Patent Application
20180021376
The invention relates to the isolation and purification of blood plasma, products derived therefrom, and methods of modifying levels of immune cells and related proteins in peripheral blood and organs of a treated individual.
The practice of administering antibodies or antitoxins acquired from exposed or vaccinated individuals or animals to a patient susceptible to the disease in question has been the underlying medical practice involved in passive immunotherapy since the late 1800s. Since the 1940s human albumin and other therapeutic proteins have been extracted from plasma to address specific clinical needs.
In comparison to most antibiotics, antibody-based therapies that use human antibodies have low toxicity and high specificity. The high specificity means that the antibody targets only the disease-causing microorganism that causes disease without affecting the host's endogenous organisms, therefore minimizing adverse reactions and the chance of the development of resistant organisms. This also means, however, that more than one antibody preparation may be required to target micro-organisms with high antigenic variation. Combination plasma products containing antibodies specific for a variety of diseases and afflictions, as well as therapies for administering the same, are therefore desired for addressing a range of diseases while minimizing damage to healthy cells.
The identification of various fractions of human plasma as well as the proteins residing therein and the unique characteristics of those proteins, has resulted in new life saving treatments for a variety of chronic and acute, hereditary and acquired diseases. Improvements in the manufacturing processes that ensure product safety and efficacy, and the identification of specific clinical applications require new advances in the technology.
Various techniques to remove protein aggregates, such as chromatography, have made some products more tolerable when administered intravenously and the addition of virus removal/inactivation steps has made the products essentially free of lipid and nonlipid enveloped viruses.
Intravenous immunoglobin (IVIG) is a blood product generally administered intravenously. IVIG is administered to patients with immunodeficiencies and its benefits for secondary ailments related to immunodeficiencies has made it an increasingly appealing first or second line treatment.
IVIG is manufactured from pooled human plasma and contains white blood cells called lymphocytes. A lymphocyte is any of three types of immune cells including: (1) natural killer cells (NK cells, which function in cell-mediated, cytotoxic innate immunity), (2) T cells (for cell-mediated, cytotoxic adaptive immunity), and (3) B cells (for humoral, antibody-driven adaptive immunity).
Antibodies are produced by the B-cells and plasma cells after exposure to antigens. They can be either immunoglobin G (IgG), IgA, IgM, IgE, or IgD, but in the case of hyper-immunes, IgG are the antibodies of interest. IgG consists of four polypeptide chains, two pairs of polypeptide chains, two pairs of heavy and light chains in a Y-shaped arrangement. The top ends of the IgG molecule, Fab or antibody binding region, are created from one heavy and one light chain, forming the antigen binding site. This fragment variable (Fv) region contains various amino acid combinations, which makes each antibody unique. Importantly, purified IVIG intravenous hyperimmune products contain human IgG protein, of which at least 96% is IgG containing specific antibodies against the specific antigen. Significantly, since the mid to late 2000s the need for more efficient manufacture of isolated purified IVIG has increased dramatically. Products regulating the percentages of B and T cells to target specific ailments and disease are also desirable.
The invention relates to isolated purified human immunoglobulin plasma products, methods of their manufacture and their use in treating diseases and infections such as hepatitis B virus. The purified human immunoglobulin plasma products are useful in treating a variety of chronic and acute, hereditary and acquired diseases by regulating the levels of immune cells and their related proteins in the treated subject.
In embodiments of the invention various purified blood plasma products are used to treat viral infections such as HBV by modifying lymphocyte proliferation in an individual.
Certain embodiments of the invention include the regulation of B and T cell levels in the peripheral blood and organs of a treated individual through prophylactic or therapeutic administration of purified blood plasma products.
Other embodiments include the regulation of granulocyte and macrophage levels in the peripheral blood and organs of a treated individual through prophylactic or therapeutic administration of purified blood plasma products.
In one embodiment of the invention a purified protein complex is obtained by purifying intravenous immunoglobulin G (IVIG) from human plasma fraction II+III paste.
In another embodiment of the invention a purified protein complex is obtained by purifying hepatitis B immune globulin (HBIG) from human plasma fraction II+III of donors having high antibody levels of the hepatitis B surface antigen.
In yet another embodiment a purified protein complex is formulated to combat the scarcity of the hepatitis B antibody.
In a related embodiment a method of manufacture for a purified protein complex comprises: following manufacturing protocol to separately manufacture normal immunoglobulin and hepatitis B antibody up to the step of obtaining non-sterile final bulk for both products, taking 80% normal immunoglobulin non-sterile final bulk and mixing with 20% hepatitis B antibody non-sterile final bulk, and performing sterile filtration for filling the final product.
In another embodiment the method of manufacture for a purified protein complex comprises: taking 80% of normal immunoglobulin fraction II+III and 20% hepatitis B antibody fraction II+III, and dissolving the fractions together in a process tank for production of the normal immunoglobulin until the final product is filled.
Embodiments of the invention include purified protein complexes containing various proteins having unique characteristics useful in treating infection and disease. A “KH” designation has been assigned to certain proteins contained in the purified protein complexes. Those designations, as well as additional information corresponding to those proteins is found in the figures below.
538 functions have been identified for the 55 KEI proteins, which provide them with unique characteristics for treating a wide range of disease, infection, and other cellular disturbances as expressed in some embodiments of the invention as described.
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = dock4 pe = 1 sv = 3
sapiens gn = mthfsd pe = 1 sv = 2
sapiens gn = mthfsd pe = 1 sv = 2
sapiens gn = mthfsd pe = 1 sv = 2
sapiens gn = mthfsd pe = 1 sv = 2
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = krt1 pe = 1 sv = 6
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
sapiens gn = actc1 pe = 1 sv = 1
Additional protein sequence information as well as sequence identifiers and accession numbers for KH proteins 1-55 are found in the table below.
The details of one or more embodiments of the present invention are set forth in the accompanying figures and the description below. Further areas of applicability will become apparent from the description provided herein. It should be understood that the description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.
Characterization of Lymphoid Tissues and Peripheral Blood in HBV Infected BALB/c Mice Treated with RAAS 105
Investigation was made into the effects of RAAS 105 on multiple cell lineages in lymphoid tissues and peripheral blood in HBV infected BALB/c mice. HBV infection and RAAS 105 treatment were performed by ID unit at Wuxi. At the termination, blood samples and lymphoid tissues were provided to us for analysis of various cell lineages by FACS.
Two independent experiments were performed. One experiment was to test therapeutic effects of RAAS 105 and the other experiment was to test prophylactic effects of RAAS 105.
Compared with the vehicle group, the differences observed in the animals treated with RAAS 105 therapeutically include: 1) percentages of T cells and B cells in peripheral blood, spleen and lymph nodes were decreased significantly; 2) CD62L was greatly downregulated on both CD4+ and CD8+ T cells in the spleen and lymph nodes; 3) granulocytes and monocytes/macrophages in peripheral blood and lymph nodes increased significantly; 4) the percentages of regulatory T cells (CD4+CD25+Foxp3+) in the spleen and lymph nodes were increased significantly.
However, prophylactic treatment with RAAS 105 led to somewhat different results. In the group treated with RAAS 105, T- and B-lymphocytes were also decreased. The percentages of monocytes and macrophages were increased albeit to a less degree.
These results suggested that administration of RAAS 105 had significant effects on the frequencies of immune cell lineages.
Peripheral blood was collected through cardiac puncture. After removing red blood cells with lysis buffer followed by two rounds of washing using 1×PBS, mononuclear cells (monocytes, macrophages, dendritic cells, and lymphocytes) and granulocytes were obtained. Spleen and lymph nodes cell suspension were obtained after filtering through 70 μm cell strainer. Cell viability and number were analyzed by Vi-CELL Cell Viability Analyzer followed by cell surface staining. Cells were centrifuged and resuspended in staining buffer (0.08% NaN3/PBS+1% FBS) containing appropriate fluorescent-conjugated antibodies. After 30 min incubation at 4° C. in the dark, cells were washed twice with 0.08% NaN3/PBS (200 μl per sample), and resuspended with 400 μl 0.08% NaN3/PBS in BD Falcon tubes (12×75 mm, 5 ml) followed by FACS analysis.
FACS data were analyzed by flowjo software.
To investigate the therapeutic and prophylactic effect of RAAS 105 on the immune system in mice infected with HBV, the study had divided into two parts.
The purpose of this study was to investigate the effect of RAAS 105 on cellular composition in lymphoid tissues and peripheral blood of HBV infected mice treated with RAAS 105.
Effect of Therapeutic Treatment with RAAS 105
Total 10 female BALB/c mice including 2 naïve mice at the same age were transferred from Infectious Disease (ID) Group of WuxiApptec. The group and the regimen information were shown by Table 1.
After removing red blood cells, T cell lineages, B cells, DCs, granulocytes, and monocytes/macrophages in peripheral blood were analyzed by FACS analysis.
Total T cells and B cells were characterized by CD3 and CD19, respectively. HBV infection did not change the percentages of CD3+ T cells compared with naïve mice. Therapeutic treatment of RAAS 105 reduced the percentages of both CD3+ T cells and CD1913 cells significantly (
Further analysis of the percentages of CD4+ and CD8+ (non-CD4+) T cell lineages were performed gating on total CD3+ T cells. The results showed there were no differences in the percentages of CD4+ and CD8+ T cells among all the groups (
Percentages of total CD11c+ dendritic cells (DC) and Gr-1+ granulocytes in peripheral blood were investigated. HBV infection reduced the percentages of CD11c+ DCs, a phenomenon which also be observed in human patients, whereas the percentages of Gr-1+ granulocytes were not affected. Therapeutic treatment of RAAS 105 did not show any effect on CD11c+ DCs, but increased the percentages of Gr-1+ granulocytes significantly (
Percentages of Monocytes were examined using surface marker CD11b. It increased significantly as same as Gr1+ granulocytes compared with the vehicle group (
Cell lineages in spleen including T cell lineages (CD4+/CD8+ T cells, naïve T cells, memory T cells and regulatory T cells), B cells, mDCs, pDCs, granulocytes and macrophages were characterized by cell surface and intracellular markers.
Percentages of total T cells and B cells in spleen were investigated. Therapeutic treatment of RAAS 105 reduced the percentages of both CD3+ T cells and CD19+ B cells significantly (
Further analysis of the percentages of CD4+ (non-CD8+) and CD8+ T cell lineages were performed gating on total CD3+ T cells. There were no differences in the percentages of CD4+ and CD8+ T cells among all the groups (
Three T cell lineages, naïve T cells (CD44lowCD62Lhigh), central memory T cells (TCMs, CD44highCD62Lhigh) and Effector memory T cells (TEMs, CD44highCD62Llow), were characterized by surface markers CD44 and CD62L. Percentages of these T cell lineages in CD4+ or CD8+ T cells were analyzed respectively. Both in CD4+ and CD8+ T cells, percentages of naïve T cells and TCMs decreased and TEMs increased after the therapeutic treatment of RAAS 105, suggesting the compound may have effect to promote the transformation of T cells from naïve T cells to memory T cells in spleen (
Regulatory T cells (Tregs) were analyzed by cell surface staining of anti-CD4 and anti-CD25 antibodies followed by intracellular staining of anti-Foxp3 antibody. Percents of Tregs in spleen increased compared with the vehicle group (
Dendritic cells, including myeloid dendritic cells (mDC, B220−CD11c+) and plasmacytoid dendritic cells (pDC, B220+CD11c+) in spleen were analyzed. No significant differences of mDCs and pDCs were observed among all groups (
CD11b+ macrophages and Gr-1+ granulocytes in spleen were analyzed. There were no significant alterations among all groups in the percentages of these cell lineages in spleen, as shown in
Cell lineages in draining lymph nodes including T cell lineages (CD4+/CD8+ T cells, naïve T cells, memory T cells and regulatory T cells), DCs, granulocytes and macrophages were characterized by cell surface and intracellular markers.
Percentages of total T cells in lymph nodes were analyzed. HBV infection did not affect the percentages of CD3+ T cells but therapeutic treatment of RAAS 105 reduced it significantly compared with vehicle group (
Further analysis of the percentages of CD4+ and CD8+ T cell lineages were performed gating on total CD3+ T cells. Percentages of CD4+ T cells tended to decrease while CD8+ T cells tended to increase, suggesting that therapeutic treatment of RAAS 105 may have effect on the ratio of CD4+/CD8+ T cells in lymph nodes (
Three T cell lineages, naïve T cells, TCMs and TEMs were characterized by surface markers CD44 and CD62L. Percentages of these T cell lineages in CD4+ or CD8+ T cells were analyzed respectively. The results in lymph nodes were comparable to those in spleen. Both in CD4+ and CD8+ T cells, percentages of naïve T cells and TCMs decreased and TEMs increased after the therapeutic treatment of RAAS 105, suggesting the compound also have effect to promote the transformation of T cells from naïve T cells to memory T cells in lymph nodes (
Regulatory T cells (Tregs) were analyzed. Percentages of Tregs in lymph node slightly increased without significant differences (
Total dendritic cells in lymph nodes were analyzed. Therapeutic treatment of RAAS 105 may reverse the reduction of DCs induced by HBV infection (
CD11b+ macrophages and Gr-1+ granulocytes in lymph nodes were analyzed. Both percentages of CD11b+ macrophages and Gr-1+ granulocytes increased significantly (
Effect of Prophylactic Treatment with RAAS 105
Total 14 female BALB/c mice including 2 naïve mice at the same age were transferred from Infectious Disease (ID) Group of Wuxi Apptec. The group and the regimen information were shown by Table 2.
After removing red blood cells, T cell lineages, B cells, DCs, granulocytes, and monocytes/macrophages in peripheral blood were analyzed by FACS analysis.
Total T cells and B cells were characterized. Unlike therapeutic treatment, prophylactic treatment of RAAS 105 had no effect on percentages of CD3+ T cells but reduced the percentages of CD19+ B cells although the statistical significance was not found (
Further analysis of the percentages of CD4+ and CD8+ (non-CD4+) T cell lineages were performed gating on total CD3+ T cells. Unlike therapeutic treatment, prophylactic treatment reduced percentages of CD4+ T cells and increased percentage of CD8+ T cells, suggesting the potential effect of RAAS 105 to reduce the ratio of CD4+/CD8+ T cells in peripheral blood (
Results of total CD11c+ dendritic cells (DC) and Gr-1+ granulocytes in peripheral blood were also different from those in therapeutic treatment. Prophylactic treatment of RAAS 105 reversed the reduction of DCs induced by HBV infection, but had no significant effect on granulocytes in peripheral blood (
Percentages of Monocytes were examined. There were no significant differences among all groups (
Cell lineages in spleen including T cell lineages (CD4+/CD8+ T cells, naïve T cells, memory T cells and regulatory T cells), B cells, mDCs, pDCs, granulocytes and macrophages were characterized by cell surface and intracellular markers.
Percentages of total T cells and B cells in spleen were investigated. Unlike therapeutic treatment, prophylactic treatment did not show effects on percentages of CD3+ T cells and CD19+ B cells (
Further analysis of the percentages of CD4+ (non-CD8+) and CD8+ T cell lineages were performed gating on total CD3+ T cells. Percentages of CD4+ T cells slightly decreased and CD8+ T cells slightly increased in spleen (
Naïve T cells, central memory T cells and Effector memory T cells were investigated. Percentages of these T cell lineages in CD4+ or CD8+ T cells in spleen were analyzed respectively. Both in CD4+ and CD8+ T cells, percentages of naïve T cells decreased and TEMs increased significantly after the prophylactic treatment of RAAS 105 (
Results of regulatory T cells (Tregs) were comparable with those in therapeutic treatment. Percentages of Tregs in spleen increased compared with the vehicle group by prophylactic treatment of RAAS 105 (
Dendritic cells, including mDCs and pDCs in spleen were analyzed. No significant differences of mDCs and pDCs were observed among all groups after prophylactic treatment (
CD11b+ macrophages and Gr-1+ granulocytes in spleen were analyzed. Percentages of macrophages and granulocytes increased, but no statistical differences were observed, as shown in
Cell lineages in draining lymph nodes including T cell lineages (CD4+/CD8+ T cells, naïve T cells, memory T cells and regulatory T cells), DCs, granulocytes and macrophages were characterized by cell surface and intracellular markers.
Percentages of total T cells in lymph nodes were analyzed. Similar with therapeutic treatment, HBV infection did not affect the percentages of CD3+ T cells but prophylactic treatment of RAAS 105 reduced it significantly compared with vehicle group (
Further analysis of the percentages of CD4+ and CD8+ T cell lineages were performed gating on total CD3+ T cells. Percentages of CD4+ T cells tended to decrease while CD8+ T cells tended to increase after prophylactic treatment, as was seen in therapeutic treatment (
Results of naïve T cells, central memory T cells and Effector memory T cells were totally difference with those in therapeutic treatment. Prophylactic treatment did not show significant effects on naïve T cells and TCMs, but increased percentages of TEMs (
Regulatory T cells were analyzed. There were no significant differences among all groups (
Results of total dendritic cells in lymph nodes were similar with those in therapeutic treatment. Prophylactic treatment of RAAS 105 also increased the percentages of DCs significantly compared with vehicle group (
CD11b+ macrophages and Gr-1+ granulocytes in lymph nodes were analyzed. Both macrophages and granulocytes increased significantly (
The effects of RAAS 105 on different cell lineages in lymphoid tissues and peripheral blood in HBV infected mice were investigated by FACS analysis. T cell lineages (including CD4+/CD8+ T cells, naïve T cells, memory T cells and regulatory T cells), B cells, dendritic cells (including mDCs, pDCs), granulocytes and monocytes/macrophages were analyzed. RAAS 105 was administered in two different time schedules for therapeutic and prophylactic treatment.
Therapeutic treatment revealed some interesting findings. The animals treated with RAAS 105 exhibited alterations in multiple immune cells and various lineages compared with that in the vehicle group, including reduction of lymphocytes and increase of granulocytes and monocytes. Prophylactic treatment led to less dramatic alterations in the immune cells.
The present patent application claims priority to and is a §371 national application of Patent Cooperative Treaty (“PCT”) application PCT/US15/32807, filed May 28, 2015, which claims priority to provisional application No. 62/003,664, filed May 28, 2014, which is incorporated by reference in its entirety. This application is also a continuation in part of non-provisional application Ser. No. 14/151,147, filed on Jan. 9, 2014, which is a continuation in part of non-provisional application Ser. No. 13/108,970, filed May 6, 2011, which is a continuation in part of non-provisional patent application Ser. No. 13/064,070, filed Mar. 4, 2011, each of which application is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/32807 | 5/28/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62003664 | May 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14151147 | Jan 2014 | US |
| Child | 15355304 | US | |
| Parent | 13108970 | May 2011 | US |
| Child | 14151147 | US | |
| Parent | 13064070 | Mar 2011 | US |
| Child | 13108970 | US |
