Compositions with reduced immunogenicity

Abstract

The present invention relates to a polyclonal antibody against at least one non-human biological pathogen or at least one molecule derived from said pathogen, against a human or non-human animal organism wherein said polyclonal antibody is free of an antigenic determinant selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and / or (ii) alpha-1, 3-galactose, and its use as a medicament.

Description

[0001] This application is a divisional application of the invention patent application filed on October 15, 2015, with application number 201580067999.4 and invention title "Composition with Reduced Immunogenicity". Technical Field

[0002] This invention relates to the field of immunology, and more particularly to polyclonal antibodies against human or animal organisms against non-human biological pathogens or molecules derived from said pathogens, and their use in medicine. Background Technology

[0003] Antibodies are Y-shaped protein molecules naturally produced by the body as part of the immune defense system. A key function of antibodies is to bind to molecules that the immune system does not recognize as part of the body. For example, the immune system produces antibodies to bind to pathogens (such as bacteria and viruses), thereby neutralizing their effects and helping the immune system clear infections.

[0004] Most antibody therapies are monoclonal antibodies. Monoclonal antibodies are antibodies that bind to a single epitope. Essentially, they are all the same antibody, not a collection of different antibodies. Researchers use a variety of known methods to produce monoclonal antibodies.

[0005] The current choice of polyclonal antibodies over monoclonal antibodies in most antibody therapies makes sense because polyclonal antibodies (pAbs) are antibodies secreted by highly diverse B cell and plasma cell lineages in the body (while monoclonal antibodies originate from a single cell lineage). Therefore, polyclonal hyperimmune antibodies are collections of highly diverse immunoglobulin molecules (various classes and isotypes that react with specific antigens, but each identifies different epitopes). Conversely, the combination of monoclonal antibodies is limited to a small number of molecules and cannot mimic the polyclonal diversity of polyclonal antibodies.

[0006] Furthermore, polyclonal antibodies exert their effects through a variety of mechanisms (particularly complement and cell-dependent cytotoxicity (CDC and ADCC), neutralization, opsonization, etc.), which are only available through various molecular targets and Ig classes and isotypes and cannot be replicated by monoclonal antibodies or even combinations of monoclonal antibodies. For example, polyclonal IgG targeting human T cells interacts with at least 50 clusters of differentiation (CD) (Popov et al., Transplantation, 2012).

[0007] Therefore, passive serum therapy using serum from animals (rabbits, horses, goats) or purified polyclonal immunoglobulins has become the first major advance in the treatment or prevention of the spread of serious infectious diseases such as plague and diphtheria.

[0008] However, despite proven efficacy, the injection of immune immunoglobulins (such as IgG or IgM) from animals into humans may still be immunogenic and cause immune complex-related diseases (ICDs) and serious undesirable adverse reactions such as serum sickness (SSD), including severe forms (such as myocarditis, nephropathy) or other immune complex manifestations such as rash, fever, headache, arthritis, or pseudomeningitis syndrome.

[0009] A human model of ICD has been established in animals (F Dixon J Exp Med 1956). The most common and identified complication in humans following the injection of animal IgG is serum sickness (SSD), which has been observed in almost 100% of young individuals with type 1 diabetes who have received thymoglobin (a purified rabbit IgG anti-T lymphocyte preparation) and have not received any other immunosuppressants (SE Giteman et al., The Lancet Diabetes & Endocrinology, 1:306, 2003).

[0010] Beyond safety concerns, passive immunotherapy for most people faces the presence of pre-existing anti-nonhuman animal Igs, which alter the bioseparability of the injected material and its potential early efficacy. Indeed, most people are known to already possess these anti-nonhuman animal Igs due to their diet and gut microbiota. Therefore, even with the efficient preparation of therapeutic nonhuman animal Igs intended for human administration, severe and highly frequent SSDs remain a major safety and potential efficacy barrier for passive immunotherapy in non-immunosuppressed recipients with serious infectious diseases. Furthermore, safety concerns may also limit the widespread use of preventative measures against potentially contaminated individuals near the patient.

[0011] More importantly, the presence of clinical manifestations of SSD is a clinical drawback to the accurate assessment of the outcomes of therapeutic or prophylactic regimens using purified polyclonal antibodies. In fact, clinical symptoms of SSD, particularly headache, fever, arthritis, or pseudomeningitis syndrome, can mislead the correct assessment of disease evolution.

[0012] Therefore, there remains a need for therapeutic polyclonal antibodies derived from non-human mammals that can effectively prevent and / or treat diseases caused by non-human pathogens and have reduced adverse reactions, including significantly lower immunogenicity compared to conventional polyclonal antibodies and ideally independent of the manifestation of IC-related diseases. Invention Summary

[0013] According to a first aspect, the present invention relates to polyclonal antibodies against at least one non-human biological pathogen or at least one molecule derived from said pathogen, preferably against human organisms, wherein said polyclonal antibodies do not contain a first antigenic determinant selected from: (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose.

[0014] According to another aspect, the present invention relates to compositions comprising at least one polyclonal antibody as defined above.

[0015] According to another aspect, the present invention relates to a method for producing polyclonal antibodies or compositions comprising polyclonal antibodies as defined above, comprising the following steps:

[0016] a) Provide a non-human mammal lacking a genetic alteration selected from the following first genes: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-1,3-galactosyltransferase (α1,3GT, GGTA1, or GT1).

[0017] b) Inoculate the genetically altered non-human mammal with at least one non-human pathogen or at least one molecular immunization derived from said pathogen, which is targeted at human or non-human animal organisms, preferably human organisms.

[0018] c) Collect antibodies contained in the bodily fluids of the non-human mammal with the genetic alterations described in step b).

[0019] According to a preferred embodiment, the genetically modified non-human mammal lacks only the gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH).

[0020] According to another preferred embodiment, the genetically altered non-human mammal lacks a gene selected from two genes characterized by: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-1,3-galactosyltransferase (α1,3GT, GGTA1, or GT1).

[0021] It is known in the art that antibodies against non-human pathogens for human or animal organisms (preferably human organisms) can be readily obtained by immunizing non-human mammals, particularly pigs, horses, or rabbits, with or without adjuvants, using an immunogenic composition (containing an antigen of the target non-human pathogen or a derivative thereof, i.e. a molecule derived from the pathogen).

[0022] Then, any class or type of polyclonal antibody against any kind of non-human biological pathogen can be obtained by immunizing a non-human mammal with the said non-human biological pathogen or its derived antigens (i.e., molecules derived from the said pathogen).

[0023] This includes therapeutic polyclonal antibodies that target non-human biological pathogens whose presence in human or non-human animal organisms, especially human organisms, is undesirable.

[0024] This includes polyclonal antibodies against microorganisms that exert harmful effects on human or non-human animal organisms, especially human organisms.

[0025] Therefore, the non-human biological pathogens used in human or animal organisms are preferably selected from bacteria, parasites, mushrooms, viruses, toxins, venoms, and combinations thereof. However, polyclonal antibodies known in the art, especially those produced in animals, particularly non-human mammals such as pigs or rabbits, remain immunogenic in humans because these known polyclonal antibodies possess α-1,3-galactose and / or N-ethylene glycol neuraminic acid (Neu5Gc) epitopes, which are recognized by cells of the human immune system.

[0026] To overcome these drawbacks of known polyclonal antibodies, the present invention envisions using genetically modified non-human mammals to generate polyclonal antibodies (or compositions comprising them) for human or animal organisms, said non-human mammals lacking a first gene selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH) and (ii) a gene encoding a functional α-1,3-galactosyltransferase, said polyclonal antibody targeting at least one non-human pathogen or at least one molecule derived from said pathogen.

[0027] The use of such genetically modified non-human mammals offers a further advantage because these mammals produce minimal amounts of anti-NeuGc antibodies on an unchanged diet. Therefore, this eliminates the need for the step of immunization absorption of the serum from these genetically modified non-human mammals before injection into human patients.

[0028] Using KO animals for at least the GT1 gene also has the following advantages: there is no further enhancement of the production of anti-Gal antibodies that may protect against pathogens (Katopodis AG et al., J. Clin. Invest., 2002).

[0029] According to another aspect, the present invention relates to the use of polyclonal antibodies or compositions comprising them as pharmaceuticals according to the invention.

[0030] The present invention also relates to the use of the polyclonal antibodies or compositions described above for the prevention and / or treatment of serious infections, particularly those selected from the following serious infections: Acinetobacter infections, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), amoebiasis, Alfa disease, anthrax, hemolytic lactobacillus infections, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infections, brassicosis, Bacillus cereus infections, bacterial pneumonia, bacterial vaginosis (BV), Bacteroidetes infections, balanitis, ascariasis, BK virus infections, black hairy nodular disease, human budsomya infections, blastomycosis, and vitiligo. Bolivian hemorrhagic fever, spirochetal infection, botulism (and infantile botulism), Brazilian hemorrhagic fever, brucellosis, plague, Burkholderia infection, brucellosis ulcer, calicivirus infection (norovirus and poxvirus), campylobacteriosis, candidiasis (candidiasis; thrush), cat bite disease, cellulitis, Chagas disease (American trypanosomiasis), chancroid, varicella, chikungunya, chlamydia, Chlamydia pneumoniae infection (Taiwan Acute Respiratory Agent or TWAR), cholera, melanocytosis, pheochromocytosis, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever (CTF), common cold (acute viral rhinitis,Acute rhinitis), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneous larvae (CLM), cyclosporidiosis, cysticercosis, cytomegalovirus infection, dengue fever, binuclear amoebiasis, diphtheria, trichomoniasis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, helminthiasis, enterobacterial infection (pinworm infection), enterococcal infection, enterovirus infection (especially enterovirus 71 (EV71)). (Huang et al., Curr. Opin. Virol., 2014, 12; 5: 98-104)), epidemic typhus, lupus erythematosus (fifth disease), roseola infantum (sixth disease), fascioliasis, fascioliasis, fatal familial insomnia (FFI), filariasis, food poisoning caused by Clostridium perfringens, free-living amoeba infection, fusobacterial infection, gas gangrene (Clostridium necrophorum), terriginosis, Jetzman- Stosterländer-Schenck syndrome (GSS), giardiasis, glanders, gnathostomiasis, gonorrhea, granuloma inguinale (urinary tract infection), group A streptococcal infection, group B streptococcal infection, Haemophilus influenzae infection, hand-foot-mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS), cardiogenic diseases, Helicobacter pylori infection, hemolytic uremic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex virus infection, histoplasmosis, hookworm infection, human bocavirus infection, human Evans elektiasis, human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human monocytic elektiasis, human papillomavirus (HPV) infection, human parainfluenza virus infection, hymenolepis taeniasis, Epstein-Barr virus infectious mononucleosis (Mono), influenza (flu), isosporidiosis.Kawasaki disease, keratitis, Agrobacterium infection, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease (Pontiac fever), leishmaniasis, leprosy, leptospirosis, Listeria infection, Lyme disease (Lyme spirochete infection), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East Respiratory Syndrome (MERS), glanders (Whitmore's disease), meningitis, meningococcal infection, metaclonorchiasis, microsporidiosis, molluscum contagiosum (MC), monkeypox, mumps, mouse typhus (endemic typhus), mycoplasma pneumonia, mycoplasma gondii, myiasis, neonatal conjunctivitis (neonatal ophthalmitis), ( New variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardia, Onchocerciasis (river blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurella multocida, Head lice, Body lice, Pubic lice, Pelvic inflammatory disease (PID), Pertussis, Plague, Pneumococcal infection, Pneumocystis pneumonia (PCP), Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis (PAM), Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rabies virus, Respiratory syncytial virus infection, Nasal spore infection, Rhinovirus infection, Rickettsia infection, Rickettsia pox, Rift Valley Rheumatoid arthritis (RVF), Rocky Mountain spotted fever (RMSF), Rotavirus infection, Rubella, Salmonella infection, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Septicemia, Shigella infection (bacterial dysentery), Herpes zoster (shingles), Smallpox, Sporothrix infection, Staphylococcus aureus food poisoning, Staphylococcus aureus infection, Strongyloides stercoralis infection, Subacute sclerosing panencephalitis, Syphilis, Tapeworm infection, Tetanus, Tinea barbae (ringworm), Tinea capitis (tinea capitis), Tinea corporis (tinea corporis), Tinea cruris (tinea cruris), Tinea manuum (tinea manuum), Tinea pedis (athlete's foot), Onychomycosis (nail fungus), Tinea versicolor (tinea versicolor), Toxocara canis (toxocara canis) Visceral larval migration syndrome (OLM), toxocariasis (VLM), trachoma, trichoccliasis, trichinosis, trichomoniasis, whipworm infection, tuberculosis, tularemia, ureaplasma infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white nodular disease (tinea capitis), plague, Yersinia pestis infection, Yersinia pestis, yellow fever, zygomycosis, or at least one serious infection induced by at least one antibiotic-resistant pathogen (DRoux et al., J. Antimicrob. Chemother., 2012; JDBerry et al., New Biotechnology, 2011, 28:489-501).

[0031] Therefore, the non-human biological pathogens considered in this invention and selected from bacteria, parasites, mushrooms, viruses, toxins, venoms, and combinations thereof are more specifically non-human biological pathogens that cause at least one serious infection as described above.

[0032] The present invention further relates to the use of the polyclonal antibodies or compositions described above for serum prophylaxis and / or serum therapy.

[0033] The present invention further relates to the use of the polyclonal antibodies or compositions described above for reducing and / or inhibiting immune complex-related diseases (ICDs) and serious adverse reactions, such as serum sickness (SSD), including severe forms (e.g., myocarditis, nephropathy) or other immune complex manifestations such as rash, fever, headache, arthritis or pseudomeningitis syndrome and immune complex manifestations induced by administration of an antibody containing at least one antigenic determinant selected from: (i) N-ethylene glycol neuraminic acid (Neu5Gc) or (ii) α-1,3-galactose, preferably containing at least both antigenic determinants (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose. Attached Figure Description

[0034] Figure 1 Immunization of double-KO pigs (porcine IgG ELISA). Each double-KO pig was injected with 700 μg EBOLA GP five times. Anti-GP specific antibodies were analyzed by ELISA from serum samples obtained from the vaccinated double-KO pigs before immunization (day 0), during immunization (days 15, 30, and 57), or after immunization (day 83). Bars represent the mean OD at each time point for each dilution. For each day (0, 15, 30, 57, and 83), from left to right (columns numbered 1-4): serum diluted to 1 / 500; 1 / 4500; 1 / 40.500; and 1 / 121.500, respectively. Mean optical density (OD) is expressed on the y-axis.

[0035] Figure 2 : Weight changes of guinea pigs during the experiment. Each number represents a group of guinea pigs. The standard deviations for each group and time point are shown: <1> >It's Mock-PBS. <2> >It is the EBOLA virus plus immune IgG D0 group.< <3> >It is EBOLA virus plus immune IgG D0+D3. <4> This is EBOLA virus plus non-immune IgG D0. Weight is represented on the y-axis. Number of days is represented on the x-axis.

[0036] Figure 3: Viral load in serum on day 3. The viral load in the serum of each animal on day 3 was evaluated by RTqPCR analysis using primers targeting the EBOLA L polymerase. The viral load of guinea pigs (n = 9) receiving anti-Ebola immune IgG from DKO was significantly lower (Mann-Whitney, p = 0.012) compared to guinea pigs (n = 5) receiving non-immune IgG. From left to right: EBOLA virus D0 without immune IgG, EBOLA virus + immune IgG D0. The viral load (y-axis) is expressed as EBOLA genomes / mL. One guinea pig receiving immune IgG (the viral load value of this animal was 3.5E7 genomes / ml) was excluded from the analysis as an outlier. The sera of animals from the control Mock PBS group did not show any viral load.

[0037] Figure 4 : Kaplan Meier survival curves. The Kaplan Meier survival curves show the percentage of guinea pigs surviving each day after a 12-day observation period of EBOLA virus exposure. The survival rate of guinea pigs (n = 10) receiving anti-Ebola immune IgG from DKO pigs was significantly higher (Log-rank, p = 0.0424*) compared to guinea pigs (n = 5) receiving non-immune IgG. The y-axis represents the survival percentage (%). The number of days is represented on the x-axis. Each curve is represented by its corresponding number: Mock-PBS is <<1>>; <<EBOLA virus + immune IgG D0+D3>> is <<2>>; <<EBOLA virus + immune IgG D0>> is <<3>>; <<EBOLA virus + non-immune IgG D0>> is <<4>>. Invention Details

[0039] 1. Definitions

[0040] To more fully understand the present invention, the following definitions are set forth. Such definitions are intended to cover grammatical equivalents.

[0041] As used herein, the term "comprising" covers "consisting of".

[0042] The term "antibody" is used herein in its broadest sense. An "antibody" refers to any polypeptide that at least comprises: (i) an Fc region and (ii) a binding polypeptide domain derived from the variable region of an immunoglobulin. Thus, antibodies include, but are not limited to: full-length immunoglobulins, antibodies, antibody conjugates, and their respective fragments. The terms "antibody" and "immunoglobulin" are used interchangeably herein.

[0043] The term "antibody" encompasses the above-mentioned polypeptides that further include at least one sugar moiety different from the antigenic determinants selected from: (i) N-ethylene glycol neuraminic acid (Neu5Gc) and / or (ii) α-1,3-galactose.

[0044] As used herein, a "polyclonal antibody" is a mixture of antibodies that recognize different epitopes of a given antigen. Polyclonal antibodies encompass those contained in or derived from the bodily fluids of non-human mammalian organisms, particularly serum or plasma.

[0045] Within the general meaning of this invention, the antibody according to the invention targets at least one non-human biological pathogen or at least one molecule derived from said pathogen, and is used in human or non-human animal organisms.

[0046] As used in this article, "natural" or "endogenous" antibodies refer to antibodies that are not derived from recombinant DNA.

[0047] In the case of human immunoglobulins, light chains are classified into κ and λ light chains. Reconnections are classified into μ, δ, γ, α, or ε chains, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively.

[0048] As used herein, “IgG” refers to a polypeptide belonging to the class of antibodies encoded by a generally accepted immunoglobulin γ gene. In humans, IgG comprises subclasses or isotypes IgG1, IgG2, IgG3, and IgG4. In mice, IgG comprises IgG1, IgG2a, IgG2b, and IgG3. Pig IgG exhibits six distinct isotypes. In pigs, IgG comprises subclasses or isotypes of IgM, IgD, IgG, IgE, and IgA antibodies, as well as a large number of IgG subclasses (Butler et al., Developmental and Comparative Immunology 30 (2006) 199–221; Butler et al., Developmental and Comparative Immunology 33 (2009) 321–333). Full-length IgG consists of two pairs of identical immunoglobulin chains, each pair having one light chain and one heavy chain. Each light chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, Cγ1 (also known as CH1), Cγ2 (also known as CH2), and Cγ3 (also known as CH3).

[0049] As used in this article, “complement-dependent cytotoxicity” (or CDC) refers to the result of different pathways of complement molecule binding or complement activation, which are associated with Ig and isotype complement.

[0050] As used herein, “antibody-dependent cell-mediated toxicity” (or ADCC) refers to a cell-mediated immune mechanism in which effector cells of the immune system actively lyse target cells that have been bound by a specific antibody. ADCC is mostly mediated by NK cells, but can also be mediated by other immune cells such as neutrophils and eosinophils. Typically, ADCC results from the activation of NK cells. NK cell activation involves the binding of its Fc receptor to the Fc region of IgG, which binds to an antigen present on the surface of the target cell. This interaction induces the release of cytokines and cytotoxic granules via NK cells. To assess the ability of an antibody to induce ADCC, an assay can be performed as described in de Romeuf et al. Br J Haematol. 2008 Mar; 140(6):635-43.

[0051] ADCC and CDC activities can be assessed using methods well known to those skilled in the art.

[0052] As used herein, the term "antigenic determinant" (or epitope) applicable to non-human mammalian polyclonal antibodies refers to a structural component of an antigen molecule, including the antigenic protein and antigenic carbohydrate, responsible for its specific interaction with antibody molecules induced by the same or related antigens. More broadly, the term "antigenic determinant" applicable to non-human mammalian polyclonal antibodies herein also refers to antigen molecules containing multiple epitopes, including conformational motifs that require a sugar motif but represent only a portion of the epitopes and are readily recognized by antibody molecules induced by the same or related antigens. Illustratively, the antigen molecule N-ethylene glycol neuraminic acid (Neu5Gc) may be referred to herein as an "antigenic determinant," although the antigen molecule may exhibit more than one epitope recognized by antibodies induced by Neu5Gc or molecules containing Neu5Gc.

[0053] "T cells" or "T lymphocytes" are a type of white blood cell known as lymphocytes and play a crucial role in cell-mediated immunity. They are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of the T cell receptor (TCR) on their cell surface. They are called T cells because they mature in the thymus.

[0054] B cells, or B lymphocytes, are a type of white blood cell known as lymphocytes, making them an important part of the humoral immune branch of the immune system, particularly the adaptive immune system. B cells are distinguished from other lymphocytes, such as T cells and natural killer cells (NK cells), by the presence of a protein called the B cell receptor (BCR) on their outer surface. This specialized receptor protein allows B cells to bind to specific antigens. The primary function of B cells is to act as antigen-presenting cells (APCs) to deliver antibodies against antigens and, upon activation by antigen-antigen interactions, to develop into memory B cells and plasma cells.

[0055] In blood, "serum" is a component derived from plasma, from which cells (white blood cells and red blood cells) and clotting factors have been removed. Serum includes all proteins and electrolytes that are not involved in blood clotting (coagulation), antibodies, antigens, hormones, and any substances that are ultimately exogenous (such as drugs and microorganisms).

[0056] As used herein, “conventional polyclonal antibody” refers to a polyclonal antibody that does not lack antigenic determinants selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose. In this regard, products commercially available under the names thymosin or venous globulin may be cited in particular.

[0057] As used herein, the term "pathogen" encompasses any substance capable of causing disease. In this context, the term refers to an infectious agent that causes disease or infection in its host, which may be selected from bacteria, parasites, mushrooms, viruses, toxins, venoms, and combinations thereof. More broadly, the term "pathogen" also encompasses antigenic portions (i.e., any molecules derived from said pathogens) derived from such infectious agents, particularly including antigenic proteins or antigenic polysaccharides.

[0058] As used herein, the term "host" encompasses both human and non-human animal organisms. This is why, according to the present invention, a non-human biological pathogen refers to a pathogen that has a harmful effect on a healthy human or animal organism.

[0059] Furthermore, as used herein, the term “non-human biological pathogen targeting human or animal organisms” encompasses all pathogens of a non-human nature that are not naturally present in healthy human or animal organisms, or that are naturally present but in a non-infectious quantity.

[0060] The term also covers saprophytic organisms that may be pathogenic when human or non-human animal organisms are immunosuppressed.

[0061] Preferably, the non-human biological pathogens targeting human or animal organisms are non-human exogenous biological pathogens targeting human or animal organisms.

[0062] As used herein, the term "molecule derived from a non-human pathogen targeting a human or animal organism" broadly refers to any antigen to which a human or animal organism can elicit an immune response. As used herein, the term "molecule" (or antigen) broadly refers to a molecule containing at least one antigenic determinant that an immune response may target. An immune response can be cell-mediated, humoral, or both. Molecules derived from non-human pathogens may be proteins, carbohydrates, lipids, or nucleic acids, or combinations of these biomolecules. Molecules derived from non-human pathogens also include molecules such as polymers.

[0063] As used herein, the term “nucleic acid” encompasses ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), including nucleic acids selected from: single-stranded deoxyribonucleic acid (ssDNA); double-stranded deoxyribonucleic acid (dsDNA); single-stranded ribonucleic acid (ssRNA); double-stranded ribonucleic acid (dsRNA); single-stranded oligodeoxyribonucleic acid (ssODNA); double-stranded oligodeoxyribonucleic acid (ssODNA); single-stranded oligoribonucleic acid (ssORNA); double-stranded oligoribonucleic acid (dsORNA); RNA-DNA duplex.

[0064] In a non-limiting manner, the nucleic acid may be in the following forms: messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe and primer.

[0065] 2. The composition according to the invention

[0066] In order to overcome the disadvantages of conventional polyclonal antibodies against undesirable pathogens or molecules derived from said pathogens, the inventors have conceived of polyclonal antibodies and compositions comprising them that are less immunogenic in human individuals and thus have a reduced ability to induce serious adverse reactions (especially immunogenic complex IC).

[0067] Furthermore, the polyclonal antibodies according to the invention are of particular interest because they possess increased complement-dependent cytotoxicity (or CDC) and antibody-dependent cell-mediated toxicity (or ADCC) activities.

[0068] The present invention relates primarily to polyclonal antibodies against human or animal organisms against at least one non-human biological pathogen or against at least one molecule derived from said pathogen, wherein said polyclonal antibodies do not contain a first antigenic determinant selected from the following: (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose.

[0069] According to a specific embodiment, the polyclonal antibody according to the invention further contains no second antigenic determinant different from the first antigenic determinant, wherein the second antigenic determinant is selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose.

[0070] This invention relates in particular to “non-human” polyclonal antibodies, such as polyclonal antibodies produced by genetically modified non-human mammals as described above.

[0071] More specifically, the present invention relates to non-human mammalian polyclonal antibodies against at least one non-human biological pathogen or against at least one molecule derived from said pathogen, wherein said polyclonal antibodies do not contain:

[0072] – The first antigenic determinant is selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose;

[0073] – The second antigenic determinant, which is different from the first antigenic determinant, is selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose.

[0074] The present invention also relates to compositions comprising at least one polyclonal antibody according to the present invention.

[0075] Compared with polyclonal antibodies and compositions comprising them currently used in the art, it is believed that the polyclonal antibodies and compositions comprising them according to the present invention have reduced immunogenicity in humans.

[0076] It is known in the art that Neu5Gc is immunogenic in humans (Noguchi A. et al., J. Biochem. Tokyo (1995), 117(1): 59-62; Scobie L et al., J. Immunol., 2012). Furthermore, it is known that patients develop severe immune complexes (ICs) after infusion of most animal immunoglobulin carrier antibodies developed targeting the Neu5Gc epitope (Merrick JM et al., Int. Allergy Appl. Immunol., 1978, Vol. 57: 477-480; Aggarwal S. et al., Nat Biotechnol. 2008; 26: 1227–1233; Arnold JN et al., Annu Rev Immunol. 2007; 25: 21–50; Durocher Y et al., Curr Opin Biotechnol. 2009; 20: 700–707; Higgins E et al., Glycoconj. J. 2009).

[0077] It is also known in the art that the enzyme α-1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α-1,3-galactose (α1,3Gal) epitopes (Galα1, 3Galβ1, 4GlcNAc-R), which are major xenoantigens that cause hyperacute rejection in pig-to-human xenotransplantation.

[0078] Therefore, polyclonal antibodies that do not contain (i) N-ethylene glycol neuraminic acid (Neu5Gc) and / or (ii) α-1,3-galactose antigenic determinants, as well as compositions that mechanically contain them, have reduced immunogenicity because these polyclonal antibodies are less immunogenic than conventional polyclonal antibodies. It is then believed that such polyclonal antibodies according to the invention and compositions containing them have reduced performance in inducing various adverse reactions following administration of conventional polyclonal antibody products, including immune complex-associated diseases (ICDs) and serious undesirable adverse reactions such as serum sickness (SSD), including severe forms (e.g., myocarditis, nephropathy) or other immune complex manifestations such as rash, fever, headache, arthritis, or pseudomeningitis syndrome.

[0079] Therefore, as illustrated herein, it is believed that the polyclonal antibodies according to the present invention and compositions comprising them reduce the risk of adverse reactions induced by conventional polyclonal antibodies, and, if they still occur, reduce their severity.

[0080] Furthermore, it is believed that the polyclonal antibodies according to the invention and compositions comprising them exhibit increased complement-dependent cytotoxicity (or CDC) and antibody-dependent cell-mediated toxicity (or ADCC) activities.

[0081] Based on the inventors' knowledge, polyclonal antibodies that do not contain antigenic determinants selected from (i) Neu5Gc and / or (ii) α-1,3-galactose for the purposes disclosed in this specification are unknown in the art.

[0082] In this respect, the polyclonal antibodies according to the invention and compositions comprising them are particularly advantageous because they precisely overcome or reduce the aforementioned adverse reactions caused by conventional polyclonal antibodies, while retaining their immunomodulatory properties and exhibiting lower toxicity at the systemic level in human or animal organisms, preferably humans or animals.

[0083] In other words, the polyclonal antibodies and compositions comprising them according to the invention have significantly lower immunogenicity, and therefore the adverse reactions observed with conventional polyclonal antibodies, particularly the occurrence of immune complex (IC)-related diseases, are expected to be significantly reduced. This beneficial effect of the lower immunogenicity of the polyclonal antibodies of the invention is particularly relevant when these polyclonal antibodies are injected into healthy individuals, i.e., individuals with an effective immune response, such as in the case of prophylactic medical instructions, for example, in prophylactic treatment (prevention) near an infected patient.

[0084] Furthermore, it is known in the art that N-glycosylation of antibodies plays a key role in regulating their effector properties, especially their pro- or anti-inflammatory properties.

[0085] Therefore, it has been identified that sialylation is the addition of N-acetylneuraminic acid (also known as Neu5Ac, NANA, N-acetylcreatine, or sialic acid) to the galactose residues of the N-glycan in the crystallizable fragment (Fc) of an antibody.

[0086] Sialization endows antibodies with particularly interesting anti-inflammatory properties (Dimitrov et al.; Nephrol. Dial. Transplant., 2007.22:1301 and WO 2007 / 117505).

[0087] Therefore, according to embodiments in which the polyclonal antibodies according to the invention do not contain at least the antigenic determinant N-ethylene glycol neuraminic acid (Neu5Gc), the polyclonal antibodies are further advantageous because they exhibit increased affinity for FcγR by allowing more physiologically acquired Fcγ receptors.

[0088] Therefore, the polyclonal antibody according to this embodiment exhibits increased complement-dependent cytotoxicity (or CDC) and antibody-dependent cell-mediated toxicity (or ADCC) activity against the considered non-human biological pathogens.

[0089] Furthermore, since the polyclonal antibody according to the invention lacks an antigenic determinant selected from (i) Neu5Gc and / or (ii) α-1,3-galactose, when administered to human subjects, the polyclonal antibody does not elicit an immune response including the production of anti-Neu5Gc or anti-GAL antibodies, which cause adverse reactions, particularly immune complex-associated diseases (ICDs) and serious undesirable adverse reactions such as serum sickness (SSD), including severe forms (e.g., myocarditis, nephropathy) or other immune complex manifestations such as rash, fever, headache, arthritis, or pseudomeningitis syndrome.

[0090] The methods used to identify or characterize the polyclonal antibodies according to the present invention are common knowledge to those skilled in the art.

[0091] Methods for identifying or characterizing the polyclonal antibodies according to the invention, which can be used by those skilled in the art, include enzyme-linked immunosorbent assays (ELISA) in which anti-Neu5Gc antibodies and / or anti-Gal antibodies are used as detection molecules. Neu5Gc or 1-3Gal specific lectins (e.g., IB4 for 1,3GAL) may also be used; these lectins are known to those skilled in the art.

[0092] As an anti-Neu5Gc antibody for evaluating the absence of the Neu5Gc antigenic determinant, chicken IgY anti-Neu5Gc or lectin and Gc-Free Basic Kit, marketed by Sialix, Inc., can be used.

[0093] As for anti-Gal antibodies used to demonstrate the absence of the α-1,3-galactose antigenic determinant, the experimental protocols disclosed in Jianq-Qiang Wang et al. (J.Am.Chem.Soc.,1999,121:8181) or those sold by Sigma-Aldrich under the name WH0051083M1 Sigma can be considered.

[0094] In ELISA methods in which specific anti-Neu5Gc and / or anti-Gal antibodies or lectins are immobilized in the wells of a microtiter plate, only antibodies selected from the antigenic determinants (i) N-ethylene glycol neuraminic acid (Neu5Gc) and / or (ii) α-1,3-galactose form complexes with the anti-Neu5Gc and / or anti-Gal antibodies, thereby maintaining binding to the wells. When using such an ELISA method, the polyclonal antibodies according to the invention are those that do not contain one or more antigenic determinants selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and / or (ii) α-1,3-galactose, and therefore do not form complexes with (i) anti-Neu5Gc antibodies, (ii) anti-Gal antibodies, or (iii) both anti-Neu5Gc antibodies and anti-Gal antibodies.

[0095] Alternatively, the polyclonal antibody to be studied according to the invention can be bound to a plate and then identified using an identification reagent. As an identification reagent, a lectin specific to Neu5Gc or 1,3Gal or anti-Neu5Gc and / or anti-Gal antibodies can be used.

[0096] The present invention also relates to a method for producing polyclonal antibodies or compositions according to the present invention as defined above, comprising the following steps:

[0097] a) Provide a non-human mammal lacking a genetic alteration selected from the following first genes: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-1,3-galactosyltransferase;

[0098] b) Inoculate the genetically altered nonhuman mammal with at least one nonhuman pathogen or at least one pathogen derived from said pathogen for human or nonhuman animal organisms;

[0099] c) Collect antibodies contained in the bodily fluids of the non-human mammal with the genetic alterations described in step b).

[0100] Preferably, step b) is carried out by implementing at least one molecule derived from at least one specific non-human biological pathogen targeting human or non-human animal organisms.

[0101] In some embodiments, the compositions according to the invention can be prepared by mixing the polyclonal antibody collected in step c) of the above method with one or more adjuvants and / or one or more pharmaceutically acceptable excipients, such as physiologically acceptable carriers, excipients or stabilizers.

[0102] In some embodiments, the polyclonal antibody is purified prior to use in the composition according to the invention.

[0103] In some embodiments, the polyclonal antibody composition according to the invention is in liquid form.

[0104] In some embodiments, the polyclonal antibody composition according to the invention is in solid form, including lyophilized form.

[0105] The compositions of the present invention can be formulated according to standard methods, such as those described in Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins; Twenty first Edition, 2005).

[0106] The compositions of the present invention may further comprise at least one adjuvant, a pharmaceutically acceptable excipient, or a combination thereof.

[0107] As used herein, the term "adjuvant" refers to any substance that enhances the immune response to an antigen with which it is mixed. Adjuvants that may be used in this invention include, but are not limited to: Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfactants such as lysophosphatidylcholine, complex polyols, polyanionic peptides, oil emulsions, keyhole hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guérin) and Corynebacterium parvum are also included.

[0108] The compositions according to the invention may use any known adjuvant, including oil-based adjuvants such as Freund's complete and incomplete adjuvants, mycoester-based adjuvants (e.g., trehalose dimycoester), bacterial lipopolysaccharides (LPS), peptidoglycans (i.e., cell wall plasmin, peptidoglycan, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), Biostim.TM. (e.g., 01K2), "Iscom" as in EP 109 942, EP 180 564, and EP 231 039, aluminum hydroxide, saponins, DEAE-glucan, neutral oils (e.g., miglyol), vegetable oils (e.g., peanut oil), liposomes, Pluronic.RTM. polyols, and Ribi adjuvant systems (see, for example, GB-A-2 189). 141), or interleukins, especially those that stimulate cell-mediated immunity. Alternatively, the adjuvant may consist of an extract of *Amycolata*, a genus of bacteria in the order Actinomycetes, as described in U.S. Patent No. 4,877,612. Suitable adjuvant mixtures are also commercially available. The adjuvant used depends in part on the receiving organism. The amount of adjuvant administered will depend on the type and size of the animal. The optimal dosage can be readily determined using conventional methods.

[0109] Suitable adjuvants include, but are not limited to: surfactants such as hexadecylamine, octadecylamine, lysophosphatidylcholine, dimethyloctadecylammonium bromide, N,N-dioctyl-N'-N-bis(2-hydroxyethyl-propanediamine), methoxyhexadecyl-glycerol, and polyether polyols; polyanionic agents such as pyran, dextran sulfate, polyIC, polyacrylic acid, and carbomer; and peptides such as muramyl dipeptide, MPL, dimethylglycine, tafocin, oil emulsions, alum, and mixtures thereof. Other potential adjuvants include B-peptide subunits of E. coli heat-labile toxin or cholera toxin. (McGhee, JR et al., “On vaccine development” Sem. Hematol., 30:3-15 (1993).)

[0110] Saponins have long been known to possess adjuvant properties, particularly their ability to enhance immunogenic antibody titers. As used herein, the term "saponin" refers to a group of plant-derived surfactant glycosides consisting of a hydrophilic region (typically several sugar chains) associated with a hydrophobic region of a steroid or triterpenoid structure. Although saponins are available from a wide variety of sources, those with useful adjuvant activity are derived from the South American tree *Gnaphalium affine* (Molina). Saponins from this source are used to isolate a "uniform" fraction known as "QuilA" (Dalsgaard, K., (1974), Arch. Gesamte Virusforsch. 44:243).

[0111] In some embodiments of the compositions according to the invention, the compositions may further comprise one or more charged inorganic carriers as pharmaceutical excipients. Examples of suitable charged organic carriers include, but are not limited to: saponins, saponin complexes, any one or more components of immunostimulatory complexes of saponins, cholesterol and lipids (e.g., saponin components and / or phospholipid components) called ISCMATRIX.TM., liposomes, or oil-in-water emulsions. (The composition and preparation of ISCMATRIX.TM. are described in detail in PCT / SE86 / 0048, Australian Patent Nos. 558258 and 632067, and EP 0 180 564, the disclosures of which are incorporated herein by reference).

[0112] Other adjuvants can be found in the book by Vogel et al. (Vogel FR, Powell MF and Alving CR, "Acompendium of vaccine adjuvants and excipients"); 2 <nd>Edition; Vogel FR and Powell MF, 1995, "A summary compendium of vaccine adjuvants and excipients. In: Powell MF, Newman MJ eds. "Vaccine design: the subunit and adjuvant approach". New York: Plenum publishing, 1995: 141-228).

[0113] Pharmaceutically acceptable excipients are available, particularly as described in the American Pharmaceutical Association's Handbook of Pharmaceutical Excipients (Pharmaceutical Press; 6th revised edition, 2009).

[0114] For the treatment of patients in need as described above, a therapeutically effective dose of the polyclonal antibody according to the invention or a composition comprising such an antibody may be administered.

[0115] "Therapeutic effective dose" refers to the dose that produces the effect required for treatment. The exact dose will depend, in particular, on the therapeutic purpose, the nature of the disease / infection, the severity of the disease / infection, and will be determined by a person skilled in the art using known techniques.

[0116] The dosage can be 0.001-100 mg or more of the polyclonal antibody according to the invention per kg body weight (mg / kg) or higher, for example 0.1, 1.0, 10 or 50 mg / kg body weight, preferably 1-10 mg / kg. The dosage and frequency of administration can be varied depending on the nature of the disease / infection, the severity of the disease / infection, the host response, and the frequency of injections due to better tolerability. Dosage and regimen may vary depending on whether the treatment or prophylactic use is desired.

[0117] Furthermore, any routine procedure can be followed after any injection to prevent and / or avoid allergic reactions.

[0118] Furthermore, the polyclonal antibodies or compositions comprising them according to the invention can be injected via a large peripheral access or, if possible, via a central catheter.

[0119] It may be necessary in the art to adjust for protein degradation, systemic and local delivery, as well as age, weight, general health, sex, diet, timing of administration, possible allergies, drug interactions and severity of illness, which can be readily determined by those skilled in the art through routine experiments.

[0120] The compositions of the present invention can be administered in various ways, including but not limited to: oral, subcutaneous, intravenous, parenteral, intranasal, intra-arterial, intraocular, rectal, vaginal, percutaneous, topical (e.g., gel), intraperitoneal, intramuscular, intrapulmonary, or intrathecal administration.

[0121] The compositions of the present invention can be applied following the Besredka method.

[0122] The compositions of the present invention can be administered concurrently with other therapeutic agents, i.e., the therapeutic agents described herein can be co-administered with other therapies or therapeutic agents, including, for example, small molecules, other biological substances, radiotherapy, surgery, etc.

[0123] In the most preferred embodiment, the composition according to the invention is in a form suitable for intravenous administration.

[0124] According to one specific embodiment, the composition according to the invention may further comprise at least one anti-inflammatory drug, such as a glucocorticoid.

[0125] 3. A method for producing polyclonal antibodies (and compositions comprising them) according to the present invention.

[0126] As described above, a method for producing polyclonal antibodies or compositions comprising them according to the invention as defined above includes the following steps:

[0127] a) Provide a non-human mammal lacking a genetic alteration selected from the following first genes: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase;

[0128] b) Inoculating the genetically altered nonhuman mammal with at least one nonhuman pathogen or at least one pathogen derived from said pathogen; and

[0129] c) Collect antibodies contained in the bodily fluids of the non-human mammal with the genetic alterations described in step b).

[0130] According to a specific implementation scheme, the genetically altered non-human mammal may further lack a second gene different from the first gene, the second gene being selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase.

[0131] Therefore, the present invention more specifically relates to a method for producing polyclonal antibodies or compositions comprising them according to the invention as defined above, comprising the following steps:

[0132] a) Provide a non-human mammal lacking the following genetic alterations:

[0133] --Selected from the following first genes (i) encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) encoding a functional α-(1,3)-galactosyltransferase; and

[0134] --The second gene, which is different from the first gene, is selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase;

[0135] b) Inoculating the genetically altered nonhuman mammal with at least one nonhuman pathogen or at least one pathogen derived from said pathogen; and

[0136] c) Collect antibodies contained in the body fluids of the genetically altered non-human mammals described in step b).

[0137] Preferably, the method of the present invention further includes step d) purifying the polyclonal antibody, or a specific type or specific isotype of the polyclonal antibody, from the body fluid.

[0138] When produced by non-human mammals with the genetic alterations described herein, the polyclonal antibodies of the present invention may also be referred to as "non-human mammal" polyclonal antibodies.

[0139] Therefore, when produced by non-human mammals with the genetic alterations described herein, the polyclonal antibodies of the present invention can be natural / endogenous non-human polyclonal antibodies.

[0140] Alternatively, when a polyclonal antibody (e.g., IgG) is produced by a non-human mammal (e.g., a cow) with genetic alterations described herein and further expresses an exogenous immunoglobulin gene (e.g., a human immunoglobulin gene), the polyclonal antibody may be a non-natural, non-human polyclonal antibody.

[0141] 3.1 Steps for providing genetically altered non-human mammals a)

[0142] To prepare the polyclonal antibody according to the invention, a first step is performed: a) providing a non-human transgenic mammal lacking a genetically modified gene selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and / or (ii) a gene encoding a functional α-(1,3)-galactosyltransferase.

[0143] Preferably, the genetically altered nonhuman mammal is a CMAH and / or GGTA1 knockout nonhuman transgenic mammal (or a CMAH and / or GGTA1 KO nonhuman mammal), including CMAH and GGTA1 double knockout nonhuman transgenic mammals.

[0144] As used in this article, "knockout (KO) nonhuman transgenic mammal" refers to a nonhuman transgenic mammal in which the alleles of one or more target genes have been altered, for example, through homologous recombination or other insertions or deletions.

[0145] In some implementations, the gene is disrupted. A "disrupted gene" refers to a portion of a gene's coding that is altered to affect the transcription and / or translation of that segment, for example, by knockout techniques or by inserting an additional gene to transmit a desired protein or by inserting a regulatory sequence that controls the transcription of an existing sequence, thus rendering that segment of coding unreadable.

[0146] In some embodiments of the present invention, all cells of a non-human transgenic mammal include disruptive genes.

[0147] In some implementations, knockout non-human transgenic mammals are non-human transgenic mammals in which the alleles of one or more target genes have lost their function.

[0148] In some embodiments, both alleles of the target gene lose function. Such embodiments include what are commonly referred to as "gene knockout," "gene knock-in," and any other modifications to one or more natural alleles of the gene that render the natural target gene nonfunctional. This non-human transgenic mammal can be used as a source for producing the compositions according to the invention.

[0149] Methods for obtaining genetic alterations in non-human mammals lacking genes selected from: (i) genes encoding functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and / or (ii) genes encoding functional α-(1,3)-galactosyltransferase.

[0150] Non-human mammals with genetic alterations that lack the gene encoding functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase are called CMAH KO non-human mammals.

[0151] Non-human mammals with genetic alterations that lack the gene encoding a functional α-(1,3)-galactosyltransferase are called GAL KO non-human mammals.

[0152] Methods for obtaining CMAH knockout non-human transgenic mammals are particularly described in WO2006 / 133356, which more specifically discloses the production of animal products for human use that do not contain 5-ethylene glycol neuraminic acid (Neu5Gc), comprising the steps of: preparing a genetically altered non-human mammal lacking the functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH) gene; and extracting at least one animal product from said genetically altered non-human mammal.

[0153] The methods used to obtain GAL knockout non-human transgenic mammals are common knowledge to those skilled in the art (Cooper DK et al., Genetically engineered pigs, Lancet 1993, 342:682; Lai L et al., Science 2002, 295:1089; Sachs DH et al., Current Opinion in Organ Transplantation, 2009, 14:148–153).

[0154] Methods for obtaining GAL knockout non-human transgenic mammals are specifically described in US 7,547,816.

[0155] According to one specific embodiment, to obtain the polyclonal antibodies according to the invention and compositions comprising them, namely polyclonal antibodies against non-human biological pathogens or against at least one molecule derived from said pathogens, wherein said polyclonal antibodies do not contain antigenic determinants selected from: (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose, involving the implementation of genetic alterations in non-human mammals lacking genes selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase.

[0156] In other words, the specific genetically altered non-human mammals are CMAH and GLA double knockout (KO) non-human transgenic mammals.

[0157] The protocol for obtaining this specific CMAH and GAL double knockout non-human transgenic mammal is described in Lutz AL et al. (Xenotransplantation, 2013; 20(1):27-35) or Conchon S. et al. (Xenotransplantation; special issue International Xenotransplantation Association IXA 2013, 2013, Vol.20, Issue 5).

[0158] As non-human transgenic mammals that can be used for genetic alteration in this invention, Ovidae, Bovidae, Suidae, Leporidae, and Equidae are particularly mentioned.

[0159] Preferably, the genetically altered non-human transgenic mammal can be a mouse, rat, guinea pig, rabbit, goat, sheep, llama, or pig, with pig being the preferred species.

[0160] In fact, pigs are preferred for obtaining the polyclonal antibodies according to the invention because they are of particular interest from an industrial point of view.

[0161] In fact, pigs offer several advantages, particularly compared to rabbits, in terms of the volume of immune serum, and thus the volume of target polyclonal antibodies, allowing for the collection of polyclonal antibodies in proportion to the animal's body weight (30 times better).

[0162] Furthermore, the procedure for legally harvesting serum is significantly accelerated because no pigs need to be euthanized at the time of serum harvesting.

[0163] In fact, 10% of the animal's blood volume can be collected each month.

[0164] For all these reasons, obtaining the composition according to the invention from genetically modified transgenic pigs is particularly economical.

[0165] 3.2 Immunization of genetically altered non-human mammals with non-human pathogens that target human or non-human animal organisms. Animal steps b)

[0166] Once a non-human transgenic mammal lacking genetic alterations selected from the following genes: i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase is obtained, an injection of a solution specifically containing at least one non-human biological pathogen or at least one molecule derived from said pathogen will produce protection against human or non-human animal organisms.

[0167] Preferably, the non-human biological pathogen in step b) can be selected from bacteria, parasites, mushrooms, viruses, toxins, venoms and combinations thereof, or at least one molecule derived from said pathogen, more specifically those that induce at least one of the specific serious infections described below.

[0168] For example, methods for obtaining molecules that, in particular, contain a specific non-human biological pathogen or at least one molecule derived from said pathogen, in relation to a particular relevant disease / infection, are common knowledge to those skilled in the art.

[0169] Preferably, the pathogenicity of the non-human biological pathogens under consideration is reduced.

[0170] For example, in an embodiment where the non-human biological pathogen is a virus, it is preferable to inject a weakened (or killed) virus or only an extract of the virus into a non-human transgenic mammal with the aforementioned genetic alterations.

[0171] Methods for obtaining attenuated pathogens, especially attenuated viruses, are common knowledge to those skilled in the art.

[0172] In other words, methods for obtaining molecules derived from specific non-human pathogens that are targeted at human or non-human animal organisms are common knowledge to those skilled in the art.

[0173] In this regard, viral culture supernatant, recombinant viruses, lysates of cells transfected or transduced with viruses, or components thereof can be used. Furthermore, in the case of toxins, the toxins can be inactivated chemically or by heat (such harmless toxins are often called "toxin-like substances").

[0174] The acquisition of good immune levels in non-human transgenic mammals relative to T cells also applies to protocols specifically described in EP 0335 804, which are similar to those for specific non-human biological pathogens or molecules derived from said pathogens targeting human or non-human animal organisms.

[0175] This scheme can specifically consist of: according to known methods, by repeatedly administering to a human or animal organism, preferably to a human organism, at least one specific non-human pathogen, preferably a unique specific non-human pathogen, or at least one molecularly immunized animal derived from said pathogen, such as a rabbit, horse, or pig, preferably a pig.

[0176] For example, it can be administered several times intravenously or subcutaneously. Preferably, the first administration is subcutaneous, with or without adjuvant, and the second administration is intravenous, each time 10 mg. 6 -10 9 Cells were administered at intervals of at least one week. Approximately two weeks after the last immunization, serum was collected from the immunized animals and isolated using known methods.

[0177] Genetically modified non-human transgenic mammals will produce antibodies against specific non-human pathogens or molecules derived from said pathogens, and depending on the nature of the genetically modified non-human transgenic mammal under consideration, said specific antibodies do not contain the antigenic determinant Neu5Gc and / or α-1,3-Gal.

[0178] 3.3 Step c) involves collecting antibodies from the bodily fluids of non-human mammals with genetic alterations as described in step b).

[0179] Then, a portion of the body fluids from the genetically altered non-human transgenic mammal is removed, and antibodies, i.e., target antibodies, are collected from it.

[0180] According to a specific implementation, the body fluid may be selected from plasma and serum.

[0181] Methods for obtaining bodily fluids, more specifically plasma and serum, are common knowledge to those skilled in the art.

[0182] 3.4 Optional step d) for purifying antibodies from body fluids in step c).

[0183] According to a preferred embodiment, as described above, the method according to the invention may further include step d) of purifying antibodies from the body fluid.

[0184] The purification step d) is advantageous because it is particularly effective in overcoming potential undesirable side effects associated with various cellular contaminants present in bodily fluids, which may be involved in the formation of corresponding contaminating antibodies by immunization of non-human mammals.

[0185] The purification step d) is advantageous because it allows for the acquisition of a composition with the desired purity.

[0186] The purification step d) is common knowledge to those skilled in the art. All possible modifications to any conventional purification protocol are also common knowledge to those skilled in the art.

[0187] As a suitable method for obtaining the polyclonal antibody according to the invention, the following methods may be specifically mentioned: fractional precipitation with ethanol, ammonium sulfate, rivanol, polyethylene glycol or octanoic acid; or by ion exchange column method; other methods may involve affinity columns for proteins A or G. The resulting antibody can then be routinely processed for intravenous administration, for example by enzymatic cleavage with plasmin, papain or pepsin.

[0188] In this regard, more specific reference can be made to the scheme used in Example 3 of EP 0 335 804, which involves ion exchange chromatography of DEAE cellulose.

[0189] According to other embodiments, the polyclonal antibody and the composition according to the invention can be a polyclonal antibody and composition in which the antibody obtained in step c) of the above method is separated from other cellular components that are not antibodies (particularly including neutrophils, monocytes, erythrocytes and platelets).

[0190] According to these other embodiments, the polyclonal antibody and the composition according to the invention can be a purified polyclonal antibody originally present in serum and a composition containing the purified polyclonal antibody, said purified polyclonal antibody being substantially free of serum protein components, or even the polyclonal antibody being substantially free of any substances originally present in the serum used as a starting product.

[0191] As suitable methods for purifying these target polyclonal antibodies, examples include those methods that purify antibodies using affinity supports against protein G or protein A conjugated with antigens, such as those sold by companies like ProteoGenix, CellBiolabs, Inc., or Clini Sciences, or disclosed in EP 1 601 697, JP 7 155 194, or US 6,870,034.

[0192] It may also be mentioned that the immunoaffinity purification of polyclonal antibodies specific to at least one particular pathogen or at least one of its components.

[0193] Purification may also involve specific types or specific isotypes of polyclonal antibodies according to the invention.

[0194] Also mentioned are affinity supports for selectively immobilizing target polyclonal antibodies from blood flow, comprising a solid support having immobilizing aptamers that specifically bind to the target antibody from blood flow. This method is specifically disclosed in WO 2010 / 094901.

[0195] Alternatively, with regard to the use of polyclonal antibodies obtained from genetically modified nonhuman mammals as described above, the present invention also covers polyclonal antibodies obtained after inoculating wild nonhuman mammals with at least one nonhuman biological pathogen or at least one molecular immunization derived from said pathogen.

[0196] The term "wild nonhuman mammal" has the opposite meaning to genetically altered nonhuman mammals. In other words, "wild nonhuman mammal" refers to a nonhuman mammal that does not lack at least one gene selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH) and / or (ii) a gene encoding a functional α-(1,3)-galactosyltransferase.

[0197] In this regard, to maintain the purpose of the present invention, the polyclonal antibodies obtained from this wild non-human mammal must be desialylated by appropriate biochemical (especially enzymatic) treatment.

[0198] In other words, the polyclonal antibody according to the present invention can be obtained from: (1) a non-human mammal with the above-mentioned genetic alteration, or (2) a polyclonal antibody obtained from a wild non-human mammal obtained through biochemical (especially enzymatic) treatment, wherein the wild non-human mammal has been inoculated with at least one non-human biological pathogen against a human or non-human animal organism, or at least one molecular immunization derived from said pathogen.

[0199] 4. Medical uses according to the present invention

[0200] As described above, according to one aspect of the invention, the invention relates to the use of genetically modified non-human mammals in the production of compositions comprising polyclonal antibodies, said genetically modified non-human mammals lacking a first gene selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH) and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase, said polyclonal antibody targeting a human or non-human animal organism, preferably targeting at least one non-human pathogen of a human organism, or at least one molecule derived from said pathogen.

[0201] According to a specific implementation, the genetically altered non-human mammal may further lack a second gene different from the first gene, the second gene being selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH) and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase.

[0202] More specifically, the present invention relates to the use of genetically modified non-human mammals in the production of polyclonal antibodies or compositions comprising them according to the invention and as defined above, wherein the genetically modified non-human mammals lack:

[0203] – The first gene is selected from: (i) the gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH) and (ii) the gene encoding a functional α-(1,3)-galactosyltransferase; and

[0204] – Unlike the first gene, the second gene is selected from: (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH) and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase.

[0205] In particular, the absence of anti-Neu5Gc antibody in a sample can be assessed according to the dosing method described by Padler-Karavani V et al. (PLoS One. 2013; 8(3):e58443).

[0206] According to another specific embodiment, the composition of the present invention may be at least one non-human biological pathogen targeting a human or non-human animal organism, preferably a human organism, or at least a serum derived from a molecule of said pathogen.

[0207] The present invention also relates to the use of the polyclonal antibodies described in the entirety of this application or compositions comprising them as pharmaceuticals.

[0208] Therefore, it can be understood from the above that the present invention also relates to the use of polyclonal antibodies or compositions comprising them according to the present invention for the preparation of pharmaceuticals.

[0209] This invention relates to the use of the polyclonal antibodies or compositions comprising them described in the entirety of this application for the prevention and / or treatment of serious infections.

[0210] The following is a non-exhaustive list of serious infections: Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), Amoebiasis, Alfa disease, Anthrax, Hemolytic Lactobacillus infections, Argentine hemorrhagic fever, Ascariasis, Aspergillus infection, Astrovirus infection, Brassicosis, Bacillus cereus infection, Bacteroides pneumonia, Bacterial vaginosis (BV), Bacteroides infection, Balanopsis infection, Ascaris infection, BK virus infection, Trichophyton mentagrophytes, Blastomycosis, Bolivian hemorrhagic fever, Leptospirosis, Botulinum toxin infection (and infantile botulism), Brassic hemorrhagic fever, Brucellosis, Plague, Burkholderia infection, brucellosis, and cucurbitacinosis. Viral infections (norovirus and poxvirus), Campylobacteriosis, Candidiasis (candidiasis; thrush), Cat bite disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chikungunya, Chlamydia, Chlamydia pneumoniae infection (Taiwan Acute Respiratory Infection or TWAR), Cholera, Chromocytosis, Methioninosis, Clostridium difficile infection, Coccidioidomycosis, Colorado tick fever (CTF), Common cold (acute viral rhinitis, acute rhinitis), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), Cryptococcosis, Cryptosporidiosis, Cutaneous larvae (CLM), Cyclosporidiosis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Binuclear amoebiasis, Diphtheria, Trichomoniasis, Draconiariasis, Ebola hemorrhagic fever, Echinococcosis, Helminthiasis, Enterobacterial infection (pinworm infection), Enterococcal infection, Enterovirus infection (especially Enterovirus 71), Epidemic typhus, Systemic lupus erythematosus (fifth disease), Roseola infantum (sixth disease), Fasciolopsis buski, Fasciolopsis buski, Fatal familial insomnia (FFI), Filariasis, Clostridium perfringens food poisoning, Free-living amoeba infection, Fusobacterium infection, Gas gangrene (Clocas necrophorum), Lystilosis, Jetzmann-Stokes-Schink syndrome (GSS), Giardiasis, Meredithiasis, Gnathostoma spinigerum infection, Gonorrhea, Granuloma inguinale (urinary tract infection), Group A Streptococcus infection, Group B Streptococcus infection, Haemophilus influenzae Bacillus infection, hand-foot-mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS), cardiogenic viral disease, Helicobacter pylori infection, hemolytic uremic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex virus infection, histoplasmosis, hookworm infection, human bocavirus infection, human Evans elektriosis, human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human mononucleotic elektriosis, human papillomavirus (HPV) infection, human parainfluenza virus infection, hymenolepis taeniasis, Epstein-Barr virus infectious mononucleosis (Mono), influenza (flu), isosporidiosis,Kawasaki disease, keratitis, *Gynostemma pentaphyllum* infection, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease (Pontiac fever), leprosy, leptospirosis, Listeria infection, Lyme disease (Lyme spirochete infection), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East Respiratory Syndrome (MERS), glanders (Whitmore's disease), meningitis, meningococcal infection, metaclonorchiasis, microsporidiosis, molluscum contagiosum (MC), monkeypox, mumps, mouse typhus (endemic typhus), mycoplasmal pneumonia, mycoplasma gondii, myiasis Neonatal conjunctivitis (neonatal ophthalmitis), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardia, Onchocerciasis (river blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurella multocida, Head lice, Body lice, Pubic lice, Pelvic inflammatory disease (PID), Pertussis, Plague, Pneumococcal infection, Pneumocystis pneumonia (PCP), Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis (PAM), Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rabies virus, Respiratory syncytial disease Viral infections, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsial pox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infection, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, schistosomiasis, sepsis, Shigella (bacterial dysentery), herpes zoster (shingles), smallpox (pox), sporotrichosis, staphylococcal food poisoning, Staphylococcus aureus infection, strongyloides stercoralis infection, subacute sclerosing panencephalitis, syphilis, tapeworm infection, tetanus, tinea barbae (ringworm), tinea capitis (tinea capitis), tinea corporis (tinea corporis), tinea cruris (tinea cruris). Tinea manuum (hand fungus), black tinea, tinea pedis (athlete's foot), onychomycosis (nail fungus), tinea versicolor, toxocariasis (ocular larval migration (OLM)), toxocariasis (visceral larval migration (VLM)), trachoma, trichinosis, trichinosis, trichomoniasis, whipworm infection, tuberculosis, tularemia, ureaplasma infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white sarcoidosis (tinea versicolor), plague, Yersinia pestis infection, Yersinia pestis, yellow fever, zygomycosis, or at least one serious infection induced by at least one antibiotic-resistant pathogen.

[0211] Therefore, this invention relates to the use of the polyclonal antibodies or compositions comprising them described in the entirety of this application for the prevention and / or treatment of Ebola hemorrhagic fever.

[0212] The present invention also relates to the use of the polyclonal antibodies described in the entirety of this application or compositions comprising them for serum prophylaxis and / or serum therapy.

[0213] As used herein, the term "serological prophylaxis" refers to the injection of immune serum obtained from an immunized animal, preferably an immunized non-human mammal, for the purpose of preventing at least one infectious disease in a human or non-human animal organism, particularly a human, said infectious disease being associated with at least one non-human pathogen.

[0214] As used herein, the term "serum therapy" refers to the injection of immune serum obtained from an immunized animal, preferably an immunized non-human mammal, for the purpose of treating at least one infectious disease in a human or non-human animal organism, particularly a human, said infectious disease being associated with at least one non-human pathogen.

[0215] Therefore, the present invention relates to the polyclonal antibodies or compositions comprising them described in the entirety of this application for the prevention and / or treatment of serious infections caused by pathogens selected from toxins, venoms, and combinations thereof.

[0216] The toxins and venoms may be selected from: botulinum toxin, tetanus toxin, shiga toxin, diphtheria toxin, pertussis toxin, neurotoxin, myotoxin, hemotoxin, cytotoxin, plant-derived toxins, fungal toxins, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emeticine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, scintillans, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, ricin, CC-1065, toxins and animal venoms derived from venomous animals; and their analogues or homologs.

[0217] Examples of animal venom include snake venom, spider venom, bee venom, fish venom, jellyfish venom, scorpion venom, snail venom; including viper venom, cobra venom, rattlesnake venom, Naja venom; and their analogues or homologs.

[0218] The present invention also relates to the use of the polyclonal antibodies or compositions comprising them described throughout this application for reducing and / or inhibiting immune complex-associated diseases (ICDs) and serious undesirable adverse reactions, such as serum sickness (SSD), including severe forms (e.g., myocarditis, nephropathy) or other immune complex manifestations, such as rash, fever, headache, arthritis or pseudomeningitis syndrome and immune complex manifestations induced by administration of an antibody comprising at least one antigenic determinant selected from: (i) N-ethylene glycol neuraminic acid (Neu5Gc) or (ii) α-1,3-galactose, preferably comprising at least both antigenic determinants (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose. Detailed Implementation

[0219] In all the following embodiments, the pigs used had an unmodified diet.

[0220] Example 1: A scheme for preparing polyclonal antibodies against Ebola hemorrhagic fever virus from double GAL / CMAH KO pigs

[0221] First, the double GAL / CMAH KO pigs used are disclosed in Lutz AL et al. (Xenotransplantation, 2013; 20(1):27-35) or in Conchon S. et al. (Xenotransplantation; special issue International Xenotransplantation Association IXA 2013, 2013, Vol.20, Issue 5).

[0222] 1) Immunization protocol for double GAL / CMAH KO pigs with Ebola hemorrhagic fever virus. Immunization of double GAL / CMAH KO pigs by administering soluble forms of these proteins released from cells expressing Ebola virus glycoproteins (GP) into the culture medium, as described by Lutz AL et al. (Xenotransplantation, 2013; 20(1):27-35) or Conchon S. et al. (Xenotransplantation; special issue International Xenotransplantation Association IXA 2013, 2013, Vol.20, Issue 5) (e.g., Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses, Nature Medecine, 2005, Jones, SM et al., or Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles, PNAS, 1996, Schnell MJ et al., or A Marzi, PLoS ONE, 7:e36192).

[0223] Therefore, immunization can be performed by administering 700 μg of Ebola virus glycoprotein.

[0224] In this embodiment, the viral antigen preparation is preferably free of Neu5Gc. The viral antigen preparation (also known as polyclonal hyperimmune serum) conforms to all legal recommendations regarding good manufacturing practices at the antigen preparation level for double-KO (CMAH and GT1) animals used for immunization and for all steps mentioned below:

[0225] a) Administer a first subcutaneous injection of a solution containing the above-mentioned Ebola virus glycoprotein in a soluble form and aluminum hydroxide as an adjuvant, followed by two or more intravenous injections (i.e., on day 14 and day 21).

[0226] b) Optionally, on the 5th day, at 10 7 -10 8 One pathogen / 10 doses are administered intravenously with 10 doses of BCG or any type of adjuvant.

[0227] c) Collect serum by bleeding on or after day 35. Collect approximately 100 ml of porcine serum.

[0228] Serum analysis (ELISA plates coated with Ebola EP and neutralization assays of Vero cells infected with Ebola GP-transfected VSV) showed titers of 1 / 10000 and 1 / 100, respectively.

[0229] Therefore, immunization can be performed as follows: a first subcutaneous injection of a solution containing the above-mentioned Ebola virus glycoprotein in a soluble form and aluminum hydroxide as an adjuvant, followed by four intravenous injections on days 14, 29, 44 and 78.

[0230] Therefore, serum can be collected by bloodletting 40 days after the last boost.

[0231] 2) A protocol for obtaining polyclonal (IgG) antibodies against administered Ebola hemorrhagic fever virus from double GAL / CMAH KO pigs.

[0232] The following provides a protocol for obtaining the polyclonal antibodies (i.e., IgG, IgM, etc.) described herein, comprising the following steps:

[0233] a) The above-mentioned porcine serum was subjected to chromatography on a Whatman cellulose DEAE plate, and then eluted with sodium dihydrogen phosphate buffer at pH 8 (1.5 g / L).

[0234] b) The obtained γ-globulin solution was purified by double precipitation with 180 g / L sodium sulfate followed by 170 g / L sodium sulfate at pH 7. The precipitate was then dissolved in 0.3 M glycine at pH 7 to obtain a volume equal to the initial volume.

[0235] Alternatively, the above purification steps can be performed via an octanoic acid precipitation stage, followed by protein A. The purification process can then be completed using an ion-exchange column. This alternative purification method yields good polyclonal IgG, with a yield of 51% and a purity of 95%.

[0236] c) Human erythrocyte particles in the solution were adsorbed twice with Hema (each time the volume of adsorbed particles was approximately equal to the volume of crude serum) to reduce the proportion of hemagglutinin. The solution was then precipitated again with sodium sulfate to remove hemoglobin. The precipitate was dissolved in 0.3M glycine buffer and percolated with a final solution of glycine 10 g / L, NaCl 2 g / L, and sorbitol 10 g / L. Protein was added to a final concentration of 5 g / L, and then the solution was lyophilized.

[0237] Measure the ELISA titers of double-KO porcine anti-Ebola EP and neutralizing anti-Ebola virus antibodies (as described above) and prevent death in guinea pigs infected with recombinant EBOV applicable at 1,000 LD50. Figure 1 .

[0238] Female guinea pigs (Hartley strain), weighing approximately 200g (Charles River), were divided into four groups. Note that the product to be injected had been pre-tested by intraperitoneal injection in two groups of five mice: one group was injected with 200 mg for 4 days, and the second group was injected with 2 mg for 4 days, with no obvious toxicity.

[0239] Each group consists of five guinea pigs, as shown below:

[0240]

[0241]

[0242] Following the challenge, monitor clinical signs of infection and body weight for two weeks. On day 3, collect a blood sample (1 ml, if possible) to measure viral load and porcine IgG concentration. Continue monitoring survivors up to 15 days post-infection. At the end of the experiment, or on the day of animal death (if applicable), analyze blood samples and organs (spleen, liver, etc.).

[0243] Comment

[0244] The results provide the following evidence (see Figure 3 Animals belonging to the Ebola virus-immune IgG D0 group (see above) showed statistically significantly lower viral replication (viral load) on day 3 compared to animals belonging to the Mock or non-immune IgG group.

[0245] The results also provide the following evidence (see Figure 4 Animals belonging to the Ebola virus-immune group survived longer than animals belonging to the Ebola virus-non-immune IgG D0 group.

[0246] Polyclonal antibodies against the proposed nonhuman pathogen (i.e., Ebola hemorrhagic fever virus) obtained from double GAL / CMAH KO pigs are of particular interest because they are significantly less immunogenic and more cytotoxic in humans (especially in the case of CDC) compared to conventional polyclonal antibodies against the same proposed nonhuman pathogen.

[0247] Therefore, these polyclonal antibodies allow for the effective treatment or prevention of disease / infection caused by Ebola hemorrhagic fever virus, while reducing the undesirable side effects that patients may experience consecutively with routine polyclonal antibody injections, such as immune complex (IC)-related illnesses and / or serum sickness. Furthermore, these polyclonal antibodies have been observed to exhibit very significant and interesting activity against Ebola hemorrhagic fever virus, due to increased ADCC and CDC.

[0248] All these advantages will inevitably increase patient well-being, especially if the disease / infection to be treated already involves severe symptoms.

[0249] Example 2: Measurement of anti-Neu5Gc antibody (or anti-Neu5Gc IgG) in double GAL.CMAH KO pigs

[0250] The ELISA assay for quantifying anti-Neu5Gc antibodies in immunized porcine serum (sampled on day 35 of the immunization protocol) of Example 1 was adapted and modified from Scobie et al., J. Immunol., 2013 to increase specificity. Briefly, plates were coated with wild-type mouse serum (containing Neu5Gc) and incubated overnight at 4°C, followed by blocking with PBS containing 1% ovalbumin and 0.05% Tween at room temperature for 2 hours. During this time, with or without the addition of 5 mM synthetic Neu5Gc (for competitive uptake of anti-Neu5Gc antibodies), the samples were pre-incubated on ice for 2 hours with serum from CMAH-KO mice (not expressing Neu5Gc). The samples were then added to ELISA plates at room temperature for 2 hours. Anti-Neu5Gc antibodies were detected using horseradish peroxidase-labeled goat anti-porcine IgG (Fc) secondary antibody (AbD Serotec, reference number: AAI41P), and the plates were developed using TMB substrate (Sigma-Aldrich). Optical density was read on an MRX plate reader (Dynatech Laboratories). The results are expressed as the difference in optical density with and without synthetic Neu5Gc suppression.

[0251] result

[0252] Therefore, the double GAL / CMAH KO pigs formed only a minimal amount of anti-NeuGc antibodies, indicating that immune serum uptake was not required.< / nd>

Claims

1. A non-human mammalian polyclonal antibody against at least one non-human pathogen or at least one molecule derived from said pathogen, wherein said polyclonal antibody does not contain: – The first antigenic determinant is selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose; and – A second antigenic determinant, different from the first antigenic determinant, is selected from (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose.

2. The polyclonal antibody of claim 1, wherein the non-human biological pathogen targeting a human or animal organism is selected from: bacteria, parasites, mushrooms, viruses, toxins, venoms, and combinations thereof.

3. A composition comprising at least the polyclonal antibody as described in claim 1 or 2, and optionally at least one adjuvant, a pharmaceutically acceptable excipient, or a mixture thereof.

4. A method for producing the polyclonal antibody of claim 1 or 2 or the composition of claim 3, comprising the following steps: a) Provide a non-human mammal lacking the following genetic alterations: - The first gene is selected from (i) the gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) the gene encoding a functional α-(1,3)-galactosyltransferase; and - A second gene that is different from the first gene is selected from (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) Genes encoding functional α-(1,3)-galactosyltransferase; b) Inoculating the genetically altered nonhuman mammal with at least one nonhuman pathogen or at least one pathogen derived from said pathogen; and c) Collect antibodies contained in the bodily fluids of the non-human mammal with the genetic alterations described in step b).

5. The method of claim 4, wherein the non-human biological pathogen targeting a human or animal organism or the molecule derived from said pathogen is selected from the group consisting of bacteria, parasites, mushrooms, viruses, and combinations thereof.

6. The method of claim 4 or 5, wherein the body fluid is selected from the group consisting of plasma and serum.

7. The method according to any one of claims 4-6, wherein the non-human mammal is a mouse, rat, guinea pig, rabbit, goat, sheep, llama, or pig, preferably a pig.

8. Use of a genetically modified non-human mammal for the production of the polyclonal antibody of claim 1 or 2 or the composition of claim 3, wherein the genetically modified non-human mammal lacks: - The first gene is selected from (i) the gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) the gene encoding a functional α-(1,3)-galactosyltransferase; - Unlike the first gene, the second gene is selected from (i) a gene encoding a functional cytidine-5'-monophosphate N-acetylneuraminic acid hydrolase (CMAH); and (ii) a gene encoding a functional α-(1,3)-galactosyltransferase.

9. The use according to claim 8, wherein the non-human biological pathogen targeting a human or animal organism or the molecule derived from said pathogen is selected from the group consisting of bacteria, parasites, mushrooms, viruses, and combinations thereof.

10. Use of the polyclonal antibody of claim 1 or 2 or the composition of claim 3 in the preparation of a medicament.

11. Use of the polyclonal antibody of claim 1 or 2 or the composition of claim 3 in the preparation of a medicament for the prevention and / or treatment of serious infections, said serious infections being particularly selected from the following: Acinetobacter infections, actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS (Acquired Immunodeficiency Syndrome), amoebiasis, Alfa disease, anthrax, hemolytic lactobacillus infections, Argentine hemorrhagic fever, ascariasis, aspergillosis, astrovirus infections, brassicosis, Bacillus cereus infections, bacterial pneumonia, bacterial vaginosis (BV), Bacteroides infections, balanitis, shellfish ascariasis, BK virus infections, black hairy nodular disease, human budding disease. Cysticercosis, blastomycosis, Bolivian hemorrhagic fever, spirochetal infection, botulism (and infantile botulism), Brazilian hemorrhagic fever, brucellosis, plague, Burkholderia infection, brucellosis ulcer, calicivirus infection (norovirus and poxvirus), campylobacteriosis, candidiasis (candidiasis; thrush), cat bite disease, cellulitis, Chagas disease (American trypanosomiasis), chancroid, chickenpox, chikungunya, chlamydia, Chlamydia pneumoniae infection (Taiwan Acute Respiratory Agent or TWAR), cholera, pigmentary hyperplasia, pheochromocytosis, Clostridium difficile infection, coccidioidomycosis, Colorado tick fever (CTF), common cold (acute viral rhinitis),Acute rhinitis), Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneous larvae (CLM), cyclosporidiosis, cysticercosis, cytomegalovirus infection, dengue fever, binuclear amoebiasis, diphtheria, trichomoniasis, dracunculiasis, Ebola hemorrhagic fever, echinococcosis, helminthiasis, enterobacterial infection (pinworm infection), enterococcal infection, enterovirus infection (especially enterovirus 71 (EV71)), epidemic typhus, lupus erythematosus (fifth disease), roseola infantum (sixth disease), fascioliasis, fascioliasis, fatal familial insomnia (FFI), filariasis, food poisoning caused by Clostridium perfringens, free-living amoeba infection, fusobacterial infection, gas gangrene (Clostridium necrophorum), terriginosis, Jetzmann-Stokes-Schink syndrome (GSS), giardiasis Glandular disease, Gnathostoma spinigerum infection, gonorrhea, granuloma inguinale (urinary tract infection), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, hand-foot-mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS), cardiogenic viral disease, Helicobacter pylori infection, hemolytic uremic syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex virus infection, histoplasmosis, hookworm infection, human bocavirus infection, human Evans elektiasis, human granulocytic anaplasmosis (HGA), human metapneumovirus infection, human mononucleotic elektiasis, human papillomavirus (HPV) infection, human parainfluenza virus infection, hymenolepis taeniasis, Epstein-Barr virus infectious mononucleosis (Mono), influenza (flu), isosporidiosis.Kawasaki disease, keratitis, *Gynostemma pentaphyllum* infection, kuru, Lassa fever, Legionnaires' disease, Legionnaires' disease (Pontiac fever), leprosy, leptospirosis, Listeria infection, Lyme disease (Lyme spirochete infection), lymphatic filariasis (elephantiasis), lymphocytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East Respiratory Syndrome (MERS), glanders (Whitmore's disease), meningitis, meningococcal infection, metaclonorchiasis, microsporidiosis, molluscum contagiosum (MC), monkeypox, mumps, mouse typhus (endemic typhus), mycoplasmal pneumonia, mycoplasma gondii, myiasis Neonatal conjunctivitis (neonatal ophthalmitis), (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardia, Onchocerciasis (river blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurella multocida, Head lice, Body lice, Pubic lice, Pelvic inflammatory disease (PID), Pertussis, Plague, Pneumococcal infection, Pneumocystis pneumonia (PCP), Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis (PAM), Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rabies virus, Respiratory syncytial disease Viral infections, rhinosporidiosis, rhinovirus infection, rickettsial infection, rickettsial pox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), rotavirus infection, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, schistosomiasis, sepsis, Shigella (bacterial dysentery), herpes zoster (shingles), smallpox (pox), sporotrichosis, staphylococcal food poisoning, Staphylococcus aureus infection, strongyloides stercoralis infection, subacute sclerosing panencephalitis, syphilis, tapeworm infection, tetanus, tinea barbae (ringworm), tinea capitis (tinea capitis), tinea corporis (tinea corporis), tinea cruris (tinea cruris). Tinea manuum (hand fungus), black tinea, tinea pedis (athlete's foot), onychomycosis (nail fungus), tinea versicolor, toxocariasis (ocular larval migration (OLM)), toxocariasis (visceral larval migration (VLM)), trachoma, trichinosis, trichinosis, trichomoniasis, whipworm infection, tuberculosis, tularemia, ureaplasma infection, valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever, white sarcoidosis (tinea versicolor), plague, Yersinia pestis infection, Yersinia pestis, yellow fever, zygomycosis, or at least one serious infection induced by at least one antibiotic-resistant pathogen.

12. The use according to claim 11, wherein the severe infection is Ebola hemorrhagic fever.

13. The use of claim 11, wherein the serious infection is caused by a pathogen selected from toxins, venoms, and combinations thereof.

14. Use of the polyclonal antibody of claim 1 or 2 or the composition of claim 3 in the preparation of a medicament for serum prophylaxis and / or serum therapy.

15. Use of the polyclonal antibody of claim 1 or 2 or the composition of claim 3 in the preparation of a medicament for reducing and / or inhibiting immune complex-associated disease (ICD) and serious undesirable adverse effects, such as serum sickness (SSD), including severe forms such as myocarditis, nephropathy or other immune complex manifestations, such as rash, fever, headache, arthritis or pseudomeningitis syndrome and immune complex-associated disease and serious undesirable adverse effects induced by administration of an antibody comprising at least one antigenic determinant selected from: (i) N-ethylene glycol neuraminic acid (Neu5Gc) or (ii) α-1,3-galactose, preferably composed of at least both antigenic determinants (i) N-ethylene glycol neuraminic acid (Neu5Gc) and (ii) α-1,3-galactose.

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