Fusion proteins for diagnosing, preventing, and treating infectious diseases

Abstract

The present invention relates to the use of phosphotidylserine or pathogenic carbohydrate-targeted therapeutics to manage and treat microbial infections, including, inter alia, Zika, Dengue, West Nile, Ebola, H1N1, enterovirus, leishmania, malaria, and the coronavirus SARS-COV. In one aspect, the invention relates to fusion constructs comprising an Ig-Fc domain or other protein scaffold, such as albumin, and a peptide, protein, or antibody fragment that binds to phosphatidylserine, and / or a peptide or protein that binds to and / or recognizes a PAMP expressed by a microorganism. Other aspects are also described.

Description

Technical Field

[0001] This specification includes a computer-readable form sequence listing submitted together with this application. The sequence listing forms part of this disclosure and is incorporated herein in its entirety.

[0002] The present invention relates to the use of phosphatidylserine or pathogenic glycan targeting therapeutics for the management and treatment of microbial infections, including Zika, Dengue, Respiratory Syncytial Virus, West Nile, Ebola, H1N1, Mycobacterium Leprae, Mycobacterium tuberculosis, Enterovirus, Leishmania, Malaria, and Coronavirus SARS-CoV.

[0003] There are provided novel therapeutic protein compositions comprising furin protease inhibitors, T cell engagers, platforms having cytotoxic functions, monovalent and multivalent molecules, drug conjugates and adjuvants, pathogen-neutralizing proteins that can be conjugated to carriers, as well as methods of administration, particularly subcutaneous, oral or nasal administration methods. The present invention further relates to companion diagnostics as a method of selecting subjects who may benefit from such treatment, and to blood biomarkers for rapidly and easily monitoring the response to treatment.

Background Art

[0004] According to the WHO, the arboviruses Zika virus (ZIKV), Chikungunya virus (CHIKV), Dengue virus (DENV), West Nile virus (WNV), as well as Ebola virus (EBLV) and SARS, are relatively recent, life-threatening rare diseases that pose a high global public health risk and are prone to pandemic spread (WHO report 2020).

[0005] There are no FDA-approved treatments for these diseases. A recombinant live vaccine consisting of the envelope glycoprotein of the Zaire Ebola virus (Ervebo, 2019), one of the Ebola strains, was recently approved by the FDA for use in adults only, but the duration of protection is not yet fully known. Another recombinant vaccine (Dengvaxia, 2019) consisting of pre-M and E proteins from all four dengue strains has also been approved, but Lim et al., 2019, reported a new mutation of DENV with different antigenic properties.

[0006] ZIKV, WNV, and DENV are flaviviruses (family Flaviviridae) (i.e., arboviruses) primarily transmitted by mosquito vectors. Zika is a positive-sense single-stranded RNA flavivirid virus primarily transmitted by Aedes mosquitoes, and since its first isolation in Uganda in 1947, numerous strains from two phylogenetic lineages (Asian and African) have been identified (Ramos da Silva, 2016). According to the WHO, the 2015-2017 outbreak caused more than 30,000 cases worldwide (World Health Organization Zika Epidemiology Update July 2019, website: www_who.int / emergencies / diseases / zika / zika-epidemiology-update-july-2O19.pdf?ua=1, accessed August 5, 2020).

[0007] According to Musso, 2015, the Zika can also spread through sexual contact (Musso, 2015), and according to Rasmussen, 2016, the Zika can spread from maternal blood to fetal blood (Rasmussen, 2016).

[0008] A broad spectrum of neurological sequelae has been reported. According to Rasmussen, 2016, a particularly serious comorbidity of Zika infection in pregnant women is severe congenital microcephaly in their offspring (Rasmussen, 2016). According to Barbi, 2018, in adults, Guillain-Barré syndrome (GBS), an autoimmune disease that destroys the myelin sheath and causes progressive ascending paralysis, is estimated to occur in 1.23% of patients (Barbi, 2018). Other reported neurological complications of ZIKV include encephalitis / meningoencephalitis, acute disseminated encephalomyelitis, myelitis, cerebrovascular complications, seizures and encephalopathy, sensory polyneuropathy and sensory neuropathy.

[0009] The main hosts of ZIKV include humans, monkeys, and mosquitoes. According to Hou, 2017, neural stem cells, fibroblasts, epithelial cells, and blood cells are susceptible to ZIKV infection (Hou, 2017).

[0010] Dengue virus (DEGV) is a negative RNA flavivirus that causes the most prevalent arthropod-derived viral disease in the world (1 million cases per year). DENV infection causes a wide range of clinically presenting human illnesses, from asymptomatic infection or a self-limiting febrile illness called dengue fever (DF) to life-threatening illnesses including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Currently, there is no cure for dengue, and there is no vaccine for people who have not previously been infected with dengue or who travel from non-endemic areas. Dengvaxia is a vaccine approved for people aged 9-16 years who have previously been infected with dengue in a laboratory and live in an endemic area.

[0011] Patent application US2009175865A1 describes an antibody modified by replacing one or more amino acids of the parent antibody with a non-crosslinked, highly reactive cysteine ​​amino acid. Among the mutations, the patent application specifically refers to A339C and S337C.

[0012] Patent application WO2015157595 describes conjugate compounds comprising antibodies and fragments thereof manipulated with one or more reactive cysteine ​​residues. Among the mutations, this patent application specifically refers to K340C.

[0013] According to Wenwen Bi et al. (IgG Fc-binding mortif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibody, PLOS Pathogens, December 5, 2019), a strategy has been developed to extend the in vivo half-life of the short HIV-1 fusion inhibitor peptide CP24 by fusing it with a human IgG Fc-binding peptide (IBP). [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] U.S. Patent Application Publication No. 2009 / 175865 [Patent Document 2] International Publication No. 2015 / 157595 [Non-patent literature]

[0015] [Non-Patent Document 1] World Health Organization, report, 2020 [Non-Patent Document 2] Ervebo,2019 [Non-Patent Document 3] Dengvaxia, 2019 [Non-Patent Document 4] Lim et al., 2019. [Non-Patent Document 5] Ramos da Silva, 2016 [Non-Patent Document 6] World Health Organization, Zika Epidemiology Update, July 2019, [searched on August 5, 2020], website <URL:www_who.int / emergencies / diseases / zika / zika-epidemiology-update-july-2O19.pdf? ua=1> [Non-Patent Document 7] Musso, 2015 [Non-Patent Document 8] Rasmussen, 2016 [Non-Patent Document 9] Barbi, 2018 [Non-Patent Document 10] Hou, 2017 [Non-Patent Document 11] Wenwen Bi et al., IgG Fc-binding mortif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibodies, PLOS Pathogens, December 5, 2019 [Summary of the Invention] [Problems to be Solved by the Invention]

[0016] (Summary of the Invention) There are still unaddressed needs for new technologies for managing emerging and re-emerging infectious diseases prone to genetic mutations.

[0017] The similarities in that the virus binds to permissive human cells, is activated in the endosome-lysosome compartment, and becomes infectious provide insights into the potential for a pan-therapeutic approach to their treatment.

[0018] Pathogenic sugars as CD209 treatment targets Glycans are essential structural and functional components of microorganisms. Among these, glucans, which are polysaccharide moieties derived from D-glucose, are major components of the cell walls of fungi, plants, and mycobacteria. High-mannose structures (mannans) are expressed by many viruses, fungi, and bacteria, while fucose structures (fucans) have been found on the surface of helminths and some bacteria (Geijtenbeek and Gringhuis, 2009; Robinson et al., 2006).

[0019] The human immune system has evolved innate pattern recognition receptors that distinguish self-glycans from non-self-glycans. While the binding of type C lectins to pathogenic sugars triggers both innate and adaptive immune events leading to pathogen elimination, this interaction may also be exploited to enhance pathogenicity.

[0020] The bone marrow cell, dendritic cell, and macrophage cell-specific C-type lectin receptor CD209 (also known as DC-SIGN) (Zelensky and Gready, 2005) is used to treat ZIKV (Perera Lecoin, 2013, Osorio and Sousa, 2011), influenza (Gillespie, 2016), DENV (Cruz-Oliveira, 2015), WNV (Davis, 2006), Ebola (Alvarez, 2002), enterovirus (REN, 2014), Mycobacterium tubercurocystis (Tailleux, 2003), and Mycobacterium leple (Barreiro, 2006), as well as SARS-CoV-2 / COVID-19 (Amraei, 2020, Cai, 2020, Jeffers, 2006). CD209 is an important host cell receptor for invasion (2004). Leshmaniasis and malaria, which are parasite-borne diseases, are nonviral pathogens that utilize CD209 for host invasion (Colmenares 2002, Morenikeji 2020). CD209 binds to both the mannan (high-mannose N-linked oligosaccharide) moiety and the fukan moiety, which contain the viral signature or "pathogen-associated molecular pattern (PAMP)". This binding occurs within a compact protein region with a unique structural fold, known as the "C-type carbohydrate recognition domain" or "C-type lectin domain (CTLD)" (Weis and Drick-Amer, 1996).

[0021] Currently, there is no cure for the global pandemic of COVID-19, and although the FDA has granted emergency use authorization for the antiviral drug remdesivir, its effectiveness against Covid-19 has not yet been demonstrated in large-scale randomized clinical trials. Early preclinical development approaches include the ACE2 decoy protein, which blocks viral attachment to host cells, and the off-label use of dexamethasone to reduce inflammation in patients on mechanical ventilation who are in the early stages of the disease but show no symptoms. Therefore, there is an urgent need for effective and safe means to treat and alleviate COVID-19 and related symptoms. Consequently, there is an urgent need for diagnostics that can accurately select patients who will benefit from specific treatments.

[0022] Phosphatidylserine (PS) lipids as a therapeutic target for TIM1 The outer viral membrane layer of some viruses is rich in the phospholipid phosphatidylserine (PS), but in host cell membranes, PS is usually confined to the inner membrane layer.

[0023] T cell immunoglobulins and the mucin domain 1 (TIM-1) human membrane receptor function as potent co-stimulatory molecules for T cell activation.

[0024] TIM1 (also known as HAVCR1) is a type I transmembrane glycoprotein containing an extracellular domain consisting of an N-terminal immunoglobulin variable (IgV)-like domain followed by a glycosylated mucin domain, a single transmembrane domain, and a short cytoplasmic tail with a tyrosine phosphorylation motif. The IgV domain of TIM1 is predicted to contain a conserved PS-binding site (Santiago et al., 2007).

[0025] TIM-1 is preferentially expressed in type 2 helper T (Th2) cells in the brain, gastrointestinal tract, liver and gallbladder, kidneys, testes, and lymphoid tissues. According to Freeman 2010, TIM-1 recognizes and attaches to exposed PS with high specificity in dying apoptotic cells, inducing phagocytosis by the immune system (Freeman 2010). TIM-1 promotes apoptotic clearance by binding to PS via metal ion-dependent ligand-binding sites (MILIBS) within its IgV domain.

[0026] Furthermore, TIM1 has been shown to be an entry factor for a wide variety of viruses, including Zika (Lee 2018), Ebola (Brunton 2019), Dengue (Chu 2019, Amara 2015), West Nile (Richard 2015), Hepatitis A, and possibly malaria (Nuchnoi 2020) (Jemielity, 2013).

[0027] These studies suggest that TIM-1 functions as a common adhesion factor for various enveloped viruses by directly interacting with the PS of the viral envelope, independently of glycoproteins.

[0028] TIM1 is the most well-known PS receptor, but other PS receptors from the TAM family of proteins, such as Tyro3, ​​Axl, and Mer, have also been described, albeit less frequently.

[0029] TIM-1 as a receptor for viral proteins According to Angiari et al. (2014), TIM-1 plays a role in the development of autoimmune and inflammatory diseases by regulating T cell adhesion through binding to P-selectin proteins via the TIM-1 mucin and IgV domain (Angiari 2014).

[0030] Kuroda et al. (2015) reported that treatment with a TIM-1-specific monoclonal antibody that inhibits the interaction between TIM-1 and Niemann-Pick disease C1 protein (NPC1) significantly suppressed filovirus infection and GP-mediated membrane fusion. This study suggests that TIM-1 may also be involved in viral membrane fusion.

[0031] According to Yuan et al. (2015), human TIM-1 directly binds to EBOV glycoprotein (GP), and the authors determined the crystal structures and binding regions of the IgV domains of hTIM-1 and hTIM-4.

[0032] According to Kondratowicza et al. (2010), TIM-1 on host epithelial cells of the trachea, cornea, and conjunctiva directly binds to the receptor-binding domain of the Zaire Ebola virus (EBOV) glycoprotein, enhancing airborne and hand-to-eye transmission. Blocking this interaction with antibodies suppressed binding and Ebola infection.

[0033] Therefore, TIM-1 may possess both protein-dependent and protein-independent functions in viral infection.

[0034] Furin protease inhibition as a therapeutic strategy Furin cleavage sites are found in the entry proteins of Zika, dengue, COVID-19 (Coutard 2020), Ebola, HIV, and hepatitis B virus (Braun 2019).

[0035] Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), including SEQ ID NO: 81, and hexa-D-arginine (D6R) are small, synthetic furin inhibitors that have been used to demonstrate reduced viral infectivity in vitro (Owczarek, 2019; Imran, 2019; Remacle, 2010; Couture, 2015). CMK is more effective than D6R in reducing hepatitis replication by inhibiting furin-mediated processing of hepatitis B e antigen (HBeAg) precursors to mature HBeAg. Dec-RVKR-cmk is a small, synthetic, irreversible, and cell permeability competitive inhibitor of all proprotein convertases (PC1, PC2, PC4, PACE4, PC5, PC7, and furin). CMK has been reported to inhibit furin-mediated cleavage and fusion activity of viral glycoproteins and acts as an antiviral agent against a variety of viruses, including human immunodeficiency virus, chikungunya virus, chronic hepatitis B virus, influenza A, Ebola virus infection, and papillomavirus. Smith et al. and Steinmetzer et al. also patented a peptide-mimicking furin inhibitor by modifying the C-terminus of dec-RVKR-cmk with a decarboxylated arginine mimetic, resulting in a highly potent furin inhibitor (Couture 2015).

[0036] A broad range of furin / proprotein inhibitors are thought to have minimal off-pathogen, on-target effects in the host, as evidenced by the highly redundant nature of proprotein convertases, as demonstrated by furin knockout mice.

[0037] CMK has been shown to possess antiflaviviral activity at non-cytotoxic concentrations (Imran 2019).

[0038] The Zika virus comprises three structural proteins (capsid-pC, envelope-pE, and membrane-prM) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Zika virus replication occurs in tolerant host cells after internalization via clathrin-mediated pH-dependent endocytosis and maturation of viral proteins in the lysosomal compartment (Owczarek, 2019). In lysosomes, furin or furin-like proteases cleave the viral surface glycoprotein prM into its active form, destabilizing the viral membrane and promoting the release of viral RNA for replication in mitochondria and the endoplasmic reticulum.

[0039] There are investigational drugs in clinical development, but there is no vaccine against ZIKV.

[0040] It has been shown that furin inhibition transports immature virions to the late compartment, where they undergo proteolytic degradation. The degradation products are then expelled from the cell via slowly recirculating vesicles (Owczarek, 2019).

[0041] Similar to ZIKV, DENV binding of the viral E protein to the cell receptor allows the viral particle to be internalized into the permissible cell via the cratrin-mediated endocytosis pathway. To release the viral RNA genome, the DENV virion undergoes acid-induced structural changes and membrane fusion. The newly synthesized viral protein, generated near the endoplasmic reticulum (ER), facilitates viral RNA genome replication, induction of membrane rearrangement, and assembly of new viral particles. To facilitate the DENV replication process, DENV interacts with various cellular components as well as inducing various host responses, such as autophagy.

[0042] T cell mediated therapy CD3 is a protein complex and a T cell co-receptor involved in T cell activation. CD3 is selectively expressed in T cells of blood, bone marrow, and lymphoid tissues, but not in other normal tissues, and does not cross-react with other animals except chimpanzees. In recent years, human CD3 transgenic mice have been created, making it easier to study anti-CD3 immunotherapy.

[0043] Anti-CD3-based therapies such as muromomab-CD3 (Janssen, orthoclone, OKT3) have been extensively studied in humans, both systemically and orally, for blocking reactive T cells and restoring ulcerative colitis and metabolic syndrome (da Cunha 2011, Ilan 2010-NCT01287195, NCT01205087). Anti-CD3 bispecific antibody platforms that bridge tumors and engage T cells, such as blinatumomab and catumakisomab, are approved for cancer treatment, and several other CD3 bispecifics are in clinical development (Suurs, 2019). [Means for solving the problem]

[0044] According to one embodiment, the present invention relates to an Ig-Fc domain or other protein scaffold, such as albumin, and a. Peptides, proteins, or antibody fragments that bind to phosphatidylserine, and / or b. Peptides or proteins that bind to and / or recognize PAMP expressed by microorganisms. Regarding fusion structures that include this.

[0045] PAMP refers to pathogen-associated molecular patterns (PAMPs) that are produced by microbial pathogens but not by the host organism, and are recognized by the host's innate immune system; these are conserved molecular structures.

[0046] In another aspect, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment Regarding fusion structures that include this.

[0047] In another aspect, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment A fusion structure that includes, Provides enhancements to ADCC, ADCP and / or CDC. Regarding fusion structures.

[0048] ADCC can be defined as antibody-dependent cell-mediated cytotoxicity. ADCP can be defined as antibody-dependent phagocytosis. CDC can be defined as complement-dependent cytotoxicity.

[0049] In another aspect, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment A fusion structure that includes, Includes additional CDR areas as described in Sequence IDs 54-59. Regarding fusion structures.

[0050] In another aspect, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment A fusion structure that includes, Further containing furin inhibitors, Regarding fusion structures.

[0051] Preferably, the furin inhibitor is selected from chloromethyl ketones and D-arginine derivatives, such as hexa-D-arginine and dec-RVKR-cmk (including SEQ ID NO: 81).

[0052] Linkers and spacers can be conjugated to the Fuhrin; see Table 7.

[0053] In another aspect, the present invention relates to a fusion construct which is an IgG3 construct, wherein the IgG3 construct includes a hinge region, and the hinge region is modified.

[0054] In another embodiment, the present invention relates to a fusion construct, fusion protein, or antibody comprising a constant region and a hinge of IgG3, wherein the hinge is preferably selected from an IgG1 or IgG4 hinge.

[0055] In another aspect, the present invention relates to an IgG3 homodimer comprising a hinge region, wherein the hinge region comprises a sequence selected from sequence numbers 6, 8, and 68.

[0056] In another aspect, the present invention relates to an IgG3 heterodimer comprising a hinge region, wherein the hinge region comprises a sequence selected from sequence numbers 6, 8, and 68.

[0057] In another aspect, the present invention relates to IgG3 comprising mutations at positions 405 and / or 409. In another aspect, the present invention relates to IgM heterodimers that can be obtained by altering the charge pair of the CH2 and / or CH4 domains.

[0058] In another embodiment, the present invention relates to an IgM heterodimer containing one or more of the mutations in Table 8.

[0059] In another aspect, the present invention relates to a fusion construct comprising an IgG3 homodimer, an IgG3 heterodimer, and / or an IgM heterodimer as described in the present invention.

[0060] In another aspect, the present invention relates to the use of the fusion construct described in the present invention for treating infectious diseases.

[0061] In another aspect, the present invention relates to a use in which the infectious disease is selected from viral infections, bacterial infections, and protozoan infections.

[0062] In another aspect, the present invention relates to a use in which the treatment includes the administration of the fusion construct in a dosage form selected from subcutaneous, intradermal, intramuscular, oral, and nasal.

[0063] In another aspect, the present invention relates to the use of IgG4 or a portion of IgG4 for payload delivery, wherein IgG4 is modified to not contain Fc, or the activity of Fc in IgG4 is inactivated or reduced by one or more mutations.

[0064] In another aspect, the present invention relates to a vaccine comprising a fusion construct described in the present invention.

[0065] In another aspect, the present invention relates to a vaccine comprising mannan, a high-mannose-containing structure, fukan, and / or the phospholipid phosphatidylserine (PS).

[0066] In another aspect, the present invention relates to a composition comprising a fusion construct according to the present invention, which optionally comprises one or more excipients such as a diluent, a binder, or a carrier.

[0067] In another aspect, the present invention relates to a method for treating and / or preventing an infectious disease in a subject, comprising the step of administering a fusion construct and / or vaccine and / or composition described in the present invention.

[0068] In another aspect, the present invention relates to a method for screening and / or monitoring the progression of a disease in a subject, the following i. A step of providing a blood sample derived from the target, ii. The step of bringing a blood sample into contact with the fusion construct described in the present invention. This includes methods.

[0069] In another aspect, the present invention relates to an isolated nucleic acid molecule encoding a fusion construct described in the present invention.

[0070] In another embodiment, the present invention relates to a recombinant vector comprising the nucleic acid molecule of the present invention.

[0071] In another aspect, the present invention relates to a host cell comprising the recombinant vector of the present invention.

[0072] In another embodiment, the present invention is The steps include culturing the host cells described in the present invention in a culture medium under conditions that enable the expression of the fusion construct, and Step to isolate the fusion construct from the culture medium. The present invention relates to a method for generating a fusion construct, including the present invention. [Modes for carrying out the invention]

[0073] Detailed disclosure According to one embodiment, the present invention relates to an Ig-Fc domain or other protein scaffold, such as albumin, and a. Peptides, proteins, or antibody fragments that bind to phosphatidylserine, and / or b. Peptides or proteins that bind to and / or recognize PAMP expressed by microorganisms. Regarding fusion structures that include this.

[0074] PAMP refers to pathogen-associated molecular patterns (PAMPs) that are produced by microbial pathogens but not by the host organism, and are recognized by the host's innate immune system; these are conserved molecular structures.

[0075] The term "protein scaffold" refers to a protein structure to which the active elements defined in a. and b. above can be bound. The protein scaffold should preferably be soluble in plasma and preferably have a long residence time in plasma, which can typically be provided when the fully fused protein is larger than the renal clearance limit, for example, larger than 60 kDa, or by selecting a protein scaffold under the influence of an activity retention system, for example, a protein that binds to plasma and is recycled via an FcRn receptor. Examples of suitable protein scaffolds include plasma proteins or fragments thereof, such as the constant region of immunoglobulins, albumin, albumin domains I, II, or III, transferrin, and lactoferrin.

[0076] According to one embodiment, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment Regarding fusion structures that include this.

[0077] According to one embodiment, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant Ig-like type V domain of human TIM1 fragment, and / or b. Recombinant C-type lectin domain of human CD209 fragment Regarding fusion structures that include this.

[0078] According to one embodiment, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Two or more recombinant Ig-like type V domains derived from one or more human TIM1 fragments, and / or b. Two or more recombinant C-type lectin domains derived from one or more human CD209 fragments. Regarding fusion structures that include this.

[0079] According to one embodiment, the present invention is a. Recombinant Ig-like V domain of human TIM1 fragment, and b. Recombinant C-type lectin domain of human CD209 fragment Regarding fusion structures that include this.

[0080] According to one embodiment, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment A fusion structure that includes, Provides enhancements to ADCC, ADCP and / or CDC. Regarding fusion structures.

[0081] ADCC can be defined as antibody-dependent cell-mediated cytotoxicity. ADCP can be defined as antibody-dependent phagocytosis. CDC can be defined as complement-dependent cytotoxicity.

[0082] According to one embodiment, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment A fusion structure that includes, Includes additional CDR areas as described in Sequence IDs 54-59. Regarding fusion structures.

[0083] According to one embodiment, the present invention relates to an IgG-Fc domain or other protein scaffold, and a. Recombinant human TIM1 fragment, and / or b. Recombinant human CD209 fragment A fusion structure that includes, Further containing furin inhibitors, Regarding fusion structures.

[0084] Preferably, the furin inhibitor is selected from chloromethyl ketones and D-arginine derivatives, such as hexa-D-arginine and dec-RVKR-cmk (including SEQ ID NO: 81).

[0085] Linkers and spacers can be conjugated to the Fuhrin; see Table 7.

[0086] According to one embodiment, the present invention relates to a fusion construct in which a peptide, protein, or antibody fragment can bind to and / or stimulate immune cells.

[0087] According to one embodiment, the present invention relates to a fusion construct in which the TIM1 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 50-180 amino acid residues, 60-160 amino acid residues, 70-140 amino acid residues, 80-130 amino acid residues, 90-120 amino acid residues, 100-120 amino acid residues, and 100-110 amino acid residues.

[0088] According to one embodiment, the present invention relates to a fusion construct in which the CD209 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 40-190 amino acid residues, 50-180 amino acid residues, 60-170 amino acid residues, 70-160 amino acid residues, 80-150 amino acid residues, 90-150 amino acid residues, 100-150 amino acid residues, 110-150 amino acid residues, 120-150 amino acid residues, and 130-140 amino acid residues.

[0089] According to one embodiment, the present invention relates to a fusion construct in which the TIM1 and / or CD209 fragments have at least 70%, 75%, 80%, 85%, 90%, or 95% sequence homology with wild-type TIM1 or CD209.

[0090] According to one embodiment, the present invention relates to a fusion structure in which TIM1 and / or CD209 fragments have intact TIM1 and / or CD209 functions.

[0091] According to one embodiment, the present invention relates to a fusion construct in which the IgG-Fc domain is an IgG3-Fc domain.

[0092] According to one embodiment, the present invention is as follows: a) IgG3, in which the hinge sequence is preferably replaced with an IgG4 hinge sequence; b) CDR areas as described in Sequence IDs 54-59; and / or c) Furin inhibitors Regarding fusion constructs that additionally include at least one of the following.

[0093] According to one embodiment, the present invention relates to a fusion construct comprising the sequence described in Sequence ID No. 1 and / or Sequence ID No. 2.

[0094] According to one embodiment, the present invention relates to a fusion construct comprising the sequence described in Sequence ID No. 3 and / or Sequence ID No. 4.

[0095] According to one embodiment, the present invention relates to a fusion construct comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, preferably at least eight disulfide bonds.

[0096] According to one embodiment, the present invention relates to a fusion construct that can bind to a target, wherein the target is mannan, a high-mannose-containing structure, fukan, the phospholipid phosphatidylserine and / or CD3.

[0097] According to one embodiment, the present invention is a. A protein fragment comprising or consisting of the sequence described in Sequence ID No. 1, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and b. Protein fragments comprising or consisting of the sequence described in Sequence ID No. 3, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity. This relates to fusion structures, including those mentioned above.

[0098] According to one embodiment, the present invention is The sequence described in ai Sequence ID No. 1 or Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. Sequences described in Sequence ID No. 9 and Sequence ID No. 43, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The first chain including, b.iii. Sequences described in Sequence ID No. 1, Sequence ID No. 2, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. Sequences described in Sequence ID No. 9, Sequence ID No. 43, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The second chain including This relates to fusion structures, including those mentioned above.

[0099] According to one embodiment, the present invention is The sequences described in ai Sequence ID No. 3 and Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. Sequences described in Sequence ID No. 9 and Sequence ID No. 43, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The first chain including, b.iii. Sequences described in Sequence ID No. 3, Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. Sequences described in Sequence ID No. 9, Sequence ID No. 43, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The second chain including This relates to fusion structures, including those mentioned above.

[0100] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. Sequences described in Sequence ID No. 11 and Sequence ID No. 45, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The first chain including, b.iii. Sequences described in Sequence ID No. 3, Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. Sequences described in Sequence ID No. 13 and Sequence ID No. 47, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The second chain including This relates to fusion structures, including those mentioned above.

[0101] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. Sequences described in Sequence ID Nos. 14, 15, and 66, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The first chain including, b.iii. Sequences described in Sequence ID No. 1, Sequence ID No. 2, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. Sequences described in Sequence IDs 16, 17, and 67, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and v. Preferably the linker sequence described in Sequence ID No. 41, and vi. A sequence described in any of the sequences selected from sequence numbers 18 to 35, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The second chain including This relates to fusion structures, including those mentioned above.

[0102] According to one embodiment, the present invention is The sequences described in ai Sequence ID No. 3 and Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. Sequences described in Sequence ID Nos. 14, 15, and 66, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The first chain including, b.iii. Sequences according to Sequence ID No. 3 and / or Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. Sequences described in Sequence IDs 16, 17, and 67, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and v. Preferably the linker sequence described in Sequence ID No. 41, and vi. A sequence described in any of the sequences selected from sequence numbers 18 to 35, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The second chain including This relates to fusion structures, including those mentioned above.

[0103] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. Sequences described in Sequence ID Nos. 14, 15, and 66, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The first chain including, b.iii. Sequences described in Sequence ID No. 3, Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. Sequences described in Sequence IDs 16, 17, and 67, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and v. Preferably the linker sequence described in Sequence ID No. 41, and vi. A sequence described in any of the sequences selected from sequence numbers 18 to 35, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The second chain including This relates to fusion structures, including those mentioned above.

[0104] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. Sequences described in Sequence IDs 16, 17, and 67, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences, iii. Preferably the linker sequence described in Sequence ID No. 41, and iv. A sequence described in any of the sequences selected from sequence numbers 18 to 35, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The first chain including, The sequences described in bv Sequence ID No. 3 and Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and vi. Preferably the linker sequence described in Sequence ID No. 41, and vii. Sequences described in Sequence IDs 14, 15, and 66, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with one of these sequences. The second chain including This relates to fusion structures, including those mentioned above.

[0105] According to one embodiment, the present invention relates to a fusion structure including a linker.

[0106] According to one embodiment, the present invention relates to a fusion construct in which the linker is selected from (GGGGS)3 linker (SEQ ID NO: 41), (GGGGS)4 linker (SEQ ID NO: 70), (GGGGS)5 linker (SEQ ID NO: 71), and (GGGGS)6 linker (SEQ ID NO: 72).

[0107] The (GGGGS) linker can be defined as the Gly-Gly-Gly-Gly-Ser linker (sequence number 69).

[0108] According to one embodiment, the present invention relates to a fusion construct comprising at least one free cysteine ​​residue, at least two free cysteine ​​residues, at least three free cysteine ​​residues, at least four free cysteine ​​residues, at least five free cysteine ​​residues, preferably at least six free cysteine ​​residues.

[0109] According to one embodiment, the present invention relates to a fusion construct in which free cysteine ​​enables interaction with a drug and / or payload.

[0110] According to one embodiment, the present invention relates to a fusion construct in which the payload is a furin inhibitor.

[0111] According to one embodiment, the present invention relates to a fusion construct comprising the A339C mutation, the S337C mutation, and / or the K340C mutation.

[0112] According to one embodiment, the present invention relates to a fusion construct comprising an array selected from any of the arrays of SEQ ID NOs: 36, 37, 38, 39, 40, 42, 44, or 46.

[0113] According to one embodiment, the present invention relates to a fusion construct which is IgG1, IgG2, IgG3, or IgG4.

[0114] According to one embodiment, the present invention relates to a fusion construct which is IgG, IgM, IgA, IgD, or IgE.

[0115] According to one embodiment, the present invention relates to a fusion structure including a null fc.

[0116] According to one embodiment, the present invention relates to a fusion construct in which null fc comprises an Ala substitution at position 234 and / or an Ala substitution at position 235, and / or an N297A mutation and / or a K322A mutation.

[0117] According to one embodiment, the present invention relates to a fusion construct comprising a heterodimerizing domain.

[0118] According to one embodiment, the present invention relates to a fusion construct in which the heterodimerizing domain comprises the sequence described in Sequence ID No. 48, 49, or 50.

[0119] According to one embodiment, the present invention relates to a fusion construct including a heterodimerizing mutation.

[0120] According to one embodiment, the present invention relates to a fusion construct in which the heterodimerizing mutation is F405L and / or K409R mutation.

[0121] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. The sequence described in Sequence ID No. 38, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The first chain including, b.iii. Sequences described in Sequence ID No. 1, Sequence ID No. 2, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. The sequence described in Sequence ID No. 38, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The second chain including This relates to fusion structures, including those mentioned above.

[0122] According to one embodiment, the present invention is The sequences described in ai Sequence ID No. 3 and Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. The sequence described in Sequence ID No. 38, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The first chain including, b.iii. Sequences described in Sequence ID No. 3, Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. The sequence described in Sequence ID No. 38, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The second chain including This relates to fusion structures, including those mentioned above.

[0123] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. The sequence described in Sequence ID No. 38, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The first chain including, b.iii. Sequences described in Sequence ID No. 3, Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and iv. The sequence described in Sequence ID No. 40, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The second chain including This relates to fusion structures, including those mentioned above.

[0124] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. The linker sequence described in Sequence ID No. 41, and iii. The sequence described in Sequence ID No. 65, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The first chain, including The sequences described in bv Sequence ID No. 1 and Sequence ID No. 2, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and vi. Linker sequence described in Sequence ID No. 41, and vii. The sequence described in Sequence ID No. 65, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The second chain including This relates to fusion structures, including those mentioned above.

[0125] According to one embodiment, the present invention is The sequences described in ai Sequence ID No. 3 and Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. The linker sequence described in Sequence ID No. 41, and iii. The sequence described in Sequence ID No. 65, or a sequence having at least 90% sequence identity with this sequence, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity. The first chain, which includes; b.iv. Sequences described in Sequence ID No. 3, Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and v. Linker sequence described in Sequence ID No. 41, and vi. The sequence described in Sequence ID No. 65, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with this sequence. The second chain including This relates to fusion structures, including those mentioned above.

[0126] According to one embodiment, the present invention is ai Sequence ID No. 1, Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and ii. The linker sequence described in Sequence ID No. 41, and iii. Sequences described in Sequence ID No. 65, wherein Sequence ID No. 65 contains one or more of the mutations in Table 8. The first chain, including b.iv. Sequences described in Sequence ID No. 3, Sequence ID No. 4, or sequences having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences, and v. Linker sequence described in Sequence ID No. 41, and vi. Sequences described in Sequence ID No. 65, wherein Sequence ID No. 65 contains one or more of the mutations in Table 8. The second chain including This relates to fusion structures, including those mentioned above.

[0127] According to one embodiment, the present invention relates to a fusion construct in which the ratio of the fusion construct to the drug and / or payload is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0128] According to one embodiment, the present invention relates to a fusion construct comprising a kappa light chain as described in Sequence ID No. 51, or a lambda light chain as described in Sequence ID No. 52 or 53.

[0129] According to one embodiment, the present invention relates to a fusion construct which is an IgG3 construct, wherein the IgG3 construct includes a hinge region, and the hinge region is modified.

[0130] According to one embodiment, the present invention relates to a fused construct in which the hinge region includes an array having at least 10% identity in total with the array described in Sequence ID No. 6 or Sequence ID No. 8, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity.

[0131] According to one embodiment, the present invention relates to a fusion construct comprising the sequences described in Sequence IDs 5, 7, 9, 10, 11, 12 and / or 13.

[0132] According to one embodiment, the present invention relates to a fusion construct in which the hinge region comprises at least one free cysteine ​​residue, at least two free cysteine ​​residues, preferably at least three free cysteine ​​residues.

[0133] According to one embodiment, the present invention relates to a fusion construct in which the hinge region contains the S228P mutation.

[0134] According to one embodiment, the present invention relates to a fusion construct in which the hinge region includes the sequence described in Sequence ID No. 6 and / or Sequence ID No. 8 and / or Sequence ID No. 68.

[0135] According to one embodiment, the present invention relates to a fusion construct used for detecting phosphatidylserine.

[0136] According to one embodiment, the present invention relates to a fusion construct used for detecting phosphatidylserine in the blood of a subject.

[0137] According to one embodiment, the present invention relates to a fusion construct comprising the sequence described in Sequence ID No. 1 and / or the sequence described in Sequence ID No. 2.

[0138] According to one embodiment, the present invention relates to a fusion construct used for detecting C-type lectins that bind to mannan or fukan moieties.

[0139] According to one embodiment, the present invention relates to a fusion construct used for detecting C-type lectins that bind to mannan or fukan portions in the blood of a target.

[0140] According to one embodiment, the present invention relates to a fusion construct comprising the sequence described in Sequence ID No. 3 and / or the sequence described in Sequence ID No. 4.

[0141] According to one embodiment, the present invention relates to a fusion construct, fusion protein, or antibody comprising a constant region and a hinge of IgG3, wherein the hinge is preferably selected from an IgG1 or IgG4 hinge.

[0142] According to one embodiment, the present invention relates to a fusion construct, fusion protein, or antibody comprising one or more heterodimerizing mutations.

[0143] According to one embodiment, the present invention relates to a fusion construct, fusion protein, or antibody containing a heterodimerizing mutation involving the 405th and / or 409th position (EU numbering).

[0144] According to one embodiment, the present invention relates to an IgG3 homodimer comprising a hinge region, wherein the hinge region comprises a sequence selected from sequence numbers 6, 8, and 68.

[0145] According to one embodiment, the present invention relates to an IgG3 heterodimer comprising a hinge region, wherein the hinge region comprises a sequence selected from sequence numbers 6, 8, and 68.

[0146] According to one embodiment, the present invention relates to IgG3 containing a mutation at position 405 and / or position 409.

[0147] According to one embodiment, the present invention relates to an IgM heterodimer that can be obtained by changing the charge pair of the CH2 and / or CH4 domains.

[0148] According to one embodiment, the present invention relates to an IgM heterodimer containing one or more of the mutations in Table 8.

[0149] According to one embodiment, the present invention relates to IgM comprising the sequence described in Sequence ID No. 64 and / or 65.

[0150] According to one embodiment, the present invention relates to a fusion construct comprising an IgG3 homodimer, an IgG3 heterodimer, and / or an IgM heterodimer as described in the present invention.

[0151] According to one embodiment, the present invention relates to a fusion structure for use in the treatment of infectious diseases.

[0152] According to one embodiment, the present invention relates to a fusion construct in which the infectious disease is caused by a virus, parasite, bacteria, fungus, or protozoan.

[0153] According to one embodiment, the present invention relates to a fusion construct in which the virus is selected from arbovirus, Zika virus, dengue virus, West Nile virus, Ebola virus, influenza virus, influenza virus H1N1, chikungunya virus, enterovirus, and coronavirus SARS-CoV.

[0154] According to one embodiment, the present invention relates to a fusion construct in which the bacteria are selected from Mycobacterium tuberculosis and Mycoplasma leple.

[0155] According to one embodiment, the present invention relates to a fusion construct in which the parasite is selected from leishmania and malaria.

[0156] According to one embodiment, the present invention relates to the use of a fusion structure described in the present invention for the treatment of infectious diseases.

[0157] According to one embodiment, the present invention relates to a use in which the infectious disease is selected from viral, bacterial, and protozoan infections.

[0158] According to one embodiment, the present invention relates to a use in which the treatment includes the administration of a fusion construct in a dosage form selected from subcutaneous, intradermal, intramuscular, oral, and nasal.

[0159] According to one embodiment, the present invention relates to the use of IgG4 or a portion of IgG4 for payload delivery, wherein IgG4 is modified to not contain Fc, or the activity of Fc in IgG4 is inactivated or reduced by one or more mutations.

[0160] According to one embodiment, the present invention relates to a use in which IgG4 comprises one or more heterodimerizing mutations.

[0161] According to one embodiment, the present invention relates to the use of IgG4 comprising one or more Cys mutations, preferably thereby enabling site-directed conjugation.

[0162] According to one embodiment, the present invention relates to the use of IgG4 containing Cys at position 339 (EU numbering).

[0163] According to one embodiment, the present invention relates to a vaccine comprising a fusion construct described in the present invention.

[0164] According to one embodiment, the present invention relates to a vaccine comprising mannan, a high-mannose-containing structure, fukan, and / or the phospholipid phosphatidylserine (PS).

[0165] According to one embodiment, the present invention relates to a vaccine further comprising a β-glucan adjuvant for enhancing the immune response.

[0166] According to one embodiment, the present invention relates to a vaccine for the prevention and / or treatment of infectious diseases.

[0167] According to one embodiment, the present invention relates to a vaccine in which an infectious disease is caused by a virus, parasite, bacteria, fungus, or protozoan.

[0168] According to one embodiment, the present invention relates to a fusion construct and / or vaccine that enables administration via a route selected from subcutaneous, intradermal, intramuscular, oral, and / or nasal administration.

[0169] According to one embodiment, the present invention relates to a composition comprising a fusion construct according to the present invention, which optionally comprises one or more excipients such as a diluent, a binder, or a carrier.

[0170] According to one embodiment, the present invention relates to a method for treating and / or preventing an infectious disease in a subject, comprising the step of administering a fusion construct and / or vaccine and / or composition described in the present invention.

[0171] According to one embodiment, the present invention is a method for screening and / or monitoring the progression of a disease in a subject, the following i. A step of providing a blood sample derived from the target, ii. The step of bringing a blood sample into contact with the fusion construct described in the present invention. This includes methods.

[0172] According to one embodiment, the present invention relates to an isolated nucleic acid molecule encoding a fusion construct described in the present invention.

[0173] According to one embodiment, the present invention relates to a recombinant vector comprising a nucleic acid molecule described in the present invention.

[0174] According to one embodiment, the present invention relates to a host cell comprising a recombinant vector described in the present invention.

[0175] According to one embodiment, the present invention relates to a method for producing a fusion construct according to the present invention, comprising the steps of culturing host cells according to the present invention in a culture medium under conditions that enable the expression of the fusion construct, and isolating the fusion construct from the culture medium.

[0176] Additional embodiments of the present invention are described below.

[0177] According to one embodiment, the present invention relates to a fusion structure including a hinge region, wherein the hinge region includes any of the following arrangements:

[0178] [Table 1]

[0179] According to one embodiment, the present invention relates to a fusion construct comprising an Fc heterodimerization sequence in residues 405-409, wherein the Fc heterodimerization sequence comprises the sequence described in Sequence ID No. 48, 49, or 50.

[0180] IgG Fc heterodimerization sequence (residues 405-409)

[0181] [Table 2]

[0182] Immunoglobulins are glycoproteins composed of one or more units, each containing four polypeptide chains: two identical heavy chains (HC) and two identical light chains (LC). The amino-terminuses of the polypeptide chains exhibit considerable diversity in amino acid composition and are called variable (V) regions to distinguish them from the relative constant (C) regions. Each light chain consists of one variable domain VL and one constant domain CL. The heavy chain consists of a variable domain VH and three constant domains CH1, CH2, and CH3. The heavy and light chains are joined together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bivalent structure. The V regions of the H and L chains contain the antigen-binding sites of the immunoglobulin (Ig) molecule. Each Ig monomer contains two antigen-binding sites and is said to be divalent.

[0183] Fab comprises one complete light chain, as well as the V and CH1 portions of one heavy chain. Fab can be further divided into a variable fragment (Fv) consisting of a VH domain and a VL domain, and a constant fragment (Fb) consisting of a CL domain and a CH1 domain.

[0184] The constant H chain domain is generally defined as CH1-CH2-CH3 (IgG, IgA, IgD), and for IgM and IgE, it is defined to include an additional domain (CH4). As mentioned above, the CH1 domain is located within the F(ab) region, while the remaining CH domains (CH2-CH3 or CH2-CH4) contain an Fc fragment. This Fc fragment defines the immunoglobulin isotype and subclass.

[0185] CH3 domain: The terms CH3 domain and CH3 region are used interchangeably herein.

[0186] CH1 domain: The terms CH1 domain and CH1 region are used interchangeably in this specification.

[0187] Hinge region: The hinge region is the heavy chain region between the first and second C domains, linked by a disulfide bond. The hinge region typically contains 10 to 30 amino acid residues.

[0188] Linker: The linker may be a peptide linker or a non-peptide linker. An example of a peptide linker is the Gly / Ser peptide linker containing the 5-amino acid residue unit GGGGS (SEQ ID NO: 71), which can be repeated an appropriate number of times. The linker may be a naturally occurring linker or a synthetically produced linker. The linker may occur spontaneously within the molecule or be added to the molecule synthetically.

[0189] Antibody Fragment: As used herein, “antibody fragment” includes, for example, a portion of an intact antibody, such as the antigen-binding or variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, an antibody fragment may include an isolated fragment consisting of heavy and light chain variable regions, an “Fv” fragment, a recombinant single-chain polypeptide molecule ("ScFv protein") in which the light and heavy chain variable regions are linked by a peptide linker, and a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region. In many embodiments, an antibody fragment contains a sufficient sequence of a parent antibody, which is a fragment that binds to the same antigen as the parent antibody. In some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include: Examples of antibody antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs). Antigen-binding fragments of antibodies can be generated by any means. For example, antibody antigen-binding fragments can be generated enzymatically or chemically by fragmentation of intact antibodies and / or recombinantly from genes encoding partial antibody sequences. Alternatively, or additionally, antibody antigen-binding fragments can be generated entirely or partially synthetically.

[0190] Antibody antigen-binding fragments may optionally contain single-chain antibody fragments. Alternatively, or additionally, antibody antigen-binding fragments may contain multiple chains linked together, for example, by disulfide bonds. Antibody antigen-binding fragments may optionally contain multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, and more typically at least about 200 amino acids.

[0191] Antibody or fragment thereof: As used herein, “antibody or fragment thereof” means the antibody or antibody fragment as defined above.

[0192] Humanized antibodies: Humanized antibodies are antibodies derived from non-human species whose protein sequences have been modified to increase their similarity to antibody variants naturally produced in humans.

[0193] IMGT: The International ImMunoGeneTics Information System is an international reference system in immunogenetics and immunoinformatics.

[0194] Single-chain Fv (scFv): Single-chain Fv (scFv) is widely known and used in the art. Single-chain Fv is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of immunoglobulins, often linked by a short linker peptide (see, for example, Benny KC Lo (ed.), Antibody Engineering—Methods and Protocols, Humana Press 2004, and the references cited therein). [Brief explanation of the drawing]

[0195] [Figure 1] This figure shows the TIM1 and CTLD structures with enhanced ADCC, ADCP, and CDC. [Figure 2] This figure shows TIM1 and CTLD constructs that exhibit T cell engager activity. [Figure 3] This figure shows TIM1 and CTLD constructs with a furin inhibitor payload. [Figure 4] This figure shows the TIM1 and CTLD structures with IgM effector functionality. [Figure 5a] This is a diagram showing the SEC-HPLC analysis of VP019. [Figure 5b] This is a diagram showing the SEC-HPLC analysis of VP020. [Figure 6a] This figure shows that VP025 is composed of four different peaks. [Figure 6b]This figure shows fraction 3 (VP025-F3) from intact mass spectrometry peak 3. [Figure 6c] This figure shows intact mass spectrometry: fraction 4 (VP025-F4) from peak 4. [Figure 7a] This figure shows a schematic diagram and purity of VP300 obtained by SEC-HPLC. [Figure 7b] This figure shows a schematic diagram and purity of VP301 obtained by SEC-HPLC. [Figure 8] This figure shows the binding of the SARS-CoV-2 S protein (D614G). [Figure 9] This figure shows the binding of VP025-CT to the viral protein. [Figure 10a] This figure shows the binding curves of VP019 and VP020 (heterodimer mixture) to select groups of biotin-phosphatidylserine and viral antigens. [Figure 10b] This figure shows the binding curves of VP025-CT (heterodimer mixture) and VP025-F4 (78% purity heterodimer) to select groups of biotin-phosphatidylserine and viral antigens. [Figure 11] This figure shows the structures of hexa-D-arginine linker compounds and decanoyl-RVKR-CMK linker compounds. [Figure 12] This figure shows the reaction scheme of the decanoyl-RVKR-CMK linker compound. [Figure 13a] This figure shows a mass spectrogram demonstrating that the conjugation of the furin inhibitor payload to VP020 was achieved. [Figure 13b] This figure shows a mass spectrogram demonstrating that the conjugation of the furin inhibitor payload to VP020 was achieved. [Figure 14a] This figure shows the neutralization assay of the selected fusion protein of the present invention. For details, please refer to Example 16. [Figure 14b]This figure shows the neutralization assay of the selected fusion protein of the present invention. For details, please refer to Example 17.

[0196] All cited references are incorporated by reference.

[0197] The accompanying drawings and examples are provided for illustrative purposes only and are not intended to limit the present invention. It will be apparent to those skilled in the art that aspects, embodiments, claims, and any other items of the present invention can be combined.

[0198] Unless otherwise specified, all percentages are weight / weight. Unless otherwise specified, all measurements are performed under standard conditions (room temperature and atmospheric pressure). Unless otherwise specified, test conditions follow European Pharmacopoeia 8.0. [Examples]

[0199] [Example 1] Selection of recombinant human TIM1 fragment Construct V-TIM1-1 was selected from residues 21-125 of the full-length TIM-1 sequence (https: / / www.uniprot.org / uniprot / Q96D42), and V-TIM1-2 was selected from residues 21-127. V-TIM1-2 contains two extra Pro residues at the C-terminal domain boundary.

[0200] [Table 3]

[0201] [Example 2] Selection of recombinant human C-type lectin domain (CTLD) fragments of DC-SIGN (CD209) Construct V-CTLD-1 was selected from residues 250-385 of the full-length DC-SIGN sequence (https: / / www.uniprot.org / uniprot / Q9NNX6), and V-CTLD-2 was selected from residues 254-383. V-CTLD-1 contains four internal disulfide bonds, while V-CTLD-2 contains three internal disulfide bonds.

[0202]

Table 4

[0203] [Example 3] Design of TIM-1 and CTLD constructs with IgG3 effector function Among all human IgG subclasses, IgG3 has the highest effector function in terms of ADCC, ADCP, and CDC (https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2014.00520 / full). IgG3 typically is not used for therapy because the long hinge region between the CH1 and CH2 domains results in a short serum half-life due to proteolytic cleavage. To utilize the potent effector function of the IgG3 subclass, a V-IgG3 construct was designed in which the IgG3 hinge (LKTPLGDTTHTPEPKSCDTPPPCPRCPAP) (SEQ ID NO: 6) was replaced with an IgG4 hinge sequence (SKYGPPCPPCPAP) (SEQ ID NO: 8) containing the IgG4 hinge S228P mutation that prevents Fab arm exchange or an IgG1-like hinge (KTGDTTHTCPRCPAP) (SEQ ID NO: 68).

[0204] The heterodimeric V-IgG3 construct was designed by including the K409R (on one heavy chain) mutation and the F405L (on the second antibody) mutation in the CH3 domain (https: / / www.nature.com / articles / nprot.2014.169). Each heavy chain was first generated as a single homodimer and then mixed together and recombined as a heterodimer under reducing and oxidizing conditions. The resulting sequences are noted as V-IgG3-A and V-IgG3-B and are paired together or noted as V-IgG3-D and V-IgG3-E that pair together. The sequences include a cleaved version containing a (GGGGS)3 linker (SEQ ID NO: 41) for replacing the CH1 domain and are confirmed in Table 3.

[0205]

Table 5

[0206] The TIM1 and CTLD fusion protein was designed using a modified IgG3-Fc domain, as shown in Figure 1 and Table 4.

[0207] [Table 6]

[0208] [Example 4] Design of TIM-1 and CTLD constructs with T cell engagement activity We designed additional constructs to engage T cell effector function by fusing TIM-1 and CTLD with a single anti-CD3 scFv. The designs are shown in Figure 2 and Table 5.

[0209] [Table 7]

[0210] [Table 8] TIFF0007883301000011.tif250166TIFF0007883301000012.tif250166TIFF0007883301000013.tif33166

[0211] [Example 5] Design of TIM-1 and CTLD constructs using furin inhibitor payload delivery Site-specific addition of a drug payload to the antibody Fc region was devised by analysis of the co-crystal structure of human IgG1 Fc and the 3-helix bundle of bacterial protein A (PDB structure 5U4Y https: / / www.rcsb.org / sequence / 5U4Y). Computational modeling revealed that A339C has a stabilizing effect on the structure, while S337C or K340C has a neutral effect on the stability of the Fc domain. A339C was selected as the site for site-specific conjugation.

[0212]

Table 9

[0213] TIM1 and CTLD fusion proteins having an Fc domain containing a payload conjugation site were designed and are shown in Figure 3 and Table 8.

[0214]

Table 10

[0215] [Example 6] Furin linker Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk) (SEQ ID NO: 81) or hexa-D-arginine (D6R) was linked to the TIM-1 and CTLD constructs using a cleavable linker such as an acid-sensitive N-acyl-hydrazone, or an enzyme-sensitive maleimide conjugate-type dipeptide, valine-alanine, valine-citrulline or phenylalanine-lysine.

[0216] Acid-sensitive linkers are cleaved in the lysosomal acidic environment after the construct has moved internally. This strategy is used in two approved ADCs, gemtuzumab ozogamicin and inotuzumab ozogamicin. Lysosomal protease-sensitive dipeptides release the drug after being cleaved by proteases such as cathepsin B-lysosomal protease. This type of linker chemistry is used in the FDA-approved brentuximab vedotin.

[0217] The antibody is conjugated to the polypeptide via a nucleophile of lysine or cysteine, either by random conjugation which produces a heterogeneous mixture of conjugates, or by site-specific conjugation to engineered cysteine ​​which reduces the heterogeneity of the product to an antibody-drug ratio (ADR) of 1 or 2.

[0218] The nucleophilic reactivity of the thiol functional group of a Cys residue towards the maleimide group is approximately 1000 times higher than that of any other amino acid functional group in a protein, such as the amino group or N-terminal amino group of a lysine residue. While thiol-specific functional groups in maleimide reagents can react with amine groups, this requires a higher pH (>9.0) and a longer reaction time (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

[0219] The first FDA-approved, engineered cysteine-based site-directed arteriovenous drug (ADC) was vadastuximabutarilin (Seattle Genetics).

[0220] [Table 11]

[0221] [Example 7] Fusion protein with IgM constant region IgM molecules, in particular, possess a potent Fc effector function with CDC. IgM molecules spontaneously homodimerize and then covalently form pentamers or hexamers. IgM does not contain a hinge region like IgG molecules, but instead contains an extra CH domain (CH1-CH2-CH3-CH4). The homodimeric heavy chain unites at the CH2 and CH4 domains. Based on the crystal structure of the mouse IgM CH2 domain (pdb 4JVU), the crystal structure of the mouse IgM CH4 domain (pdb 4JVW), and a visual analysis of sequence alignment with mouse sequences homologous to human IgM CH2 and CH4 sequences, we designed mutations to induce IgM heavy chain heterodimerization by inducing a charge difference at the homodimerization interface.

[0222] Sequence of the human IgM constant region from residue numbers 1 to 453 according to uniprot (www_uniprot.org / uniprot / P01871):

[0223] [ka]

[0224] IgM CH2-CH3-CH4 sequences that can be used for fusion to antibody fragments (Fab, scFv, VHH, etc.) or targeting proteins (TIM-1, CTLD / DC-SIGN) to add effector function to IgM (residues 105-453):

[0225] [ka]

[0226] Structural analysis confirmed that the underlined residues K131 and Q135 are in close proximity at the CH2:CH2 interface, and residues T354 and E385 are in close proximity at the CH4:CH4 interface. The following mutations were made to alter the charge patterns of V-IGM-A and V-IGM-B, induce A:B heterodimer formation, and avoid the formation of A:A or B:B.

[0227] [Table 12]

[0228] We designed a TIM1 and CTLD fusion protein with IgM effector function, which is shown in Figure 4 and Table 11.

[0229] [Table 13]

[0230] Example 8: Expression and purification of VP011-VP020 The ten proteins shown in [Table 1 and Figures 1-3] were expressed and batch purified from 2.5 mL of ExpiCHO culture using Protein A / G magnetic agarose beads. Expression yield and monomer purity % are shown in Table 12.

[0231] [Table 14]

[0232] VP011 (100 mL), VP012 (100 mL), VP013 (100 mL), VP014 (100 mL), VP019 (1 L), and VP020 (250 mL) were prepared on a larger scale using ExpiCHO cells. VP011, VP019, and VP020 were purified by MabSelect SuRe Protein A resin column chromatography. VP012, VP013, and VP014 were purified by HiTrap Protein G resin column chromatography. Expression yields and monomer purity % are shown in Table 13.

[0233] [Table 15]

[0234] The sequences of the expressed recombinant proteins are shown in Table 14.

[0235] [Table 16] TIFF0007883301000025.tif245166TIFF0007883301000026.tif98166

[0236] Example 9: Preparation of VP019 fraction and VP025 heterodimer SEC-HPLC analysis of VP019 (Figure 5a) shows that VP019 forms the expected homodimer (fraction F6) and a polymer of approximately 600 kDa (fraction F2). These fractions were purified and their functional activity was evaluated separately (named VP019-F6 and VP019-F2, respectively).

[0237] Figure 5b: SEC-HPLC analysis of VP020.

[0238] The protein product VP025, a heterodimer of VP019 and VP020, was generated by co-expressing the genes TM-G4-A-DC and CT-G4-B-DC in ExpiCHO cells and purified by MabSelect SuRe Protein A resin column chromatography. The resulting co-transfected sample product was named VP025-CT. Figure 6a shows that VP025-CT contains four distinct peaks, which were purified and evaluated separately by intact mass spectrometry. Fractions from peaks 1 and 2 showed many species. Fraction 3 (VP025-F3) from peak 3 showed a mass of 86,721 Da, corresponding to the VP020 homodimer (Figure 6b). Fraction 4 (VP025-F4) from peak 4 showed a mass of 83,881 Da, corresponding to a well-formed VP019 / VP020 heterodimer with a purity of approximately 78% (Figure 6c).

[0239] Example 10: Expression and purification of VP300 and VP301 Two additional bispecific molecules containing TIM-1 and CTLD domains on a single polypeptide chain (Table 15) were generated in 100 mL of ExpiCHO cells and purified by MabSelect SuRe Protein A resin column chromatography. A schematic diagram and purity measured by SEC-HPLC are shown in Figures 7a and 7b.

[0240] The sequences of the expressed recombinant proteins are shown in Table 14.

[0241] [Table 17]

[0242] Example 11: Binding of VP019, VP020, and VP025 to the SARS-CoV-2 S protein The binding of VP019-F2, VP020, and VP025-CT to SARS-CoV-2 S D614G was investigated by ELISA. This protein is representative of the SARS-CoV-2 strain that was dominant in early 2020. Since DC-SIGN is known to use calcium at its binding site, all ELISA assays in this and subsequent examples were performed in the presence of 2.5 mM CaCl2. The binding curves are shown in Figure 8, and the EC50 values ​​are shown in Table 16. VP019-F2 showed unexpectedly high binding to the SARS-CoV-2 S protein, while VP020 showed only slight binding.

[0243] [Table 18]

[0244] Example 12: Binding of VP025-CT to a wide range of viral antigens. The binding of VP025-CT to a wide variety of viral surface protein antigens was investigated by ELISA. The binding curves are shown in Figure 9, and the EC50 values ​​are shown in Table 17.

[0245] [Table 19]

[0246] Example 13: Binding of VP019, VP020, VP025-CT, and VP025-F4 to phosphatidylserine and viral antigens. The binding of VP019, VP020, VP025-CT (heterodimer mixture), and VP025-F4 (78% pure heterodimer) to biotin-phosphatidylserine and selected viral antigens (influenza A H1N1 HA, human RSV glycoprotein G, Zika virus envelope protein, and SARS-CoV-2 S D614G) was investigated by ELISA. Binding curves are shown in Figures 10a and 10b, and EC50 values ​​are shown in Tables 18 and 19. VP025-F4 showed stronger binding to all antigens than VP025-CT.

[0247] [Table 20]

[0248] [Table 21]

[0249] Example 14: Synthesis of a furin inhibitor linker payload Solvents and reagents were purchased from Sigma-Aldrich, VWR, or Fisher Scientific and used without further purification. The reactions were monitored by thin-layer chromatography (TLC) or by analytical liquid chromatography-mass spectrometry (LC-MS) using a Waters Acquity Ultra Performance LC system and a Synapt high-resolution mass spectrometer. 1 The 1H NMR spectra were recorded with a Varian Unity INOVA spectrometer (500 MHz). All chemical shifts are reported in ppm, and the coupling constant J is reported in Hertz (Hz). The NMR solvent peaks were referenced as follows: 1(H NMR) CDCl3: 7.27 ppm, DMSO-d6: 2.50 ppm. The compounds were purified by flash column chromatography using a Teledyne ISCO Combi-Flash system with normal-phase silica gel (SiliCycle Inc.) or reverse-phase (Teledyne Gold-C18 or C18-Aq) pre-packed columns. The purity of the compounds was determined by analytical HPLC (Waters Acquity Ultra Performance) using an Acquity UPLC CSH C18 1.7 μm (50 mm × 2.1 mm) column and a flow rate of 0.3 mL / min. Gradient conditions: Solvent A (0.05% formic acid in water) and solvent B (0.05% formic acid in acetonitrile): 0-0.1 min 95% A, 0.1-4.0 min 5-95% B (linear gradient), 4.0-5.0 min 95% B, UV detection at 254 nm and 220 nm.

[0250] The reaction scheme is shown in Figure 12.

[0251] N-methylmorpholine (13.8 μL, 0.126 mmol) was added under an argon atmosphere to a solution of hexa-D-Arg (D-arginine amide D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-alanine; Ambeed, cat# A333458) (30 mg, 0.0314 mmol), MC-Val-Cit-PAB-PNP (BroadPharm Cat#:BP-23292, CAS:159857-81-5) (46.4 mg, 0.0629 mmol) and HOBt.H2O (1-hydroxybenzotriazole monohydrate; 5.3 mg, 0.0345 mmol) in anhydrous DMF (1 mL). The solution was stirred at room temperature for 18 hours. The reaction mixture was diluted with 10 mL of 1:1 ACN / water (0.05% HCO2H), purified by reverse-phase C18-Aq flash chromatography (gradient elution; 100% water - 100% ACN containing 0.05% HCO2H as mobile phase additive), and lyophilized to obtain MC-VC-PAB-(D-Arg)e-NH2 (12 mg, 0.00773 mmol, 25%) as a white solid. 1H NMR (500 MHz, DMSO-d6) d 10.09 (s, 1H), 8.65 (br. s, 6H), 8.47 (s, 6H), 8.32 - 8.45 (m, 4H), 8.12 - 8.20 (m, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.50 - 7.80 (m, 18H), 7.29 (d, J = 7.8 Hz, 2H), 7.17 (s, 1H), 7.01 (s, 2H), 6.08 - 6.10 (m, 1H), 5.43 - 5.50 (s, 2H), 4.89 - 5.01 (m, 2H), 4.36 - 4.41 (m, 1H), 4.12 - 4.30 (m, 6H), 4.01 - 4.09 (m, 1H), 3.35 - 3.40 (m, 2H), 2.91 - 3.12 (m, 12H), 2.09 - 2.21 (m, 2H), 1.93 - 2.00 (m, 1H), 1.65 - 1.75 (m, 6H), 1.40 - 1.62 (m, 28H), 1.30 - 1.40 (m, 1H), 1.15 - 1.25 (m, 2H), 0.80 - 0.89 (m, 6H);MS(ESI)m / z:1552.4[M+H] + .

[0252] The structure is shown in Figure 11.

[0253] Example 15: Conjugation of a furin inhibitor payload to VP020 The conjugation test of the hexa-D-arginine linker compound to VP020 was performed by reacting VP025 with 4 equivalents of TCEP and incubating at 37°C for 1 hour to reduce free cysteine. The sample was passed through a Zeba column to remove TCEP and the buffer was replaced with 1×PBS containing 1 mM DTPA pH 6.5. This sample was then reacted with 2.5 equivalents of the payload (Mc-VC-PAB-(D-Arg6)-CONH2) at room temperature for 1 hour. The final product was analyzed by mass spectrometry (see Figure 13) and showed a mass of 88,136 Da, close to the expected mass of 88,138 Da.

[0254] Example 16: RSV neutralization assay of VP019, VP020, and VP025 Microneutralization assays were performed to determine the antiviral properties of three compounds (VP019-F2, VP020, and VP025-F4) against RSV. After incubating each virus with each antibody for 1 hour, the mixture was added to A549 cells (human lung cancer cell line). Antiviral activity was determined after 24 hours using an immunofluorescence-based assay. After 24 hours, the infection plates were washed with PBS, fixed with 4% formaldehyde for 30 minutes, washed again with PBS, and stored in PBS at 4°C until staining. All residual formaldehyde was quenched with 50 mM ammonium chloride, and the cells were then permeabilized (0.1% Triton X100) and stained with an antibody that recognizes the RSV fusion protein (GeneTex GTX40697). Primary antibodies were detected with Alexa-488 conjugate secondary antibodies (Life Technologies, A21244 and A11001), and the nuclei were stained with Hoechst. The images were acquired using the CellInsight CX5 High Content Platform (Thermo Scientific), and the infection rate was calculated using CellInsight CX5 software (infected cells / total cells × 100).

[0255] The test substance was used at a concentration of 0.5 μM, and the sample was tested in triplicate. The results are shown in Figure 14. For RSV, VP019-F2 inhibited 97% of the virus, while VP025-F5 inhibited 55% and VP020 inhibited 28%.

[0256] Example 17: ZIKV neutralization assay of VP025 The second neutralization assay was performed for Zika virus using VP025-F4 with the following procedure.

[0257] Preparation of cell cultures Vero E6 cells were maintained in DMEM supplemented with 2% FBS (and confirmation with 1% pen-strep-Allen) and stored at 37°C in 5% CO2. The cells were placed in a 48-well plate at a rate of 8.0 × 10⁶ cells per well. 4 Cells were seeded at a concentration of one cell and allowed to adhere overnight. On the morning of infection, the cell monolayer was examined to confirm a density of 90-95%.

[0258] Zika virus neutralization test VP025-F4 was serially diluted in three series using infection medium in a 1:3 ratio for a total of eight dilutions (220 to 0.1 μg / mL). Zika virus (ZIKV) strain MEX-1-44 was incubated at a MOI of 1.0 (8.0 × 10⁻⁶). 4 Add the FFU to each dilution, mix, and incubate at 37°C and 5% CO2 for 1 hour.

[0259] Neutralization of positive control VP025: Positive control samples were incubated simultaneously with the incubation of ZIKV. Mouse α-ZIKV MIAF (mouse immunoassay antibody) was diluted to 1:500, 1:1000, and 1:1500 and combined with ZIKV using the same virus concentration as the test wells in a triple well setup.

[0260] Plate infection After incubating VP025-F4:ZIKV and α-ZIKV MIAF:ZIKV, the Vero E6 well plates were removed from the incubator. The culture medium was aspirated from the cells, and the test samples and positive control samples were transferred to the Vero E6 plates. The plates were then returned to the incubator, and the cells were infected with the non-neutralizing viruses for 1 hour.

[0261] Negative control focus formation assay Simultaneously with the incubation of the plate infection, serially dilute ZIKV (10 -2 ~10 -5 The sample was used to infect Vero E6 cells in a triple chain.

[0262] Focus reduction neutralization assay After incubating Vero E6 cells for 1 hour, a 0.8% methylcellulose overlay was added to all wells and maintained at 37°C and 5% CO2 for approximately 60 hours. The plate was removed from the incubator, the overlay was aspirated, and the cells were gently washed twice with phosphate-buffered saline. The virus was inactivated with a 1:1 fixative mixture of methanol and acetone, and the plate was fixed for 30 minutes. After inactivation, the fixative was removed, and the plate was air-dried until the fixative was completely absorbed.

[0263] All incubation and washing were performed at room temperature, and the plates were placed in a plate locker. Cells were permeabilized with 0.5% Triton in PBS and washed with 0.02% Tween 20 in PBS (PBST). A blocking solution in PBST containing BSA and normal goat serum was prepared and incubated in all wells for 1 hour. The primary antibody, mouse α-ZIKV MIAF, was diluted in PBST containing BSA and stored on ice until use. The blocking solution was removed, and the primary antibody was incubated for 1 hour. The antibody was removed, and the plates were washed with PBST. The secondary antibody, goat α-mouse IgG (high and low chain) HRP conjugate type, was diluted in PBST containing BSA. The antibody was incubated in the wells for 1 hour, and then the wells were washed with PBST containing BSA. Vector Labs ImmPACT AMEC developing solution was prepared according to the kit instructions and added to each well. The plates were incubated in the dark, but staining was checked periodically. After the lesions were clearly visible (approximately 15 minutes), the wells were rinsed with deionized water and the plates were dried.

[0264] result No lesions were observed in the VP025-F4 test well or the positive control well (see Figure 14b). Lesions developed as expected in the negative control focus formation assay well, and the lesions were shown to decrease with each sequential viral dilution. This suggests that the VP025 test material was able to neutralize ZIKV at dilutions of (220–0.1 μg / mL).

Claims

1. Ig-Fc domains or other protein scaffolds, such as albumin or antibody fragments that bind to albumin, and a. A peptide, protein, or fragment that binds to phosphatidylserine and contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, and b. A peptide or protein that binds to and / or recognizes PAMP expressed by a microorganism, and comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3 or SEQ ID NO:

4. A fusion structure that includes this.

2. IgG-Fc domain or other protein scaffolds, a. i. Recombinant human TIM1 fragment, and ii. Recombinant human CD209 fragment b. i. Recombinant Ig-like V-domain of human TIM1, and ii. Recombinant C-type lectin domain of human CD209 c. i. Two or more recombinant Ig-like type V domains derived from one or more human TIM1, and ii. Two or more recombinant C-type lectin domains of human CD209 d. i. Recombinant Ig-like V-domain of human TIM1, and ii. Recombinant C-type lectin domain of human CD209 fragment e. i. Recombinant human TIM1 fragment, and ii. Recombinant human CD209 fragment f. i. Recombinant human TIM1 fragment, and ii. Recombinant human CD209 fragment (In this case, the fusion structure further comprises an immunoglobulin heavy chain variable domain including a heavy chain CDR1 containing the sequence described in SEQ ID NO: 57, a heavy chain CDR2 containing the sequence described in SEQ ID NO: 58, and a heavy chain CDR3 containing the sequence described in SEQ ID NO: 59, and an immunoglobulin light chain variable domain including a light chain CDR1 containing the sequence described in SEQ ID NO: 54, a light chain CDR2 containing the sequence described in SEQ ID NO: 55, and a light chain CDR3 containing the sequence described in SEQ ID NO: 56.) or g. i. Recombinant human TIM1 fragment, and ii. Recombinant human CD209 fragment (In this case, the fusion structure further includes a furin inhibitor.) A fusion structure according to claim 1, including the above.

3. The fusion construct according to claim 1 or 2, wherein the IgG-Fc domain is an IgG3-Fc domain.

4. below a) IgG3, in which the hinge arrangement is preferably replaced with an IgG4 hinge arrangement or an IgG1 hinge arrangement; b) Immunoglobulin heavy chain variable domains comprising heavy chain CDR1 containing the sequence described in SEQ ID NO: 57, heavy chain CDR2 containing the sequence described in SEQ ID NO: 58, and heavy chain CDR3 containing the sequence described in SEQ ID NO: 59, and immunoglobulin light chain variable domains comprising light chain CDR1 containing the sequence described in SEQ ID NO: 54, light chain CDR2 containing the sequence described in SEQ ID NO: 55, and light chain CDR3 containing the sequence described in SEQ ID NO: 56; and / or c) Furin inhibitors A fusion structure according to any one of claims 1 to 3, further comprising at least one of the following.

5. A fusion construct according to any one of claims 1 to 4, comprising the sequence described in Sequence ID No. 1 and / or Sequence ID No. 2, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences.

6. A fusion construct according to any one of claims 1 to 5, comprising the sequence described in Sequence ID No. 3 and / or Sequence ID No. 4, or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity with one of these sequences.

7. A fusion construct according to any one of claims 1 to 6, wherein the fusion construct can bind to a target, the target being mannan, a high-mannose-containing structure, fukan, the phospholipid phosphatidylserine and / or CD3.

8. A fusion construct according to any one of claims 1 to 7, wherein the fusion construct comprises a free cysteine ​​residue, the free cysteine ​​enabling interaction with a drug and / or payload.

9. The fusion construct according to claim 8, wherein the payload is a furin inhibitor.

10. A fusion structure according to any one of claims 1 to 9, comprising null fc.

11. A composition comprising a fused structure according to any one of claims 1 to 10 for use in the treatment of infections caused by viruses, bacteria, fungi, or parasitic protozoa.

12. The composition according to claim 11, wherein the virus is selected from arbovirus, Zika virus, dengue virus, West Nile virus, Ebola virus, influenza virus, influenza virus H1N1, chikungunya virus, enterovirus, coronavirus SARS-CoV-2 and coronavirus SARS-CoV, RSV, and HIV; the bacterium is selected from Mycobacterium tuberculosis and Mycoplasma leprae; and the parasite is selected from Leishmania and malaria.

13. A composition comprising a fusion structure according to any one of claims 1 to 10, optionally comprising one or more excipients such as a diluent, a binder, or a carrier.

14. A composition comprising a fusion construct according to any one of claims 1 to 10 for the treatment or diagnosis of microbial infections in humans or animals.

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