Antithymocyte globulin

Abstract

This invention provides a human anti-thymocyte globulin (ATG) product, as well as a method for producing and using the same. [Solution] This disclosure provides polyclonal immunoglobulins derived from ungulates having a population of fully human or substantially human immunoglobulins. The population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes. Such compositions can be produced by immunizing transgenic animals having the human Ig locus with human thymocytes. This method produces polyclonal immunoglobulins with yield, purity, and antigen specificity that enable the use of this product in medical applications.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority to U.S. Application No. 62 / 975,649, filed on 12 February 2020, which is incorporated herein by reference in its entirety.

[0002] Inclusion by referencing the sequence list This application is submitted along with an electronic sequence listing. The sequence listing is provided as a file titled A2BI_001_01WO_SeqList.txt, created on September 8, 2021, and is 70 kilobytes in size. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] The present invention generally relates to a method for producing antithymocyte globulin for biomedical applications. [Background technology]

[0004] ATG (anti-thymocyte globulin) is a polyclonal immunoglobulin approved by the FDA for use in organ transplantation. ATG is also used in the treatment of graft-versus-host disease and type 1 diabetes (T1D), and is being used in clinical trials. In T1D, ATG is used as monotherapy to maintain β-cell function and as an immunosuppressant in non-myeloablative hematopoietic stem cell transplantation.

[0005] Current ATG preparations are manufactured by immunizing rabbits or horses to produce polyclonal xenoimmunoglobulins. ATG therapy exposes patients to the risk of serum sickness because the recipient's immune system reacts to the xenoimmunoglobulins in the ATG. Furthermore, readmission becomes problematic due to the immune response to ATG. Additionally, since glucocorticoids impair the function of the patient's β-cells, serum sickness in T1D cannot be managed with glucocorticoids.

[0006] Therefore, there remains a need in the art for improved methods of manufacturing ATG, as well as related compositions and methods of use. [Overview of the Initiative]

[0007] This disclosure relates to anti-thymocyte globulin (ATG) generally produced in transgenic animals possessing a fully human (or at least partially human) immunoglobulin locus. The resulting composition has fully human (or substantially human) immunoglobulin. Surprisingly, immunization of transgenic animals with human thymocytes produces ATG with potency comparable to or greater than that of a reference ATG product. Therefore, the method disclosed herein produces ATG in sufficient yield and potency to enable the production of a human ATG product that solves the problem of serum sickness caused by heterologous immunoglobulins.

[0008] In one embodiment, the disclosure provides an ungulate polyclonal immunoglobulin composition having a population of fully human or substantially human immunoglobulins. The population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

[0009] In another embodiment, the disclosure provides a composition produced by immunizing a transgenic ungulate with human thymocytes. This composition comprises a population of fully human or substantially human immunoglobulins. The population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

[0010] In yet another embodiment, the present disclosure provides a method for producing anti-thymocyte globulin (ATG), comprising the step of administering human thymocytes to a transgenic ungulate. The genome of the transgenic ungulate has a human immunoglobulin locus. The transgenic ungulate produces a polyclonal immunoglobulin having anti-thymocyte globulin (ATG).

[0011] In a further embodiment, the Disclosure provides a method for providing anti-thymocyte globulin (ATG) therapy to a subject in need thereof, the method comprising the step of administering to the subject: i) a polyclonal immunoglobulin composition according to the Disclosure; ii) a composition according to the Disclosure; or iii) a polyclonal immunoglobulin composition manufactured in accordance with the Disclosure. The Method provides an effective amount of anti-thymocyte globulin (ATG) to the subject.

[0012] In yet another aspect, the disclosure provides a pharmaceutical composition comprising a population of whole or substantially human immunoglobulins and one or more pharmaceutically acceptable excipients. The population of whole or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

[0013] Additional embodiments, features, and advantages of the present invention will become apparent from the following detailed description and through the implementation of the invention. [Brief explanation of the drawing]

[0014] [Figure 1]Figures 1A-1H show the construction of the isHAC and isKcHACΔ vectors. Figure 1A shows the flow chart for the construction of the isHAC and isKcHACΔ vectors. The bovine vector pCC1BAC-isHAC is BAC-based (the backbone is the pCC1BAC vector), consisting of 10.5kb of genomic DNA as the long arm and 2kb as the short arm. 9.7kb of bovine genomic DNA covering bovine Iγ1-Sγ1 and its surrounding region replaces the region corresponding to 6.8kb of human Iγ1-Sγ1, the FRT sequence, and the chicken β-actin promoter-driven neo gene adjacent to the DT-A gene. After target bovine transformation, the neo cassette is removed by FLP introduction. Figure 1B shows detailed information of the target vector pCC1BAC-isHAC. The 2kb Afe I-Bam HI fragment for the short arm and the 10.5kb Apa I-Hpa I fragment for the long arm were obtained from clones h10 and h18 / h20, respectively, from a λ phage genome library constructed from CHO cells containing κHAC, by screening with probes around the human Iγ1-Sγ1 region. The 9.7kb fragment (from the 5' end to Bsu36 I) was obtained from clone b42 from the λ phage bovine genome library. Figure 1C shows the senotyping of the bovine Iγ1-Sγ1 region. As shown, five sets of genomic PCR were performed. iscont1-F1 / R1 is a positive PCR specific to homologous recombination. iscont1-F1×hIgG1-R10 is a negative PCR prohibited by the presence of the neo cassette. isHAC-Sw-dig-F5 / R3 and isHAC-TM-dig-F3 / R2 are for checking the structural integrity of the corresponding regions digested by Bam HI+Pvu II and Age I, Sma I, or Pvu II, respectively. bNeo 5'-R×bIgG1-5'-seq-R6 is for confirming the presence of the FRT sequence. Figure 1D shows the genotyping of the neo cassette after FLP-FRT deletion. Figure 1E shows extensive genomic PCR for genotyping of the isHAC vector. The position of each genomic PCR primer pair is shown in relation to the isHAC vector structure.Figure 1F shows CGH analysis between three different CHO clones containing the isHAC vector. DNA from isC1-133 was used as a reference. There were no apparent structural differences of isHAC among the three cell lines. Figure 1G shows extensive genomic PCR for determining the genotype of the isKcHACΔ vector. The position of each genomic PCR primer pair is depicted in relation to the isKcHACΔ vector structure. Figure 1H shows CGH analysis between three different CHO clones containing the isKcHACΔ vector. DNA from isKCDC15-8 was used as a reference. There were no apparent structural differences of isKcHACΔ among the three cell lines. [Figure 2] Figure 2 shows the flow cytometry evaluation of the binding of TcB-derived ATG products to human PBMCs. [Figure 3] Figures 3A and 3B show the levels of regulatory T (Treg) cells treated with equine (Ho-ATG), rabbit (Rb-ATG), or TcB (SAB-ATG) products. Figure 3A shows the percentage of CD4+CD25+Foxp3+ cells. Figure 3B shows the percentage of CD4+CD25+Foxp3+ cells relative to the amount of immunoglobulin G (** indicates a two-sided p-value less than 0.001). [Figure 4] Figures 4A-4B show the levels of activated conventional T (Tconv) cells treated with equine (Ho-ATG), rabbit (Rb-ATG), or TcB (SAB-ATG) products. Figure 4A shows the percentage of CD4+CD25+Foxp3- cells. Figure 4B shows the percentage of CD4+CD25+Foxp3- cells relative to the amount of immunoglobulin G (two-sided p-values; **=<0.05; **=<0.001; ***=<0.0001). [Figure 5] Figures 5A and 5B show the levels of naive conventional T (Tconv) cells treated with equine (Ho-ATG), rabbit (Rb-ATG), or TcB (SAB-ATG) products. Figure 5A shows the percentage of CD4+CD25-Foxp3- cells. Figure 5B shows the percentage of CD4+CD25-Foxp3- cells relative to the amount of immunoglobulin G (two-sided p-values; **=<0.05; **=<0.001; ***=<0.0001). [Modes for carrying out the invention]

[0015] The inventors have developed a human ATG product that overcomes the limitations of animal ATG. Transgenic animals in which the endogenous Ig locus is replaced with a human artificial chromosome encoding the human Ig locus express fully human polyclonal antibodies. Immunization of such transgenic animals with human thymocytes produces polyclonal immunoglobulins with yield, purity, and antigen specificity that enable their use in medical applications. Various embodiments of the present invention are provided in the following description.

[0016] definition All cited documents are hereby incorporated by reference in their entirety. Unless otherwise specified herein, the techniques utilized herein can be found in any of the following well-known documents: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, Calif.), "Guide to Protein Purification" in Methods in Enzymology (M. P. Deutscher, ed., (1990) Academic Press, Inc.), PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990, Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987, Liss, Inc., New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), Ambion 1998 Catalog (Ambion, Austin, Tex.).

[0017] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, "and" is used interchangeably with "or" unless expressly stated otherwise.

[0018] All embodiments of any aspect of the present invention can be used in combination, unless the context clearly indicates otherwise.

[0019] Throughout this specification and the claims, unless the context clearly requires otherwise, words such as "comprise", "comprising", etc. are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, in the sense of "including, but not limited to". Also, words using the singular or plural number shall include the plural and the singular respectively. Further, the terms "this specification", "above", "below", and similar terms, as used in this application, refer to the entire application as a whole and not to a particular part of the application.

[0020] The term "ungulate" refers to any suitable ungulate including, but not limited to, cows, pigs, horses, donkeys, zebras, deer, bulls, goats, sheep, and serows.

[0021] The term "transgenic" means that a cell of an ungulate has one or more polynucleotides encoding a foreign gene (e.g., an immunoglobulin locus). Such polynucleotides may be part of an artificial chromosome. Alternatively, or in addition to an artificial chromosome, one or more polynucleotides encoding the foreign gene(s) may be integrated into the genome of the cell of the ungulate.

[0022] The terms “polyclonal,” “polyclonal serum,” “polyclonal plasma,” or “polyclonal immunoglobulin” refer to a population of immunoglobulins that have a common constant region but diverse variable regions. However, the term polyclonal does not exclude immunoglobulins derived from a single B cell precursor or a single recombination event, such as when a dominant immune response occurs. Polyclonal serum or plasma contains soluble forms (e.g., IgG) of the immunoglobulin population. The term “purified polyclonal immunoglobulin” refers to polyclonal immunoglobulin purified by serum or plasma. Methods for purifying polyclonal immunoglobulin include, but are not limited to, caprylic acid fractionation and adsorption by red blood cells (RBCs).

[0023] The “population” of immunoglobulins refers to immunoglobulins with diverse sequences, as opposed to samples containing multiple copies of a single immunoglobulin. Similarly, the term population excludes immunoglobulins secreted from a single B cell, plasma cell, or hybridoma in culture, or from host cells transformed with recombinant polynucleotides encoding a single pair of heavy-chain and light-chain immunoglobulin sequences.

[0024] The term “immunoglobulin” refers to a protein complex comprising at least two heavy chains and at least two light chains in a 1:1 ratio, and includes one of five classes of immunoglobulins—IgM, IgG, IgA, IgD, and IgE. In variations, immunoglobulins are manipulated in any of a variety of ways known or to be discovered in the art, including, but not limited to, mutations to alter glycosylation patterns and / or mutations to increase or decrease complement-dependent cell-mediated cytotoxicity.

[0025] An immunoglobulin is "fully human or substantially human" if its protein sequence is sufficiently similar to that of native human immunoglobulins, and when administered to a subject, it produces an anti-immunoglobulin immune response similar to, or not significantly worsening than, the immune response to native human immunoglobulins. Fully human immunoglobulins may include one or more substitutions, insertions, and deletions in the variable region, consistent with recombination, selection, and affinity maturation of the immunoglobulin sequence. In modifications, fully human or substantially human immunoglobulins may be manipulated in any of the various methods known or to be discovered in the art, including, but not limited to, mutations to alter glycosylation patterns and / or mutations to increase or decrease complement-dependent cytotoxicity.

[0026] The terms “thymocyte,” “T cell,” “B cell,” and “monocyte” have their common meanings in the art. Thymocytes are hematopoietic progenitor cells present in the thymus. In the methods of the disclosure, the step of administering (human) thymocytes may, in some embodiments, refer to the step of administering a mixed population of cells containing thymocytes, where thymocytes are present in sufficient quantity and purity to generate an anti-thymocyte immune response in transgenic ungulates. Modifications of the methods of the disclosure may use non-human thymocytes, such as thymocytes from non-human primates.

[0027] The proportion of immunoglobulins "by total immunoglobulin mass" (e.g., immunoglobulins that specifically bind to human thymocytes) is defined as the concentration of the target immunoglobulin population divided by the total immunoglobulin concentration in the sample, multiplied by 100. The concentration of the target immunoglobulin can be determined, for example, by affinity purification of the target immunoglobulin (e.g., using an affinity column made of thymocytes or thymocyte cell membranes) followed by concentration measurement.

[0028] The terms "about" or "approximately" mean a range of tolerance for a particular value as determined by those skilled in the art, which will depend in part on the method by which the value is measured or determined, for example, on the limits of the measuring system. For example, "about" can mean within or greater than one standard deviation. Alternatively, "about" can mean plus or minus a range of up to 20%, up to 10%, or up to 5%.

[0029] The terms “immunization” and “immunizing” mean administering to a subject (e.g., a transgenic ungulate) a sufficient amount of composition to induce a desired immune response (e.g., a thymocyte-specific polyclonal immunoglobulin response) after one or more administration steps. Administration can be carried out by intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route. Immunization may consist of one to ten or more administrations (e.g., injections) of the composition, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more administrations. The initial administration may not induce a detectable immune response, as each subsequent administration generally enhances the immune response produced by the previous administration.

[0030] The term "target antigen" refers to any antigen used to induce a desired immune response. Target antigens used to generate ATG products may be thymocytes, cells that share one or more endogenous protein markers with thymocytes, cells that recombinantly express one or more thymocyte proteins, recombinant thymocyte proteins, or nucleic acids (e.g., RNA, linear DNA, or plasmid DNA) that encode thymocyte proteins.

[0031] The term "purify" refers to separating target cells or molecules (e.g., a population of immunoglobulins, thymocytes) from other substances present in a composition. Immunoglobulins can be purified by plasma fractionation, by affinity (e.g., protein A or protein G binding, or other capture molecules), by charge (e.g., ion exchange chromatography), by size (e.g., size exclusion chromatography), or otherwise. Purifying a population of immunoglobulins may involve treating the composition containing the immunoglobulin population with one or more of the following: acids, bases, salts, enzymes, heat, cold, coagulation factors, or other suitable agents. Purification may further include adsorption of the composition containing target cells or molecules and impurities onto non-target cells or molecules (e.g., red blood cells) to partially or completely remove the impurities. Purification may further include serum or plasma pretreatment, e.g., caprylic acid fractionation.

[0032] The terms “to treat” and “to cure” mean one or more of the actions of alleviating, reducing, delaying, decreasing, reversing, improving, or managing at least one symptom of the condition in question. The term “to treat” may also mean one or more of the actions of preventing, delaying (i.e., the period before the clinical manifestation of) a symptom, or reducing the risk of developing or worsening a symptom.

[0033] The term "pharmaceutically acceptable" means that it is biologically or pharmacognitively suitable for use in vivo in animals or humans and may mean that it is approved by a federal or state regulatory agency or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, particularly humans.

[0034] The term "hyperimmunization" refers to an immunization regimen that generates a higher immune response in a target than is required to produce a desired titer (e.g., binding titer) after dilution of the immunoglobulin produced by the target. For example, if the desired titer is 1:100, the animal can be hyperimmunized with 1, 2, 3 or more boost immunizations to produce a titer of 1:1,000 or higher in the target, and the immunoglobulin produced by the target can be diluted in the production of a biopharmaceutical to confer the desired titer to the biopharmaceutical.

[0035] The terms “specific” or “preferentially binding” (as used interchangeably herein) to a target (e.g., thymocytes or thymocyte antigens) are well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates with a particular cell or substance more frequently, more rapidly, more persistently, and / or with higher affinity than it reacts with other cells or substances. “Specifically binding” of an immunoglobulin to a particular cell or substance means that it binds with higher affinity, higher binding activity, more readily, and / or for a longer duration than it binds to other particular cells or substances. For example, an immunoglobulin that specifically or preferentially binds to thymocytes is an immunoglobulin that binds to thymocytes with higher affinity, higher binding activity, more readily, and / or for a longer duration than it binds to other cells. An immunoglobulin that specifically binds to a first cell or substance may or may not specifically or preferentially bind to a second cell or substance. Thus, "specific bonding" does not necessarily require (though it can include) exclusive bonding. Generally, references to bonding imply specific bonding, but this is not always the case.

[0036] The term "HAC vector" refers to a vector having at least a centromere sequence, a telomere sequence, and an origin of replication derived from a human chromosome, and may include other sequences depending on the intended use. When present in a host cell, the HAC vector exists independently of the host cell's chromosomes and nucleus. Any suitable method can be used to prepare the HAC vector and insert the nucleic acid of interest into the HAC, including, but not limited to, those described in the following examples. As is known to those skilled in the art, the HAC vector is a double-stranded DNA vector.

[0037] Embodiment A method for producing anti-thymocyte globulin (ATG) is provided, comprising the step of administering human thymocytes to a transgenic ungulate. Thymocytes are hematopoietic progenitor cells present in the thymus. Thymic tissue must be excised from a patient and is available from various sources, such as from cardiac surgery in children and young adults that would normally be discarded. Live human thymocytes can be used because they better preserve the conformation of surface antigens. In some embodiments, the method comprises the step of administering an effective amount of human thymocytes. In embodiments, the effective amount is at least about 1 × 10⁻⁶ 8 , at least about 5 × 10 8 , at least about 1 × 10 9 , at least about 5 × 10 9 , at least about 1 × 10 10 , or at least about 5 × 10 11 These are thymic cells.

[0038] In variations, non-human thymocytes (e.g., thymocytes from livestock such as dogs, cats, or sheep) are used. In such cases, the transgenic ungulate may have an artificial chromosome encoding the Ig gene locus of the non-human species, which can then be used to produce antibodies of that species.

[0039] In some embodiments, thymocytes are administered before, during, or after the administration of one or more adjuvants. In some embodiments, thymocytes and one or more adjuvants are administered together in a single composition having optionally one or more pharmaceutically acceptable excipients.

[0040] Exemplary adjuvants include aluminum salt adjuvants, oil-in-water emulsions (e.g., oil-in-water emulsions containing squalene such as MF59 or AS03), TLR7 agonists (e.g., imidazoquinoline or imiquimod), or combinations thereof. Suitable aluminum salts include hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates) (see, for example, Chapters 8 and 9 of Vaccine Design (1995) eds. Powell & Newman. ISBN: 030644867X. Plenum), or mixtures thereof. Further exemplary adjuvants include, but are not limited to, Adju-Phos, Adjumerlm, albumin-heparin microparticles, algal glucans, algamurin, alum, antigen preparations, AS-2 adjuvant, autologous dendritic cells, autologous PBMCs, Avridine®, B7-2, BAK, BAY R1005, bupivacaine, bupivacaine-HCl, BWZL, calcitriol, calcium phosphate gel, CCR5 peptide, CFA, cholera holotoxin (CT) and cholera toxin B subunit (CTB), cholera toxin A1 subunit-protein A D-fragment fusion protein, CpG, CRL1005, cytokine-containing liposomes, D-mura palmitin, DDA, DHEA, diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC / alum complex, fowlpox, Freund's complete adjuvant, gamma inulin, Gerbu adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12 (N222L), hTNF-alpha, IFA, IFN gamma of pcDNA3, IL-12 DNA, IL-12 plasmid, IL-12 / GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2 / Ig plasmid, IL-2 / Ig protein, IL-4, IL-4 in pcDNA3, imiquimod, ImmTher®, immunoliposomes containing antibodies against costimulatory molecules, interferon-gamma, interleukin-1 beta, interleukin-12, interleukin-2, interleukin-7, ISCOM®®, Iscoprep 7.0.3(Trademark), MONTANIDE(Trademark) ISA-25, Keyhole Limpet Hemocyanin, Lipid-Based Adjuvant, Liposome, Loxoribine, LT(R192G), LT-OA or LT Oral Adjuvant, LT-R192G, LTK63, LTK72, MF59, MONTANIDE ISA51, MONTANIDE ISA 720, MPL.TM., MPL-SE, MTP-PE, MTP-PE Liposome, Muramethide, Murapalmitin, NAGO, nCT Natural Cholera Toxin, Nonionic Surfactant Vesicle, Non-toxic variant E1 of Cholera Toxin mCT-E112K 12K, p-hydroxybenzoate methyl ester, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, peptomer-NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic L121, PMMA, PODDS (trademark), Poly rA:Poly rU, polysorbate 80, protein cocreate, QS-21, quadriA saponin, Quil-A, ISA-25 / Quil-A, hydrogel HPA, hydrogel LV, RIBI, Ribilike adjuvants (MPL, TMD, CWS) This product contains S-28463, SAB-adj-1, SAB-adj-2, SAF-1, Sclavo peptide, Sendai proteoliposomes, Sendai-containing lipid matrix, Span85, Specol, Squalane 1, Squalene 2, Stearyl tyrosine, Tetanus toxoid (TT), Ceramide (trademark), Threonyl muramyl dipeptide (TMDP), Ty particles, and Walter Reed liposomes.

[0041] Immunization can be carried out by subcutaneously, intravenously, or intraperitoneally administering human thymocytes containing a suitable adjuvant, such as complete Freund's adjuvant or aluminum hydroxide gel, and a Bordetella pertussis vaccine, to transgenic ungulates. In one embodiment, immunization involves hyperimmunization. In various embodiments, human thymocytes are administered 1 to 10 times at intervals of 1 to 4 weeks after the initial administration. Blood is collected from the animals 1 to 14 days after each administration, and the antibody titer of the serum is measured.

[0042] In some embodiments, human thymocytes are administered 3, 4, 5, 6, or more times. Administration of human thymocytes may be performed, for example, every 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, or 6-7 weeks, or at longer intervals, for example, every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. After each immunization, serum and / or plasma may be collected once or multiple times from the transgenic ungulate. For example, the method may include 2-3 control blood collections at intervals of approximately 7-14 days.

[0043] In some embodiments, the antigen used to generate the ATG product may be a cell that shares one or more endogenous protein markers with a thymocyte, one or more thymocyte proteins, recombinant thymocyte proteins, or nucleic acids encoding thymocyte proteins (e.g., RNA, linear DNA, or plasmid DNA), rather than thymocytes.

[0044] In embodiments of the methods of this disclosure, the genome of a transgenic ungulate has a human immunoglobulin locus. Exemplary methods are provided in U.S. Patents 9,902,970, 9,315,824, 7,652,192, 7,429,672, and 7,253,334, the disclosures of which are incorporated herein by reference for all purposes. Further exemplary methods are provided in Kuroiwa, Y., et al. (2009) Nat Biotechnol. 27(2):173-81 and Matsushita et al. (2015) PLoS ONE 10(6):e0130699.

[0045] This disclosure is, (a) One or more human antibody heavy chains, wherein each gene encoding the antibody heavy chain is operatively linked to a class switch control element, (b) One or more human antibody light chains, and (c) One or more human antibody surrogate light chains and / or ungulate-derived IgM heavy chain constant regions We provide a human artificial chromosome (HAC) vector containing a gene encoding a gene, Here, at least one class switch regulatory element of one or more genes encoding human antibody heavy chains is replaced with a class switch regulatory element derived from ungulates.

[0046] The HAC vectors of this disclosure can be used, for example, in the mass production of fully human antibodies by transgenic animals, as described in the methods of the present invention. The HAC vectors of this disclosure have one or more genes encoding human antibody heavy chains. Any human antibody heavy chain or combination of human antibody heavy chains may be encoded by one or more nucleic acids on the HAC. In various embodiments, all 1, 2, 3, 4, 5, 6, 7, 8, or 9 human antibody heavy chains IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgD may be encoded on the HAC in one or more copies. In one embodiment, the HAC has a human IgM antibody heavy chain encoding gene, either alone or in combination with 1, 2, 3, 4, 5, 6, 7, or 8 other human antibody chain encoding genes. In one preferred embodiment, the HAC has a gene encoding at least a human IgG1 antibody heavy chain; in this embodiment, it is more preferable that the HAC has a gene encoding a human IgM antibody heavy chain, or a gene encoding a human IgM antibody heavy chain that is chimeric to encode an ungulate-derived IgM heavy chain constant region (e.g., bovine heavy chain constant region). In another embodiment, the HAC has a gene encoding at least a human IgA antibody heavy chain; in this embodiment, it is more preferable that the HAC has a gene encoding a human IgM antibody heavy chain, or a gene for a human IgM antibody heavy chain that is chimeric to encode an ungulate-derived IgM heavy chain constant region (e.g., bovine heavy chain constant region). In yet another preferred embodiment, the HAC has genes encoding all nine antibody heavy chains, more preferably the gene encoding the human IgM antibody heavy chain is chimeric to encode an ungulate-derived IgM heavy chain constant region. In yet another embodiment, the HAC may have a portion of human chromosome 14 encoding a human antibody heavy chain. The variable and constant region genes of the human antibody heavy chain form a cluster, and the human heavy chain locus is located at 14q32 of human chromosome 14. In one embodiment, the region of human chromosome 14 inserted into the HAC has both the variable and constant regions of the human antibody heavy chain, originating from the 14q32 region of human chromosome 14.

[0047] In some embodiments of the HAC vectors of this disclosure, at least one class switch regulatory element of the human antibody heavy chain coding nucleic acid is replaced with a class switch regulatory element derived from an ungulate. The class switch regulatory element points to a nucleic acid located at the 5' end of the antibody heavy chain constant region. Each heavy chain constant region gene is operatively linked (i.e., under its control) to its own switch region associated with its own I-exon. The class switch regulatory element controls the recombination of the class switch and determines the isotype of the Ig heavy chain. Germline transcription of each heavy chain isotype is driven by promoter / enhancer elements located exactly 5' of the I-exon, and these elements are cytokine or other activator-responsive. In a simple model of class switching, a specific activator and / or cytokine induces germline transcription of each heavy chain isotype from its class switch regulatory element (i.e., activator / cytokine-responsive promoter and / or enhancer). Prior to class switching, transcription of the I-exon takes place from the switch region associated with each Ig heavy (IGH) locus. Each heavy chain constant region gene is linked to its respective switch region.

[0048] Any suitable class switch regulatory element derived from an ungulate can be used. For example, the isotypes of the human heavy chain genes shown below are the following class switch regulatory elements: IgM:Iμ-Sμ, IgG1:Iγ1-Sγ1, IgG2:Iγ2-Sγ2, IgG3:Iγ3-Sγ3, IgG4:Iγ4-Sγ4, IgA1:Iα1-Sα1, IgA2:Iα2-Sα2, and IgE:Iε-Sε It holds.

[0049] In various embodiments, one, one or more, or all of the human antibody heavy chain genes on the HAC have their class switch regulatory elements replaced with ungulate-derived class switch regulatory elements, including but not limited to ungulate Iμ-Sμ, Iγ-Sγ, Iα-Sα, or Iε-Sε class switch regulatory elements. In one embodiment, the Iγ1-Sγ1 human class switch regulatory element (e.g., sequence ID number: 1) of the human IgG1 heavy chain encoding the nucleic acid on the HAC is replaced with an ungulate Iγ1-Sγ1 class switch regulatory element. Exemplary ungulate Iγ1-Sγ1 class regulatory regulatory elements include the bovine IgG1 Iγ1-Sγ1 class switch regulatory element (sequence ID number: 2), the horse Iγ1-Sγ1 class switch regulatory element (sequence ID number: 3), and the pig Iγ1-Sγ1 class switch regulatory element (sequence ID number: 4). However, it is not necessary to replace the human class switch regulatory elements with ungulate class switch regulatory elements from the corresponding heavy chain isotype. Therefore, for example, the Iγ3-Sγ3 human class switch control element of the human IgG3 heavy chain encoding nucleic acid on the HAC can be replaced with an ungulate Iγ1-Sγ1 class switch control element. As will be apparent to those skilled in the art based on the teachings herein, any such combination can be used in the HAC of this disclosure.

[0050] In another embodiment, the HAC has at least one ungulate enhancer element to replace enhancer elements associated with one or more human antibody heavy chain constant regions encoding one or more nucleic acids on the HAC. There are two 3' enhancer regions (alpha 1 and alpha 2) associated with the human antibody heavy chain gene. The enhancer element is located at 3' of the heavy chain constant region and also helps to regulate class switching. Any suitable ungulate enhancer can be used, including but not limited to 3'Eα enhancers. Non-limiting examples of usable 3'Eα enhancers include 3'Eα, 3'Eα1, and 3'Eα2. Exemplary bovine-derived 3'Eα enhancer elements that can be used in HACs and replace human enhancers include, but are not limited to, the bovine HS3 enhancer (sequence ID number: 5), the bovine HS12 enhancer (sequence ID number: 6), and the bovine enhancer HS4. This embodiment is particularly preferred in which the HAC consists of a variable region and a constant region of a human antibody heavy chain from the 14q32 region of human chromosome 14.

[0051] The HAC vectors of this disclosure may have one or more genes encoding human antibody light chains. Any suitable gene encoding a human antibody light chain can be used in the HAC vector of the present invention. A human antibody light chain comprises two genes, namely, a kappa / K chain gene and a lambda / L chain gene. In one embodiment, the HAC comprises one or more copies of the genes encoding both kappa and lambda. The variable and constant regions of the κ chain are located at 2p11.2–2p12 on human chromosome 2, and the λ chain forms a cluster located at 22q11.2 on human chromosome 22. Thus, in one embodiment, the HAC vector of the present invention comprises a human chromosome 2 fragment containing a κ chain gene cluster in the 2p11.2–2p12 region. In another embodiment, the HAC vector of the present invention comprises a human chromosome 22 fragment containing a lambda chain gene cluster in the 22q11.2 region.

[0052] In another embodiment, the HAC vector has at least one gene encoding a human antibody surrogate light chain. The gene encoding the human antibody surrogate light chain means a gene that encodes a transient antibody light chain that binds to an antibody heavy chain produced by gene rearrangement in human pro-B cells to constitute a pre-B cell receptor (preBCR). Any suitable human antibody surrogate light chain encoding gene is available, including but not limited to the VpreB1 (sequence ID: 7), VpreB3 (sequence ID: 8), and λ5 (also known as IgLL1, sequence ID: 9) human antibody surrogate light chains, and combinations thereof. The VpreB and λ5 genes are located in the human antibody λ chain locus at 22q11.2 on human chromosome 22. Thus, in one embodiment, the HAC may consist of the 22q11.2 region of human chromosome 22 containing the VpreB and λ5 genes. The present invention provides either or both of the human VpreB gene, VpreB1 gene (sequence ID number: 7) and VpreB3 gene (sequence ID number: 8), and in one embodiment, both the VpreB1 gene and the VpreB3 gene are provided.

[0053] In yet another embodiment, the HAC vector contains a gene encoding an IgM heavy chain constant region derived from an ungulate. In this embodiment, the IgM heavy chain constant region is expressed as a chimera with the human IgM antibody heavy chain variable region. Any suitable ungulate IgM heavy chain antibody constant region encoding nucleic acid is available, including but not limited to bovine IgM (sequence ID number: 10), horse IgM (sequence ID number: 11), sheep IgM (sequence ID number: 12), and porcine IgM (sequence ID number: 13). In one embodiment, the chimeric IgM consists of the sequence of sequence ID number: 14. Pre-BCR / BCR signaling via the IgM heavy chain molecule promotes B cell proliferation and development by interacting with B cell membrane molecules Ig-alpha / Ig-beta to induce intracellular signal transduction. The transmembrane region and other constant regions of IgM are thought to play important roles in signal transduction by interacting with Ig-α / Ig-β. Examples of constant regions of the IgM heavy chain include constant region domains such as CH1, CH2, CH3, and CH4, and nucleic acids encoding B cell transmembrane domains and cytoplasmic domains such as TM1 and TM2. The nucleic acids encoding the IgM heavy chain constant region derived from ungulates included in the human artificial chromosome vector of the present invention are not particularly limited as long as they are within a range that can sufficiently induce B cell receptor signaling or B cell proliferation and development in the above-mentioned IgM heavy chain constant region. In one embodiment, the nucleic acid encoding the IgM heavy chain constant region derived from ungulates provides transmembrane domains and cytoplasmic TM1 and TM2 domains derived from ungulates, and in other embodiments, the CH2, CH3, CH4, TM1, and TM2 domains derived from ungulates or the CH1, CH2, CH3, CH4, TM1, and TM2 domains derived from ungulates are encoded.

[0054] In one embodiment, the gene encoding the bovine IgM heavy chain constant region is a gene encoding the bovine IgM heavy chain constant region (derived from IGHM) located within the IGHM region where the bovine endogenous IgM heavy chain gene is located, or a gene encoding the bovine IgM heavy chain constant region located within the IGHML1 region (derived from IGHML1). In another embodiment, the gene encoding the bovine IgM heavy chain constant region is located within the IGHM region.

[0055] In further embodiments, the HAC is a gene encoding a human antibody heavy chain, which also has a gene encoding a human heavy chain (e.g., a human IgG heavy chain such as IgG1), and a certain region of the human heavy chain gene's transmembrane and intracellular domains are replaced with a heavy chain derived from an ungulate (e.g., an ungulate IgG heavy chain such as IgG1), the transmembrane domain of the certain region gene, and the intracellular domain of the certain region gene. In one embodiment, a gene encoding the transmembrane and intracellular domains of the constant region of an ungulate-derived (e.g., bovine) IgG (e.g., IgG1) heavy chain is used to replace the corresponding region of the human IgG heavy chain gene. In another embodiment, a gene encoding the TM1 and TM2 domains of the constant region of an ungulate-derived (e.g., bovine) IgG (e.g., IgG1) heavy chain is used to replace the corresponding region of the human IgG heavy chain gene. In yet another embodiment, a gene encoding the CH1-CH4 domain and / or one or more of the TM1 and TM2 domains of the constant region of an ungulate-derived (e.g., bovine) IgG (e.g., IgG1) heavy chain is used to replace the corresponding region of the human IgG heavy chain gene.

[0056] This disclosure further provides transgenic ungulates having HAC vectors in any embodiment or combination of embodiments of this disclosure. A transgenic ungulate having the HAC vector of the present invention refers to an animal into which the human artificial chromosome vector of the present invention has been introduced. A transgenic ungulate having the HAC of the present invention is not particularly limited as long as it is an animal into which human artificial chromosome fragments can be introduced into its cells, and non-human animals, such as ungulates such as cattle, horses, goats, sheep, and pigs, may be used. In one embodiment, the transgenic ungulate is a cattle. A transgenic ungulate having the HAC vector of the present invention can be constructed by introducing the HAC vector of the present disclosure into the oocyte of a host animal using any suitable technique, such as those described herein. The HAC vector of the present invention may be introduced into somatic cells derived from a host ungulate, for example, by microcell fusion. Subsequently, an animal having the HAC vector can be constructed by transplanting the nucleus or chromatin aggregate of the cell into an oocyte, and then transplanting the oocyte or the embryo formed from the oocyte into the uterus of a host animal and allowing it to give birth. Whether the animals constructed by the above method possess a human artificial chromosome vector can be confirmed by the method of Kuroiwa et al. (Kuroiwa et al., Nature Biotechnology, 18, 1086-1090, 2000 and Kuroiwa et al., Nature Biotechnology, 20, 889-894).

[0057] This disclosure is, (a) One or more human antibody heavy chains, wherein each gene encoding the antibody heavy chain is operatively linked to a class switch control element, (b) One or more human antibody light chains, and (c) One or more human antibody surrogate light chains and / or ungulate-derived IgM heavy chain constant regions Further providing transgenic ungulates having genes incorporated into the genome encoding, Here, at least one class switch regulatory element of one or more genes encoding human antibody heavy chains is replaced with a class switch regulatory element derived from ungulates.

[0058] In such embodiments, the transgenic ungulate may have any embodiment or combination of embodiments of the nucleic acid as described herein for HAC, but which is integrated into the ungulate's chromosome rather than being present in the HAC.

[0059] The present disclosure is a method for producing human antibodies, the method comprising (a) administering human thymocytes or other target antigens of the present disclosure to a transgenic ungulate in any embodiment or combination of embodiments of the present disclosure to generate and accumulate a population of human immunoglobulins specific to human thymocytes (or T cells, B cells, and / or monocytes) in the serum or plasma of the ungulate; and optionally (b) isolating, recovering, and / or purifying the population of human immunoglobulins specific to human thymocytes (or T cells, B cells, and / or monocytes) from the serum or plasma of the ungulate.

[0060] Polyclonal serum or plasma, or human immunoglobulins purified from polyclonal serum or plasma, can be used as ATG preparations.

[0061] In a modified example, the Disclosure provides a method for recovering the protein sequence of a human antibody, the method comprising (i) isolating lymphocytes from a transgenic ungulate, (ii) generating a human monoclonal antibody-producing hybridoma from the lymphocytes, and (iii) recovering a human monoclonal antibody specific to human thymocytes from the hybridoma. In another embodiment, lymphocytes from a transgenic ungulate are isolated from the lymph nodes of the transgenic ungulate. In a further embodiment, the transgenic ungulate is hyperimmunized with human thymocytes or other target antigens of the Disclosure.

[0062] Thymocyte-specific human immunoglobulins can also be manufactured by immunizing transgenic ungulates possessing HAC vectors with human thymocytes or other target antigens of the present disclosure to produce thymocyte-specific human immunoglobulins in the serum or plasma of the transgenic ungulates, and then recovering the thymocyte-specific human immunoglobulins from the serum or plasma of the transgenic ungulates.

[0063] Examples of methods for detecting and measuring thymocyte-specific human immunoglobulins in a composition include binding measurements using enzyme-linked immunosorbent assays. The amount of human immunoglobulin bound can be measured by incubating a composition containing human immunoglobulin with cells (e.g., thymocytes, T cells, B cells and / or monocytes, or recombinant protein antigens) and using an antibody that specifically recognizes human immunoglobulin.

[0064] In a modified form, the method of the present disclosure is used to generate monoclonal antibodies. Methods for preparing and utilizing various types of antibodies are well known to those skilled in the art and would be suitable for implementation of the present invention (see, for example, Harlow, et al: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Kohler and Milstein, Nature 256:495 (1975)). An example of a method for preparing hybridomas includes (1) immunizing a transgenic ungulate with thymocytes; (2) collecting antibody-producing cells from the transgenic ungulate (i.e., from lymph nodes); (3) fusing the antibody-producing cells with myeloma cells; (4) selecting hybridomas from the fused cells obtained in the above step that produce monoclonal antibodies specific to thymocytes; and optionally (5) selecting hybridomas from the selected hybridomas that produce monoclonal antibodies specific to thymocytes.

[0065] In embodiments of the methods for producing anti-thymocyte globulin (ATG) according to the present disclosure, a transgenic ungulate produces human anti-thymocyte globulin (ATG). The method may include the step of collecting polyclonal serum and / or polyclonal plasma from the transgenic ungulate. In some embodiments, the ungulate is a cattle. In some embodiments, the polyclonal immunoglobulin composition has a population of fully human immunoglobulins or a population of substantially human immunoglobulins.

[0066] Some embodiments of the methods and related compositions of this disclosure have the remarkable advantage that thymocyte-specific immunoglobulins are produced in high yield, high purity, and / or as a high proportion of total immunoglobulins present in the serum or plasma of a transgenic ungulate. In some embodiments, the ungulate is a cattle.

[0067] In some embodiments of the methods and compositions of this disclosure, the polyclonal serum or polyclonal plasma contains, by mass, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, and at least 1.7% of total immunoglobulins in the polyclonal serum or polyclonal plasma. , at least 1.8%, at least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6.0%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6. 8%, at least 6.9%, at least 7.0%, at least 7.1%, at least 7.2%, at least 7.3%, at least 7.4%, at least 7.5%, at least 7.6%, at least 7.7%, at least 7.8%, at least 7.9%, at least 8.0%, at least 8.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, at least 8.6%, at least 8.7%, at least 8.8%, at least 8.8%, at least 9.0%, at least 9.1%, at least 9.2%, at least 9.It contains 3%, at least 9.4%, at least 9.5%, at least 9.6%, at least 9.7%, at least 9.8%, at least 9.8%, at least 9.9%, or at least 10% of fully human (or substantially human) immunoglobulin.

[0068] In some embodiments of the methods and compositions of this disclosure, the polyclonal serum or polyclonal plasma is present in the following proportions of total immunoglobulins in the polyclonal serum or polyclonal plasma: 0.1-0.6%, 0.2-0.7%, 0.3-0.8%, 0.4-0.9%, 0.5-1%, 0.6-1.1%, 0.7-1.2%, 0.8-1.3%, and 0.9%. ~1.4%, 1~1.5%, 1.1~1.6%, 1.2~1.7%, 1.3~1.8%, 1.4~1.9%, 1.5~2%, 1.6~2.1%, 1.7~2.2%, 1.8~2.3%, 1.9~2.4%, 2~2.5%, 2.1~2.6%, 2.2~2.7%, 2.3~2.8%, 2.4~2.9%, 2.5~3%, 2.6~3.1%, 2.7~ 3.2%, 2.8-3.3%, 2.9-3.4%, 3-3.5%, 3.1-3.6%, 3.2-3.7%, 3.3-3.8%, 3.4-3.9%, 3.5-4%, 3.6-4.1%, 3.7-4.2%, 3.8-4.3%, 3.9-4.4%, 4-4.5%, 4.1-4.6%, 4.2-4.7%, 4.3-4.8%, 4.4-4.9%, 4.5 It contains ~5%, 4.6~5.1%, 4.7~5.2%, 4.8~5.3%, 4.9~5.4%, 5~5.5%, 5.1~5.6%, 5.2~5.7%, 5.3~5.8%, 5.4~5.9%, 5.5~6%, 5.6~6.1%, 5.7~6.2%, 5.8~6.3%, or 5.9~6.4% fully human (or substantially human) immunoglobulins.

[0069] In some embodiments of the methods and compositions of the present disclosure, the polyclonal serum or polyclonal plasma contains, by mass of total immunoglobulins in the polyclonal serum or polyclonal plasma, 0-0.5%, 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, 9.5-10%, or more of full human (or substantially human) immunoglobulins.

[0070] In some embodiments of the methods and compositions of the present disclosure, the polyclonal serum or polyclonal plasma contains 0-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, or more by mass of total immunoglobulins in the polyclonal serum or polyclonal plasma, which are full human (or substantially human) immunoglobulins.

[0071] In some embodiments of the methods and compositions of the present disclosure, the polyclonal serum or polyclonal plasma contains 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, or more of complete human (or substantially human) immunoglobulins by mass of total immunoglobulins in the polyclonal serum or polyclonal plasma.

[0072] In some embodiments of the methods and compositions of the present disclosure, the polyclonal serum or polyclonal plasma contains 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, or more of complete human (or substantially human) immunoglobulins by mass of total immunoglobulins in the polyclonal serum or polyclonal plasma.

[0073] In some embodiments of the methods and compositions of the present disclosure, the polyclonal serum or polyclonal plasma contains at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% by mass of total immunoglobulins in the polyclonal serum or polyclonal plasma of full human (or substantially human) immunoglobulins.

[0074] In some embodiments of the methods and compositions of the present disclosure, the polyclonal serum or polyclonal plasma contains 1-4%, 2-5%, 3-6%, 4-7%, 5-8%, 6-9%, or 7-10% by mass of total immunoglobulins in the polyclonal serum or polyclonal plasma of fully human (or substantially human) immunoglobulins.

[0075] In some embodiments of the methods and compositions of this disclosure, the polyclonal immunoglobulin is present in the polyclonal immunoglobulin in an amount of at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, at least 1.6%, at least 1.7%, at least 1.8%, and less than 1. At least 1.9%, at least 2%, at least 2.1%, at least 2.2%, at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4. 4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6.0%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%, at least 6.9 %, at least 7.0%, at least 7.1%, at least 7.2%, at least 7.3%, at least 7.4%, at least 7.5%, at least 7.6%, at least 7.7%, at least 7.8%, at least 7.9%, at least 8.0%, at least 8.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, at least 8.6%, at least 8.7%, at least 8.8%, at least 8.8%, at least 9.0%, at least 9.1%, at least 9.2%, at least 9.3%, at least 9.It contains 4%, at least 9.5%, at least 9.6%, at least 9.7%, at least 9.8%, at least 9.8%, at least 9.9%, or at least 10% fully human (or substantially human) immunoglobulin.

[0076] In some embodiments of the methods and compositions of this disclosure, polyclonal immunoglobulins are present in amounts of 0.1-0.6%, 0.2-0.7%, 0.3-0.8%, 0.4-0.9%, 0.5-1%, 0.6-1.1%, 0.7-1.2%, 0.8-1.3%, 0.9-1.4%, and 1-1% of total immunoglobulin in the polyclonal immunoglobulin. 5%, 1.1-1.6%, 1.2-1.7%, 1.3-1.8%, 1.4-1.9%, 1.5-2%, 1.6-2.1%, 1.7-2.2%, 1.8-2.3%, 1.9-2.4%, 2-2.5%, 2.1-2.6%, 2.2-2.7%, 2.3-2.8%, 2.4-2.9%, 2.5-3%, 2.6-3.1%, 2.7-3.2%, 2. 8-3.3%, 2.9-3.4%, 3-3.5%, 3.1-3.6%, 3.2-3.7%, 3.3-3.8%, 3.4-3.9%, 3.5-4%, 3.6-4.1%, 3.7-4.2%, 3.8-4.3%, 3.9-4.4%, 4-4.5%, 4.1-4.6%, 4.2-4.7%, 4.3-4.8%, 4.4-4.9%, 4.5-5%, It contains 4.6-5.1%, 4.7-5.2%, 4.8-5.3%, 4.9-5.4%, 5-5.5%, 5.1-5.6%, 5.2-5.7%, 5.3-5.8%, 5.4-5.9%, 5.5-6%, 5.6-6.1%, 5.7-6.2%, 5.8-6.3%, or 5.9-6.4% fully human (or substantially human) immunoglobulins.

[0077] In some embodiments of the methods and compositions of the present disclosure, the polyclonal immunoglobulin contains, by mass of total immunoglobulins, 0-0.5%, 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, 4.5-5%, 5-5.5%, 5.5-6%, 6-6.5%, 6.5-7%, 7-7.5%, 7.5-8%, 8-8.5%, 8.5-9%, 9-9.5%, 9.5-10%, or more of complete human (or substantially human) immunoglobulin.

[0078] In some embodiments of the methods and compositions of the present disclosure, the polyclonal immunoglobulin contains 0-1%, 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10%, or more of complete human (or substantially human) immunoglobulin by mass of total immunoglobulin in the polyclonal immunoglobulin.

[0079] In some embodiments of the methods and compositions of the present disclosure, the polyclonal immunoglobulin contains 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, or more of complete human (or substantially human) immunoglobulin by mass of total immunoglobulin in the polyclonal immunoglobulin.

[0080] In some embodiments of the methods and compositions of the present disclosure, the polyclonal immunoglobulin contains 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, or more of complete human (or substantially human) immunoglobulin by mass of total immunoglobulin in the polyclonal immunoglobulin.

[0081] In some embodiments of the methods and compositions of the present disclosure, the polyclonal immunoglobulin contains at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% by mass of total immunoglobulins in the polyclonal immunoglobulin, which is full human (or substantially human) immunoglobulin.

[0082] In some embodiments of the methods and compositions of the present disclosure, the polyclonal immunoglobulin contains 1-4%, 2-5%, 3-6%, 4-7%, 5-8%, 6-9%, or 7-10% by mass of total immunoglobulins in the polyclonal immunoglobulin, which is full human (or substantially human) immunoglobulin.

[0083] In some embodiments of the methods and compositions of this disclosure, the polyclonal immunoglobulin has at least 5% complete human immunoglobulin by mass of total immunoglobulins in the polyclonal immunoglobulin.

[0084] In some embodiments of the methods and compositions disclosed herein, the polyclonal immunoglobulin contains 2% to 5% by mass of complete human immunoglobulin in the polyclonal immunoglobulin.

[0085] In some embodiments, the ungulate-derived polyclonal immunoglobulin has a “chimeric” human immunoglobulin (referred to as “cIgG”) having a human heavy chain and an ungulate kappa light chain. In some embodiments, the polyclonal immunoglobulin has cIgG in percentages of total protein concentration of less than about 0.5%, less than about 0.75%, less than about 1.0%, less than about 1.25%, less than about 1.5%, less than about 1.75%, about 2.0%, less than about 2.25%, less than about 2.5%, less than about 2.75%, less than about 3.0%, less than about 3.25%, less than about 3.5%, less than about 3.75%, or less than 4.0%. In some embodiments, the polyclonal immunoglobulin contains about 0.5% to about 1.0%, about 1.0% to about 1.5%, about 1.5% to about 2.0%, about 2.0% to about 2.5%, or about 2.5% to about 3.0% of the total protein concentration as a percentage.

[0086] In some embodiments, the polyclonal immunoglobulins of this disclosure are more potent in complement-dependent cell-mediated cytotoxicity (CDC) assays than reference products (e.g., thymoglobulin or ATGAM). In some embodiments, the polyclonal immunoglobulins of this disclosure are at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 150%, or at least about 200% or more potent in complement-dependent cell-mediated cytotoxicity (CDC) assays than reference products (e.g., thymoglobulin or ATGAM).

[0087] In some embodiments, the polyclonal immunoglobulins of this disclosure produce higher toxicity to CD8+ cells than a reference product (e.g., thymoglobulin or ATGAM). In some embodiments, the polyclonal immunoglobulins of this disclosure exhibit at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 100%, or at least about 200% higher efficacy in CD8+ cell killing assays than a reference product (e.g., thymoglobulin or ATGAM).

[0088] In some embodiments, the polyclonal immunoglobulins of this disclosure produce a lower rate of CD4+ T cell apoptosis than reference products (e.g., thymoglobulin or ATGAM). In some embodiments, the polyclonal immunoglobulins of this disclosure are at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 100%, or at least about 200% less toxic in CD4+ cell apoptosis assays than reference products (e.g., thymoglobulin or ATGAM).

[0089] In some embodiments, the polyclonal immunoglobulins of this disclosure are more effective than reference products (e.g., thymoglobulin or ATGAM) in conventional T cell delivery. reg To better preserve. In some embodiments, the polyclonal immunoglobulins of this disclosure are T reg It is at least approximately 5%, at least approximately 10%, at least approximately 25%, at least approximately 50%, at least approximately 100%, at least approximately 150%, or at least approximately 200% less toxic to cells.

[0090] In some embodiments of the methods and compositions of this disclosure, a population of fully human immunoglobulins (or substantially human) specifically binds to human thymocytes, T cells, B cells, and / or monocytes. In some embodiments, a population of fully human (or substantially human) immunoglobulins specifically binds to human thymocytes.

[0091] This disclosure further provides compositions produced by immunizing transgenic ungulates with human thymocytes, wherein the compositions have a population of fully human or substantially human immunoglobulins, the population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

[0092] In some embodiments, the genome of a transgenic ungulate has a human immunoglobulin locus.

[0093] In some embodiments, transgenic ungulates are immunized three, four, five, or more times.

[0094] In some embodiments, a population of fully human or substantially human immunoglobulins is purified from the serum of transgenic ungulates after immunization.

[0095] This disclosure provides a method for providing anti-thymocyte globulin (ATG) therapy to a subject in need thereof, the method comprising the step of administering polyclonal immunoglobulin according to this disclosure to the subject. In some embodiments, the method provides an effective amount of anti-thymocyte globulin (ATG) to the subject. In some embodiments, the subject has type 1 diabetes. In some embodiments, the subject is an organ transplant recipient. In some embodiments, the subject has or is at risk of graft-versus-host disease. In some embodiments, the subject is a stem cell transplant recipient.

[0096] This disclosure provides a method for providing anti-thymocyte globulin (ATG) therapy to a subject in need thereof, comprising the step of administering a composition produced by immunizing a transgenic ungulate with human thymocytes to the subject. In some embodiments, the method provides an effective amount of anti-thymocyte globulin (ATG) to the subject. In some embodiments, the subject has type 1 diabetes. In some embodiments, the subject is an organ transplant recipient. In some embodiments, the subject has or is at risk of graft-versus-host disease. In some embodiments, the subject is a stem cell transplant recipient.

[0097] This disclosure provides a method for providing anti-thymocyte globulin (ATG) therapy to a subject in need thereof, the method comprising the step of administering polyclonal immunoglobulin produced in accordance with this disclosure to the subject. In some embodiments, the method provides an effective amount of anti-thymocyte globulin (ATG) to the subject. In some embodiments, the subject has type 1 diabetes. In some embodiments, the subject is an organ transplant recipient. In some embodiments, the subject has or is at risk of graft-versus-host disease. In some embodiments, the subject is a stem cell transplant recipient.

[0098] Exemplary methods of treatment with ATG are described, for example, in the following literature: Voltarelli, JC, et al. (2007) Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus. JAMA. 297(14):1568-76. Couri, CE, et al. (2009) C-peptide levels and insulin independence following autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus. JAMA. 301(15):1573-9. Haller, MJ, et al., (2015) Anti-thymocyte globulin / G-CSF treatment preserves beta cell function in patients with established type 1 diabetes. J Clin Invest. 125(1):448-55. Haller, MJ, et al., (2018) Low-Dose Anti-Thymocyte Globulin (ATG) Preserves beta-Cell Function and Improves HbA1c in New-Onset Type 1 Diabetes. Diabetes Care. 41(9):1917-1925

[0099] This disclosure further provides pharmaceutical compositions comprising a population of fully human or substantially human immunoglobulins and one or more pharmaceutically acceptable excipients. In some embodiments, the population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

[0100] In some embodiments, the pharmaceutical composition contains at least about 1 mg / mL, at least about 50 mg / mL, at least about 100 mg / mL, or at least about 1,000 mg / mL of fully human immunoglobulin or substantially human immunoglobulin. In some embodiments, the pharmaceutical composition contains at least about 100 μg / mL, at least about 250 μg / mL, at least about 500 μg / mL, at least about 750 μg / mL, or at least about 1,000 μg / mL of fully human immunoglobulin or substantially human immunoglobulin.

[0101] In some embodiments, fully human or substantially human immunoglobulins are produced in ungulates. In some embodiments, the ungulate is a cattle.

[0102] In some embodiments, the pharmaceutical composition contains at least 5% by mass of total immunoglobulins in the pharmaceutical composition, which is complete human immunoglobulin.

[0103] In some embodiments, the pharmaceutical composition contains 2% to 5% by mass of total immunoglobulins in the pharmaceutical composition, which is full human immunoglobulin.

[0104] Examples The following specific embodiments are to be interpreted merely as examples and do not limit the remainder of this disclosure.

[0105] Example 1 Production of human polyclonal ATG in transchromosomal cattle (TcB) strains We report the development of a novel human polyclonal ATG product (hereinafter referred to as "TcB product") that overcomes the known limitations of animal-derived ATGs. Using the diversitAb® platform technology and the transchromosomic bovine (TcB) system, we enabled cattle in which the bovine Ig gene locus was replaced with a human artificial chromosome to express fully human polyclonal antibodies.

[0106] Human thymocytes and an adjuvant were immunized at intervals of 3 to 5 weeks for the TcB target. Hyperimmune plasma was collected after the 3rd to 5th vaccinations (V3 - V5). The design of the immunization test is summarized in Table 1. The amount of hyperimmune plasma collected from the test animals on the 7th, 11th, and 14th days after the 5th vaccination (V5) was 2.1% of the plasma based on the animal's weight (BW).

Table 1

[0107] Complement-dependent cytotoxicity Complement-dependent cytotoxicity (CDC) was equivalent to ATGAM and thymoglobulin, and the efficacy of SAB-ATG increased from V3 / V4 to V5. The immunoglobulin concentration in each sample was measured with a NanoDrop (trademark) spectrophotometer that measures total protein at a wavelength of 260 nm.

[0108] The CDC assay is a cytometry-based assay that incubates serum, plasma, in-process or purified antibody products with human PBMCs and then with rabbit complement. Antibodies specific for human lymphocytes bind to the cells, and complement binds to both the immunoglobulin and the cells. Complement, when bound to the cells, is a cascade of proteins that ultimately leads to cell lysis. Cell death is measured using a cell viability dye such as ViaCount reagent (registered trademark). When the sample is read on a flow cytometer, the percentage of viable cells is calculated. By plotting the cell viability percentage against the antibody concentration, LT 25 values can be calculated. This value indicates the amount of antibody required to kill 25% of the cells. The lower this value, the higher the efficacy or activity of the ATG. This value can be standardized using CF 25 if necessary.

[0109] The results of the CDC assay using rabbit complement are shown in Table 2. When using rabbit complement, the TcB product had CDC activity equivalent to rabbit-derived thymoglobulin (registered trademark). [Table 2] LT 25 This refers to the IgG concentration at which 25% of human PBMCs dissolve in the presence of rabbit complement. CF 25 is 25 th This refers to percentile cytotoxicity factors. 25 This has been standardized to account for assay variability, (Sample LT 25 / Reference LT 25 )×Reference LT 25 It is calculated as follows.

[0110] Further CDC assays involved adsorbing hyperimmune plasma, administered with vaccine 3 (V3) or vaccine 5 (V5), onto human erythrocytes (RBCs). The efficacy of CDC was determined using rabbit complement. The results of the rabbit complement-based CDC assay are shown in Table 3. Lower values ​​indicate stronger efficacy. [Table 3] LT50 refers to the IgG concentration at which 50% of human PBMCs are soluble in the presence of rabbit complement. The LT50 value is the average value obtained from a two-day assay.

[0111] In human complement, the TcB product exhibited similar CDC efficacy to rabbit-derived thymoglobulin (registered trademark) (data not shown). Biochemical characterization To measure the concentration of human immunoglobulin and confirm the absence of bovine immunoglobulin in the plasma sample, a portion of the product was adsorbed onto human red blood cells (RBCs).

[0112] Size exclusion chromatography and SDS-PAGE confirmed that the sample mainly contained well-folded, unaggregated pairs of heavy and light chain IgG molecules. Approximately 5.6% of the total protein (=(62.1-58.6) / 62.1) adsorbed to red blood cells, indicating that a small portion of the immunoglobulin cross-reacts with red blood cells even without adsorption to red blood cells and subsequent purification.

[0113] Binding to human PBMCs Flow cytometry evaluation of binding to human PBMCs: pan-T cells, CD4+ and CD8+ conventional T cells, Treg, NK T cells, B cells, and neutrophils (Figure 2) revealed that TcB products exhibit the same specificity to T cells, B cells, and / or monocytes as equine-derived (ATGAM) and rabbit-derived (thymoglobulin) ATG. There were no single positive cells, and the mean fluorescence intensity (MFI) was also very similar. The rare red blood cells stained with TcB products (~0.4%) were also stained with ATGAM and thymoglobulin, confirming that TcB products do not possess inherent RBC specificity that might contraindicate their use in humans.

[0114] T cell killing ATG preparations exert their medical effects by killing T cells. Therefore, in vitro T cell elimination is commonly used as a substitute for the in vivo efficacy of ATG preparations.

[0115] Under inactivation conditions, the TcB product surprisingly exhibited significantly higher toxicity to CD8+ cells compared to thymoglobulin, while ATGAM was the least potent. Again, surprisingly, TcB treatment induced apoptosis in CD4+ T cells less effectively, preserved more CD4+ cells than thymoglobulin, and increased Treg preservation compared to conventional T cells. Results in inactivated T cells are shown in Table 4 (percentages indicate cell population, + / - standard deviation). [Table 4]

[0116] After cell activation, the TcB product exhibited cytotoxicity to both CD8+ and CD4+ cells (more pronounced against CD4+ cells). Surprisingly, the TcB product was more potent than other ATGs. Unexpectedly, apoptotic CD8+ and CD4+ cells were less frequent with higher concentrations of the TcB product than with other ATGs. This suggests that the cytotoxicity mediated by the TcB product is more rapid and involves additional biochemical pathways. Results in PHA-activated T cells are shown in Table 5 (percentages indicate cell population, + / - standard deviation). [Table 5]

[0117] From the above, it was found that SAB-ATG exhibits binding affinity and in vitro cytotoxicity equivalent to or better than commercially available ATG products.

[0118] T cell survival The effects of TcB products on regulatory T (Treg) cell survival were compared with those of thymoglobulin® and ATGAM®. Figures 3A-3B show the levels of regulatory T (Treg) cells treated with horse (Ho-ATG), rabbit (Rb-ATG), or TcB (SAB-ATG) products. Treg cells were maintained at similar levels with TcB products as with thymoglobulin®.

[0119] Similar assays were performed with conventional T(Tconv) cells. The results for activated Tconv cells and naive Tconv cells are shown in Figures 4A-4B and 5A-5B, respectively. The TcB product induced T cell activation at the same level as thymoglobulin®. The TcB product reduced naive T cells at the same level as thymoglobulin®.

[0120] * * * * While embodiments of the present invention have been shown and described herein, those skilled in the art will understand that such embodiments are provided only as examples. Numerous modifications, alterations, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be adopted when carrying out the present invention. The following claims define the scope of the present invention, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be covered thereby.

Claims

1. A polyclonal immunoglobulin composition derived from ungulates having a population of fully human or substantially human immunoglobulins, A composition wherein the population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

2. A composition according to claim 1 or 2, wherein the composition has at least the same efficacy as a reference product in a CDC assay.

3. A composition according to claim 1 or 2, wherein the composition is at least about 10% more potent than the reference product in a CDC assay.

4. A composition according to any one of claims 1 to 3, wherein the composition is at least about 10% more potent than a reference product in a CD8+ cell killing assay.

5. A composition according to any one of claims 1 to 3, wherein the composition is at least about 10% less than the reference product in a CD4+ cell apoptosis assay.

6. A composition according to any one of claims 2 to 5, wherein the reference product is rabbit-derived ATG, optionally thymoglobulin.

7. A composition according to any one of claims 2 to 5, wherein the reference product is horse-derived ATG, optionally ATGAM.

8. A composition according to any one of claims 1 to 7, wherein the composition comprises at least 2% by mass of total immunoglobulins in the composition of complete human or substantially human immunoglobulins.

9. A composition according to any one of claims 1 to 8, wherein the ungulate is a cattle.

10. A composition produced by immunizing a transgenic ungulate with human thymocytes, wherein the composition has a population of fully human or substantially human immunoglobulins, and A composition wherein the population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

11. A composition according to claim 10, wherein the composition has at least the same efficacy as a reference product in a CDC assay.

12. A composition according to claim 10, wherein the composition is at least about 10% more potent than the reference product in a CDC assay.

13. A composition according to any one of claims 10 to 12, wherein the composition is at least about 10% more potent than a reference product in a CD8+ cell killing assay.

14. A composition according to any one of claims 10 to 12, wherein the composition is at least about 10% less than the reference product in a CD4+ cell apoptosis assay.

15. A composition according to any one of claims 11 to 14, wherein the reference product is rabbit-derived ATG, optionally thymoglobulin.

16. A composition according to any one of claims 11 to 14, wherein the reference product is horse-derived ATG, optionally ATGAM.

17. A composition according to any one of claims 10 to 16, wherein the composition comprises at least 2% by mass of total immunoglobulins in the composition of whole human or substantially human immunoglobulins.

18. A composition according to any one of claims 10 to 17, wherein the ungulate is a cattle.

19. A composition according to any one of claims 10 to 18, wherein the genome of the transgenic ungulate has a human immunoglobulin gene locus.

20. A composition according to any one of claims 10 to 19, wherein the transgenic ungulate is immunized 3, 4, 5 or more times.

21. A composition according to any one of claims 10 to 20, wherein the population of fully human or substantially human immunoglobulins is purified from the serum of the immunized transgenic ungulate.

22. A method for producing anti-thymocyte globulin (ATG), comprising the step of administering human thymocytes to a transgenic ungulate, wherein the transgenic ungulate has a genome having a human immunoglobulin locus or an artificial chromosome having a human immunoglobulin locus, and the transgenic ungulate produces human anti-thymocyte globulin (ATG).

23. A method according to claim 22, comprising the step of administering the human thymocytes three, four, five or more times.

24. A method according to claim 22 or 23, comprising the step of collecting serum or plasma from the transgenic ungulate.

25. A method according to any one of claims 22 to 24, wherein the serum or plasma has a population of complete human immunoglobulins.

26. A method according to any one of claims 22 to 25, wherein the population of complete human immunoglobulins is at least about 10% more potent than the reference product in a CDC assay.

27. A method according to any one of claims 22 to 26, wherein the population of fully human immunoglobulins is at least about 10% more potent than a reference product in a CD8+ cell killing assay.

28. A method according to any one of claims 22 to 27, wherein the population of complete human immunoglobulins is at least about 10% less than the reference product in a CD4+ cell apoptosis assay.

29. A method according to any one of claims 25 to 28, wherein the human polyclonal immunoglobulin specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

30. A method according to any one of claims 26 to 28, wherein the reference product is rabbit-derived ATG, optionally thymoglobulin.

31. A method according to any one of claims 26 to 28, wherein the reference product is horse-derived ATG, optionally ATGAM.

32. A method for providing anti-thymocyte globulin (ATG) therapy to a subject in need thereof, wherein the subject i) The composition according to any one of claims 1 to 9, ii) The composition according to any one of claims 10 to 21, iii) A composition produced by the method according to any one of claims 22 to 31 The process includes administering The method described above is a method for providing an effective amount of anti-thymocyte globulin (ATG) to the subject.

33. The method according to claim 32, wherein the subject is suffering from type 1 diabetes.

34. The method according to claim 32, wherein the subject is an organ transplant recipient.

35. The method according to claim 32, wherein the subject is suffering from or at risk of suffering from graft-versus-host disease.

36. The method according to claim 32, wherein the subject is a stem cell transplant recipient.

37. A pharmaceutical composition comprising a population of fully human or substantially human immunoglobulins and one or more pharmaceutically acceptable excipients, A pharmaceutical composition wherein the population of fully human or substantially human immunoglobulins specifically binds to human thymocytes, T cells, B cells, and / or monocytes.

38. A pharmaceutical composition according to claim 37, wherein the pharmaceutical composition has at least the same efficacy as a reference product in a CDC assay.

39. A pharmaceutical composition according to claim 37, wherein the pharmaceutical composition is at least about 10% more potent than the reference product in a CDC assay.

40. A pharmaceutical composition according to any one of claims 37 to 39, wherein the pharmaceutical composition is at least about 10% more potent than a reference product in a CD8+ cell killing assay.

41. A pharmaceutical composition according to any one of claims 37 to 40, wherein the pharmaceutical composition is at least about 10% less than the reference product in a CD4+ cell apoptosis assay.

42. A pharmaceutical composition according to any one of claims 38 to 41, wherein the reference product is rabbit-derived ATG, optionally thymoglobulin.

43. A pharmaceutical composition according to any one of claims 38 to 41, wherein the reference product is horse-derived ATG, or optionally ATGAM.

44. A pharmaceutical composition according to any one of claims 37 to 43, wherein the pharmaceutical composition contains at least 2% by mass of total immunoglobulins in the pharmaceutical composition of complete human or substantially human immunoglobulins.

45. A pharmaceutical composition according to any one of claims 35 to 44, wherein the pharmaceutical composition contains at least about 0.5 mg / mL, at least about 1 mg / mL, at least about 50 mg / mL, at least about 100 mg / mL, or at least about 1000 mg / mL of whole human or substantially human immunoglobulin.

46. A pharmaceutical composition according to any one of claims 35 to 45, wherein the fully human or substantially human immunoglobulin is produced in an ungulate.

47. A pharmaceutical composition according to any one of claims 35 to 46, wherein the ungulate is a cattle.

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