NO 6, 11, or 12.

18. The method of claim 17, wherein the amino acid sequence of said CD24 protein consists of the sequence set forth in SEQ ID No. 6, 11, or 12.

19. The method of any one of claims 1-18, wherein the CD24 protein is soluble.

20. The method of any one of claims 1-19, wherein the CD24 protein is glycosylated.

21. The method of any one of claims 1-20, wherein the CD24 protein is produced using a eukaryotic expression system.

22. The method of claim 21, wherein the eukaryotic expression system comprises expression from a vector in a mammalian cell.

23. The method of claim 22, wherein the mammalian cell is a chinese hamster ovary cell.

Use of a CD24 protein in the manufacture of a medicament for treating or preventing Graft Versus Host Disease (GVHD) in a subject.

25. The use of claim 24, wherein said use reduces the risk of grade III-IV acute GVHD in said subject.

Use of a CD24 protein in the manufacture of a medicament for treating or preventing mucositis in a subject.

27. The use of any one of claims 24-26, wherein the subject has cancer.

28. The use of claim 27, wherein the cancer is Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Myelogenous Leukemia (CML), myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML).

29. The use of any one of claims 24-28, wherein the CD24 protein is administered at a dose of 240mg or 480 mg.

30. The use of any one of claims 24-29, wherein the subject will undergo or has undergone hematopoietic stem cell transplantation (HCT).

31. The use of claim 30, wherein said CD24 protein is administered before or after said HCT.

32. The use of claim 31, wherein said CD24 protein is administered the day before said HCT.

33. The use of claim 31 or 32, wherein the CD24 protein is administered more than once.

34. The use of claim 33, wherein said CD24 protein is administered in a once-three-two-week dose comprising a dose one day before said HCT, a dose 14 days after said HCT, and a dose 28 days after said HCT.

35. The use of claim 34, wherein the dose of CD24 protein is 480mg, 240mg, and 240mg, respectively.

36. The use of any one of claims 24-35, wherein said CD24 protein comprises a mature human CD24 polypeptide fused at its N-or C-terminus to the Fc region of a mammalian immunoglobulin (Ig) protein.

37. The use of claim 36, wherein the mature human CD24 polypeptide comprises the sequence set forth in SEQ ID No. 1 or 2.

38. The use of claim 37, wherein the Ig protein is human, and wherein the Fc region comprises a hinge region and CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, or IgA.

39. The use of claim 37, wherein the Ig protein is human, and wherein the Fc region comprises a hinge region and CH2, CH3, and CH4 domains of IgM.

40. The use of claim 38, wherein said CD24 protein comprises the sequence set forth in SEQ ID NO 6, 11, or 12.

41. The use of claim 40, wherein the amino acid sequence of said CD24 protein consists of the sequence shown in SEQ ID NO 6, 11 or 12.

42. The use of any one of claims 24-41, wherein the CD24 protein is soluble.

43. The use of any one of claims 24-42, wherein the CD24 protein is glycosylated.

44. The use of any one of claims 24-43, wherein the CD24 protein is produced using a eukaryotic expression system.

45. The use of claim 44, wherein said eukaryotic expression system comprises expression from a vector in a mammalian cell.

46. The use of claim 45, wherein the mammalian cell is a Chinese hamster ovary cell.

Technical Field

The present invention relates to compositions and methods for the prevention and treatment of graft versus host disease and mucositis.

Background

Allogeneic hematopoietic stem cell transplantation (HCT) is the only established cure for a wide range of high-risk leukemias and myelodysplasias in adults. An important function of allogeneic transplantation is to eliminate allogeneic leukemia cells using donor T cells, a function known as GVL. However, Graft Versus Host Disease (GVHD) is a life-threatening complication that occurs when immune competent cells from a donor stem cell graft immune attack the host. Activated donor T cells damage host epithelial cells following an inflammatory cascade initiated with a preparatory protocol. The exact risk depends on the source of the stem cells, the age, condition of the patient and the GVHD prevention used. The incidence is directly related to the degree of Human Leukocyte Antigen (HLA) differentiation. Median onset of acute GVHD is usually 21 to 25 days post-transplant. The incidence ranges from 30% to 65% in recipients of total histocompatibility-related donor grafts to 60% to 80% in recipients of mismatched hematopoietic cells or hematopoietic cells from unrelated donors. Cord blood transplantation is associated with slower recovery of neutrophils, lower morbidity and later onset of acute GVHD. Factors that increase morbidity include the use of peripheral blood rather than bone marrow as a source of hematopoietic cells and the age of the larger recipient. Median time for diagnosis of chronic GVHD was 4.5 months after HLA sibling transplantation and 4 months after unrelated donor transplantation. After 2 years of allogeneic HCT development, new chronic GVHD never developed.

For more than twenty years, the combination of calcineurin inhibitors (e.g., cyclosporine and tacrolimus) with methotrexate (methotrexate) remains the standard of care for the prevention of GVHD. Despite routine administration of immunoprophylaxis, clinically significant GVHD (grade II-IV) occurs in about 30% to 65% of patients undergoing HLA-matched associated HCT and 60% to 80% of patients receiving unrelated donor HCT. Acute GVHD is an early event after HCT with a median time to onset of approximately 25 to 30 days. In patients with very severe GVHD, mortality rates exceed 90%. One explanation for this is that, once established, more than 50% of patients respond inefficiently to first-line treatment with high doses of corticosteroids. For patients who show steroid refractory or require long-term treatment, survival rates are significantly reduced. Even with success, high doses of corticosteroids are a major source of morbidity because infection is increased and deregulated (deregulation), putting patients at significant risk of TRM.

Host tissue damage caused by HCT conditioning (conditioning) protocols, including high dose chemotherapy and/or Total Body Irradiation (TBI), is considered the first step in the development of acute GVHD. Host tissue damage caused by regulatory regimes leads to the release of pro-inflammatory cytokines (e.g., TNF- α, IL-1 β, and IL-6), as well as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Both DAMPs and PAMPs can activate Antigen Presenting Cells (APCs), such as Dendritic Cells (DCs), by binding to Pattern Recognition Receptors (PRRs). The host APC subsequently activates donor T cells and the immune cascade, leading to the release of pro-inflammatory cytokines and the expansion of antigen-specific alloreactive T cells that target host tissues, thereby producing GVHD. Therefore, it is of great interest to explore whether GVHD can be alleviated by targeting the host's response to tissue damage and preventing activation of APC (a key process that initiates GVHD).

To date, treatment and prevention of GVHD has focused primarily on the pharmacological inhibition or depletion of T cells by in vivo or ex vivo approaches to inhibit the expansion of alloreactive T cells that mediate tissue damage. While non-selective T cell depletion strategies (e.g., anti-thymocyte globulin) are effective in preventing GVHD, they do not improve survival by offsetting the risk of relapse, infection, and graft rejection. In contrast, more selective inhibition by targeting a single pro-inflammatory cytokine has not shown clinical benefit for treatment of GVHD. As a result, no biologic has been approved for GVHD other than T cell depleting antibodies, and the combination of tacrolimus and methotrexate remains the standard of care for prevention of GVHD. There is an unmet significant medical need to provide more selective biological products for the prevention and treatment of GVHD.

Mucositis is a common and painful side effect of chemotherapy and radiotherapy to treat cancer, which results in inflammation and ulceration of the mucosa lining the digestive tract. Which is the result of tissue damage caused by radiation/Radiation Therapy (RT) or chemotherapy. Mucositis can occur anywhere along the Gastrointestinal (GI) tract, but oral mucositis refers to specific inflammation and ulceration occurring in the oral cavity. Oral and Gastrointestinal (GI) mucositis affects almost all patients receiving cytarabine and high dose 5-fluorouracil, alkylating agents and platinum-based compounds high dose chemotherapy, as well as most patients with head and neck malignancies receiving radiation therapy. Radiation-induced oral mucositis (RIOM) occurs in 100% of patients with head and neck cancer with varying fractionated radiation therapy. Patients with oral mucositis can experience severe pain, inflammation, ulceration and bleeding, which can severely impede the patient's ability to swallow, which can also limit tumor control due to discontinuation of cancer therapy. Oral mucositis is therefore an important adverse reaction seen with chemotherapy and/or radiotherapy of the head and neck in cancer patients. Mucositis of the digestive tract increases mortality and morbidity and leads to increased medical costs. In the united states, the rim economic cost for each patient with head and neck cancer is estimated to $ 17,000.00.

Mucositis burden is deepest due to regulatory regimens for hematopoietic stem cell transplantation (HCT). Treatment regimens for HCT include preconditioning regimens, including highly mucotoxic chemotherapy with or without systemic irradiation (TBI), which require killing of recipient cancerous hematopoietic cells prior to transplantation. This preconditioning treatment can result in severe damage to the entire digestive tract. Lower mucositis includes mucosal erythema and patchy ulcers or pseudomembranes (pseudomembranes). Severe mucositis (> grade 3) is accompanied by confluent ulcerations or pseudomembranes and bleeding from minor trauma, which may progress to tissue necrosis, significant spontaneous bleeding and life threatening consequences. Another indication associated with mucositis in patients receiving HCT is oral Graft Versus Host Disease (GVHD), a form of chronic GVHD. Like chemotherapy and radiation-induced mucositis, oral GVHD includes mucosal erythema, ulceration and painful, desquamating oral lesions. However, there is a lack of a true clinical case definition of oral acute GVHD, as several factors, especially the presence or absence of concurrent radiotherapy-mediated chemotherapy, contribute to the development of oral lesions during the first 28 days after HCT.

As noted, GVHD, mucositis and related indications all involve elements of tissue damage. To address these challenges, there is a need in the art for effective methods of treating and preventing GVHD and mucositis.

Summary of The Invention

Provided herein are methods of preventing or treating graft versus host disease (GvHD) or mucositis in a subject in need thereof, which can include administering CD24 protein to the subject. Also provided herein is the use of a CD24 protein in the manufacture of a medicament for preventing or treating GvHD or mucositis in a subject. The method or use may reduce the risk of a subject developing grade III-IV acute GvHD. The subject may be a human. The subject may have undergone or has undergone hematopoietic stem cell transplantation (HCT). The subject may have cancer, which may be Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Myelogenous Leukemia (CML), Myelodysplastic syndrome (MDS), or Chronic Myelomonocytic Leukemia (CMML).

The CD24 protein may be administered in a dose of 240mg or 480 mg. The CD24 protein may be administered before or after HCT, and may be administered the day before HCT. The CD24 protein may be administered more than once, and may be administered in a dose once every two weeks. The dose may include a dose one day before HCT, a dose 14 days after HTC, and a dose 28 days after HCT, and the doses may be 480mg, 240mg, and 240mg, respectively.

The CD24 protein may comprise a mature human CD24 polypeptide fused at its N-or C-terminus to the Fc region of a mammalian immunoglobulin (Ig) protein. The mature human CD24 polypeptide may comprise the sequence shown in SEQ ID NO 1 or 2. The Ig protein may be human. The Fc region may comprise a hinge region and CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, or IgA. The Fc region may comprise a hinge region and CH2, CH3, and CH4 domains of IgM. The CD24 protein may comprise the sequence shown in SEQ ID NO 6, 11 or 12. The amino acid sequence of the CD24 protein can consist of the sequence shown in SEQ ID NO 6, 11 or 12. The CD24 protein may be soluble and may be glycosylated. The CD24 protein can be prepared using eukaryotic expression systems, including expression from vectors in mammalian cells. The cell may be a chinese hamster ovary cell.

Brief Description of Drawings

FIG. 1A shows the amino acid composition of the full-length CD24 fusion protein, CD24Fc (also referred to herein as CD24Ig) (SEQ ID NO: 5). The underlined 26 amino acids are the signal peptide of CD24 (SEQ ID NO:4), which is excised from the cell expressing the protein during secretion, and thus deleted from the processed version of the protein (SEQ ID NO: 6). The bold part of the sequence is the extracellular domain of the mature CD24 protein (SEQ ID NO:2) for the fusion protein. To avoid immunogenicity, the last amino acid (a or V) normally present in the mature CD24 protein has been deleted from the construct. The non-underlined, non-bold letters are the sequence of IgG1Fc, including the hinge region and the CH1 and CH2 domains (SEQ ID NO: 7). FIG. 1B shows CD24^VFc (SEQ ID NO:8), wherein the mature human CD24 protein (bold) is a valine polymorphic variant of SEQ ID NO: 1. FIG. 1C shows CD24^AFc (SEQ ID NO:9), wherein the mature human CD24 protein (bold) is an alanine polymorphic variant of SEQ ID NO: 1. The various portions of the fusion proteins in FIGS. 1B and 1C are as indicated in FIG. 1A and the variant valine/alanine amino acids are double underlined.

FIG. 2 shows the amino acid sequence variation between mature CD24 proteins from mouse (SEQ ID NO:3) and human (SEQ ID NO: 2). Potential O-glycosylation sites are shown in bold, and N-glycosylation sites are underlined.

Pharmacokinetic WinNonlin compartment (comparative) model analysis of CD24IgG1(CD24 Fc). Open circles represent the average of 3 mice and the line is the predicted pharmacokinetic curve. Figure 3a.i.v. injection of 1mg CD24IgG 1. Figure 3b.s.c. injection of 1mg CD24IgG1(CD24 Fc). Figure 3c comparison of total amount of antibody in blood measured by area under the curve (AUC), half-life, and maximum blood concentration. Note that overall, the AUC and Cmax for the s.c injection were approximately 80% of the i.v. injection, although the differences were not statistically significant.

The interaction of CD24-Siglec G (10) can distinguish between PAMP and DAMP. FIG. 4A. host response to PAMP is not affected by CD24-Siglec G (10) interaction. FIG. 4B CD24-Siglec G (10) interaction may inhibit host response to DAMP by SHP-1 associated with Siglec G/10.

Fig. 5A-c.cd24 Fc binds to Siglec10 and HMGB1 and activates Siglec G: mouse homolog of human Siglec 10. Figure 5a. affinity measurement of cd24fc-Siglec 10 interaction. Figure 5b. cd24fc specifically interacts with HMGB-1 in a cation-dependent manner. CD24Fc was mixed with HMGB1 in the presence or absence of the cation chelator EDTA at 0.1mM CaCl₂And MgCl₂And (4) carrying out incubation. CD24Fc was pulled down together with protein G microbeads and the amount of HMGB1, CD24Fc or control Fc was determined by Western blot (Western blot). Cd24fc activates mouse siglecgs by inducing tyrosine phosphorylation (middle panel) and associating with SHP-1 (upper panel). The amount of Siglec G is shown in the lower graph. Stimulation of CD24 with 1. mu.g/ml of CD24Fc, control Fc or vehicle (PBS) control^-/-Splenocytes for 30 minutes. Siglec G was then immunoprecipitated and probed with anti-phosphotyrosine or anti-SHP-1.

FIG. 6A-B. CD24Fc inhibits the production of TNF- α and IFN- γ by human T cells activated by anti-CD 3. Human PBML was stimulated with anti-CD 3 for 4 days in the presence or absence of CD24Fc and the amounts of IFN-. gamma.and TNF-. alpha.released in the cell culture supernatants were measured by ELISA. Data shown are the average of three replicates. Error bars, SEM.

Cd24 inhibits the production of inflammatory cytokines by human macrophages. FIG. 7 A.silencing of ShRNA by CD24 results in spontaneous production of TNF- α, IL-1 β and IL-6. THP1 cells were transduced with lentiviral vectors encoding either a scrambled (scrambled) or two independent CD24 shRNA molecules. The transduced cells were differentiated into macrophages by culturing with PMA (15ng/ml) for 4 days. After washing away PMA and non-adherent cells, the cells were cultured for an additional 24 hours to measure inflammatory cytokines by cytokine bead array. Fig. 7b is the same as fig. 7A except that CD24Fc or control IgG Fc at a given concentration was added to the macrophages over the last 24 hours. The data shown in fig. 4A are the mean and s.d from three independent experiments, while those in fig. 4B represent at least 3 independent experiments.

Figure 8 shows a graph of mean plasma CD24Fc concentrations (+ -SD) in a population evaluable by treating PK in human subjects. PK ═ pharmacokinetics; SD-standard deviation.

FIG. 9 shows CD24Fc C for PK evaluable populations_maxDose proportion graph with respect to dose.

FIG. 10 shows the CD24Fc AUC for PK evaluable populations_0-42dDose proportion graph with respect to dose.

FIG. 11 shows the CD24Fc AUC for PK evaluable populations_0-infDose proportion graph with respect to dose.

Figure 12 shows a trial design for a randomized, placebo-controlled phase IIa dose escalation trial to assess the addition of CD24Fc in the standard of care for preventing acute GVHD in cancer patients receiving allogeneic myelosuppressive (myeloablative) hematopoietic stem cell transplantation (HCT).

Figure 13 shows the dosing schedule for the single and multiple dose groups in the phase IIa trial.

Figure 14 shows the median time to transplant for patients enrolled in the trial.

FIG. 15 shows the chimerism of bone marrow donors in patients enrolled in the trial.

Figure 16 shows the incidence of grade II-IV and grade III-IV acute GVHD in the treatment (CD24Fc) group.

Figure 17 shows the cumulative incidence of grade III-IV aGVHD 180 days post HCT in patients receiving methotrexate/tacrolimus + CD24Fc compared to contemporary control patients receiving methotrexate/tacrolimus.

FIG. 18 shows a Kaplan-Meier survival assay comparing 180 days of grade III-IV aGVHD without recurrence survival in patients receiving CD24Fc or placebo control.

FIG. 19 shows a Kaplan-Meier survival assay comparing 180 day grade III-IV aGVHD with no recurrence survival in patients receiving CD24Fc and contemporary controls.

FIG. 20 shows a Kaplan-Meier survival assay comparing overall survival for patients receiving CD24Fc or placebo control at 800 days post-transplantation.

FIG. 21 shows a Kaplan-Meier survival assay comparing the overall survival of patients receiving CD24Fc or contemporary controls at 800 days post-transplantation.

Fig. 22A-B show PK data from the 240 and 480mg single dose groups. Figure 22a is a graph of mean (standard deviation) plasma CD24Fc concentration (ng/mL) versus time on a linear scale. Figure 22b is a graph of mean (standard deviation) plasma CD24Fc concentration (ng/mL) versus time on a semilog scale.

Fig. 23A-B show PK data from multiple dose groups. Figure 23a is a graph of mean (standard deviation) plasma CD24Fc concentration (ng/mL) versus time on a linear scale. Figure 23b is a graph of mean (standard deviation) plasma CD24Fc concentration (ng/mL) versus time on a semilog scale.

FIGS. 24A-B show the effect of CD24Fc treatment on oral mucositis. Figure 24a. mucositis composite score. To measure the effect on mucositis, we generated a number of days with patients with severe (grade 3 or 4) mucositis, and the numbers are provided in bar graphs and the number of patients with mucositis is indicated in parentheses. Figure 24b dose-dependent reduction in mucositis score by cd24fc. The mean and standard error of the individual mucositis scores are shown. The correlation coefficient and P-value between patient drug dose and mucositis score were determined using the pearson method. R-0.9983, P-0.0009.

FIGS. 25A-B show 180 days grade III-IV GVHD free Survival (free Survival) for CD24Fc compared to placebo (FIG. 25A) and contemporary control (FIG. 25B).

Fig. 26A-B show the relapse free survival rate of the CD24Fc group compared to the placebo control group (fig. 26A) and the contemporary control group (fig. 26B).

Detailed Description

Tissue damage can result in the release of proinflammatory cytokines, such as TNF- α, IL-1 β, and IL-6, as well as damage-related molecular patterns (DAMPs) and pathogen-related molecular patterns (PAMPs). Both DAMPs and PAMPs can activate Antigen Presenting Cells (APCs), such as Dendritic Cells (DCs), by binding to Pattern Recognition Receptors (PRRs). The host APC subsequently activates donor T cells and immune cascades leading to the release of pro-inflammatory cytokines and the expansion of antigen-specific alloreactive T cells that target host tissues. It is these events that lead to the development of GVHD and exacerbate the effects of mucositis. For example, RIOM begins with acute inflammation of the oral mucosa, tongue and pharynx following radiation therapy, which coincides with the recruitment of various inflammatory cells and the release time of inflammatory cytokines, chemokines and growth factors.

The involvement of tissue damage in mucositis and GVHD suggests that the prospect of down-regulating host response to DAMP by CD24Fc may be explored for GVHD treatment. Preclinical studies by the inventors have shown that CD24Fc specifically targets DAMP-mediated inflammation and is able to prevent GVHD in mouse models, including humanized mouse models. Importantly, this drug has advantages over conventional immunosuppressive agents in that it does not cause general immunosuppression, and the use of high doses of CD24Fc does not block the antibody response in non-human primates. The data also indicate that CD24Fc prevents GVHD, but retains the graft-versus-leukemia (GVL) effect, making it an ideal drug for preventing GVHD in leukemic patients. Finally, studies by the inventors in non-human primates showed that CD24Fc does not inhibit antigen-specific immune responses, indicating that CD24Fc is unlikely to increase the risk of infection.

The inventors have found that soluble forms of CD24 are highly effective for the prevention of Graft Versus Host Disease (GVHD) and related conditions such as mucositis and for the prevention of leukemia relapse after HCT. The inventors have also found that CD24Fc produces a dose-dependent reduction in severe mucositis (. gtoreq.3 grade) in patients receiving HCT treatment. These effects can be mediated by DAMPs. Pattern recognition involves inflammatory responses triggered by PAMPs and DAMPs. The inventors have recognized that recent studies indicate that an exacerbated host response to DAMPs may play a role in the pathogenesis of inflammatory and autoimmune diseases. DAMP was found to promote the production of inflammatory cytokines and autoimmune diseases and was found in animal models, and therefore inhibitors of DAMP such as HMGB1 and HSP90 were found to alleviate Rheumatoid Arthritis (RA). TLRs, RAGE-R, DNGR (encoded by Clec 9A) and Mincle have been shown to be receptors responsible for mediating inflammation triggered by a variety of DAMPs.

Recent work by the inventors has shown that CD24-Siglec G interaction can distinguish between innate immunity to DAMP and PAMP. Siglec proteins are members of the membrane-associated immunoglobulin (Ig) superfamily, which recognize a variety of sialic acid-containing structures. Most Siglecs have intracellular immunotyrosine inhibitory motifs (ITIMs) associated with SHP-1, -2 and Cbl-b to control key regulators of the inflammatory response. The inventors have reported that CD24 acts as the first natural ligand for Siglec (Siglec G in mice and Siglec10 in humans). Siglec G interacts with sialylated CD24 through SHP-1/2 signaling mechanisms to inhibit TLR-mediated host responses to DAMPs (e.g., HMGB 1).

Human CD24 is a small GPI-anchored molecule encoded by a 240 base pair open reading frame in the CD24 gene. Of these 80 amino acids, the first 26 constitute the signal peptide, while the last 23 serve as cleavage signals to allow GPI tail attachment. As a result, the mature human CD24 molecule has only 31 amino acids. One of these 31 amino acids is polymorphic in the human population. The C to T transition at nucleotide 170 of the open reading frame results in a substitution of alanine (A) for valine (V) at residue 31 of the mature protein. Since this residue is immediately N-terminal to the cleavage site, and since the substitutions are non-conserved, the two alleles can be expressed at different efficiencies on the cell surface. Indeed, transfection studies with cDNA revealed CD24^vAlleles are more efficiently expressed on the cell surface. In accordance therewith, CD24^v/vPBLs express higher levels of CD24, especially on T cells.

The present inventors have demonstrated that CD24 negatively regulates host response to cellular DAMPs released as a result of tissue or organ damage, and that at least two overlapping mechanisms may explain this activity. First, CD24 binds to several DAMPs, including HSP70, HSP90, HMGB1, and nucleolin, and suppresses host responses to these DAMPs. To this end, it is speculated that CD24 may capture inflammatory stimuli to prevent interaction with its receptor, TLR or RAGE. Secondly, use p-BThe inventors demonstrated that CD24 provides a powerful negative regulation of host responses to tissue injury through interaction with its receptor Siglec G. To achieve this activity, CD24 can bind and stimulate signaling through Siglec G, where Siglec G-associated SHP1 triggers negative regulation. The two mechanisms may act synergistically because mice with targeted mutations of either gene produce a much stronger inflammatory response. Indeed, when stimulated with HMGB1, HSP70 or HSP90, never from CD24^-/-Or Siglec G^-/-Bone marrow cultured DCs from mice produced higher levels of inflammatory cytokines. To the best of the inventors' knowledge, CD24 is the only inhibitory DAMP receptor capable of turning off the inflammation triggered by DAMP, and there are currently no drugs available to specifically target the host inflammatory response to tissue injury. Furthermore, the inventors have demonstrated the ability of exogenous soluble CD24 protein to alleviate DAMP-mediated autoimmune disease using mouse models of RA, MS and GvHD.

1. Definition of

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

With respect to recitation of numerical ranges herein, each intervening number between which the same degree of accuracy is recited is explicitly contemplated. For example, for the range of 6-9, numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

A "peptide" or "polypeptide" is a linked sequence of amino acids, and may be natural, synthetic, or a modification or combination of natural and synthetic.

"substantially identical" may mean that the first amino acid sequence and the second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical over a region of 1, 2,3, 4,5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acids.

When referring to protecting an animal from a disease, "treating" or "treating" means preventing, inhibiting, suppressing, or completely eliminating the disease. Prevention of disease involves administering the compositions of the present invention to an animal prior to the onset of disease. Inhibiting a disease involves administering a composition of the invention to an animal after induction of the disease but prior to its clinical manifestation. Suppression of disease involves administering a composition of the invention to an animal after clinical manifestation of the disease.

"variant" may mean a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Representative examples of "biological activity" include the ability to bind to toll-like receptors and be bound by specific antibodies. A variant may also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., substitutions of amino acids with different amino acids having similar properties (e.g., hydrophilicity, extent, and distribution of charged regions) are well known in the art to generally involve minor variations. As understood in the art, these minor changes may be identified, in part, by considering the hydropathic index of amino acids. Kyte et al, j.mol.biol.157: 105-132(1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids having a hydropathic index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins that retain biological function. In the context of peptides, the consideration of the hydrophilicity of amino acids allows the calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated herein by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions may be performed with amino acids having hydrophilicity values within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with this observation, amino acid substitutions compatible with biological function are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other characteristics.

2.CD24

Provided herein are CD24 proteins, which may comprise mature CD24 or variants thereof. Mature CD24 corresponds to the extracellular domain (ECD) of CD 24. Mature CD24 may be derived from a human or another mammal. As described above, the mature human CD24 protein is 31 amino acids long and has variable alanine (a) or valine (V) residues at its C-terminus. The mature CD24 protein may comprise the following sequence:

SETTTGTSSNSSQSTSNSGLAPNPTNATTK(V/A)(SEQ ID NO:1)

the C-terminal valine or alanine may be immunogenic and may be omitted from the CD24 protein, thereby reducing its immunogenicity. Thus, the CD24 protein may comprise the amino acid sequence of mature human CD24 lacking the C-terminal amino acid:

SETTTGTSSNSSQSTSNSGLAPNPTNATTK(SEQ ID NO:2)

despite considerable sequence variation in the amino acid sequences of mature CD24 proteins from mice and humans, they are functionally equivalent, as human CD24Fc has been shown to be active in mice. The amino acid sequence of human CD24 ECD shows some sequence conservation with mouse proteins (39% identity; Genbank accession No. NP-033976). However, it is not surprising that the percent identity is not high, since CD24 ECD is only 27-31 amino acids in length, depending on the species, and binding to some of its receptors (e.g., Siglec10/G) is mediated by sialic acid and/or galactose of the glycoprotein. The amino acid sequence identity between the extracellular domain of human Siglec-10(GenBank accession No. AF310233) and its murine homolog, Siglec-G (GenBank accession No. NP _766488), receptor protein was 63% (fig. 2). Since sequence conservation between mouse and human CD24 is primarily in the C-terminus and abundant glycosylation sites, significant variations in mature CD24 protein may be tolerated when using CD24 protein, especially if these variations do not affect conserved residues at the C-terminus, or do not affect glycosylation sites from mouse or human CD 24. Thus, the CD24 protein may comprise the amino acid sequence of mature murine CD 24:

NQTSVAPFPGNQNISASPNPTNATTRG(SEQ ID NO:3)。

the amino acid sequence of human CD24 ECD showed more sequence conservation with cynomolgus monkey protein (52% identity; UniProt accession No. UniProtKB-I7GKK1) than with mice. Again, since ECD is only 29-31 amino acids in length in these species, and the role of sugar residues in binding to its receptor, it is not surprising that the percent identity is not high. The amino acid sequence of the cynomolgus monkey Siglec-10 receptor has not been determined, but the amino acid sequence identity between the human and rhesus monkey Siglec-10(GenBank accession XP-001116352) proteins is 89%. Thus, the CD24 protein may also comprise the amino acid sequence of mature cynomolgus monkey (or rhesus monkey) CD 24:

TVTTSAPLSSNSPQNTSTTPNPANTTTKA(SEQ ID NO:10)

the CD24 protein may be soluble. The CD24 protein may also contain an N-terminal signal peptide to allow secretion of the protein from cells expressing the protein. The signal peptide sequence may comprise the amino acid sequence MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO: 4). Alternatively, the signal sequence may comprise any of those found on other transmembrane or secreted proteins, or those modified by existing signal peptides known in the art.

a. Fusion

The CD24 protein may be fused at its N-terminus or C-terminus to a protein tag, which may comprise a portion of a mammalian Ig protein, which may be human or mouse or from another species. This portion may comprise the Fc region of the Ig protein. The Fc region may comprise at least one of the hinge region, CH2, CH3, and CH4 domains of an Ig protein. The Ig protein may be human IgG1, IgG2, IgG3, IgG4, or IgA, and the Fc region may comprise the hinge region and CH2 and CH3 domains of Ig. The Fc region can comprise human immunoglobulin G1(IgG1) isotype SEQ ID NO 7. The Ig protein may also be IgM, and the Fc region may comprise the hinge region and CH2, CH3, and CH4 domains of IgM. The protein tag may be an affinity tag that facilitates purification of the protein, and/or a solubility enhancing tag that enhances solubility and recovery of the functional protein. Protein tags may also increase the potency of CD24 protein. The protein tag may also comprise GST, His, FLAG, Myc, MBP, NusA, Thioredoxin (TRX), small ubiquitin protein-like modifier (SUMO), ubiquitin (Ub), albumin, or camelid Ig. Methods for preparing fusion proteins and purifying fusion proteins are well known in the art.

Based on preclinical studies, for the construction of the fusion protein CD24Fc identified in the examples, a truncated form of the 30 amino acid native CD24 molecule has been used, which lacks the final polymorphic amino acid preceding the GPI signal cleavage site (i.e., the mature CD24 protein having SEQ ID NO: 2). The mature human CD24 sequence was fused to the human IgG1Fc domain (SEQ ID NO: 7). The sequence of the full-length CD24Fc fusion protein is provided in SEQ ID No. 5 (fig. 1A), and the sequence of the processed form of the CD24Fc fusion protein secreted from the cell (i.e., lacking the signal sequence that is cleaved off) is provided in SEQ ID No. 6. The processing polymorphic variant of mature CD24 (i.e., mature CD24 protein having SEQ ID NO: 1) fused to IgG1Fc can comprise the amino acid sequence set forth in SEQ ID NO:11 or 12.

b. Production of

CD24 protein may be highly glycosylated and may be involved in CD24 functions, such as co-stimulation of immune cells and interaction with injury-associated molecular pattern molecules (DAMPs). The CD24 protein can be produced using eukaryotic expression systems. Expression systems may require expression from vectors in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The system may also be a viral vector, such as a replication-defective retroviral vector that may be used to infect eukaryotic cells. The CD24 protein can also be stabilizedCell lines are generated that express the CD24 protein from a vector or a portion of a vector that has been integrated into the genome of the cell. Stable cell lines can express CD24 protein from integrated replication defective retroviral vectors. The expression system may be GPEx^TM。

c. Pharmaceutical composition

The CD24 protein may be contained in a pharmaceutical composition that may comprise a pharmaceutically acceptable amount of CD24 protein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise a solvent, which may stabilize the CD24 protein for an extended period of time. The solvent may be PBS, which is stable at-20 ℃ (-15 to-25 ℃) for at least 66 months for CD24 protein. The solvent may be capable of holding the CD24 protein in combination with another drug.

The pharmaceutical compositions may be formulated for parenteral administration, including but not limited to by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents including, but not limited to, suspending, stabilizing and dispersing agents. The compositions may also be provided in powder form for reconstitution with a suitable vehicle, including, but not limited to, sterile pyrogen-free water.

The pharmaceutical compositions may also be formulated as a slow-acting formulation, which may be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). Formulations for subcutaneous injection may be particularly relevant for indications such as lupus and its associated manifestations and complications.

3. Method of treatment

a.GVHD

Provided herein are methods of preventing, alleviating, or treating Graft Versus Host Disease (GVHD) in a subject in need thereof by administering to the subject a CD24 protein. The subject may have or be at risk of developing GVHD. The subject may have undergone or has undergone hematopoietic stem cell transplantation (HCT). The CD24 protein can be used prophylactically to prevent GVHD in subjects undergoing HCT. GVHD may be acute GVHD. The CD24 protein can reduce the risk of developing grade III-IV acute GVHD in a subject. GVHD can be chronic GVHD, including oral GVHD.

The subject may have cancer. The cancer may be Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Myelogenous Leukemia (CML), myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML).

b. Mucositis

Provided herein are methods of preventing, reducing, or treating mucositis in a subject in need thereof by administering to the subject CD24 protein. Also described herein are methods of preventing, alleviating, or treating other disorders associated with GVHD or HCT in a subject in need thereof by administering to the subject a CD24 protein. The associated condition may be mucositis, which may be oral mucositis. Mucositis is a painful inflammation and ulceration of the lining mucosa of the digestive tract, often as an adverse reaction to preconditioning chemotherapy and/or radiation therapy regimens of HCT. Mucositis can occur anywhere in the gastrointestinal tract. Oral mucositis refers to specific inflammation and ulceration occurring in the oral cavity. Oral mucositis is a common and often debilitating complication of cancer therapy. Many hematopoietic stem cell transplant recipients experience mucositis, with oral mucositis being the most common and most debilitating.

The subject may have, or be at risk of developing, mucositis, which may be oral mucositis. The subject may be undergoing or may be undergoing at least one of chemotherapy and radiation therapy. The CD24 protein can be used prophylactically to prevent mucositis in a subject. The CD24 protein may reduce the risk of developing severe mucositis in a subject.

c. Medicine

Also provided is the use of CD24 protein in the manufacture of a medicament for use as described herein.

d. Dosage regimen

The dose of CD24 protein administered may be from 0.01mg/kg to 1000mg/kg, or from 1 to 500mg/kg, depending on the desired effect on GVHD or mucositis and the route of administration. The CD24 protein may be administered by Intravenous (IV) infusion or by subcutaneous, intramural (i.e., within the wall of a cavity or organ) or intraperitoneal injection. The dose may be 10-1000mg, 10-500mg, 240mg or 480mg, particularly suitable where the subject is a human.

The CD24 protein may be administered before or after stem cell transplantation. The CD24 protein may be administered 1-4 days, particularly 1 day, prior to stem cell transplantation. The CD24 protein may also be administered in multiple doses before or after stem cell transplantation. The CD24 protein may be administered in 2,3, 4,5 or 6 doses every two weeks. Each dose of CD24 protein may be 240mg or 480 mg. The first dose may be administered on days-4 to 0, and particularly may be administered on day-1, relative to the day of stem cell transplantation (day 0). Subsequent doses may be administered every 9-19 or 11-17 days thereafter. The second dose may be administered on days +9 to +19 or days +11 to +17, especially days +14, relative to the stem cell transplantation day. The third dose may be administered on days +18 to +38, +23 to +33, or +22 to +34, particularly +28, relative to the stem cell transplantation day. In particular, the CD24 protein may be administered in three biweekly administrations of 480mg, 240mg, and 240mg on days-1, +14, and +28, respectively, relative to the stem cell transplantation day. The CD24 protein may in particular be CD24 Fc.

The CD24 protein may be administered before or during at least one of chemotherapy and radiation therapy administered to the subject. The CD24 protein may be administered so that it is present when the DAMP is released.

e. Combination therapy

The CD24 protein can be administered to a subject in combination with a standard of care for prevention of GVHD. The standard of care for prevention of GVHD may comprise administration of methotrexate plus a calcineurin inhibitor (such as tacrolimus (Prograf, FK506) or cyclosporin (Sandimmune, Neoral)). Tacrolimus may be administered on day-3 relative to the day of stem cell transplantation, and may be administered by IV or PO (oral). For IV administration as a continuous infusion, the starting dose may be 0.03 mg/kg/day based on adjusted body weight. For oral administration, the starting dose may be 0.045 mg/kg/dose twice daily. If the subject is unable to tolerate tacrolimus, the subject may be administered cyclosporine IV at a dose 100 times the IV infusion dose of tacrolimus (e.g., 3 mg/kg/day starting dose). Cyclosporin may also be administered orally at a dose 3 times the IV dose. When using the Neoral brand, cyclosporin may be administered orally in 2 times the IV dose due to higher bioavailability.

In the absence of GVHD, therapeutic doses of tacrolimus levels can only be monitored over the first 100 days post-transplantation. The therapeutic target trough level (trough level) of tacrolimus may be 5-15 ng/mL. Tacrolimus levels should be monitored a minimum of 3 times (e.g., once every 48-72 hours) the first week after infusion of CD24 protein (day 0 to day 7). In the absence of GVHD or relapse, tacrolimus may be gradually reduced starting +100 days post-transplantation. In the presence of GVHD, tacrolimus may continue to be used at therapeutic doses.

Methotrexate can be used in combination with tacrolimus for standard GVHD prevention. Methotrexate may be present at 15mg/m on day 1 after HCT²Dose/dose was administered intravenously once daily at 10mg/m on days 3, 6 and 11 after HCT²Dose/dose the dose was administered intravenously.

For mucositis, the CD24 protein may be administered with at least one immunomodulator (i.e., other than the CD24 protein) to minimize the worsening inflammatory component. For subjects treated with 5-fluorouracil, CD24 protein can be administered in combination with allopurinol.

The CD24 protein may be administered in combination with one or more of the following myelosuppressive preconditioning regimens, and may be administered prior to or during the preconditioning regimens. The CD24 protein may be administered such that it is present when the DAMP is released in response to at least one of a myelosuppressive preconditioning regimen and HCT.

Busulfan and fludarabine

Busulfan may be administered on days-5 to-2 relative to the stem cell transplantation day. The dosage may be 3.2 mg/kg/day or 130mg/m²Daily, and may be administered intravenously. The total dose may be 12.8mg/kg or 520mg/m². Fludarabine can be administered on days-5 to-2 relative to the day of stem cell transplantation. The dosage can beSo as to be 30-45mg/m²The total dose can be 120-180mg/m². The sequence and timing of busulfan and fludarabine may be performed according to institutional standards known in the art for myelosuppressive preconditioning.

Busulfan and cyclophosphamide

Busulfan may be administered on days-7 to-4 relative to the stem cell transplantation day. The dosage may be 3.2 mg/kg/day or 130mg/m²Daily, and may be administered intravenously. The total dose may be 12.8mg/kg or 520mg/m². Cyclophosphamide may be administered on days-3 to-2 relative to the stem cell transplantation day. The dose may be 60 mg/kg/day and the total dose may be 120 mg/kg.

Cyclophosphamide and whole body irradiation

With respect to stem cell transplantation, systemic irradiation (TBI) may be administered on days-7 to-4. Cyclophosphamide may be administered on days-3 to-2 relative to the stem cell transplantation day. The dose may be 60 mg/kg/day and the total dose may be 120 mg/kg. The order of administration of cyclophosphamide, TBI and TBI for myelosuppressive regimens can be performed according to institutional standards known in the art for myelosuppressive preconditioning.

The CD24 protein may also be administered with myelosuppressive therapy alone or in combination as described above.

Example 1: CD24 pharmacokinetics in mice

1mg of CD24Fc (CD24Fc) was injected into naiveBlood samples were collected in C57BL/6 mice and at different time points (5 minutes, 1 hour, 4 hours, 24 hours, 48 hours, 7 days, 14 days and 21 days), with 3 mice per time point. Serum was diluted 1: 100 and levels of CD24Fc were detected using a sandwich ELISA, using purified anti-human CD24 (3.3. mu.g/ml) as capture antibody and peroxidase-conjugated goat anti-human IgG Fc (5. mu.g/ml) as detection antibody. As shown in fig. 3a. The decay curve of CD24Fc reveals a typical biphasic decay of proteins. The first biodistribution phase had a half-life of 12.4 hours.The second phase follows a model of first order elimination from the central compartment. The half-life of the second phase was 9.54 days, which is similar to the half-life of antibodies in vivo. These data suggest that the fusion protein is very stable in the bloodstream. In another study in which the fusion protein was injected subcutaneously, almost the same half-life of 9.52 days was observed (fig. 3 b). More importantly, although it took approximately 48 hours for CD24Fc to reach peak levels in blood, the total amount of fusion protein in blood was essentially the same by either route of injection as measured by AUC. Thus, from a therapeutic point of view, the use of different routes of injection should not affect the efficacy of the drug. This observation greatly simplifies the experimental design for primate toxicity and clinical trials.

Example 2: CD24-Siglec 10 interaction in host response to tissue injury

Matzinger proposed what was commonly referred to as a theory of risk nearly two decades ago. Essentially, she thinks the immune system is open when she perceives a danger in the host. Although the nature of the risk was not well defined at the time, it was determined that necrosis was associated with the release of intracellular components such as HMGB1 and heat shock proteins (which are known as DAMPs) for risk-related molecular patterns. DAMPs were found to promote the production of inflammatory cytokines and autoimmune diseases. In animal models, inhibitors of HMGB1 and HSP90 were found to improve RA. The involvement of DAMP offers the prospect that negative regulation of host response to DAMP can be explored for RA therapy.

Using acetaminophen-induced hepatic necrosis and ensuring inflammation, it was observed that CD24 provides a powerful negative regulation of host responses to tissue injury through Siglec G interactions. CD24 is a GPI-anchored molecule that is widely expressed in hematopoietic cells and other tissue stem cells. Genetic analysis of various autoimmune diseases in humans, including multiple sclerosis, systemic lupus erythematosus, RA, and giant cell arthritis, shows a significant correlation between the CD24 polymorphism and the risk of autoimmune disease. Siglec G is a member of the I-lectin family, defined by its ability to recognize sialic acid containing structures. Siglec G recognizes sialic acid containing structures on CD24 and down-regulates the production of inflammatory cytokines by dendritic cells. Human Siglec10 and mouse Siglec G are functionally equivalent in terms of their ability to interact with CD 24. However, it is not clear whether there is a one-to-one correlation between mouse and human homologues. Although this mechanism has not been fully elucidated, it seems reasonable that SiglecG-related SHP1 may be involved in negative regulation. These data lead to a new model in which the CD24-Siglec G/10 interaction may play a key role in distinguishing pathogen-associated molecular patterns (PAMPs) from DAMPs (fig. 4).

At least two overlapping mechanisms may explain the function of CD 24. First, by binding to various DAMPs, CD24 may capture inflammatory stimuli to prevent their interaction with TLRs or RAGE. This view is supported by the observation that CD24 associates with several DAMP molecules, including HSP70, 90, HMGB1, and nucleolin. Second, CD24 may stimulate signaling through Siglec G, possibly after association with DAMP. Both mechanisms can act synergistically because mice with targeted mutations of either gene produce a much stronger inflammatory response. Indeed, DCs cultured from bone marrow from CD 24-/-or Siglec G-/-mice produced much higher inflammatory cytokines when stimulated with HMGB1, HSP70 or HSP 90. In contrast, in their comparison to PAMPs such as LPS and PolyI: no effect was found in the response of C. These data not only provide a mechanism for the innate immune system to differentiate pathogens from tissue damage, but also suggest CD24 and Siglec G as potential therapeutic targets for diseases associated with tissue damage.

Example 3: CD24Fc interacts with HMGB1, Siglec10 and induces an association between Siglec G and SHP-1

To measure the interaction between CD24Fc and Siglec10, we immobilized CD24Fc on CHIP and measured the binding of different concentrations of Siglec-10Fc using Biacore. As shown in FIG. 5a, CD24Fc was assigned a designation of 1.6X10^-7The Kd of M binds to Siglec 10. This is 100-fold higher affinity than control Fc. The interaction between CD24Fc and HMGB1 was confirmed by a pull-down experiment using CD24 Fc-bound protein G beads, followed by western blotting with anti-IgG or anti-HMGB 1. These data demonstrate that CD24Fc, but not Fc, binds to HMGB1, and thatBinding was cation-dependent (fig. 5 b). To determine whether CD24Fc is an agonist of Siglec G (the mouse counterpart of human Siglec 10), we stimulated CD24 with CD24Fc, control Fc, or vehicle (PBS) control^-/-Splenocytes for 30 minutes. Siglec G was then immunoprecipitated and probed with anti-phosphotyrosine or anti-SHP-1. As shown in fig. 5c, CD24Fc induced extensive phosphorylation of Siglec G and association of SHP-1, a well-known inhibitor of adaptive and innate immunity.

In vitro efficacy studies of CD24 Fc.

To investigate the effect of CD24Fc on inflammatory cytokines produced by human T cells, mature T cells in human PBML were activated by anti-CD 3 antibody (OKT3), a common agonist of T cell receptors in the presence of various concentrations of CD24Fc or human IgG1 Fc. After 4 days, supernatants were collected and activation was confirmed by measuring IFN-. gamma.and TNF-. alpha.production by enzyme-linked immunosorbent assay (ELISA). The results in fig. 6 demonstrate that CD24Fc from two different preparation batches significantly reduced the production of IFN- γ and TNF- α from activated human PBML compared to the control IgG Fc control. In addition, cytokine production was inhibited in a dose-dependent manner when CD24Fc was added. Thus, CD24Fc can inhibit anti-CD 3-induced activation of human PBML in vitro. This study not only suggests that the mechanism of action of CD24Fc may be through inhibition of T cell activation, but also establishes a reliable bioassay for drug efficacy and stability testing.

To determine whether CD24Fc modulates the production of inflammatory cytokines in human cell lines, we first silenced CD24 in the human acute monocytic leukemia THP1 cell line using RNAi and then induced their differentiation into macrophages by treating them with PMA. As shown in FIG. 7a, silencing of CD24 dramatically increased the production of TNF α, IL-1 β, and IL-6. These data demonstrate the important role of endogenous human CD24 in limiting inflammatory cytokine production. Importantly, CD24Fc restored inhibition of TNF α in CD24 silenced cell lines (FIG. 7b), as well as inhibition of IL-1 β and IL-6. These data not only demonstrate the relevance of CD24 in the inflammatory response of human cells, but also provide a simple assay to assess the biological activity of CD24 Fc.

Taken together, these data demonstrate that CD24Fc is able to inhibit cytokine production triggered by adaptive and innate stimuli. However, since this drug is more effective in reducing cytokine production by innate effectors, we believe that the main mechanism of its preventive function is to prevent inflammation caused by tissue damage at the early stage of transplantation.

Example 4: pharmacokinetics of CD24 in humans

This example shows the pharmacokinetic analysis of CD24 protein in humans. This was derived from phase I, randomized, double-blind, placebo-controlled, single ascending dose studies to evaluate the safety, tolerability, and PK of CD24Fc in healthy male and female adult subjects. A total of 40 subjects in 5 cohorts of 8 subjects each were recruited in the study. 6 of 8 subjects in each group received study medication and 2 subjects received placebo (0.9% sodium chloride, saline). The first group was dosed with 10 mg. Subsequent cohorts received 30mg, 60mg, 120mg and 240mg of CD24Fc or matched placebo and were administered at least 3 weeks apart to allow review of safety and tolerability data for each of the previous cohorts. Only if sufficient safety and tolerability has been demonstrated, the next higher dose is allowed to be administered to a new group of subjects.

In each cohort, the first 2 subjects were 1 study drug recipient and 1 placebo recipient on day 1. Subjects 3 to 5 and 6 to 8 were administered after day 7 (with a minimum of 24 hours between subgroups). Each subject in the same subgroup was administered at least 1 hour apart. If necessary, the dosing of the remaining subjects is delayed, awaiting review of any significant safety issues that may have occurred during the post-dose period involving the first subgroup or the second subgroup in the cohort. Subsequent groups were administered at least 3 weeks after the previous group.

And (3) screening period:

screening visits (visit 1) occurred up to 21 days before the start of the active treatment period. After providing informed consent, the subjects underwent a screening procedure for eligibility.

The treatment period is as follows:

subjects were enrolled in Clinical Pharmacology Units (CPU) on day-1 (visit 2) and the randomized treatment period started on day 1 after an overnight fast for a minimum of 10 hours. Subjects were randomized to treatment with CD24Fc or placebo as a single dose. Subjects remained closed until the morning of day 4.

Follow-up:

all subjects returned to the CPU on days 7, 14, 21, 28 and 42 (± 1 day) for follow-up visits (visit 3, visit 4, visit 5, visit 6 and visit 7). Visit 7 was the last visit of all subjects.

Duration of treatment:the total study duration for each subject was up to 63 days. Single dose administration occurred on day 1.

Number of subjects:

planning to be used: 40 subjects

Screening: 224 subjects

Randomized: 40 subjects

And (3) completing: 39 subjects

Interrupted: 1 subject

Diagnostic and primary criteria for inclusion:the population used in this study was healthy males and females between 18 and 55 years of age (inclusive), with 18kg/m²To 30kg/m²Body mass index (inclusive).

Study product and comparator information:

CD24 Fc: administering a single dose of 10mg, 30mg, 60mg, 120mg, or 240mg via IV infusion; batch number: 09 MM-036. CD24Fc is a fully humanized fusion protein consisting of the mature sequence of human CD24 and the crystallizable fragment region of human immunoglobulin G1(IgG1 Fc). CD24Fc was supplied as a sterile, clear, colorless, preservative-free aqueous solution for IV administration. CD24Fc was formulated as a single dose injection solution at a concentration of 10mg/mL and a pH of 7.2. Each vial of CD24Fc contained 160mg of CD24Fc, 5.3mg of sodium chloride, 32.6mg of sodium phosphate dibasic heptahydrate, and 140mg of sodium phosphate monobasic monohydrate in 16 mL. + -. 0.2mL of CD24 Fc. CD24Fc was supplied in clear borosilicate glass vials with chlorobutyl rubber stoppers and aluminum flip seals.

Administration of matched placebo (0.9% sodium chloride, saline) via IV infusion; batch number: p296855, P311852, P300715 and P315952.

The intent-to-treat (ITT) population consisted of all subjects receiving at least 1 dose of study drug. The ITT population is the main analytical population for subject information and safety assessment.

Clinical laboratory assessments (chemistry, hematology and urinalysis) were summarized by treatment and visit. Changes from baseline are also summarized. Vital signs (blood pressure, heart rate, respiratory rate and temperature) are summarized by treatment and time points. Changes from baseline are also summarized. All physical examination data are listed. Electrocardiographic parameters and changes from baseline are summarized. The general explanation is listed.

Plasma CD24Fc concentration

As shown in figure 8, the mean plasma concentration of CD24Fc increased in proportion to the dose of CD24Fc administered. For all dose groups except 120mg, the maximum mean plasma concentration of CD24Fc was reached 1 hour after the dose. The maximum mean plasma concentration for CD24Fc in the 120mg group was reached 2 hours after the dose. By day 42 (984 hours), the mean plasma concentration of CD24Fc for all groups had decreased to 2% to 4% of the maximum mean plasma concentration.

Table 1 summary plasma CD24Fc PK parameters by treatment for PK evaluable populations.

Table 1 summary of plasma CD24Fc pharmacokinetic parameters by treatment-PK evaluable population

Plasma CD24Fc dose proportionality analysis

FIG. 9 shows CD24Fc C_maxDose proportion graph relative to dose for PK evaluable populations. FIG. 10 shows the CD24Fc AUC_0-42dDose proportion graph relative to dose for PK evaluable populations. FIG. 11 shows the CD24Fc AUC_0-infDose proportion graph relative to dose for PK evaluable populations. Table 2 shows the dose-ratiometric efficacy analysis.

C_maxThe slope estimate was 1.172 with 90% CI from 1.105 to 1.240. AUC_0-42dThe slope estimate was 1.088, with 90% CI from 1.027 to 1.148. AUC_0-infThe slope estimate was 1.087 with 90% CI ranging from 1.026 to 1.1.

Pharmacokinetic conclusions

C of plasma CD24Fc_maxAnd AUC increased in proportion to the dose administered in mice, monkeys, and humans. Plasma CD24Fc reached T between 1.01 and 1.34 hours_max. T of plasma CD24Fc_1/2Ranging between 280.83 and 327.10 hours.

Example 5: CD24 can be used for treating graft versus host disease in human subjects

A multicenter (multicenter), predictive (pro-active), double-blind, randomized, placebo-controlled phase IIa dose escalation trial was performed to evaluate the addition of CD24 protein, CD24Fc, to the standard of care for the prevention of acute GVHD in cancer patients undergoing allogeneic myelosuppressive hematopoietic stem cell transplantation (HCT). The experimental design is shown in figure 12.

The primary objective of the phase IIa study included assessing the safety and tolerability of CD24Fc in combination with methotrexate and tacrolimus prevention in patients with unrelated donor HCTs matched after myelosuppressive preconditioning to determine the recommended phase 2 dose (RP2D) or Maximum Tolerated Dose (MTD). In addition, secondary efficacy goals for the phase IIa study included:

determination of whether addition of CD24Fc to standard GVHD prophylaxis methotrexate and tacrolimus reduces the cumulative incidence of grade II-IV aGVHD on days 100 post HCT

Estimate stage II-IV aGVHD disease free (free) survival (GFS) 180 days after HCT

Describe the incidence of cGVHD (cGVHD) at 1 year

Describe the incidence of one-year relapse of HCT

Morbidity describing transplant-related mortality (TRM) one year after HCT

Describe the infection rate on day 100 after HCT

Assessment of Overall Survival (OS), grade III-IV GVHD loss and relapse-free survival one year after HCT

Assessment of preconditioning toxicity, including oral mucositis and organ failure

Other objectives include assessing the Pharmacokinetic (PK) profile of CD24Fc, examining the immune cell profile and functional response of APC and T cells after HCT in CD24Fc and placebo groups, and assessing the plasma concentrations of Pharmacokinetic (PD) biomarkers, such as pro-inflammatory cytokines, DAMPs, lipids, and GVHD biomarkers in CD24Fc and placebo groups.

The trial recruited patients undergoing allogeneic HCT, receiving grafts from matched unrelated donors, according to institutional practice. Patients between 18-70 years of age received matched unrelated donor allogeneic HCT due to hematologic malignancy, with a carnofsky performance score of 70%, qualifying for the study. 8/8HLA allele matching between unrelated donors and recipients of HLA-A, HLA-B, HLA-C and HLA-DRB1 is required. Given the high incidence of aGVHD class II-IV (60-80%) and aGVHD class III-IV (20-35%) in this population, it is expected to limit the study to patients receiving HCT from unrelated donors to limit heterogeneity and facilitate statistical estimation of aGVHD incidence for subsequent efficacy assessment.

This trial used only myelosuppressive preconditioning regimens and standard of care (SOC) prophylaxis comprising tacrolimus and methotrexate, as these patients experienced the most severe tissue damage, and in this case the drugs would likely have the strongest biological effects. All patients received myelosuppressive modulation of methotrexate and tacrolimus and standard of care for prevention of GVHD according to the phase IIa regimen. At the discretion of the treating physician, patients received a myelosuppressive conditioning regimen consisting of fludarabine and busulfan (Flu/Bu 4) or cyclophosphamide and total body irradiation (Cy/TBI) followed by infusion of stem cells on day 0. GVHD was administered prophylactically to all patients and was combined with tacrolimus (starting on day-3 before transplantation) and methotrexate (starting on day +1 after transplantation) with CD24Fc in the treatment group or normal saline in the placebo group. In the absence of GVHD, tacrolimus was gradually reduced starting on day + 100. The source of donor stem cells is Peripheral Blood Stem Cells (PBSC) or Bone Marrow (BM).

The phase IIa clinical trial contained two single ascending dose groups (240mg and 480mg) and one single multi-dose CD24Fc group, in addition to preventing SOC GVHD, as described in Table 3 below. As shown in figure 13, study agent CD24Fc was administered intravenously on day-1 relative to the day of stem cell transplantation in the single dose group. In the multiple dose group, patients received 480mg (day-1), 240mg (day + 14), and 240mg (day + 28) of CD24Fc once three two weeks. Based on the PK data for CD24Fc, this bi-weekly dosing period would allow for more than two half-lives. The dose is based on a fixed amount, not on weight or BSA. Random 3: a 1 ratio (6 subjects CD24Fc and 2 placebo subjects) was designed to recruit 8 subjects for a total of 24 patients.

TABLE 32 phase a dose escalation plan

Table 4 lists the demographic information and clinical characteristics of the patients in the CD24Fc and placebo cohorts, which are relatively balanced among various risk factors (e.g., age, malignancy, and complications). The most common malignancy among the CD24Fc and placebo cohorts was AML/MDS (66.7% and 83.3%). 72% of patients in the CD24Fc group and 50% of patients in the placebo group had a moderate or high complication index. In both groups, PBSC were used more frequently as a source of grafts than bone marrow, and Flu/Bu 4 was the most common regulatory scheme in both groups. Four patients in the overall CD24Fc cohort received Cy/TBI modulation.

TABLE 42 stage a patient characteristics

BM is bone marrow; Cy/TBI ═ cyclophosphamide/whole body irradiation; d ═ donor; Flu/Bu 4 ═ fludarabine/busulfan; r ═ recipient

The main objectives of this study were: assessing the safety and tolerability of CD24Fc in subjects undergoing myelosuppressive allogeneic Hematopoietic Cell Transplantation (HCT); and determining the recommended phase II dose (RP2D) or the Maximum Tolerated Dose (MTD) of CD24Fc for patients receiving HCT.

All patients enrolled into the study had completed a treatment period, which was the first day of treatment with CD24Fc, up to 30 days after a single dose group of HCTs or 60 days after multiple dose groups of HCTs (the exact number of days may vary depending on the last day of study drug administration, not constitute a bias), which was the evaluation and reporting period for Adverse Events (AEs) including dose-limiting toxicities that may be associated with study drugs. Table 5 provides a summary of the toxicity observed in the phase 2a trial. Overall, this study shows that up to 480mg of CD24Fc administered IV is generally well tolerated in the intent-to-treat (ITT) population. No infusion toxicity, Dose Limiting Toxicity (DLT) or SAE attributed or likely attributed to the study drug was observed, nor was the patient withdrawn from the study.

As shown in fig. 14, all 24 subjects were implanted after transplantation. Median values for neutrophil engraftment after HCT were 13.0 and 15.5 days in CD24Fc exposed and placebo patients, respectively. Median implanted platelets were 13.0 and 15.0 days after HCT in CD24Fc exposed and placebo patients, respectively, but only one patient in the placebo group had no implanted platelets and died on day 49. There was no case of migration failure. The median CD3 chimerism rate was 82.5% (range 38-100%) on day +30 in patients with CD24Fc exposure, compared to 82.0% (range 62-91%) in the placebo group (fig. 15). In patients exposed to CD24Fc, the median donor CD3 chimerism rate increased to 86% (range 42% -100%) on day 100, while the placebo group increased to 84% (range 17% -100%). In the CD24Fc and placebo groups, the chimerism rate of donor CD33 was 100% at day 30 and day 100.

Table 5 summary of toxicity

The efficacy analysis of the phase 2a study was considered secondary and included the following: describe grade III-IV acute GVHD survival (GFS) without 180 days post HCT; describe the cumulative incidence of grade II-IV acute GVHD on day 100 post HCT; grade III-IV GVHD at day 180 post HCT with no recurrence survival is described; describe grade II-IV acute GFS 180 days after HCT; describe the incidence of chronic GVHD one year after HCT; describe the incidence of one-year relapse after HCT; describe the incidence of transplant-related mortality (TRM) one year after HCT; describe infection rate on day 100 post HCT; the Overall Survival (OS) and disease-free survival (DFS) one year after HCT were evaluated.

In addition to the placebo arm included in the phase IIa study, the same myelosuppressive modulation and GVHD prevention protocol (minus experimental treatment CD24Fc) was used from month 2012 to month 11 2017, with contemporaneous control (N92) data collected from the same institution receiving paired unrelated donor HCT. A cohort control group was included, considering fewer patients in the placebo control group. Table 6 summarizes the demographic data of 92 adult patients in the cohort control cohort.

Table 6 characteristics of patients enrolled into the cohort

Tables 7 and 8 provide a summary of the clinical results of the Ph 2a study. Acute GVHD was graded according to common guidelines used by the international cimbtr registry and clinical trial networks for blood and bone marrow transplantation and recorded weekly. Patients were evaluated for aGVHD from day 0 after HCT to day 100 after HCT.

Table 7 summary of clinical results, including cumulative incidence of aGVHD grade II-IV and grade III-IV.

Death after day 180 (n ═ 2): group 1 pneumonia 2/2 infection (d 210) and group 2CMML relapse (d 196)

Table 8 summary of clinical results

By day 100, incidence of grade II to IV acute graft versus host disease

Table 9 summarizes the cumulative incidence of grade II to IV acute GVHD in the mITT population at day 100. By day 100, a total of 7 (38.9%) patients receiving CD24Fc (2 patients [ 33.3% ] in the 240mg CD24Fc single dose group, 3 [ 50.0% ] in the 480mg CD24Fc single dose group and 2 patients [ 33.3% ] in the 960mg CD24Fc multiple dose group) and 1 (16.7%) patient with grade II to IV acute GVHD receiving placebo. Furthermore, by day 100, 1 (16.7%) of the patients receiving placebo died without developing grade II to IV acute GVHD. Patients who survive day 100 and do not develop grade II to IV acute GVHD will be censored at or before day 100 when acute GVHD is assessed the last time. At least 50.0% of patients in each treatment group were reviewed.

TABLE 9 cumulative incidence of grade II to IV acute graft versus host disease in day 100-mITT population

Overall, the cumulative incidence of grade II to IV acute GVHD on day 100 (with 95% CI) was 38.9% (16.8%, 60.7%) for the CD24Fc treated group versus 16.7% (0.5%, 54.9%) for the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 2.6(0.5, 14.7). In contemporary controls, the cumulative incidence of aGVHD grade II-IV was 50%. In the CD24Fc treatment group, four cases of grade II aGVHD were skin only, and two cases were skin and upper Gastrointestinal (GI) tract. There were no cases of aGVHD class II in the placebo group.

By day 180, survival without grade II-IV acute graft versus host disease

Table 10 summarizes grade II to IV acute GFS for the mITT population up to 180 days. Median acute GFS Kaplan-Meier estimates of grade II to IV were not achieved in any of the treatment groups. Overall, the incidence of grade II to IV acute GFS (with 95% CI) was 61.1% (35.3%, 79.2%) in the CD24 Fc-treated group on day 180 compared to 50.0% (11.1%, 80.4%) in the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.8(0.3, 2.5). Patients who were still alive at the date of data expiration and who had not recorded the development of grade II to IV acute GVHD were reviewed at or before day 180 on the last date of acute GVHD assessment. At least 50.0% of patients in each treatment group were censored except for small sample amounts.

TABLE 10 grade II to IV acute graft versus host disease-disease free survival by day 180-mITT population

By day 180, grade III to IV acute GFS in the mITT population

As shown in table 11, a total of 1 (5.6%) of the patients receiving CD24Fc (1 [ 16.7% ] patient in the 480mg CD24Fc single dose group) and 2 (33.3%) of the patients receiving placebo had grade III to IV acute GVHD by day 180. Overall, the incidence of grade III to IV acute GFS (with 95% CI) was 94.4% (66.6%, 99.2%) in the CD24 Fc-treated group on day 180 compared to 50.0% (11.1%, 80.4%) in the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.1(0.0, 0.7). Patients who were alive at the date of data expiration and had not recorded developed grade III to IV acute GVHD were reviewed at the last date of the acute GVHD assessment (i.e., day 180 or earlier). At least 50.0% of patients in each treatment group were examined. In the contemporary control group, the grade III to IV acute GFS rate was 24% on day 180. Figure 25 shows 180 days of no grade III-IV GVHD survival in CD24Fc group compared to placebo control group (figure 25A) and contemporary control group (figure 25B).

TABLE 11 grade III to IV acute graft versus host disease-disease free survival by day 180-mITT population

All patients who developed aGVHD in the data-cut-off study responded to steroid treatment compared to the 50% response rate observed in cohort controls. After the first one hundred days post HCT, patients were evaluated quarterly for late aGVHD (defined as the onset of acute GVHD after day 100) or cGVHD until one year post HCT. No other aGVHD events were observed in the CD24Fc cohort after day 100 post-transplantation.

Figure 16 shows the cumulative incidence of grade II-IV and grade III-IV acute GVHD in the treatment (CD24Fc) cohort. In particular, only one patient was observed who involved grade III GVHD with the lower GI and no liver GVHD. In the CD24Fc group, the trend for cumulative incidence of aGVHD grade III-IV at day 180 post HCT was lower than that of aGVHD grade III-IV at day 180 in the contemporary control group (P ═ 0.097). In placebo patients, there was another case of aGVHD class III on day 182, resulting in death on day 184. The patient presented with leukemia relapse on day 145. These results indicate that, in addition to methotrexate and tacrolimus prophylactic treatment, CD24Fc also reduces the risk of developing more severe aGVHD grade III and IV in patients undergoing HCT after receiving myelosuppressive modulation.

Disease-free survival 1 year after hematopoietic stem cell transplantation

Table 12 summarizes disease-free survival (DFS) 1 year after HCT in the mITT population. Any treatment group did not reach the median value of the DFS Kaplan-Meier estimates. Overall, the DFS rate 1 year after HCT (with 95% CI) was 83.3% (56.8%, 94.3%) for the CD24 Fc-treated group versus 50.0% (11.1%, 80.4%) for the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.2(0.1, 0.9). Patients who were still alive at the end of the follow-up period and who did not experience disease recurrence were reviewed at the last date of the assessment. At least 50.0% of patients in each treatment group were reviewed. Fig. 26 shows relapse-free survival of the CD24Fc group compared to the placebo control group (fig. 26A) and the contemporary control group (fig. 26B).

TABLE 12 disease-free survival-mITT population 1 year after hematopoietic stem cell transplantation

1 year overall survival after hematopoietic stem cell transplantation

Table 13 summarizes Overall Survival (OS) 1 year after HCT in the mITT population. The median OS time Kaplan-Meier estimate was not reached for any of the treatment groups. Overall, the 1 year OS rate (with 95% CI) was 83.3% (56.8%, 94.3%) for the CD24Fc treated group and 50.0% (11.1%, 80.4%) for the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.2(0.1, 1.0). Patients who were still alive at the end of the follow-up period were reviewed on the last date they were known to be still alive. At least 50.0% of patients in each treatment group were reviewed.

TABLE 13 disease-free survival-mITT population 1 year after hematopoietic stem cell transplantation

The Overall Survival (OS) estimates for patients in the IIa phase study about 800 days after HCT are also encouraging. The Overall Survival (OS) was approximately 80% for patients in the CD24Fc group, 50% for placebo group (p 0.06) (fig. 20), and 50% for cohort control patients (p 0.05) (fig. 21). The results, in support of the above-mentioned other findings, demonstrate that co-administration of CD24Fc with methotrexate and tacrolimus significantly improves the prognosis of patients receiving HCT after myelosuppressive modulation, in patients with exposure to CD24Fc compared to placebo and cohort control patients.

Survival without acute graft versus host disease and relapse by day 180 (aGRFS)

Therapeutic strategies aimed at preventing GVHD may lead to increased recurrence of leukemia due to a decrease in the effects of graft-versus-leukemia (GVL). As shown in table 7, the incidence of leukemia recurrence was lower in patients exposed to CD24Fc on day 180 post HCT (11%) compared to placebo (33%) and contemporary control (23%). One subject in the 480mg CD24Fc group experienced a CMML relapse at day 146, while one subject in the multiple dose 960mg CD24Fc group experienced an ALL relapse at 100 days post HCT. CMML patients died from leukemia at day 196. Patients with ALL relapse treated with bonatumomab (blinatumomab) achieved complete remission, surviving as the data cutoff of 8/2018. In the placebo cohort, one patient experienced a relapse of CMML at day 94, and one patient with MDS experienced a relapse at day 146 (CMML patients died at day 316, MDS patients died at day 184). These results indicate that CD24Fc does not interfere with the beneficial graft-versus-tumor (GVT) process and may even reduce the risk of leukemia recurrence.

On day 180 post-transplantation, the CD24Fc group had fewer deaths than the placebo and contemporary controls (Table 7). On day 180 post-HCT, there were no deaths in any of the CD24Fc groups, 1 deaths due to pneumonia in the placebo group (16.7%), and 22 deaths in the contemporary control group (23.9%). An improvement in the composite endpoint of GVHD grade III-IV Relapse Free Survival (RFS) was observed in the CD24Fc group (83%) compared to the placebo group (33%) on day 180 post-HCT (P ═ 0.011, see fig. 18) and the contemporary control group (53%) (P ═ 0.017, see fig. 19) on day 180 post-HCT. This endpoint has become increasingly prevalent because, theoretically, it encompasses not only the effects of intervention on GVHD inhibition, but also the potential effects of toxicity, infection and relapse. In support of the above observations, the improvement in aGVHD, RFS grade III-IV indicates that administration of CD24Fc in combination with standard care methotrexate and tacrolimus following a myelosuppressive conditioning regimen is beneficial for patients to prevent aGVHD from occurring without affecting the GVL effect of the graft.

The aGRFS by day 180 after HCT is the post-hoc composite endpoint, with events including grade III to IV acute GVHD, relapse, or death from any cause. Table 14 summarizes grade III to IV acute GRFS from the mITT population to 180 days.

For the CD24Fc treated group, Kaplan-Meier estimates of median grade III to IV acute GRFS were not reached. For the placebo group, the median grade III to IV acute GRFS Kaplan-Meier estimate (with 95% CI) was 120.0(46.0, not estimable). Overall, the CD24Fc treated group had a grade III to IV acute GRFS rate of 83.3% (56.8%, 94.3%) on day 180 (with 95% CI) compared to 33.3% (4.6%, 67.6%) in the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.2(0.0, 0.6). On the final date of evaluation, patients who are still present and have not documented the development of grade III to IV acute GVHD, chronic GVHD requiring systemic immunosuppressive therapy or relapse on the date of data expiration were reviewed.

TABLE 14 grade III to IV acute graft versus host disease-disease-free survival and relapse-free survival by day 180-mITT population

Incidence of 1-year relapse after hematopoietic stem cell transplantation

Table 15 summarizes the cumulative incidence of HCT relapse one year after the mITT population. Overall, the cumulative incidence of relapse at 1 year (with 95% CI) after HCT in the CD24 Fc-treated group was 11.1% (1.7%, 30.4%) compared to 33.3% (2.9%, 71.1%) in the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.3(0.1, 1.4). Patients who survived at the end of the follow-up period (day 365 [1 year ]) and did not relapse were reviewed on the last date of the assessment. At least 50.0% of patients in each treatment group were reviewed.

TABLE 15 cumulative incidence of 1-year relapse after hematopoietic stem cell transplantation-mITT population

Survival and relapse free survival (GRFS) for graft-free versus host disease 1 year after hematopoietic stem cell transplantation

GRFS is a compound endpoint within 1 year after HCT, with events including grade III to IV acute GVHD, chronic GVHD requiring systemic immunosuppressive therapy, relapse or death for any reason. Table 16 summarizes grade III to IV acute GRFS 1 year after HCT in the mITT population.

Kaplan-Meier estimates (with 95% CI) for the entire CD24Fc treatment group were 229.0 days (141.0, not estimable): the single dose group of 240mg CD24Fc was 247.0 days (129.0, not estimated), the single dose group of 480mg CD24Fc was 287.0 days (24.0, not estimated), and the multiple dose group of 960mg CD24Fc was 193.5(100.0, not estimated). The Kaplan-Meier estimated median GRFS (with 95% CI) for the placebo group was 120.0 days (46.0, not estimable). Overall, the GRFS rate 1 year after HCT (with 95% CI) was 32.4% (12.7%, 54.0%) for the CD24Fc treated group versus 33.3% (4.6%, 67.6%) for the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.7(0.3, 1.7). On the final date of evaluation, patients who are still present and have not documented the development of grade III to IV acute GVHD, chronic GVHD requiring systemic immunosuppressive therapy or relapse on the date of data expiration were reviewed.

TABLE 16 survival of graft-free versus host disease 1 year after hematopoietic stem cell transplantation and relapse-free survival-mITT population

Incidence of non-recurrent death 1 year after hematopoietic stem cell transplantation

Table 17 summarizes the cumulative incidence of NRM after one year HCT in the mITT population. Overall, the cumulative incidence of NRM (with 95% CI) was 5.6% (0.3%, 23.1%) for 1 year in the CD24Fc treated group versus 16.7% (0.5%, 54.9%) in the placebo group. The risk ratio of CD24Fc to placebo (with 90% CI) was 0.3(0.0, 2.8). Surviving patients who had not relapsed at the end of the follow-up period (day 365 [1 year ]) were reviewed on the last date of known survival. At least 50.0% of patients in each treatment group were examined. The cumulative incidence of NRM (with 95% CI) at 180 days was 0.0% in the CD24Fc treated group versus 16.7% (0.5%, 54.9%) in the placebo group.

TABLE 17 cumulative incidence of non-recurrent deaths within 1 year after hematopoietic stem cell transplantation-mITT population

Incidence of chronic graft-versus-host disease of hematopoietic stem cells one year later

Table 18 summarizes the cumulative incidence of chronic GVHD one year after HCT in the mITT population. Overall, the cumulative incidence of chronic GVHD 1 year after HCT in CD24 Fc-treated group (with 95% CI) was 63.3% (34.1%, 82.4%) compared to 33.3% (2.5%, 72.0%) in placebo. The risk ratio of CD24Fc to placebo (with 90% CI) was 2.1(0.6, 7.4). There were 3 moderate chronic GVHD in the single dose group of 240mg CD24Fc, 3 mild and 1 moderate chronic GVHD in the single dose group of 480mg CD24Fc, and 2 mild and 3 moderate chronic GVHD in the multiple dose group of 960mg CD24 Fc. There were 2 patients with mild chronic GVHD in the placebo group. Overall, there were no serious cases of chronic GVHD. Patients who survived at the end of the follow-up period (day 365 [1 year ]) and did not experience chronic GVHD were reviewed at the last date of the assessment.

TABLE 18 cumulative incidence of chronic graft versus host disease 1 year after hematopoietic stem cell transplantation-mITT population

Infection rate on day 100

As with the effects on GVL, therapeutic strategies aimed at preventing GVHD through global immunosuppression may result in increased rates of infection, including bacterial infection and CMV reactivation.

Table 19 summarizes the incidence of infection in the mITT population up to day 100. A total of 13 (72.2%) patients receiving CD24Fc (240mg CD24Fc single dose group 5 [ 83.3% ] patients, 480mg CD24Fc single dose group 2 [ 33.3% ] patients and 960mg CD24Fc multiple dose group 6 [ 100.0% ] patients) and two (33.3%) patients receiving placebo developed infections up to day 100.

Most infections are considered to be controllable and resolvable. Patient 103-001 died from pneumonia in the placebo group. Patient 102-002 in the placebo group had conjunctivitis, which was reported to have recovered/alleviated. Patients 101-010 in the 480mg CD24Fc monodose group and 101-011 in the 480mg CD24Fc monodose group both had papulopustules and were reported to be incurable/unresolved. Patients 102-006 in the 960mg CD24Fc multi-dose group had upper respiratory infection and clostridium difficile colitis, reported to be continuously intervening.

The majority of infections were either bacterial (9 patients receiving CD24Fc [ 50.0% ] patients, 2 patients receiving placebo [ 33.3% ] patients) or viral (7 patients receiving CD24Fc [ 38.9% ], 1 patient receiving placebo [ 16.7% ]). Most infections occur in blood (8 [ 44.4% ] patients receiving CD24Fc, 1 [ 16.7% ] patients receiving placebo), urine (4 [ 22.2% ] patients receiving CD24Fc, patients not receiving placebo) or feces (2 [ 11.1% ] patients receiving CD24Fc and 2 [ 33.3% ] patients receiving placebo). Most of the bacteria recovered from blood cultures are common skin inhabitants and low virulence pathogens (i.e. coagulase-negative staphylococci).

TABLE 19 to 100 day infection incidence summary-mITT population

As shown in Table 20, 9 patients in the CD24Fc group had a high risk of CMV reactivation (donor/recipient CMV status before HCT: D +/R +, 5; D-/R +, 3; unknown D/R +, 1). One patient with D +/R-in the CD24Fc group had an intermediate risk of CMV reactivation. In the CD24Fc group 8 patients had D-/R-status, which was considered to be low risk. Two D-/R + patients had CMV reactivation on days 42 and 48, representing a cumulative incidence of 22.2% CMV reactivation on day 100 in the high risk group. Both patients received systemic steroid therapy before reactivation of CMV was detected. In contrast, 2 patients in the placebo group had a high risk of CMV reactivation (D +/R +, 1; D-/R +, 1). One patient in the placebo group had CMV reactivation on day 47 prior to systemic steroid treatment of acute GVHD (50.0% in the high risk group).

The rate of CMV infection in HCT patients was graded by the CMV status of the donor and recipient prior to transplantation. D ═ donor, R ═ recipient, + positive, -negative, and U is unknown.

Cytomegalovirus status	CD24Fc group	Placebo group
			D+,R+	5	1
D+,R-	1	0
			D-,R+	3	1
D-,R-	8	4
			DU,R+	1	0

Overall, the phase IIa study was well-tolerated for CD24 Fc. There were no infusion related toxicities. In the 480mg hyperglycemic group, there may be a drug-related TEAE ≧ grade III in patients exposed to CD24Fc, which is insulin-controlled. A dose-limiting toxicity (DLT) was observed in the placebo group, whereas DLT was not observed in the CD24Fc group. Patients administered CD24Fc had no adverse events leading to death within 180 days (at least 150 days after the last CD24Fc dose). The placebo group had an adverse pneumonia event on day 48, resulting in the death of one subject. 1 patient in the CD24Fc group died 7 months after HCT, although it was determined that this death was unlikely to be related to study medication. At any time on day 100 post-HCT, no anti-drug antibody (ADA) was detected in any of these 24 patients.

The most common TEAE ≧ class III (> 10%) included a decrease in platelet count (83.3% placebo and 94.4% CD24Fc), a decrease in WBC count (66.7% placebo and 88.9% CD24Fc), a decrease in neutrophil count (50% placebo and 83.3% CD24Fc), a decrease in lymphocyte count (50% placebo and 77.8% CD24Fc), anemia (50% placebo and 66.7% CD24Fc), stomatitis (83.3% placebo and 50% CD24Fc), and nausea (0% placebo and 11.1% CD24 Fc). These SAEs are consistent with the known safety of the myelosuppressive regulatory regimens used in HCT.

Myelosuppressive regulation of HCT is often associated with severe regimen-related toxicity, including organ failure. Organ failure is the most common cause of early-onset transplant-related mortality (TRM) or non-recurring mortality (NRM). None of the 18 patients in the CD24Fc group died within the first 100 days after HCT, while 1 of the 6 patients in the placebo group died by respiratory failure in 48 days.

Pharmacokinetic results

FIGS. 22-23 show PK data from three escalation cohorts of phase 2a trials. The half-lives (fig. 22) from the 240 and 480mg single dose groups were approximately 14 days, which is consistent with the data observed in healthy subjects. At the 480mg dose, the cmx was higher, but the exposure did not really increase after 14 days (i.e. the time for the development and transplantation of peep (peek) GVHD). In the last multiple dose group, an increase in exposure to 60 days was expected (fig. 23), during which the patients were most prone to GVHD.

Table 21 summarizes the plasma PK parameters for CD24Fc for the PK population in the single dose group. Geometric mean C for the 240 and 480mg CD24Fc single dose groups_max，-1dThe values were 52,145.41 and 84,155.08ng/mL, respectively, and the geometric mean AUC_{0-last, -1d}The values were 10,156,549.9 and 15,522,686.2ng h/mL, respectively, and the geometric mean AUC_0-42dThe values were 9,275,562.3 and 13,903,718.4ng h/mL, respectively, and the geometric mean AUC_0-infThe values were 10,383,503.9 and 15,716,616.4ng h/mL, respectively. Median t for the 240 and 480mg CD24Fc single dose groups_max,-1dIt is 2.10 h. For the 240 and 480mg CD24Fc single dose groups, t_1/2Are 414.739h and 406.648h, respectively, and λ z is 0.0018 and 0.0017h-1, respectively. For the 240 and 480mg CD24Fc single dose groups, the mean Vz value was 13.83L and 18.18L, respectively, and the mean CL value was 0.024L/h and 0.031L/h, respectively.

Table 20 summary of plasma pharmacokinetic parameters for CD24 Fc-PK population-single dose group

Table 22 summarizes the CD24Fc plasma PK parameters for the PK population in the multiple dose groups at day-1, day 28, and day-1 to day 100. Geometric mean C for the 960mg CD24Fc multiple dose group_max,-1dAnd C_max,28dThe values were 96,942.71ng/mL and 62,563.05ng/mL, respectively. Geometric mean AUC for the 960mg CD24Fc multiple dose group_{0-last, -1d}、AUC_0-14d、AUC_0-100dAnd AUC_0-lastThe total values are 12,317,971.2ng h/mL, 9,688,933.9ng h/mL and 37,736,555.1n respectivelyg h/mL and 37,363,953.5ng h/mL. Median t for the 960mg CD24Fc multiple dose group_max,-1dAnd t_max,28d2.13h and 2.52h respectively.

Table 21 plasma pharmacokinetic parameters of CD24Fc summary-PK population-multiple dose group-day-1, day 28 and days-1 to 100

Clinical evidence from phase IIa studies strongly suggests that administration of CD24Fc in combination with methotrexate and tacrolimus can greatly improve the prognosis of leukemia patients receiving myelosuppressive allo-HCT by reducing the likelihood of severe aGVHD (grade III-IV) and the likelihood of leukemia recurrence. As described above, the cumulative incidence of aGVHD grade III-IV was 5.6% in patients exposed to CD24Fc compared to 16.7% in the placebo group (saline plus methotrexate and tacrolimus) and 24% in the contemporary control group (methotrexate and tacrolimus only). These data indicate that administration of CD24Fc in combination with methotrexate and tacrolimus as prophylaxis reduces the risk of aGVHD grade III-IV, the most severe grade of aGVHD in HCT patients, associated with increased risk of non-recurring mortality. A trend towards a reduced recurrence rate (11.1%) in patients receiving CD24Fc compared to patients not receiving CD24Fc was observed compared to placebo (33.3%) and contemporary control (23%), indicating that CD24Fc does not affect the GVT effect of the graft and may even reduce the risk of leukemia recurrence. The NRM was better (5.6%), better overall survival for patients exposed to CD24Fc (89% versus 50%, CD24Fc versus placebo control), statistically significant improvement in aGVHD class III-IV RFS (83% versus 33%, CD24Fc versus placebo control, respectively), severe mucositis dose-dependent reduction, and good safety profile observed in the study with only one drug-related TEAE (class III) compared to placebo group (16.7%), further supporting the benefit of incorporating CD24Fc into standard GVHD prophylactic regimen.

Prophylactic agents that reduce the risk of aGVHD and leukemia recurrence would be novel and very beneficial to leukemia patients who receive allogeneic-HCT after myelosuppressive modulation. As mentioned above, early clinical data in this application strongly suggest that administration of CD24Fc in combination with methotrexate and tacrolimus can significantly improve existing clinical-meaningful prevention regimens for the prevention of aGVHD grade III-IV and leukemia relapse, and therefore should qualify for breakthrough recognition. The effect of CD24Fc observed in phase IIa part of the clinical study, which was designed to confirm the efficacy of prophylactic CD24Fc administration of aGVHD grade III-IV and leukemia relapse in leukemia patients experiencing allogeneic-HCT following myelosuppressive regulation, will be further studied in phase IIb part.

Example 6

CD24 is useful for reducing mucositis in subjects experiencing HCT

Myelosuppressive regulation of HCT is often associated with severe regimen-related toxicities, including mucositis grade 3-4. HCT patients have reported that severe oral mucositis is the most distressing symptom they have experienced. As a measure of the effect of CD24Fc treatment on mucositis in subjects in the phase IIa GVHD prevention trial described in example 5, we generated a combined mucositis scoring system to study the results. The data is shown in figure 24A and contains multiples of the number of days that the patient experienced severe (grade 3 or 4) mucositis. Scores are provided in bar graphs, number of patients with mucositis within brackets. Oral mucositis was scored according to CTCAE 4.01. As shown in figure 24B, a dose-dependent reduction in mucositis grade-daily score was observed in all CD24Fc groups compared to placebo (R ═ -0.9983; P ═ 0.0009), and a statistically significant reduction was observed in the multiple dose group (placebo vs 960mg multiple doses: P ═ 0.035, student's t test).

Sequence listing

<110> Oncoinmmone, Inc.)

Liu, Yang

Zheng, Pan

Devenport, Martin

<120> method of using CD24 for prevention and treatment of graft versus host disease and mucositis

<130> 060275.0403.01PC00

<150> 62/680,218

<151> 2018-06-04

<150> 62/739,742

<151> 2018-10-01

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