IL-6 peptide immunogen and its preparation for immunotherapy of IL-6 disorder diseases

Abstract

The present invention relates to a single peptide immunogen construct targeting a portion of the interleukin 6 (IL-6) protein, a composition comprising the construct, an antibody produced using the construct, and methods of making and using the construct and its composition. The IL-6 peptide immunogen construct of the present invention comprises a B cell epitope from IL-6, which is directly or via an optional heterologous spacer linked to a heterologous T helper cell (Th) epitope. The IL-6 peptide immunogen construct can stimulate the production of highly specific antibodies against the IL-6 receptor (IL-6R) binding site to prevent and / or treat diseases affected by IL-6 dysregulation.

Description

Technical Field

[0001] The present invention relates to peptide immunogen constructs targeting interleukin 6 (IL-6) and their preparations for use in immunotherapy of diseases affected by IL-6 dysregulation. Background Art

[0002] IL-6 is a small (~25 kD) secreted glycoprotein composed of 184 amino acids (Table 1) characterized by a four-helix bundle structure. It is produced by several cell types, including leukocytes (T and B lymphocytes, monocytes, macrophages), fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells, adipocytes, skeletal muscle cells, cardiomyocytes, brain cells (astrocytes, microglia, neurons), and some tumor cells in response to various stimuli such as lipopolysaccharide and other bacterial products, viruses, cytokines (TNF-α, IL-1, transforming growth factor (TGF)-β), adenosine triphosphate, parathyroxine, vitamin D3, homocysteine, and type II angiotensin (Sebba, 2008).

[0003] Circulating IL-6 is present in the blood of healthy individuals at low concentrations (≤1 pg / mL) and increases significantly during inflammatory responses, reaching concentrations in the ng / mL range during sepsis. IL-6 makes an important contribution to host defense against infection and tissue damage by stimulating acute phase immune responses and hematopoiesis. In addition, it regulates metabolic, regenerative, and neural processes under physiological conditions. Once released, IL-6 exerts its pleiotropic biological effects by activating the unique IL-6 receptor (IL-6R) signaling system, which includes IL-6R and downstream signaling molecules.

[0004] IL-6R is composed of two chains: (1) the IL-6 binding chain or IL-6Rα, which exists in two forms, namely (a) an 80 kD transmembrane IL-6Rα (mIL-6Rα) and (b) a 50-55 kD soluble IL-6Rα (sIL-6Rα), and (2) a 130 kD signaling chain named IL-6Rβ (or gp130).

[0005] Membrane IL-6Rα (or mIL-6Rα) is expressed on the surface of a limited number of cell types, namely hepatocytes, megakaryocytes, and leukocytes (including monocytes, macrophages, neutrophils, and T and B lymphocytes). Soluble IL-6Rα (or sIL-6Rα) is present in human plasma (25-75 ng / mL) and tissue fluids and can be generated by proteolytic cleavage (shedding) of mIL-6Rα by metalloproteinases, disintegrin metalloproteinases (i.e., ADAMs), or, to a lesser extent, by alternative splicing that deletes the transmembrane domain. Membrane IL-6Rβ is ubiquitously expressed on all human cells (Sebba, 2008).

[0006] After binding to IL-6Rα (mIL-6Rα or sIL-6Rα), IL-6 induces homodimerization of the IL-6Rα / IL-6Rβ chain, leading to the formation of a hexamer (containing two IL-6, two IL-6Rα, and two IL-6Rβ proteins), which then triggers the downstream signaling cascade (Rose-John, et al., 2017).

[0007] Cell activation by IL-6 binding to mIL-6Rα is called "classic signaling". All other cells that do not express mIL-6Rα obtain the IL-6 signal through "trans-signaling": in which IL-6 binds to circulating sIL-6Rα, and this complex forms a signaling complex with IL-6Rβ on the cell surface. Trans-signaling can occur in a wide range of human cells and therefore helps to explain the pleiotropic activities of IL-6. It is currently understood that the homeostatic and regenerative activities of IL-6 are mediated by classical signaling, while the pro-inflammatory effects are mainly caused by the activation of the trans-signaling pathway. There is increasing evidence that IL-6 trans-signaling is particularly involved in the development of the disease. A soluble form of sIL-6Rβ (or sgp130) has also been detected in the circulation at relatively high concentrations, which is mainly produced by alternative splicing. Since sIL-6Rβ can bind to the IL-6 / sIL-6Rd complex, it acts as a natural and specific inhibitor of IL-6-mediated trans-signaling, while conventional signaling is not affected by sIL-6Rβ.

[0008] Although IL-6Rd is the unique binding receptor for IL-6, the IL-6Rβ (or gp130) signaling chain is shared by IL-6 family members (including leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, IL-11, cardiotrophin-1, neurogenin-1, IL-27, and IL-35).

[0009] Following IL-6 binding, receptor homodimerization promotes interaction between the IL-6Rβ (or gp130) chain and the tyrosine kinase JAK (Janus kinase), leading to their mutual transactivation. Subsequently, JAK activation triggers three major intracellular signal transduction pathways through phosphorylation of two key proteins, namely (1) Src homology domain-containing protein tyrosine phosphatase-2 (SHP-2), and (2) signal transducer and activator of transcription proteins (STAT1-STAT3). After being phosphorylated, SHP-2 can interact with Grb2 (growth factor receptor-bound protein 2), leading to activation of the Ras / ERK / MAPK (rat sarcoma protein / extracellular signal-regulated kinase / mitogen-activated protein kinase) cascade; and / or activation of the PI3K / Akt (phosphoinositide-3 kinase / protein kinase B) pathway. Conversely, phosphorylation of STATs proteins induces the formation of heterodimers (STAT1 / STAT3) or homodimers (STAT1 / STAT1 and / or STAT3 / STAT3), which subsequently translocate to the nucleus. In all cases, activation of these intracellular pathways leads to the induction of transcription of multiple target genes that are responsible for the pleiotropic biological activities of IL-6 (Lazzerini, et al., 2016).

[0010] IL-6 exhibits a wide range of biological activities, critically involved in the activation of acute inflammatory responses and the switch from innate to adaptive immunity. IL-6 has a variety of other roles in a variety of other processes, including metabolism, cognitive function, and embryonic development.

[0011] IL-6 has been studied for its effects on the activation of acute inflammatory responses. When infection or tissue damage occurs from various factors, a systemic acute phase response is rapidly induced to neutralize the pathogen and prevent its further invasion, minimize tissue damage and promote wound healing. This acute phase response, which consists of fever and acute phase proteins produced by hepatocytes, is primarily driven by IL-6.

[0012] To this end, IL-6 increases body temperature by acting on neurons in the preoptic hypothalamic region involved in thermoregulation and stimulates the synthesis of acute phase proteins in the liver (e.g., C-reactive protein (CRP), fibrinogen, complement factor C3, serum amyloid A, hepcidin, hemopexin, α1-acid glycoprotein, α1-antitrypsin, α1-antichymotrypsin, and ceruloplasmin), while the production of albumin, transferrin, fibronectin, transthyretin, and retinol-binding protein (“negative” acute phase proteins) is inhibited.

[0013] In addition, IL-6 promotes the differentiation of monocytes into macrophages, stimulates the maturation of myeloid precursor cells and megakaryocytes leading to neutrophilia and thrombocytosis, induces angiogenesis through the production of vascular endothelial growth factor, enhances the trafficking of lymphocytes and neutrophils by upregulating the expression of adhesion molecules on endothelial cells (particularly, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1)), increases antibody production by B lymphocytes, and enhances the proliferation of T helper (TH) lymphocytes promoting their differentiation into TH2 or TH17 cells. In all cases, these changes contribute through different but synergistic mechanisms to achieve an overall response that ultimately serves as host defense.

[0014] In addition to its critical involvement in immune-inflammatory responses, IL-6 plays important roles in physiological conditions by regulating many multisystem functions, such as embryogenesis, glucose and lipid metabolism, bone remodeling, liver regeneration, neural tissue homeostasis, cognitive function, sleep, memory, pain, and emotional behavior.

[0015] Knowledge beyond these immunoinflammatory effects may help explain the pathogenic mechanisms of some of the systemic manifestations observed in rheumatoid arthritis (RA) and other chronic inflammatory diseases characterized by persistently elevated IL-6 levels.

[0016] In RA patients, there is particular interest in the effects of this cytokine on metabolism and bone homeostasis, both pathophysiologically and clinically.

[0017] Adipose tissue contributes significantly to the production of IL-6 under physiological conditions, accounting for about 35% of the circulating IL-6 level. In long-term exercise, contraction of skeletal muscle becomes the main source of IL-6, and its plasma level increases up to 100 times. IL-6 stimulates lipolysis in adipocytes (and inhibits lipogenesis), and by increasing the expression of very low-density lipoprotein receptors, increases the intake of cholesterol and triglycerides by peripheral tissues, promotes weight loss and reduces serum lipid levels. In addition, IL-6 enhances the insulin sensitivity of liver cells and muscle cells, improves glucose utilization and tolerance. Although these effects indicate that this cytokine may be part of the physiological mechanism of increased insulin activity induced by exercise, thereby enhancing endurance. However, the chronic increase of IL-6 caused by, for example, long-term excessive exercise can lead to insulin resistance in liver and adipocytes.

[0018] Regarding the effects on bone tissue, IL-6 affects bone resorption and formation required for skeletal development, growth, and maintenance by regulating the differentiation and activity of osteoblasts, osteoclasts, and chondrocytes. IL-6 enhances the expression of receptor activator of nuclear factor-kB ligand (RANKL) on the surface of matrix / osteoblasts, thereby stimulating osteoclast differentiation and bone resorption, which can promote bone remodeling and has a potential positive effect on bone homeostasis under physiological conditions. In RA, its exaggerated and persistent activation induces abnormal osteoclastogenesis, leading to osteoporosis and bone destruction.

[0019] A recent review on IL-6, IL-6 receptor, IL-6 signaling, pleiotropic biological effects of IL-6, impact on immunoinflammatory responses, effects beyond immunoinflammatory, and the role of IL-6 in various pathological states including rheumatoid arthritis, development of autoimmune processes, joint damage, extra-articular manifestations is cited here as a reference (Lazzerini, P., et al., 2016), where supporting documents can be found within the statements in the background section above.

[0020] Because IL-6 is a pleiotropic cytokine that participates in the physiology of nearly every organ system and plays a major role in the response to injury or infection, aberrant expression of IL-6 has been implicated in a variety of human diseases, especially inflammatory and autoimmune diseases, coronary and neurological disorders, pregnancy problems, and tumors.

[0021] There is interest in developing antibodies that inhibit IL-6 from binding to the IL6R (i.e., IL-6Rα and IL-6Rβ / or gp130) complex as a therapy for many of these diseases. The first such antibody developed was Tocilizumab, followed by Sarilumab, both of which target IL-6R and have been approved for the treatment of rheumatoid arthritis, Castleman's disease, and systemic juvenile idiopathic arthritis. Siltuximab is a monoclonal antibody targeting IL-6, which is currently the only therapy approved by the U.S. FDA for idiopathic Multicentric Castleman's disease (MCD). Sirukumab is another high-affinity anti-IL-6 monoclonal antibody designed to block the IL-6 pathway in adults with moderate to severe rheumatoid arthritis, but was not approved by the FDA's Arthritis Advisory Committee due to the increased mortality in patients taking this drug. Many other anti-IL-6 or anti-IL-6R monoclonal antibodies have been actively explored for this purpose (discussed in Rose-John, et al., 2017). Although many monoclonal anti-IL-6 or anti-IL-6 receptor antibodies may prove effective in the immunotherapy of certain diseases, they are expensive and must be administered frequently and chronically to maintain adequate suppression of serum IL-6 and the clinical benefit derived therefrom.

[0022] Several vaccination approaches have also been described for combating IL-6-related immune diseases. One approach uses a human IL-6 variant with seven amino acid substitutions, Sant1 (De Benedetti, et al., 2001; U.S. Patent No. 6,706,261), as an immunogen. However, this work has not led to any clinical development since its initial disclosure nearly two decades ago. Another team (Desallais, L., et al., 2014) reported the use of five randomly selected IL-6 peptides (these peptides cover more than 40% of the IL-6 protein with a length of 184 residues) that were linked to the large carrier protein KLH using a complex chemical coupling procedure to enhance the immunogenicity of each peptide. The antibodies produced by the vaccine prepared under this method are mainly directed against the non-desired carrier protein KLH, and only a small part is targeted to IL-6. In addition, this vaccine requires Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (ICFA) in the formulation, which is not ideal for clinical and industrial applications.

[0023] As described above, there are many limitations and problems with current IL-6 vaccine designs (e.g., complex chemical coupling procedures for immunogen preparation, use of KLH as a carrier protein where most of the elicited antibodies are directed against the carrier protein, use of Freund's complete adjuvant, which is not clinically permitted, to enhance the immunogenicity of vaccination, weak immunogenicity against the target IL-6 in vaccinated animals despite the most aggressive immunization schedules, unclear mechanisms of action, etc.). Therefore, there is a clear need to develop an effective immunotherapeutic vaccine that can elicit a highly specific immune response against IL-6, is easy to administer to patients, can be manufactured under strict good manufacturing practices (GMP), and is cost-effective for worldwide use to treat patients with diseases affected by IL-6 dysregulation.

[0024] The main purpose of the present invention is to produce / develop IL-6 peptide immunogen constructs and vaccine preparations thereof, wherein the B cell epitope of the designed peptide immunogen construct is very similar to the IL-6R binding site on IL-6, and such peptide immunogen constructs and preparations thereof can generate a strong immune response in the vaccinated host to allow breaking through the immune tolerance to the self-protein IL-6 and generating effective antibodies targeting the IL-6R binding site for the treatment of diseases affected by IL-6 disorder. Summary of the invention

[0025] The present invention relates to single peptide immunogen constructs targeting interleukin 6 (IL-6) and their formulations for use in immunotherapy of diseases affected by IL-6 dysregulation.

[0026] These individual IL-6 peptide immunogen constructs are 30 or more amino acids in length and contain functional B cell epitopes derived from the IL-6 receptor binding region E42-C83 (SEQ ID NO: 16) or N144-I166 (SEQ ID NO: 19) or fragments thereof, which are linked to heterologous T helper cell (Th) epitopes derived from pathogen proteins via spacer residues. These IL-6 peptide constructs (containing designed B cell and Th cell epitopes acting together) stimulate the production of highly specific antibodies against the IL-6R binding region, providing a therapeutic immune response for individuals susceptible to or suffering from diseases affected by IL-6 dysregulation.

[0027] In some embodiments, the IL-6 peptide immunogen construct of the present invention comprises a hybrid peptide having a B cell epitope derived from an IL-6R binding region or a fragment thereof (SEQ ID NOs: 5-20, 72-74, as shown in Table 1), which is linked to a heterologous Th epitope derived from a pathogen protein (SEQ ID NOs: 78-106 and 216-226, as shown in Table 2), which together act to stimulate the production of highly specific antibodies that cross-react with recombinant human IL-6 (SEQ ID NO: 1) or IL-6 of other species, such as macaque (SEQ ID NO: 2), mouse (SEQ ID NO: 3), and rat IL-6 (SEQ ID NO: 4).

[0028] References

[0029] 1. CHANG, JCC, et al., "Adjuvant activity of incomplete Freund's adjuvant," Advanced Drug Delivery Reviews, 32(3): 173-186 (1998)

[0030] 2.CILIBERTO, G., et al., "Compositions and methods comprising immunogenic muteins of interleukin-6", US Patent No. 6,706,261 (2004)

[0031] 3. DE BENEDETTI, F., et al., "In Vivo Neutralization of Human IL-6 (hIL-6) Achieved by Immunization of hIL-6-Transgenic Mice with a hIL-6ReceptorAntagonist", J. Immunol. 166: 4334-4340 (2001)

[0032] 4. DESALLAIS, L., et al., "Method for treating chronic colitis and systemic sclerosis for eliciting protective immune reaction against human IL-6". US Patent No. 9, 669, 077 (2017)

[0033] 5.DESALLAIS,L.,et al.,“Immunization against an IL-6peptide inducesanti-IL-6antibodies and modulates the Delayed-Type-Hypersensitivity reactionin cynomolgus monkeys”,Scientific Reports.6:19549;doi:10.1038 / srep19549(2016)

[0034] 6.DESALLAIS,L.,et al.,“Targeting IL-6by both passive and activeimmunization strategies prevents bleomycin-induced skin fibrosis”,ArthritisResearch and Therapy.16:R157(2014)

[0035] 7.FIELDS,G.B.,et al.,Chapter 3 in Synthetic Peptides:A User’s Guide,ed.Grant,W.H.Freeman&Co.,New York,NY,p.77(1992)

[0036] 8.LAZZERINI,P.,et al.,“Spotlight on sirukumab for the treatment ofrheumatoid arthritis:the evidence to date”,Drug Design,Development andTherapy,10:3083-3098;website:doi.org / 10.2147 / DDDT.S99898)(2016)

[0037] 9.MIHARA,M.,et al.,“IL-6 / IL-6receptor system andits role inphysiological and pathological conditions”,Clinical Science,122(4):143-159(2012)

[0038] 10.ROSE-JOHN, S., et al., "The role of IL-6 in host defense against infections: immunobiology and clinical implications", Nature ReviewsRheumatology, 13: 399-409 (2017)

[0039] 11.SEBBA, A., "Tocilizumab: The first interleukin-6-receptor inhibitor", American Journal of Health-System Pharmacy, 65(15): 1413-1418(2008)

[0040] 12.TRAGGIAI, E., et al., "An efficient method to make human monoclonalantibodies from memory B cells: potent neutralization of SARS coronavirus", Nature Medicine, 10(8): 871-875(2004) BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A sequence alignment of IL-6 sequences from human (SEQ ID NO: 227), cynomolgus (SEQ ID NO: 228), mouse (SEQ ID NO: 229) and rat (SEQ ID NO: 230) species is shown. Intramolecular loops at amino acid positions 44-50 and 73-83 are shown with shaded cysteines and brackets.

[0042] Figure 2 A flow chart showing the development process for identifying commercialization (industrialization) of pharmaceutical compositions directed to selected targets according to specific embodiments of the present invention herein. The present invention includes IL-6 peptide immunogen design, IL-6 peptide composition design, IL-6 pharmaceutical dosage form design, in vitro IL-6 functional antigenicity study design, in vivo IL-6 immunogenicity and efficacy study design, and IL-6 therapeutic clinical trial protocol design. Detailed evaluation and analysis of each step has led to a series of experiments that will ultimately lead to the commercialization of safe and effective IL-6 pharmaceutical compositions.

[0043] Figure 3A-3D shows graphs illustrating the kinetics of antibody responses over a 12-week period in guinea pigs immunized with different IL-6 peptide immunogen constructs. Specifically, antibody responses to SEQ ID NOs: 107, 112-114, and 116-118 peptide immunogen constructs are shown in Figure 3A; antibody responses to SEQ ID NOs: 124-130 peptide immunogen constructs are shown in Figure 3B; antibody responses to SEQ ID NOs: 131-137 peptide immunogen constructs are shown in Figure 3C; and antibody responses to SEQ ID NOs: 138-145 peptide immunogen constructs are shown in Figure 3D. ELISA plates were coated with recombinant human IL-6. Sera were diluted from 1:100 to 1:4.19x10 by 4-fold serial dilution. 8 The titer of the test serum was calculated using nonlinear regression with a four-parameter logistic curve-fit and expressed as Log 10 (EC 50 ).

[0044] Figures 4A-4B show the cross-reactivity of various purified polyclonal anti-IL-6 antibodies against different IL-6 peptide immunogen constructs. Specifically, the results for SEQ ID NOs: 107, 116, 118, 124-128 are shown in Figure 4A, while the results for SEQ ID NOs: 129-133 are shown in Figure 4B. ELISA plates were coated with recombinant human, monkey, mouse or rat IL-6 protein. Polyclonal anti-IL-6 IgG antibodies purified from guinea pig serum by protein A chromatography were diluted from 10 μg / mL to 0.00238 ng / mL using a 4-fold serial dilution. EC were calculated using nonlinear regression with a 4-parameter logit curve fit. 50 .

[0045] Figure 5ANeutralizing activity of various purified polyclonal anti-IL-6 antibodies generated using different IL-6 peptide immunogen constructs (SEQ ID NOs: 107, 116, 118, 124, 132, 133, 124 and 137 and GP IgG) against the interaction of IL-6 and IL-6R in an ELISA-based assay is shown. ELISA plates were coated with recombinant human IL-6R protein. Human IL-6 at a concentration of 10 ng / ml and various polyclonal anti-IL-6 IgG antibodies (various polyclonal anti-IL-6 IgG antibodies were prepared in decreasing concentrations from 100 to 0.412 μg / mL using 3-fold serial dilutions) were premixed and then added to the IL-6R-coated wells. Captured IL-6 was detected using biotinylated rabbit anti-IL-6 antibody and then detected using streptavidin poly-HRP. IC was calculated by nonlinear regression using 4-parameter logit curve fitting. 50 .

[0046] Figure 5B Neutralizing activity of various purified polyclonal anti-IL-6 antibodies generated using different IL-6 peptide immunogen constructs (SEQ ID NOs: 128, 129, 134 and 135 and GP IgG) against the interaction of IL-6 / IL-6R and gp130 in an ELISA-based assay is shown. ELISA plates were coated with recombinant gp130-Fc fusion protein. Preformed IL-6 / IL-6R complexes and various polyclonal anti-IL-6 IgG antibodies (various polyclonal anti-IL-6 IgG antibodies were prepared in decreasing concentrations from 100 to 0.412 μg / mL using 3-fold serial dilutions) at predetermined ratios were premixed and then added to the gp130-Fc coated wells. Captured IL-6 / IL-6R complexes were detected using biotinylated rabbit anti-IL-6 antibody and then detected using streptavidin poly-HRP. IC was calculated using nonlinear regression with 4-parameter logit curve fitting. 50 .

[0047] Figure 6 Neutralizing activity of various purified polyclonal anti-IL-6 antibodies generated using different IL-6 peptide immunogen constructs (SEQ ID NOs: 116, 124, 127, 128, 129, 134, 135 and 137 and GP IgG) against IL-6-dependent STAT3 phosphorylation in an ELISA-based assay is shown. RMPI 8226 cells were incubated with human IL-6 (at a concentration of 10 ng / mL) and various polyclonal anti-IL-6 IgG antibodies (at a concentration of 100 μg / mL) at 37° C., 5% CO2 for 30 minutes and then lysed to determine the level of phosphorylated STAT3 in an ELISA-based assay.

[0048] Figures 7A-7B show the neutralizing activity of various purified polyclonal anti-IL-6 antibodies generated using different IL-6 peptide immunogen constructs against IL-6-dependent cell proliferation. The neutralizing activity of SEQ ID NOs: 116, 118, 124-129, 131, 132 and 133 is shown in Figure 7A, while the neutralizing activity of SEQ ID NOs: 134-145 is shown in Figure 7A. Figure 7B TF-1 cells were incubated with human IL-6 (at a concentration of 10 ng / mL) and various polyclonal anti-IL-6 IgG antibodies at the indicated concentrations for 72 h at 37°C, 5% CO2. Cell viability was monitored by measuring the amount of ATP in metabolically active cells.

[0049] Figures 8A-8B Neutralizing activity of various purified polyclonal anti-IL-6 antibodies generated using different IL-6 peptide immunogen constructs against IL-6-induced MCP-1 secretion is shown. The neutralizing activity of SEQ ID NOs: 116, 118, 124-134 and 136 is shown in Figure 8A, while the neutralizing activity of SEQ ID NOs: 138-145 is shown in Figure 8B. Figure 8B U937 cells were incubated with human IL-6 (at a concentration of 10 ng / mL) and various polyclonal anti-IL-6 IgG antibodies at the indicated concentrations for 24 hours at 37°C, 5% CO2. The culture supernatants were collected to determine the MCP-1 level.

[0050] Fig. 9 The experimental design for the evaluation of the efficacy of rat IL-6 peptide constructs in collagen-induced arthritis (CIA) in rats using a preventive model is shown. Female Lewis rats (n=7 per group) were immunized intramuscularly with a dose of 45 μg of the peptide immunogen construct SEQ ID NOs: 148 or 157 on days -31, -10, and 4. To induce arthritis, animals were challenged intradermally with bovine type II collagen / IFA emulsion on days 0 and 7.

[0051] Fig.10 The kinetics of antibody responses over a 28-day period are shown in rats immunized with different IL-6 peptide immunogen constructs (SEQ ID NOs: 148 and 157). ELISA plates were coated with recombinant rat IL-6. Sera were diluted from 1:100 to 1:4.19 x 10 8 The titer of the test serum was calculated using nonlinear regression with a 4-parameter logit curve fit and expressed as Log 10 (EC 50 ).

[0052] Fig.11 Shown is a reduction in arthritis scores in collagen-induced rats previously immunized with different IL-6 peptide immunogen constructs (SEQ ID NOs: 148 and 157).

[0053] Fig.12 Shown is a reduction in hind paw swelling in collagen-induced rats previously immunized with different IL-6 peptide immunogen constructs (SEQ ID NOs: 148 and 157).

[0054] Fig.13 Shown is the reduction of blood neutrophilia in collagen-induced rats previously immunized with different IL-6 peptide immunogen constructs (SEQ ID NOs: 148 and 157).

[0055] Fig.14 The experimental design for the evaluation of the efficacy of rat IL-6 peptide immunogen constructs in collagen-induced arthritis (CIA) in rats using a therapeutic model is shown. Female Lewis rats (n=6 or 7 per group) were immunized intramuscularly with SEQ ID NO: 148 at a dose of 45 μg on days -7, 7, 14, 21, and 28. To induce arthritis, animals were induced intradermally with bovine type II collagen / IFA emulsion on days 0 and 7.

[0056] Fig.15 The kinetics of antibody responses over a 43-day period are shown in rats immunized with SEQ ID NO: 148 formulated with ISA 51 or ISA 51 / CpG. ELISA plates were coated with recombinant rat IL-6. Sera were diluted from 1:100 to 1:4.19 x 10 8 The titer of the test serum was calculated using nonlinear regression with a 4-parameter logit curve fit and expressed as Log 10 (EC 50 ).

[0057] Fig.16 Shown is a reduction in arthritis scores and hind paw swelling in collagen-induced rats previously immunized with SEQ ID NO: 148 formulated with ISA 51 or ISA 51 / CpG.

[0058] Fig.17 Shown is a reduction in hepatic C-reactive protein (CRP) in rats previously immunized with collagen induced with SEQ ID NO: 148 formulated with ISA 51 or ISA 51 / CpG.

[0059] Fig.18Shown is a reduction in ankle joint destruction in rats previously immunized with collagen induced with SEQ ID NO: 148 formulated with ISA 51 or ISA 51 / CpG.

[0060] Fig.19 Shown is a reduction in tissue inflammatory cytokine (TNF-α, IL-17 and MCP-1) production in collagen-induced rats previously immunized with SEQ ID NO: 148 formulated with ISA 51 or ISA 51 / CpG.

[0061] Fig. 20 Shown are the kinetics of antibody responses over a 43-day period in rats immunized with different doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG. Fig.17 The same experimental design was used for this study. Recombinant rat IL-6 was used to coat the ELISA plates. The serum was diluted from 1:100 to 1:4.19x10 8 The titer of the test serum was calculated by incorporating a cutoff value of 0.45 into a 4-parameter logit curve for each serum sample generated using nonlinear regression and expressed as Log 10 .

[0062] Fig.21 Shown is a reduction in collagen-induced weight loss in rats immunized with increasing doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG.

[0063] Fig. 22 Shown is a reduction in collagen-induced swelling in rats immunized with increasing doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG.

[0064] Fig.23 Shown are macroscopic observations of the hind paws at day 24 of collagen-primed rats immunized with increasing doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG.

[0065] Fig.24 Shown is a reduction in arthritis scores in collagen-induced rats immunized with increasing doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG.

[0066] Fig.25Shown is the reduction of blood neutrophilia in collagen-induced rats immunized with increasing doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG.

[0067] Fig.26 Shown is a reduction in collagen-induced platelet release in rats immunized with increasing doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG.

[0068] Fig. 27 Shown is a reduction in the increase in blood AST induced by collagen in rats immunized with increasing doses of SEQ ID NO: 148 formulated with ISA 51 / CpG or ADJU-PHOS / CpG.

[0069] Fig.28 Shown are the kinetics of antibody responses over a 12-week period in guinea pigs immunized with different IL-6 peptide immunogen constructs (SEQ ID NOs: 102, 114, 115, 117, and 118) formulated with the indicated adjuvants. ELISA plates were coated with recombinant human IL-6. Sera were diluted from 1:100 to 1:4.19x10 8 The titer of the test serum was calculated using nonlinear regression with a 4-parameter logit curve fit and expressed as Log 10 (EC 50 ).

[0070] Implementation

[0071] The present invention relates to single peptide immunogen constructs targeting interleukin 6 (IL-6) and their formulations for use in immunotherapy of diseases affected by IL-6 dysregulation.

[0072] The IL-6 peptide immunogen constructs of the present invention have 30 or more amino acids, comprising functional B cell epitopes derived from the IL-6 receptor (IL-6R) binding region E42-C83 (SEQ ID NO: 16) or N144-I166 (SEQ ID NO: 19) or fragments thereof (SEQ ID NOs: 5-19), which are linked to heterologous T helper cell (Th) epitopes derived from pathogen proteins (SEQ ID NOs: 78-106 and 216-226) via an optional heterologous spacer. These IL-6 peptide constructs (containing designed B cell and Th cell epitopes acting together) stimulate the production of highly specific antibodies against the IL-6R binding region, providing a prophylactic and / or therapeutic immune response for patients suffering from or susceptible to diseases affected by IL-6 dysregulation. The IL-6 peptide immunogen construct of the present invention may comprise a hybrid peptide having a B cell antigenic site derived from the IL-6R binding region or a fragment thereof (SEQ ID NOs: 5-20; 72-75), which is linked to a heterologous Th epitope derived from a pathogen protein (e.g., SEQ ID NOs: 78-106 and 216-226 of Table 2), which together act to stimulate the production of highly specific antibodies that cross-react with recombinant human IL-6 (SEQ ID NO: 1) or IL-6 of other species (e.g., macaque (SEQ ID NO: 2), mouse (SEQ ID NO: 3), and rat IL-6 (SEQ ID NO: 4)).

[0073] In some embodiments, the IL-6 peptide immunogen construct (e.g., SEQ ID NOs: 107-186 in Table 3) contains a hybrid peptide having a B cell antigenic site, such as C73-C83 (SEQ ID NO: 5), which is connected to a heterologous Th epitope derived from proteins of various pathogens (SEQ ID NOs: 78-106 and 216-226), and can elicit antibodies that cross-react with recombinant human IL-6 (SEQ ID NO: 1), and it has cross-reaction with IL-6 of other species (SEQ ID NOs: 2, 3, 4). Among the heterologous Th epitopes used to enhance B cell antigenic sites, idealized artificial Th epitopes derived from natural pathogens EBV BPLF1 (SEQ ID NO: 105), EBV CP (SEQ ID NO: 102), Clostridium tetani 1, 2, 4 (SEQ ID NOs: 78, 99-101), cholera toxin (SEQ ID NO: 85), Schistosoma mansoni (SEQ ID NO: 84) and those derived from measles virus fusion protein (MVF 1 to 5) and hepatitis B surface antigen (HBsAg 1 to 3) are particularly useful in such B cell antigenic enhancement through immunogenicity screening tests in the form of single sequences or combined sequences (e.g., SEQ ID NOs: 79, 86-92).

[0074] The present invention also relates to peptide compositions comprising a mixture of IL-6 peptide immunogen constructs and heterologous Th epitopes derived from different pathogens. Compositions comprising a mixture of IL-6 peptide immunogen constructs can be used to allow coverage of a wide range of genetic backgrounds in patients, resulting in a higher percentage of response rates after immunization, for the prevention and / or treatment of diseases affected by IL-6 disorders. When a peptide composition containing more than one IL-6 peptide immunogen construct is used, a synergistic enhancement of the immune response can be observed.

[0075] The antibody responses derived from this peptide immunogen construct were mostly (>90%) focused on the desired cross-reactions against the IL-6R binding region peptides (SEQ ID NOs: 5-19), with little, if any, cross-reactions against the heterologous Th epitopes used for immunogenicity enhancement. This is in stark contrast to standard approaches using conventional carrier proteins such as KLH, toxoids or other biological carriers for antigenic enhancement of such peptides.

[0076] The present invention also relates to pharmaceutical compositions, including formulations for preventing and / or treating diseases affected by IL-6 dysregulation. In some embodiments, the pharmaceutical composition comprises a stabilized immunostimulatory complex formed by mixing a CpG oligomer with a peptide composition containing a mixture of IL-6 peptide immunogen constructs through electrostatic binding to further enhance the immunogenicity of the IL-6 peptide, which contributes to the desired cross-reactivity with full-length human IL-6 (SEQ ID NO: 1) or with IL-6 of other species, such as macaque (SEQ ID NO: 2), mouse (SEQ ID NO: 3) and rat (SEQ ID NO: 4).

[0077] In other embodiments, the pharmaceutical composition comprises a peptide composition in which the IL-6 peptide immunogen construct is contacted with a mineral salt (including ALHYDROGEL or ADJU-PHOS) to form a suspension or contacted with MONTANIDE ISA51 or 720 as an adjuvant to form a mixture of a water-in-oil emulsion for administration to a patient to prevent and / or treat a disease affected by IL-6 disorder.

[0078] In addition, the present invention also provides methods for low-cost manufacturing and quality control of IL-6 peptide immunogen constructs and preparations thereof, which are used to prevent and / or treat animals and patients suffering from diseases affected by IL-6 disorder.

[0079] The present invention also relates to antibodies against the IL-6 peptide immunogen constructs of the present invention. In particular, the IL-6 peptide immunogen constructs of the present invention are capable of stimulating the production of highly specific antibodies that cross-react with the IL-6R binding site on the IL-6 molecule. The antibodies of the present invention utilize high specificity to bind to the IL-6R binding site, and there are not many, if any, heterologous Th epitopes for immunogenic enhancement, which is in sharp contrast to antibodies made using conventional proteins or other biological protein carriers for antigenic enhancement of such peptides. Therefore, compared to other peptide or protein immunogens, the IL-6 peptide immunogen constructs of the present invention are capable of breaking immune tolerance to self-IL-6 proteins and have a high response rate.

[0080] In certain embodiments, the antibody is directed against and specifically binds to the IL-6R binding site on the human IL-6 molecule (SEQ ID NO: 1). Highly specific antibodies elicited by the IL-6 peptide immunogen construct can inhibit the binding of IL-6 and IL-6R, as well as downstream events (e.g., IL-6-induced STAT3 phosphorylation, IL-6-dependent cell proliferation, IL-6-induced MCP production and other IL-6-related pathological states), resulting in effective treatment of patients with diseases affected by IL-6 dysregulation.

[0081] In another aspect, the present invention provides human antibodies (polyclonal and monoclonal) against IL-6 induced in patients receiving a composition containing an IL-6 peptide immunogen construct of the present invention. Traggiai et al. (2004) describe an efficient method for preparing human monoclonal antibodies from B cells isolated from the blood of human patients, the disclosure of which is incorporated herein by reference in its entirety.

[0082] Based on their unique characteristics and properties, the antibodies of the present invention elicited by the IL-6 peptide immunogen constructs can provide an immunotherapeutic approach to treat patients suffering from diseases affected by IL-6 dysregulation.

[0083] The present invention also relates to methods for preparing the IL-6 peptide immunogen constructs, compositions and antibodies of the present invention. The methods of the present invention provide for low-cost manufacturing and quality control of IL-6 peptide immunogen constructs and compositions containing the constructs, which can be used in methods for treating patients suffering from diseases affected by IL-6 disorders. The methods of the present invention can provide for low-cost manufacturing and quality control of IL-6 peptide immunogen constructs and compositions containing the constructs, which can be used in methods for treating patients suffering from diseases affected by IL-6 disorders.

[0084] The present invention also includes methods for preventing and / or treating diseases affected by IL-6 disorders in individuals using the IL-6 peptide immunogen constructs of the present invention and / or antibodies against the IL-6 peptide immunogen constructs. The methods of the present invention include the step of administering to an individual a composition containing the IL-6 peptide immunogen construct of the present invention. In some embodiments, the composition used in the method contains the IL-6 peptide immunogen construct of the present invention, and the IL-6 peptide immunogen construct is electrostatically bound to a negatively charged oligonucleotide (e.g., a CpG oligomer) to form a stable immunostimulatory complex, which can be further added with an adjuvant for administration to an individual susceptible to or suffering from a disease affected by IL-6 disorders.

[0085] The methods of the present invention also include dosing regimens, dosage forms, and routes of administration for administering the peptide immunogenic constructs to prevent and / or treat diseases affected by IL-6 dysregulation.

[0086] definition

[0087] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.All references, or portions of references, cited in this application are expressly incorporated herein by reference in their entirety for any purpose.

[0088] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Unless the context clearly indicates otherwise, the words "a", "an" and "the" include plural forms. Similarly, the expression "or" means including "and", unless the context clearly indicates otherwise. Therefore, "comprising A or B" means including A, or B, or A and B. It should be further understood that all amino acid sizes and all molecular weights or molecular mass values ​​for a given polypeptide are approximate and are provided as descriptions. However, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods of the present invention, suitable methods and materials described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference as a whole. In the event of a conflict, this specification (including the explanation of the terms) shall prevail. In addition, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0089] IL-6 peptide immunogen constructs

[0090] The present invention provides peptide immunogen constructs containing B cell epitopes having an amino acid sequence from the IL-6 receptor (IL-6R) binding region E42-C83 (SEQ ID NO: 16) or N144-I166 (SEQ ID NO: 19) or a fragment thereof (e.g., SEQ ID NOs: 5-19). The B cell epitope is covalently linked directly or through an optional heterologous spacer to a heterologous T helper cell (Th) epitope derived from a pathogen protein. These constructs (containing designed B cell and Th cell epitopes acting together) stimulate the production of highly specific antibodies against the IL-6R binding region on IL-6, providing a therapeutic immune response for patients susceptible to or suffering from a disease affected by IL-6 dysregulation.

[0091] The term "IL-6 peptide immunogen construct" or "peptide immunogen construct" as used herein refers to a peptide having a length of more than 30 amino acids, which contains (a) a B cell epitope having about 10 or more consecutive amino acid residues from the IL-6R binding region of full-length human IL-6 (SEQ ID NO: 1) (represented by peptide E42-C83 (SEQ ID NO: 16) or N144-I166 (SEQ ID NO: 19) or a fragment thereof (e.g., SEQ ID NOs: 5-19); (b) a heterologous Th epitope; and (c) an optional heterologous spacer.

[0092] In certain embodiments, the IL-6 peptide immunogen construct can be represented by the following molecular formula:

[0093] (Th)m -(A) n -(IL-6R binding region of IL-6 or a fragment thereof)-X

[0094] or

[0095] (IL-6R binding region of IL-6 or a fragment thereof)-(A) n -(Th) m -X

[0096] or

[0097] (Th) m -(A) n -(IL-6R binding region of IL-6 or a fragment thereof)-(A) n -(Th) m -X

[0098] in

[0099] Th is a heterologous T helper cell epitope;

[0100] A is a heterologous spacer;

[0101] (IL-6R binding region of IL-6 or a fragment thereof) is a B cell epitope peptide having 7 to 42 amino acid residues from the IL-6R binding region of IL-6 (SEQ ID NO: 1);

[0102] X is α-COOH or α-CONH2 of an amino acid;

[0103] m is from 1 to about 4; and

[0104] n is 0 to about 10.

[0105] The IL-6 peptide immunogen constructs of the present invention were designed and selected based on a number of theoretical bases, including:

[0106] i. The IL-6 B cell epitope peptide itself is non-immunogenic to avoid autologous T cell activation;

[0107] ii. IL-6 B cell epitope peptides can be made immunogenic by using protein carriers or effective T helper cell epitopes;

[0108] iii. When the IL-6 B cell epitope peptide becomes immunogenic and is administered to a host, the peptide immunogen construct:

[0109] a. Induce high titer antibodies that preferentially target IL-6 peptide sequences (B cell epitopes) rather than protein carrier or T helper cell epitopes;

[0110] b. destroying immune tolerance in the immunized host and producing highly specific antibodies that cross-react with IL-6 (SEQ ID NO: 1);

[0111] c. Produce highly specific antibodies that can inhibit the binding of IL-6 and IL-6R and downstream events (e.g., IL-6-induced STAT3 phosphorylation, IL-6-dependent cell proliferation, IL-6-induced MCP production); and

[0112] d. Produce highly specific antibodies that can lead to the in vivo reduction of other IL-6-related pathologies.

[0113] The IL-6 peptide immunogen constructs and formulations thereof of the present invention can effectively function as pharmaceutical compositions to prevent and / or treat individuals susceptible to or suffering from diseases affected by IL-6 dysregulation.

[0114] The various components of the IL-6 peptide immunogen constructs of the present invention are described in further detail below.

[0115] a. B cell epitope peptide from the IL-6R binding region

[0116] The present invention relates to a novel peptide composition for producing high titer antibodies, which are specific for human recombinant IL-6 protein and cross-reactive for IL-6 protein from macaque, mouse and rat species. The site specificity of the peptide composition minimizes the production of antibodies against irrelevant sites located in other regions on IL-6 or irrelevant sites located on carrier proteins, thereby providing a high safety factor.

[0117] As used herein, the term "IL-6" refers to the full-length IL-6 protein from human (UniProtKB P05231; GenBank Accession No. NP_000591.1) and full-length IL-6 proteins from other species with cross-reactivity, including macaque (UniProtKBAOA1D5QM02-1; GenBank Accession No. NP_001274245.1), mouse (UniProtKB P08505; GenBank Accession No. NP_112445.1), and rat (UniProtKB P20607; GenBank Accession No. NP_036721.1). The amino acid sequence alignment of the full-length IL-6 sequences from human (SEQ ID NO: 227), macaque (SEQ ID NO: 228), mouse (SEQ ID NO: 229), and rat (SEQ ID NO: 230) is shown below. Figure 1 shown.

[0118] More specifically, the term "IL-6" as used herein refers to the amino acid sequence of the full-length, mature IL-6 protein with the N-terminal signal peptide (containing about 24 to 28 amino acids, depending on the species) removed. The amino acid sequences of the full-length, mature IL-6 proteins from humans (SEQ ID NO: 1), macaques (SEQ ID NO: 2), mice (SEQ ID NO: 3) and rats (SEQ ID NO: 4) are shown in Table 1. Throughout this application, the numbering of amino acid positions within the IL-6 protein is based on the full-length, mature sequence of IL-6, in which the N-terminal signal sequence is removed, represented by SEQ ID NOs: 1-4, as shown in Table 1.

[0119] IL-6R is composed of two chains: (1) the IL-6 binding chain or IL-6Rα, which exists in two forms, namely (a) 80 kD transmembrane IL-6Rα (mIL-6Rα) (UniProtKB: P08887; GenBank Accession No. NP_000556.1) and (b) 50-55 kD soluble IL-6Rα (sIL-6Rα) (UniProtKB: P08887 or P08887-2), and (2) a 130 kD signaling chain named IL-6Rβ or gp130 (UniProtKB: P40189; GenBank Accession No. NP_002175.2).

[0120] Membrane IL-6Rα (or mIL-6Rα) is expressed on the surface of a limited number of cell types, namely hepatocytes, megakaryocytes, and leukocytes (including monocytes, macrophages, neutrophils, and T and B lymphocytes). Soluble IL-6Rα (or sIL-6Rα) is present in human plasma (25-75 ng / mL) and tissue fluids and can be generated by proteolytic cleavage (shedding) of mIL-6Rα by metalloproteinases, disintegrin metalloproteinases (i.e., ADAMs), or, to a lesser extent, by alternative splicing that deletes the transmembrane domain.

[0121] Membrane IL-6Rβ or gp130 is ubiquitously expressed on all human cells (Sabba, 2008).

[0122] In traditional signal transduction, IL-6 binds to the membrane-bound IL-6 receptor (mIL-6Rα), and the IL-6-mIL-6Rα complex binds to the IL-6Rβ subunit (gp130), inducing gp130 dimerization and intracellular signal transduction. Alternatively, IL-6 can bind to soluble IL-6Rα (sIL-6Rα), which is produced by cleavage of mIL-6Rα by disintegrin metalloproteinase 17 (ADAM17). The IL-6-sIL-6Rα complex then binds to the membrane-bound IL-6Rβ subunit (gp130) and induces trans-signaling even on cells that do not express IL-6R. Therefore, upon binding to mIL-6Rα or sIL-6Rα, IL-6 induces the formation of a hexameric protein containing two IL-6, two IL-6Rα, and two IL-6Rβ (gp130), which then triggers downstream signaling cascades (Rose-John, et al., 2017).

[0123] Cell activation by IL-6 binding to mIL-6Rα is called "conventional signaling". All other cells that do not express mIL-6Rα obtain their IL-6 signal through "trans-signaling": in which IL-6 binds to circulating sIL-6Rα, and this complex forms a signaling complex with IL-6Rβ or gp130 on the cell surface. Trans-signaling can occur in a wide range of human cells and therefore contributes to the pleiotropic activities of IL-6. It is currently understood that the homeostatic and regenerative activities of IL-6 are mediated by conventional signaling, while the pro-inflammatory effects are mainly caused by the activation of the trans-signaling pathway. There is increasing evidence that IL-6 trans-signaling is particularly involved in the development of the disease. Soluble forms of IL-6Rβ (sIL-6Rβ) or gp130 (sgp130) are also detected in the circulation at relatively high concentrations, which are mainly produced by alternative splicing. Since sgp130 can bind to the IL-6 / sIL-6Rα complex, it acts as a natural and specific inhibitor of IL-6-mediated trans-signaling, while conventional signaling is not affected by sgp130.

[0124] Although IL-6Rα is the unique binding receptor for IL-6, the IL-6Rβ (or gp130) signaling chain is shared by IL-6 family members (including leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, IL-11, cardiotrophin-1, neurogenin-1, IL-27, and IL-35).

[0125] The IL-6 B cell epitope portion of the IL-6 peptide immunogen construct targets the IL-6R binding region on the IL-6 molecule. The B cell epitope peptide contains about 7 to about 42 amino acid residues derived from E42-C83 (SEQ ID NO: 16) or N144-I166 (SEQ ID NO: 19) of the full-length mature IL-6 protein (SEQ ID NOs: 1-4). The IL-6 B cell epitopes were selected after extensive serological screening using IL-6 protein fragments, some of which contain naturally occurring intramolecular loop structures in the protein, such as Figure 1 Cysteine ​​is shown shaded in the figure.

[0126] In certain embodiments, the B cell epitope peptide screened and selected based on the design theory contains an amino acid sequence from the internal intramolecular loop structure of IL-6 formed by endogenous cysteine ​​(e.g., C73-C83 (SEQ ID NO: 5) or C44-C50 (SEQ ID NO: 15)) according to the numbering of the full-length mature IL-6 protein sequence (SEQ ID NO: 1), or an amino acid sequence from the carboxyl terminus of the IL-6 molecule (including an amino acid sequence from N144-I166 (SEQ ID NO: 19)). In some embodiments, the B cell epitope has an amino acid sequence of SEQ ID NOs: 5-19, as shown in Table 1.

[0127] The IL-6 B cell epitope peptides of the present invention also include immunologically functional analogs or homologs of IL-6 peptides (SEQ ID NOs: 5-19). The immunologically functional analogs or homologs of IL-6 B cell epitope peptides include variants that retain substantially the same immunogenicity as the original peptide. The immunologically functional analogs may have conservative substitutions at amino acid positions, changes in overall charge, covalent linkages with other functional groups, or additions, insertions or deletions of amino acids and / or any combination thereof (e.g., IL-6 peptides of SEQ ID NOs: 72-76).

[0128] b. Heterologous T helper cell epitopes (Th epitopes)

[0129] The present invention provides peptide immunogen constructs comprising a B cell epitope from IL-6 covalently linked to a heterologous T helper cell (Th) epitope, either directly or via an optional heterologous spacer.

[0130] Heterologous Th epitopes in the IL-6 peptide immunogen construct can enhance the immunogenicity of the IL-6 fragment, which promotes the generation of specific high-titer antibodies against the optimized target B cell epitope peptide (i.e., IL-6 fragment) screened and selected based on design theory.

[0131] The term "heterologous" as used herein refers to an amino acid sequence derived from an amino acid sequence that is not part of the wild-type sequence of IL-6 or homologous thereto. Therefore, a heterologous Th epitope is a Th epitope derived from an amino acid sequence that is not naturally present in IL-6 (i.e., the Th epitope is not autologously derived for IL-6). Because the Th epitope is heterologous to IL-6, when the heterologous Th epitope is covalently linked to the IL-6 fragment, the native amino acid sequence of IL-6 does not extend toward the amino terminal or carboxyl terminal.

[0132] The heterologous Th epitope of the present invention can be any Th epitope that does not have an amino acid sequence naturally present in IL-6. The Th epitope may also have a promiscuous binding motif for class 2 MHC molecules of various species. In certain embodiments, the Th epitope comprises a plurality of promiscuous class 2 MHC binding motifs to allow maximum activation of T helper cells, thereby leading to the initiation and regulation of an immune response. The preferred Th epitope itself is non-immunogenic (i.e., antibodies generated using IL-6 peptide immunogen constructs are rarely, if ever, directed against Th epitopes), thus allowing a very focused immune response against the target B cell epitope peptide of IL-6.

[0133] The Th epitopes of the present invention include, but are not limited to, amino acid sequences derived from foreign pathogens, as exemplified in Table 2 (SEQ ID NOs: 78-106 and 216-226). In addition, the Th epitopes include idealized artificial Th epitopes and combined idealized artificial Th epitopes (e.g., SEQ ID NOs: 79, 86, 91, 92 and 79, 86-92). Heterologous Th epitope peptides are presented in combinatorial sequences (e.g., SEQ ID NOs: 87-90), comprising a mixture of amino acid residues represented at specific positions within the peptide backbone based on the variable residues of homologs of a specific peptide. A collection of combinatorial peptides can be synthesized in a single process by adding a mixture of selected protected amino acids at specific positions during the synthesis process, rather than one specific amino acid. Such a combinatorial heterologous Th epitope peptide collection can allow for a wide range of Th epitope coverage for animals with different genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 87-90 as shown in Table 2. The Th epitope peptides of the present invention provide broad reactivity and immunogenicity to animals and patients from genetically diverse populations.

[0134] c. Heterologous spacers

[0135] The IL-6 peptide immunogen constructs of the present invention optionally comprise a heterologous spacer that covalently links the IL-6 B cell epitope peptide to a heterologous T helper cell (Th) epitope.

[0136] As described above, the term "heterologous" refers to an amino acid sequence derived from an amino acid sequence that is not part of or homologous to the native sequence of IL-6. Therefore, when a heterologous spacer is covalently linked to the IL-6 B cell epitope peptide, the native amino acid sequence of IL-6 will not extend in the amino-terminal or carboxyl-terminal direction because the spacer is heterologous to the IL-6 sequence.

[0137] A spacer is any molecule or chemical structure that can link two amino acids and / or peptides together. Depending on the application, the length or polarity of the spacer may vary. Spacer linkages may be through amide or carboxyl linkages, but other functional groups are also possible. Spacers may include chemical compounds, naturally occurring amino acids, or non-naturally occurring amino acids.

[0138] The spacer can provide construct features for the IL-6 peptide immunogen construct. Structurally, the spacer provides physical separation of the Th epitope from the B cell epitope of the IL-6 fragment. Any artificial secondary structure generated by connecting the Th epitope to the B cell epitope can be destroyed by physical separation of the spacer. In addition, the physical separation of the epitope by the spacer can eliminate the interference between the Th cell and / or B cell response. In addition, the spacer can be designed to produce or modify the secondary structure of the peptide immunogen construct. For example, the spacer can be designed as a flexible hinge to enhance the separation of the Th epitope and the B cell epitope. The flexible hinge spacer can also allow for more efficient interaction between the presented peptide immunogen and the appropriate Th cells and B cells to enhance the immune response to the Th epitope and the B cell epitope. The example of the sequence encoding the flexible hinge is found in the hinge region of the immunoglobulin heavy chain, which is usually rich in proline. A particularly useful flexible hinge for use as a spacer is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 76), wherein Xaa is any amino acid, preferably aspartic acid.

[0139] Spacers can also provide functional features for IL-6 peptide immunogen constructs. For example, spacers can be designed to change the total charge of IL-6 peptide immunogen constructs, which can affect the solubility of peptide immunogen constructs. In addition, changing the total charge of IL-6 peptide immunogen constructs can affect the ability of peptide immunogen constructs to bind to other compounds and reagents. As discussed in further detail below, IL-6 peptide immunogen constructs can form stable immunostimulatory complexes with highly charged oligonucleotides (e.g., CpG oligomers) by electrostatic binding. The total charge of IL-6 peptide immunogen constructs is important for forming these stable immunostimulatory complexes.

[0140] Chemical compounds that can be used as spacers include, but are not limited to, (2-aminoethoxy)acetic acid (AFA), 5-aminovaleric acid (AVA), 6-aminohexanoic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, mini-PEG1), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (mini-PEG3), trioxatridecan-succinamic acid (Ttds), 12-aminododecanoic acid, Fmoc-5-amino-3-oxovaleric acid (O1Pen), etc.

[0141] Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0142] Non-naturally occurring amino acids include, but are not limited to, ε-N lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), 3-mercaptopropionic acid (MPA), 3-nitrotyrosine, pyroglutamic acid, and the like.

[0143] The spacer in the IL-6 peptide immunogen construct can be covalently linked to the amino or carboxyl terminus of the Th epitope and the IL-6 B cell epitope peptide. In some embodiments, the spacer is covalently linked to the carboxyl terminus of the Th epitope and the amino terminus of the IL-6 B cell epitope peptide. In other embodiments, the spacer is covalently linked to the carboxyl terminus of the IL-6 B cell epitope peptide and the amino terminus of the Th epitope. In certain embodiments, more than one spacer can be used, for example, when there are more than one Th epitope in the IL-6 peptide immunogen construct. When more than one spacer is used, each spacer can be the same or different from each other. In addition, when there are more than one Th epitope in the IL-6 peptide immunogen construct, the Th epitope can be separated by a spacer, and the spacer can be the same or different, and the Th epitope is separated from the IL-6 B cell epitope peptide by a spacer. There is no restriction on the arrangement of the spacer relative to the Th epitope or the IL-6 B cell epitope peptide.

[0144] In certain embodiments, the heterologous spacer is a naturally occurring amino acid or a non-naturally occurring amino acid. In other embodiments, the spacer comprises more than one naturally occurring or non-naturally occurring amino acid. In specific embodiments, the spacer is Lys-, Gly-, Lys-Lys-Lys-, (d, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 77) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 231).

[0145] d. Specific Examples of IL-6 Peptide Immunogen Constructs

[0146] In certain embodiments, the IL-6 peptide immunogen construct can be represented by the following formula:

[0147] An IL-6 peptide immunogen construct having a length of about more than 30 amino acids, represented by the molecular formula:

[0148] (Th) m -(A) n -(IL-6R binding region of IL-6 or a fragment thereof)-X

[0149] or

[0150] (IL-6R binding region of IL-6 or a fragment thereof)-(A) n -(Th) m -X

[0151] or

[0152] (Th) m -(A) n -(IL-6R binding region of IL-6 or a fragment thereof)-(A) n -(Th) m -X

[0153] in

[0154] Th is a heterologous T helper cell epitope;

[0155] A is a heterologous spacer;

[0156] (IL-6R binding region of IL-6 or a fragment thereof) is a B cell epitope peptide having about 7 to about 42 amino acid residues from the IL-6R binding region of IL-6 (SEQ ID NO: 1);

[0157] X is α-COOH or α-CONH2 of an amino acid;

[0158] m is from 1 to about 4; and

[0159] n is 0 to about 10.

[0160] In some embodiments, (IL-6R binding region of IL-6 or a fragment thereof) is a B cell epitope peptide having an amino acid sequence selected from any one of SEQ ID NOs: 5-19. In certain embodiments, the B cell epitope has an amino acid sequence of E42-C83 (SEQ ID NO: 16) or N144-I166 (SEQ ID NO: 19) or a fragment thereof from IL-6 (SEQ ID NOs: 1-4). In a specific embodiment, (IL-6R binding region of IL-6 or a fragment thereof) is a B cell epitope comprising at least one naturally occurring intramolecular loop structure from C73-C83 (SEQ ID NO: 5) and / or C44-C50 (SEQ ID NO: 15), such as Figure 1 shown.

[0161] In certain embodiments, the heterologous Th epitope in the IL-6 peptide immunogen construct has an amino acid sequence selected from any one of SEQ ID NOs: 78-106, 216-226 or a combination thereof, as shown in Table 2. In some embodiments, the IL-6 peptide immunogen construct comprises more than one Th epitope.

[0162] In certain embodiments, the optional heterologous spacer is selected from any one of Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 76), ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 77), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 231), and any combination thereof, wherein Xaa is any amino acid, but preferably aspartic acid. In a specific embodiment, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 77) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 231).

[0163] In certain embodiments, the IL-6 B cell epitope peptide has about 7 to about 42 amino acid residues from the full-length mature IL-6 protein of SEQ ID NO: 1. In specific embodiments, the IL-6 B cell epitope peptide comprises an amino acid sequence from an intramolecular loop structure contained in IL-6 (E42-C83, SEQ ID NO: 16). In specific embodiments, the IL-6 B cell epitope peptide comprises an internal loop structure of IL-6 (e.g., SEQ ID NOs: 5-8, 10, 12, 15-17) from amino acids C73-C83 (SEQ ID NO: 5) or IL-6 C44-C50 (SEQ ID NO: 15), as shown in Table 1.

[0164] In certain embodiments, the IL-6 peptide immunogen construct has an amino acid sequence selected from any one of SEQ ID NOs: 107-215 as shown in Table 3. In a specific embodiment, the IL-6 peptide immunogen construct has an amino acid sequence selected from any one of SEQ ID NOs: 107-160.

[0165] IL-6 peptide immunogen constructs comprising Th epitopes can be produced simultaneously in a single solid phase peptide synthesis in tandem with an IL-6 fragment. Th epitopes can also include immunological analogs of Th epitopes. Immune Th analogs include immunopotentiating analogs, cross-reactive analogs, and fragments of any of these Th epitopes that are sufficient to enhance or stimulate an immune response to the IL-6 B cell epitope peptide.

[0166] The Th epitope in the IL-6 peptide immunogen construct can be covalently linked to the amino terminus or carboxyl terminus of the IL-6 B cell epitope peptide. In certain embodiments, the Th epitope is covalently linked to the amino terminus of the IL-6 B cell epitope peptide. In other embodiments, the Th epitope is covalently linked to the carboxyl terminus of the IL-6 B cell epitope peptide. In certain embodiments, more than one Th epitope is covalently linked to the IL-6 B cell epitope peptide. When more than one Th epitope is linked to the IL-6 B cell epitope peptide, each Th epitope can have the same amino acid sequence or different amino acid sequences. In addition, when more than one Th epitope is linked to the IL-6 B cell epitope peptide, the Th epitope can be arranged in any order. For example, the Th epitope can be continuously linked to the amino terminus of the IL-6 B cell epitope peptide, or continuously linked to the carboxyl terminus of the IL-6 B cell epitope peptide, or when different Th epitopes are covalently linked to the carboxyl terminus of the IL-6 B cell epitope peptide, the Th epitope can be covalently linked to the amino terminus of the IL-6 B cell epitope peptide. The arrangement of the Th epitope relative to the IL-6 B cell epitope peptide is not limited.

[0167] In some embodiments, the Th epitope is covalently linked directly to the IL-6 B cell epitope peptide. In other embodiments, the Th epitope is covalently linked to the IL-6 fragment via a heterologous spacer.

[0168] e. Variants, homologues and functional analogs

[0169] Variants and analogs of the above-mentioned immunogenic peptide constructs can also be used, which can induce antibodies and / or cross-react with antibodies, and this antibody is directed to the preferred IL-6 B cell epitope peptide. Analogs (including alleles, species and induced variants) are usually different from naturally occurring peptides at one, two or several positions, usually due to conservative substitutions. Analogs usually show at least 80 or 90% sequence identity with natural peptides. Some analogs also include non-natural amino acids or modifications of amino-terminal or carboxyl-terminal amino acids at one, two or several positions.

[0170] Variants that are functional analogs may have conservative substitutions at amino acid positions, changes in overall charge, covalent linkages to other functional groups or additions, insertions or deletions of amino acids and / or any combination thereof.

[0171] A conservative substitution is when one amino acid residue is replaced by another amino acid residue with similar chemical properties. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0172] In a specific embodiment, the functional analog has at least 50% identity to the original amino acid sequence. In another embodiment, the functional analog has at least 80% identity to the original amino acid sequence. In yet another embodiment, the functional analog has at least 85% identity to the original amino acid sequence. In yet another embodiment, the functional analog has at least 90% identity to the original amino acid sequence.

[0173] Variants also include changes in phosphorylated residues. For example, a variant may include different residues within a phosphorylated peptide. Variant immunogenic IL-6 peptides may also include pseudo-phosphorylated peptides. Pseudo-phosphorylated peptides are produced by replacing one or more phosphorylated serine, threonine, and tyrosine residues of an IL-6 peptide with acidic amino acid residues (e.g., glutamic acid and aspartic acid).

[0174] Functional immunological analogs of Th epitope peptides are also effective and are included as part of the present invention. Functional immunological Th analogs may contain conservative substitutions, additions, deletions and insertions of from 1 to about 5 amino acid residues in the Th epitope that do not substantially alter the Th stimulatory function of the Th epitope. As described above for the IL-6 B cell epitope peptides, conservative substitutions, additions and insertions may be accomplished using natural or unnatural amino acids. Table 2 identifies another variant of functional analogs of Th epitope peptides. Specifically, SEQ ID NOs: 79 and 86 of MvF1 and MvF2 Th are functional analogs of SEQ ID NOs: 89 and 91 of MvF4 and MvF5 because their amino acid backbones differ by deleting (SEQ ID NOs: 79 and 86) or inserting (SEQ ID NOs: 89 and 91) two amino acids each at the amino and carboxyl termini. The differences between these two series of similar sequences do not affect the function of the Th epitopes contained in these sequences. Thus, functional immune Th analogs include various versions of Th epitopes derived from the measles virus fusion protein MvF1-4Ths (SEQ ID NOs: 79, 86, 87 and 89) and from the hepatitis surface protein HBsAg 1-3Ths (SEQ ID NOs: 88, 90 and 92).

[0175] Composition

[0176] The present invention also provides a composition comprising the IL-6 immunogenic peptide construct of the present invention.

[0177] a. Peptide Composition

[0178] The composition comprising the IL-6 peptide immunogen construct of the present invention may be in liquid or solid / lyophilized form. The liquid composition may include water, buffer, solvent, salt and / or any other acceptable agent that does not change the structural or functional properties of the IL-6 peptide immunogen construct. The peptide composition may contain one or more IL-6 peptide immunogen constructs of the present invention.

[0179] b. Pharmaceutical compositions

[0180] The present invention also relates to a pharmaceutical composition comprising the IL-6 peptide immunogen construct of the present invention.

[0181] The pharmaceutical composition may contain a carrier and / or other additives in a pharmaceutically acceptable delivery system. Therefore, the pharmaceutical composition may contain a pharmaceutically effective dose of the IL-6 peptide immunogen construct and a pharmaceutically acceptable carrier, adjuvant and / or other excipient (e.g., diluent, additive, stabilizer, preservative, cosolvent, buffer, etc.).

[0182] The pharmaceutical composition may contain one or more adjuvants, which function to accelerate, prolong or enhance the immune response to the IL-6 peptide immunogen construct without having any specific antigenic effect itself. The adjuvants used in the pharmaceutical composition may include oils, oil emulsions, aluminum salts, calcium salts, immunostimulatory complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, polymer particles. In certain embodiments, the adjuvant may be selected from alum (potassium aluminum phosphate), aluminum phosphate (e.g. ), aluminum hydroxide (e.g. ), calcium phosphate, Freund's incomplete adjuvant (IFA), Freund's complete adjuvant, MF59, adjuvant 65, Lipovant, ISCOM, liposyn, saponin, squalene, L121, Monophosphoryl lipid A (MPL), Quil A, QS21, ISA 35, ISA 50V, ISA 50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycan, lipopolysaccharide (LPS), AS01, AS02, AS03, AS04, AF03, lipophilic phospholipids (lipid A), gamma inulin, algammulin, glucan, dextran, glucomannan, galactomannan, fructan, xylan, dioctadecyldimethylammonium bromide (DDA), and other adjuvants and emulsifiers.

[0183] In some embodiments, the pharmaceutical composition contains MONTANIDE TM ISA 51 (an oily adjuvant composition consisting of vegetable oil and mannide oleate for the preparation of a water-in-oil emulsion), 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion with EmulsIL-6n or EmulsIL-6n D as an adjuvant.

[0184] The pharmaceutical composition may also include pharmaceutically acceptable additives or excipients. For example, the pharmaceutical composition may contain antioxidants, binders, buffers, bulking agents, carriers, chelating agents, colorants, diluents, disintegrating agents, emulsifiers, fillers, gelling agents, pH buffers, preservatives, cosolvents, stabilizers, etc.

[0185] The pharmaceutical composition can be formulated into an immediate release or sustained release dosage form. In addition, the pharmaceutical composition can be formulated for inducing systemic or local mucosal immunity by encapsulation of immunogens and co-administration with microparticles. Such delivery systems are easily determined by those with ordinary knowledge in the art.

[0186] The pharmaceutical composition can be formulated as an injection in the form of a liquid solution or suspension. A liquid carrier containing an IL-6 peptide immunogen construct can also be prepared before injection. The pharmaceutical composition can be administered using any suitable method, such as id, iv, ip, im, intranasal, oral, subcutaneous, etc., and can be administered in any suitable delivery device. In certain embodiments, the pharmaceutical composition can be formulated for intravenous, subcutaneous, intradermal or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration can also be prepared, including oral and intranasal applications.

[0187] The pharmaceutical composition can also be formulated in a suitable dosage unit form. In some embodiments, the pharmaceutical composition contains about 0.1 μg to about 1 mg of IL-6 peptide immunogen construct per kilogram of body weight. The effective dose of the pharmaceutical composition depends on many different factors, including the mode of administration, the target, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is for prevention or treatment. Usually, the patient is human, but non-human mammals including transgenic mammals can also be treated. When delivered in multiple doses, the pharmaceutical composition can be conveniently divided into the appropriate amount of each dosage unit form. As is well known in the therapeutic field, the dosage administered depends on the age, weight and general health of the subject.

[0188] In some embodiments, the pharmaceutical composition contains more than one IL-6 peptide immunogen construct. Pharmaceutical compositions containing a mixture of more than one IL-6 peptide immunogen constructs allow for synergistic enhancement of the immunological efficacy of the constructs. Pharmaceutical compositions containing more than one IL-6 peptide immunogen constructs may be more effective in a larger genetic population due to broad class 2 MHC coverage, thus providing an improved immune response to the IL-6 peptide immunogen constructs.

[0189] In some embodiments, the pharmaceutical composition contains an IL-6 peptide immunogen construct selected from SEQ ID NOs: 107-215 (Table 3), as well as homologs, analogs and / or combinations thereof.

[0190] In certain embodiments, IL-6 peptide immunogen constructs (SEQ ID NOs: 170-172) with combined heterologous Th epitopes derived from MVF and HBsAg (SEQ ID NOs: 87-90) are mixed in an equimolar ratio and used in the formulation to allow for maximum coverage of host populations with different genetic backgrounds. Synergistic enhancement in IL-673-83 immunogen constructs (SEQ ID NOs: 170, 172) was observed in the peptide compositions of the present invention.

[0191] In addition, the IL-6 peptide immunogen construct (e.g., having SEQ ID NO: 91) 1) The antibody response elicited was mostly (>90%) focused on the desired cross-reactivity against the B cell epitope peptide of IL-6 (SEQ ID NO: 5), with little, if any, cross-reactivity against the heterologous Th epitopes used for immunogenicity enhancement (Example 6, Table 8). This is in stark contrast to conventional proteins (e.g., KLH) or other biological protein carriers used for antigenicity enhancement of such IL-6 peptides.

[0192] In other embodiments, a pharmaceutical composition comprising a peptide composition, such as an IL-6 peptide immunogen construct mixture, is contacted with a mineral salt as an adjuvant (including ALHYDROGEL or ADJUPHOS) to form a suspension dosage form for administration to a host.

[0193] The pharmaceutical composition containing the IL-6 peptide immunogen construct can be used to induce an immune response and produce antibodies in a host after administration.

[0194] c. Immunostimulatory complex

[0195] The present invention also relates to a pharmaceutical composition containing an IL-6 peptide immunogen construct that forms an immunostimulatory complex with a CpG oligonucleotide. Such an immunostimulatory complex is particularly suitable as an adjuvant and a peptide immunogen stabilizer. The immunostimulatory complex is in the form of microparticles that can effectively present the IL-6 peptide immunogen to cells of the immune system to produce an immune response. The immunostimulatory complex can be formulated into a suspension for parenteral administration. The immunostimulatory complex can also be formulated into a water-in-oil (w / o) emulsion form, as a suspension combined with a mineral salt or an in situ gel polymer, for effectively delivering the IL-6 peptide immunogen construct to cells of the host immune system after parenteral administration.

[0196] The stabilized immunostimulatory complex can be formed by complexing the IL-6 peptide immunogen construct with an anionic molecule, oligonucleotide, polynucleotide, or a combination thereof by electrostatic binding. The stabilized immunostimulatory complex can be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0197] In certain embodiments, the IL-6 peptide immunogen construct is designed to include a cationic portion that has a positive charge at a pH in the range of 5.0 to 8.0. The net charge of the cationic portion of the IL-6 peptide immunogen construct or mixture of constructs is calculated based on each lysine (K), arginine (R) or histidine (H) having a charge of +1, each aspartic acid (D) or glutamic acid (E) having a charge of -1, and the charge of the other amino acids in the sequence being 0. The charges in the cationic portion of the IL-6 peptide immunogen construct are added and expressed as a net average charge. Suitable peptide immunogens have cationic portions with a net average positive charge of +1. Preferably, the peptide immunogen has a net positive charge in the range of greater than +2. In some embodiments, the cationic portion of the IL-6 peptide immunogen construct is a heterologous spacer. In certain embodiments, when the spacer sequence is (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 77), the cationic portion of the IL-6 peptide immunogen construct has a charge of +4.

[0198] As used herein, "anionic molecules" refer to any molecule with a negative charge at a pH in the range of 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge calculation on the oligomer or polymer is based on the fact that each phosphodiester or phosphorothioate group in the oligomer carries a -1 charge. Suitable anionic oligonucleotides are single-stranded DNA molecules with 8 to 64 nucleotide bases, with the number of repeats of the CpG motif in the range of 1 to 10. Preferably, the CpG immunostimulatory single-stranded DNA molecule contains 18 to 48 nucleotide bases, with the number of repeats of the CpG motif in the range of 3 to 8.

[0199] More preferably, the anionic oligonucleotide can be of the formula 5'X 1 CGX 2 3' means, wherein C and G are unmethylated; and X 1 is selected from the group consisting of A (adenine), G (guanine) and T (thymine); and X 2 is C (cytosine) or T (thymine). Alternatively, anionic oligonucleotides may have a molecular formula of 5'(X 3 )2CG(X 4 )23' represents, wherein C and G are unmethylated; and X 3 is selected from the group consisting of A, T or G; and X 4is C or T. In specific embodiments, the CpG oligonucleotide has the following sequence: CpG1: 5'TCgTCg TTT TgTCgT TTT gTCgTTTTgTCg TT 3' (fully phosphorothioated) (SEQ ID NO: 232), CpG2: 5' phosphoTCgTCg TTT TgTCgT TTT gTCgTT 3' (fully phosphorothioated) (SEQ ID NO: 233) or CpG3: 5'TCgTCg TTT TgTCgT TTT gTCgTT 3' (fully phosphorothioated) (SEQ ID NO: 234).

[0200] The resulting immunostimulatory complex is in particulate form, the size of which is typically in the range of 1-50 microns and is a function of many factors, including the relative charge stoichiometry and molecular weight of the interacting components. Microparticulate immunostimulatory complexes have the advantage of providing adjuvantation and upregulation of specific immune responses in vivo. In addition, the stabilized immunostimulatory complex is suitable for use in the preparation of pharmaceutical compositions by a variety of methods, including water-in-oil emulsions, mineral salt suspensions, and polymeric gels.

[0201] The present invention also relates to pharmaceutical compositions for preventing and / or treating diseases affected by IL-6 disorder, including formulations. In some embodiments, the pharmaceutical composition comprises a stabilized immunostimulatory complex formed by mixing a CpG oligomer and a peptide composition comprising a mixture of IL-6 peptide immunogen constructs (e.g., SEQ ID NOs: 107-215) to further enhance the immunogenicity of the IL-6 peptide immunogen constructs and induce antibodies that cross-react with the IL-6 protein of SEQ ID NOs: 1-4, which are directed against the IL-6R binding region (Example 6).

[0202] In another embodiment, the pharmaceutical composition contains a mixture of IL-6 peptide immunogen constructs (e.g., any combination of SEQ ID NOs: 107-215), which form a stabilized immunostimulatory complex with a CpG oligomer. Preferably, the immunostimulatory complex is mixed with a mineral salt as an adjuvant with a high safety factor (including alum gel (ALHYDROGEL) or aluminum phosphate (ADJUPHOS)) to form a suspension dosage form for administration to a host.

[0203] Antibody

[0204] The present invention also provides antibodies elicited using the IL-6 peptide immunogen constructs of the present invention.

[0205] The present invention provides IL-6 peptide immunogen constructs and preparations thereof, which are cost-effective in manufacturing, and which are optimally designed to elicit high titer antibodies targeting the IL-6R binding region on the IL-6 molecule, which are capable of breaking immune tolerance to the self-protein IL-6 at a high response rate in the immunized host. Antibodies generated using the IL-6 peptide immunogen constructs have high affinity for the IL-6R binding region.

[0206] In some embodiments, the IL-6 peptide immunogen construct for eliciting antibodies comprises a hybrid of an IL-6 peptide having a B cell epitope (the B cell epitope contains about 7 to about 42 amino acids, covering the IL-6Rα and IL-6Rβ binding regions having an intramolecular loop structure optionally derived from the IL-6 peptide C73-C83 (SEQ ID NO: 5) or C44-C50 (SEQ ID NO: 15) located within IL-6 (see Table 1, Figure 1 , and SEQ ID NOs: 1 and 227)), the B cell epitope is linked to a heterologous Th epitope derived from a pathogen protein (e.g., derived from the measles virus fusion (MVF) protein and other proteins (SEQ ID NOs: 78-106 and 216-226)) via an optional spacer. The B cell epitope and the Th epitope of the IL-6 peptide immunogen construct act together to stimulate the production of highly specific antibodies that cross-react with the IL-6R binding region of the IL-6 protein (SEQ ID NO: 1).

[0207] Conventional methods for enhancing the immunogenicity of peptides, such as by chemical coupling to carrier proteins, such as keyhole limpet hemocyanin (KLH) or other carrier proteins, such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins, generally result in the production of large amounts of antibodies directed against the carrier protein. Thus, a major drawback of such peptide-carrier protein compositions is that the majority (>90%) of the antibodies generated using such immunogens are non-functional antibodies directed against the carrier proteins KLH, DT or TT, which can result in epitope suppression.

[0208] Different from the conventional methods for enhancing the immunogenicity of peptides, the antibodies generated by the IL-6 peptide immunogen constructs of the present invention (e.g., SEQ ID NO: 142) can bind to IL-6 B cell epitope peptides (e.g., SEQ ID NOs: 5-19) with high specificity, and there are not many, if any, antibodies against heterologous Th epitopes (e.g., SEQ ID NO: 91 in Table 8) or optional heterologous spacers. Specifically, the polyclonal antibodies elicited in the immunized animals can bind to the central IL-6R binding region (SEQ ID NO: 107) with high specificity, which leads to inhibition of IL-6 and IL-6R interaction via cis-signaling, as shown in FIG. 5A .

[0209] method

[0210] The present invention also relates to methods for making and using IL-6 peptide immunogenic constructs, compositions and pharmaceutical compositions.

[0211] a. Method for producing IL-6 peptide immunogen construct

[0212] The IL-6 peptide immunogen constructs of the present invention can be prepared using chemical synthesis methods well known to those of ordinary skill (see, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman & Co., New York, NY, 1992, p. 77). The IL-6 peptide immunogen constructs can be synthesized using automated Merrifield solid phase synthesis, using side chain protected amino acids, d-NH2 chemically protected with t-Boc or F-moc, for example, on an Applied Biosystems Peptide Synthesizer Model 430A or 431. Preparation of IL-6 peptide immunogen constructs containing combinatorial database peptides of Th epitopes can be achieved by providing a mixture of alternative amino acids for coupling at a given variable position.

[0213] After the desired IL-6 peptide immunogen construct is assembled, the resin is treated according to standard procedures to cleave the peptide from the resin and remove the functional groups on the amino acid side chains. The free peptide can be purified by HPLC and characterized by, for example, amino acid analysis or sequencing. Methods for purification and characterization of peptides are well known to those of ordinary skill in the art to which the present invention belongs.

[0214] The quality of the peptides produced by this chemical process can be controlled and determined, and as a result the reproducibility, immunogenicity and yield of the immunogen constructs can be guaranteed. A detailed description of the manufacture of IL-6 peptide immunogen constructs by solid phase peptide synthesis is shown in Example 1.

[0215] The range of construct variation that allows retention of desired immunological activity has been found to be more inclusive than that that allows retention of specific drug activity of small molecule drugs or desired activity and undesired toxicity in large molecules co-produced with biologic drug products.

[0216] Thus, peptide analogs that have similar chromatographic and immunological properties to the desired peptide, whether intentionally designed or produced as a mixture of unavoidable byproducts of deleted sequences due to errors in the synthetic process, generally have the same efficacy as the purified preparation of the desired peptide. Mixtures of designed and unintended analogs are also effective as long as a rigorous QC program is established to monitor the manufacturing process and product evaluation process to ensure the reproducibility and efficacy of the final product using these peptides.

[0217] IL-6 peptide immunogen constructs can also be prepared using recombinant DNA technology including nucleic acid molecules, vectors and / or host cells. Therefore, nucleic acid molecules encoding IL-6 peptide immunogen constructs and immunologically functional analogs thereof are also included in the present invention as part of the present invention. Similarly, vectors (including expression vectors) containing nucleic acid molecules and host cells containing vectors are also included in the present invention as part of the present invention.

[0218] Various exemplary embodiments also include methods for making IL-6 peptide immunogen constructs and immunologically functional analogs thereof. For example, the method may include the step of culturing a host cell under conditions of expressing the peptide and / or analogs, the host cell comprising an expression vector containing a nucleic acid molecule encoding the IL-6 peptide immunogen construct and / or its immunologically functional analogs. Longer synthetic peptide immunogens can be synthesized using well-known recombinant DNA techniques. These techniques can be provided in well-known standard manuals with detailed experimental plans. In order to construct a gene encoding a peptide of the present invention, the amino acid sequence is reversely translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably using the most suitable codons for the organism in which the gene to be expressed is present. Next, a synthetic gene is usually made by synthesizing oligonucleotides encoding the peptide and any regulatory factors (if necessary). The synthetic gene is inserted into a suitable selection vector and transfected into a host cell. The peptide is then expressed under suitable conditions suitable for the selected expression system and host. The peptide is purified using standard methods and its characteristics are described.

[0219] b. Method for producing immunostimulatory complex

[0220] Various exemplary embodiments also include methods for making immunostimulatory complexes comprising IL-6 peptide immunogen constructs and CpG oligodeoxynucleotide (ODN) molecules. Stabilized immunostimulatory complexes (ISCs) are derived from the cationic portion of the IL-6 peptide immunogen construct and the polyanionic CpG ODN molecules. The self-assembling system is driven by electrostatic neutralization of charges. The stoichiometry of the molar charge ratio of the cationic portion of the IL-6 peptide immunogen construct to the anionic oligomer determines the degree of association. The non-covalent electrostatic binding of the IL-6 peptide immunogen construct and the CpG ODN is a completely reproducible process. This peptide / CpG ODN immunostimulatory complex aggregate helps to present to the "professional" antigen presenting cells (APC) in the immune system, thereby further enhancing the immunogenicity of the complex. During the manufacturing process, the characteristics of these complexes can be easily characterized to control quality. The peptide / CpG ISC has good tolerance in vivo. This novel microparticle system comprising CpG ODN and IL-6 peptide immunogen constructs was designed to exploit the generalized B cell mitogenicity associated with the use of CpG ODN, but to promote a balanced Th-1 / Th-2 type response.

[0221] The CpG ODN in the pharmaceutical composition of the present invention is 100% bound to the immunogen in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized particles. The microparticle form allows a significant reduction in the CpG dose conventionally used in CpG adjuvants, a lower likelihood of adverse innate immune responses, and promotes alternative immunogen processing pathways including antigen presenting cells (APCs). Therefore, this dosage form is conceptually novel and offers potential advantages by promoting the stimulation of immune responses through alternative mechanisms.

[0222] c. Method for producing a pharmaceutical composition

[0223] Various exemplary embodiments also include pharmaceutical compositions containing the IL-6 peptide immunogen constructs. In certain embodiments, the pharmaceutical compositions are in the form of water-in-oil emulsions and suspensions with mineral salts.

[0224] In order to make the pharmaceutical composition available to the general public, safety becomes another important factor to be considered. Although water-in-oil emulsions have been used in many clinical trials, alum is still the main adjuvant used in preparations based on its safety. Therefore, alum or its mineral salt aluminum phosphate (ADJUPHOS) is often used as an adjuvant in preparations for clinical use.

[0225] Other adjuvants and immunostimulants include 3De-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids, such as polyglutamic acid or polylysine. Such adjuvants may be used with or without other specific immunostimulants, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) THERAMIDE TM ), or other bacterial cell wall components. The oil-in-water emulsion includes MF59 (see patent application WO 90 / 14837 of Van Nest et al., which is incorporated herein by reference in its entirety), including 5% squalene, 0.5% TWEEN 80, and 0.5% Span 85 (optionally containing different amounts of MTP-PE), formulated into submicron particles using a microfluidizer; SAF, including 10% squalene, 0.4% TWEEN 80, 5% pluronic-block copolymer L121, and thr-MDP, formed into a submicron emulsion using microfluidization or using vortexing to produce a large particle emulsion; and RIBI TM Adjuvant system (RAS) (Ribi ImmunoChem, Hamilton, Mont.) comprising 2% squalene, 0.2% TWEEN 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox TM Other adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), and tumor necrosis factor (TNF-α).

[0226] The choice of adjuvant depends on the stability of the immunogen preparation containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant to the species to be immunized, and in humans, a pharmaceutically acceptable adjuvant refers to an adjuvant that has been approved or can be approved for human administration by relevant regulatory agencies. For example, alum, MPL or Freund's incomplete adjuvant (Chang, et al., 1998), which is incorporated herein by reference in its entirety) alone or optionally all combinations thereof are suitable for human administration.

[0227] The composition may include a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a carrier commonly used to formulate a pharmaceutical composition for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the composition. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or dosage form may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0228] Pharmaceutical compositions may also include large, slowly metabolized macromolecules (e.g., proteins, polysaccharides (e.g., chitin), polylactic acid, polyglycolic acid, and copolymers (e.g., latex functionalized sepharose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). In addition, these carriers may act as immunostimulants (i.e., adjuvants).

[0229] The pharmaceutical composition of the present invention may further comprise a suitable delivery vehicle. Suitable delivery vehicles include, but are not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen microspheres and cochleates.

[0230] d. Methods of using the pharmaceutical composition

[0231] The present invention also includes methods of using the pharmaceutical compositions comprising the IL-6 peptide immunogen constructs.

[0232] In certain embodiments, pharmaceutical compositions comprising IL-6 peptide immunogen constructs can be used to treat diseases affected by IL-6 dysregulation.

[0233] In some embodiments, the method comprises administering a pharmaceutically effective dose of a pharmaceutical composition comprising an IL-6 peptide immunogen construct to a host in need thereof. In certain embodiments, the method comprises administering a pharmaceutically effective dose of a pharmaceutical composition comprising an IL-6 peptide immunogen construct to a warm-blooded animal (e.g., a human, a cynomolgus monkey, a mouse) to elicit highly specific antibodies that cross-react with human IL-6 protein (SEQ ID NO: 1) or IL-6 proteins from other species (SEQ ID NOs: 2-4).

[0234] In certain embodiments, pharmaceutical compositions containing IL-6 peptide immunogen constructs can be used to treat diseases affected by dysfunctional regulation of IL-6, as demonstrated in in vitro assays and in vivo disease models.

[0235] e. In vitro functional analysis and in vivo proof-of-concept studies

[0236] Antibodies elicited by the IL-6 peptide immunogen construct in the immunized host can be used for in vitro functional analysis. These functional analyses include but are not limited to:

[0237] (1) In vitro binding to IL-6 protein (SEQ ID NO: 1) as a recombinant protein;

[0238] (2) In vitro inhibition of IL-6 cis-binding to IL-6Rd;

[0239] (3) In vitro inhibition of trans-binding of IL-6 / IL-6Rα and IL-6Rβ (Example 3);

[0240] (4) In vitro inhibitory effect on IL-6-induced TF-1 proliferation (Examples 3 and 7);

[0241] (5) In vitro inhibition of IL-6-induced STAT3 phosphorylation (Examples 3 and 7);

[0242] (6) In vitro inhibition of IL-6-induced MCP-1 production in human U937 cells (Examples 3 and 7);

[0243] (7) In vivo inhibitory effect on the rat collagen-induced arthritis (CIA) model;

[0244] (8) In vivo inhibition / reduction of the release of rat neutrophils from the bone marrow into the circulatory system;

[0245] (9) In vivo inhibition of arthritis symptoms as shown by the rat arthritis score, which is a measure of:

[0246] (i) Inflammation-induced liver secretory proteins;

[0247] (ii) ankle joint destruction;

[0248] (iii) tissue production of TNF-α, IL-17, and MCP;

[0249] (iv) reversal of weight loss;

[0250] (v) swelling of the hind paw;

[0251] (vi) reduction of neutrophilia;

[0252] (vii) Decreased platelet release. Specific embodiments

[0253] (1) An IL-6 peptide immunogen construct having about 30 or more amino acids, which is represented by the following molecular formula:

[0254] (Th) m -(A) n -(IL-6R binding region of IL-6 or a fragment thereof)-X

[0255] or

[0256] (IL-6R binding region of IL-6 or a fragment thereof)-(A) n -(Th) m -X

[0257] or

[0258] (Th) m -(A) n -(IL-6R binding region of IL-6 or a fragment thereof)-(A) n -(Th) m -X

[0259] in

[0260] Th is a heterologous T helper cell epitope;

[0261] A is a heterologous spacer;

[0262] (IL-6R binding region of IL-6 or a fragment thereof) is a B cell epitope peptide having about 7 to about 42 amino acid residues from the IL-6R binding region of IL-6 (SEQ ID NO: 1);

[0263] X is α-COOH or α-CONH2 of an amino acid;

[0264] m is from 1 to about 4; and

[0265] n is 0 to about 10.

[0266] (2) The IL-6 peptide immunogen construct as described in (1), wherein the IL-6R binding region or fragment thereof is selected from the group consisting of SEQ ID NOs: 5-19.

[0267] (3) The IL-6 peptide immunogen construct as described in any one of (1) or (2), wherein the heterologous T helper cell epitope is selected from the group consisting of SEQ ID NOs: 78-106 and 216-226.

[0268] (4) The IL-6 peptide immunogen construct as described in (1), wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107-215.

[0269] (5) An IL-6 peptide immunogen construct comprising:

[0270] a B cell epitope comprising about 7 to about 42 amino acid residues of the IL-6 sequence from SEQ ID NOs: 1 to 4;

[0271] A bT helper cell epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 78-106, 216-226, and any combination thereof; and

[0272] c. an optional heterologous spacer selected from the group consisting of amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 77), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 231), and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 76), and any combination thereof,

[0273] wherein the B cell epitope is covalently linked to the T helper cell epitope directly or via an optional heterologous spacer.

[0274] (6) The IL-6 peptide immunogen construct as described in (5), wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 5 to 19.

[0275] (7) The IL-6 peptide immunogen construct as described in (5), wherein the T helper cell epitope is selected from the group consisting of SEQ ID NOs: 78-106.

[0276] (8) The IL-6 peptide immunogen construct as described in (5), wherein the optional heterologous spacer is (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 77), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 231) or Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 76), wherein Xaa is any amino acid, and aspartic acid is preferred.

[0277] (9) The IL-6 peptide immunogen construct as described in (5), wherein the T helper cell epitope is covalently linked to the amino terminus of the B cell epitope.

[0278] (10) The IL-6 peptide immunogen construct as described in (5), wherein the T helper cell epitope is covalently linked to the amino terminus of the B cell epitope via an optional heterologous spacer.

[0279] (11) A composition comprising the IL-6 peptide immunogen construct as described in (1).

[0280] (12) A pharmaceutical composition comprising:

[0281] a. a peptide immunogen construct as described in (1); and

[0282] b. Pharmaceutically acceptable delivery vehicles and / or adjuvants.

[0283] (13) The pharmaceutical composition according to (12), wherein

[0284] a. the IL-6R binding region or a fragment thereof is selected from the group consisting of SEQ ID NOs: 5-19;

[0285] b. the heterologous T helper cell epitope is selected from the group consisting of SEQ ID NOs: 78-106 and 216-226; and

[0286] c. the heterologous spacer is selected from the group consisting of amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 77), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 231) and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 76), and any combination thereof; and

[0287] The IL-6 peptide immunogen construct is mixed with CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

[0288] (14) The pharmaceutical composition according to (12), wherein

[0289] a. the IL-6 peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 107-215; and

[0290] The IL-6 peptide immunogen construct was mixed with CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

[0291] (15) A method for producing antibodies against IL-6 in an animal, comprising administering the pharmaceutical composition according to (12) to the animal.

[0292] (16) An isolated antibody or epitope-binding fragment thereof, which specifically binds to the IL-6R binding region of IL-6 or a fragment thereof located in the IL-6 peptide immunogen construct as described in (1).

[0293] (17) The isolated antibody or epitope-binding fragment thereof as described in (16), which is bound to an IL-6 peptide immunogen construct.

[0294] An isolated antibody or epitope-binding fragment thereof, which specifically binds to the B cell epitope peptide of the IL-6 peptide immunogen construct as described in any one of (1) to (10).

[0295] (18) A composition comprising the isolated antibody or epitope-binding fragment thereof as described in (16).

[0296] (19) A method for preventing and / or treating a disease affected by IL-6 disorder in an animal, comprising administering the pharmaceutical composition according to (12) to the animal.

[0297] Example 1. Synthesis of IL-6 related peptides and preparation of their preparations

[0298] a. Synthesis of IL-6 related peptides

[0299] Methods for synthesizing IL-6 related peptides included in the development of IL-6 peptide immunogen constructs are described. Peptides synthesized in small-scale quantities are used for serological analysis, laboratory tests and field trials, and peptides synthesized in large-scale (kilogram) quantities are used for industrial / commercial production of pharmaceutical compositions. For epitope mapping, and for screening and selecting the best peptide immunogen constructs for use in pharmaceutical compositions that effectively target IL-6, a large number of IL-6 related antigenic peptides with sequences of about 7 to 70 amino acids in length are designed.

[0300] Representative full-length IL-6 (SEQ ID NOs: 1-4), IL-6 peptide fragments, and 10-mer peptides (SEQ ID NOs: 5-75) for epitope mapping in various serological analyses of human, mouse, rat and macaque species are listed in Table 1.

[0301] The selected IL-6 B cell epitope peptides were synthetically linked to carefully designed T helper cell (Th) epitope peptides derived from pathogen proteins, including measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), peptide influenza virus, Clostridium tetani, and Epstein-Barr virus (EBV) (as shown in Table 2 (SEQ ID NOs: 78-106 and 216-226)) to make IL-6 peptide immunogen constructs. The Th epitope peptides were used in the form of single sequences (SEQ ID NOs: 78-86 and 91-106) or combinatorial databases (SEQ ID NOs: 87-90) to enhance the immunogenicity of their respective IL-6 peptide immunogen constructs.

[0302] Representative IL-6 peptide immunogen constructs selected from hundreds of peptide constructs are identified in Table 3 (SEQ ID NOs: 107-215).

[0303] All peptides used for immunogenicity studies or related serological tests for detection and / or measurement of anti-IL-6 antibodies were synthesized on a small scale using F-moc chemistry on an Applied Biosystems Peptide Synthesizer Model 430A, 431 and / or 433. Each peptide was prepared by independent synthesis on a solid support with F-moc protection at the amino terminus and side chain protecting groups of the trifunctional amino acids. The intact peptide was cleaved from the solid support and the side chain protecting groups were removed using 90% trifluoroacetic acid (TFA). The synthesized peptide products were evaluated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to determine the correct amino acid composition. Each synthesized peptide was also evaluated by reverse phase HPLC (RP-HPLC) to confirm the synthesis state and concentration of the product. Despite strict control of the synthesis process (including step-by-step monitoring of coupling efficiency), peptide analogs may still be generated due to certain unexpected events during the extension cycle, including insertion, deletion, substitution and premature termination of amino acids. Therefore, the synthesis products generally include multiple peptide analogs and the target peptide.

[0304] Despite the inclusion of these unintended peptide analogs, the final synthetic peptide products can still be used for immunological applications, including immunodiagnosis (as antibody capture antigen) and pharmaceutical compositions (as peptide immunogens). In general, as long as a rigorous QC program is developed to monitor the manufacturing process and product quality assessment procedures to ensure the reproducibility and efficacy of the final product using these peptides, such peptide analogs, including mixtures of byproducts that are deliberately designed or generated during the synthesis process, are usually as effective as the purified product of the desired peptide. Large quantities of peptides ranging from hundreds to several kilograms can be synthesized using a custom-made automated peptide synthesizer UBI2003 or similar models at a scale of 15 mmole to 150 mmole.

[0305] For active ingredients used in final pharmaceutical compositions for clinical trials, IL-6 related peptide immunogen constructs can be purified using preparative RP-HPLC under a shallow elution gradient and characterized for purity and consistency using MALDI-TOF mass spectrometry, amino acid analysis, and RP-HPLC.

[0306] b. Preparation of compositions containing IL-6 peptide immunogen constructs

[0307] Dosage forms using water-in-oil emulsions and suspensions with mineral salts are prepared. In order to design pharmaceutical compositions for use by a wide range of populations, safety becomes another important factor to consider. Although water-in-oil emulsions are used in clinical trials of many pharmaceutical compositions in humans, alum remains the main adjuvant used in pharmaceutical compositions based on its safety. Therefore, alum or its mineral salt ADJUPHOS (aluminum phosphate) is often used as an adjuvant for clinical application preparations.

[0308] Briefly, the dosage forms specified in each experimental group described below generally contain all types of specially designed IL-6 peptide immunogen constructs. More than 200 specially designed IL-6 peptide immunogen constructs were carefully evaluated in guinea pigs for their relative immunogenicity using corresponding IL-6 peptides representing immunogenic B cell epitope peptides. Epitope mapping and serological cross-reactivity were analyzed in various homologous peptides using ELISA assays using well plates coated with peptides selected from SEQ ID NOs: 1-75.

[0309] If specified, use Seppic MONTANIDE, an oil approved for human use TM ISA 51 is used as an oil-in-water emulsion or mixed with mineral salts ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum) to prepare different amounts of IL-6 peptide immunogen constructs. The IL-6 peptide immunogen construct is usually dissolved in water at a concentration of about 20 to 800 μg / mL and mixed with MONTANIDE TMISA 51 is formulated as a water-in-oil emulsion (1:1 volume), or with mineral salts ADJUPHOS or ALHYDROGEL (alum) (1:1 volume) to make the composition. The composition is placed at room temperature for about 30 minutes and mixed by vortexing for about 10 to 15 seconds before immunization. Animals are immunized with 2 to 3 doses of a specific composition, which are administered at time 0 (primary immunization) and 3 weeks after the primary immunization (wpi) (boost immunization), and optionally a second boost immunization at 5 or 6 wpi, administered by the intramuscular route. Sera from the immunized animals are then tested with selected B cell epitope peptides to evaluate the immunogenicity of various IL-6 peptide immunogen constructs present in the dosage form, as well as the cross-reactivity of the corresponding sera with the IL-6 protein. Those IL-6 peptide immunogen constructs that were initially found to be highly immunogenic in the guinea pig screen were further tested in in vitro experiments for the functional properties of their corresponding sera. The selected candidate IL-6 peptide immunogen constructs were then prepared in water-in-oil emulsions, mineral salts, and alum-based formulations and administered according to the immunization schedule for the specific periods specified.

[0310] In preparation for the submission of an Investigational New Drug Application for clinical trials in patients with diseases affected by IL-6 dysregulation, only the most promising IL-6 peptide immunogen constructs will undergo further extensive evaluation before inclusion in the final dosage form for GLP-guided preclinical studies for immunogenicity, duration, toxicity, and efficacy studies.

[0311] The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0312] Example 2. Serological Tests and Reagents

[0313] The serological assays and reagents used to assess the functional immunogenicity of IL-6 peptide immunogen constructs and formulations thereof are described in detail below.

[0314] a. ELISA assay based on IL-6 or IL-6 peptide fragments for antibody specificity analysis

[0315] The ELISA assays developed and described below in the following examples for evaluating immune serum samples were used to coat the wells of a 96-well plate individually with IL-6 or IL-6 fragment peptides (SEQ ID NOs: 1 to 20, 72 to 75) at a concentration of 2 μg / mL (unless otherwise stated) in 10 mM sodium bicarbonate buffer, pH 9.5 (unless otherwise stated) in a volume of 100 μL at 37° C. for 1 hour.

[0316] The wells coated with IL-6 or IL-6 fragment peptides were reacted with 250 μL of 3 wt % gelatin in PBS at 37°C for 1 hour to block nonspecific protein binding sites, followed by 0.05 vol % The wells were washed three times with PBS containing 20% ​​normal goat serum, 1% gelatin and 0.05% 20% PBS was used to dilute the test serum at a ratio of 1:20 (unless otherwise specified). 100 μL of the diluted sample (e.g., serum, plasma) was added to each well and reacted at 37°C for 60 minutes. Wash the wells 6 times at 20°C to remove unbound antibody. Use horseradish peroxidase (HRP) conjugated species-specific (e.g., guinea pig or rat) goat polyclonal anti-IgG antibody or protein A / G as a labeled tracer to bind to the antibody / peptide antigen complex formed in the positive wells. Prepare 100 μl of HRP-labeled detection reagent at the pre-titrated optimal dilution in a solution containing 1% normal goat serum and 0.05% 20% PBS was added to each well and reacted at 37°C for another 30 minutes. The wells were washed 6 times with 20% PBS to remove unbound antibodies and reacted for another 15 minutes with 100 μL of a substrate mixture containing 0.04 weight percent 3', 3', 5', 5'-tetramethylbenzidine (TMB) and 0.12 volume percent hydrogen peroxide in sodium citrate buffer. The substrate mixture was used to detect the peroxidase label by forming a colored product. The reaction was terminated by adding 100 μL of 1.0 M sulfuric acid and the absorbance at 450 nm (A 450 To determine the antibody titer of animals immunized with various peptide preparations, sera were diluted 10-fold from 1:100 to 1:10,000 or from 1:100 to 1:4.19 x 10 8 The test was performed with 4-fold serial dilutions of serum and A 450 A with a critical value of 0.5 480 The titer of the test serum was calculated by linear regression analysis, with Log 10 express.

[0317] b. Evaluation of antibody reactivity against Th peptides using ELISA assay based on Th peptides

[0318] The wells of a 96-well ELISA plate were individually coated with 100 μL of Th peptide at a concentration of 2 μg / mL (unless otherwise stated) in 10 mM sodium bicarbonate buffer, pH 9.5 (unless otherwise stated) for 1 hour at 37°C in a similar ELISA method and performed as described above. To determine the antibody titer of immunized animals receiving various IL-6 peptide preparations, 10-fold serial dilutions of sera from 1:100 to 1:10,000 were tested and A 450 A with a critical value of 0.5 450 The titer of the test serum was calculated by linear regression analysis, with Log 10 express.

[0319] c. Detailed specificity analysis of target IL-6 B cell epitope peptides by epitope mapping using ELISA assay based on 10-mer peptides of B cell epitope clusters

[0320] Anti-IL-6 antibodies from hosts immunized with IL-6 peptide immunogen constructs were finely analyzed for specificity using epitope mapping using an ELISA assay based on 10-mer peptides of B cell epitope clusters. Briefly, according to the steps of the above antibody ELISA method, the wells of a 96-well plate were coated with 0.5 μg of a single IL-6 10-mer peptide (SEQ ID NOs: 21-71) per 0.1 mL per well in duplicate, and then 100 μL of serum sample (prepared in PBS, diluted 1:100) was reacted in the 10-mer plate. For specificity confirmation, anti-IL-6 antibodies from immunized hosts were finely analyzed for specificity related to the target B cell epitope using the corresponding IL-6 peptide or an irrelevant control peptide.

[0321] d. Immunogenicity assessment

[0322] Pre-immune and immune serum samples from animals or human subjects were collected according to the experimental procedures and heated at 56°C for 30 minutes to inactivate serum complement factors. After administration of the formulation, blood samples were obtained according to the procedures and evaluated for immunogenicity against specific targets using ELISA tests based on the corresponding IL-6 B cell epitope peptides. Serially diluted sera were tested and the reciprocal of the dilution factor was taken as the logarithm (Log 10 ) to indicate a positive titer. The immunogenicity of a particular formulation was assessed for its ability to elicit a high titer antibody response specific for the desired epitope within the target antigen and highly cross-reactive with the IL-6 protein, while maintaining low to negligible antibody reactivity against "T helper cell epitopes" that provide the desired B cell response enhancement.

[0323] e. Immunoassay for assessing C-reactive protein (CRP) levels in rat serum

[0324] The levels of rat C-reactive protein (CRP) were determined by sandwich ELISA using polyclonal rabbit anti-rat CRP antibody (Sino Biological) as the capture antibody and biotin-labeled rabbit anti-rat CRP antibody (Assaypro LLC) as the detection antibody. Briefly, polyclonal rabbit anti-rat CRP antibody was prepared in a coating buffer (15mM sodium carbonate, 35mM sodium bicarbonate, pH 9.6) at 50ng / well to be fixed on a 96-well plate and reacted overnight at 4°C. 200μL / well of assay diluent (PBS containing 1% bovine serum albumin, 0.05% TWEEN-20 and 0.01% liquid biological preservative ProClin300) was reacted at room temperature for 1 hour to block the coated wells. The microplate was washed 3 times with 200μL / well of washing buffer (PBS containing 0.05% TWEEN-20 and 0.01% ProClin 300). Recombinant rat CRP (SinoBiological) was used to generate a standard curve in the assay diluent (by 2.5-fold serial dilution, ranging from 450 to 1.84 ng / mL). 100 μL of diluted serum (1: 30,000) and standards were added to the coated wells. Reacted for 2 hours at room temperature. All wells were blotted dry and washed 5 times with 200 μL / well of wash buffer. The captured CRP was reacted with 100 μL of detection antibody solution (containing 100 ng / ml biotin-labeled rabbit anti-rat CRP antibody in the assay diluent) at room temperature for 1 hour. Then, streptavidin poly-HRP (1: 10,000 dilution, Thermo Fisher Scientific) was used to detect the bound biotin-labeled antibody for 1 hour (100 μL / well). All wells were blotted dry and washed 6 times with 200 μL / well of wash buffer. Finally, the holes were developed using 100 μL / well NeA-Blue TMB substrate (Clinical Science Products), and the reaction was terminated by adding 100 μL / well 1M sulfuric acid. The absorbance of the development was determined by VersaMax ELISA microplate reader (Molecular Devices), and a 4-parameter logit curve was fitted using SoftMax Pro software (Molecular Devices) to generate a standard curve, which was used to calculate the CRP concentration in all test samples. Data were compared using Student t test using Prism software (GraphPad Software).

[0325] Example 3. Evaluation of the functional properties of antibodies induced by IL-6 peptide immunogen constructs and their preparations in animals

[0326] The immune sera or purified anti-IL-6 antibodies were further tested in immunized animals for their ability to: (1) block the interaction between IL-6 and its receptors IL-6R (IL-6α and IL-6Rβ / gp130) and (2) inhibit IL-6-induced STAT3 phosphorylation in RPMI 8226 cells and (3) inhibit IL-6-dependent TF-1 cell proliferation and (4) inhibit monocyte chemoattractant protein-1 (MCP-1) production in the U937 cell line.

[0327] a. Cells

[0328] (1) RPMI 8226 cell line was purchased from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 4.5 g / L L-glutamine, sodium pyruvate, and 1% penicillin / streptomycin in a humidified incubator at 37°C containing 5% CO2.

[0329] (2) TF-1 cells were placed in a humidified incubator at 37°C containing 5% CO2 and maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 1% sodium pyruvate (NaP), 2 ng / ml human granulocyte macrophage colony stimulating factor (human GM-CSF), 10% FBS, and 1% penicillin / streptomycin.

[0330] (3) The U937 cell line was placed in a humidified incubator containing 5% CO2 at 37°C and maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 1% NaP, 10% FBS and 1% penicillin / streptomycin.

[0331] b. Binding of IL-6 to IL-6Rα chain (cis binding)

[0332] Purified IgG polyclonal antibodies from pooled immune sera of guinea pigs previously immunized with different IL-6 peptide immunogen constructs were examined by ELISA for their relative ability to inhibit IL-6 binding to IL-6Rα. The wells of a 96-well plate were coated separately with a volume of 50 μL of recombinant His-tagged human IL-6Rα (GenScript) (prepared in coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6) at a concentration of 4 μg / mL) and reacted overnight at 4°C. The coated wells were reacted at room temperature for 1 hour with 200 μL / well of assay diluent (PBS containing 1% bovine serum albumin, 0.05% TWEEN-20, and 0.01% ProClin 300). The microplate was washed 3 times with 200 μL / well of wash buffer (PBS containing 0.05% TWEEN-20 and 0.01% ProClin 300). 100 μL of the mixture (the mixture consists of human IL-6 (GenScript) at a concentration of 10 ng / mL and purified guinea pig IgG polyclonal antibodies at different concentrations) was pre-reacted at room temperature for 1 hour and then added to the coated wells. React at room temperature for 1 hour. All wells were aspirated and washed 3 times with 200 μL / well of wash buffer. 100 μL / well of biotin-labeled rabbit anti-IL-6 antibody (1:1,000 dilution, R&D Systems) was reacted at room temperature for 1 hour to detect the captured IL-6. Then, the bound biotin-labeled antibody was detected for 1 hour (100 μL / well) using streptavidin poly-HRP (1:40,000 dilution, Thermo Fisher Scientific). All wells were aspirated and washed 3 times with 200 μL / well of wash buffer. Finally, the wells were developed using 100 μL / well of OptEIA TMB substrate (BD Biosciences), and the reaction was terminated by adding 100 μL / well of 1 M sulfuric acid. The developed absorbance was measured by a VersaMax ELISA microplate reader (Molecular Devices), and the half-maximal inhibitory concentration (IC) was calculated in Prism 6 software (GraphPad Software) by fitting the output response curve using a 4-parameter logit curve. 50 ).

[0333] c. Binding of the IL-6 / IL-6Rα chain complex to the IL-6Rβ chain / gp130 (trans binding)

[0334] The wells of a 96-well plate were coated with 50 μL of recombinant human gp130-Fc chimeric protein (R&D systems) at a concentration of 300 ng / mL in coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6) and reacted overnight at 4°C. The coated wells were blocked with 200 μL / well of assay diluent (PBS containing 1% bovine serum albumin, 0.05% TWEEN-20, and 0.01% ProClin 300) at room temperature for 1 hour. The microplate was washed three times with 200 μL / well of wash buffer (PBS containing 0.05% TWEEN-20 and 0.01% ProClin 300). Prior to the assay, His-tagged human IL-6Rd (4 μg / mL, GenScript) was reacted with IL-6 (100 ng / mL, GenScript) at room temperature for 1 hour to form a sIL-6Rα / IL-6 complex (with a molar ratio of IL-6 to sIL-6Rd of 1:20). 10 μL of the preformed complex solution was reacted with purified guinea pig IgG polyclonal antibody at different concentrations in a total volume of 100 μL at room temperature for 1 hour, and then the mixture was added to the gp130-Fc-coated wells. The reaction was allowed to proceed for 1 hour at room temperature. All wells were aspirated and washed three times with 200 μL / well of wash buffer. The captured IL-6 was detected by reacting with 100 μL / well of biotin-labeled rabbit anti-IL-6 antibody (1:1,000 dilution, R&D Systems) at room temperature for 1 hour. Then, bound biotin-labeled antibody was detected using streptavidin poly-HRP (1:40,000 dilution, Thermo Fisher Scientific) for 1 hour (100 μL / well). All wells were aspirated and washed three times with 200 μL / well of wash buffer. Finally, wells were developed with 100 μL / well of OptEIA TMB substrate (BD Biosciences), and the reaction was terminated by adding 100 μL / well of 1 M sulfuric acid. The developed absorbance was measured by a VersaMax ELISA microplate reader (Molecular Devices), and the half-maximal inhibitory concentration (IC) was calculated in Prism 6 software (GraphPad Software) by fitting the output response curve using a 4-parameter logit curve. 50 ).

[0335] d. IL-6-dependent TF-1 cell proliferation analysis

[0336] Human erythroleukemia TF-1 cells are able to proliferate in response to human IL-6. 5 x 10 cells were cultured in RPMI 1640 medium supplemented with 2.5% FBS in a total volume of 100 μL per well at 37°C in an atmosphere of 5% CO2 in the presence of various concentrations of purified guinea pig IgG polyclonal antibodies. 3 The cells were cultured with human recombinant IL-6 at a final concentration of 10 ng / mL for 72 hours for this analysis. Tocilizumab, an anti-IL-6 receptor antibody, was also included as a control for the study. Cell growth and survival were determined by adding 40 μL of CellTiterGlo reagent (Promega) to each well and then reacting at room temperature for 10 minutes. The resulting luminescence was measured using a SpectraMax i3x multi-function microplate reader (Molecular Devices), and the output response curve was fitted using a 4-parameter logit curve, and the half-maximal inhibitory concentration (IC) was calculated in Prism6 software (GraphPad Software). 50 ).

[0337] e. Analysis of IL-6-induced STAT3 phosphorylation

[0338] The human myeloma cell line RPMI 8226, which does not constitutively activate STAT3 phosphorylation, requires exposure to IL-6 to activate STAT3. To investigate whether purified IgG could inhibit IL-6-induced STAT3 phosphorylation in RPMI 8226 cells, 8x10 50 μL of RPMI 8226 medium were cultured in the presence of guinea pig polyclonal antibody (at a concentration of 100 μg / mL) at 37°C in an atmosphere of 5% CO2. 5Cells were cultured with IL-6 at a final concentration of 10 ng / mL for 30 minutes. The anti-IL-6 receptor antibody Tocilizumab was included as a study control. Phosphorylated STAT3 levels were measured using the PathScan p-Stat3 ELISA kit (Cell Signaling). Briefly, cell lysates were prepared by suspending cells in 30 μL of cell lysis buffer (Cell Signaling) supplemented with 1% phosphatase inhibitor cocktail 3 (Phosphatase Inhibitor Cocktail 3, Sigma-Aldrich), and centrifuged at 12,000 x g for 10 minutes at 4 ° C to remove cell debris. According to the supplier's instruction manual, 10 μg of clarified cell lysate was used to measure the phosphorylated STAT3 content. The absorbance value was determined by the VersaMax ELISA microplate reader (Molecular Devices).

[0339] f. IL-6-induced MCP-1 production

[0340] U937 is a pro-monocyte cell line that can be induced to differentiate into mature macrophages by various agents. IL-6 can promote the production of MCP-1 in monocytes. Anti-IL-6 antibodies elicited by IL-6 peptide construct immunogens can regulate IL-6-dependent MCP-1 secretion in U937 cell lines. In an environment of 37°C and 5% CO2, 8x10 3 Cells, human recombinant IL-6 at a final concentration of 10 ng / mL, and purified guinea pig IgG polyclonal antibodies at different concentrations were cultured for 24 hours for this analysis. Tocilizumab, an anti-IL-6 receptor antibody, was also included as a control for the study. Clarified supernatants were prepared by centrifuging the culture medium at 300 x g for 10 minutes and stored at -30°C. According to the supplier's instructions, 100 μL of the diluted supernatant (1:100 dilution) was applied to the human MCP-1 quantitative ELISA kit (Thermo Fisher). The absorbance of the developed image was determined by a VersaMax ELISA microplate reader (M0lecular Devices), and a standard curve was generated using a 4-parameter logit curve fit generated by SoftMax Pro software (M0lecular Devices), and used to calculate the MCP-1 concentration in all test samples. The response curve was generated using a 4-parameter logit curve fit, and the half-maximal inhibitory concentration (IC) was calculated in Prism 6 software (GraphPad Software). 50 ).

[0341] Example 4. Animals used for safety, immunogenicity, toxicity and efficacy studies

[0342] Guinea Pig:

[0343] In mature, unexposed or unstimulated Immunogenicity studies were performed in adult male and female Duncan-Hartley guinea pigs (300-350 g / BW). At least three guinea pigs were used in each group in the experiment. The experimental plan involving Duncan-Hartley guinea pigs (8-12 weeks old; Covance Research Laboratories, Denver, PA, USA) was performed in an animal facility contracted by United Biomedical (UBI) as the trial sponsor in accordance with an approved IACUC application.

[0344] Rats:

[0345] Lewis rats were used for the induction of collagen-induced arthritis (CIA). Female Lewis rats aged 8-12 weeks were purchased from Biolasco and weighed approximately 180 g. The animals were housed in the animal facility of the UBI Asia laboratory and adapted to the environment for 1 week under fixed temperature (22°C), humidity (72%), and 12-hour light / 12-hour dark cycle. Rats were given free access to rat food and water. All experimental plans followed the principles of laboratory animal care. Collagen-induced injections were administered at the base of the tail on days 0 and 7 via the intradermal route. Blood was collected as described in the experimental plan. Clinical observations were evaluated 3 times a week using a scoring system to assess the severity of arthritis in the CIA mouse model until day 35. Antibody titers were detected for anti-IL-6 (rat) using ELISA analysis. Hematological tests of relevant inflammatory biomarkers (e.g., CRP) and blood WBC counts were evaluated.

[0346] Example 5. Providing a formulation for evaluating the immunogenicity of IL-6 peptide constructs in guinea pigs

[0347] The pharmaceutical compositions and formulations used in each experiment are described in more detail as shown below. In short, the dosage form specified in each experimental group generally contains all types of specially designed IL-6 peptide immunogen constructs, which have IL-6 B cell epitope peptide fragments, which are connected by different types of spacers (e.g., εLys (εK) or lysine-lysine-lysine (KKK) to enhance the solubility of the peptide construct) and promiscuous T helper cell epitopes, and the promiscuous T helper cell epitopes include two groups of artificial T helper cell epitopes derived from measles virus fusion protein and hepatitis B surface antigen. The IL-6 B cell epitope peptide is connected to the amino or carboxyl terminus of the specially designed peptide construct. Initially, hundreds of specially designed IL-6 peptide immunogen constructs were evaluated in guinea pigs for their relative immunogenicity with the corresponding IL-6 B cell epitope peptide. As specified, different amounts of IL-6 peptide immunogen constructs were formulated in water-in-oil emulsions using Seppic MONTANIDE ISA 51, an oil approved for human vaccine use, or in suspensions using mineral salts (ADJUPHOS) or ALHYDROGEL (Alum). The formulations were generally prepared by dissolving the IL-6 peptide constructs in water at a concentration of about 20 to 800 μg / mL and formulating with MONTANIDE ISA 51 as a water-in-oil emulsion (1:1 volume), or with mineral salts (ADJUPHOS) or ALHYDROGEL (Alum) (1:1 volume). The formulations were left at room temperature for about 30 minutes and mixed by vortexing for about 10 to 15 seconds prior to immunization.

[0348] Some animals are immunized with 2 to 3 doses of a particular formulation, administered at time 0 (primary immunization) and 3 weeks after the primary immunization (wpi) (boost immunization), with an optional second boost immunization at 5 or 6 wpi, administered by the intramuscular route. These immunized animals are then evaluated for their cross-reactivity with recombinant IL-6 for the immunogenicity of the corresponding IL-6 peptide immunogen constructs used in the individual formulations. Those IL-6 peptide immunogen constructs that were strongly immunogenic in the initial screening of guinea pigs were further tested in macaques in water-in-oil emulsions, mineral salts, and alum-based formulations for dosing regimens within a specific period as specified in the immunization protocol.

[0349] Only the most promising IL-6 peptide immunogen construct candidates will be further extensively evaluated for their ability to break through immune tolerance in mice or rats using the corresponding mouse or rat IL-6 peptide immunogen constructs. The IL-6 peptide immunogen constructs with the best immunogenicity in rats can elicit anti-IL-6 antibody titers against endogenous IL-6; in particular, they have the ability to inhibit blood inflammatory factors and reduce clinical symptoms of rheumatoid arthritis in the CIA-induced Lewis rat model, or have the ability to inhibit blood neutrophilia induced by subcutaneous administration of exogenous IL-6 in cynomolgus monkeys. The optimized IL-6 peptide immunogen constructs will be incorporated into the final dosage form and provided for use in GLP-guided studies of immunogenicity, duration, toxicity and efficacy demonstration in preparation for the submission of an investigational new drug application and in clinical trials in patients with autoimmune rheumatoid arthritis.

[0350] Example 6. Design rationale, screening, identification, evaluation of functional properties, and optimization of multi-component formulations containing IL-6 peptide immunogen constructs for the treatment of autoimmune rheumatoid arthritis

[0351] IL-6, a cytokine, was selected as the target molecule for design and content of the present invention. Figure 1 Shown is an alignment of IL-6 sequences from species human (SEQ ID NO: 227), macaque (SEQ ID NO: 228), mouse (SEQ ID NO: 229), and rat (SEQ ID NO: 230). Figure 2 A general overview of the invention and development steps is described, with a flow chart identifying the development process leading to the commercialization (industrialization) of IL-6 formulations. Detailed evaluation and analysis of each step, with pleasant and unpleasant surprises, has led to countless experiments in the past, and will ultimately lead to the commercialization of safe and effective IL-6 formulations.

[0352] a. Design History

[0353] Each peptide immunogen construct or immunotherapy product requires its own design focus and approach, which is based on its specific disease mechanism and the target protein required for intervention. The design is based on the target IL-6 molecule, which is a cytokine. The process from research to commercialization usually takes one to several decades to complete. The identification of IL-6 B cell epitope peptides related to the functional site for intervention is the key to the design of immunogen constructs. Continuous pilot immunogenicity studies are conducted in guinea pigs, and various T helper cell supports (T helper support) (carrier protein or suitable T helper cell peptides) are included in various formulations to evaluate the functional properties of the induced antibodies. After extensive serological validation, the candidate IL-6 B cell epitope peptide immunogen constructs are then further tested in the target species or non-human primates to further verify the immunogenicity and direction of the IL-6 peptide immunogen design. The selected IL-6 peptide immunogen constructs are then formulated with different mixtures to evaluate the subtle differences in functional properties related to each interaction between peptide constructs when used in combination. After additional evaluations, the final peptide constructs, peptide compositions and their formulations, as well as individual physical parameters of the formulations are determined, leading to the development process of the final product.

[0354] b. Design and validation of IL-6-derived peptide immunogen constructs for use in pharmaceutical compositions with the potential to treat patients with diseases affected by IL-6 dysregulation, including autoimmune rheumatoid arthritis

[0355] In order to generate the most effective peptide constructs for inclusion into pharmaceutical compositions, a repertoire of human IL-6 B cell epitope peptides (SEQ ID NOs: 5-19) and promiscuous T helper cell epitopes derived from various pathogens or artificial T helper cell epitopes (SEQ ID NOs: 78-106 and 216-226) were further designed and made into IL-6 peptide immunogen constructs (SEQ ID NOs: 107-215) and provided initially for immunogenicity studies in guinea pigs.

[0356] i) Design of IL-6 B cell epitope peptide sequence from the region containing two intramolecular loop structures

[0357] Among many other tested regions, a region located between and including two intramolecular loop structures was selected for further B cell epitope peptide design. This region was found to be near the α and β or gp130 chains of IL-6R. After IL-6 binds to IL-6R, IL-6R will transmit activation signals within the cell, leading to subsequent major cellular events. In SEQ ID NO: 1 or Figure 1As shown in SEQ ID NO: 227, the two loop structures are C73-C83 (SEQ ID NO: 5) and C44-C50 (SEQ ID NO: 15), and the region between the two loop structures is located between 3 and 4 α-helical bundles.

[0358] Initially, the mouse and rat counterpart loop structures (e.g., SEQ ID NOs: 20 and 74) were selected as B cell epitopes for IL-6C73-C83 (SEQ ID NO: 5) to design 3T helper cell epitope peptide (SEQ ID NO: 89) and linker (SEQ ID NO: 77) connected to the IL-6 peptide immunogen structure. Two IL-6 peptide immunogen constructs were formulated using ISA 51 and CpG for primary immunization in guinea pigs at a concentration of 400 μg / 1 mL and boosted at a concentration of 100 μg / 0.25 mL (3, 6 and 9 wpi). To test the immunogenicity in guinea pigs, an ELISA assay was used, and guinea pig immune sera were diluted from 1:100 to 1:10000 in a 10-fold serial dilution. ELISA microtiter plates were coated with human IL-6 peptide (SEQ ID NO: 5), mouse or rat peptides (SEQ ID NOs: 20 and 74) at 0.5 μg of peptide per well. Using A 450 A with a critical value of 0.5 450nm The titer of the test serum was calculated by linear regression analysis, with Log 10 ELISA results showed that the two peptide immunogen constructs from human IL-6 73-83 (SEQ ID NO: 107) and mouse IL-6 72-82 (SEQ ID NO: 146) not only induced high immunogenicity titers against their own B cell epitope peptides human IL-6 C73-C83 (SEQ ID NO: 5) and mouse B cell epitope peptide (SEQ ID NO: 20), as shown in Table 4, the two antisera were also found to have moderate cross-reactivity against their homologous B cell epitope peptides from human and mouse IL-6. The study showed that the designed two peptide immunogens were able to induce specific antibodies that were cross-reactive against the human IL-6 C73-C83 peptide and its mouse counterpart peptide. In addition, as shown in Table 5A, IL-6 peptide immunogen constructs 124, 125, 126 and 132 (cyclic) and 133 (non-cyclic), which have sequences extending beyond IL-6 73-83 to the amino-terminal part of the cyclic structure, were also tested for immunogenicity and their cross-reactivity with human IL-6 protein, and the results showed that they had high immunogenicity and moderate cross-reactivity.

[0359] Subsequently, another loop structure from IL-6 C44 to C50 was designed. B cell epitope peptides of different sizes covering the C44-C50 loop structure were selected to construct IL-6 peptide immunogens. 1T helper cell epitope peptide (SEQ ID NO: 91) and a short linker εK or a longer linker KKK-εK (SEQ ID NO: 77) to construct a new human IL-6 immunogen construct. For the target B cell epitope peptide, 1T helper cell epitope peptide and linker sequence were placed at the amino or carboxyl terminus or both ends of the structure. Seven human IL-6 immunogen constructs (SEQ ID NO: 128, 129, 131 and 134-137) from three different sized B cell epitopes IL-644-50 (SEQ ID NO: 15), IL-642-57 (SEQ ID NO: 12), IL-642-72 (SEQ ID NO: 10) were designed and used for immunogenicity studies. Each peptide immunogen was formulated with ISA51 and CpG to immunize guinea pigs, using a dose of 400 μg / ml for primary immunization and a dose of 100 μg / ml for boosting at 3, 6, and 9 wpi, with 3 guinea pigs in each group. ELISA assays were performed to evaluate the immunogenicity of the designed IL-6 peptide immunogens. The wells of a microtiter plate were coated with IL-6 B cell epitope peptides and human IL-6 protein (SEQ ID NO: 1) as target peptides. Guinea pig immune serum was diluted in a 10-fold serial dilution from 1:100 to 1:100000. 450 A with a critical value of 0.5 450nm The titer of the test serum was calculated by linear regression analysis, with Log 10 All eight peptide immunogens induced strong immunogenic titers against the B cell epitope peptides coated in the microplate wells. The ELISA results showed that these seven peptide immunogen constructs not only induced high immunogenic titers against the corresponding IL-6 B cell epitope peptides, but also that these antisera had moderate cross-reactivity to human IL-6 protein (SEQ ID NO: 1), as shown in Table 5B.

[0360] In addition, two other B cell epitope peptides were designed, which have sequences between the two loop structures of SEQ ID NO: 13 and SEQ ID NO: 9 (i.e., IL-6 61-75 and IL-6 52-72). 1T helper cell epitope peptide (SEQ ID NO: 91) and a short linker εK or a longer linker KKK-εK (SEQ ID NO: 77) to construct new human IL-6 immunogen constructs (SEQ ID NOs: 127, 138-145). For the target B cell epitope peptide, 1T helper cell epitope peptide and linker sequence were placed at the amino or carboxyl terminus of the construct. Nine human IL-6 immunogen constructs (SEQ ID NO: 127, 138-145) from three different sized B cell epitopes IL-652-72 (SEQ ID NO: 9), IL-661-75 (SEQ ID NO: 13), IL-661-72 (SEQ ID NO: 14) were designed and used for immunogenicity studies. Each peptide immunogen was formulated with ISA51 and CpG to immunize guinea pigs, using a dose of 400 μg / ml for primary immunization and a dose of 100 μg / ml for boosting at 3, 6, and 9 wpi, with 3 guinea pigs in each group. ELISA assays were performed to evaluate the immunogenicity of the designed IL-6 peptide immunogens. IL-6 B cell epitope peptides and human IL-6 protein (SEQ ID NO: 1) were used to coat the wells of microtiter plates as target peptides. Guinea pig immune serum was diluted in a 10-fold serial dilution from 1:100 to 1:100000. 450 A with a critical value of 0.5 450nm The titer of the test serum was calculated by linear regression analysis, with Log 10 All nine peptide immunogens induced strong immunogenic titers against the B cell epitope peptides coated in the microplate wells. The ELISA results showed that these eight peptide immunogen constructs not only induced high immunogenic titers against the corresponding IL-6 B cell epitope peptides, but also that these antisera had moderate cross-reactivity to human IL-6 protein (SEQ ID NO: 1), as shown in Table 5C.

[0361] In addition to the peptide constructs containing endogenous internal loop structures as described above, IL-6 B cell epitope peptide designs were also directed against an epitope located on human IL-6 associated with the monoclonal antibody Olokizumab. Olokizumab is known to inhibit the binding of IL-6 / sIL-6R to gp130. Two peptides of different sequence sizes covering a portion of the Olokizumab-associated conformational epitope were designed to construct IL-6 peptide immunogen constructs. 1T helper cell epitope peptide (SEQ ID NO: 91) and a longer linker εK-KKK (SEQ ID NO: 77) to construct a new human IL-6 immunogen construct. The 1T helper cell epitope and linker sequence were placed at the amino terminus or carboxyl terminus or both of the B cell epitope peptides. Five human IL-6 immunogen constructs (SEQ ID NOs: 112-117) from two different sized B cell epitopes (SEQ ID NOs: 18 and 19) were designed and used for immunogenicity studies. Another IL-6 73-83 construct (SEQ ID NO: 118) of 1 (Group 6) was used as a control group for comparison of immunogenicity and immunospecificity. Each peptide immunogen was formulated with ISA51 and CpG to immunize guinea pigs, using a dose of 400 μg / ml for primary immunization and a dose of 100 μg / ml for booster immunization at 3, 6, and 9 wpi, with 3 guinea pigs in each group. ELISA assay was performed to evaluate the immunogenicity of these designed peptide immunogens. Three B cell epitope peptides (IL-6C73-C83 (SEQ ID NO: 5), IL-6150-162 (SEQ ID NO: 18), and IL-6144-166 (SEQ ID NO: 19)) were used to coat the wells of microtiter plates as target peptides. Guinea pig immune sera were diluted from 1:100 to 1:100000 in a 10-fold serial dilution. Using A 450 A with a critical value of 0.5 450nm The titer of the test serum was calculated by linear regression analysis, with Log 10 All six peptide immunogens induced strong immunogenic titers against their own B cell epitope peptides coated in the microplate wells. ELISA data showed that there was cross-reactivity between the five immunogen constructs (SEQ ID NOs: 112-117) because they shared the same helix-turn-helix structure from sequences 144 to 166 as shown in Table 5D. Table 5E shows the cross-reactivity of immune sera from these constructs to human IL-6 protein, indicating the potential of this site to interfere with IL-6 binding, which will be tested in other functional assays of IL-6 induction.

[0362] ii) Ranking of pathogen-derived heterologous T helper cell epitopes and their inclusion in the design of IL-6 peptide immunogen constructs to enhance the immunogenicity of selected IL-6 B cell epitope peptides

[0363] Table 2 lists a total of 29 heterologous Th epitopes (SEQ ID NOs: 78-106 and 216-226) that have been tested for relative potency in multiple species including mice, rats, guinea pigs, baboons and macaques to enhance the immunogenicity of B cell epitopes.

[0364] For immunogenicity studies in guinea pigs, a representative study of IL-6 peptide immunogen constructs containing the IL-6C73-C83 B cell epitope peptide (SEQ ID NO: 5) linked to each promiscuous T helper epitope via an εK spacer was performed to rank the relative potency of each heterologous T helper epitope as shown in Table 6. Results obtained at 6 weeks after primary immunization (6wpi) were used to rank the different IL-6 peptide immunogen constructs. While all selected Th epitopes had the ability to enhance the immunogenicity of the IL-6 B cell epitope peptide, the most potent construct was found to be the construct of SEQ ID NO: 119.

[0365] Careful calibration of the immunogenicity of each and all IL-6 peptide immunogen constructs in different species, including primates, ensures the success of the final Th peptide selection and final formulation development.

[0366] iii) Evaluation of the immunogenicity of IL-6 peptide immunogen constructs with respect to their antibody reactivity with recombinant IL-6

[0367] Figure 3 The kinetics of antisera over a 12-week period in guinea pigs immunized with 25 different IL-6 peptide immunogen constructs (SEQ ID NOs: 107, 112-114, 116-118, and 124-145) are further illustrated. Guinea pig antisera collected at 0, 3, 6, 8 / 9, and 12 wpi were diluted from 1:100 to 1:4.19 x 10 8 ELISA microtiter plates were coated with 50 ng of human IL-6 (GenScript) per well. The titers of the test sera were calculated by 4-parameter logit curve fitting and expressed as Log (EC 50 ) to obtain the half of maximal effect concentration (EC 50 )). All 25 immunogen constructs were able to elicit a certain degree of cross-reactivity with native human IL-6, suggesting that the anti-IL-6 antibodies raised by them may have the potential to neutralize IL-6 activity.

[0368] These data provide useful information for further animal testing to investigate whether the designed human IL-6 peptide immunogens can elicit cross-reactive antibodies from different animal species. 8- or 9-wpi sera induced with 29 different immunogen constructs (SEQ ID NOs: 107, 112-114, 116-118, and 124-145) were selected for IgG purification by protein A affinity chromatography. Figure 4 illustrates that purified polyclonal guinea pig IgG induced with SEQ ID NOs: 107, 116, 118, and 124-133 cross-react with human, monkey, and rat recombinant IL-6 proteins (all purchased from GenScript). Among them, the peptides of (SEQ ID NOs: 107, 118 and 124-126) contain the IL-6 73-83 loop structure in different peptide constructs, the peptides (SEQ ID NOs: 128, 129 and 131) contain the IL-6 44-50 loop structure, and SEQ ID NO: 132 contains two loop structures.

[0369] iv) Identification of endogenous / autologous Th epitopes for exclusion in IL-6 B cell epitope peptide design

[0370] Identification of potential endogenous / self Th epitopes present in target proteins may provide benefits for the design of compositions for immunotherapeutic interventions, as the presence of T helper cell epitope structural features in peptide immunogen constructs may cause undesirable inflammation upon boosting due to activation of autologous T cells, as in the previous Alzheimer's vaccine AN1792. As shown in Table 7, despite being formulated in a potent water-in-oil emulsion formulation, free IL-6 B cell epitope peptides IL-6 62-83 (SEQ ID NO: 6), IL-6 58-83 (SEQ ID NO: 7), IL-6 52-83 (SEQ ID NO: 8), IL-6 52-72 (SEQ ID NO: 9), and IL-6 42-72 (SEQ ID NO: 10) provided a clean background in immunogenicity testing, indicating that they qualify as IL-6 B cell epitope peptide candidates for use in constructing IL-6 peptide immunogen constructs for use in IL-6 formulations.

[0371] v) Focused antibody responses elicited using IL-6 peptide immunogen constructs targeting only IL-6 B cell epitopes

[0372] It is well known that all carrier proteins (such as keyhole limpet hemocyanin (KLH) or other carrier proteins (such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins)) used to enhance the immune response against target B cell epitope peptides can elicit more than 90% of antibodies against the enhancing carrier protein and less than 10% of antibodies against the target B cell epitope in the immunized host by chemically conjugating such B cell epitope peptides to the respective carrier proteins. It is therefore important to evaluate the specificity of the IL-6 peptide immunogen constructs of the present invention. A series of eight IL-6 peptide immunogen constructs (SEQ ID NOs: 138-145 from Table 3) have B cell epitopes of different lengths, and the B cell epitopes are linked to heterologous T helper cell epitopes via spacer sequences. 1 (SEQ ID NO: 91), was prepared for antigenicity evaluation. 1 (T helper cell epitope peptide for B cell epitope immunopotency) was coated on a microtiter plate, and immune serum from guinea pigs was used to test the peptide for immunopotency. In contrast to the high immunogenicity of these constructs to the corresponding target IL-6 B cell epitope peptides (as indicated by the high titers of antibodies generated against the IL-6 B cell epitopes), most, if not all, immune sera were found to cross-reactive with the IL-6 B cell epitope peptides as shown in Table 8. 1 peptide was non-reactive.

[0373] In summary, a simple peptide immunogen design (comprising a target IL-6 B cell epitope peptide linked to a carefully selected T helper cell epitope) can generate a focused and clean immune response only to the corresponding IL-6 B cell epitope peptide. For pharmaceutical composition design, the more specific the immune response it generates, the higher the safety it provides for the composition. Therefore, the IL-6 peptide immunogen constructs of the present invention are highly specific, but highly effective for their target.

[0374] vi) Immunogenicity assessment of IL-6 peptide immunogen constructs for their inhibition of IL-6 and IL-6R interaction

[0375] IL-6 signals through the heterotrimeric IL-6R / gp130 complex, which is involved in activating downstream signal transduction. Neither IL-6 nor IL-6R alone can activate downstream signal transduction. Further studies were conducted to investigate whether candidate IL-6 peptide immunogen constructs can elicit antibodies in guinea pigs, and the elicited antibodies can neutralize IL-6 to block the interaction between IL-6 and IL-6 receptors (IL-6R) (Rose-John, et al., 2017).

[0376] Purified guinea pig IgGs (guinea pig IgGs were purified from immune sera of guinea pigs immunized with 25 respective candidate IL-6 peptide immunogen constructs (SEQ ID NOs: 107, 116, 118, 124-145)) were used in ELISA assays to evaluate: (a) their relative immunogenicity by ELISA using the corresponding IL-6 B cell epitope peptides as solid phase antigen coating as described in Example 3; (b) their relative ability to cross-react with IL-6 proteins from human, monkey and rodent species; if both (a) and (b) gave positive results, then whether these purified antibodies could neutralize IL-6 protein and thereby inhibit the interaction between IL-6 and IL-6Ra (i.e., cis signaling) or the interaction between IL-6 / IL-6Ra and IL-6Rb / or gp130 (i.e., trans signaling) was investigated.

[0377] like Figure 3 As shown in Figure 2, all purified antibodies from guinea pigs immunized with carefully designed candidate IL-6 peptide immunogen constructs showed significant antibody titers over a time course that matched the immunization schedule. Figure 4A and 4B As shown, all purified antibodies from immune sera derived from immunization with these IL-6 peptide immunogen constructs showed high reactivity with human IL-6 protein and also had moderate cross-reactivity with monkey (macaque) and rodent IL-6 proteins.

[0378] In addition, as shown in Figure 5A, representative antibodies (representative antibodies purified from immune sera of guinea pigs immunized with various candidate IL-6 peptide immunogen constructs (e.g., those having SEQ ID NOs: 107, 116, 118, 124, 132-134 and 137 sequences)) can competitively inhibit the interaction between IL-6 and IL-6Ra in a dose-dependent manner through a cis signal transduction mode.

[0379] In addition, as shown in Figure 5B, representative antibodies (representative antibodies purified from immune sera of guinea pigs immunized with various candidate IL-6 peptide immunogen constructs (e.g., those having SEQ ID NOs: 128, 129, 134 and 135 sequences)) can competitively inhibit the interaction of IL-6-IL-6Ra complex with IL-6Rb / gp130 in a dose-dependent manner through a trans-signaling mode.

[0380] In contrast, antibodies purified from immune sera derived from a peptide immunogen construct (SEQ ID NO: 130) comprising a prior art B cell epitope peptide sequence (SEQ ID NO: 11) were unable to inhibit either cis or trans signaling pathways.

[0381] vii) Epitope mapping for fine specificity using immune sera (9 wpi) elicited by various IL-6 peptide immunogen constructs

[0382] The design of the IL-6 composition containing the IL-6 peptide immunogen construct is focused on the region containing two intramolecular loop structures C44-C50 (SEQ ID NO: 15) and C73-C83 (SEQ ID NO: 5) near the IL-6R binding site. This construct-based design aims to retain at least one natural intramolecular loop structure as an immunogenic target.

[0383] Eight representative IL-6 B cell epitope peptides 62-83 (SEQ ID NO: 124), 58-83 (SEQ ID NO: 125), 52-83 (SEQ ID NO: 126), 52-72 (SEQ ID NO: 127), 42-72 (SEQ ID NO: 128) have Th located at the amino terminus of the B cell epitope, 42-72 (SEQ ID NO: 129 have Th located at the carboxyl terminus of the B cell epitope), 50-67 (SEQ ID NO: 130) and 73-83 (SEQ ID NO: 107).

[0384] IL-662-83 (SEQ ID NO: 6), 58-83 (SEQ ID NO: 7), 52-83 (SEQ ID NO: 8), 52-72 (SEQ ID NO: 9), 42-72 (SEQ ID NO: 10), 50-67 (SEQ ID NO: 11) and 73-83 (SEQ ID NO: 5) were used to design B cell epitope peptides. The B cell epitope peptides were fused to the amino or carboxyl terminal of the B cell epitope peptides. 1 (SEQ ID NO: 91) was connected to form a prototype peptide immunogen. An εK linker or εK-KKK (SEQ ID NO: 77) spacer was used between the B cell epitope and the Th epitope to form the peptide immunogen constructs shown in Table 3 (SEQ ID NOs: 124-130, 107). All B cell epitope peptides located within amino acids (aa) 42-83, 42-72, and 73-83 were designed to have a constrained ring structure of C44-C50 or C73-C83 by cyclization.

[0385] Individual IL-6 B cell epitope peptides C73-C83 (SEQ ID NO: 5) and E42-G72 (SEQ ID NO: 10) were used for microplate coating for ELISA assays to assess the antibody reactivity of hyperimmune sera obtained from guinea pigs immunized with IL-6 peptide immunogen constructs (SEQ ID NOs: 124-130, 107). The results showed that structures SEQ ID NOs: 124, 125, 126 and 107 containing the C73-C83 cyclic construct can induce high-titer antibodies against IL-6 B cell epitope peptide C73-C83 (SEQ ID NO: 5), while guinea pig antisera induced by IL-6 peptide immunogen constructs SEQ ID NOs: 127-130 containing the C44-C50 cyclic structure have antibody reactivity with the B cell epitope peptide E42-C72 (SEQ ID NO: 10), and only little or no cross-reactivity to the C73-C83 cyclic structure (SEQ ID NO: 5), indicating high specificity of immunogenicity, that is, the designed immunogen construct can induce specific antibodies to react with the B cell epitope functional block corresponding to IL-6 (Table 9).

[0386] In fine epitope mapping studies (Table 9), the antibody binding site was localized to specific residues within the target region, and 51 overlapping 10-mer peptides (SEQ ID NOs: 21 to 71) were synthesized, which covered the IL-6 sequence region from amino acid 32 to amino acid 91. These 10-mer peptides were coated as solid phase immunosorbents on 96-well microtiter plates. Pooled guinea pig antisera prepared in sample dilution buffer at a dilution ratio of 1:100 were added to the microtiter plate wells coated with 2.0 μg / mL of 10-mer peptides, followed by incubation at 37°C for 1 hour. After washing the microtiter plate wells with washing buffer, horseradish peroxidase (HRP)-conjugated recombinant protein A / G was added and incubated for 30 minutes. After washing again with PBS, the substrate was added to the wells, and the absorbance at 450 nm was measured using an ELISA microtiter plate reader, and the samples were analyzed in duplicate. The binding of immune sera elicited by the IL-6 peptide immunogen to the corresponding IL-6 B cell epitope peptide-coated wells represents the maximum antibody binding signal.

[0387] The results of fine epitope mapping showed that the pooled guinea pig sera from the IL-6 peptide immunogen constructs SEQ ID NOs: 124, 125, 126 and 107 containing the C73-C83 loop structure could induce high-titer antibodies mainly against the 10mer peptide cluster from amino acids 69-78 (SEQ ID NO: 58) to amino acids 76-85 (SEQ ID NO: 65), which had high cross-reactivity to the peptide with amino acids 35-44 (SEQ ID NO: 24), and occasionally had moderate activity to the amino terminus slightly beyond the loop structure.

[0388] Surprisingly, pooled guinea pig sera from IL-6 peptide immunogen constructs SEQ ID NOs: 127-129 containing a C44-C50 loop structure induced high titer antibodies primarily against a 10mer peptide cluster from amino acids 61-70 (SEQ ID NO: 50) to amino acids 67-76 (SEQ ID NO: 56) outside the C44-C50 loop structure, and IL-6 peptide construct 129 had a more broadly dispersed antibody reactivity extending to the amino-terminal portion of the B cell epitope peptide 41-50 (SEQ ID NO: 30), 45-54 (SEQ ID NO: 34), 57-66 (SEQ ID NO: 46), and 58-67 (SEQ ID NO: 47). Notably, immune sera generated using IL-6 peptide immunogen constructs 128 and 129 showed preferential trans-inhibition in competitive IL-6 / IL-6Rα cis and (IL-6 / IL-6Rα complex) / IL-6Rβ trans-competitive binding inhibition studies by respective ELISAs.

[0389] In summary, the designed synthetic IL-6 peptide immunogen construct (IL-6 peptide immunogen construct is with 1 The ring structure C44-C50 and C73-C83 located in IL-6 connected by epitope peptides (represented by C44-C50 and C73-C83) can induce a strong immune response and produce polyclonal antibodies against different 10mer peptide clusters close to the IL-6R binding region to allow binding inhibition (IL-6 / IL-6Rα-mediated cis or (IL-6 / IL-6Rα complex) / IL-6Rβ (or Gp130)-mediated trans-competitive binding inhibition (see Figures 5A and 5B)), which should have important medical significance.

[0390] Example 7. Evaluation of the functional properties of antibodies elicited by IL-6 peptide immunogen constructs and their preparations in an in vitro model

[0391] After demonstrating that antibodies purified from immune sera of guinea pigs immunized with carefully selected candidate IL-6 peptide immunogen constructs were highly immunogenic and cross-reactive, the following studies were designed to evaluate whether each purified IgG from these immune sera could (a) inhibit IL-6-induced STAT3 phosphorylation; (b) inhibit cell proliferation in the TF-1 cell line; and (c) inhibit IL-6-induced MCP-1 production in U937 cells, all experiments being performed in vitro.

[0392] Inhibition of IL-6-induced STAT3 phosphorylation by anti-IL-6 antibody

[0393] The IL-6 signal transduction pathway initially involves the formation of the IL-6 / IL-6Ra / IL6Rb (or Gp130) complex on the cell membrane, followed by phosphorylation of the downstream protein STAT3 in the cytoplasm. The RPMI 8226 cell line was used to evaluate the ability of these purified anti-IL-6 antibodies derived from guinea pig immune sera immunized with carefully selected candidate IL-6 peptide immunogen constructs to inhibit IL-6-induced STAT3 phosphorylation, because this 8226 cell line does not constitutively express phosphorylated STAT3.

[0394] First, cultured cells were treated with IL-6 (10 ng / ml) and purified IgGs at different concentrations. Anti-IL-6R monoclonal antibodies, such as Figure 6 As shown, anti-IL-6 IgGs elicited with representative immunogens (SEQ ID NOs: 128, 129, 134, 135 and 137) can reduce STAT3 phosphorylation at an IgG concentration of 100 μg / mL. IgG from immune sera elicited with a peptide construct (SEQ ID NO: 130) containing a prior art B cell epitope sequence (SEQ ID NO: 11) cannot inhibit STAT3 phosphorylation.

[0395] Inhibition of IL-6-dependent cell proliferation in TF-1 cells

[0396] Human erythrocytic leukemia TF-1 cells are able to proliferate in response to human IL-6. To investigate whether purified IgGs from immune sera of guinea pigs immunized with carefully selected candidate IL-6 peptide constructs (SEQ ID NOs: 116, 118, 124-129, 131-145) could inhibit IL-6-dependent cell proliferation in TF-1 cell lines, all TF-1 cell cultures were treated simultaneously with IL-6 (10 ng / ml) and purified guinea pig IgGs. TF-1 cells not treated with IL-6 and TF-1 cells treated with IL-6 alone without antibodies served as controls. As shown in Figures 7A and 7B, TF-1 cells were more proliferative in the presence of IL-6 alone than in all other groups, and their cell proliferation was twice that of cells in the group not treated with IL-6. TF-1 cell growth in the presence of anti-IL-6 IgG antibodies elicited with representative candidate IL-6 peptide immunogen constructs (SEQ ID NOs: 116, 118, 124-245, 127-129, 131-145) could be inhibited to some extent (Figures 7A and 7B). IgG from immune sera elicited with a peptide construct (SEQ ID NO: 130) containing a prior art B cell epitope sequence (SEQ ID NO: 11) was unable to inhibit IL-6-induced cell proliferation.

[0397] Inhibition of IL-6-induced MCP-1 production

[0398] MCP-1 plays an important role in acute and chronic inflammatory processes. MCP-1 is a chemokine that attracts monocytes and basophils in the pathogenesis of the disease. IL-6 can induce the expression of MCP-1 in the pro-monocytic cell line U937. In order to investigate whether anti-IL-6 antibodies elicited in guinea pigs using IL-6 peptide immunogen constructs can inhibit IL-6-dependent MCP-1 secretion in the U937 cell line, all cell culture groups were treated with IL-6 cytokine at a concentration of 10 ng / ml to induce the production of MCP-1. Representative preparations of purified IgGs (purified Ig6s are from guinea pig immune sera elicited using candidate IL-6 peptide immunogen constructs (SEQ ID NOs: 116, 118, 124-134, 136138-145)) were added to the test groups at different concentrations, and Tocilizumab was also included as a positive control group. U937 cell cultures in the presence of only IL-6 without the addition of antibodies were set up as negative controls. As Fig. 8A and 8BAs shown, with the exception of IgG from immune serum elicited by a peptide construct (SEQ ID NO: 130) comprising a prior art B cell epitope sequence (SEQ ID NO: 11) (see FIG. 8A ), varying degrees of antibody concentration-dependent inhibition of MCP-1 production were observed in groups treated with purified IgGs antibodies elicited by representative candidate peptide constructs in a dose-dependent manner.

[0399] The above-mentioned in vitro functional studies indicated that these representative IL-6 peptide immunogen constructs showed inhibitory effects on IL-6-induced inflammatory processes and pathogenic mechanisms, indicating that they have the potential to treat diseases affected by IL-6 dysregulation (including autoimmune rheumatoid arthritis).

[0400] Example 8. Evaluation of IL-6 peptide immunogen constructs in a preventive setting in the Lewis rat collagen-induced arthritis (CIA) model

[0401] Rheumatoid arthritis was targeted in a prevention study as described below to evaluate the effects of IL-6 peptide immunogen constructs on the rat collagen-induced arthritis (CIA) model.

[0402] Human IL-6 and rat IL-6 share approximately 40% amino acid sequence similarity. Based on the rat IL-6 protein sequence, rat peptide immunogen constructs (SEQ ID NOs: 148 and 157) were designed with homologs of human IL-6 B cell epitope peptides IL-6 73-83 and IL-6 144-166, and the B cell epitope peptides were used to enhance the T helper cell epitope. (SEQ ID NO: 89) and εK-KKK (SEQ ID NO: 77) as a linker were connected to the amino terminus or carboxyl terminus of the IL-6 B cell epitope peptide, respectively.

[0403] Use Fig. 9 The experimental scheme briefly shown uses Lewis rats for this study. A total of 21 rats were divided into 3 groups, and the placebo group was injected with adjuvant only. The rats in the experimental group were injected with IL-6 peptide immunogen constructs, which were formulated with ISA51 and CpG as 45μg / 0.5mL doses for primary and booster immunizations. A total of three doses were administered on days -31, -10 and 4. All rats were injected at the base of the tail using bovine type II collagen / IFA emulsion (100μg collagen per rat in a volume of 100μL) via the intradermal route 4 days before the third administration (day 0), and supplemented 3 days after the third administration (day 7). Rats were bled on days -31, -10, 0, 7, 14, 21, 26, 28 and 35. The immunogenicity titer of rat recombinant IL-6 protein was determined by ELISA analysis.

[0404] ELISA results showed that no detectable antibody titers were observed in any group before immunization on day -31. After three immunizations, placebo-treated rats showed no detectable antibody titers against anti-rat recombinant IL-6. Compared with other groups (SEQ ID NO: 157), the peptide immunogen targeting the IL-673-83 B cell epitope (SEQ ID NO: 148) during CIA elicited stronger anti-IL-6 antibody titers, Log (EC 50 ) is about 3.0( Fig.10 ).

[0405] Evaluation of the effects of IL-6 immunotherapy in a preventive model of CIA in rats

[0406] For clinical signs and symptoms of arthritis, rats immunized with rat IL-6 peptide constructs (having SEQ ID NOs 148 or 157) and then subjected to CIA arthritis were carefully examined. Collagen (bovine type II collagen, Chondrex Inc.) was injected into rats to rapidly develop CIA-induced arthritis. About 2 weeks after collagen induction, clinical inflammatory signs of acute arthritis were found in the hind paws (including erythema and joint swelling, with each paw scored 0-4 (total score between 0 and 16)). The highest arthritis severity scores and the most severe paw swelling ( Fig.11 and 12 ). The therapeutic efficacy of different IL-6 immunogen constructs was evaluated by arthritis severity scores. Compared with other tested immunogen constructs (SEQ ID NO: 157), the group immunized with SEQ ID NOs: 148 showed lower arthritis severity scores (reduced arthritis severity) and milder paw swelling, and there was a statistically significant difference compared with the placebo group during this in vivo immunotherapy study ( Fig.11 and 12 and Tables 10 and 11).

[0407] To observe whether IL-6 immunogen can reduce the release of neutrophils from the bone marrow into the circulation during the rat CIA induction study, the results showed that the number of neutrophils released from the bone marrow gradually increased from day 0 and reached its peak on day 14. Rat IL-6 immunogen (SEQ ID NO: 148) effectively reduced the release of neutrophils from the bone marrow into the circulation ( Fig.13 and Table 12). This suggests that both designed IL-6 immunogen constructs play an important role in reducing the inflammatory process.

[0408] The results of this study indicate that the rat IL-6 B cell epitope peptide IL-6 72-82 represents a good candidate for human IL-6 peptide immunogen constructs incorporating IL-6 73-83 as B cell epitope peptides to treat diseases affected by IL-6 dysregulation in a preventive mode, where induced polyclonal antibodies against the IL-6 molecule can neutralize the blood-circulating cytokine IL-6 to block / inhibit its signal transduction, thereby reducing clinical inflammatory pathological processes.

[0409] CIA rats were injected 3 times intramuscularly with rat IL-6 peptide immunogen constructs or with adjuvant alone. These animals had good overall tolerance to the candidate rat IL-6 formulation at a dose of 45 μg / 0.5 mL. The candidate rat IL-6 peptide immunogen with SEQ ID NO: 148 showed greater efficacy in antibody response and reduction of arthritis severity compared to that with SEQ ID NO: 157.

[0410] Example 9. Effect of IL-6 peptide immunogen constructs and formulations thereof on the treatment of rheumatoid arthritis as shown in a therapeutic paradigm in the Lewis rat CIA model

[0411] Proof of Concept (POC) Study of IL-6 Peptide Immunogen Constructs in the Lewis Rat Collagen-Induced Arthritis (CIA) Model

[0412] To confirm the efficacy of the IL-6 immunogen construct (SEQ ID NO: 148), a POC study was performed in the Lewis rat CIA model, where two different adjuvant formulations were evaluated in this efficacy study, such as Fig.14 As shown. Seven animals were assigned to each of the two treatment groups and six animals were assigned to the placebo group. The animals in the two treatment groups were injected with a peptide immunogen construct (SEQ ID NO: 148) formulated with ISA 51 alone or ISA51 / CpG at 45 μg / 0.5 mL / dose for primary and booster immunizations (boost immunizations were performed on days -7, 7, 14, 21 and 28). At the same injection time point as the treatment groups, an adjuvant vehicle without a peptide immunogen construct was used alone to inject the placebo group. All groups were injected at the base of the tail via the intradermal route on days 0 and 7 using a bovine type II collagen / IFA emulsion (100 μg of collagen in a volume of 100 μL per rat) to induce arthritis. This study was terminated on day 35.

[0413] The immunogenicity titers of sera from immunized rats against rat IL-6 recombinant protein were evaluated by ELISA. The results indicated that both treatment groups (the two treatment groups were treated with the same IL-6 peptide immunogen construct formulated with different adjuvants) produced high antibody titers against rat IL-6 in a steadily increasing manner after immunization. The titers of both treatment groups reached a level of 3Log (EC 50 ) and remained stable until the end of the study on day 35 ( Fig.15 ). This result further confirmed that the peptide immunogen construct (SEQ ID NO: 148) had comparable immunogenicity and was able to break through immune tolerance to induce specific polyclonal antibodies against rat IL-6, thereby effectively enhancing antibody production, under the conditions of using two adjuvant formulations.

[0414] Before and after immunization with IL-6 immunogen construct and induction of CIA arthritis, clinical evaluation was performed between the treatment group and the placebo group for CIA-induced arthritis in Lewis rats. Based on the clinical signs of arthritis severity during the study, the severity of arthritis was graded as 0-4 for each paw (total score between 0 and 16). The results showed that CIA-induced arthritis developed rapidly in rats after induction with collagen. The adjuvant placebo group reached the highest arthritis score of 9 on the 14th day. On the contrary, both treatment groups showed milder arthritis severity, and the scores of both groups at the same time point on the 14th day were less than 6, which was statistically significant (p < 0.01). Since then, from the 14th day to the 35th day, it was observed that the arthritis scores in all groups monitored every 2 to 3 days decreased, and a total of 9 assessments were performed until the end of the study. Results from each assessment showed that from day 14 to day 35, both treatment groups had significantly lower arthritis severity scores than the placebo group, and this was statistically significant (p < 0.01 or P < 0.001 in most cases). By the end of the study on day 35, the placebo group had a score of approximately 6, while both treatment groups had a score of approximately 3. Fig.16 As shown. Clinical signs of CIA located in the hind paws were also evaluated, and the results showed that the volume of the hind paws increased in all rats with arthritis from day 14, which is a consequence of joint inflammation. However, similar results were observed, and the hind paw volumes of both treatment groups were much less than the placebo group on days 14, 21, 28, and 35, respectively, which was statistically significant (mostly p < 0.01 to P < 0.001). By the end of the study on day 35, the hind paw volumes of both treatment groups had approached normal volumes, while the placebo group maintained a higher volume, as shown. Fig.16 All these findings indicate that both adjuvants showed similar clinical efficacy in this study, but the ISA51+CpG combination was slightly better than ISA51.

[0415] Serum IL-6 levels are positively correlated with the degree and severity of joint involvement; while other downstream serum inflammatory biomarkers such as C-reactive protein (CRP) are also indicators for assessing the severity of inflammation. ELISA was used to measure serum levels of CRP. Rats in the placebo group (adjuvant vehicle-treated CIA) had significantly higher serum CRP levels (p < 0.05) compared to both treatment groups ( Fig.17 ). The mean serum CRP value in CIA rats treated with the immunogen (SEQ ID NO: 148) on day 21 was close to the normal value, which was significantly lower than that of the placebo group.

[0416] Histopathological examination studies were performed to evaluate the effect of IL-6 peptide immunogen on the changes in ankle joint tissue destruction. CIA rats (7 / treatment group, 6 / placebo group) were sacrificed on day 35, and ankle joint tissues were removed for fixation, decalcification and paraffin embedding of tissue sections. Tissue sections were prepared and stained with H&E. Histopathological examination showed Fig.18 , where the normal control group showed healthy joint spaces and normal tissue. In contrast, the placebo group showed typical features of arthritis, characterized by significant synovial and periarticular inflammation, synovial hyperplasia, and bone erosion. Joint pathology of CIA rats immunized with SEQ ID NO: 148 showed milder inflammation (with milder cellular infiltration, milder synovial hyperplasia, and bone erosion), indicating that the destruction of the ankle joint was alleviated by the peptide immunogen construct (SEQ ID NO: 148). Fig.18 Also shown is a comparison of pathological scores among the three different groups, which uses a modified Mankin scoring system to evaluate articular cartilage, by grading cartilage structure from 0 to 6, cellular morphology from 0 to 3, safranin O staining from 0 to 4, and synovial inflammation and hyperplasia from 0 to 4 (Clin Immunol. 124: 244-257). The peptide immunogen construct (SEQ ID NO: 148) treatment group significantly reduced the pathological score to 6 points compared to 11 points in the placebo group.

[0417] Inflammatory cytokines are thought to play an important role in the pathogenesis of RA. Immunohistochemical staining was used to evaluate inflamed ankle tissue. Briefly, formalin-fixed paraffin-embedded tissue sections were dewaxed in xylene, immersed in decreasing concentrations of ethanol, and rehydrated in water. All sections were processed for microwave-enhanced antigen retrieval. Slide-mounted sections immersed in antigen retrieval citrate solution (Scytek) were heated to boiling at maximum power in a microwave oven and then cooled to room temperature for 30 minutes. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide / PBS for 10 minutes. Sections were pre-reacted with Ultravision Protein Block (ThermoFisher) at room temperature for 1 hour. The sections were then reacted with primary rabbit anti-rat IL-17 (Abbiotec, diluted 1:100 in PBST), anti-rat TNF-α (Abcam, diluted 1:100 in PBST), or anti-rat MCP-1 (Abcam, diluted 1:200 in PBST) at 4°C overnight, washed with TBST (Scytek), and then developed (using Polink-2 Plus HRP Rabbit with DAB Kit (GBILabs)). The sections were counterstained with hematoxylin (Leica Biosystems), dehydrated, and mounted in Surgipath Micromount mounting medium (Leica Biosystems). Fig.19 A significant increase in tissue TNF-α, IL-17, and MCP-1 was shown in the placebo group. However, the production of these cytokines was greatly suppressed in CIA rats treated with IL-6 immunogen.

[0418] This study showed that immunization with the IL-6 peptide immunogen construct significantly reduced the incidence of inflammatory arthritis and protected bone and cartilage from destruction. These findings strongly support the clinical application of IL-6 peptide immunogen immunization in vivo to treat or prevent rheumatoid arthritis and other autoimmune diseases.

[0419] Evaluation of the effects of dose and adjuvant on immune responses elicited by IL-6 peptide immunogen constructs in the CIA model

[0420] The POC study in CIA rats showed that the designed peptide immunogen constructs had high immunogenicity and therapeutic efficacy against IL-6-induced pathogenic mechanisms, suggesting its potential immunotherapeutic application in rheumatoid arthritis and other autoimmune diseases. The following studies will focus on the optimization of peptide immunogen constructs and the selection of adjuvants as well as dose determination in CIA Lewis rats.

[0421] MONTANIDE ISA 51 and ADJU-PHOS were evaluated as different adjuvants formulated with the same peptide immunogen (SEQ ID NO: 148) and CpG in a rat CIA immunization study. Five rats were assigned to each of five groups, which received one of the two adjuvant formulations, for a total of 10 groups for the two different adjuvants. All animals in the treatment groups were injected with different doses of 5, 15, 45, 150 μg in 0.5 volume by the im route at the primary immunization and booster immunization (boost immunization was performed on days -7, 7, 14, 21 and 28) and clinical observation was performed until day 35. Two different adjuvant placebo groups without peptide immunogen were injected only with the adjuvant vehicle in the formulation. Anti-IL-6 titer, body weight, hind paw swelling examination, arthritis severity score, blood neutrophil, platelet count and liver function were evaluated in the following studies.

[0422] Anti-IL-6 titers were measured by ELISA against rat IL-6 recombinant protein coated in microtiter wells. The results showed that no detectable anti-IL-6 antibody titers were found in both placebo groups injected with the two different adjuvant carriers by ELISA, while all treatment groups immunized with the IL-6 immunogen construct (SEQ ID NO: 148) with both adjuvant formulations produced antibodies against rat IL-6. In general, the results showed that a dose-dependent manner was observed, especially for the groups using the ISA 51 formulation ( Fig. 20 ) In immunized rats, ISA 51 formulation induced a higher immune response than ADJUPHOS formulation, with immunogenicity logs of 1.0 and 1.0, respectively, at all doses. 10 The values ​​are more than 3 respectively.

[0423] Body weight was monitored every 7 days during the 35-day study. Fig.21 Depicting the weight change pattern of rats receiving immunization, the weight loss of experimental CIA rats began on the 14th day in each group compared to normal rats, reached the lowest point on the 21st day, and then gradually increased in weight. At the end of the study (the 35th day), all CIA rats still showed about 10% weight loss compared to the normal control group. The data also pointed out that the weight change was dose-dependent, and lower weight loss was observed in the higher dose group. Regardless of the adjuvant used, the dosage group with a dose of 150 μg increased more weight than the other dosage groups. In contrast, the weight gain of the dosage group with a dose of 150 μg using ADJUPHOS on the 28th and 35th days was higher than any other group, and the weight gain exceeded 200g (Table 13).

[0424] The clinical severity of CIA-induced inflammation and destruction in rats was also assessed by quantifying changes in paw volume. Visual observation indicated that IL-6 immunogen constructs (SEQ ID NO: 148) formulated with ISA 51 or ADJUPHOS protected against the development of CIA in the rat model. Acute clinical signs of swelling and redness in the paws of rats were recorded during the study following collagen induction. From day 7 to day 21, all immunized rats showed an increase in paw swelling, which then gradually recovered as inflammation decreased, as shown in Table 1. Fig. 22 and as shown in Table 14. When compared with the normal group on day 24, the placebo group showed obvious changes in swelling and redness in macroscopic observation ( Fig.23 ). The peptide immunogen construct (SEQ ID NO: 148) reduced paw redness and swelling in a dose-dependent manner. By quantitative analysis, the greatest reduction in inflammation was found in the 150 μg dose group on day 24. The effect of ADJU-PHOS was slightly better than that of MONTANIDE ISA 51.

[0425] The clinical severity of arthritis in each paw was graded on a scale of 0-4 based on inflammatory changes in erythema and swelling signs (scoring criteria). Animals were examined every two or three days and measurements were presented as mean ± standard error to assess the severity of arthritis ( Fig.24 and Table 15). Initial signs of arthritis development caused by collagen challenge were visible on day 14. Among the treatment and placebo groups, the arthritis score in the CIA group increased rapidly, reaching a maximum score of 5-9 at about day 20, and then the inflammatory manifestations gradually weakened from day 21 to day 35. However, during the study, all treatment groups using the immunogen construct (SEQ ID NO: 148) caused a greater reduction in arthritis than the placebo group as scored by clinical signs. A peptide immunogen dose-dependent manner was also observed, with lower arthritis scores in the higher dose groups in all treatment groups (using adjuvants ISA51 or ADJUPHOS). The two placebo groups using two different adjuvant carriers were found to be more severe in clinical arthritis signs, as their clinical sign scores were higher than all treatment groups. When compared to the 5 and 15 μg doses using ISA 51 and ADJUPHOS formulations, the optimal dose levels were found to be 45 and 150 μg, which significantly reduced the signs and symptoms of arthritis. On days 33 and 35, the ADJUPHOS formulation group showed more significant arthritis score reductions than the ISA 51 group at the 150 μg dose level (61% and 63% arthritis score reductions for the ADJUPHOS formulation group, respectively, and 31% and 45% arthritis score reductions for the ISA 51 formulation group, respectively).

[0426] After the first collagen injection, neutrophil counts increased rapidly from day 0 to day 7 and then gradually increased after the second induction until day 14. The increased neutrophil counts could be rapidly reduced by immunization in a dose-dependent manner ( Fig.25 and Table 16). It was found that all immunogen-treated groups had a greater reduction in neutrophil counts than the placebo group at each time point. In the IL-6 immunogen-treated group, the two higher doses significantly reduced neutrophilia, but the ADJUPHOS formulation significantly reduced neutrophil counts to 1.55±0.23x10 at the 45 and 150 μg doses, respectively. 3 per μL and 1.36 ± 0.25 x 10 3 per μL (p < 0.001), which was more effective than the preparation using ISA 51 at the same dose level.

[0427] Collagen-induced arthritis was also associated with a significant increase in platelet counts. In the CIA rats tested, the mean platelet counts in all groups showed a steady increase after the first collagen injection and then gradually decreased ( Fig.26 And Table 17). Dose dependence was also observed, showing that platelet counts were lower when using higher doses. In particular, the IL-6 composition formulated with ADJUPHOS significantly reduced platelet levels to near normal values ​​at 45 and 150 μg doses, while the placebo group had higher blood platelet counts at each time point.

[0428] Liver damage was quantified by measuring serum aspartate transaminase (AST) levels using the conventional human AST test on a Hitachi 7080 chemistry analyzer (Hitachi). Fig. 27 and Table 18). Compared to the normal rat group, rats treated with IL-6 peptide immunogen construct formulations and collagen resulted in a modest increase in serum AST levels from day 0 to day 7. AST concentrations remained stable until day 21 and then slowly declined until the end of the study. Dose dependence was also observed in AST levels. Rats treated with the 150 μg dose showed significantly lower AST levels in both formulations. At the 45 μg dose, significantly lower AST levels were only shown in the ADJUPHOS formulation, but not in the ISA 51 formulation.

[0429] In summary, the II-6 peptide immunogen construct (SEQ ID NO: 148) formulated with an adjuvant is able to induce IL-6 antibodies to neutralize excess IL-6, thereby reducing the severity of arthritis and inhibiting the number of inflammatory factors (such as blood neutrophils and platelets) and protecting liver function. A similar dose-dependent response pattern to the composition was observed in each IL-6 peptide immunogen construct treatment group. The results showed that animals receiving 150 μg per dose produced the highest immune response, followed by animals receiving 45 μg. In addition, when used in combination with the IL-6 peptide immunogen construct, both adjuvant delivery systems (ISA51 and ADJUPHOS) showed the ability to reduce arthritis symptoms. However, the adjuvant ADJU-PHOS performed slightly better than MONTANIDE ISA 51 in all pathological parameters related to arthritis. Therefore, the highest dose (150 μg immunogen per 0.5 mL ADJUPHOS) is considered to be the optimal dose for rat immunization and will be used as a guide to explore immunogenicity in different species.

[0430] Example 10. Treatment of chronic inflammatory diseases by immunization using IL-6 peptide immunogen constructs and their preparations

[0431] IL-6 is involved in a wide range of biological events, such as immune responses, hematopoiesis, and acute phase responses. However, overproduction of IL-6 is associated with the pathogenic mechanisms of a variety of diseases, including a variety of chronic inflammatory diseases and cancers. The use of inhibitors for IL-6 signaling should provide important information to better understand the molecular mechanisms of diseases affected by IL-6 disorders, which will help develop new therapeutic interventions for these diseases. The clinical use of IL-6 peptide immunogen constructs and their preparations of the present invention as pharmaceutical compositions for disease prevention and / or treatment is described in Examples 11 to 15. A review article on the clinical use of IL-6 inhibitors for IL-6 signals in diseases is provided as a reference (Mihara, et al., 2012).

[0432] Anemia of Chronic Inflammatory Diseases (ACD)

[0433] Anemia is commonly observed in patients with chronic inflammatory diseases such as RA, inflammatory bowel disease, and cancer and is referred to as ACD (anemia of chronic disease). ACD is characterized by hypoferremia in the absence of adequate iron stores. Inflammatory cytokines are thought to play an important role in ACD.

[0434] The anemia observed in collagen-induced arthritis in monkeys is characterized by decreased serum iron and transferrin saturation and elevated serum ferritin. The severity of the anemia correlates with serum IL-6 levels. Hepcidin is a major regulator of iron homeostasis in humans and other mammals. It inhibits iron absorption in the small intestine and the release of circulating iron from macrophages, effectively reducing iron delivery to mature erythrocytes in the bone marrow. Mice genetically engineered to overproduce hepcidin die shortly after birth from severe iron deficiency.

[0435] IL-6 induces hepcidin production in hepatocytes. Administration of TCZ, a monoclonal antibody against the IL-6 receptor, to monkeys with collagen-induced arthritis rapidly improved anemia and caused a rapid but transient decrease in serum hepcidin. Serum from arthritic monkeys was more potent in inducing hepcidin mRNA expression than that from healthy animals, which could be inhibited by administration of TCZ. These evidences suggest that TCZ can improve anemia in monkey arthritis by inhibiting IL-6-induced hepcidin production.

[0436] Instead of expensive antibody therapy, the IL-6 peptide immunogen constructs and preparations thereof of the present invention are administered to patients to induce IL-6R binding site antibodies to interfere with the binding of IL-6 and IL-6R, thereby treating the disease.

[0437] Example 11. Treatment of cancer by immunization using IL-6 peptide immunogen constructs and their preparations

[0438] Chronic inflammation in human carcinogenesis

[0439] Chronic inflammation plays an important role in human carcinogenesis. There are many reports describing that IL-6 levels are elevated in the serum of cancer patients, which is related to the severity and prognosis of the disease. IL-6 is related to the growth and differentiation regulation of many cancers. It is also found that IL-6 elevation is associated with the poor prognosis of renal cell carcinoma, ovarian cancer, lymphoma, melanoma and prostate cancer. IL-6 stimulates tumor cell proliferation by activating ERK1 / 2. IL-6 is an important regulator of cell survival, providing a mechanism for tumor cells to escape cell death induced by stress and cytotoxic drugs. In addition, it has been shown that the physiological effects of IL-6 not only promote tumor proliferation, but also promote the transfer and symptoms of cachexia.

[0440] Multiple Myeloma (MM)

[0441] MM is a malignant tumor of plasma cells and is the most common malignant lymphoma in adults. It is characterized by tumor cells located in the bone marrow, where these cells spread and induce bone diseases. The interaction between MM cells and stromal cells in the bone marrow microenvironment stimulates the production of cytokines, growth factors and adhesion molecules. Together, they play an important role in the proliferation and localization of bone marrow MM cells. MM cells cause osteolysis, leading to bone pain and hypercalcemia. IL-6 is the main growth factor for MM cells. The proliferation of cultured MM cells observed in about half of MM patients is mediated by an autocrine loop, and it is now well known that IL-6 produced by the bone marrow environment is a major cytokine involved in the growth and survival of MM cells. In addition, it is well known that IL-6 is an element of MM cell survival because it prevents MM cell apoptosis induced by different stimuli such as dexamethasone, Fas and serum deprivation. Compared to IL-6 alone, the IL-6-sIL-6R complex is more effective in upregulating Bcl-xL and Mc1-1 in natural MM cells, which do not express IL-6R on the cell surface. Therefore, it is important to enable the composition containing the IL-6 peptide immunogen construct to elicit antibodies against sites that interfere with trans-signaling (i.e., at the level of IL-6 / IL-6Rα and IL-6Rβ / iegp130 complex formation). Therefore, the IL-6 composition of the present invention is suitable for the treatment of MM.

[0442] Prostate cancer

[0443] The expression of IL-6 and IL-6R in prostate cancer and the role of IL-6 as a growth factor have been well documented. IL-6 is responsible for resisting apoptosis and increasing the levels of anti-apoptotic members of the Bcl-2 family in the advanced prostate cancer cell line LNCaP. Since the growth of prostate cancer cells depends on the presence of androgens, almost all patients with advanced prostate cancer will initially respond to androgen deprivation and anti-androgen therapy. Because IL-6 stimulates the synthesis of androgens and the expression of ARs (androgen receptors) in prostate cancer cells, IL-6 may reduce the efficacy of anti-androgen therapy in prostate cancer. On the other hand, in AR-negative prostate cancer cells, IL-6 is known as an inhibitor of apoptosis. The IL-6 composition of the present invention will allow the production of anti-IL-6 antibodies in patients receiving immunization to neutralize the negative effects of IL-6 in these cancer patients.

[0444] Cancer-related loss of appetite and cachexia

[0445] Cancer-related anorexia and cachexia are serious complications associated with malignant diseases. Cachexia is characterized by anemia, abnormal liver function, fatigue and vomiting. Elevated serum IL-6 has been observed in pancreatic cancer patients and is associated with cachexia. As mentioned above, IL-6 is associated with iron metabolism. In addition, IL-6 also has regulatory effects associated with excess glucose metabolism and muscle loss. In a syngeneic mouse model, IL-6 is also necessary for cancer cachexia, in which treatment with anti-IL-6 antibodies prevents the induction of cancer cachexia. In addition, in syngeneic mice, injection of Lewis lung cancer cells transfected with IL-6 cDNA can result in unchanged net tumor growth rate, but can lead to weight loss and shortened survival. It has been reported that anti-human IL-6 antibodies (ALD518) can reverse fatigue and reduce the loss of lean body mass in patients with advanced non-small cell lung cancer (-0.19 kg for patients taking ALD518 and -1.50 kg for patients taking placebo). In these patients, ALD518 increased hemoglobin, hematocrit, mean hemoglobin content, and albumin levels, and raised hemoglobin levels to ≥12 g / dl in 58% of patients whose baseline hemoglobin levels were ≤11 g / dl. Therefore, anti-IL-6 antibodies (the anti-IL-6 antibodies can be monoclonal antibodies or antibodies elicited by immunizing patients with compositions containing IL-6 peptide immunogen constructs) may be non-erythropoietic-stimulating agents that can be used to treat cancer-related anemia.

[0446] Patients with long-standing ulcerative colitis have a much higher risk of developing colon cancer, suggesting a role for the immune system as a tumor promoter in the colon. One study showed that IL-6 is produced in innate immune cells within the lamina propria in response to intestinal injury, which can enhance the proliferation of tumor-initiating cells and protect normal and precancerous intestinal epithelial cells from apoptosis induced during acute colitis and CAC (colitis-associated cancer). In addition, in azoxymethane-induced colon tumors in an ulcerative colitis model, the appearance of tumors was accompanied by the co-appearance of the F4 / 80+CDllbhighGrllow (M2) macrophage subset, which is a source of tumor-promoting factors (including IL-6). These results suggest that IL-6 blockade may be a method for treating colitis-associated cancers.

[0447] Instead of expensive antibody therapy, used to interfere with IL-6 and IL-6 receptor interaction and reduce IL-6 serum levels, leading to the treatment and improvement of cancer (including multiple myeloma (MM), androgen-dependent or androgen-independent prostate cancer, non-small cell lung cancer, cancer-related loss of appetite and cachexia, cancer-related anemia, and colitis-related cancer), immunization with IL-6 peptide immunogen constructs and their formulations will be suitable for the treatment of these devastating diseases.

[0448] Example 12. Treatment of rheumatoid arthritis by immunization using IL-6 peptide immunogen constructs and their preparations

[0449] Rheumatoid arthritis (RA)

[0450] Rheumatoid arthritis (RA) is a chronic, progressive, autoimmune inflammatory disease of unknown etiology that particularly affects the joints of the hands and feet. The synovial tissue of the affected joints is infiltrated by inflammatory cells (such as macrophages and lymphocytes), resulting in proliferation with neovascularization, which in turn causes joint swelling, stiffness, and pain. This process ultimately leads to cartilage destruction and bone resorption in the joints, with some patients suffering from permanent disability. The biological activity of IL-6 and its elevation in the serum and synovial fluid of RA patients indicate that IL-6 is one of the key cytokines involved in the development of RA. Seven Phase III clinical trials using the anti-IL-6R monoclonal antibody TCZ (tocilizumab) conducted in Japan and worldwide have shown that it is effective as a monotherapy or in combination with DMARDs (disease-modifying anti-rheumatic drugs) in adult patients with moderate to severe RA. Furthermore, the SAMURAI (Study of Monotherapy with Active Controls, IL-6 Inhibitors in Rheumatoid Arthritis) and LITHE (Safety and Structural Joint Damage Prevention Trial of Tocilizumab) trials both demonstrated that TCZ treatment significantly inhibited radiographic damage to joints. As a result, TCZ is now approved for the treatment of RA in many countries.

[0451] Systemic juvenile idiopathic arthritis (sJIA)

[0452] Systemic juvenile idiopathic arthritis (sJIA) is a subtype of chronic childhood arthritis that can lead to joint destruction and dysfunction, accompanied by systemic inflammation. This long-term inflammation can also cause spike fever, anemia and growth failure. The acute complication of sJIA is called macrophage activation syndrome, which is associated with severe morbidity. IL-6 has been reported to be significantly elevated in the blood and joint fluid of patients, and IL-6 levels have been shown to be associated with disease activity. TCZ showed excellent efficacy in 56 sJIA patients who were refractory to conventional treatment in a randomized, double-blind, placebo-controlled withdrawal phase III clinical trial. It was approved in Japan in 2008 as the first biological drug for sJIA.

[0453] As shown above, instead of expensive antibody therapy, which is used to intervene in the level of IL-6 and IL-6 receptor interaction to reduce IL-6 serum levels and improve sJIA disease, immunization with the IL-6 peptide immunogen constructs and preparations thereof of the present invention will be suitable for treating sJIA disease.

[0454] Example 13. Treatment of Castleman's disease by immunization using IL-6 peptide immunogen constructs and their preparations

[0455] Castleman's disease is a lymphoproliferative disease with benign hyperplastic lymph nodes, characterized by follicular hyperplasia and microvascular hyperplasia accompanied by endothelial hyperplasia. IL-6 is produced in large quantities in hyperplastic lymph nodes, and IL-6 is a key factor in causing various clinical symptoms. Two open clinical trials showed that the anti-IL-6R antibody TCZ, administered at 8 mg / kg body weight every 2 weeks, had a significant effect on clinical symptoms, laboratory test results, and improvements determined by histology. In addition, TCZ treatment induced a rapid decrease in serum hepcidin-25 in patients with Castleman's disease. After the initiation of TCZ treatment, a long-term reduction was observed, accompanied by a gradual normalization of iron-related parameters and symptom improvement, indicating that IL-6 plays an important role in inducing hepcidin in Castleman's disease. TCZ was approved in Japan in 2005 as an orphan drug for Castleman's disease.

[0456] Instead of expensive antibody therapy to interfere with the level of IL-6 and IL-6 receptor interaction, thereby reducing IL-6 serum levels and improving Castleman's disease, immunization with IL-6 peptide immunogen constructs and their preparations will be suitable for treating Castleman's disease.

[0457] Example 14. Treatment of depression by immunization using IL-6 peptide immunogen constructs and their formulations

[0458] The connection between the immune system and the brain may provide new mechanistic understanding and insights into the treatment of depression. Cytokine-mediated communication between the immune system and the brain has been implicated in the pathogenesis of depression. In patients infected with hepatitis C virus, major depression is common (one in four) after interferon treatment, which is a potent cytokine inducer. Experimental immune activation in healthy volunteers can lead to depressive symptoms and reduced cognitive ability. Meta-analysis of cross-sectional studies has confirmed that circulating inflammatory cytokine levels are elevated in patients with depression. Longitudinal studies have shown that serum cytokine levels are elevated first, and therefore may cause depressive symptoms. In addition, activation of the inflammatory system is considered to be the basis for the development of antidepressant drug resistance, emphasizing the relationship between inflammation and treatment response. Based on these findings, it would be most meaningful to target inflammatory cytokines, especially IL-6, using the IL-6 peptide immunogen constructs of the present invention and their preparations, which can provide therapeutic benefits for patients with depression and pain, especially for patients with chronic inflammatory states.

[0459] Table 1. Amino acid sequences of IL-6 and its fragments used in serological tests

[0460]

[0461]

[0462] *Cysteine ​​residues substituted at the amino-terminal and / or carboxyl-terminal amino acids of the IL-6 fragments are underlined.

[0463] Table 2. Amino acid sequences of pathogen protein-derived Th epitopes including idealized artificial Th epitopes used in the design of IL-6 peptide immunogen constructs

[0464]

[0465] Table 3. Amino acid sequences of IL-6 peptide immunogen constructs

[0466]

[0467] Table 3. (Continued)

[0468]

[0469] Table 3. (Continued)

[0470]

[0471] * The polypeptide is cyclized by forming disulfide bonds between cysteines, which are shown in bold italics. The cysteines used to replace the amino and / or carboxyl terminal amino acids of the IL-6 fragment are underlined.

[0472] 1: SEQ ID NO: 91

[0473] 2: SEQ ID NO: 92

[0474] 3: SEQ ID NO: 89

[0475] Table 4. Evaluation of immunogenicity of vaccine formulations containing IL-6-derived peptide immunogen constructs targeting the IL-6R binding site in guinea pigs

[0476]

[0477] Table 5A-5E. Immunogenicity evaluation of vaccine formulations containing IL-6-derived peptide immunogen constructs targeting the IL-6 / IL-6Rα / IL-6Rβ (or gp130) binding site

[0478] (Table 5A)

[0479]

[0480] (Table 5B)

[0481]

[0482] (Table 5C)

[0483]

[0484] (Table 5D)

[0485]

[0486] (Table 5E)

[0487]

[0488]

[0489] Table 7. Absence of endogenous IL-6Th epitopes in selected IL6R binding site B epitope sequences

[0490]

[0491] Table 8. Evaluation of immunogenicity of IL-6 peptide immunogen constructs against Th epitope localization in guinea pigs

[0492]

[0493]

[0494]

[0495] Table 10. Arthritis scores in CIA rats immunized with vaccines containing IL-6R binding site peptide constructs

[0496]

[0497] *, p<0.05 **, p<0.01 ***, p<0.001 ****, p<0.0001

[0498] Table 11. Hind paw swelling in CIA rats immunized with vaccines containing IL-6R binding site peptide constructs

[0499] Group Day 14 Day 21 Day 26 Day 35 Placebo 1.5±0.1 2.3±0.2 2.2±0.1 1.9±0.1 SEQ ID NO: 148 1.4±0.1 1.6±0.3 1.6±0.4 1.6±0.3 SEQ ID NO: 157 1.5±0.1 2.0±0.3 1.9±0.3 1.6±0.3

[0500] *, p<0.05 **, p<0.01 ***, p<0.001

[0501] Table 12. Neutrophil levels in CIA rats immunized with vaccines containing IL-6R binding site peptide constructs

[0502] Group Day 0 Day 7 Day 14 Day 21 Day 26 Placebo 2.0±0.4 3.8±0.9 6.6±0.8 4.0±0.7 4.7±0.8 SEQ ID NO: 148 2.4±0.4 3.9±0.8 4.2±1.2 2.8±0.6 3.1±0.6 SEQ ID NO: 157 2.3±0.3 4.3±1.0 5.0±1.1 3.0±0.6 3.7±1.1

[0503] *, p<0.05 **, p<0.01

[0504] Table 13. Body weight of CIA rats immunized with different dose levels of vaccine formulations containing IL-6R binding site derived peptide structure (SEQ ID NO: 148)

[0505]

[0506] *, p<0.05 **, p<0.01

[0507] Table 14. Hind paw swelling in CIA rats immunized with different dose levels of vaccine formulations containing IL-6R binding site peptide construct (SEQ ID NO: 148)

[0508]

[0509] *, p<0.05 **, p<0.01 ***, p<0.001

[0510] Tables 15A-15B. Arthritis scores in CIA rats immunized with different dose levels of vaccine formulations containing IL-6R binding site peptide construct (SEQ ID NO: 148)

[0511] (Table 15A) Formulations with ISA 51 / CpG

[0512]

[0513] *, p<0.05 **, p<0.01 ***, p<0.001

[0514] (Table 15B) Formulations with ADJU-PHOS / CpG

[0515]

[0516] *, p<0.05 **, p<0.01 ***, p<0.001

[0517] Table 16A-16B. Neutrophil levels in CIA rats immunized with different dose levels of vaccine formulations containing IL-6R binding site peptide construct (SEQ ID NO: 148)

[0518] (Table 16A) Formulations with ISA 51 / CpG

[0519]

[0520] *, p<0.05 **, p<0.01

[0521] (Table 16B) Formulations with ADJU-PHOS / CpG

[0522]

[0523] *, p<0.05 **, p<0.01 ***, p<0.001 ****, p<0.0001

[0524] Table 17A-17B. Platelet release in CIA rats immunized with different dose levels of vaccine formulations containing IL-6R binding site peptide construct (SEQ ID NO: 148)

[0525] (Table 17A) Formulations with ISA 51 / CpG

[0526]

[0527] *, p<0.05 **, p<0.01

[0528] (Table 17B) Formulations with ADJU-PHOS / CpG

[0529]

[0530] *, p<0.05 **, p<0.01 ***, p<0.001

[0531] Tables 18A-18B. AST in CIA rats immunized with vaccine formulations containing IL-6-derived peptide constructs (SEQ ID NO: 148) at different dose levels

[0532] (Table 18A) Formulations with ISA 51 / CpG

[0533]

[0534] *, p<0.05 **, p<0.01

[0535] (Table 18B) Formulations with ADJU-PHOS / CpG

[0536]

[0537] *, p<0.05 **, p<0.01

[0538] Table 19. Cross-reactivity of IgGs from immune sera targeting human IL-6R binding site peptide immunogen constructs with macaque and rodent IL-6 proteins

[0539]

[0540] Table 20A-20B. Neutralizing activity of IgGs induced by IL-6 peptide immunogen constructs against cis / trans binding

[0541] (Table 20A) IC of inhibition of cis-binding 50

[0542]

[0543] (Table 20B) IC for inhibition of trans-binding 50

[0544]

[0545] Table 21. Neutralizing activity of IgGs induced by IL-6 peptide immunogen constructs against IL-6-induced TF-1 proliferation

[0546]

[0547] Table 22. Cross-reactivity of IgGs induced by IL-6 peptide immunogen constructs to human, monkey and rodent IL-6

[0548]

Claims

1. An IL-6 peptide immunogen construct, wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NO: 112-114, 116-118, 124-129, 131-146, 148 and 157.

2. A composition comprising the IL-6 peptide immunogen construct as claimed in claim 1.

3. A pharmaceutical composition comprising: a. The IL-6 peptide immunogen construct as claimed in claim 1; and b. Pharmaceutically acceptable delivery vehicles and / or adjuvants.

4. The pharmaceutical composition according to claim 3, wherein The IL-6 peptide immunogen construct is mixed with CpG oligodeoxynucleotides to form a stabilized immunostimulatory complex.

5. Use of the pharmaceutical composition according to claim 3 in the preparation of a medicament for preventing and / or treating rheumatoid arthritis in an animal, comprising administering the pharmaceutical composition to the animal.

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