Recombinant polyclonal proteins and use methods thereof

Abstract

To provide recombinant polyclonal proteins and use methods thereof.SOLUTION: Provided herein is a composition comprising recombinant polyclonal proteins (RPPs) derived from mammalian plasma cells or plasmablasts. Methods for using the RPPs are also provided. The RPPs are recombinant, and their sequences are derived from peripheral blood plasma cells or plasmablasts. The peripheral blood plasma cells or plasmablasts are mobilized by, e.g., a vaccine administered to a donor, and the peripheral blood plasma cells or plasmablasts are specifically separated from other peripheral blood cells. The peripheral blood cells can come from any mammal, for example a mouse, a rat, a human, a monkey, a horse, or a cow.SELECTED DRAWING: None

Description

Technical Field

[0001] 1. Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Application No. 62 / 841,097, filed on April 30, 2019, the entire content of which is incorporated herein by reference for all purposes. 2. Sequence Listing

[0002] This application includes a sequence listing related to the sequence of 120158 submitted via EFS - Web, which is incorporated herein by reference in its entirety. The ASCII copy created on April 30, 2020, is named GGN035WOsequencelisting.txt and has a size of 30.3 MB. 3. Field

[0003] Provided herein are recombinant polyclonal antibody proteins, recombinant hyperimmune globulins, or recombinant polyclonal proteins (RPPs), also simply called recombinant hyperimmune, having binding specificity for antigens including vaccines, and libraries and compositions containing such RPPs, including pharmaceutical compositions. Also provided are methods for making RPPs and methods for using RPPs, for example, for therapeutic purposes.

Background Art

[0004] 4. Background The widespread use of active vaccines has greatly reduced the incidence of preventable infectious diseases, but vaccine failure due to low or no vaccine-induced immune responses remains an important problem. Certain populations, including the elderly or individuals with congenital humoral immunodeficiency, are particularly at risk of infection, and these weakened immune systems prevent the induction of an appropriate immune response to vaccine antigens. (D'Acremont et al., 2006; Jilkova et al., 2009; Weinberger et al., 2010; Langley et al., 2011; Cramer et al., 2016; Bader, 2007; Goldacker et al., 2007; van Assen et al., 2010). Poor responders ) are at very high risk of infection and will be hospitalized at a high rate, require antibiotic or antiviral therapy, or result in long-term illness or death. These patients would benefit from antibody replacement therapy that provides protective immunity as an alternative to failed vaccine modalities.

[0005] Passive immunization (McDonagh, 1966) provides an alternative defense strategy for individuals with immune deficiencies who do not respond to active vaccines. For example, intravenous immunoglobulin (IVIg) is a broad-spectrum polyclonal antibody therapy derived from the plasma of thousands of human donors. IVIg is used as antibody replacement therapy for patients with humoral immunodeficiency (Lucas et al., 2010; Resnick et al., 2012). However, IVIg has low titers for antibodies against many common pathogens and causes significant morbidity and mortality in immunocompromised patients (Orange et al., 2010). To increase anti-pathogen titers, some groups have developed high-titer plasma-derived antibodies, often referred to as hyperimmune. Hyperimmune is generally It is derived from the plasma of donors immediately after administration of an active vaccine such as HyperHEP B (Grifols), which has a high titer against hepatitis viruses.

[0006] Hyperimmune derived from donors recently administered an active vaccine is an excellent option for passive immunity, but scaling up such products commercially is challenging (Kreil et al., 2012). Importantly, it can be difficult to identify strong responders who consent to being vaccinated and repeatedly donate plasma. Thus, hyperimmune manufacturing lots are necessarily derived from different donor sets, resulting in variability between lots. The anti-pathogen titers vary considerably by hyperimmunization, from about 2- to 3-fold lower (Schampera et al., 2017) to about 50-fold higher (Kreil et al., 2012). Thus, in some cases, physicians may simply administer higher doses of IVIg (Polilli et al., 2012). Physicians and patients would benefit from a more consistent, higher-titer hyperimmune that is easier to manufacture on a large scale. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] 5. Summary ​​For example, provided herein are a novel library of RPPs (recombinant polyclonal antibody proteins, recombinant hyperimmune globulins, or recombinant polyclonal proteins also referred to as recombinant hyperimmune), having binding specificity for an antigen, such as a vaccine, and methods of using such RPPs, for example, as human therapeutics. RPPs are recombinants and these sequences are derived from peripheral blood plasmocytes or plasmablasts. Peripheral blood plasmocytes or plasmablasts are mobilized, for example, by a vaccine administered to a donor and are specifically separated from other peripheral blood cells. The peripheral blood cells can be derived from any mammal, such as a mouse, rat, human, monkey, horse, or cow.

[0008] RPPs specifically bind to an antigen. Examples include, but are not limited to, polysaccharides of Haemophilius influenzae type b, pneumococcal polysaccharides, hepatitis B virus antigen, or human thymocytes. Some RPP compositions are derived from plasmocytes or plasmablasts mobilized by a vaccine, for example, containing a protein antigen derived from a virus. In some embodiments, the vaccine is a mammalian cell, such as an immune cell or a cancer cell. In other embodiments, the vaccine is a killed or inactivated pathogen, such as a bacterium or a virus. In other embodiments, the vaccine is a bacterial polysaccharide. In some embodiments, the vaccine is an agent approved by the US Food and Drug Administration for the prevention of an infectious disease. In all embodiments, the vaccine mobilizes plasmocytes or plasmablasts in peripheral blood or causes plasmocytes or plasmablasts to be mobilized in peripheral blood.

[0009] The RPP library includes a mixture of RPPs, such as antibodies, and can be referred to as a polyclonal antibody. The mixture of antibodies can include 10, 100, 1,000, 10,000, 100,000 or more distinct antibody sequences. In some embodiments, the library includes RPPs having the homologous heavy chain CDR3 and light chain CDR3 sequences disclosed herein.

[0010] In some embodiments, the antibody is chimeric. In some embodiments, the antibody is humanized. In some embodiments, the antibody is a human antibody. In some embodiments, the RPP comprises a mixture of antibody fragments. In some embodiments, the RPP comprises a mixture of single-chain variable fragments (scFv). In some embodiments, the RPP comprises full-length antibodies. In some embodiments, the antibody is IgG, IgA, or IgM.

[0011] The RPPs provided herein can induce various biological effects related to the binding of the RPPs to antigens including vaccines. In some embodiments, the RPPs provided herein prevent the binding of the virus to cells and thereby prevent the entry of the virus into cells. In some embodiments, the provided RPPs bind to the cell surface of bacteria and enable the lysis of the bacteria by the immune system. In some embodiments, the RPP binds to the cell surface of the patient's cells to eliminate cells associated with the disease state. In some embodiments, the RPP binds to the surface of T cells to eliminate T cells associated with autoimmune diseases or graft-versus-host disease in transplantation.

[0012] Also provided are isolated polynucleotides encoding the RPPs provided herein and portions thereof. In some aspects, the invention provides a mixture of polynucleotides encoding the RPPs provided herein. In other aspects, the invention provides a mixture of vectors comprising the isolated polynucleotides. In other aspects, the invention provides a mixture of host cell clones comprising a mixture of polynucleotides or vectors.

[0013] Also provided are methods of producing RPPs using the polynucleotides, vectors or host cells provided herein. Some aspects of the invention relate to methods of producing RPPs comprising expressing an antibody in a host cell using a library of polynucleotide vectors and isolating the RPP.

[0014]

[0015] There is also provided a pharmaceutical composition comprising RPP and a pharmaceutically acceptable excipient.

[0016] Also provided are methods of using the RPP provided herein, for example, methods of treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the RPP provided herein or a pharmaceutical composition comprising such RPP. In some embodiments, the disease or condition is cancer or Alzheimer's disease. In some embodiments, the disease or condition is a viral or bacterial infection. In some embodiments, the method further comprises administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an immunostimulant or immunosuppressant. In some embodiments, the RPP is used to modulate the xenograft-versus-host or host-versus-xenograft response in a transplantation setting. In some embodiments, the RPP is used to modulate a viral disease in a transplantation setting.

[0017] In some embodiments, the RPP is in an amount sufficient to be a prophylactic against infectious diseases when administered to a subject. In some embodiments, the RPP is in an amount sufficient to clear an infection in an individual actively fighting an infection.

[0018] In yet a further aspect, the present invention is a method for generating a library of recombinant antibodies, comprising the steps of injecting an antigen related to hepatitis B virus (HBV) into a mammalian donor, isolating plasmablasts or plasma cells of the donor, and generating a library of recombinant antibodies from the plasmablasts or plasma cells, wherein the activity of the library of recombinant antibodies is at least 10-fold greater than the serum activity titer of the donor against the antigen. The mammalian donor may comprise two or more individuals. In one embodiment, the mammalian donor may be a human, mouse, humanized mouse, rat, humanized rat, horse, or cow. The method of the present invention can generate at least 100 recombinant antibodies, for example, at least 1,000 recombinant antibodies, for example at least 10,000 recombinant antibodies. In one embodiment, the method of the present invention can generate at least 100,000 recombinant antibodies.

[0019] In connection with the method of the present invention, the activity titer can be measured by an in vitro pathogen neutralization assay. Alternatively, the activity titer can be measured by an in vitro binding assay against the antigen. In one embodiment, the activity titer can be measured by an in vivo efficacy assay.

[0020] In one embodiment, the method of the present invention comprises the steps of obtaining a plurality of first linear polynucleotides, each of the first linear polynucleotides comprising a first sequence encoding a heavy chain variable domain derived from a cognate pair from a single plasma cell or plasmablast; and a second sequence encoding a light chain variable domain derived from the cognate pair; and a third sequence linking the first sequence and the second sequence and containing a restriction site; obtaining a second linear polynucleotide not operably linked to the first polynucleotide, the second linear polynucleotide comprising a fourth sequence homologous to a part of the first polynucleotide; circularizing each of the plurality of first polynucleotides with the second polynucleotide to generate a library of polynucleotides encoding a library of recombinant antibodies, wherein the circularization is effected by Gibson Assembly; and expressing the library of recombinant antibodies in mammalian cells containing the library of polynucleotides encoding the recombinant antibodies, thereby generating a library of recombinant antibodies.

Brief Description of Drawings

[0021] 6. Brief Description of Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0028] 7. DETAILED DESCRIPTION 7.1. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context dictates otherwise, singular terms shall include pluralities and plural terms shall include singulars. Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, as well as the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, are those well known and commonly used in the art. The methods and techniques of the present invention, unless otherwise indicated, generally are carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold See Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990) It is desired that these references be incorporated herein by reference. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, as commonly practiced in the art or as described herein. The terminology used in connection with the analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein, as well as the experimental procedures and techniques of the analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein, are well-known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0029] The following terms shall be construed to have the following meanings unless otherwise indicated:

[0030] The term "immunoglobulin" refers to a class of structurally related proteins that generally contain two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains. In "intact immunoglobulin", all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins is well-characterized. See, for example, Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain has a heavy chain variable region (V H ) and and generally includes a heavy chain constant region (C H ). The heavy chain constant region generally includes three domains, abbreviated as C H1 , C H2 and C H3 . Each light chain generally includes a light chain variable region (V L ) and a light chain constant region. The light chain constant region generally includes one domain, abbreviated as C L .

[0031] The term "recombinant polyclonal protein" (RPP) refers to a protein that includes two or more antigen-binding domains that specifically bind to an antigen or epitope(s). In some embodiments, the antigen-binding domains bind to the antigen or epitope with the same specificity and affinity as those of a naturally occurring antibody. In some embodiments, the RPP includes an antibody. In some embodiments, the RPP consists of an antibody. In some embodiments, the RPP consists essentially of an antibody. In some embodiments, the RPP includes an alternative scaffold. In some embodiments, the RPP consists of an alternative scaffold. In some embodiments, the RPP consists essentially of an alternative scaffold. In some embodiments, the RPP includes an antibody fragment. In some embodiments, the RPP consists of an antibody fragment. In some embodiments, the RPP consists essentially of an antibody fragment.

[0032] The term "antibody" is used herein in its broadest sense and includes a particular type of immunoglobulin molecule that includes one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is an antigen-binding domain formed by a V H -V L dimer. An antibody includes one type of RPP.

[0033] The term "alternate scaffold" refers to a molecule capable of diversifying one or more regions to generate one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds to an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. Exemplary alternate scaffolds include those derived from fibronectin (e.g., Adnectins™), those derived from β-sandwich (e.g., iMab), those derived from lipocalin (e.g., Anticalins®), those derived from EETI-II / AGRP, those derived from BPTI / LACI-D1 / ITI-D2 (e.g., the knotted domain), those derived from thioredoxin peptide aptamers, those derived from protein A (e.g., Affibody®), those derived from ankyrin repeats (e.g., DARPin), those derived from gamma-B-crystallin / ubiquitin (e.g., affilin), those derived from CTLD3 (e.g., tenectin), those derived from finomer, and those derived from (LDLR-A molecules) (e.g., avimer). Further information on alternate scaffolds is provided in Binz et al., Nat. Biotechnol., 2005 23:1257-1268; Skerra, Current Opin. in Biotech., 2007 18:295-304; and Silacci et al., J. Biol. Chem., 2014, 289:14392-14398, each of which is hereby incorporated by reference in its entirety. An alternate scaffold includes one type of RPP.

[0034] The term "antigen-binding domain" means the portion of an antibody capable of specifically binding to an antigen or epitope.

[0035] The terms "full-length antibody", "intact antibody", and "whole antibody" are used synonymously herein to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having a heavy chain that includes an Fc region.​

[0036] The term "Fc region" means, in the context of a naturally occurring antibody, the C-terminal region of the immunoglobulin heavy chain that interacts with certain proteins of the Fc receptor and the complement system. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125:S41-52, which is hereby incorporated by reference in its entirety. The F c region may be a naturally occurring Fc region or an Fc region modified as described elsewhere in this disclosure.

[0037] V H and V L regions can be further subdivided into regions of hypervariability (also called "hypervariable regions (HVRs)" or "complementary determining regions (CDRs)") in which more conserved regions are interspersed. The more conserved regions are called framework regions (FRs). V H and V L each generally contains three CDRs and four FRs, which are arranged in the following order (from the N-terminus to the C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, which is hereby incorporated by reference in its entirety.

[0038] Light chains from any vertebrate species can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the sequence of their constant domain.

[0039] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes), namely IgA, IgD, IgE, IgG and IgM. These classes are also denoted as α, δ, ε, γ and μ, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0040] One of ordinary skill in the art can determine the amino acid sequence boundaries of the CDRs using any of several known numbering schemes, such as Kabat et al., supra (the “Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the “Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the “Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the “IMGT” numbering sch eme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the “A Ho” numbering scheme), each of which is hereby incorporated by reference in its entirety.

[0041] Table 1 shows the CDR1-L (CDR1 of V L ), CDR2-L (CDR2 of V L ), CDR3-L (CDR3 of V L ), CDR1-H (CDR1 of V H ), CDR2-H (CDR2 of V H ), and CDR3-H (CDR3 of V HIt provides the positions of (CDR3). For CDR1-H, residue numbering is provided using both the Kabat numbering scheme and the Chothia numbering scheme.

[0042] The CDRs can be specified using antibody numbering software such as Abnum, which is described in Abhinandan and Martin, Immunology, 2008, 45:3832 - 3839, available, for example, at www.bioinf.org.uk / abs / abnum / , and the entire content is incorporated herein by reference.

Table 1

[0043] The "EU numbering scheme" is generally used when referring to residues in the constant region of the antibody heavy chain (e.g., as reported by Kabat et al., supra).

[0044] An "antibody fragment" includes a portion of an intact antibody, for example, the antigen - binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab’)2 fragments, Fab’ fragments, scFv (sFv) fragments, and scFv - Fc fragments.

[0045] An "Fv" fragment includes a dimer non - covalently linked by one heavy - chain variable domain and one light - chain variable domain.

[0046] A "Fab" fragment includes, in addition to the heavy - and light - chain variable domains, the constant domain of the light chain, and the first constant domain (C H1 ) of the heavy chain. A Fab fragment can be prepared, for example, by recombinant methods or by papain digestion of a full - length antibody.

[0047] The "F(ab’)2" fragment contains two Fab’ fragments that are linked by a disulfide bond and are located near the hinge region. The F(ab’)2 fragment can be prepared, for example, by recombinant methods or by pepsin digestion of intact antibodies. The F(ab’) fragment can be dissociated, for example, by treatment with β-mercaptoethanol.

[0048] The "single-chain Fv" or "sFv" or "scFv" antibody fragment contains a V H domain and a V L domain within a single polypeptide chain. The V H and V L are generally linked by a peptide linker. See Pluckthun A. (1994). In some embodiments , the linker is (GGGGS) n (SEQ ID NO: 5). In some embodiments, n = 1, 2, 3, 4, 5 or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore G.P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, which is hereby incorporated by reference in its entirety.

[0049] The "ScFv-Fc" fragment contains an scFv that is bound to an Fc domain. For example, the Fc domain may be bound to the C-terminus of the scFv. Depending on the orientation of the variable domains within the scFv, the Fc domain may follow the V H or V L (i.e., V H -V L or V L -V H ). Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain includes an IgG4 Fc domain.

[0050] The term "single domain antibody" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen in the absence of other variable domains. Single domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem.Sci., 2001, 26:230-245, and each of these references is hereby incorporated by reference in its entirety.

[0051] "Monospecific RPP" is an RPP that contains a binding site that specifically binds to a single epitope. An example of a monospecific RPP is a naturally occurring IgG molecule that is bivalent but recognizes the same epitope in each antigen-binding domain. The binding characteristics can exist with any suitable valence.

[0052] "Multispecific RPP" is an RPP that contains a binding site that non-specifically binds to two or more epitopes. An example of a multispecific RPP is a mixture of antibodies that bind to different serotypes of pneumococcal bacteria.

[0053] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. A substantially homogeneous population of antibodies includes antibodies that are substantially similar and that bind to the same epitope, excluding variants that may typically arise during the production of monoclonal antibodies. Such variants generally exist only in very small amounts. Monoclonal antibodies are generally obtained by a process that involves the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a pool of multiple clones, such as hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further engineered, for example, to improve its affinity for the target ("affinity maturation"), humanize the antibody, improve its production in cell culture, and / or reduce its immunogenicity in a subject.

[0054] The term "polyclonal antibody" refers to a mixture of at least two monoclonal antibodies. A polyclonal antibody may be monospecific or multispecific.

[0055] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0056] The "humanized" form of a non-human antibody is a chimeric antibody that contains the minimal sequence derived from the non-human antibody. Generally, a humanized antibody is a human antibody (recipient antibody) in which the residue(s) from one or more CDRs are replaced by the residue(s) from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken or non-human primate antibody having the desired specificity, affinity or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. A humanized antibody may also contain residues not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated herein by reference in its entirety.

[0057] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an amino acid sequence corresponding to an amino acid sequence utilizing a human antibody repertoire or a sequence encoding a human antibody (e.g., obtained from a human source or newly designed) from a non-human source. A human antibody specifically does not include a humanized antibody.

[0058] "Isolated RPP" or "isolated nucleic acid" is an RPP or nucleic acid that has been separated and / or recovered from the components of its natural environment. The components of the natural environment can include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In some embodiments, the isolated RPP is purified to a sufficient extent to obtain at least 15 residues of the N-terminal or internal amino acid sequence, for example, by using a spinning cup sequenator. In some embodiments, the isolated RPP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions using detection with Coomassie blue or silver stain. The isolated RPP includes the RPP in its original location within a recombinant cell because at least one component of the natural environment of the RPP is absent. In some aspects, the isolated RPP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, the isolated RPP or isolated nucleic acid is purified to at least 80 wt%, 85 wt%, 90 wt%, 95 wt% or 99 wt%. In some embodiments, the isolated RPP or isolated nucleic acid is purified to at least 80 vol%, 85 vol%, 90 vol%, 95 vol% or 99 vol%. In some embodiments, the isolated RPP or isolated nucleic acid is provided as a solution containing at least 85 wt%, 90 wt%, 95 wt%, 98 wt%, 99 wt% to 100 wt% RPP or nucleic acid. In some embodiments, the isolated RPP or isolated nucleic acid is provided as a solution containing at least 85 vol%, 90 vol%, 95 vol%, 98 vol%, 99 vol% to 100 vol% RPP or nucleic acid.

[0059] "Affinity" refers to the total strength of the non-covalent interactions between a single binding site of a molecule (e.g., RPP) and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity representing a 1:1 interaction between members of a binding pair (e.g., RPP and antigen or epitope). The affinity of molecule X for its partner Y is expressed as the dissociation equilibrium constant (K D) can be represented by. The kinetic components contributing to the dissociation equilibrium constant will be described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. For example, surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®) can be used to determine affinity.

[0060] With respect to the binding of RPP to a target molecule, the terms "binds to", "specifically binds to", "specifically binds with", "specific for", "selectively binds to", and "selective for" a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean a binding that is clearly distinct from non-specific or non-selective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring the binding to the target molecule and comparing it to the binding to non-target molecules. Specific binding can also be determined by competition with a control molecule that mimics the recognized epitope on the target molecule. In that case, specific binding is indicated when the binding of RPP to the target molecule is competitively inhibited by the control molecule.

[0061] The term "k d " (per second -1 ) when used herein refers to the dissociation rate constant of a particular ABP-antigen interaction. This value is also referred to as the k off value.

[0062] The term "k a " (M -1 × per second -1 ) when used herein refers to the association rate constant of a particular ABP-antigen interaction. This value is also referred to as the k on value.

[0063] The term "K D " (M) when used herein refers to the dissociation equilibrium constant of a particular ABP-antigen interaction. K D = k d / k a .

[0064] The term "K" A "(M" -1 ) when used in this specification refers to the association equilibrium constant of a particular ABP-antigen interaction. K A = k a / k d .

[0065] An "immunoconjugate" is an RPP conjugated with one or more heterologous molecules.

[0066] "Effector function" refers to biological activities mediated by the Fc region of an antibody, and these activities can vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding for activating complement-dependent cytotoxicity (CDC), and Fc receptor binding for activating antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP).

[0067] As used herein in connection with two or more RPPs, the terms "compete with" or "cross-compete with" indicate that two or more RPPs compete for binding to an antigen (e.g., a pneumococcal polysaccharide). In one exemplary assay, a surface is coated with a pneumococcal polysaccharide, contacted with a first pneumococcal polysaccharide RPP, and then a second pneumococcal polysaccharide RPP is added. In another exemplary assay, a surface is coated with a first pneumococcal polysaccharide RPP, contacted with a pneumococcal polysaccharide, and then a second pneumococcal polysaccharide RPP is added. If the presence of the first pneumococcal polysaccharide RPP reduces the binding of the second pneumococcal polysaccharide RPP in either assay, these RPPs compete. The term "compete with" also includes combinations of RPPs where one RPP reduces the binding of another RPP, but no competition is observed when the RPPs are added in the reverse order. However, in some embodiments, the first and second RPPs inhibit each other's binding regardless of the order in which they are added. In some embodiments, one RPP reduces the binding of another RPP to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. One of ordinary skill in the art can select the concentration of the antibodies used in the competition assay based on the affinity of the RPP for the pneumococcal polysaccharide and the valence of the RPP. The assays described in this definition are for illustrative purposes, and one of ordinary skill in the art can utilize any suitable assay to determine whether the antibodies compete with each other. Suitable assays are described in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358, and this Each of these is hereby incorporated by reference in its entirety.

[0068] The term "epitope" means the portion of an antigen that specifically binds to RPP. Epitopes often consist of surface-exposed amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational epitopes and non-conformational epitopes are distinguished by the fact that binding to the former conformational epitopes may be lost in the presence of a denaturing solvent, while binding to the latter non-conformational epitopes is not. An epitope can include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. The epitope to which RPP binds can be determined using known epitope determination techniques, such as testing for RPP binding to pneumococcal polysaccharide serotypes.

[0069] The "percent identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignments for determining amino acid sequence identity percentage can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0070] "Conservative substitution" or "conservative amino acid substitution" refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Tables of conservative substitutions that result in similar amino acids are well known in the art. As an example, the groups of amino acids provided in Tables 2 - 4 are considered to be conservative substitutions for one another in some embodiments.

Table 2-1

Table 2-2

Table 3

Table 4

[0071] Additional conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY. An RPP generated by making one or more conservative substitutions of amino acid residues in the parent RPP is referred to as a "conservatively modified variant".

[0072] The term "treating" (and its inflected forms, e.g., "treat" or "treatment") refers to a clinical intervention performed with the purpose of altering the natural course of a disease or condition in a subject in need thereof. Treatment may be performed for disease prevention or during the course of a clinical pathology examination. Desirable effects of treatment include preventing the occurrence or recurrence of a disease, alleviating symptoms, ameliorating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the condition, and remission, or improvement of prognosis.

[0073] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an RPP or pharmaceutical composition provided herein that is effective to treat a disease or disorder when administered to a subject.

[0074] As used herein, the term "subject" means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, female cows, horses, camels, goats, rabbits and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with the RPP provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection.

[0075] The term "package insert" is used to refer to the instructions customarily included within the commercial package of a therapeutic or diagnostic product (e.g., a kit) that contain information regarding indications, usage, dosage, administration, combination therapies, contraindications and / or warnings regarding the use of such therapeutic or diagnostic product.

[0076] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0077] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include "anti-hormonal agents" or "endocrine therapies" that act to modulate, decrease, block or inhibit the effects of hormones that can promote cancer growth.

[0078] The term "cytostatic agent" refers to a compound or composition that inhibits cell growth either in vitro or in vivo. In some embodiments, the cytostatic agent is an agent that reduces the percentage of cells in the S phase. In some embodiments, the cytostatic agent reduces the percentage of cells in the S phase by at least about 20%, at least about 40%, at least about 60%, or at least about 80%.

[0079] The term "tumor" refers to any neoplastic cell growth and proliferation, whether malignant or benign, and also refers to any pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cancer proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive when referred to in this specification. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with a degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer.

[0080] The term "pharmaceutical composition" refers to a preparation in a form that enables the biological activity of the active ingredient contained therein to be effective for the treatment of a subject, and that does not contain additional ingredients that are unacceptably toxic to the subject.

[0081] The terms "modulate" and "modulation" refer to reducing or inhibiting the variable element described, or alternatively, activating or increasing it.

[0082] The terms "increase" and "activate" refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater of the variable element described.

[0083] The terms "decrease" and "inhibit" refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% of the variable element described to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100 or greater.

[0084] The term "activate" refers to the activation of receptor signaling to induce a biological response associated with the activation of a receptor. An "agonist" is an entity that binds to and activates a receptor.

[0085] The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is an entity that binds to a receptor and antagonizes it.

[0086] The term "effector T cell" includes helper T (i.e., CD4 + ) cells and cytotoxic (i.e., CD8 + ) T cells. CD4 + effector T cells contribute to the occurrence of several immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. CD8 + effector T cells destroy virus-infected cells and tumor cells. For further information regarding effector T cells, see Seder and Ahmed, Nature Immunol., 2003, 4:835-842, which is hereby incorporated by reference in its entirety.

[0087] The term "regulatory T cell" includes cells that regulate immunological tolerance, for example by suppressing effector T cells. In some embodiments, regulatory T cells have a CD4 + CD25 + Foxp3 + phenotype. In some embodiments, regulatory T cells have a CD8 + CD25 + phenotype. For further information regarding regulatory T cells, see Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099, which is hereby incorporated by reference in its entirety. See.

[0088] The term "dendritic cell" refers to professional antigen-presenting cells that can activate naive T cells and can stimulate the growth and differentiation of B cells.

[0089] The term "plasma cell" refers to white blood cells that secrete large amounts of antibodies. These are transported by the plasma and the lymphatic system. B cells (either germinal center naive B cells or memory B cells, for example) differentiate into plasma cells and produce antibody molecules that precisely model the receptors of precursor B cells. Once released into the blood and lymph, these antibody molecules bind to target antigens (foreign substances in the body) and initiate their neutralization or destruction. Plasma cells that have differentiated to the end express relatively few surface antigens and do not express common pan-B cell markers such as CD19 and CD20. Instead, plasma cells are identified by flow cytometry by the additional expression of CD138, CD78, and the interleukin 6 receptor. In humans, CD27 is an excellent marker for plasma cells, naive B cells are CD27-, memory B cells are CD27+, and plasma cells are CD27++. The surface antigen CD138 (syndecan-1) is expressed at high levels. Another important surface antigen is CD319 (SLAMF7). This antigen is expressed at high levels in normal human plasma cells. This antigen is also expressed in malignant plasma cells in multiple myeloma. Compared with CD138, which rapidly disappears ex vivo, the expression of CD319 is quite stable.

[0090] The term "plasmablast" refers to antibody-secreting cells in peripheral blood that differentiate from activated B cells such as memory B cells upon stimulation by an antigen. Plasmablasts are the most immature blood cells considered to be of the plasma cell lineage. Plasmablasts secrete more antibodies than B cells but fewer than plasma cells. Plasmablasts divide rapidly and are still able to internalize antigens and present them to T cells. The cells remain in this state for several days and then either die or irreversibly differentiate into mature, fully differentiated plasma cells. The differentiation of mature B cells into plasma cells depends on the transcription factors Blimp-1 / PRDM1 and IRF4.

[0091] The term "memory B cell" refers to a B cell subtype formed within germinal centers after a primary infection, which is important in generating an accelerated and more potent antibody-mediated immune response (also known as the secondary immune response) upon reinfection. Memory B cells do not secrete antibodies until they are activated by their specific antigen.

[0092] The term "naive B cell" refers to a B cell that has not been exposed to an antigen. Once exposed to an antigen, a naive B cell will become either a memory B cell or a plasma cell that secretes antibodies specific to the antigen it originally bound to. Plasma cells do not persist long in the circulating blood, in contrast to memory cells which persist for a very long time.

[0093] The term "titer" refers to a measure of how much antibody a living organism produces that recognizes a specific epitope or antigen, and is expressed as the reciprocal of the highest dilution (in serial dilutions) that still gives a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common means of determining antibody titers.

[0094] The term "peripheral blood" refers to the blood that moves through the peripheral blood vessels. Peripheral blood is generally obtained by venipuncture (also called phlebotomy), or by pricking a finger with a needle for a small amount.

[0095] The term "vaccine" refers to an agent that stimulates the body's immune system, is recognized by the immune system as a threat, is destroyed, and further recognizes and destroys any of the microorganisms associated with that agent that the body may encounter in the future. The term "vaccine" can refer to a biological preparation that confers active acquired immunity against a specific disease. Vaccines often contain an agent that resembles the disease-causing microorganism and are often made from a weakened or killed form of the microorganism, its toxin, or one of its surface proteins. Vaccines can be prophylactic (e.g., preventing or ameliorating the effects of future infection by a natural or "wild" pathogen) or therapeutic (e.g., vaccines against cancer are being studied). More generally, the term "vaccine" can refer to any agent that induces an immune response. For example, cancer cells can be used to vaccinate an individual against a specific cancer antigen. Some vaccines contain inactivated, formerly toxic microorganisms that have been destroyed by chemicals, heat, or radiation. Examples include the polio vaccine, hepatitis A vaccine, rabies vaccine, and some influenza vaccines. Some vaccines contain live, attenuated microorganisms. Many of these are active viruses that are cultured under conditions that either disable their toxic properties or use closely related but less dangerous organisms and produce a broad immune response. The most attenuated vaccines are viral, but in fact, some are bacterial. Examples include the viral diseases yellow fever, measles, mumps, and rubella, and the bacterial disease typhoid fever. The live Mycobacterium tuberculosis vaccine developed by Calmette and Guerin does not contain an infectious strain but contains a strain called "BCG" that has been modified by a virulent method used to induce an immune response to the vaccine. A live attenuated vaccine containing the Yersinia pestis EV strain is used for immunization against the plague. Attenuated vaccines have several advantages and disadvantages.Attenuated vaccines generally elicit a more sustained immunological response and are a preferred type for healthy adults. However, attenuated vaccines may not be safe for use in immunocompromised individuals and, very rarely, mutate into a virulent form and cause disease. Toxoid vaccines are made from inactivated toxin compounds that cause disease rather than the microorganisms themselves. Examples of toxoid-based vaccines include tetanus and diphtheria. Toxoid vaccines are known for their effectiveness. Not all toxoids are against microorganisms; for example, a Crotalus atrox toxoid is used to vaccinate dogs against rattlesnake bites. In protein subunit vaccines, rather than introducing inactivated or attenuated microorganisms (which would constitute a "whole-agent" vaccine) to the immune system, fragments thereof can elicit an immune response. Examples include subunit vaccines against hepatitis B virus composed only of the virus's surface proteins (previously extracted from the sera of chronically infected patients but now produced by recombinant of viral genes into yeast), or edible algal vaccines, virus-like particle (VLP) vaccines against human papillomavirus (HPV) composed of the virus's major capsid protein, and hemagglutinin and neuraminidase subunits of influenza virus. As conjugate vaccines, certain bacteria have a polysaccharide outer coat that is poorly immunogenic. By linking these coats to proteins (e.g., toxins), the immune system can be made to recognize the polysaccharide as if it were a protein antigen. This approach is used in the Haemophilus influenzae type B vaccine. Dendritic cell vaccines combine dendritic cells with antigens in order to present the antigens to white blood cells in the body and thereby stimulate an immune reaction. These vaccines have shown some positive preliminary results regarding the treatment of brain tumors and are also being tested in malignant melanoma. In recombinant vector vaccines, combining the physiology of one microorganism with the DNA of another can confer immunity against diseases with complex infection processes.An example is the RVSV-ZEBOV vaccine, authorized for Merck, which was used in 2018 to fight Ebola hemorrhagic fever in the Congo. An experimental approach to vaccination, called DNA vaccination, is being developed that is made from the DNA of the vector. The proposed mechanism is the insertion (and expression, enhanced by the use of electroporation, which induces recognition by the immune system) of viral or bacterial DNA into human or animal cells. Some cells of the immune system that recognize the expressed proteins initiate an attack against these proteins and the cells that express them. Since these cells survive for a very long time, pathogens that normally express these proteins will be encountered at a later time and will be immediately attacked by the immune system. One possible advantage of DNA vaccines is that they are very easy to produce and store. Vaccines can be monovalent (also called univalent) or multivalent (also called polyvalent). Monovalent vaccines are designed to immunize against a single antigen or a single microorganism. Multivalent or polyvalent vaccines are designed to immunize against two or more strains of the same microorganism or against two or more microorganisms. The valence of a multivalent vaccine may be indicated by a Greek or Latin prefix (e.g., tetravalent or quadrivalent). In certain cases, monovalent vaccines may be preferred for quickly eliciting a strong immune response.

[0096] The term "hyperimmune" refers to a polyclonal antibody preparation similar to intravenous immunoglobulin (IVIg), except that it is prepared from the plasma of donors with high - titer antibodies against specific organisms or antigens. The term "hyperimmune" is often used interchangeably with the terms "hyperimmune gamma globulin" and "hyperimmune globulin". Some of the agents for which hyperimmune globulin can be utilized include hepatitis B, rabies, tetanus toxin, varicella - zoster, etc. Administration of hyperimmune globulin provides "passive" immunity in the patient against the agent. This is in contrast to vaccines, which provide "active" immunity. However, while vaccines take much longer to achieve this goal, hyperimmune globulin provides immediate and "passive" short - term immunity.

[0097] The term "in vivo" is interpreted as "in the living body" and refers to scientific studies in which the effects of various biological entities are tested in living organisms or whole cells, usually animals, including humans, and plants, as opposed to tissue extracts or dead organisms. This should not be confused with experiments conducted in vitro ( "in the test tube"), i.e., in a laboratory environment using test tubes, Petri dishes, etc. Examples of in vivo investigations include the etiology of diseases by comparing the effects of a bacterial infection with the effects of a purified bacterial toxin; generally, the development of non - antibiotics, antiviral drugs, and new drugs; and new surgical procedures. In conclusion, animal testing and clinical trials are the main elements of in vivo investigations. In vivo testing is used more frequently than in vitro because it is more suitable for observing the overall effects of experiments on living subjects.

[0098] The term "activity" refers to the quantitative measurement of RPP or antibodies against an antigen, vaccine, protein, epitope, cell, bacterium, or virus. Activity can be evaluated using in vivo or in vitro methods.

[0099] The term "recombinant" refers to a protein that results from the expression of recombinant DNA in living cells. Recombinant DNA is a general name for a single DNA created by the combination of at least two separate segments of DNA.

[0100] The term "in vitro" is interpreted as "in a test tube" and refers to scientific studies conducted on microorganisms, cells, or biomolecules outside of their normal biological contexts. The so-called "test tube experiments," these studies in biology and the sub-disciplines of that field of study are traditionally carried out within experimental apparatuses such as test tubes, flasks, Petri dishes, and microtiter plates. Studies conducted using components of organisms isolated from their normal biological environments allow for more detailed or more convenient analysis than can be done on the whole organism, however, the results obtained from in vitro experiments may sometimes not be able to fully or accurately predict the effects on the whole organism. In contrast to experiments in vitro, studies in vivo are conducted on animals, including humans, and on whole plants.

[0101] The term "neutralization" refers to the ability of specific antibodies to block the sites that viruses use to enter their target cells. The effects of neutralizing antibodies may be negligible even when these are produced in excess if they lack specificity for this antigen. The production of specific antibodies can be learned for a more rapid response upon subsequent exposure. The reduction or destruction of the agent of the same source may be partial or complete and can render it no longer infectious or pathogenic to other cells.

[0102] A "variant" of a polypeptide (e.g., an antibody) contains an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted in the amino acid sequence as compared to the native polypeptide sequence, and retains essentially the same biological activity as the native polypeptide. The biological activity of a polypeptide can be measured using standard techniques in the art (e.g., if the variant is an antibody, its activity can be tested by the binding assays described herein). Variants of the invention include fragments, analogs, recombinant polypeptides, synthetic polypeptides, and / or fusion proteins.

[0103] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation with another chemical moiety such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term "antibody" includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutant proteins thereof, examples of which are described below.

[0104] A nucleotide sequence is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can, for example, exert its effect directly on the nucleic acid being regulated or can exert its effect through the action of one or more molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

[0105] A "host cell" is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the invention. The host cell can be a prokaryote, e.g., E. coli, or a eukaryote, e.g., a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells) or a hybridoma. Examples of host cells include CS-9 cells, the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or derivatives thereof, e.g., Veggie CHO and related cell lines that grow in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31 cells to be irradiated), HeLa cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line (ATCC CCL 70) derived from African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10:2821), human fetal-derived kidney cells , for example, 293, 293 EBNA or MSR 293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, primary tissues, cell lines derived from in vitro culture of primary explants, HL-60, U937, Hak or Jurkat cells. Typically, the host cell is a cultured cell that can be transformed with or transfected with a nucleic acid encoding a polypeptide, and thus such nucleic acid can be expressed in the host cell.

[0106] The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed with or transfected with a nucleic acid to be expressed. The host cell can be a cell that contains the nucleic acid but does not express the nucleic acid at the desired level unless regulatory sequences are introduced into the host cell such that the regulatory sequences are operably linked to the nucleic acid. It will be understood that the term host cell refers not only to a particular target cell but also to the progeny or potential progeny of such a cell. For example, such progeny may not actually be identical to the parental cell due to mutations or environmental influences that may occur in subsequent generations, but nevertheless are still included within the scope of this term as used herein. 7.2. Other Interpretation Provisions

[0107] The ranges described in this specification are to be construed as a shorthand for all values within the range including the recited groups. For example, the range of 1 to 50 is to be construed as including any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.

[0108] Unless otherwise indicated, reference to a compound having one or more stereocenters is intended to include each stereoisomer of the compound, and all combinations of the stereoisomers of the compound. 7.3.RPP and RPP libraries

[0109] Each member of the RPP libraries described herein is a polypeptide that specifically binds to an antigen, e.g., an antibody or an antibody fragment. In some embodiments, the RPP includes cognate pairs of the heavy and light chain CDR3 sequences disclosed herein. In some embodiments, the RPP is a scFv. In some embodiments, the RPP is a full-length antibody.

[0110] In some embodiments, the RPP is an antibody fragment. A Fab fragment is a monovalent fragment having V L , V H , C L and C H1 domains; an F(ab’)2 fragment is a divalent fragment having two Fab fragments linked by disulfide bridges in the hinge region; an Fd fragment has V H and C H1 domains; an Fv fragment has V L and V H domains of a single arm of an antibody; a dAb fragment has V H domain, V L domain, or V H or V LHaving antigen-binding fragments of domains (U.S. Patent Nos. 6,846,634, 6,696,245, U.S. Patent Application Publication Nos. 2005 / 0202512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958, Ward et al., Nature 341:544-546, 1989).

[0111] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions, also called complementarity-determining regions or CDRs. Both light and heavy chains, from the N-terminus to the C-terminus, contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain follow the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Department of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.

[0112] The term "human antibody", also referred to as a "fully human antibody", includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (fully human antibodies). These antibodies can be prepared by a variety of methods, including immunization of a mouse genetically modified to express antibodies derived from human heavy and / or light chain-encoding genes with the antigen of interest, examples of which are described below.

[0113] A humanized antibody has a sequence that differs from the sequence of an antibody derived from a non-human species in terms of one or more amino acid substitutions, deletions, and / or additions. Thus, a humanized antibody, when administered to a human subject, is likely to induce a lower and / or less intense immune response compared to a non-human species antibody. In one embodiment, certain amino acids within the framework and constant domains of the heavy and / or light chains of a non-human species antibody are mutated to produce a humanized antibody. In another embodiment, a constant region from a human antibody is fused to the variable domain of a non-human species. In another embodiment, one or more amino acid residues within one or more CDR sequences of a non-human antibody are changed to reduce the putative immunogenicity of the non-human antibody when it is administered to a human subject, and the amino acid residues being changed are not critical for the immunospecific binding of the antibody to its antigen or the changes to the added amino acid sequence are conservative changes, and thus the binding of the humanized antibody to its antigen is not significantly worse than the binding of the non-human antibody to its antigen. Examples of methods for making humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0114] One of ordinary skill in the art can readily prepare antibody fragments or analogs according to the teachings herein and using techniques well known in the art. The preferred amino and carboxy termini of the fragments or analogs are located near the boundaries of the functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or private sequence databases. Using computerized comparison methods, sequence motifs or predicted protein three-dimensional structural domains present in other proteins of known structure and / or function can be identified. Methods for identifying protein sequences that fold into known three-dimensional structures are also known. See, for example, Bowie et al., 1991, Science 253:164.

[0115] The RPP can be any synthetic protein or genetically engineered protein. For example, antibody fragments include isolated fragments consisting of the light chain variable region; "Fv" fragments consisting of the variable regions of the heavy and light chains; and recombinant single-chain polypeptide molecules (scFv proteins) in which the light and heavy chain variable regions are connected by a peptide linker.

[0116] Another form of antibody fragment is a peptide that contains one or more complementarity-determining regions (CDRs) of an antibody. The CDRs (also called "minimal recognition units" or "hypervariable regions") can be incorporated into a molecule, either covalently or non-covalently, to make that molecule an antigen-binding protein. CDRs can be obtained by constructing a polynucleotide that encodes the CDR of interest. Such polynucleotides can be prepared, for example, by using the mRNA of antibody-producing cells as a template and synthesizing the variable region using the polymerase chain reaction (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995)).

[0117] The variable region domain of the RPP may be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain created using recombinant DNA engineering techniques. Such engineered versions include those created, for example, from a particular antibody variable region by insertions, deletions or changes in or to the amino acid sequence of a particular antibody. Particular examples include engineered variable region domains that contain at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.

[0118] The variable region domain can be covalently linked to the C-terminal amino acid of at least one other antibody or fragment thereof. Thus, for example, the V H The V domain can be linked to an immunoglobulin CH1 domain or a fragment thereof. L The domain can be linked to a CK domain or a fragment thereof. Thus, for example, an antibody can be provided in which the antigen-binding domain is linked to the associated V H and V L The fragment may be a Fab fragment containing the CH1 and CK domains, the C-terminus of which is covalently linked to the CH1 and CK domains, respectively. The CH1 domain may be extended with additional amino acids to provide, for example, a hinge region or a portion of a hinge region domain as found in a Fab' fragment, or to provide additional domains such as antibody CH2 and CH3 domains.

[0119] As described herein, the RPPs include cognate pairs of the heavy and light chain CDR3 sequences disclosed herein. For example, the CDRs can be incorporated into known antibody framework regions (such as IgG1, IgG2, etc.), or conjugated to a suitable vehicle to extend its half-life. Suitable vehicles include, but are not limited to, Fc, polyethylene glycol (PEG), albumin, transferrin, and the like. These and other suitable vehicles are known in the art. Such conjugated CDR peptides can be in monomeric, dimeric, trimeric or other forms. In one embodiment, one or more water-soluble polymers are attached at one or more specific positions of the binder, for example, at the amino terminus.

[0120] In certain embodiments, the antibody in the RPPs includes one or more water-soluble polymers that are attached and include, but are not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337. In certain embodiments, the derivative binder includes one or more of methoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), and polyvinyl alcohol, and mixtures of such polymers. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains. In certain embodiments, PEG can act to improve the therapeutic ability of a binder such as an antibody. One such method is discussed, for example, in U.S. Pat. No. 6,133,426, which is hereby incorporated by reference herein for all purposes.

[0121] The RPP may have, for example, the structure of a naturally occurring immunoglobulin. An "immunoglobulin" is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, with each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 to 110 or more amino acids that is mainly responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is mainly responsible for effector functions. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids. Generally, see Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light chain / heavy chain pair form an antibody binding site such that a native immunoglobulin has two binding sites. For all purposes).

[0122] Various RPPs can bind to various domains of a disease target and act by different mechanisms of action. In particular, as shown herein, the domain regions are designed to include groups unless otherwise indicated. For example, amino acids 4 - 12 refer to 9 amino acids: the amino acids at positions 4 and 12, and 7 intervening amino acids in the sequence. Another example is an antigen-binding protein that inhibits the binding of a pathogen to its target cell, i.e., neutralizing activity. The antigen-binding protein does not need to completely inhibit binding to the target cell for use in the present invention.

[0123] The RPPs described herein may include an FC region, e.g., a dimeric Fc polypeptide. One preferred Fc polypeptide is described in PCT application WO93 / 10151 (incorporated herein by reference) and is a single-chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent No. 5,457,035 and Baum et al., 1994, EMBO J. It is the Fc mutein described in 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence shown in WO93 / 10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. The mutein shows a reduced affinity for the Fc receptor.

[0124] Antigen-binding fragments of the RPPs of the present invention can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F(ab’)2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also contemplated.

[0125] Additional embodiments include chimeric antibodies, e.g., humanized versions of non-human (e.g., murine) monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and offer the advantage of reduced immunogenicity when administered to humans. In one embodiment, the humanized antibody comprises the variable domain (or all or a portion of its antigen-binding site) of a murine antibody and the constant domain derived from a human antibody. Alternatively, the humanized antibody fragment may comprise the antigen-binding site of a murine antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered antibodies include those described in Riechmann et al., 1988, Nature 332:323, Liu et al., 1987, Proc. Nat. Acad. Sci. USA 84:3439, Larrick et al., 1989, Bio / Technology 7:934, and Winter et al., 1993, TIPS 14:139 . In one embodiment, the chimeric antibody is a CDR-grafted antibody. Techniques for humanizing antibodies are discussed, e.g., in U.S. Pat. Nos. 5,869,619, 5,225,539, 5,821,337, 5,859,205, 6,881,557, Padlan et al., 1995, FASEB J. 9:133-39, and Tamura et al., 2000, J. Immunol. 164:1432-41

[0126] The procedures have been developed to generate human or partially human antibodies in non-human animals. For example, mice have been prepared in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes are introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animals are incorporated into human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animals. In one embodiment, a non-human animal such as a transgenic mouse is immunized with a vaccine such that antibodies directed against the vaccine antigen are produced in the animal.

[0127] Examples of techniques for the production of human or partially human antibodies and the use of transgenic animals for their production are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, Davis et al., 2003, Production of human antibodies from transgenic mice in Lo, ed. Antibody Engineering: Methods and Protocols, Humana Press, NJ:191-200, Kellermann et al., 2002, Curr Opin Biotechnol. 13:593-97, Russel et al., 2000, Infect Immun. 68:1820-26, Gallo et al., 2000, Eur J Immun. 30:534-40, Davis et al., 1999, Cancer Metastasis Rev. 18:421-25, Green, 1999, J Immunol Methods. 231:11-23, Jakobovits, 1998, Advanced Drug Delivery Reviews 31:33-42, Green et al., 1998, J Exp Med. 188:483-95, Jakobovits A, 1998, Exp. Opin. Invest. Drugs. 7:607-14, Tsuda et al., 1997, Genomics. 42:413-21, Mendez et al., 1997, Nat Genet. 15:146-56, Jakobovits, 1994, Curr Biol. 4:761-63, Arbones et al., 1994, Immunity. 1:247-60, Green et al., 1994, Nat Genet. 7:13-21, Jakobovits et al., 1993, Nature. 362:255-58, Jakobovits et al., 1993, Proc Natl Acad Sci U S A 90:2551-55, Chen, J., M. Trounstine, F. W. Alt, F. Young, C. Kurahara, J. Loring, D. Huszar. Inter'l Immunol. 5(1993): 647-656, Choi et al., 1993, Nature Genetics 4: 117-23, Fishwild et al., 1996, Nature Biotech. 14: 845-51, Harding et al., 1995, Annals of the New York Academy of Sciences, Lonberg et al., 1994, Nature 368: 856-59, Lonberg, 1994, Transgenic Approaches to Human Monoclonal Antibodies in Handbook of Experimental Pharmacology 113: 49-101, Lonberg et al., 1995, Internal Review of Immunology 13: 65-93, Neuberger, 1996, Nature Biotechnology 14: 826, Taylor et al., 1992, Nucleic Acids Res. 20: 6287-95, Taylor et al., 1994, Inter'l Immunol. 6: 579-91, Tomizuka et al., 1997, Nature Genetics 16: 133-43, Tomizuka et al., 2000, Pro. Nat'l Acad. Sci. USA 97: 722-27, Tuaillon et al., 1993 Pro.Nat'l Acad.Sci. USA 90: 3720-24, and Tuaillon et al., 1994, J.Immunol. 152: 2912-20.

[0128] The RPPs of the present invention (e.g., antibodies, antibody fragments, and antibody derivatives) may include any constant region known in the art. The light chain constant region may be, for example, a kappa or lambda light chain constant region, such as a human kappa or lambda light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu heavy chain constant region. In one embodiment, the light chain or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.

[0129] Techniques for inducing antibodies of various subclasses or isotypes from a target antibody, i.e., subclass switching, are known. Thus, an IgG antibody may, for example, be derived from an IgM antibody, and vice versa. Such techniques enable the preparation of new antibodies having the antigen-binding properties of a given antibody (parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques may be used. Cloned DNA encoding a specific antibody polypeptide, for example, DNA encoding the constant domain of an antibody of a desired isotype, can be used in such procedures. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16. Please refer to.

[0130] A single-chain antibody (scFv) can be formed by linking heavy and light chain variable domain (Fv region) fragments by an amino acid bridge (a short peptide linker, e.g., a synthetic sequence of amino acid residues) to obtain a single polypeptide chain. Such single-chain Fv (scFv) is prepared by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (V L and V H ). Depending on the length of the flexible linker between the two variable domains, the resulting polypeptide can fold itself to form an antigen-binding monomer or form multimers (e.g., dimers, trimers, or tetramers) (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108, Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Polypeptides containing various V L and V H can be combined By doing so, multimeric scFvs that bind to various epitopes can be formed (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). For the production of single-chain antibodies Techniques developed include those described in U.S. Patent No. 4,946,778, Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544; de Graaf et al., 2002, Methods Mol Biol. 178:379-87.

[0131] In certain embodiments, the invention includes RPPs generated from libraries of antibodies encoding expression vectors. The RPPs include 10, 100, 1,000, 10,000, or more than 100,000 distinct antibody sequences. In certain embodiments, the RPPs are generated from mammalian cells recombinantly engineered using antibody sequences encoded by a single plasma cell or plasmablast. In certain embodiments, the RPPs are polyvalent in that they include antibodies having various antigen-binding properties. In some embodiments, the RPPs bind to multiple epitopes on a target antigen. In some embodiments, the RPPs bind to multiple antigens. 7.4. CDR3 Sequences of RPPs

[0132] CDR3H (heavy chain immunoglobulin) and CDR3L (light chain immunoglobulin) polypeptide sequences, each member of 12 RPPs generated using the methods described herein, are provided in the Sequence Listing. A summary of the sequences is provided in Table 5. The sequences can be found in the Sequence Listing submitted with this application. The RPPs provided herein are generated from humanized mice that fully express human V(D)J antibody sequences using human thymocytes or human T cells as immunogens. The RPPs provided herein are generated from vaccinated human donors using pneumococcal polysaccharides, influenza A virus antigens, hepatitis B virus antigens, or polysaccharides of Haemophilus influenzae type B. The RPPs contain between 1,141 and 10,537 unique antibodies. [Table 5]

[0133] An oligopeptide or polypeptide is within the scope of the invention if it has an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at least one of the CDRs provided herein.

[0134] 7.5. Nucleic Acids

[0135] In one aspect, the present invention provides an isolated nucleic acid molecule. The nucleic acid encodes, for example, all or part of an RPP, such as one or both chains of the antibodies of the present disclosure, or fragments, derivatives, mutant proteins, or variants thereof; a polynucleotide sufficient for use as a hybridization probe, PCR primer, or sequencing primer to identify, analyze, mutate, or amplify a polynucleotide encoding a polypeptide; an antisense nucleic acid for inhibiting the expression of a polynucleotide; and complementary sequences of the foregoing. The nucleic acid can be of any length. The nucleic acid can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 nucleotides or longer in length, and / or can include one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. The nucleic acid can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0136] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, CDRs only, or full length) can be isolated from B cells of mice immunized with a vaccine. The nucleic acids can be isolated by conventional procedures such as polymerase chain reaction (PCR).

[0137] The polypeptide sequences of CDR3 derived from the variable regions of the heavy and light chain variable regions are shown herein. Due to the degeneracy of the genetic code, it will be understood by those skilled in the art that each of the polypeptide sequences disclosed herein is encoded by a number of other nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding each RPP of the present invention.

[0138] Methods for hybridizing nucleic acids are well known in the art. See, for example, Curr. Prot. in Mol. Biol., John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization ation conditions are a prewash solution containing 5× sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of approximately 50% formamide, 6× SCC, and a hybridization temperature of 55° C. (or other similar hybridization solutions, e.g., those containing approximately 50% formamide, and a hybridization temperature of 42° C.); and washing conditions of 0.5× SSC, 0.1% SDS at 60° C. Stringent hybridization conditions are hybridization in 6× SSC at 45° C., followed by washing one or more times in 0.1× SSC, 0.2% SDS at 68° C. Furthermore, one of ordinary skill in the art can increase or decrease the stringency of hybridization such that nucleic acids containing nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% identical to each other generally maintain a hybridized state with each other by manipulating the hybridization and / or washing conditions. Basic parameters that affect the choice of hybridization conditions, and guidance for devising suitable conditions, are provided, for example, by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Curr. Prot. in Mol. Biol. 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and one of ordinary skill in the art can use, for example, D It can be easily determined based on the length and / or base composition of NA.

[0139] Mutations can be introduced into the nucleic acid, thereby causing changes in the amino acid sequence of the polypeptide encoded by the nucleic acid (e.g., RPP). Any technique known in the art can be used to introduce mutations. In one embodiment, for example, site-directed mutagenesis protocols are used to change one or more specific amino acid residues. In another embodiment, for example, random mutagenesis protocols are used to change one or more randomly selected residues. However it is done, mutant polypeptides can be expressed and screened for desired properties (e.g., binding to a virus).

[0140] In another aspect, the present invention provides a nucleic acid molecule suitable for use as a primer or hybridization probe for the detection of the nucleic acid sequences of the present invention. The nucleic acid molecules of the present invention may comprise only a portion of the nucleic acid sequence encoding the full-length polypeptide of the present invention, e.g., a fragment that can be used as a probe or primer, or a fragment encoding the active portion (e.g., the virus-binding portion) of the polypeptide of the present invention.

[0141] Using a probe based on the sequence of the nucleic acid of the present invention, nucleic acids encoding the polypeptides of the present invention or similar nucleic acids, such as transcripts, can be detected. The probe can include a labeling group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells that express the polypeptide.

[0142] In another aspect, the present invention provides a library of nucleic acids encoding a library of antibody proteins derived from plasmablasts and plasma cells. These libraries of nucleic acids are generated by isolating plasmablasts and plasma cells in a single cell reaction vessel, where they are lysed and the antibody-specific nucleic acids are purified or captured on a solid support such as beads, for example. The present invention provides, in parallel, a method for performing transcript capture from millions of single cells. After transcript capture, the nucleic acids encoding the heavy and light chain immunoglobulins are amplified and then ligated to form a library of fusion constructs encoding both the heavy chain immunoglobulin and the light chain immunoglobulin. In such libraries, the natural pairing of the heavy chain immunoglobulin and the light chain immunoglobulin is maintained as originally seen in the input plasmablasts and plasma cells. Such methods are performed in parallel in millions of single cells such that the library obtained for the nucleic acids of the fused heavy chain immunoglobulin and light chain immunoglobulin contains sequences that are naturally paired for millions of single cells. Such methods are described elsewhere (Adler et al., Mabs 9, 1282-1996, 2017). 7.6. Vectors and Expression Vectors

[0143] The present invention provides a vector comprising a nucleic acid encoding a polypeptide of the present invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors.

[0144] In another aspect of the invention, there are also provided expression vectors containing the nucleic acid molecules and polynucleotides of the invention, host cells transformed with such vectors, and methods for producing polypeptides. The term "expression vector" refers to a plasmid, phage, virus or vector for expressing a polypeptide from a polynucleotide sequence. A vector for polypeptide expression contains minimally the sequences necessary for vector propagation and for expression of the cloned insert. An expression vector contains a transcription unit comprising an assembly of (1) one or more genetic elements (singular or plural) that play a regulatory role in gene expression, such as a promoter or enhancer, (2) a sequence encoding a polypeptide and protein that is transcribed into mRNA and translated into protein, and (3) appropriate transcription start and termination sequences. These sequences may further include a selectable marker. Vectors suitable for expression in host cells are readily available, and the nucleic acid molecules are inserted into the vectors using standard recombinant DNA techniques. Such vectors can include promoters that function in a particular tissue, and viral vectors for expression of polypeptides in target human or animal cells.

[0145] The recombinant expression vectors of the invention can contain the nucleic acids of the invention in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors include one or more regulatory sequences selected based on the host cell to be used for expression, which are operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., the SV40 early gene enhancer, the Rous sarcoma virus promoter and the cytomegalovirus promoter), those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, which are hereby incorporated by reference in their entirety into this specification those that are rare), and those that direct the inducible expression of nucleotide sequences in response to specific treatments or conditions (e.g., the metallothionin promoter in mammalian cells, as well as the tet-responsive and / or streptomycin-responsive promoters in both eukaryotic and prokaryotic cell lines (see the reference)). It will be understood by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the expression level of the desired protein, etc. The expression vector of the present invention can be introduced into a host cell, thereby producing a protein or peptide encoded by the nucleic acid described herein, such as a fusion protein or peptide.

[0146] The present invention further provides a method for producing a polypeptide, e.g., RPP. Various other expression / host systems can be utilized. Vector DNA can be introduced into prokaryotic or eukaryotic cell lines by conventional transformation or transfection techniques. These systems include microorganisms such as bacteria (e.g., E. coli) transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell lines infected with virus expression vectors (e.g., baculovirus); plant cell lines transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell lines, but are not limited thereto. Mammalian cells useful for the production of recombinant proteins include VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, or their derivatives, e.g., Veggie CHO and related cell lines that grow in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) (i) or CHO cell line DX-B11 lacking DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), COS cells, such as the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), W138, BHK, HepG2, 3T3 (ATCC CCL 163), RIN, MDCK, A549, PC12, K562, L cells, C127 cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human fetal-derived kidney cells, such as 293, 29 3 EBNA or MSR 293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, primary tissues, cell lines derived from in vitro culture of primary explants, HL-60, U937, HaK or Jurkat cells, but not limited to these. Mammalian expression enables the production of secreted or soluble polypeptides, and these polypeptides can be recovered from the growth medium.

[0147] For the stable transfection of mammalian cells, it is known that only some cells can incorporate foreign DNA into their genomes, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selectable marker (e.g., for resistance to an antibiotic) is generally introduced into the host cell along with the gene of interest. Transforming such cells with a vector containing not only the desired expression cassette but also a selectable marker allows these cells to be grown in enriched medium, for example, before switching the enriched medium to a selective medium. Selectable markers are designed to allow the growth and recovery of cells that successfully express the introduced sequences. Resistant aggregates of stably transformed cells can be propagated using tissue culture techniques suitable for the cell line utilized. An overview of the expression of recombinant proteins can be found in Methods of Enzymology, v. 185, Goeddell, D.V., ed., Academic Press (1990). Preferred Examples of selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. Cells in which the introduced nucleic acid has been stably transfected can be identified, among other ways, by drug selection (e.g., cells into which the selectable marker gene has been integrated will survive while other cells will die).

[0148] The transformed cells can be cultured under conditions that promote the expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification procedures (as defined above).

[0149] In some cases, for example, in the case of expression using a prokaryotic cell system, it may be necessary to "refold" the polypeptide expressed by the present invention, oxidize it to an appropriate three-dimensional structure, and generate disulfide bonds so that it becomes biologically active. Refolding can be accomplished using several procedures well known in the art. Such methods include, for example, exposing the solubilized polypeptide to a pH usually higher than 7 in the presence of a chaotropic agent. The choice of chaotrope is similar to that used for inclusion body solubilization, but the chaotrope is generally used at a lower concentration. Exemplary chaotropic agents are guanidine and urea. In most cases, the refolding / oxidation solution will also contain a reducing agent and its oxidized form in a specific ratio to produce a specific redox potential that allows disulfide shuffling to occur for the formation of cysteine crosslinks. Some commonly used redox pairs include cysteine / cystamine, glutathione / dithio-bisGSH, cupric chloride, dithiothreitol DTT / dithiane DTT, and 2-mercaptoethanol (bME) / dithio-bME. In many cases, a co-solvent can be used to increase the efficiency of refolding. Commonly used co-solvents include glycerol, polyethylene glycols of various molecular weights, and arginine.

[0150] In addition, polypeptides can be synthesized in solution or on a solid support according to the prior art. Various automated synthesizers are commercially available and can be used according to known protocols. For example, Stewart and Young, Solid Phase Peptide Synthesis, 2d.Ed., Pierce Chemical Co. (1984); Tam et al., J Am Chem Soc, See 105:6442, (1983); Merrifield, Science 232:341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; Barany et al., Int J Pep Protein Res, 30:705-739 (1987).

[0151] The polypeptides and proteins of the present invention can be purified according to protein purification techniques well known to those skilled in the art. These techniques include, at one level, a crude fractionation of proteinaceous and non-proteinaceous fractions. Once the peptide polypeptide is separated from other proteins, chromatographic and electrophoretic techniques can be used to further purify the peptide or polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). The term "purified polypeptide", as used herein, is intended to refer to a composition isolatable from other components, in which the polypeptide is purified to any degree compared to its naturally obtainable state. Thus, a purified polypeptide also refers to a polypeptide that has been separated from the environment in which it naturally occurs. Generally, "purified" will refer to a polypeptide composition that has been subjected to fractionation to remove various other components and that substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation will refer to a polypeptide or peptide composition in which the polypeptide or peptide forms the major component of the composition, e.g., constitutes about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins in the composition.

[0152] Various techniques suitable for use in purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation) and the like, or by heat denaturation, followed by centrifugation; chromatography, such as affinity chromatography (protein A column), ion exchange, gel filtration, reverse phase, hydroxylapatite, hydrophobic interaction chromatography, isoelectric focusing, gel electrophoresis, and combinations of these techniques. As is generally known in the art, the order in which various purification steps are performed may be varied or certain steps may be omitted, and still a method suitable for the preparation of a substantially purified polypeptide is considered to be obtained. Exemplary purification steps are provided in the examples below.

[0153] Various methods for quantifying the degree of purification of a polypeptide will be known to those skilled in the art in view of the present disclosure. These methods include, for example, determining the specific binding activity of the active fraction or assessing the amount of peptide or polypeptide in the fraction by SDS / PAGE analysis. A preferred method for assessing the purity of a polypeptide fraction is to calculate the binding activity of the fraction, compare it to the binding activity of the initial extract, and thus calculate the degree of purification, which is herein assessed by the "purification fold". The actual units used to represent the amount of binding activity will, of course, depend on the particular assay technique chosen to follow the purification and whether the polypeptide or peptide exhibits detectable binding activity.

[0154] In one aspect, the invention includes a library of antibodies encoding nucleic acid vectors for site-specific integration into the mammalian genome. Such vectors include plasmids, retroviruses, and lentiviruses. These libraries of vectors encode libraries of antibody sequences and thus are used to engineer mammalian cells for the production of RPPs. The library of nucleic acid vectors can include sequences encoding 10, 100, 1,000, 10,000, or more than 100,000 different antibodies. These sequences are derived from plasmablasts and plasma cells. These libraries of nucleic acids are generated by isolating plasmablasts and plasma cells in a single cell reaction vessel where they are lysed and the antibody-specific nucleic acids are purified or captured on a solid support such as beads. The invention, in parallel, provides a method for performing transcript capture from millions of single cells. After transcript capture, the nucleic acids encoding the heavy and light chain immunoglobulins are amplified and then the nucleic acids are ligated to form a library of fusion constructs encoding both the heavy chain immunoglobulin and the light chain immunoglobulin. In such a library, the natural pairing of the heavy chain immunoglobulin and the light chain immunoglobulin is maintained as originally seen in the input plasmablasts and plasma cells. Such a method is performed in parallel in millions of single cells such that the library obtained for the nucleic acids of the fused heavy chain immunoglobulin and the light chain immunoglobulin contains sequences that are naturally paired for millions of single cells. These paired fusion amplicons are then engineered into full-length antibody constructs using Gibson assembly, restriction endonucleases, or other recombinant DNA techniques. Manipulations to the full-length antibody constructs are carried out en masse with respect to the entire library, such that the antibody sequence content and the number of antibody sequences of the library are essentially maintained through this process. In some embodiments, the library of expression vectors is manipulated in two steps, such that the scFv amplicons are subcloned into an intermediate vector and then additional domains of the antibody are engineered into the linker of the scFv using Gibson assembly, restriction enzyme digestion, or other recombinant techniques for the second time (USPTO 14 / 734,953). The natural pairing of the heavy-chain immunoglobulin and the light-chain immunoglobulin is essentially maintained through the process of manipulation to the full-length expression vector library. The vectors are designed in various orientations, for example, two separate promoters drive the expression of the heavy-chain immunoglobulin and the light-chain immunoglobulin, or one promoter drives the expression of both the heavy-chain immunoglobulin and the light-chain immunoglobulin, or a translational skip motif is used to separately translate the heavy-chain immunoglobulin and the light-chain immunoglobulin into separate polypeptides. In some embodiments, the expression vector contains sequences for site-specific integration into mammalian production cells, for example, for CRISPR-Cas9, Flp-In, Cre / Lox, or zinc finger recombination methods. Site-specific integration ensures that each mammalian production cell encodes a single antibody sequence and reduces the variability of expression levels among single production cells.

[0155] 7.7.RPP, e.g., method for producing an antibody

[0156] RPP can purify an antibody from a host cell transfected with a gene encoding the antibody by elution of the filtered supernatant of the host cell culture using a heparin HP column, using a salt gradient, or using protein A resin.

[0157] Fully human monoclonal antibodies can be generated by a number of techniques well known to those of skill in the art. Such methods include, but are not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures known in the art and based on the disclosure herein. For example, fully human monoclonal antibodies may be obtained from transgenic mice engineered to produce specific human antibodies in response to antigen challenge. Methods for obtaining fully human antibodies from transgenic mice are described, for example, in Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; U.S. Patent No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al., 1995 Ann. N. Y. Acad. Sci. 764:525-35. In this technique, elements of the human heavy and light chain loci are introduced into a strain of mice derived from an embryonic stem cell line containing targeted disruption of the endogenous heavy and light chain loci (Bruggemann et al., Curr. (See also Opin. Biotechnol. 8:455-58 (1997).) For example, the human immunoglobulin transgene may be a mini-gene construct on a yeast artificial chromosome or a transgene locus that undergoes B-cell specific DNA rearrangement and high-frequency mutation in mouse lymphoid tissue. Fully human monoclonal antibodies can be obtained by immunizing transgenic mice that are then capable of producing human antibodies specific for the antigen target(s). According to the methods described herein, lymphoid cells from immunized transgenic mice can be used to generate hybridomas that secrete human antibodies.

[0158] Another method for generating human antibodies of the present invention involves immortalizing human peripheral blood cells by EBV transformation. See, for example, U.S. Patent No. 4,464,456. Such immortalized B-cell lines (or lymphoblastoid cell lines) that produce an RPP that specifically binds to the target(s) can be identified by the immunoassay methods provided herein, e.g., by ELISA, and then isolated by standard cloning techniques. The stability of the lymphoblastoid cell line producing the RPP can be improved by fusing the transformed cell line with mouse myeloma cells to generate a mouse-human hybrid cell line according to methods known in the art (see, for example, Glasky et al., Hybridoma 8: 377-89(1989)). Yet another method for generating human RPP involves in vitro immunization, which includes priming human spleen B cells with an antigen target and then fusing the primed cells with a heterohybrid fusion partner. See, for example, Boerner et al., 1991 J. Immunol. 147:86-95.

[0159] In certain embodiments, B cells producing the RPP are selected, and the light and heavy chain variable regions are known in the art (WO92 / 02551; U.S. Patent No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)), and cloned from the B cells according to the molecular biology techniques described herein. B cells from immunized animals may be isolated from the spleen, lymph nodes, or peripheral blood samples by selecting cells that produce antibodies that specifically bind to the antigen target. B cells may be isolated from humans, for example, from peripheral blood samples.

[0160] For example, methods for detecting a single B cell producing an antibody with the desired specificity, such as by plaque formation, fluorescence-activated cell sorting, in vitro stimulation, and subsequent detection of specific antibodies, are well known in the art. Methods for selection of B cells producing specific antibodies include, for example, preparing a single cell suspension of B cells in soft agar containing the antigen target. Binding of the specific antibody produced by the B cells to the antigen results in the formation of a complex that can be visualized as an immunoprecipitate.

[0161] In some embodiments, B cells that produce specific antibodies are selected by using methods that enable the identification of naturally paired antibodies. For example, the methods described in Adler et al., A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library, MAbs (2018), which is incorporated herein by reference in its entirety, can be used. The method combines microfluidic technology, molecular genomics, yeast single-chain variable fragment (scFv) display, fluorescence-activated cell sorting (FACS), and deep sequencing. Briefly, B cells can be isolated from immunized animals and then pooled. The B cells are encapsulated into droplets using oligo dT beads and a lysis solution, the beads bound with mRNA are purified from the droplets, and then injected into a second emulsion containing an OE-RT-PCR amplification mix that generates DNA amplicons encoding scFvs in which the heavy and light chain Igs are naturally paired. Next, a library of naturally paired amplicons is electroporated into yeast for scFv display. FACS is used to identify high-affinity scFvs. Finally, deep sequencing of the antibodies can be used to identify all clones in the scFv library before and after selection.

[0162] After selecting the B cells that produce the desired antibody, the specific antibody gene may be cloned by isolating and amplifying DNA or mRNA according to methods known in the art and described herein.

[0163] The methods for obtaining the antibodies of the present invention can also employ various phage display techniques known in the art. For example, Winter et al., 1994 Annu. Rev. Immunol. See 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. Combinatorial libraries of human or murine immunoglobulin variable region genes can be generated in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to RPP or variants or fragments thereof. For example, U.S. Patent No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66 ; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited therein. See, for example, libraries containing multiple polynucleotide sequences encoding Ig variable region fragments may be inserted in frame with sequences encoding phage coat proteins into the genome of filamentous bacteriophages such as M13 or variants thereof. The fusion protein may be a fusion of the coat protein with the light chain variable region domain and / or the heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments can also be displayed on the phage particle (see, for example, U.S. Patent No. 5,698,426).

[0164] In one embodiment, in a hybridoma, the variable region of the gene expressing the desired monoclonal antibody is amplified using nucleotide primers. These primers may be synthesized by those skilled in the art or purchased from commercial sources. (See, for example, Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including, inter alia, primers for the V Ha , V Hb , V Hc , V Hd , C H1 , V L and C L regions). Using these primers, the heavy or light chain variable regions may be amplified and then inserted into vectors such as ImmunoZAP™ H or ImmunoZAP™ L (Stratagene), respectively. These vectors may then be introduced into E. coli, yeast, or mammalian-based systems for expression. Large amounts of single-chain proteins containing fusions of the V H and V L domains may be produced using these methods (see Bird et al., Science 242:423-426, 1988).

[0165] Once cells producing the antibodies according to the present invention are obtained using any of the above immunization and other techniques, the gene for the specific antibody may be cloned by isolating and amplifying the DNA or mRNA therefrom by standard procedures described herein. The antibodies produced therefrom are sequenced to identify the CDRs, and the DNA encoding the CDRs is manipulated as previously described according to the present invention to generate other antibodies.

[0166] The RPP of the present invention preferably has activity in the cell-based assays and / or in vivo assays described herein and / or binds to one or more of the domains described herein. Thus, such binders can be identified using the assays described herein.

[0167] Other antibodies according to the present invention may be obtained by conventional immunization and cell fusion procedures described herein and known in the art.

[0168] Also, using molecular evolution of the complementarity determining regions (CDRs) at the center of the antibody binding site, antibodies with increased affinity, for example, antibodies having increased affinity for c-erbB-2 as described in Schier et al., 1996, J. Mol. Biol. 263:551 have been isolated.

[0169] Human antibodies, partial human antibodies, or humanized antibodies are suitable for many applications, particularly those involving the administration of antibodies to human subjects. However, other types of antigen-binding proteins are suitable for certain specific applications. The non-human antibodies of the present invention can be derived from, for example, any antibody-producing animal such as a mouse, rat, rabbit, goat, donkey, or non-human primate (such as a monkey (e.g., cynomolgus monkey or rhesus monkey) or an ape (e.g., chimpanzee)). The non-human antibodies of the present invention can be used, for example, in in vitro and cell culture-based applications, or in any other application where an immune response to the antibodies of the present invention does not occur, is not important, cannot be inhibited, is not involved, or is desirable. In one embodiment, the non-human antibodies of the present invention are administered to non-human subjects. In another embodiment, the non-human antibodies do not induce an immune response in non-human subjects. In another embodiment, the non-human antibodies are derived from the same species as the non-human subject. For example, the mouse antibodies of the present invention are administered to mice. Antibodies from a particular species can be prepared, for example, by immunizing animals of that species with a desired immunogen or using an artificial system for generating antibodies of that species (e.g., a system based on bacteria or phage display for generating antibodies of a particular species), or by converting an antibody derived from one species into an antibody derived from another species by, for example, replacing the constant region of the antibody with the constant region of another species, or by replacing one or more amino acid residues of the antibody so that the antibody approximates the sequence of an antibody derived from another species. In one embodiment, the antibody is a chimeric antibody comprising amino acid sequences derived from antibodies from two or more different species.

[0170] The antigen-binding protein may be prepared by any of several conventional techniques and screened for desired properties. Some techniques involve isolating the nucleic acid encoding the polypeptide chain (or portion thereof) of the RPP of interest and manipulating the nucleic acid by recombinant DNA techniques. The nucleic acid may be fused to another nucleic acid of interest or may be modified, for example, by adding, deleting, or substituting one or more amino acid residues (e.g., by mutagenesis or other conventional techniques). Further, the antigen-binding proteins may be purified from cells that naturally express them (e.g., an antibody can be purified from the hybridoma that produces it) or may be produced in a recombinant expression system using any of the techniques known in the art. For example, see Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0171] The recombinant polypeptide of the present invention can be produced using any expression system known in the art. The expression systems are described in detail above generally. Generally, a host cell is transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Among the host cells that may be used are prokaryotes, yeasts or cells of higher eukaryotes. Examples of prokaryotes include gram-negative or gram-positive organisms, such as E. coli or Bacilli. Examples of higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell line, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) described in McMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning vectors and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). The production of cell lines for monoclonal antibodies (mAbs) is generally produced by randomly inserting an expression construct into the genome of mammalian production cells, such as the CHO genome (Rita Costa et al., 2010). However, this standard method inserts into the CHO genome

[0172] ​Produce a cell line that has multiple copies of the mAb produced. When the inventors randomly inserted their polyclonal antibody construct library into the CHO genome, many clones would express multiple antibodies, resulting in a high frequency of unnatural pairings between the heavy chain Ig and the light chain Ig. Furthermore, different genomic positions have different transcriptional activity levels (Kito et al., 2002), which can lead to heterogeneous, inconsistent, and / or unstable bioproduction. Thus, in some aspects, the present invention provides a CHO cell line having a stably engineered Flp recombinase recognition target (FRT) landing pad in the genome. Such a site-specific genomic integration cell line is then used for stable expression of the RPP.

[0173] It will be appreciated that the antibodies of the present invention may have at least one amino acid substitution as long as the antibody retains its binding specificity. Thus, modifications of the antibody structure are encompassed within the scope of the present invention. These may include amino acid substitutions, which may be conservative or non-conservative, that do not disrupt the binding ability of the antibody, including the RPP. Conservative amino acid substitutions may typically involve the incorporation of non-naturally occurring amino acid residues that are incorporated by chemical peptide synthesis rather than by synthesis in a biological system. These include peptidomimetics and other inverted or reversed forms of amino acid moieties. Conservative amino acid substitutions may involve the substitution of a canonical residue of a natural amino acid residue such that it has little or no effect on the polarity or charge of the amino acid residue at that position.

[0174] Non-conservative substitutions may also involve the exchange of a member of one class of amino acids or amino acid mimics for a member derived from another class having different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of the human antibody that are homologous to the non-human antibody or into non-homologous regions of the molecule.

[0175] Furthermore, one of ordinary skill in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those of ordinary skill in the art. Information on suitable variants can be collected using such variants. For example, if it is discovered that a change to a particular amino acid residue results in disrupted, undesirably reduced, or otherwise unsuitable activity, variants having such a change may be avoided. In other words, based on the information collected from such routine experimentation, one of ordinary skill in the art can readily determine which additional substitutions, alone or in combination with other mutations, should be avoided for an amino acid.

[0176] One of ordinary skill in the art will be able to determine suitable variants of the polypeptides shown herein using well-known techniques. In certain embodiments, one of ordinary skill in the art can identify suitable areas of the molecule that can be altered without disrupting activity by targeting regions that are not thought to be important for activity. In certain embodiments, residues and portions of the molecule that are conserved among similar polypeptides can be identified. In certain embodiments, even areas that may be important for biological activity or structure may be subjected to conservative amino acid substitutions without disrupting biological activity or having an adverse effect on the polypeptide structure.

[0177] Furthermore, one of ordinary skill in the art can examine structure - function studies for identifying residues in similar polypeptides that are important for activity or structure. In view of such comparisons, the importance of the amino acid residues of a protein corresponding to amino acid residues important for the activity or structure of a similar protein can be predicted. One of ordinary skill in the art may select amino acid substitutions that are chemically similar to such predicted important amino acid residues.

[0178] One of ordinary skill in the art can also analyze the structure and amino acid sequence related to the three-dimensional structure of similar polypeptides. In view of such information, one of ordinary skill in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. In certain embodiments, since amino acid residues predicted to be present on the surface of a protein can be involved in important interactions with other molecules, one of ordinary skill in the art can select such residues so that drastic changes do not occur.

[0179] Several scientific papers have contributed to the prediction of secondary structure. Moult J., Curr. Op. in Biotech., 7(4):422-427(1996), Chou et al., Biochem., 13(2):222-245(1974) ;Chou et al., Biochem., 113(2):211-222(1974);Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148(1978);Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979) are referred to. Further, computer programs are currently available to facilitate the prediction of secondary structure. One method of predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins having more than 30% sequence identity, or more than 40% similarity, often have similar structural topologies. Recent developments in the Protein Data Bank (PDB), including the possible number of foldings within the structure of a polypeptide or protein, have led to enhanced predictability of secondary structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247(1999). It has been suggested that there are a limited number of foldings for a given polypeptide or protein, and that once the critical number of structures is elucidated, the prediction of structure becomes dramatically more accurate (Brenner et et al., Curr. Op. Struct. Biol., 7(3):369-376(1997)).

[0180] As additional methods for predicting secondary structure, "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87(1997); Sippl et al., Structure, 4(1):15-19(1996)), "profile analysis" (Bowie et al., Science, 253:164-170(1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358(1987)), and "evolutionary linking" (see Holm, supra (1999), and Brenner, supra (1997)) are mentioned. as examples.

[0181] In certain embodiments, variants of the antibody include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the variant contains a greater or lesser number of N-linked glycosylation sites than the native protein. N-linked glycosylation sites are characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue represented by X may be any amino acid residue other than proline. Substitution of the amino acid residues that create this sequence results in new possible sites for the addition of N-linked carbohydrate chains. Alternatively, substitution that eliminates this sequence will result in the removal of existing N-linked carbohydrate chains. Rearrangement of the N-linked carbohydrate chains is also effected, where one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or replaced with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine variants can be useful, for example, when the antibody has to refold into a biologically active conformation after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein and typically have an even number to minimize interactions arising from unpaired cysteines.

[0182] According to certain embodiments, preferred amino acid substitutions are those that (1) decrease susceptibility to proteolysis, (2) decrease susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and / or (4) confer or modify other physiochemical or functional properties of such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (conservative amino acid substitutions in certain embodiments) may be made in a naturally occurring sequence (in certain embodiments, the portion of the polypeptide outside of the domain forming intermolecular contacts). In certain embodiments, conservative amino acid substitutions typically need not substantially change the structural characteristics of the parent sequence (e.g., the amino acid replacement should not tend to break a helix present in the parent sequence or disrupt other types of secondary structure that characterize the parent sequence). Examples of secondary and tertiary polypeptide structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991), each of which is incorporated herein by reference.

[0183] In certain embodiments, the antibodies of the invention may be chemically conjugated to a polymer, lipid, or other moiety.

[0184] The binder may comprise at least one of the CDRs described herein, incorporated within a biocompatible framework structure. In one example, the biocompatible framework structure can display one or more sequences of amino acids that bind an antigen (e.g., a CDR, variable region, etc.) in a localized surface region, a three-dimensional structure-stable structural support, or framework, or scaffold-forming polypeptide or portion thereof that is sufficient. Such structures may be naturally occurring polypeptides or polypeptide "foldings" (structural motifs), or may have one or more modifications such as addition, deletion or substitution of amino acids relative to naturally occurring polypeptides or foldings. These scaffolds can be derived from polypeptides of any species (or two or more species) such as human, other mammals, other vertebrates, invertebrates, plants, bacteria or viruses.

[0185] Typically, the biocompatible framework structure is based on a protein scaffold or backbone other than an immunoglobulin domain. For example, those based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, P ST1, coiled coil, LACI-D1, Z domain and tendamistat domain can be used (see, for example, Nygren and Uhlen, 1997, Curr. Opin. In Struct. Biol., 7, 463-469).

[0186] It will be appreciated that the antibodies of the present invention include humanized antibodies as described herein. Humanized antibodies such as those described herein can be produced using techniques known to those of skill in the art (Zhang, W., et al., Molecular Immunology. 42(12):1445-1451, 2005; Hwang W. et al., Methods. 36(1):35-42, 2005; Dall'Acqua WF, et al., Methods 36(1):43-60, 2005; and Clark, M., Immunology Today. 21(8):397-402, 2000).

[0187] The antibody comprises one or more of the above CDR1-H, CDR2-H, CDR3-H, CDR1-L, CDR2-L and CDR3-L, and the antibody may be obtained by expression from a host cell containing DNA encoding these sequences. The DNA encoding each CDR sequence is determined based on the amino acid sequence of the CDR, and may be synthesized together with any desired antibody variable region framework and constant region DNA sequence using oligonucleotide synthesis techniques, site-directed mutagenesis and polymerase chain reaction (PCR) techniques as appropriate. DNA encoding the variable region framework and constant region is widely available to those of skill in the art from gene sequence databases such as GenBank®.

[0188] Once synthesized, the DNA or fragment thereof encoding the antibody of the present invention can be amplified and expressed according to any of a variety of well-known procedures for excision, ligation, transformation of nucleic acids, and transfection using any of a number of known expression vectors. Thus, in certain embodiments, expression of antibody fragments may be preferred in prokaryotic hosts such as Escherichia coli (see, for example, Pluckthun et al., 1989 Methods Enzymol. 178:497-515). In certain other embodiments, Expression of the antibody or its fragment may be preferred in eukaryotic host cells including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), animal cells (including mammalian cells) or plant cells. Examples of suitable animal cells include, but are not limited to, myeloma cells (such as mouse NSO line), COS cells, CHO cells, or hybridoma cells. Examples of plant cells include cells of tobacco, corn, soybean, and rice.

[0189] A replicable expression vector containing DNA encoding the variable and / or constant regions of the antibody is prepared and can be used to transform an appropriate cell line, e.g., a non-producing myeloma cell line such as the mouse NSO line where antibody production occurs or bacteria such as E. coli. To obtain efficient transcription and translation, the DNA sequence in each vector should include a leader sequence operably linked to appropriate regulatory sequences, particularly a promoter and the variable domain sequence. Specific methods for producing such antibodies are generally well-known and routinely used. For example, the procedures of basic molecular biology are described in Maniatis et al. (Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory, New York, (2001) should also be referred to). DNA sequencing can be carried out as described in Sanger et al. (PNAS 74:5463, (1977)) and the Amersham International plc sequencing handbook, and site-directed mutagenesis is within the skill It can be carried out according to methods known in the art (Kramer et al., Nucleic Acids Res. 12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92(1985); Kunkel et al., Methods in Enzymol. 154:367-82(1987); the Anglian Biotechnology Ltd. handbook). Furthermore, a number of publications describe techniques suitable for the manipulation of DNA, the construction of expression vectors, and the preparation of antibodies by the transformation and culture of appropriate cells (Mountain A and Adair, J R in Biotechnology and Genetic Engineering Reviews(ed. Tombs, M P, 10, Chapter 1, 1992, Intercept, Andover, UK); "Current Protocols in Molecular Biology", 1999, F.M. Ausubel(ed.), Wiley Interscience, New York).

[0190] If it is desirable to improve the affinity of the antibody according to the present invention, containing one or more of the above-described CDRs involves maintaining the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), the use of mutant strains of E. coli. (Low et al., J. Mol. Biol., 250, 350-368, 1996), DNA shuffling (Patten et al., (Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 7-88, 1996) and sexual PCR (Crameri, et al., Nature, 391, 288-291, 1998). All of these methods of affinity maturation are discussed by Vaughan et al. (Nature Biotech., 16, 535-539, 1998).

[0191] It is understood by those skilled in the art that some proteins, such as antibodies, may undergo various post-translational modifications. The type and extent of these modifications often vary depending on the host cell line and culture conditions used to express the protein. Such modifications can include changes in glycosylation, methionine oxidation, diketopiperidine formation, aspartic acid isomerization and asparagine deamidation. A frequently occurring modification is the loss of basic residues (e.g., lysine or arginine) at the carboxy terminus due to the action of carboxypeptidase (as described in Harris, R.J. Journal of Chromatography 705:129-134, 1995). 7.8. Pharmaceutical Compositions

[0192] Also provided are pharmaceutical compositions containing the RPP of the present invention. Such compositions contain a therapeutically or prophylactically effective amount of a polypeptide or protein in a mixture comprising a pharmaceutically acceptable substance and a physiologically acceptable formulation substance.

[0193] The pharmaceutical composition may contain, for example, formulation substances for modifying, maintaining or preserving the pH, osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition.

[0194] Suitable pharmaceutical substances include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids, etc.); bulking agents (such as mannitol or glycine, etc.), chelating agents (such as ethylenediaminetetraacetic acid (EDTA), etc.); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin, etc.); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrin, etc.); proteins (such as serum albumin, gelatin or immunoglobulins, etc.); coloring agents; flavoring agents and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone, etc.); low molecular weight polypeptides; salt-forming counterions (such as sodium, etc.); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid or hydrogen peroxide, etc.); solvents (such as glycerin, propylene glycol or polyethylene glycol, etc.); sugar alcohols (such as mannitol or sorbitol, etc.); suspending agents; surfactants or wetting agents (such as polysorbates such as Pluronic (registered trademark), PEG, sorbitan esters, polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.); stability enhancers (such as sucrose or sorbitol, etc.); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. Neutral buffered saline or saline mixed with homologous serum albumin are examples of suitable diluents. According to appropriate industrial standards, preservatives such as benzyl alcohol may also be added. The composition may be formulated as a lyophilized product using a suitable excipient solution (such as sucrose) as a diluent. Suitable components are non-toxic to the recipient at the dosages and concentrations used.Further examples of components that can be used in pharmaceutical formulations are found in Remington's Pharmaceutical Sciences, 16th th Ed. (1980) and 20th th Ed. (2000), Mack Publishing Company, Easton, PA.

[0195] Optionally, the composition further comprises one or more physiologically active agents such as anti-angiogenic agents, chemotherapeutic agents (e.g., capecitabine, 5-fluorouracil, or doxorubicin), analgesic agents, etc. (non-exclusive examples of these are provided herein). In various specific embodiments, the composition comprises 1, 2, 3, 4, 5, or 6 physiologically active agents in addition to the RPP.

[0196] In another embodiment of the present invention, the compositions disclosed herein may be formulated in neutral or salt form. Exemplary pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of proteins), which are formed with, for example, inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups may be derived from, for example, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution will be administered in a therapeutically effective amount in a manner compatible with the dosage formulation.

[0197] The carrier may further contain all kinds of solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic agents and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well-known in the art. Their use in therapeutic compositions is contemplated, except where any conventional medium or agent is incompatible with the active ingredient. Supplementary active ingredients may be introduced into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic reaction or similar adverse reaction when administered to humans.

[0198] Optimal pharmaceutical compositions will be determined by those skilled in the art, for example, according to the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions can affect the physical state, stability, in vivo release amount, and in vivo clearance amount of the polypeptide. For example, suitable compositions may be water for injection, an aqueous physiological saline solution for parenteral administration. 7.8.1. Content of pharmaceutically active ingredient

[0199] In typical embodiments, the active ingredient (i.e., the proteins and polypeptides of the present invention) is present in the pharmaceutical composition at a concentration of at least 0.01 mg / ml, at least 0.1 mg / ml, at least 0.5 mg / ml, or at least 1 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, or 25 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml or 50 mg / ml. 7.8.2. General formulation

[0200] The pharmaceutical composition may be in any form suitable for human or vertebrate medicine, including a liquid, oil, emulsion, gel, colloid, aerosol, or solid.

[0201] The pharmaceutical composition can be formulated for administration by any route suitable for human or vertebrate medicine, including enteral and parenteral routes of administration.

[0202] In various embodiments, the pharmaceutical composition is formulated for administration by inhalation. In certain of these embodiments, the pharmaceutical composition is formulated for administration by nebulizer. In certain of these embodiments, the pharmaceutical composition is formulated for administration by vaporizer. In certain of these embodiments, the pharmaceutical composition is formulated for administration by aerosolizer.

[0203] In various embodiments, the pharmaceutical composition is formulated for oral, buccal, or sublingual administration.

[0204] In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, or subcutaneous administration.

[0205] In some embodiments, the pharmaceutical composition is formulated for intrathecal or intraventricular administration.

[0206] In some embodiments, the pharmaceutical composition is formulated for topical administration. 7.8.3. Pharmaceutical Composition Compatible with Injection

[0207] For intravenous, cutaneous or subcutaneous injection, or injection into the site of the disease, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can adequately prepare suitable solutions using isotonic vehicles such as, for example, sodium chloride injection solution, Ringer's injection solution, lactated Ringer's injection solution, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included if necessary.

[0208] In various embodiments, the unit dosage form is a vial, an ampoule, a bottle, or a pre-filled syringe. In some embodiments, the unit dosage form contains a pharmaceutical composition of 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 25 mg, 50 mg, 75 mg, or 100 mg. In some embodiments, the unit dosage form contains a pharmaceutical composition of 125 mg, 150 mg, 175 mg, or 200 mg. In some embodiments, the unit dosage form contains a pharmaceutical composition of 250 mg.

[0209] In typical embodiments, the pharmaceutical composition in the unit dosage form is in liquid form. In various embodiments, the unit dosage form contains a pharmaceutical composition between 0.1 mL and 50 mL. In some embodiments, the unit dosage form contains a pharmaceutical composition of 1 mL, 2.5 mL, 5 mL, 7.5 mL, 10 mL, 25 mL, or 50 mL.

[0210] In certain embodiments, the unit dosage form is a vial containing 1 mL of a pharmaceutical composition at a concentration of 0.01 mg / mL, 0.1 mg / mL, 0.5 mg / mL, or 1 mg / mL. In some embodiments, the unit dosage form is a vial containing 2 mL of a pharmaceutical composition at a concentration of 0.01 mg / mL, 0.1 mg / mL, 0.5 mg / mL, or 1 mg / mL.

[0211] In some embodiments, the pharmaceutical composition in the unit dosage form is in solid form, such as a lyophilized product suitable for solubilization.

[0212] Embodiments of unit dosage forms suitable for subcutaneous, intradermal, or intramuscular administration include pre-filled syringes, autoinjectors, and autoinject pens, each containing a predetermined amount of the pharmaceutical composition described above herein.

[0213] In various embodiments, the unit dosage form is a prefilled syringe comprising a syringe and a predetermined amount of a pharmaceutical composition. In certain embodiments of the prefilled syringe, the syringe is adapted for subcutaneous administration. In certain embodiments, the syringe is suitable for self - administration. In specific embodiments, the prefilled syringe is a single - use syringe.

[0214] In various embodiments, the prefilled syringe contains from about 0.1 mL to about 0.5 mL of the pharmaceutical composition. In certain embodiments, the syringe contains about 0.5 mL of the pharmaceutical composition. In specific embodiments, the syringe contains about 1.0 mL of the pharmaceutical composition. In certain embodiments, the syringe contains about 2.0 mL of the pharmaceutical composition.

[0215] In certain embodiments, the unit dosage form is an auto - injection pen. The auto - injection pen includes an auto - injection pen containing the pharmaceutical composition described herein. In some embodiments, the auto - injection pen delivers a predetermined volume of the pharmaceutical composition. In other embodiments, the auto - injection pen is configured to deliver a volume of the pharmaceutical composition by the user.

[0216] In various embodiments, the auto - injection pen contains from about 0.1 mL to about 5.0 mL of the pharmaceutical composition. In specific embodiments, the auto - injection pen contains about 0.5 mL of the pharmaceutical composition. In certain embodiments, the auto - injection pen contains about 1.0 mL of the pharmaceutical composition. In other embodiments, the auto - injection pen contains about 5.0 mL of the pharmaceutical composition. 7.8.4. Mixtures of IVIg in plasma with recombinant hyper - immunity

[0217] In some embodiments, the recombinant hyperimmune is spiked into conventional plasma IVIg to increase the anti-pathogen titer of the IVIg. In some embodiments, several anti-pathogen recombinant hyperimmunes are spiked into conventional plasma IVIg. For example, hyperimmunes against Hib, pneumococcus, influenza A virus, and tetanus are simultaneously spiked into plasma IVIg to treat patients with primary immunodeficiency. The spike in the hyperimmune increases the titer of antibodies directed against pathogens to which patients with primary immunodeficiency are particularly susceptible. Multiple spike-ins can be mixed with plasma IVIg to generate increased titers against multiple pathogens.

[0218] In some embodiments, the spike-in recombinant hyperimmune is mixed with plasma IVIg by a pharmacist. In some embodiments, the spike-in recombinant hyperimmune is mixed with plasma IVIg by the manufacturer. 7.9. Unit Dosage Forms

[0219] The pharmaceutical composition may, for convenience, be present in unit dosage forms.

[0220] Unit dosage forms will typically be suitable for one or more specific routes of administration of the pharmaceutical composition.

[0221] In various embodiments, the unit dosage form is suitable for administration by inhalation. In certain of these embodiments, the unit dosage form is suitable for administration by nebulizer. In certain of these embodiments, the unit dosage form is suitable for administration by vaporizer. In certain of these embodiments, the unit dosage form is suitable for administration by aerosolizer.

[0222] In various embodiments, the unit dosage form is suitable for oral, buccal, or sublingual administration.

[0223] In some embodiments, the unit dosage form is suitable for intravenous, intramuscular, or subcutaneous administration.

[0224] In some embodiments, the unit dosage form is suitable for intrathecal or intraventricular administration.

[0225] In some embodiments, the pharmaceutical composition is formulated for topical administration.

[0226] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect. 8. RPP activity

[0227] RPP, for example, an antibody according to the present invention, may be less than or equal to 5×10 -7 M, or less than or equal to 1×10 -7 M, or less than or equal to 0.5×10 -7 M, or less than or equal to 1×10 -8 M, or less than or equal to 1×10 -9 M, or less than or equal to 1×10 -10 M, or less than or equal to 1×10 -11 M, or less than or equal to 1×10 -12 M, and may have binding affinity for the antigen target.

[0228] The affinity of the RPP and the degree to which the antibody inhibits binding are determined using conventional techniques, such as those described in Scatchard et al. (Ann. N.Y. Acad. Sci. 51:660-672 (1949)). Alternatively, it can be determined by those skilled in the art by surface plasmon resonance (SPR; BIAcore, Biosensor, Piscataway, NJ). In surface plasmon resonance, the target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase flowing along a flow cell. When the ligand binds to the immobilized target, a local refractive index change occurs, resulting in a change in the SPR angle, and the change in the SPR angle can be monitored in real time by detecting the change in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to obtain the apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al., Cancer Res. 53:2560-65 (1993)). 9. Method of treating a disease responsive to RPP

[0229] In another aspect, a method for treating a subject having a disease responsive to RPP is shown. The disease may be cancer, AIDS, Alzheimer's disease or a viral or bacterial infection. In certain aspects, RPP is used to induce tolerance during transplantation of an organ, tissue, or cell population from a donor to a host.

[0230] The terms “treatment,” “treating,” etc. are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in terms of completely or partially preventing a disease, condition, or symptom thereof, and / or therapeutic in terms of partial or complete cure of a disease or condition and / or adverse effects such as symptoms caused by the disease or condition. “Treatment,” as used herein, encompasses any treatment of a disease or condition in a mammal, particularly a human: (a) preventing a disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition; (b) inhibiting a disease or condition (e.g., preventing its onset); or (c) alleviating a disease or condition (e.g., regressing the disease or condition, effecting improvement of one or more symptoms). Improvement of any condition can be readily evaluated according to standard methods and techniques known in the art. For the disease, the population of subjects treated by the method includes subjects suffering from an undesirable condition or disease and subjects at risk of developing the condition or disease.

[0231] The term “therapeutically effective dose” or “effective amount” means a dose or amount that produces a desired effect on the thing to which it is administered. The exact dose or amount will vary depending on the purpose of the treatment and will be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0232] The term “sufficient amount” means an amount sufficient to produce a desired effect.

[0233] The term “therapeutically effective amount” is an amount effective to improve the symptoms of a disease. A therapeutically effective amount may be a “prophylactically effective amount” since prevention can be considered treatment.

[0234] The term "ameliorate" refers to any therapeutically beneficial outcome in the treatment of a disease state, such as a neurodegenerative disease state (including its prevention, alleviation of severity or progression, remission, or cure).

[0235] In vivo and / or in vitro assays can be used as needed to help identify the optimal dosage range. The exact dosage used in the formulation will also vary depending on the route of administration and the severity of the condition and should be determined according to the judgment of the practicing physician and the circumstances of each subject. The effective dosage may be estimated from a dose-response curve derived from in vitro or animal model test systems.

[0236] The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the protein aggregation disease being treated. The determination of the treatment prescription, such as the dosage, etc., is within the responsibility of the general practitioner and other physicians and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practicing physician. Examples of the techniques and protocols described above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0237] In some embodiments, the pharmaceutical composition is administered by inhalation, orally, by buccal administration, by sublingual administration, by injection, or by topical administration.

[0238] In some embodiments, the pharmaceutical composition is administered in an amount sufficient to modulate neuron survival or dopamine release. In some embodiments, the major cannabinoid is administered in an amount of less than 1 g, less than 500 mg, less than 100 mg, or less than 10 mg per dose.

[0239] In some embodiments, the pharmaceutical composition is administered once a day, 2 - 4 times a day, 2 - 4 times a week, once a week, or once every two weeks.

Example

[0240] 10. Example The following are examples of specific embodiments for practicing the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Although efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, temperatures, etc.), some experimental errors and variations should, of course, be tolerated.

[0241] In the practice of the present invention, unless otherwise indicated, conventional methods in protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology will be used within the scope of the art. Such techniques are well described in the references. For example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992). 10.1.1. (Example 1) Generation of an RPP library having activity against human thymocytes or T cells

[0242] Four libraries of RPPs targeting human thymocytes or T cells, i.e., recombinant human antithymocyte globulin (rhATG), were produced. Both in vitro and in vivo studies were used to demonstrate the functional similarity between rhATG and commercially available rabbit ATG (Thymoglobulin, Sanofi). The sequences of the heavy and light chain CDR3s are provided for RPP10 - 13 in Table 5 above.

[0243] Commercially available antithymocyte globulin (ATG, (Thymoglobulin, Sanofi)) is useful for inducing transplant tolerance and is produced by immunizing New Zealand rabbits with human thymocytes; blood is collected from thousands of animals and the antibodies are purified from plasma. The RPPs disclosed herein, i.e., the libraries of rhATG, combine the advantage of the efficacy of polyclonal ATG with the advantage of the safety of a fully human recombinant RPP library.

[0244] First, transgenic mice having the inserted human immunoglobulin gene were immunized with human thymocytes or human T cells. Two Trianni mice were given plantar injections twice a week for three weeks, and then boosted for the next two weeks. One to two million thymocytes were injected into each mouse at each time point. Prior to the final boost, the serum titers of the antibodies to the thymocytes were assessed by flow cytometry using a dilution series of the sera of each animal starting at 1:200 and ending at 1:145,000. The inventors observed a strong serum response in both animals, with one animal showing a slightly stronger response. After sacrifice, the lymph nodes (popliteal, inguinal, axillary, and mesenteric) were surgically removed. They were disrupted by hand and then passed through a 70 μm filter to create a single cell suspension for each animal. Next, B cells were isolated from each sample using the EasySep™ Mouse Pan-B Cell Isolation Kit (Stemcell Technologies), a negative selection kit. The lymph node B cell population was quantified by counting with a C-Chip hemocytometer (Incyto), and viability was assessed using trypan blue. The cells were then diluted to 5,000 - 6,000 cells per mL in phosphate buffered saline (PBS) containing 12% OptiPrep™ Density Gradient Medium (Sigma). This cell mixture was used for microfluidic encapsulation. The inventors ran approximately one million B cells from each of six animals through the platform of emulsion droplet microfluidics.

[0245] Using a platform of emulsion droplet microfluidics or vortex emulsions, a DNA library encoding scFvs from single-cell RNA was generated in which natural heavy-chain and light-chain Igs were intact and paired. The method for generating the DNA library was divided into 1) poly(A)+ mRNA capture, 2) multiplexed overlap extension reverse transcriptase polymerase chain reaction (OE-RT-PCR), and 3) removal of artifacts and nested PCR for adding adapters for deep sequencing or yeast display libraries. The scFv library was made from approximately 1 million B cells from each animal that reached a positive titer.

[0246] For poly(A)+ mRNA capture, a custom-designed parallel flow emulsion droplet microfluidic chip made from glass (Dolomite) was used. The microfluidic chip had two input channels for fluorocarbon oil (Dolomite), one input channel for the above cell suspension mix, and one input channel for oligo-dT beads (NEB) at 1.25 mg / ml in cell lysis buffer (20 mM Tris pH 7.5, 0.5 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Tween®-20, and 20 mM dithiothreitol). The input channels were narrowed to 150 μm for most of the length of the chip and 55 μm at the droplet junction, etched to 50 μm, and coated with hydrophobic Pico-Glide (Dolomite). Three Mitos P-Pump pressure pumps (Dolomite) were used to pump the liquid through the chip. The droplet size varied with pressure, but typically droplets with a diameter of about 45 μm were the most stable. The emulsion was collected in a cooled 2 ml microcentrifuge tube and incubated at 40 °C for 15 min for mRNA capture. Pico-Break (Dolomite) was used to extract the beads from the droplets. In some embodiments, a vortex is used to create an emulsion that divides similar single cells.

[0247] For multiplex OE-RT-PCR, a glass Telos droplet emulsion microfluidic chip (Dololomite) was used. Beads bound with mRNA were resuspended in the OE-RT-PCR mix and injected into the microfluidic chip at a pressure that generated 27-μm droplets, together with a mineral oil-based surfactant mix (commercially available from GigaGen). The OE-RT-PCR mix contains 2× one-step RT-PCR buffer, 2.0 mM MgSO4, SuperScript III reverse transcriptase, and Platinum Taq (Thermo Fisher Scientific), along with a mixture of primers targeting the IgK C region, IgG C region, and all V regions. The overlap region is a DNA sequence encoding a Gly-Ser rich scFv linker sequence. A droplet disruption solution (commercially available from GigaGen) was used to recover the DNA fragments from the droplets, which were then purified using the QIAquick PCR Purification Kit (Qiagen). In some embodiments, a similar OE-RT-PCR emulsion was made using a vortex.

[0248] In nested PCR, first, the purified OE-RT-PCR product was run on a 1.7% agarose gel at 150 V for 80 min. A band of 1200–1500 base pairs (bp) corresponding to the ligated product was excised and purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey Nagel). Next, PCR was performed with addition of adapters for Illumina sequencing or yeast display; for sequencing, 7-nucleotide randommers were added to increase the accuracy of base calling in the next next-generation sequencing step. Nested PCR was carried out using 2× NEBNext High-Fidelity amplification mix (NEB) containing either an Illumina adapter with primers or primers for cloning into a yeast expression vector. The nested PCR product was run on a 1.2% agarose gel at 150 V for 50 min. An 800–1100 bp band was excised and purified using the NucleoSpin Gel and PCR Clean-up Kit (Macherey Nagel).

[0249] To convert the GigaLink (trademark) scFv library into a full-length CHO expression library, nested outer PCR primers were used to add adapters with overhangs for Gibson assembly to the 5' and 3' ends of the scFv library. Then, the NEBuilder HiFi DNA Assembly Master Mix (NEB, Ipswich, MA, USA) was used to insert the scFv library into a vector containing a single promoter, a secretion leader sequence for the light chain Ig, and the remainder of the IgG1 constant region, creating a cloned scFv library. This intermediate library was transformed into E. coli, spread on LB-ampicillin plates, and 500,000 to 1,000,000 colonies were scraped and pooled, and the plasmid was purified using ZymoPURE II Plasmid Maxiprep Kits (Zymo Research, Irvine, CA, USA). To create the full-length antibody library, the product of GA1 was linearized with BamHI-HF (NEB, Ipswich, MA, USA), and a second Gibson assembly was performed by using it as a vector to insert a synthetic amplicon containing a part of the light chain Ig constant region, a poly(A) signal for the light chain Ig, a promoter for the IgG gene, and a secretion leader sequence for the IgG gene. Then, the full-length library was transformed into E. coli and spread on LB-ampicillin plates. More than 500,000 colonies were scraped and the plasmid was purified using ZymoPURE II Plasmid Maxiprep Kits (Zymo Research), creating a full-length recombinant high-titer maxiprep library for transfection.

[0250] Subsequently, the Flp-In™-CHO cell line was adapted to suspension culture using FRT sites (Thermo Fisher Scientific, Waltham, MA, USA) integrated into the genome. In all steps of the adaptation process, "Ham's F-12" refers to Ham's F-12 (containing L-glutamine, Thermo Fisher Scientific, Waltham, MA, USA) and 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), and "BalanCD" refers to BalanCD CHO Growth A (Irvine Scientific) containing 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA). To adapt this cell line to suspension, cells were first passaged in a 50% mixture of Ham's F-12 and 50% BalanCD in a T-flask. Next, the cells were passaged in 25% Ham's F-12 and 75% BalanCD and switched to a shaking Erlenmeyer flask. Then, the cells were passaged in 10% Ham's F-12, 90% BalanCD + 0.2% anti-clumping agent (Irvine Scientific, Santa Ana, CA, USA) and stored for future use.

[0251] Using the Amaxa Nucleofector 4D (SG buffer, pulse DU133; Lonza, Basel, Switzerland), 100 million adapted Flp-In CHO cells were transfected per recombinant high immune library. These cells were plated in a shaking Erlenmeyer flask and allowed to recover in an incubator at 37 °C and 125 rpm for 48 hours. After 48 hours, the cells were counted to determine viability, seeded at 1 million cells per mL, and selection was initiated using 600 μg / mL hygromycin B (Gemini Bio, West Sacramento, CA, USA) in fresh medium. The cells were counted and the medium was changed every 2 - 3 days during the 7-day selection. The library was maintained in 600 μg / mL hygromycin B (Gemini Bio, West Sacramento, CA, USA) during expansion until viability exceeded 95%. When the cells were more than 95% viable and doubling every 24 hours, the cell line was stored for liquid nitrogen storage.

[0252] CHO cells stably expressing the antibody library were grown in a medium consisting of 90% BalanCD CHO Growth A Medium (Irvine Scientific, Santa Ana, CA), 9% Ham’s F-12 (Thermo Fisher Scientific, Waltham, MA, USA), 1% FBS (ThermoFisher Scientific), 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), 0.2% anti-aggregation agent (Irvine Scientific, Santa Ana, CA, USA). For small-scale production, 1 × 10 6Individual cells were seeded into 50 mL of medium in a 250 mL Erlenmeyer flask and grown at 37 °C, 5% CO2, and 125 rpm. The cells were continuously grown under these conditions and supplemented with 7.5 mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) on days 2, 4, and 7 of production runs. On day 8, the supernatant was recovered by centrifugation followed by filtration through a 0.22 μm 250 mL filter bottle (EMD Millipore, Burlington, MA, USA) with a 1 μm prefilter (EMD Millipore, Burlington, MA, USA). The recovered cell culture fluid (HCCF) was stored at 4 °C until protein A purification. For large-scale production of plasmacytoma recombinant hyperimmunization, the cells were grown in the same medium except that some modifications were made to the production conditions. Using the seed line, the cultures were scaled up at 37 °C from 2×10 7 cells to 1.2×10 10 cells. Then, 1×10 6 cells per mL were seeded into 2 L out of 5 L flasks (in triplicate; day 0). On day 2, the temperature was changed from 37 °C to 33 °C. On days 2, 4, 6, 8, 10, and 13 of the culture, 300 mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) was supplied to each flask. On day 14, the supernatant was recovered.

[0253] After recovery, HCCF was purified using MabSelect SuRe Protein A resin (GE Life Sciences, Marlborough, MA, USA) with the following buffers: Equilibration, Trapping, Wash 2 (25 mM Tris, 150 mM NaCl, pH 7.4), Wash 1 (25 mM Tris, 1 M NaCl, pH 7.4), Elution (20 mM Citric acid, pH 3.0), Neutralization (100 mM Tris, pH 8.0 for small scale and 1 M Tris, pH 9.0 for large scale). The column was sanitized with 0.1 N NaOH before and after use. For large scale production of plasma cell recombinant hyperimmune, an additional Wash 3 consisting of 0.5 M Arginine, pH 7.4 was used and further washed with Wash 2 before elution. The order of the purification steps was as follows: Equilibration, Load, Trapping, Wash 1, Wash 2 (Large scale: Wash 3, Wash 2), Elution, Neutralization (manually added into the tubes used for collection of elution fractions). Recombinant hyperimmune (RPP) was concentrated using a Vivaspin 20, 30 kDa molecular weight cut-off spin concentrator (Sartorius, Gottingen, Germany) and formulated in PBS (for small scale production) or 0.2 M Glycine, pH 4.5 (for large scale production) and then filtered at 0.22 μm.

[0254] Using ELISA, the binding of rhATG, i.e., the RPP of anti-T cells and anti-thymocytes, to antigens known to be expressed on the surface of T cells and thymocytes was tested. ELISA showed binding to CD4, CD45, and CD81. The antigen was coated on the ELISA plate at 1 μg / mL. Using a 1 / 3 serial dilution starting at 100 μg / mL for each antibody, a dose-setting curve was performed to determine the EC50. Since various secondary detection antibodies were used, the EC50 values could not be directly compared between rabbit ATG and rhATG. However, within each library, it was determined that the antigen had a stronger binding than its respective background. The antibody response was broadly reactive against many T cell antigens for both rhATG and rabbit ATG, and any binding was very strong against CD45 and CD5, and weaker binding against CD4, CD11, and CD81 (data not shown).

[0255] In vivo validation studies were performed. Using an in vivo model of GvHD (graft-versus-host disease), the functional efficacy of the delay induced by ATG treatment against GvHD was demonstrated. 1 × 10 7 human PBMC from one donor were engrafted into NSG mice. In this study, 6 mice per group were used and the drugs tested: rhATG (RPP), commercially available rabbit ATG, and vehicle control were injected IV. At a single time point 7 days after engraftment, the animals were treated (6 mg / kg). Additionally, a positive control group (8 mice) received abatacept, a drug commonly used to prevent GvHD, which was administered intraperitoneally (IP) every other day from day 5 until the end of the study. Immune cells were measured by flow cytometry for expansion indicating progression to GvHD, and the animals were monitored for the clinical picture of GvHD resulting in weight loss and death.

[0256] Forty-two days after PBMC engraftment, all surviving animals were withdrawn, and survival analysis was completed for each treatment group. There was no significant delay for rhATG (p = 0.2, Mantel-Cox), and only a slight delay for rabbit ATG against GvHD was observed (p = 0.01, Mantel-Cox) (data not shown). Flow cytometry was used to measure engrafted PBMC before treatment, two days after treatment, and nine days after treatment. As seen two days after treatment, rhATG and rabbit ATG depleted CD45+ cells caused a delay in the complete engraftment of CD45+ cells, however, by day 9, there was no significant difference between any of the groups (data not shown).

[0257] These results demonstrate that rhATG (RPP library) has an antigen-specific antibody binding profile similar to that of currently marketed rabbit ATG, although some differences were observed. In addition, rhATG has a similar performance to marketed rabbit ATG in delaying the progression to GvHD in mice using various dosing regimens. 10.1.2. (Example 2) Generation of an RPP library with activity against Haemophilus influenzae type b (Hib) from human donors

[0258] Both in vitro and in vivo studies were conducted to test a library of polyclonal antibody pools (pAbs), i.e., RPPs, having activity against Haemophilus influenzae type b (Hib). Anti-Hib pAbs generated from four different B cell subtypes collected from donors vaccinated with Pedvax-HIB conjugate vaccine were tested. The four subtypes tested were CD43+ plasmablasts, CD27+ memory B cells, peripheral CD138+ plasma cells, and pan B cells (all B cells). All four pAbs were first tested in vitro. The pAb generated from CD138+ plasma cells was the most potent in vitro and thus this product was tested in an in vivo challenge model compared to IVIG.

[0259] SEQ ID numbers of the heavy and light chain CDR3 sequences of the RPPs are provided in RPP3 - 6 of Table 5 above.

[0260] CRO (BloodCenter Wisconsin, Milwaukee, WI, USA) was used to vaccinate two donors (donor 1, a 26-year-old Caucasian female, and donor 2, a 21-year-old Asian male) with PedvaxHIB vaccine (Merck, Kenilworth, NJ, USA). Leukapheresis was performed 8 or 9 days later to obtain PBMCs. In parallel, plasma was isolated from separate blood draws on the day of leukapheresis, prior to vaccination. For plasma samples, the response to the vaccine was confirmed by ELISA against Hib (Alpha Diagnostics, San Antonio, TX, USA; see the method below) compared to plasma from the same donors prior to vaccination. The sample collection protocol was approved for GigaGen by the Institutional Review Board (IRB) protocol number PRO00028063 (Medical College of Wisconsin / Froedtert Hospital IRB). Informed consent was obtained from all participants and samples were transported anonymously to GigaGen.

[0261] To isolate naïve B cells, the inventors used the Human EasySep Pan - B Cell Enrichment Kit (Stemcell number 19554, Vancouver, BC, Canada). To isolate CD43+ cells, the inventors used positive selection beads against naïve B cells and CD43 (Miltenyi number 130 - 091 - 333, Bergisch Gladbach, Germany). To isolate CD27+ cells, the inventors applied CD27 positive selection beads (Miltenyi number 130 - 051 - 601, Bergisch Gladbach, Germany) to the negative fraction from CD43+ selection. For plasmablasts, the inventors applied the EasySep Human CD138 Positive Selection Kit (Stemcell number 18357, Vancouver, BC, Canada) to PBMC. After isolation, antibody - producing cells were cryopreserved using CryoStor® CS10 (Stemcell Technologies, Vancouver, BC, Canada). Immediately before generating the paired heavy - and light - chain libraries, the cells were thawed, washed in cold DPBS + 0.5% BSA, viability was assessed using trypan blue with a Countess™ cell counter (Thermo Fisher Scientific, Waltham, MA, USA), and then resuspended at 5,000 - 6,000 cells per μl in 12% OptiPrep® Density Gradient Medium (Sigma, St. Louis, MO, USA). This cell mixture was used for microfluidic encapsulation as described in the next section.

[0262] Generation of scFv libraries from antibody - producing cells (Adler et al., Mabs 9, 1282 - 1996, 2017) consists of three steps: (i) poly(A)+ mRNA capture, (ii) multiplexing Overlap extension reverse transcriptase polymerase chain reaction (OE-RT-PCR), and (iii) nested PCR to remove artifacts and add adapter sequences for deep sequencing or yeast display libraries.

[0263] To convert the GigaLink™ scFv library into a full-length CHO expression library, the inventors first used nested outer PCR primers to add adapters with overhangs for Gibson assembly to the 5' and 3' ends of the scFv library. Next, the scFv library was inserted into a vector containing a single promoter, a secretion leader sequence for the light chain Ig, and the remaining part of the IgG1 constant region using NEBuilder HiFi DNA Assembly Master Mix (NEB, Ipswich, MA, USA) to create a cloned scFv library. This intermediate library was transformed into E. coli, spread on LB-ampicillin plates, and 500,000 to 1,000,000 colonies were scraped and pooled, and the plasmid was purified using ZymoPURE II Plasmid Maxiprep Kits (Zymo Research, Irvine, CA, USA). To generate the full-length antibody library, the product of GA1 was linearized with BamHI-HF (NEB, Ipswich, MA, USA), and it was used as a vector to insert a synthetic amplicon containing a part of the light chain Ig constant region, a poly(A) signal for the light chain Ig, a promoter for the IgG gene, and a secretion leader sequence for the IgG gene, and a second Gibson assembly was performed. Then, the full-length library was transformed into E. coli and spread on LB-ampicillin plates. The inventors generally scraped more than 500,000 colonies, purified the plasmid using ZymoPURE II Plasmid Maxiprep Kits (Zymo Research), and created a full-length recombinant high-titer maxiprep library for transfection.

[0264] The inventors adapted the adherent Flp-In™-CHO cell line to suspension culture using FRT sites (Thermo Fisher Scientific, Waltham, MA, USA) integrated into the genome. At all steps in the adaptation process, "Ham's F-12" refers to Ham's F-12 (with L-glutamine, Thermo Fisher Scientific, Waltham, MA, USA) and 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), and "BalanCD" refers to BalanCD CHO Growth A (Irvine Scientific) containing 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA). To adapt this cell line to suspension, the cells were first passaged into a 50% mixture of Ham's F-12 and 50% BalanCD in a T-flask. Next, the cells were passaged into 25% Ham's F-12 and 75% BalanCD and switched to a shaking Erlenmeyer flask. Then, the cells were passaged into 10% Ham's F-12, 90% BalanCD + 0.2% anti-agglutinin (Irvine Scientific, Santa Ana, CA, USA) and stored for future use.

[0265] One hundred million adapted Flp-In CHO cells were transfected per recombinant hyperimmune library using the Amaxa Nucleofector 4D (SG buffer, pulse DU133; Lonza, Basel, Switzerland). These cells were plated in shaking Erlenmeyer flasks and allowed to recover in an incubator at 37 °C and 125 rpm for 48 h. After 48 h, the cells were counted to determine viability, seeded at 1 million cells per mL, and selection was initiated using 600 μg / mL hygromycin B (Gemini Bio, West Sacramento, CA, USA) in fresh medium. The cells were counted and the medium was changed every 2–3 days during 7 days of selection. The library was maintained in expansion with 600 μg / mL hygromycin B (Gemini Bio, West Sacramento, CA, USA) until viability exceeded 95%. If the cells were more than 95% viable and doubling every 24 h, the cell line was stored for liquid nitrogen storage.

[0266] CHO cells stably expressing the antibody library were grown in a medium consisting of 90% BalanCD CHO Growth A Medium (Irvine Scientific, Santa Ana, CA), 9% Ham’s F-12 (Thermo Fisher Scientific, Waltham, MA, USA), 1% FBS (ThermoFisher Scientific), 4 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), 0.2% anti-clumping agent (Irvine Scientific, Santa Ana, CA, USA). For small-scale production, 1 × 10 6Individual cells were seeded into 50 mL of medium in a 250 mL Erlenmeyer flask and grown at 37 °C, 5% CO2, and 125 rpm. Cells were continuously grown under these conditions and supplemented with 7.5 mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) on days 2, 4, and 7 of production runs. On day 8, the supernatant was recovered by centrifugation followed by filtration through a 0.22 μm 250 mL filter bottle (EMD Millipore, Burlington, MA, USA) with a 1 μm prefilter (EMD Millipore, Burlington, MA, USA). The recovered cell culture fluid (HCCF) was stored at 4 °C until protein A purification. For large-scale production of plasmacytoma recombinant hyperimmunization, cells were grown in the same medium except that some modifications were made to the production conditions. Using the seed line, the culture was scaled up at 37 °C from 2×10 7 cells to 1.2×10 10 cells. Then, 1×10 6 cells per mL were seeded into 2 L out of 5 L flasks (in triplicate; day 0). On day 2, the temperature was changed from 37 °C to 33 °C. On days 2, 4, 6, 8, 10, and 13 of the culture, 300 mL of CHO Feed 1 (Irvine Scientific, Santa Ana, CA, USA) was supplied to each flask. On day 14, the supernatant was recovered.

[0267] After recovery, HCCF was purified using MabSelect SuRe Protein A resin (GE Life Sciences, Marlborough, MA, USA) with the following buffers: Equilibration, Trapping, Wash 2 (25 mM Tris, 150 mM NaCl, pH 7.4), Wash 1 (25 mM Tris, 1 M NaCl, pH 7.4), Elution (20 mM Citric acid, pH 3.0), Neutralization (100 mM Tris, pH 8.0 for small scale, 1 M Tris, pH 9.0 for large scale). Before and after use, the column was sanitized with 0.1 N NaOH. For large scale production of plasmacytoma recombinant hyperimmune, the inventors used an additional Wash 3 consisting of 0.5 M arginine, pH 7.4 and further washed with Wash 2 before elution. The order of the purification steps was as follows: Equilibration, Loading, Trapping, Wash 1, Wash 2 (large scale: Wash 3, Wash 2), Elution, Neutralization (manually added into the tubes used for collection of elution fractions). A Vivaspin 20, 30 kDa molecular weight cut-off spin concentrator (Sartorius, Gottingen, Germany) was used to concentrate the recombinant hyperimmune and formulate it in PBS (for small scale production) or 0.2 M glycine, pH 4.5 (for large scale production), and then filtered at 0.22 μm.

[0268] Imaged capillary isoelectric focusing (iCIEF) was performed using a Maurice imaging cIEF analyzer (Protein Simple, San Jose, CA, USA). Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) was performed under reducing and non-reducing conditions using a LabChip GX II Touch HT (Perkin Elmer, Waltham, MA, USA). Endotoxin levels were measured using an Endosafe nexgen-PTS (Charles River, Wilmington, MA, USA).

[0269] The inventors observed a 92.2% HBV RPP yield in the inventors' Protein A step. Under non-reducing conditions, the inventors observed a single peak (>99%) of 166.2 kDa by CE-SDS. Under reducing conditions, the RPP showed >99% pure IgG monomer and <1% other proteins, while plasma IVIg showed approximately 3.1% unknown proteins, suggesting that recombinant hyperimmune can be produced with a higher purity of IgG than plasma IVIg. Analysis of the recombinant hyperimmune purified by iCIEF revealed a broad spectrum of isoelectric point species, while plasma IVIg showed a considerably broader range of isoelectric point species. The inventors speculate that plasma IVIg has a more diverse range of isoelectric point species because plasma IVIg contains a more diverse set of antibodies, including various IgG isotypes as well as IgL (recombinant hyperimmune is only IgG1). Finally, the endotoxin level was <0.5 endotoxin units (EU) per mg, which is a typical criterion for recombinant mAb therapeutics.

[0270] The deep antibody sequencing library was quantified using the quantitative PCR Illumina Library Quantification Kit (KAPA, Wilmington, MA, USA) and diluted to 17.5 pM. According to the manufacturer's instructions, the library was sequenced on a MiSeq (Illumina, San Diego, CA, USA) using the 500-cycle MiSeq Reagent Kit v2. To generate the sequencing library, the inventors added Illumina sequencing adapters to the 5' and 3' ends of the construct of interest using tail-end PCR. The inventors then obtained 340 cycles of forward reads and 162 cycles of reverse reads. This resulted in forward and reverse reads that overlapped in part with the CDR3-H and VH genes, increasing the reliability of nucleotide calls. Sequence analysis was performed using the bioinformatics pipeline previously reported by the inventors (Adler et al., Mabs 9, 1282-1996, 2017). Pearson correlation was performed.

[0271] Each of the four HBV RPPs was derived from 1.12 to 1.39 million input cells. After subjecting the repertoire to the inventors' library generation pipeline, the recombinant hyperimmune clonal diversities were all less than 2,000 antibody clones (range of 880 - 1,659), capturing a substantial portion of the diversity of the input antibodies. All four recombinant hyperimmunizations had a median germline IgHV identity of 93%, consistent with previous analysis for individuals vaccinated with the Hib vaccine (Truck et al., 2015), and for any cell type It was suggested that antibodies with deliberately high affinity were not generated. The clonal diversity was not strongly skewed towards the most frequently occurring antibodies in any of the mixtures. The most common antibodies were present at a frequency of 3.5% (plasma cell hyperimmunization). Poly B recombinant hyperimmunization had the least skewed clonal diversity (the top 20 antibodies were 12.7% of all antibodies), and plasma cell recombinant hyperimmunization had the most skewed clonal diversity (the top 20 antibodies were 26.6% of all antibodies).

[0272] The inventors investigated the genetic diversity of four recombinant hyperimmunization libraries. Overlap analysis revealed that 11.8% or fewer clones were shared between any given two recombinant hyperimmunization libraries. Pearson correlation analysis was not significant for any of the pairwise comparisons (p < 0.01). All four recombinant hyperimmunization libraries contained various IgGV-J gene pairings, which included a high frequency of antibodies related to the IgHV3-23 and IgHJ4 genes, which is also seen in the anti-Hib repertoire, among others (Silverman & Lucas, 1991; Adderson et al., 1993; Lucas et al., 2003; Truck et al., 2015). Other common IgHV gene sons included IgHV3-30, IgHV1-69, and IgHV3-7. All libraries also contained complementarity-determining region (CDR) 3 sequences containing either the peptide GYGFD or GYGMD, which have been previously observed in the anti-Hib repertoire (Lucas et al., 2003; Truck et al., 2015). The inventors concluded that all four libraries contained standard anti-Hib sequences, as well as similar levels of divergence from the germline and genetic diversity. However, the four libraries contained distinct antibody mixtures, which may have different functional characteristics.

[0273] ​The Human Anti-Hib-PRP IgG ELISA kit (Alpha Diagnostics number 980-100-PHG, San Antonio, TX, USA) was used for anti-Hib ELISA titers. Serial dilutions of the antibody preparation were performed in Low NSB (non-specific binding) sample diluent. Quantitative measurements were performed at 450 nm in a plate reader (Molecular Devices, Fremont, CA, USA). The EC50 value was calculated using SoftMax Pro (Molecular Devices, Fremont, CA, USA). The inventors also determined the plasma pools from both donors before and after vaccination with the Hib active vaccine, and the anti-Hib RPP antibody titers regarding IVIg. In plasmablast, pan-B, and plasmablast recombinant hyperimmunization, much higher Hib binding titers were obtained (in the range of 160× to 2,323×), and the highest titer was obtained in plasmablast hyperimmunization. The post-vaccination plasma was only 3.7× the anti-Hib titer of IVIg, but under the tested conditions, no anti-Hib titer was detected in memory B cell recombinant hyperimmunization. Overall, these data indicate that by simply selecting the appropriate cell type from vaccinated donors by the inventors' manufacturing process, the anti-Hib titer can be significantly increased.

[0274] In vitro neutralization studies were performed by a CRO (ImQuest Frederick, MD, USA). The Haemophilus influenzae type b Eagan strain was obtained as a frozen glycerol stock from Zeptometrix (number 0801679, Buffalo, NY, USA) and stored at -80 °C. The Haemophilus influenzae strain ATCC 10211 was obtained as a frozen stock from the American Type Culture Collection (ATCC, Frederick, MD, USA) and amplified as recommended by the supplier. Colonies from an overnight incubation on chocolate agar plates were inoculated into growth medium (Brain Heart Infusion, or BHI broth, BD BBL 299070, San Jose, CA, USA, containing 2% Fildes enrichment, Remel number R45037, San Diego, CA, USA) and an optical density of approximately 0.4 at 625 nm (OD 625 ) was achieved. The culture was adjusted to an OD 8 of 0.15, equal to approximately 5×10 625 colony forming units (CFU) / mL. The culture was further diluted in dilution buffer (Hanks Balanced Salt Solution, Gibco, Waltham, MA, USA number 14025-092, containing 2% Fildes enrichment) to 5×10 4 CFU / mL. The density of the bacterial culture used in this assay was confirmed by plating 50 μL of the 5×10 3 and 5×10 2 dilutions in duplicate on chocolate agar and counting the colonies after incubation at 37 °C / 5% CO2 for 24 hours.

[0275] The test article was started at 200 μg / mL and serially diluted 3-fold in dilution buffer so that a total of 10 dilutions were evaluated. 10 μL of each dilution of the test article was added in duplicate to a 96-well microtiter plate. Then, approximately 5×10 4Eagan or ATCC 10211 bacteria at a concentration of CFU / mL were added to plates with a volume of 20 μL such that the total bacterial concentration in the wells was 1×10 4 CFU / 20 μL. After incubation at 37 °C / 5% CO2 for 15 minutes, 25 μL of rabbit complement (Pel-Freez number 31061-1, Rogers, AR, USA) and 25 μL of dilution buffer were added to each well. The plates were incubated at 37 °C / 5% CO2 for 60 minutes. After incubation, 5 μL of each reaction mixture was diluted in 45 μL of dilution buffer, and the entire 50 μL was plated onto chocolate agar plates. The plates were incubated at 37 °C / 5% CO2 for approximately 16 hours. After incubation, the bacterial colonies were counted. The concentration of the test substance at which more than 50% of the bacteria were killed was the SBI.

[0276] As expected from the ELISA data, memory B cell recombinant hyperimmunization was unable to neutralize any of the Hib strains at any of the concentrations tested. With plasmablast recombinant hyperimmunization, again, the highest titers were obtained, and for the Eagan and ATCC 10211 strains, the SBIs were 81 and 243, respectively. Pan-B and plasmablast recombinant hyperimmunization were one-ninth as effective as plasmablast recombinant hyperimmunization. No neutralization was detected for IVIg at any of the concentrations tested. The inventors conclude that plasmablast recombinant hyperimmunization is the most effective among the four cell types tested.

[0277] All vertebrate experiments were conducted in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies, Performed under the supervision and approval of either the Institutional Animal Care and Use Committee of Sinclair Research Center, LLC, Missouri (USA) in accordance with the standards incorporated in the Eighth Edition or the National Committee of Animal Ethics, Denmark in accordance with the standards of EU Directive 2010 / 63 / EU (Permit Number: 2014-15-0201-00171). Performed under the supervision and approval of either the Institutional Animal Care and Use Committee of Sinclair Research Center, LLC, Missouri (USA) in accordance with the standards incorporated in the Eighth Edition or the National Committee of Animal Ethics, Denmark in accordance with the standards of EU Directive 2010 / 63 / EU (Permit Number: 2014-15-0201-00171).

[0278] For acute toxicity, Balb / cJ mice (Charles River, Wilmington, MA, USA) were randomly divided by a CRO (Sinclair Research, Auxvasse, MO, USA) into seven groups of six animals per group. Three of these groups were administered a single dose of recombinant hyperimmune at 30 mg / kg, 100 mg / kg, or 300 mg / kg. The negative control group was administered a single dose of saline vehicle. The three remaining groups were administered a single dose of IVIg in plasma (Gammagard; Grifols, Sant Cugat, Catalonia) at 30 mg / kg, 100 mg / kg, or 300 mg / kg. The test article sample was diluted in 0.2 M glycine, pH 4.5. Administration of the test article was performed intravenously via the tail vein. The dosing volume was calculated based on the most recent body weight of each individual animal. The mice were then observed twice daily for 8 days for general health, response at the test article administration site, morbidity and mortality, body weight, and a general physical examination (skin, mucosa, eyes, ears, nose, and respiration). After 3 days, the animals were euthanized with CO2 and a terminal serum biochemical examination was performed, including albumin, globulin, glucose, total protein, blood urea nitrogen, and several other metrics.

[0279] The inventors did not observe any findings related to the test article in any of the test groups. The inventors conclude that the no-observed-adverse-effect level (NOAEL) for a single intravenous administration of the plasmablast recombinant hyperimmune is 300 mg / kg. IVIg is generally administered to immunocompromised patients at approximately 300 mg / kg for protection against Hib and other pathogens, and since the Hib hyperimmune product is thousands of times more potent, the inventors conclude that the plasmablast recombinant hyperimmune has no observable toxicity at minimally effective doses.

[0280] For pharmacokinetics, a contract research organization (CRO) (Sinclair Research, Auxvasse, MO, USA) administered the plasmablast recombinant hyperimmune as a single intravenous tail vein dose of 100 mg / kg to 20 male Balb / cJ mice (Charles River, Wilmington, MA, USA). A sparse blood sampling procedure was performed so that no mouse received more than 2 of the planned 7 PK blood samplings. The inventors then measured human IgG in serum using a sandwich ligand binding assay (LBA) and Meso Scale Discovery (MSD; Rockville, MD, USA) electrochemiluminescence (ECL) technology. A capture antibody (SouthernBiotech number 2049-01, Birmingham, AL, USA) was coated onto 96-well plates (MSD, Rockville, MD, USA). Serum samples were diluted in PBS / T containing 1% BSA (PBS / T / BSA) to a minimum dilution ratio (MRD) of 1:100. The diluted samples were then added to the designated wells. After another wash step, PBS / T / BSA containing 1 mg / mL biotinylated goat anti-human IgG (SouthernBiotech number 2049-08, Birmingham, AL, USA) was inoculated into the wells. After incubation, streptavidin-SULFO-TAG and then 2× read buffer T (MSD, Rockville, MD, USA) were added. MSD QuickPlex SQ Using a 120 instrument, ECL units were measured. Further, for each run using recombinant hyperimmune based on plasma cells, a standard curve was created. Using Discovery Workbench software (MSD, Rockville, MD, USA), the data was fit using a four-parameter logistic (4-PL) curve fit of mean ECL units versus nominal IgG standard values. The inventors removed two animals with readings of 1100 ng / mL or lower at the 1-hour time point from further analysis under the assumption that intravenous administration had failed. Then, the inventors applied non-compartmental analysis to the concentration-time data using the PKNCA package of R (Denney et al., 2015) to estimate the maximum plasma concentration (C max ), the time to reach the maximum plasma concentration (T max ), and the half-life (t 1 / 2 ).

[0281] The maximum plasma concentration was 12,360 ng / mL (C max ), observed 1 hour after administration (T max ). The half-life (t 1 / 2 ) of the recombinant hyperimmune was approximately 34.5 hours. Combining these data with the ELISA titer data, the inventors estimate that the maximum value of the anti-Hib trough level was 861 IU / mL for a single 100 mg / kg intravenous dose.

[0282] The Haemophilus influenza strain ATCC10211 was grown overnight on chocolate agar plates at 35 °C and 5% CO2. Overnight colonies were resuspended in sterile saline to 1.5×10 8 CFU / mL. This suspension was diluted in BHI broth containing 5% mucin and 2% hemoglobin to approximately 1×10 6 CFU / mL and further diluted 10-fold to 10 CFU / mL.

[0283] Balb / cJ mice (Taconic, Denmark; n = 6 per group) were given 104 , 10 5 , or 10 6 The mice were inoculated with a single 0.5 mL intraperitoneal administration of Hib bacteria (strain ATCC 10211) at 10

[0284] Hib infection was lethal to all but one of the mice at all inoculation doses in the vehicle control group. In contrast, in the recombinant hyperimmune treatment group (10 6 CFU inoculation group), only 1 out of 18 mice was severely infected. IVIg was much less protective than recombinant hyperimmunity, with 5 / 6 of the mice in the 10 5 CFU and 10 6 CFU inoculation groups, and 10 4Two out of six mice in the CFU inoculation group suffered severely from the infection. Analysis of the bacterial load in the blood showed that recombinant hyperimmunization eliminated Hib from the bloodstream of all animals, while IVIg treatment resulted in a significantly lower bacterial load than the vehicle control in only one of the inoculation groups, and no significant decrease was demonstrated in the two inoculation groups (Dunnett's multiple comparison test, p < 0.05). In peritoneal perfusion, recombinant hyperimmunization significantly reduced the bacterial load again compared to the vehicle control group (Dunnett's multiple comparison test, p < 0.05). However, Hib bacteria were undetectable in the peritoneal perfusion of surviving animals treated with ciprofloxacin, while Hib bacteria were detectable in the peritoneal perfusion of 6 / 17 surviving animals treated with recombinant hyperimmunization (in the range of 23 - 77 CFU / mL). This probably suggests a difference in the efficacy of recombinant hyperimmunization between the peritoneum and the blood due to the bioavailability of the drug or complement in the peritoneum.

[0285] In some embodiments, Hib hyperimmunization is spiked into conventional plasma IVIg to increase the anti-Hib titer of IVIg. In some embodiments, several anti-pathogen hyperimmunizations are spiked into conventional plasma IVIg; for example, hyperimmunizations against Hib, pneumococcus, influenza A virus, and tetanus are spiked into plasma IVIg to treat patients with primary immunodeficiency. The spike in hyperimmunization increases the titer of antibodies against pathogens to which patients with primary immunodeficiency are particularly sensitive. Multiple spike-ins can be mixed with plasma IVIg to produce increased titers against multiple pathogens.

[0286] A series of in vitro and in vivo experiments were used to determine the following. In Hib, plasmablasts after vaccination produce the most effective RPP. Plasmablast Hib RPP was more than 2,300× more effective than IVIG in plasma (by ELISA). Plasmablast Hib RPP strongly protected against Hib infection in an in vivo challenge model. The use of germinal center B cells and pan B cells also resulted in effective RPP in vitro, although less potent than plasmablasts. For this antigen, RPP generated from memory B cells had only undetectable levels of effectiveness in in vitro assays. 10.1.3. (Example 3) Generation of an RPP library with activity against the capsular polysaccharide of Streptococcus pneumoniae

[0287] Streptococcus pneumoniae is the cause of pneumococcal pneumonia. A recombinant polyclonal antibody (pAb) with activity against Streptococcus pneumoniae, i.e., the RPP library "GG-Pnc", was generated. GG-Pnc was tested in vitro. The results demonstrate the functional effectiveness and potency in vitro of GG-Pnc with activity against the capsular polysaccharide of Streptococcus pneumoniae. The library was analyzed by ELISA for bulk pneumococcal polysaccharide, serotype-specific ELISA, and serotype-specific opsonization assay.

[0288] The sequence numbers of the heavy and light chain CDR3 sequences of GG-Pnc are provided in Table 5 above (RPP1).

[0289] Using the recombinant techniques described in Examples 1 and 2, GG-Pnc, i.e., an RPP library, was prepared. This library was prepared from three donors vaccinated with Pneumovax-23. Pneumovax-23 consists of capsular polysaccharides derived from 23 pneumococcal serotypes. All three donors showed an increase in titer against pneumococcal capsular polysaccharides after vaccination when measured by ELISA. The rpAb was made from a mixture of all B cell subtypes isolated from the donors.

[0290] The response of bulk polysaccharide-specific antibodies against the 23 pneumococcal polysaccharides found in the Pneumovax-23 vaccine was measured by Alpha Diagnostics ELISA, and this was used to measure the EC50 of the RPP library. An 8-step 3-fold dilution series was performed and a 4-parameter logistic analysis was performed to calculate the EC50. The RPP library, GG-Pnc, was approximately 100-fold more effective than IVIG.

[0291] Serotype multiplex ELISA was performed to evaluate the antibody diversity of the GG-Pnc RPP library compared to IVIG. Twenty pneumococcal serotypes were measured by ELISA. Using international standards for pneumococcal-specific responses, the antibody-specific responses in GG-Pnc and IVIG (Gamunex) were measured. GG-Pnc had concentrations similar to or higher than IVIG against all serotypes except serotype 6A.

[0292] Serotype-specific opsonophagocytosis assays were performed to evaluate the killing function induced by the antibodies. Fourteen pneumococcal serotypes were measured by opsonophagocytic response using GG-Pnc and IVIG (Gamunex). Consistent with the multiplex ELISA, GG-Pnc was similar to or more effective than IVIG against all serotypes except 6A.

[0293] Since it was not included in previous analyses, a serotype 2-specific ELISA was performed to determine the ability of GG-Pnc to bind to this serotype, which is an option available for the in vivo mouse model. An 8-step three-fold dilution series was performed and a four-parameter logistic analysis was used to calculate the EC50; since IVIG had only minimal binding to serotype 2, only GG-Pnc had a value even at very high concentrations.

[0294] The GG-Pnc RPP library strongly bound to a diverse set of pneumococcal serotypes and was able to neutralize all serotypes tested based on an in vitro opsonization assay. GG-Pnc was similar or more effective than IVIG against all but one serotype (for both binding and killing), using all B cells isolated from vaccinated donors without performing a serotype-specific enrichment procedure. GG-Pnc also strongly bound to serotype 2. 10.1.4. (Example 4) Generation of an RPP library with activity against influenza A antigen

[0295] An RPP library (RPP1) with activity against influenza A antigen was generated using the recombinant method described herein.

[0296] The SEQ ID numbers of the heavy and light chain CDR3 sequences of RPP1 are provided in Table 5 above (RPP1). 10.1.5. (Example 5) Generation of an RPP library with activity against hepatitis B virus antigen (Engerix, GSK)

[0297] Two libraries of RPPs (RPP8 and RPP9) with activity against hepatitis B virus antigen were generated using the recombinant method described herein.

[0298] Provide the SEQ ID NOs of the RPP9 and the heavy and light chain CDR3 sequences of RPP9 in Table 5 above (RPP1). 11. Incorporation by reference

[0299] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document had been individually indicated to be incorporated by reference for all purposes. 12. Equivalents

[0300] Although various specific embodiments have been illustrated and described, the above specification is not limiting. It will be recognized that various changes may be made without departing from the spirit and scope of the invention. Many of these changes will be apparent to those skilled in the art in light of this specification. The present invention provides, for example, the following items. (Item 1) A library of recombinant polyclonal proteins (RPPs) that specifically bind to an antigen, a. wherein the antigen is a polysaccharide of Haemophilius influenzae type b, and the library comprises at least 100 to 6,597 RPPs each having a pair of homologous sequences of heavy chain CDR3 and light chain CDR3 selected from the sequences of SEQ ID NOs: 33,981 to 47,174, or b. wherein the antigen is a polysaccharide of Haemophilius influenzae type b, and the library comprises at least 100 to 8,583 RPPs each having a pair of homologous sequences of heavy chain CDR3 and light chain CDR3 selected from the sequences of SEQ ID NOs: 47,175 to 64,340, or c. wherein the antigen is a polysaccharide of Haemophilius influenzae type b, and the library comprises at least 100 to 7,956 RPPs each having a pair of homologous sequences of heavy chain CDR3 and light chain CDR3 selected from the sequences of SEQ ID NOs: 64,341 to 80,252, or d. The antigen is a polysaccharide of Haemophilus influenzae type b, and the library contains at least 100 to 10,187 RPPs, each having a homologous pair of the sequences of the heavy-chain CDR3 and the light-chain CDR3 selected from the sequences of SEQ ID NOs: 80,253 to 100,626, or e. The antigen is a polysaccharide of Streptococcus pneumoniae, and the library contains at least 100 to 10,537 RPPs, each having a homologous pair of the sequences of the heavy-chain CDR3 and the light-chain CDR3 selected from the sequences of SEQ ID NOs: 1 to 21,074, or f. The antigen is a hepatitis B virus antigen, and the library contains at least 100 to 1,617 RPPs, each having a homologous pair of the sequences of the heavy-chain CDR3 and the light-chain CDR3 selected from the sequences of SEQ ID NOs: 100,627 to 103,860, or g. The antigen is a hepatitis B virus antigen, and the library contains at least 100 to 1,260 RPPs, each having a homologous pair of the sequences of the heavy-chain CDR3 and the light-chain CDR3 selected from the sequences of SEQ ID NOs: 103,861 to 106,380, or h. The antigen contains human thymocytes, and the library is an RPP library containing at least 100 to 6,889 RPPs, each having a homologous pair of the sequences of the heavy-chain CDR3 and the light-chain CDR3 selected from the sequences of SEQ ID NOs: 106,381 to 12,015. (Item 2) The RPP library according to Item 1, wherein each RPP is a scFv. (Item 3) The RPP library according to Item 1, wherein each RPP is a full-length antibody. (Item 4) The RPP library according to Item 1, wherein each RPP is a full-length antibody and is produced in CHO cells. (Item 5) The RPP library according to any one of Items 1 to 4, wherein each RPP is recombinantly produced using a sequence derived from a plasmacyte or a plasmablast from at least one donor injected with the antigen. (Item 6) Each RPP is recombinantly produced using sequences derived from plasma cells or plasmablasts from at least one donor injected with the antigen, and the activity of the RPP library is at least 10-fold greater than the serum titer activity of the donor against the antigen. The RPP library according to any one of items 1 to 4. (Item 7) The RPP library according to item 6, wherein the activity is measured by an in vitro pathogen neutralization assay, an in vitro binding assay to an antigen, or an in vivo efficacy assay. (Item 8) The RPP library according to any one of items 5 to 7, wherein the donor is human. (Item 9) The RPP library according to any one of items 1 to 8, comprising at least 100, at least 1000, at least 10,000 or at least 100,000 RPPs. (Item 10) A pharmaceutical composition comprising the RPP library according to any one of items 1 to 9 and a pharmaceutically acceptable excipient. (Item 11) A method of treating a subject in need, comprising administering to the subject an effective amount of the RPP library according to any one of items 1 to 9 or the pharmaceutical composition according to item 10. (Item 12) The method according to item 11, wherein the subject has immunodeficiency, cancer, Alzheimer's disease, a viral infection, a bacterial infection, or has undergone a solid organ or cell transplantation. (Item 13) A method comprising administering to a subject an effective amount of the RPP library according to any one of items 1 to 9 or the pharmaceutical composition according to item 10. (Item 14) The method according to any one of items 11 to 13, further comprising administering one or more drugs. (Item 15) A plurality of isolated polynucleotides, wherein each polynucleotide encodes one member of the RPP library according to any one of items 1 to 9. (Item 16) A plurality of isolated vectors, wherein each vector contains a polynucleotide encoding one member of the RPP library according to any one of items 1 to 9. (Item 17) The plurality of isolated vectors according to item 16, which are expression vectors. (Item 18) A plurality of isolated host cells comprising the plurality of isolated polynucleotides according to item 15 or the plurality of isolated vectors according to item 16 or item 17. (Item 19) A method for producing the RPP library according to any one of items 1 to 9, the method comprising incubating the isolated host cell according to item 18 under conditions for expression of the RPP library, and isolating the RPP. (Item 20) The method according to item 19, wherein the RPP is a full-length antibody and the isolated host cell is a CHO cell.

Claims

【Claim 1】 The invention described in the specification.

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