Custom recombinant polyclonal proteins and methods of use thereof

Abstract

Provided herein is a recombinant polyclonal protein library (RPP) generated, at least in part, by an in silica method. Also provided are methods of using the RPP.

Description

CUSTOM RECOMBINANT POLYCLONAL PROTEINS AND METHODS OF USE THEREOF1. FIELD

[0001] Provided herein is a polyclonal library comprising recombinant proteins (“a recombinant polyclonal protein library” or RPP), also called recombinant polyclonal antibody proteins, recombinant hyperimmune globulins, or simply recombinant hyperimmunes, with binding specificity for a target molecule or complex of molecules (also referred to herein as custom recombinant polyclonal proteins, custom RPP, or cRPP). Included are therapeutics, vaccines, and libraries, and compositions comprising such RPP, including pharmaceutical compositions. Also provided are methods of making an RPP, and methods of using an RPP, for example, for therapeutic purposes.2. BACKGROUND

[0002] Many diseases, such as those caused by infectious viruses or bacteria with many variants or serotypes, are best treated by drugs that target multiple epitopes. An established therapeutic modality is multispecific (multivalent) antibodies derived from human or animal plasma, such as intravenous immunoglobulin (IVIG). Polyclonal antibody drugs with higher potency, known as hyperimmune globulins, are often derived from the plasma of recently vaccinated human donors, for example, HepaGam B against hepatitis B virus (HBV) and BabyBIG against infant botulism. In diseases for which human vaccination is not possible, hyperimmune globulins can be generated by immunizing animals, for example, rabbit- derived thymoglobulin (‘rabbit- ATG’) against human thymocytes for transplant tolerance. For rapid response to emerging pathogens with poorly characterized neutralizing epitopes, many groups have developed hyperimmune globulins derived from immunized animal plasma or convalescent human serum, for example, Zika virus hyperimmune globulin or severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Polyclonal antibodies have low-affinity' nonspecific binding to various targets. Because polyclonal antibodies bind to multiple targets, they result in faster clearance.

[0003] Plasma-derived antibody therapeutics have substantial drawbacks. First, demand for normal and convalescent donor plasma often outstrips supply. Plasma-derived drugs have suffered from impurities, including infectious viruses and clotting factors, that have resulted in serious adverse events. Antibody drugs derived from animal plasma occasionally cause allergic reactions, lead to antidrug antibodies and have suboptimal effector properties.Because they are derived from naturally occurring proteins, plasma-derived drugs are not easily engineered; for example, it is not possible to modify Fc sequences to improve mechanism of action or drug half-life. Finally, each batch of plasma-derived drug is usually derived from a different cohort of human donors or animals, resulting in batch-to-batch variation.

[0004] Many of these problems could be solved by generating multivalent hyperimmune globulins using recombinant DNA technology.3. SUMMARY

[0005] Provided herein are RPPs designed, at least in part, by an in silica method. Generation of RPPs using the in silica method has several significant advantages. First, use of in silica methods allows for a high degree of control over the antibody design process. It is possible to adjust numerous parameters to target specific aspects of an antigen and optimize properties such as binding affinity, specificity, and stability . Additionally, by narrowing down the possibilities to a smaller set of highly promising candidates before moving on to actual synthesis and testing, in silica design can help to significantly reduce the costs associated with antibody development. With in silica design, researchers can predict potential issues related with cross-reactivity, immunogenicity, stability^, and manufacturability, thereby mitigating risks in early stages of development.

[0006] The present disclosure provides various methods for in silica design of RPPs and RPPs generated by the in silica approach. The RPPs comprises recombinant ABPs, and their sequences can be derived from any number of suitable sources, including human or other mammalian samples, known antibodies, synthetic antibodies, antibody database and any combinations thereof.

[0007] The in silica methods disclosed herein involve analysis and selection of 10, 100, 1,000, 10,000, 100,000, or more than 100.000 distinct ABPs for generation of an RPP comprising a mixture of antigen-binding proteins (ABPs), e.g., antibodies, and can be termed a polyclonal antibody. In some embodiments, the ABPs of an RPP described herein are selected based on one or more characteristics (including, e.g., physical characteristics and functional characteristics) and / or one or more library properties. The characteristics and library properties can include those determined experimentally or by an in silica process.

[0008] In some embodiments, the ABPs of the RPP are antibodies. In some embodiments, the antibodies are chimeric. In some embodiments, the antibodies are humanized. In someembodiments, the antibodies are human. In some embodiments, the RPP comprises a mixture of antibody fragments. In some embodiments, the RPPs comprises a mixture of single-chain variable fragments (scFvs). In some embodiments, the RPPs comprise full-length antibodies. In some embodiments, the antibodies are IgGs, IgAs, or IgMs.

[0009] The RPP provided herein can induce various biological effects associated with binding to a target. In some embodiments, an RPP provided herein prevents binding of a virus to a cell, which therein prevents entry of the virus into the cell. In some embodiments, the RPP binds to the cell surface of a patient’s cells, in order to eliminate cells associated with a pathology.

[0010] Also provided are isolated polynucleotides encoding the RPPs provided herein, and portions thereof. In some aspects, the present invention provides a mixture of polynucleotides encoding the RPP provided herein. In other aspects, the present invention provides a mixture of vectors comprising the isolated polynucleotides. In other aspects, the present invention provides a mixture of host cell clones comprising the mixture of polynucleotides or vectors.

[0011] Also provided are methods of producing the RPP using the polynucleotides, vectors, or host cells provided herein. Some aspects of the present invention are related to a method of producing RPPs. comprising: expressing the antibodies in host cells using a library of polynucleotide vectors, and isolating the RPP.

[0012] Also provided are pharmaceutical compositions comprising the RPPs and a pharmaceutically acceptable excipient.

[0013] Also provided are methods of using the RPPs provided herein, e.g., methods of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of an RPP provided herein, or a pharmaceutical composition comprising such RPP. In some aspects, the disease or condition is a viral disease, e.g., a COVID-19 infection. In some aspects the method further comprises administering one or more additional therapeutic agents. In some aspects, the additional therapeutic agent is an immune stimulatory or suppressive agent.

[0014] In some embodiments, the RPP is in an amount sufficient as prophylaxis against infectious disease when administered to a subject. In some embodiments, the RPP is an amount sufficient to clear infectious disease in an individual actively fighting infection.

[0015] In some embodiments, provided herein is a method of generating a recombinant polyclonal protein (RPP) specific for a target molecule or complex of target molecules,comprising: (1) obtaining an input antigen binding protein (ABP) library dataset including an ABP profile for each of a plurality of ABPs, wherein (a) each ABP of the plurality of ABPs specifically binds a target antigen associated with the target molecule or complex; and (b) each ABP profile comprises a reference to the respective ABP and a plurality of characteristic descriptors for the respective ABP selected from: (i) a binding affinity of the respective ABP for the respective target antigen; (ii) an effector activity7of the respective ABP against the target molecule or complex; (iii) a solubility score of the respective ABP; (iv) an aggregation score of the respective ABP; (v) a hydrophobicity score of the respective ABP; (vi) an isoelectric point of the respective ABP; (vii) a stability score of the respective ABP; (viii) a molecular weight of the respective ABP; (ix) a number of unpaired cysteine residues in the respective ABP; (x) an abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen; (xi) a fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, as compared to the abundance frequency prior to enrichment; (xii) a partial or full sequence of the respective ABP; (xiii) a number of non-canonical glycosylation sites in the respective ABP; (xiv) a number of cleavage sites in the respective ABP; (xv) a number of deamidation sites in the respective ABP; (xvi) a number of isomerization sites in the respective ABP; (xvii) a number of oxidation sites in the respective ABP; (xviii) CDR3H length of the respective ABP; and (xix) binding specificity' of the respective ABP; (2) generating a filtered ABP library dataset corresponding to a subset of the plurality of ABPs and comprising a reference to each of the subset of the plurality of ABPs, wherein (a) each ABP in the subset of the plurality of ABPs has at least one of the plurality of characteristic descriptors having a value within a predetermined range for the characteristic; and (b) the subset of the plurality7of ABPs have one or more preferred library7properties selected from: (i) the set of heavy7chain CDR3 sequences contained in the subset of the plurality of ABPs comprises at least about 10, 20, 50. 100, 200, or 1000 unique sequences; (ii) the subset of the plurality7of ABPs specifically bind to at least two unique epitopes associated with the target molecule or complex; (iii) the subset of the plurality of ABPs is capable of modulating at least two target antigen variants; (iv) the set of heavy7chain V genes represented in the subset of the plurality of ABPs comprises at least two unique V genes; (v) the set of light chain V genes represented in the subset of the plurality of ABPs comprises at least two unique V genes; (vi) the set of heavy7chain J genes represented in the subset of the plurality7of ABPs comprises at least two unique J genes; (vii) the set of lightchain J genes represented in the subset of the plurality of ABPs comprises at least two unique J genes; (viii) the average percent germline identity of heavy chain V genes represented in the subset of the plurality of ABPs is between about 50% and about 100%; (ix) the average percent germline identity of light chain V genes represented in the subset of the plurality of ABPs is between about 50% and about 100%; (x) the average percent germline identity of heavy chain J genes represented in the subset of the plurality of ABPs is between about 50% and about 100%; and (xi) the average percent germline identity of light chain J genes represented in the subset of the plurality of ABPs is between about 50% and about 100%; and (3) providing the filtered ABP library dataset for generation of a composition comprising ABPs corresponding to the ABP references in the filtered ABP library dataset, thereby generating the RPP.

[0016] In some aspects, the techniques described herein relate to a method of generating a recombinant polyclonal protein library (RPP) specific to a target molecule or complex of target molecules, including: (1) obtaining a dataset for an input antigen binding protein (ABP) library’ including at least 100 candidate ABPs, wherein the input ABP library dataset includes an ABP profile for each of the at least 100 candidate ABPs, wherein (a) each candidate ABP of the input ABP library is capable of specific binding to a target antigen associated with the target molecule or complex; and (b) each ABP profile includes a reference to a respective candidate ABP and a plurality of characteristic descriptors for the respective candidate ABP selected from: (i) a binding affinity of the respective candidate ABP to the respective target antigen; (ii) an effector activity of the respective candidate ABP against the target molecule or complex; (iii) a binding pattern of the respective candidate ABP to the respective target antigen and its variants; (iv) an abundance frequency of the respective candidate ABP following sorting the input ABP library’ or a subset thereof to enrich for binding to the respective target antigen; and (v) a fold-change of the increase in the abundance frequency of the respective candidate ABP following sorting the input ABP library’ or a subset thereof to enrich for binding to the respective target antigen, as compared to the abundance frequency prior to enrichment: (2) generating a dataset for a filtered ABP library including selected ABPs. wherein the dataset includes a reference to each of the selected ABPs and (a) the filtered ABP library includes at least 10 selected ABPs which is a subset of the at least 100 candidate ABPs; and (b) each selected ABP has at least one of the plurality of characteristic descriptors meets a preferred criteria selected from: (i) a binding affinity to the respective target antigen is ranked at least top 25% among all the candidateABPs in the input ABP library: (ii) an effector activity against the target molecule or complex is ranked at least top 25% among all the candidate ABPs in the input ABP library: (iii) a binding pattern for the respective target antigen and its variants is shared among less than 20% of candidate ABPs in the input ABP library; (iv) an abundance frequency of the selected ABP following sorting the input ABP library or a subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof; and (v) a foldchange of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof; and (3) providing the dataset for the filtered ABP library for generation of a composition including selected ABPs, thereby generating the RPP.

[0017] In some embodiments, the techniques described herein relate to a method, wherein the plurality of characteristic descriptors in (2)(b) includes the binding affinity to the respective target antigen.

[0018] In some embodiments, the techniques described herein relate to a method, wherein the binding affinity is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% among all the candidate ABPs in the input ABP library, optionally wherein the binding affinity is determined by surface plasmon resonance (SPR) or biolayer interferometry' (BLI).

[0019] In some embodiments, the techniques described herein relate to a method, wherein the plurality of characteristic descriptors in (2)(b) includes the effector activity against the target molecule or target molecule complex.

[0020] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria is that the effector activity against the target molecule or complex is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% among all the candidate ABPs in the input ABP library.

[0021] In some embodiments, the techniques described herein relate to a method, wherein the target molecule or target molecule complex includes a virus, and the effector activity is a neutralization activity determined by a pseudovirus neutralization assay or a live virus neutralization assay.

[0022] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria for the effector activity is neutralization activity corresponding to (a) an IC50 from about 0.08 pg / mL to about 900 pg / mL when determined by pseudovirusneutralization assay or (b) an IC50 from about 0.003 pg / rnL to about 1350 pg / mL when determined by live virus neutralization assay.

[0023] In some embodiments, the techniques described herein relate to a method, wherein the target molecule or target molecule complex includes a bacterium, and the effector activity is a bactericidal activity' determined by a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPKA).

[0024] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria for the bactericidal activity corresponds to a concentration yvhere 50% bactericidal activity is observed from about 0.08 pg / ml to about 3600 pg / ml.

[0025] In some embodiments, the techniques described herein relate to a method, wherein the plurality of characteristic descriptors in (2)(b) includes an abundance frequency or foldchange of the increase in the abundance frequency’ of the respective ABP folloyving a sorting process to enrich for binding to the respective target antigen, optionally wherein the sorting process is fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) sorting, further optionally yvherein the sorting is carried by yeast display.

[0026] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria for the post-sort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% within a pool of ABPs obtained after the sorting process.

[0027] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria for the post-sort abundance frequency is between 0.01% and 50%, betyveen 1% and 50%, between 5% and 45%, between 10% and 40%, between 15% and 35%, or between 20% and 30%.

[0028] In some embodiments, the techniques described herein relate to a method, yvherein the preferred criteria for the post-sort fold-change is greater than about any of 1.5, 2, 2.5, 3, 4, 5. 6, 7, 8, 9. 10. 50. 100, 200, 300, 400, 500, 600, 700 or greater.

[0029] In some embodiments, the techniques described herein relate to a method, yvherein the preferred criteria for the post-sort fold-change ranges from 1.5 to 1000, from 10 to 500, from 10 to 400, from 10 to 1000, from 100 to 1000. from 200 to 1000, from 300 to 1000, from 400 to 1000, or from 500 to 1000.

[0030] In some embodiments, the techniques described herein relate to a method, yvherein the enrichment is performed by FACS or MACS.

[0031] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria for the post-sort abundance frequency or the post-sort fold-change is that the corresponding ABP is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% within the input ABP library.

[0032] In some embodiments, the techniques described herein relate to a method, wherein the plurality of characteristic descriptors in (2)(b) includes the binding pattern, optionally wherein the binding pattern corresponds to a subset of the respective target antigen and its variants capable of binding to the respective ABP, further optionally wherein the binding pattern is determined by a Poly Map assay.

[0033] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria is that the binding pattern of the respective ABP is shared among less than 15%, less than 10%, less than 5%, or less than 3% candidate ABPs in the input ABP library.

[0034] In some embodiments, the techniques described herein relate to a method, wherein the preferred criteria is that the binding pattern of the respective ABP is shared with less than 100, less than 50, less than 10 or less than 5 other candidate ABPs in the input ABP library.

[0035] In some embodiments, the techniques described herein relate to a method, wherein the selected ABPs in the filtered ABP library as a group meet 1, 2, 3, 4, 5 or 6 different preferred criteria, selected from: (2)(b)(i) to (2)(b)(v).

[0036] In some embodiments, the techniques described herein relate to a method, wherein the selected ABPs in the filtered ABP library meet one or more additional preferred criteria related to: a solubility score of the respective ABP; an aggregation score of the respective ABP; a hydrophobicity score of the respective ABP; an isoelectric point of the respective ABP: a stability score of the respective ABP; a molecular weight of the respective ABP; a number of unpaired cysteine residues in the respective ABP; a partial or full sequence of the respective ABP; a number of non-canonical glycosylation sites in the respective ABP; a number of cleavage sites in the respective ABP; a number of deamidation sites in the respective ABP; a number of isomerization sites in the respective ABP; a number of oxidation sites in the respective ABP; CDR3H length of the respective ABP; or binding specificity of the respective ABP.

[0037] In some embodiments, the techniques described herein relate to a method, wherein the filtered ABP library meets one or more preferred properties selected from: the set of heavy chain CDR3 sequences contained in the filtered ABP library includes at least about 10, 20,50, 100, 200, or 1000 unique sequences; the selected ABPs in the filtered ABP library as a group specifically bind to at least two unique epitopes associated with the target molecule or complex; the selected ABPs in the filtered ABP library as a group are capable of modulating at least two target antigen variants; the set of heavy chain V genes represented in the ABPs in the filtered ABP library as a group includes at least two unique V genes; the set of light chain V genes represented in the ABPs in the filtered ABP library' as a group includes at least two unique V genes; the set of heavy chain J genes represented in the ABPs in the filtered ABP library as a group includes at least two unique J genes; the set of light chain J genes represented in the ABPs in the filtered ABP library as a group includes at least two unique J genes; the average percent germline identity of heavy chain V genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; the average percent germline identity of light chain V genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; the average percent germline identity' of heavy chain J genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; and the average percent germline identity' of light chain J genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%.

[0038] In some embodiments, the techniques described herein relate to a method, wherein each candidate ABP of the input ABP library includes a cognate pair of heavy chain and light chain variable regions from a single cell out of a blood sample from at least one donor previously exposed to the target molecule or complex.

[0039] In some embodiments, the techniques described herein relate to a method, wherein the at least one donor is at least two. three, four, five, six. seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more donors.

[0040] In some embodiments, the techniques described herein relate to a method, wherein the at least one donor has been previously vaccinated with a vaccine derived from the target molecule or complex.

[0041] In some embodiments, the techniques described herein relate to a method, wherein the blood sample includes cells purified from peripheral blood mononuclear cells (PBMCs) of the donor.

[0042] In some embodiments, the techniques described herein relate to a method, wherein the single cell is a B cell, plasma cell, or plasmablast.

[0043] In some embodiments, the techniques described herein relate to a method, wherein the target molecule or complex is associated with a pathogen, optionally wherein the pathogen is a virus, bacteria, or toxin.

[0044] In some embodiments, the techniques described herein relate to a method, wherein the input ABP library is a pool of ABPs, wherein each of the ABPs have been selected for a selection criteria, wherein the selection criteria is (i) binding or binding affinity to a target antigen or its variant, (ii) activity against the pathogen, or (iii) effector activity. .

[0045] In some embodiments, the techniques described herein relate to a method, wherein the input ABP library includes at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900. at least 1000. at least 1100. at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 3000, at least 4000, at least 5000, at least 10,000, at least 50,000 or more ABPs.

[0046] In some embodiments, the techniques described herein relate to a method, wherein the RPP includes at least 10, at least 25, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600. at least 700, at least 800. at least 900, or at least 1000 ABPs.

[0047] In some embodiments, the techniques described herein relate to a method, wherein each ABP of the RPP includes full-length antibodies or functional fragments thereof.

[0048] In some embodiments, the techniques described herein relate to a method, wherein the full-length antibodies or functional fragments thereof are of the human IgGl, IgG2, IgG3, or IgG4 subtype.

[0049] In some embodiments, the techniques described herein relate to a method, further including generating the composition including ABPs corresponding to the ABP references in the filtered ABP library' dataset.

[0050] In some embodiments, the techniques described herein relate to a method, wherein the binding affinity or the binding pattern is determined by a Poly Map assay including the steps of: providing a library of target-decorated cells, wherein each of the target-decorated cells presents the target molecule or complex on the membrane; contacting the library of targetdecorated cells with a plurality' of ABP-ribosome-mRNA (ARM) complexes corresponding to the one or more of the plurality of ABPs, thereby inducing binding between the targetdecorated cells and the ARM complexes; generating a plurality of monodisperse orpoly disperse emulsion microdroplets, wherein each microdroplet contains a single cell out of the target-decorated cells, one or more ARM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; capturing RNA released from the single cell on a solid surface or within a semi-permeable shell; generating a library of hybrid polynucleic acids that include a sequence from a transcript of the single cell and / or a sequence from the mRNA of the ARM complex; sequencing the library of hybrid polynucleic acids; and determining a presence or absence of binding of each of the one or more of the plurality of ABPs to their respective target antigen.

[0051] In some aspects, the techniques described herein relate to an RPP generated by the method.

[0052] In some aspects, the techniques described herein relate to a plurality of polynucleotides, wherein each polynucleotide encodes an ABP disclosed herein.

[0053] In some aspects, the techniques described herein relate to a plurality of polynucleotides, wherein each polynucleotide is cloned into an expression vector.

[0054] In some aspects, the techniques described herein relate to a plurality of host cells comprising a plurality of the polynucleotides disclosed herein.

[0055] In some aspects, the techniques described herein relate to a method comprising culturing the host cells disclosed herein for expression of the ABPs and isolating the ABPs.

[0056] In some aspects, the techniques described herein relate to a pharmaceutical composition comprising the RPP and a pharmaceutically acceptable excipient.

[0057] In some aspects, the techniques described herein relate to a method of treating a patient in need thereof by administering an effective amount of the pharmaceutical composition.

[0058] In some embodiments, the techniques described herein relate to a method, wherein the patient has been exposed to the target molecule or complex or a variant thereof.

[0059] In some embodiments, the techniques described herein relate to a method, wherein the patient has a disease associated with the target molecule or complex or a variant thereof.

[0060] In some embodiments, the techniques described herein relate to a method, wherein the pharmaceutical composition is administered intramuscularly, subcutaneously, intravenously, intradermally, orally, or through inhalation.

[0061] In some embodiments, the techniques described herein relate to a method, wherein the effective amount is sufficient to treat the disease associated with the target molecule or complex or a variant thereof.

[0062] Aspects of the present disclosure provides a method of generating a recombinant polyclonal protein library' (RPP) specific to a target molecule or complex of target molecules, comprising: (1) obtaining a dataset for an input antigen binding protein (ABP) library’ comprising at least 100 candidate ABPs, wherein the input ABP library dataset comprises an ABP profile for each of the at least 100 candidate ABPs, wherein (a) each candidate ABP of the input ABP library is capable of specific binding to a target antigen associated with the target molecule or complex; and (b) each ABP profile comprises a reference to a respective candidate ABP and a plurality of characteristic descriptors for the respective candidate ABP selected from: (i) a binding affinity of the respective candidate ABP to the respective target antigen; (ii) an effector activity of the respective candidate ABP against the target molecule or complex; (iii) a binding pattern of the respective candidate ABP to the respective target antigen and its variants; (iv) an abundance frequency of the respective candidate ABP following sorting the input ABP library or a subset thereof to enrich for binding to the respective target antigen; and (v) a fold-change of the increase in the abundance frequency7of the respective candidate ABP following sorting the input ABP library or a subset thereof to enrich for binding to the respective target antigen, as compared to the abundance frequencyprior to enrichment; (2) generating a dataset for a filtered ABP library comprising selected ABPs, wherein the dataset comprises a reference to each of the selected ABPs and (a) the filtered ABP library7comprises at least 10 selected ABPs which is a subset of the at least 100 candidate ABPs; and (b) each selected ABP has at least one of the plurality of characteristic descriptors meets a preferred criteria selected from: (i) a binding affinity to the respective target antigen is ranked at least top 25% among all the candidate ABPs in the input ABP library; (ii) an effector activity7against the target molecule or complex is ranked at least top 25% among all the candidate ABPs in the input ABP library-; (iii) a binding pattern for the respective target antigen and its variants is shared among less than 20% of candidate ABPs in the input ABP library; (iv) an abundance frequency of the selected ABP following sorting the input ABP library or a subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library' or the subset thereof; and (v) a fold-change of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library- orthe subset thereof; and(3) providing the dataset for the filtered ABP library for generation of a composition comprising selected ABPs, thereby generating the RPP.

[0063] In some embodiments, the plurality of characteristic descriptors in (2)(b) comprises the binding affinity to the respective target antigen. In some embodiments, the binding affinity is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% among all the candidate ABPs in the input ABP library, optionally wherein the binding affinity is determined by surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0064] In some embodiments, the plurality' of characteristic descriptors in (2)(b) comprises the effector activity’ against the target molecule or target molecule complex.

[0065] In some embodiments, the preferred criteria is that the effector activity against the target molecule or complex is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% among all the candidate ABPs in the input ABP library.

[0066] In some embodiments, the target molecule or target molecule complex comprises a virus, and the effector activity is a neutralization activity' determined by a pseudovirus neutralization assay or a live virus neutralization assay. In some embodiments, the preferred criteria for the effector activity is neutralization activity corresponding to (a) an IC50 from about 0.08 pg / mL to about 900 pg / mL yvhen determined by' pseudovirus neutralization assay or (b) an IC50 from about 0.003 pg / mL to about 1350 pg / mL yvhen determined by live virus neutralization assay.

[0067] In some embodiments, the target molecule or target molecule complex comprises a bacterium, and the effector activity' is a bactericidal activity' determined by a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPKA).

[0068] In some embodiments, the preferred criteria for the bactericidal activity' corresponds to a concentration where 50% bactericidal activity' is observed from about 0.08 pg / ml to about 3600 pg / ml. In some embodiments, the plurality' of characteristic descriptors in (2)(b) comprises an abundance frequency or fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, optionally yvherein the sorting process is fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) sorting, further optionally yvherein the sorting is carried by yeast display.

[0069] In some embodiments, the preferred criteria for the post-sort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% wi thin a pool of ABPs obtained after the sorting process.

[0070] In some embodiments, the preferred criteria for the post-sort abundance frequency is between 0.01% and 50%, between 1% and 50%, between 5% and 45%, between 10% and 40%, between 15% and 35%, or between 20% and 30%.

[0071] In some embodiments, the preferred criteria for the post-sort fold-change is greater than about any of 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700 or greater. In some embodiments, the preferred criteria for the post-sort fold-change ranges from 1.5 to 1000, from 10 to 500, from 10 to 400. from 10 to 1000. from 100 to 1000, from 200 to 1000, from 300 to 1000, from 400 to 1000, or from 500 to 1000.

[0072] In some embodiments, the enrichment is performed by FACS or MACS.

[0073] In some embodiments, the preferred criteria for the post-sort abundance frequency or the post-sort fold-change is that the corresponding ABP is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% within the input ABP library’.

[0074] In some embodiments, the plurality of characteristic descriptors in (2)(b) comprises the binding pattern, optionally wherein the binding pattern corresponds to a subset of the respective target antigen and its variants capable of binding to the respective ABP, further optionally wherein the binding pattern is determined by a Poly Map assay.

[0075] In some embodiments, the preferred criteria is that the binding pattern of the respective ABP is shared among less than 15%, less than 10%, less than 5%, or less than 3% candidate ABPs in the input ABP library. In some embodiments, preferred criteria is that the binding pattern of the respective ABP is shared with less than 100, less than 50, less than 10 or less than 5 other candidate ABPs in the input ABP library’.

[0076] In some embodiments, the selected ABPs in the filtered ABP library as a group meet 1, 2, 3, 4, 5 or 6 different preferred criteria, selected from: (2)(b)(i) to (2)(b)(v).

[0077] In some embodiments, the selected ABPs in the filtered ABP library meet one or more additional preferred criteria related to: a solubility score of the respective ABP; an aggregation score of the respective ABP; a hydrophobicity score of the respective ABP; an isoelectric point of the respective ABP; a stability' score of the respective ABP; a molecular weight of the respective ABP; a number of unpaired cysteine residues in the respective ABP; a partial or full sequence of the respective ABP; a number of non-canonical glycosylationsites in the respective ABP; a number of cleavage sites in the respective ABP; a number of deamidation sites in the respective ABP; a number of isomerization sites in the respective ABP; a number of oxidation sites in the respective ABP; CDR3H length of the respective ABP; or binding specificity of the respective ABP.

[0078] In some embodiments, the filtered ABP library' meets one or more preferred properties selected from; the set of heavy chain CDR3 sequences contained in the filtered ABP library comprises at least about 10. 20. 50. 100, 200, or 1000 unique sequences; the selected ABPs in the filtered ABP library as a group specifically bind to at least two unique epitopes associated with the target molecule or complex; the selected ABPs in the filtered ABP library as a group are capable of modulating at least two target antigen variants; the set of heavy chain V genes represented in the ABPs in the filtered ABP library as a group comprises at least two unique V genes; the set of light chain V genes represented in the ABPs in the filtered ABP library as a group comprises at least two unique V genes; the set of heavy chain J genes represented in the ABPs in the filtered ABP library as a group comprises at least two unique J genes; the set of light chain J genes represented in the ABPs in the filtered ABP library as a group comprises at least two unique J genes; the average percent germline identity of heavy chain V genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; the average percent germline identity of light chain V genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; the average percent germline identity of heavy chain J genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; and the average percent germline identity of light chain J genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%.

[0079] In some embodiments, each candidate ABP of the input ABP library comprises a cognate pair of heavy chain and light chain variable regions from a single cell out of a blood sample from at least one donor previously exposed to the target molecule or complex. In some embodiments, the at least one donor is at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more donors.

[0080] In some embodiments, the at least one donor has been previously vaccinated with a vaccine derived from the target molecule or complex. In some embodiments, the bloodsample comprises cells purified from peripheral blood mononuclear cells (PBMCs) of the donor.

[0081] In some embodiments, the single cell is a B cell, plasma cell, or plasmablast.

[0082] In some embodiments, the target molecule or complex is associated with a pathogen, optionally wherein the pathogen is a virus, bacteria, or toxin. In some embodiments, the input ABP library is a pool of ABPs, wherein each of the ABPs have been selected for a selection criteria, wherein the selection criteria is (i) binding or binding affinity to a target antigen or its variant, (ii) activity against the pathogen, or (iii) effector activity .

[0083] In some embodiments, the input ABP library comprises at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800. at least 1900, at least 2000, at least 3000, at least 4000, at least 5000, at least 10.000, at least 50,000 or more ABPs. In some embodiments, the RPP comprises at least 10, at least 25, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 ABPs.

[0084] In some embodiments, each ABP of the RPP comprises full-length antibodies or functional fragments thereof. In some embodiments, the full-length antibodies or functional fragments thereof are of the human IgGl, IgG2, IgG3, or IgG4 subtype.

[0085] In some embodiments, the method further comprises generating the composition comprising ABPs corresponding to the ABP references in the filtered ABP library dataset.

[0086] In some embodiments, the binding affinity or the binding pattern is determined by a PolyMap assay comprising the steps of: providing a library of target-decorated cells, wherein each of the target-decorated cells presents the target molecule or complex on the membrane; contacting the library' of target-decorated cells with a plurality' of ABP-ribosome-mRNA (ARM) complexes corresponding to the one or more of the plurality of ABPs, thereby inducing binding between the target-decorated cells and the ARM complexes; generating a plurality of monodisperse or poly disperse emulsion microdroplets, wherein each microdroplet contains a single cell out of the target-decorated cells, one or more ARM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; capturing RNA released from the single cell on a solid surface or within a semi-permeable shell; generating a library of hybrid polynucleic acids that comprise a sequence from a transcript of the single celland / or a sequence from the mRNA of the ARM complex; sequencing the library of hybrid polynucleic acids; and determining a presence or absence of binding of each of the one or more of the plurality of ABPs to their respective target antigen.

[0087] An aspect of the present disclosure provides an RPP generated by any of the methods of the present disclosure. An aspect of the present disclosure provides a plurality of polynucleotides, wherein each polynucleotide encodes an ABP in the RPP described herein. An aspect of the present disclosure includes a plurality of polynucleotide constructs, comprising the plurality of polynucleotides of the present disclosure cloned into an expression vector. An aspect of the present disclosure includes a plurality of host cells comprising the plurality of polynucleotides or the plurality of polynucleotide constructs of the present disclosure.

[0088] An aspect of the present disclosure includes a method of producing an RPP, the method comprising culturing the plurality of host cells of the present disclosure under conditions for expression of the ABPs and isolating the ABPs. An aspect of the present disclosure includes a pharmaceutical composition comprising the RPP of the present disclosure and a pharmaceutically acceptable excipient.

[0089] An aspect of the present disclosure includes a method of treating a patient in need thereof by administering an effective amount of the pharmaceutical composition of the present disclosure. In some embodiments, the patient has been exposed to the target molecule or complex or a variant thereof. In some embodiments, the patient has a disease associated with the target molecule or complex or a variant thereof.

[0090] In some embodiments, the pharmaceutical composition is administered intramuscularly, subcutaneously, intravenously, intradermally, orally, or through inhalation. In some embodiments, the effective amount is sufficient to treat the disease associated with the target molecule or complex or a variant thereof.4. BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

[0091] FIG. 1 illustrates an exemplary' process of generating a recombinant polyclonal protein library (RPP) specific for a target molecule or complex of target molecules using an in silico method disclosed herein.

[0092] FIG. 2 provides a heatmap visualizing FACS-enriched clones in COVID antibody libraries.

[0093] FIG. 3 shows V and J gene usage in COVID antibody libraries.

[0094] FIG. 4 shows COVID antibody V / J percent identity to germline sequences.

[0095] FIG. 5 shows PolyMap profile of a COVID antibody I ibrary .

[0096] FIG. 6 shows the distribution of computationally determined developability parameters for COVID antibodies, alongside the reference distribution of clinical antibodies as reported by Jain et al. (Biophysical properties of the clinical-stage antibody landscape. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):944-949).

[0097] FIG. 7 shows % of reads in the gRNA or RNA sequencing data from the transfected cells. This shows transfection (gRNA) and expression (RNA) efficacy of each clone (left) or different pools (Top 50, Top 25, Top 10, GIGA2050).

[0098] FIG. 8 shows the developability parameters (aggregation, hydrophobicity, skade_solubility) of the tested antibodies relative to clinical antibodies.

[0099] FIG. 9 shows titers of different antibodies selected based on developability criteria (antibodies selected for “good” developability, “bad” developability, or “others”).

[0100] FIG. 10 shows Tm (melting Temperature, °C), Tagg (Aggregation Temperature, °C) or Z-ave diameter (the average size of the antibody particles in solution, measured using dynamic light scattering, nm) of different pools of antibodies selected based on developability criteria (antibodies selected for “good” developability, “bad” developability, or “all”).

[0101] FIG. 11 provides SEC MALS results from an accelerated stability study conducted to evaluate the stability of matched custom pools under various temperature conditions up to a six-week period.

[0102] FIG. 12 provides ELISA results from an accelerated stability study conducted to evaluate the stability of matched custom pools under various temperature conditions up to a six-week period.

[0103] FIG. 13 provides data from the binding ELISA 1 assay described in Example 8. The graph shows the raw absorbance values of each sample's titration curves normalized for ease of comparison.

[0104] FIG. 14 provides EC50 values determined from the binding ELISA 1 assay described in Example 8.

[0105] FIG. 15 provides data from the binding ELISA 2 assay described in Example 8. The graph shows the raw absorbance values of each sample's titration curves normalized for ease of comparison.

[0106] FIG. 16 provides data from the pseudoviral neutralization assay targeting the Wuhan- Hu- 1 strain.

[0107] FIG. 17 shows that a pool with ten clones selected using the PolyMap approach demonstrated neutralization potency that was comparable to, or in some cases exceeded, that of the top ten clones from the GIGA-2050 pool.

[0108] FIG. 18 provides pseudoviral neutralization (IC50) measured in IVIG, strain GIGA 2025, Pool 1 through 9 and Pool 16 and set of 4 mAbs against Wuhan variant.

[0109] FIG. 19 provides data from the neutralization potency assay tested against the Omicron variant and subsequent variants.

[0110] FIG. 20 provides the neutralization data from FIG. 19, further normalized to the Wuhan strain (set as 1).5. DETAILED DESCRIPTIONDefinitions[OHl] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology7, 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 are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., 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 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. Enzymaticreactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery', and treatment of patients.

[0112] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0113] The term "‘recombinant polyclonal protein library” or “RPP” refers to more than one recombinant antigen binding proteins (ABPs), collectively comprising more than one antigen-binding domains that specifically bind to an antigen or epitope, or multiple antigens and epitopes. The recombinant polyclonal protein or RPP can be antibodies or variants or derivatives thereof. In some embodiments, the antigen-binding domains bind an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, an RPP comprises antibodies. In some embodiments, the RPP consists essentially of antibodies. In some embodiments, an RPP is a mixture of antibodies. In some embodiments, an RPP comprises scFvs. In some embodiments, the RPP comprises an alternative scaffold. In some embodiments, the RPP consists of alternative scaffolds. In some embodiments, the RPP consists essentially of alternative scaffolds. In some embodiments, the RPP comprises an antibody fragment. In some embodiments, the RPP consists of antibody fragments. In some embodiments, the RPP consists essentially of antibody fragments.

[0114] The term “antigen binding protein” or “ABP” as used herein refers to a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment.

[0115] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes intact antibodies (e.g.. intact immunoglobulins), antibody fragments, and multi-specific antibodies. One example of an antigen-binding domain is an antigen-binding domain formed by a Vn -VL dimer.

[0116] The term “alternative scaffold” refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity' and affinity similar to that of naturally occurring antibodies.Exemplary alternative scaffolds include those derived from fibronectin (e.g, Adnectins™), the |3-sandwich (e.g., iMab), lipocalin (e.g., Anticalins®), EETI-II / AGRP, BPTI / LACI- D1 / ITI-D2 (e.g., Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins). gamma-B-crystallin / ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), Fynomers, and (LDLR-A module) (e.g., Avimers). Additional information on alternative 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 incorporated by reference in its entirety'. Alternative scaffolds comprise one ty pe of RPP.

[0117] The term “antigen-binding domain” means the portion of an antibody that is capable of specifically binding to an antigen or epitope.

[0118] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably' to refer to an antibody having a structure substantially similar to a naturally occurring antibody structure and having heavy chains that comprise an Fc region.

[0119] The term “immunoglobulin” refers to a class of structurally' related proteins, e.g., antibodies, generally' comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain ty pically comprises a heavy chain variable region (Vn) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI, Cm, and Cm. Each light chaintypically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0120] The term '‘Fc region’’ means the C-terminal region of an immunoglobulin heavy chain that, in naturally occurring antibodies, interacts with Fc receptors and certain proteins of 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, incorporated by reference in its entirety. The Fc region may be a naturally occurring Fc region, or an Fc region modified as described elsewhere in this disclosure.

[0121] The VH and VL regions may be further subdivided into regions of hypervariability (‘’hypervariable regions (HVRs)” also called “complementarity determining regions” (CDRs)) interspersed with regions that are more conserved. The more conserved regions are called framework regions (FRs). Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen binding, and influence 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, incorporated by reference in its entirety.

[0122] The light chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (X), based on the sequence of its constant domain.

[0123] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 8, s, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0124] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732- 745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated by reference in its entirety.

[0125] Table 1 provides the positions of CDR1-L (CDR1 of VL), CDR2-L (CDR2 of VL), CDR3-L (CDR3 of VL), CDR1-H (CDR1 of VH), CDR2-H (CDR2 of VH), and CDR3-H (CDR3 of VH), as identified by the Kabat and Chothia schemes. For CDR1-H, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0126] CDRs may be assigned, for example, using antibody numbering software, such as Abnum, available at www.bioinf.org.uk / abs / abnum / , and described in Abhinandan and Martin. Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety.* The C-terminus of CDR1-H. when numbered using the Kabat numbering convention, varies between 32 and 34, depending on the length of the CDR.

[0127] The “EU numbering scheme’’ is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra).

[0128] An "antibody fragment” comprises a portion of an intact antibody, such as 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.

[0129] “Fv” fragments comprise a non-covalently -linked dimer of one heavy chain variable domain and one light chain variable domain.

[0130] “Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0131] “F(ab’)2” fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds. F(ab’)2 fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab’) fragments can be dissociated, for example, by treatment with B -mercaptoethanol.

[0132] “Single-chain Fv” or “sFv” or “scFv’' antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Pliickthun A. (1994). In some embodiments, the linker is a (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). Spring er-Verlag, New York, incorporated by reference in its entirety.

[0133] “scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH -VL or VL -VH ). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0134] The term “single domain antibody"’ refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. 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, each of which is incorporated by reference in its entirety.

[0135] The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes 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 plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0136] The term “polyclonal antibody” refers to a mixture of at least two monoclonal antibodies. Polyclonal antibodies may be either monospecific or polyspecific.

[0137] 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.

[0138] “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues 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 a desired specificity, affinity , or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may 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 by reference in its entirety.

[0139] A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0140] An “isolated RPP” or “isolated nucleic acid” is an RPP or nucleic acid that has been separated and / or recovered from a component of its natural environment. Components of the natural environment may include enzy mes, hormones, and other proteinaceous or nonproteinaceous materials. In some embodiments, an isolated RPP is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example by use of a spinning cup sequenator. In some embodiments, an isolated RPP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or nonreducing conditions, with detection by Coomassie blue or silver stain. An isolated RPP includes an RPP in situ within recombinant cells, since at least one component of the RPP'snatural environment is not present. In some aspects, an isolated RPP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, an isolated RPP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, an isolated RPP or isolated nucleic acid is purified to at least 80%. 85%, 90%, 95%, or 99% by volume. In some embodiments, an isolated RPP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% RPP or nucleic acid by weight. In some embodiments, an isolated RPP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% RPP or nucleic acid by volume.

[0141] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g. an RPP) and its binding partner (e.g., an antigen or epitope). Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., RPP and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are descnbed in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology7(e.g., BIACORE®) or biolayer interferometry (e.g, FORTEBIO®).

[0142] With regard to the binding of an RPP to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., wi th a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the RPP to the target molecule is competitively inhibited by the control molecule.

[0143] The term “kd” (sec'1), as used herein, refers to the dissociation rate constant of a particular ABP -antigen interaction. This value is also referred to as the koff value.

[0144] The term “ka” (M'Usec'1), as used herein, refers to the association rate constant of a particular ABP -antigen interaction. This value is also referred to as the konvalue.

[0145] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular ABP -antigen interaction. KD = ka / ka.

[0146] The term “KA” (M-1), as used herein, refers to the association equilibrium constant of a particular ABP -antigen interaction. KA = ka / kd.

[0147] An “immunoconjugate’' is an RPP conjugated to one or more heterologous molecule(s).

[0148] “Effector functions'’ refer to those biological activities mediated by the Fc region of an antibody, which activities may vary’ depending on the antibody isotype. Examples of antibody effector functions include Clq binding to activate complement dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity’ (ADCC), and antibody dependent cellular phagocytosis (ADCP).

[0149] When used herein in the context of two or more RPPs, the term “competes with” or “cross-competes with” indicates that the two or more RPPs compete for binding to an antigen (e.g., pneumococcus polysaccharide). In one exemplary assay, an antigen is coated on a surface and contacted with a first RPP against the antigen, after which a second RPP against the antigen is added. In another exemplary assay, a first RPP against an antigen is coated on a surface and contacted with the antigen, and then a second RPP against the antigen is added. If the presence of the first RPP against an antigen reduces binding of the second RPP, in either assay, then the RPPs compete. The term “competes with” also includes combinations of RPPs where one RPP reduces binding of another RPP, but where no competition is observed when the RPPs are added in the reverse order. However, in some embodiments, the first and second RPPs inhibit binding of each other, regardless of the order in which they are added. In some embodiments, one RPP reduces 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%. A skilled artisan can select the concentrations of the antibodies used in the competition assays based on the affinities of the RPPs for pneumococcus polysaccharide and the valency’ of the RPPs. The assays described in this definition are illustrative, and a skilled artisan can utilize any suitable assay to determine if antibodies compete with each other. Suitable assays are described, for example, 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; each of which is incorporated by reference in its entirety.

[0150] The term '‘epitope” means a portion of an antigen the specifically binds to an RPP or an ABP. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an RPP or an ABP binds can be determined using known techniques for epitope determination such as. for example, testing for RPP or an ABP binding to an antigen.

[0151] 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, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR). CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0152]

[0153] The term treating " (and variations thereof such as “treat” or “treatment ) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and dunng the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminish of any direct or indirect pathological consequences of the disease, preventing reinfection or associated symptom, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Improvements in any conditions can be readily assessed according to standard methods and techniques known in the art. The population of subjects treated by themethod of the disease includes subjects suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease.

[0154] As used herein, the term ‘therapeutically effective amount” or ‘ effective amount” refers to an amount of an RPP or pharmaceutical composition provided herein that, when administered to a subject, is effective to produces the desired effect for which it is administered. The exact dose or amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g.. Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy. The term “sufficient amount” means an amount sufficient to produce a desired effect.

[0155] As used herein, the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, 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 an RPP provided herein. In some aspects, the disease or condition is a cancer. In some aspects, the disease or condition is a viral infection.

[0156] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic or diagnostic products.

[0157] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject.

[0158] The term “plasma cell” refers to white blood cells that secrete large volumes of antibodies. They are transported by the blood plasma and the lymphatic system. B cells (for example, either germinal center naive B cells or memory B cells) differentiate into plasma cells that produce antibody molecules closely modelled after the receptors of the precursor B cell. Once released into the blood and lymph, these antibody molecules bind to the target antigen (foreign substance) and initiate its neutralization or destruction. Terminally differentiated plasma cells express relatively few surface antigens, and do not expresscommon pan-B cell markers, such as CD 19 and CD20. Instead, plasma cells are identified through flow cytometry by their additional expression of CD138, CD78, and the Interleukin-6 receptor. In humans, CD27 is a good marker for plasma cells, naive B cells are CD27-. memory B-cells are CD27+ and plasma cells are CD27++. The surface antigen CD 138 (syndecan-1) is expressed at high levels. Another important surface antigen is CD319 (SLAMF7). This antigen is expressed at high levels on normal human plasma cells. It is also expressed on malignant plasma cells in multiple myeloma. Compared with CD138, which disappears rapidly ex vivo, the expression of CD319 is considerably more stable.

[0159] The term “plasmablast” refers to antibody-secreting cells in the peripheral blood, which differentiate from activated B cells, such as memory B cells, upon stimulation with an antigen. The most immature blood cell that is considered of plasma cell lineage is the plasmablast. Plasmablasts secrete more antibodies than B cells, but less than plasma cells. They divide rapidly and are still capable of internalizing antigens and presenting them to T cells. A cell may stay in this state for several days, and then either die or irrevocably differentiate into a mature, fully differentiated plasma cell. Differentiation of mature B cells into plasma cells is dependent upon the transcription factors Blimp- 1 / PRDM1 and IRF4.

[0160] The term “memory B cell” refers to a B cell sub-ty pe that are formed within germinal centers following primary infection and are important in generating an accelerated and more robust antibody-mediated immune response in the case of re-infection (also known as a secondary immune response). Memory B cells do not secrete antibody until activated by their specific antigen.

[0161] The term “naive B cell” refers to a B cell that has not been exposed to an antigen. Once exposed to an antigen, the naive B cell either becomes a memory B cell or a plasma cell that secretes antibodies specific to the antigen that was originally bound. Plasma cells do not last long in the circulation, this is in contrast to memory cells that last for very long periods of time.

[0162] The term “peripheral blood” refers to blood which travels through peripheral vessels. Peripheral blood is typically obtained by venipuncture (also called phlebotomy), or by finger prick for small quantities.

[0163] The term “plasma hyperimmune” refers to a polyclonal antibody preparation similar to intravenous immunoglobulin (IVIg), except that it is prepared from the plasma of donors with high titers of antibody against a specific organism or antigen. The term hyperimmune isoften used interchangeably with the terms “hyperimmune gammaglobulin” and “hyperimmune globulin”. Some agents against which hyperimmune globulins are available include hepatitis B, rabies, tetanus toxin, varicella-zoster, etc. Administration of hyperimmune globulin provides "passive" immunity to the patient against an agent. This is in contrast to vaccines that provide "active" immunity. However, vaccines take much longer to achieve that purpose while hyperimmune globulin provides instant "passive" short-lived immunity.

[0164] The term activity refers to a Quantitative measurement of an RPP or antibody against an antigen, vaccine, protein, epitope, cell, bacterium, or virus. Activity can be assessed using in vivo or in vitro methods.

[0165] The term “recombinant” refers to proteins that result from the expression of recombinant DNA within living cells. Recombinant DNA is the general name for a piece of DNA that has been created by the combination of at least two separate segments of DNA.

[0166] The term “neutralization” refers to the ability' of specific antibodies to block the site(s) on viruses that they use to enter their target cell. The effect of a neutralizing antibody can be negligible even with large excesses of antibody production if they lack specificity to this antigen. The production of specific antibodies can be learned for a faster response at next exposition. The reduction or destruction of a homologous infectious agent can be partial or complete and can make it no longer infectious or pathogenic to other cells.

[0167] A “variant” of a polypeptide (e.g., an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to the native polypeptide sequence, and retains essentially the same biological activity' as the native polypeptide. The biological activity of the polypeptide can be measured using standard techniques in the art (for example, if the variant is an antibody, its activity may be tested by binding assays, as described herein). Variants of the invention include fragments, analogs, recombinant polypeptides, synthetic polypeptides, and / or fusion proteins.

[0168] A “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g, human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0169] A nucleotide sequence is “operably linked” to a regulatory sequence if the regulatory sequence affects the expression (e.g, the level, timing, or location of expression) of the nucleotide sequence. A “regulatory sequence” is a nucleic acid that affects the expression (e.g. , the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g, polypeptides that bind to the regulatory' sequence and / or the nucleic acid). Examples of regulator}' sequences include promoters, enhancers and other expression control elements (e.g., poly adenylation signals). Further examples of regulatory sequences are described in, for example, 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.

[0170] A “host cell” is a cell that can be used to express a nucleic acid, e.g, a nucleic acid of the invention. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g, a tobacco or tomato plant cell), an animal cell (e.g. , a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) 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 their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, the 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 embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.Other interpretational conventions

[0171] Ranges recited herein are understood to be shorthand for all of the values w ithin the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood toinclude 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.

[0172] Unless otherwise indicated, reference to a compound that has one or more stereocenters intends each stereoisomer, and all combinations of stereoisomers, thereof.Custom recombinant polyclonal protein library (RPP)

[0173] The present disclosure provides a custom RPP comprising a plurality of ABPs having specific properties. The custom RPP can be generated by employing any of the in silica processes described herein.

[0174] In some embodiments, ABPs in the RPP specifically bind a target molecule or complex of molecules (such as molecules or complexes of molecules associated with a pathogen, including, e.g., viral and bacterial pathogens). The RPP comprises a plurality' of ABPs that specifically bind to the target molecule or complex and that are selected for having one or more characteristics (including, e.g., physical characteristics and functional characteristics) and / or one or more library properties (i.e., properties of the library of ABPs that make up the RPP). The characteristics and library properties can include those determined experimentally or by an in silica process.

[0175] Accordingly, the present disclosure relates to methods of selecting ABPs having desired properties for generation of an RPP. The present invention also relates to the RPP generated by any of the methods disclosed herein.ABP Characteristics

[0176] In some embodiments, an ABP of an RPP described herein is selected based on a plurality of characteristics selected from binding affinity, effector activity (e.g., neutralization activity or killing activity), solubility, aggregation, hydrophobicity, isoelectric point, stability, molecular weight, number of cysteine residues, abundance frequency and fold-change in abundance following sorting of a library of ABPs. number of glycosylation sites, number of cleavage sites, number of deamidation sites, number of isomerization sites, number of oxidation sites, CDR3H length, binding specificity, and full or partial amino acid sequence.

[0177] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on a binding affinity of the ABP to a respective target antigen. In some embodiments, the binding affinity is expressed in KD, and the ABP is selected for having a KD less thanabout 100 pM, 10 pM, 1 pM, 100 nM, 10 nM, 1 nM, or lower. In some embodiments, the ABP has a KD less than about 100 pM (such as less than about any of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM. 30 pM, 20 pM, 10 pM, 9 pM. 8 pM, 7 pM. 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 900 nM, 800 nM. 700 nM, 600 nM, 500 nM. 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 1 pM, or lower). In some embodiments, the ABP has a KD from about 100 pM to about 1 pM (such as about any of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM. 20 pM. 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or 1 pM, including any ranges between any of these values). In some embodiments, such a binding affinity is determined by surface plasmon resonance (SPR) or biolayer interferometry (BLI). In some embodiments, the binding affinity is expressed qualitatively as being able to bind or not being able to bind, and the ABP is selected for being able to bind. In some embodiments, the APB of an RPP described herein is selected based, at least in part, on a binding pattern of the respective candidate ABP to the respective target antigen and its variants. In some embodiments, such a binding affinity or binding pattern is determined by a PolyMap assay as described herein and in PCT / US2024 / 012238, which is incorporated by reference in its entirety herein.

[0178] In some embodiments, binding affinity is ranked at least top 20%, at least top 15%. at least top 10%, or at least top 5% among all the candidate ABPs in the input ABP library. In some embodiments, binding affinity is ranked at least top 50%, at least top 45%, at least top 40%, at least top 35%, at least top 30%, at least top 25%, or at least top 20% among all the candidate ABPs in the input ABP library .

[0179] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on an effector activity' of the ABP. In some embodiments, the target molecule or complex comprises a virus, and the effector activity is a neutralization activity. In some embodiments, the neutralization activity is determined by a pseudovirus neutralization assay or a live virus neutralization assay. In some embodiments, the neutralization activity is determined by a pseudovirus neutralization assay and corresponds to an IC50 from about 1 ng / mL to about 500 mg / mL (such as about any of 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL. 40 ng / mL, 50 ng / mL, 60 ng / mL. 70 ng / mL, 80 ng / mL, 90 ng / mL. 100 ng / mL, 200 ng / mL. 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1pg / mL, 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, 1 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 200 mg / mL. 300 mg / mL, 400 mg / mL, or 500 mg / mL, including any ranges between any of these values). In some embodiments, the neutralization activity7is an ICso from about 0.08 pg / mL to about 900 pg / mL. In some embodiments, the neutralization activity is determined by a live virus neutralization assay and corresponds to an ICso from about 1 ng / mL to about 500 mg / mL (such as about any of 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1 pg / mL, 10 pg / mL, 20 pg / mL. 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL. 80 pg / mL. 90 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, 1 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL. 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, including any ranges between any of these values). In some embodiments, the neutralization activity7is an ICso from about 0.003 pg / mL to about 1350 pg / mL. In some embodiments, the target molecule or complex comprises a bacterial protein, and the effector activity7is a bactericidal activity'. In some embodiments, the bactericidal activity' is determined by a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPKA). In some embodiments, the bactericidal activity corresponds to a concentration where 50% bactericidal activity7is observed from about 1 ng / ml to about 500 mg / ml (such as about any7of 1 ng / mL, 2 ng / mL, 3 ng / rnL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 20 ng / mL. 30 ng / mL, 40 ng / mL, 50 ng / mL. 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL. 200 ng / mL, 300 ng / mL, 400 ng / mL. 500 ng / mL, 600 ng / mL, 700 ng / mL. 800 ng / mL, 900 ng / mL, 1 pg / mL, 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, 1 mg / mL. 10 mg / rnL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, or 500 mg / mL, including any ranges between any of these values). In some embodiments, the bactericidal activity is a concentration where 50% bactericidal activity' is observed from about 0.08 pg / ml to about 3600 pg / ml.

[0180] In some embodiments, an ABP of an RPP described herein is selected for having an effector activity' against the target molecule or complex ranked at least top 30% among all the candidate ABPs in the input ABP library’. In some embodiments, an ABP of an RPP described herein is selected for having an effector activity against the target molecule or complex ranked at least top 25% among all the candidate ABPs in the input ABP library. In some embodiments, an ABP of an RPP described herein is selected for having an effector activity against the target molecule or complex ranked at least top 20% among all the candidate ABPs in the input ABP library. In some embodiments, an ABP of an RPP described herein is selected for having an effector activity7against the target molecule or complex ranked at least top 15% among all the candidate ABPs in the input ABP library7. In some embodiments, an ABP of an RPP described herein is selected for having an effector activity against the target molecule or complex ranked at least top 10% among all the candidate ABPs in the input ABP library. In some embodiments, an ABP of an RPP described herein is selected for having an effector activity7against the target molecule or complex ranked at least top 5% among all the candidate ABPs in the input ABP library.

[0181] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on solubility of the ABP. In some embodiments, the ABP is assigned a solubility7score. In some embodiments, the solubility7score is determined using SKA.DE. See, e.g., Raimondi, D., Orlando, G., Fariselli, P., & Moreau, Y. (2020). Insight into the protein solubility driving forces with neural attention. PLoS computational biology, 16( ). el007722. In some embodiments, the solubility7score is greater than about 0.5 (such as greater than about any of 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89. 0.9, 0.91, 0.92, 0.93. 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99). In some embodiments, the solubility score is from about 0.5 to about 0.8 (such as about any of 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, or 0.8, including any ranges between any of these values). In some embodiments, the solubility score is greater than about 0.5. or between 0.5 and 0.8.

[0182] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on aggregation (e.g., predicted aggregation) of the ABP. In some embodiments, the ABP is assigned an aggregation score. In some embodiments, the aggregation score corresponds to the number of residues predicted to have a propensity to aggregate. In someembodiments, the number of residues predicted to have a propensity to aggregate is determined by a method comprising the steps of: determining a 3D structure of the ABP (e.g., using AbodyBuilder2); and determining the aggregation score based on the 3D structure (e.g., using Aggrescan3D). In some embodiments, the aggregation score is fewer than 50 (such as fewer than any of 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) aggregation-prone sites. In some embodiments, the aggregation score is fewer than 20, 15, or fewer aggregation-prone sites.

[0183] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on hydrophobicity (e.g., predicted hydrophobicity) of the ABP. In some embodiments, the ABP is assigned a hydrophobicity score. In some embodiments, the hydrophobicity score is determined as the grand average of hydropathy (GRAVY). In some embodiments, the hydropathy value of each amino acid is calculated using the Eisenberg scale. In some embodiments, the hydrophobicity score is less than about 0. 1 (such as less than about any of 0.1, 0.095, 0.09, 0.085, 0.08, 0.075, 0.07, 0.065, 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025. 0.02. 0.015, 0.01, 0.005. or lower). In some embodiments, the hydrophobicity score is less than 0.03, less than 0.02, or less than 0.015.

[0184] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on an isoelectric point of the ABP. In some embodiments, the isoelectric point is determined as EMBOSS pK values. In some embodiments, the isoelectric point is between about 6.5 and about 9.5 (such as about any of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1. 9.2, 9.3, 9.4, or 9.5, including any ranges between any of these values). In some embodiments, the isoelectric point is between about 7.0 and about 9.0, or between about 8.0 and about 8.5.

[0185] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on a stability (e.g.. predicted stability) of the ABP. In some embodiments, the ABP is assigned a stability score. In some embodiments, the stability score is determined by calculating an aliphatic index by determining the relative volume of A, V, L, and I residues, wherein the stability score corresponds to the aliphatic index. In some embodiments, the stability score is from about 60 to about 80 (such as about any of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, including any ranges between any of these values). In some embodiments, the stability score is from about 65 to about 73.

[0186] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the molecular weight of the ABP. In some embodiments, the molecular weight is less than about 250 kDa (such as less than about any of 250 kDa, 245 kDa, 240 kDa, 235 kDa. 230 kDa, 225 kDa. 220 kDa. 215 kDa. 210 kDa. 205 kDa. 200 kDa. 195 kDa. 190 kDa, 185 kDa, 180 kDa, 175 kDa, 170 kDa, 165 kDa, 160 kDa, 155 kDa, 150 kDa, 145 kDa, 140 kDa, 135 kDa, 130 kDa, 125 kDa, 120 kDa, 115 kDa, 110 kDa, 105 kDa, 100 kDa, or less). In some embodiments, the molecular weight is less than about 170 kDa, less than about 160 kDa, less than about 150 kDa, less than about 140 kDa, less than about 130 kDa, less than about 120 kDa, or lower.

[0187] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the number of cysteine residues in the ABP. In some embodiments, the selection is based on the number of unpaired cysteine residues. In some embodiments, the number of unpaired cysteine residues is less than 5, 4, 3, 2, or 1.

[0188] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on an abundance frequency of the ABP following sorting of a library of ABPs to enrich for binding to the respective target antigen. In some embodiments, the sorting is by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). In some embodiments, the sorting is carried out by yeast display. The term "‘frequency" as described herein, refers to a measure of how often the ABP occurs within a defined population of ABPs in a library or sub-library.

[0189] In some embodiments, an ABP is selected when its abundance frequency following sorting the input ABP li brary or a subset thereof is ranked at least top 30% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when its abundance frequency following sorting the input ABP library' or a subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when its abundance frequency following sorting the input ABP library’ or a subset thereof is ranked at least top 20% among all the candidate ABPs in the input ABP library' or the subset thereof. In some embodiments, an ABP is selected when its abundance frequency following sorting the input ABP library or a subset thereof is ranked at least top 15% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when its abundance frequency following sorting the input ABP library or a subset thereof is rankedat least top 10% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when its abundance frequency following sorting the input ABP library’ or a subset thereof is ranked at least top 5% among all the candidate ABPs in the input ABP library or the subset thereof.

[0190] In some embodiments, the ABP of an RPP described herein is selected based, at least in part, on binding specificity. In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on polyreactivity. In some embodiments, binding specificity is determined at least in part, by assessing the polyreactivity of the ABP. In some embodiments, the ABP of an RPP described herein is selected based, at least in part, on cross-reactivity. In some embodiments, polyreactivity is assessed by performing binding assays. In some embodiments, polyreactivity is assessed by performing a FACS-based assay. In some embodiments, the polyreactivity assay requires one or more reagents e.g., that are used before or during cell sorting.

[0191] In some embodiments, an ABP is selected when it has specific binding to a target antigen or its variant. In some embodiments, an ABP is selected when it has polyreactivity. In some embodiments, an ABP is excluded when it is highly specific or highly polyreactive. In some embodiments, an ABP is selected when it has specificity to a target antigen or its variant with a ranking in the range of 10% to 90% within the input ABP library. In some embodiments, an ABP is selected when it has specificity to a target antigen or its variant with a ranking in the range of 20% to 80% within the input ABP library. In some embodiments, an ABP is selected when it has specificity’ to a target antigen or its variant with a ranking in the range of 25% to 75% within the input ABP library-. In some embodiments, an ABP is selected when it has specificity to a target antigen or its variant with a ranking in the range of 30% to 70% within the input ABP library.

[0192] In some embodiments, the post-sort abundance frequency is greater than about 0.01% (such as greater than about any of 0.010%, 0.015%. 0.020%, 0.025%, 0.030%, 0.035%, 0.040%, 0.045%, 0.050%, 0.055%, 0.060%, 0.065%, 0.070%, 0.075%, 0.080%, 0.085%, 0.090%, 0.095%, 0.100%, or greater) within a pool of ABPs obtained after the sorting process. In some embodiments, the post-sort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or greater within a pool of ABPs obtained after the sorting process. In some embodiments, the post-sort abundance frequency is greater than about 0.01% (such as greater than about any of 1%, 2%, 5%, 10%, 15%, 16%, 17%, 18%,19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% within a pool of ABPs obtained after the sorting process. In some embodiments, the post-sort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or greater within a pool of ABPs, obtained after the sorting process.

[0193] In some embodiments, the APB of an RPP described herein is selected based, at least in part, on an abundance frequency of the respective candidate ABP following sorting the input ABP library or a subset thereof to enrich for binding to the respective target antigen. In some embodiments, the APB of an RPP described herein is selected based, at least in part, on a fold-change of the increase in the abundance frequency of the respective candidate ABP following sorting the input ABP library of subset thereof to enrich for binding to the respective target antigen, as compared to the abundance frequency prior to enrichment. In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on a fold-change in an abundance frequency of the ABP following sorting of a library of ABPs to enrich for binding to the respective target antigen. In some embodiments, the sorting is by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). In some embodiments, the sorting is carried out by yeast display. In some embodiments, the post-sort fold-change in abundance frequency is greater than about any of 0.5 (such as greater than about any of 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2,4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, or greater). In some embodiments, the post-sort foldchange in abundance frequency is greater than about any of 1, 1.5, 2, 2.5. 3, 4, or greater. In some embodiments, the sorting is carried out by yeast display. In some embodiments, the post-sort fold-change in abundance frequency is greater than about any of 0.5 (such as greater than about any of 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8. 4.0, 4.2,4.4, 4 6, 4.8, 5.0. 5.2, 5.4, 5.6, 5.8. 6.0, 6.5, 7, 7.5. 8, 85. 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, or greater). In some embodiments, the post-sort fold-change in abundance frequency is greater than about any of 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2. 4.4, 4.6, 4.8, 5.0. 5.2, 5.4, 5.6, 5.8. 6.0, 6.5, 7, 7.5. 8, 85. 9,9.5, 10. 15. 20. 25. 30. 35. 40. 45, 50, 75, 100. 125, 150, 175. 200, 250. 300, 350, 400. 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, or greater.

[0194] In some embodiments, an ABP is selected based on a fold-change of the increase in the abundance frequency of the ABP following sorting the input ABP library or a subset thereof. In some embodiments, an ABP is selected when a fold-change of the increase in theabundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 30% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when a fold-change of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when a fold-change of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 20% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when a fold-change of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 15% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, an ABP is selected when a fold-change of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 10% among all the candidate ABPs in the input ABP library' or the subset thereof. In some embodiments, an ABP is selected when a fold-change of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 5% among all the candidate ABPs in the input ABP library or the subset thereof.

[0195] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the number of glycosylation sites (e.g., predicted glycosylation sites) in the ABP. In some embodiments, the selection is based on the number of non-canonical glycosylation sites. In some embodiments, the number of non-canonical glycosylation sites is less than 5, 4, 3, 2, or 1 .

[0196] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the number of cleavage sites (e.g., predicted cleavage sites) in the ABP. In some embodiments, the number of cleavage sites is less than 5, 4, 3, 2, or 1 . In some embodiments, the cleavage sites comprise a DP motif in the variable heavy or variable light chain region of the respective ABP.

[0197] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the number of deamidation sites (e.g., predicted deamidation sites) in the ABP. In some embodiments, the number of deamidation sites is less than 5, 4, 3, 2, or 1. In someembodiments, the deamidation sites comprise an NG, NS, or NA motif in CDR2H or CDR1L of the respective ABP.

[0198] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the number of isomerization sites (e.g., predicted isomerization sites) in the ABP. In some embodiments, the number of isomerization sites is less than 5, 4, 3, 2, or 1. In some embodiments, the isomerization sites comprise a DG or DS motif in CDR2H, CDR3H. or CDR1L of the respective ABP.

[0199] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the number of oxidation sites (e.g., predicted oxidation sites) in the ABP. In some embodiments, the number of oxidation sites is less than 5, 4, 3, 2. or 1. In some embodiments, the oxidation sites comprise a W or M residue in the CDRHs or CDRLs of the respective ABP.

[0200] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the CDR3H length in the ABP. In some embodiments, the CDR3H length is from about 10 to about 14 amino acids.

[0201] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the binding specificity of the ABP. For example, in some embodiments, the ABP is capable of binding to at least 1 (such as at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) variant of a target antigen (e.g., a variant of a viral polypeptide). In some embodiments, the binding specificity is determined by a Poly Map assay as described herein. In some embodiments, the binding specificity is further determined by a polyreactivity assay.

[0202] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on its binding pattern a target antigen or variants thereof. In some embodiments, an ABP is selected for a frequent binding pattern. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among less than 30% of candidate ABPs in the input ABP library.

[0203] In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among less than 25% of candidate ABPs in the input ABP library. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among less than 20% of candidate ABPs in the input ABP library. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected forhaving a binding pattern shared among less than 15% of candidate ABPs in the input ABP library7. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among less than 10% of candidate ABPs in the input ABP library. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among less than 5% of candidate ABPs in the input ABP library.

[0204] In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among more than 75% of candidate ABPs in the input ABP library. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among more than 80% of candidate ABPs in the input ABP library. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among more than 85% of candidate ABPs in the input ABP library7. In some embodiments, an ABP is selected for a rare binding pattern. In some embodiments an ABP is selected for having a binding pattern shared among more than 90% of candidate ABPs in the input ABP library.

[0205] In some embodiments, an ABP of an RPP described herein is selected based, at least in part, on the full or partial amino acid sequence of the ABP. In some embodiments, the selection is based on the full amino acid sequence of the ABP. In some embodiments, the selection is based on a partial amino acid sequence of the ABP, including, e.g., CDRs of the ABP. In some embodiments, the selection is based on the CDR3 (heavy and / or light chain) sequences of the ABP. In some embodiments, the selection is based on the CDR1, CDR2, and / or CDR3 (heavy and / or light chain) sequences of the ABP. In some embodiments, the selection is based on the CDR1, CDR2, and CDR3 (heavy and / or light chain) sequences of the ABP. In some embodiments, the selection is based on the heavy chain and light chain CDR1, CDR2, and CDR3 sequences of the ABP.

[0206] In some embodiments, an ABP of an RPP described herein is selected based on at least any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the characteristics described herein. The selection characteristics may be individually chosen for each ABP in the RPP, or may be the same for all ABPs in the RPP. For example, in some embodiments, a first ABP of an RPP may be selected based on its binding affinity, solubility, and hydrophobicity7, while a second ABP of the RPP may be selected based on its aggregation,isoelectric point, and stability. In other embodiments, all of the ABPs in an RPP may be selected based on their binding affinity, solubility, hydrophobicity', aggregation, isoelectric point, and stability.

[0207] In some embodiments, one or more of the characteristics of an ABP described herein may be determined (e.g., predicted) in silico. For example, in some embodiments, where an ABP characteristic selected from solubility, aggregation, hydrophobicity, isoelectric point, stability, number of cysteine residues, number of glycosylation sites, number of cleavage sites, number of deamidation sites, number of isomerization sites, and number of oxidation sites is used as a basis for selection to be included in an RPP, the ABP characteristic can be determined in silico. Such in silico determinations can be carried out using any convenient means known in the art, such as by using a full or partial sequence of the ABP or a nucleic acid encoding the ABP as an input.

[0208] The method can comprises generating a dataset for a filtered ABP library comprising the ABPs selected for the selection criteria. In some embodiments, the filtered ABP library comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more ABPs selected for their binding affinity. In some embodiments, the filtered ABP library comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more ABPs selected for their effector activity'. In some embodiments, the filtered ABP library comprises at least 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 15, 20, or more ABPs selected for their binding pattern. In some embodiments, the filtered ABP library comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more ABPs selected for their abundance frequency following sorting the input ABP library' or a subset thereof to enrich for binding to a target antigen. In some embodiments, the filtered ABP library comprises at least 1, 2, 3, 4, 5, 6, 7, 8. 9, 10, 15, 20, or more ABPs selected for their fold-change of the increase in the abundance frequency following sorting the input ABP library or a subset thereof to enrich for binding to a target antigen, as compared to the abundance frequency prior to enrichment.ABP Library Properties

[0209] In some embodiments, the ABPs of an RPP described herein are selected based on one or more ABP library properties selected from number of unique CDR3H sequences, number of unique epitopes, number of antigen variants targeted, number of unique heavy’ chain V genes, number of unique light chain V genes, number of unique heavy chain J genes, number of unique light chain J genes, heavy chain V gene average percent germline identity,light chain V gene average percent germline identity, heavy chain J gene average percent germline identity, and light chain J gene average percent germline identity7.

[0210] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the number of unique CDR3H sequences in the ABPs. In some embodiments, the library7of ABPs in the RPP comprises at least 2 (such as at least any of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or more) unique CDR3H sequences.

[0211] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the number of unique epitopes targeted by ABPs in the library7of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP targets at least 2 (such as at least any of 3, 4, 5. 6. 7, 8, 9. 10. 20. 30. 40, 50, 60, 70, 80, 90, 100. 200, or more) unique epitopes.

[0212] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the number of antigen variants targeted by the ABPs. In some embodiments, the library of ABPs in the RPP targets at least 1 (such as at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) variant of an antigen targeted by the RPP. For example, where the RPP targets a particular viral spike protein, the library of ABPs in the RPP may target one or more variants of the viral spike protein.

[0213] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the number of unique heavy chain V genes represented in the library of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents at least 2 (such as at least any of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more) unique heavy chain V genes.

[0214] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the number of unique light chain V genes represented in the library7of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents at least 2 (such as at least any of 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, or more) unique light chain V genes.

[0215] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the number of unique heavy chain J genes represented in the library7of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents at least 2 (such as at least any of 3, 4, 5, or 6) unique heavy chain J genes.

[0216] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the number of unique light chain J genes represented in the library of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents at least 2 (such as at least any of 3. 4, 5, 6. 7. 8, or 9) unique light chain J genes.

[0217] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the heavy chain V gene average percent germline identity represented in the library of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents a heavy chain V gene average percent germline identity between about 50% and about 100% (such as about any of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%. 72%, 73%. 74%. 75%. 76%. 77%. 78%. 79%. 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% including any ranges between any of these values).

[0218] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the light chain V gene average percent germline identity represented in the library of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents a light chain V gene average percent germline identity between about 50% and about 100% (such as about any of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%. 61%. 62%. 63%. 64%. 65%. 66%. 67%. 68%. 69%. 70%. 71%. 72%. 73%. 74%. 75%. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, including any ranges between any of these values).

[0219] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the heavy chain J gene average percent germline identity7represented in the library of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents a heavy chain J gene average percent germline identity between about 50% and about 100% (such as about any of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%. including any ranges between any of these values).

[0220] In some embodiments, the ABPs of an RPP described herein are selected based, at least in part, on the light chain J gene average percent germline identity represented in the library of ABPs that make up the RPP. In some embodiments, the library of ABPs in the RPP represents a light chain J gene average percent germline identity between about 50% and about 100% (such as about any of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%. including any ranges between any of these values).

[0221] In some embodiments, the ABPs of an RPP described herein are selected based on at least any 1. 2, 3, 4. 5, 6, 7, 8. 9, 10, or 11 of the library properties described herein.ABP Source

[0222] ABPs for generation of an RPP can be obtained from various sources (e.g.. input ABP library). In some embodiments, a naturally occurring ABP isolated from a donor sample is used. In some embodiments, the input ABP library comprises ABPs originated from one or more donors. In some embodiments, an ABP synthesized based on known sequences is used. In some embodiments, an ABP having an artificially generated sequence is used. In some embodiments, an ABP having a sequence from an antibody sequence database (e.g., UniProt, IMGT, abYsis, The ABCD database, SabDab, Thera-SabDab, Aho’s Amazing Atlas of Antibody Anatomy, Observed Antibody Space database, cAb-Rep) is used.

[0223] In some embodiments, an RPP comprises ABPs originated from the same source. In some embodiments, an RPP comprises ABPs originated from multiple different sources.

[0224] In some embodiments, ABPs are originated from one or more donor samples. In some embodiments, the ABPs obtained from one or more sources are analyzed and selected to be included in the RPP.

[0225] In some embodiments, an ABP of an RPP described herein comprises a cognate pair of heavy chain and light chain variable regions from a single cell out of a blood sample from at least one (such as at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) donor previously exposed to the target molecule or complex (e.g., target molecule or complex associated with a virus or bacterium). In some embodiments, the cognate pair of heavy chain and light chain variable regions from a single cell is generated by the method described in Adler et al, high-affinity anti-pathogen antibodies from humanrepertoires, discovered using microfluidics and molecular genomics. Mabs. 2017 Nov / Dec;9(8): 1282-1296, which is incorporated by reference in its entirety.

[0226] In some embodiments, the at least one donor has been previously vaccinated with a vaccine derived from the target molecule or complex. In some embodiments, the blood sample comprises cells purified from peripheral blood mononuclear cells (PBMCs) of the donor. In some embodiments, the single cell is a B cell (e.g., memory B cell), plasma cell, or plasmablast. In some embodiments, the target molecule or complex is associated with a virus. In some embodiments, the target molecule or complex is a viral protein or complex of viral proteins. In some embodiments, the target molecule or complex is associated with a bacterium. In some embodiments, the target molecule or complex is a bacterial protein or complex of bacterial proteins. In some embodiments, the subject is a human. In some embodiments, the subject is a transgenic animal (including, e.g., mice, rats, and chickens) expressing human antibody sequences.

[0227] Fully human monoclonal antibodies may be generated by any number of techniques with which those having ordinary skill in the art will be familiar. 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 as known in the art and based on the disclosure herein. For example, fully human monoclonal antibodies may be obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. Methods for obtaining fully human antibodies from transgenic mice are described, for example, by 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 locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human immunoglobulin transgenes may be mini-gene constructs, or transloci on yeast artificial chromosomes, which undergo B-cell-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue. Fully human monoclonal antibodies may be obtained by immunizing the transgenic mice, which may then produce human antibodiesspecific for the antigen target or targets. Lymphoid cells of the immunized transgenic mice can be used to produce human antibody-secreting hybridomas according to the methods described herein.

[0228] Another method for generating human antibodies of the invention includes immortalizing human peripheral blood cells by EBV transformation. See, e.g., U.S. Patent No. 4,464,456. Such an immortalized B-cell line (or lymphoblastoid cell line) producing an ABP that specifically binds to target or targets can be identified by immunodetection methods as provided herein, for example, an ELISA, and then isolated by standard cloning techniques. The stability of the lymphoblastoid cell line producing an ABP may be improved by fusing the transformed cell lines with a murine myeloma to produce a mouse-human hybrid cell line according to methods known in the art {see, e.g., Glasky et al. , Hybridoma 8:377-89 (1989)). Still another method to generate human ABPs is in vitro immunization, which includes priming human splenic B-cells with antigen targets, followed by fusion of primed with a heterohybrid fusion partner. See, e.g., Boemer et al., 1991 J. Immunol. 147:86-95.

[0229] In certain embodiments, B-cells that are producing an ABP are selected and the light chain and heavy chain variable regions are cloned from the B-cell according to molecular biology techniques known in the art (WO 92 / 02551; U.S. Patent 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. B-cells from an immunized animal may be isolated from the spleen, lymph node, or peripheral blood sample by selecting a cell that is producing an antibody that specifically binds to the antigen target. B-cells may also be isolated from humans, for example, from a peripheral blood sample.

[0230] Methods for detecting single B-cells that are producing an antibody with the desired specificity are well known in the art, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibody, and the like. Methods for selection of specific antibody -producing B-cells include, for example, preparing a single cell suspension of B-cells in soft agar that contains the antigen target. Binding of the specific antibodies produced by the B-cell to the antigen results in the formation of a complex, which may be visible as an immunoprecipitate.

[0231] In some embodiments, specific antibody-producing B-cells are selected by using a method that allows identification natively paired antibodies. For example, a method 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 byreference in its entirety herein, can be employed. The method combines microfluidic technology, molecular genomics, yeast single-chain variable fragment (scFv) display, yeast Fab display, fluorescence-activated cell sorting (FACS) and deep sequencing. In short. B cells can be isolated from immunized animals and then pooled. The B cells are encapsulated into droplets with oligo-dT beads and a lysis solution, and mRNA-bound beads are purified from the droplets, and then injected into a second emulsion with an OE-RT-PCR amplification mix that generates DNA amplicons that encode scFv or Fab with native pairing of heavy and light chain Ig. Libraries of natively paired amplicons are then electroporated into yeast for scFv or Fab display. FACS is used to identify high affinity scFv or Fab. Finally, deep antibody sequencing can be used to identify all clones in the pre- and post-sort scFv or Fab libraries.

[0232] After the B-cells producing the desired antibody are selected, the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA according to methods known in the art and described herein.

[0233] The methods for obtaining antibodies of the invention can also adopt various phage display technologies known in the art. See, e.g., Winter et al., 1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57: 191-280. Human or murine immunoglobulin variable region gene combinatorial libraries may be created in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind specifically to a target antigen. See, e.g., 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. Nall. Acad. Sci. USA 88:4363-66; Hoogenboom et al.. 1992 Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited therein. For example, a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments may be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, in frame with the sequence encoding a phage coat protein. A fusion protein may be a fusion of the coat protein with the light chain variable region domain and / or with the heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments may also be displayed on a phage particle (see, e.g., U.S. Patent No. 5,698,426).

[0234] In one embodiment, in a hybridoma the variable regions of a gene expressing a monoclonal antibody of interest are amplified using nucleotide primers. These primers maybe synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. (See, e.g., Stratagene (La Jolla, California), which sells primers for mouse and human variable regions including, among others, primers for Vm, Vnb, VHC, Vna, CHI, VL and CL regions.) These primers may be used to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAP1MH or ImmunoZAPIML (Stratagene), respectively. These vectors may then be introduced into E. coli, yeast, or mammalian-based systems for expression. Large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced using these methods (see Bird et al., Science 242:423-426, 1988).

[0235] Once cells producing antibodies according to the invention have been obtained using any of the above-described immunization and other techniques, the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures as described herein. The antibodies produced therefrom may be sequenced and the CDRs identified and the DNA coding for the CDRs may be manipulated as described previously to generate other antibodies according to the invention.

[0236] Other antibodies according to the invention may be obtained by conventional immunization and cell fusion procedures as described herein and known in the art.

[0237] Molecular evolution of the complementarity determining regions (CDRs) in the center of the antibody binding site also has been used to isolate antibodies with increased affinity, for example, antibodies having increased affinity7for c-erbB-2, as described by Schier et al., 1996, J. Mol. Biol. 263:55 Lit will be appreciated that an antibody of the present invention may have at least one amino acid substitution, providing that the antibody retains binding specificity. Therefore, modifications to the antibody structures are encompassed within the scope of the invention. These may include amino acid substitutions, which may be conservative or non-conservative that do not destroy the binding capability of an antibody comprising the RPP. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. A conservative amino acid substitution may also involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.

[0238] Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid mimetics for a member from another class with different physical properties (e.g. size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.

[0239] Moreover, one skilled in the art may 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 skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0240] A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify' residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity' or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0241] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity' or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0242] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. In certain embodiments, one skilled in the art may choosenot to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules.

[0243] A number of scientific publications have been devoted to the prediction of secondary structure. See 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). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology' modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity' greater than 40% often have similar structural topologies. The recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide’s or protein’s structure. See Holm et al., Nucl. Acid. Res., 27(l):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. Biol., 1(3 .369-316 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a cntical number of structures have been resolved, structural prediction will become dramatically more accurate.

[0244] Additional methods of predicting secondary structure include “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 cd., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and “evolutionary linkage” (See Holm, supra (1999). and Brenner, supra (1997)).

[0245] In certain embodiments, variants of antibodies include glycosylation variants wherein the number and / or ty pe of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide. In certain embodiments, variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylationsites ('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 wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g.. serine) as compared to the parent amino acid sequence. Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the 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 resulting from unpaired cysteines.

[0246] According to certain embodiments, preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity' for forming protein complexes, (4) alter binding affinities, and / or (4) confer or modify other physiochemical or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution ty pically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary’ and tertiary structures 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), which are each incorporated herein by reference.

[0247] In certain embodiments, ABPs of the invention may be chemically bonded with polymers, lipids, or other moieties.

[0248] The binding agents may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In one example, the biocompatible framework structure comprises a polypeptide or portion thereof that is sufficient to form a conformationally stable structural support, or framework, or scaffold, which is able to display one or more sequences of amino acids that bind to an antigen (e.g., CDRs, a variable region, etc.) in a localized surface region. Such structures can be a naturally occurring polypeptide orpolypeptide ‘’fold” (a structural motif), or can have one or more modifications, such as additions, deletions or substitutions of amino acids, relative to a naturally occurring polypeptide or fold. These scaffolds can be derived from a polypeptide of any species (or of more than one species), such as a human, other mammal, other vertebrate, invertebrate, plant, bacteria or virus.

[0249] Typically, the biocompatible framework structures are based on protein scaffolds or skeletons other than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CPI zinc finger, PST1, coiled coil, LACI- Dl, Z domain and tendamistat domains may be used (See e.g., Nygren and Uhlen, 1997, Curr. Opin. in Struct. Biol., 7, 463-469).

[0250] It will be appreciated that the ABPs of the invention include the humanized antibodies described herein. Humanized antibodies such as those described herein can be produced using techniques known to those skilled 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(l):43-60, 2005; and Clark, M., Immunology Today. 21(8): 397-402, 2000).ABP Format

[0251] In some embodiments, the RPP comprises scFvs. In some embodiments, the RPP consists of scFvs. In some embodiments, the RPP comprises antibody fragments. In some embodiments, the RPP consists of antibody fragments. In some embodiments, the RPP comprises recombinant full-length antibodies. In some embodiments, the RPP consists of recombinant full-length antibodies. In some embodiments, the RPP comprises human antibodies. In some embodiments, the RPP comprises humanized antibodies. In some embodiments, the RPP comprises monospecific ABPs. In some embodiments, the RPP comprises bispecific ABPs. In some embodiments, the RPP consists of ABPs (individually or in combination) of a human IgG subtype including IgGl, IgG2, IgG3, and IgG4. In some embodiments, the RPP comprises IgM, IgD, IgG, IgA, IgE, or a combination thereof.

[0252] In some embodiments, the RPP comprises antibody fragments. The ABPs of the RPP can be a Fab fragment, a F(ab’)2 fragment an Fv fragment, or a combination thereof. A Fab fragment is a monovalent fragment having the VL, VH, CL and CHI domains; a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VH and CHI domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain.or an antigen-binding fragment of a VH or VL domain (US Pat. No. 6,846,634, 6,696,245, US App. Pub. No. 05 / 0202512, 04 / 0202995, 04 / 0038291, 04 / 0009507, 03 / 0039958, Ward et al., Nature 341:544-546. 1989).

[0253] Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity’ determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2. CDR2. FR3. CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5thEd., US Dept, of Health and Human Services. PHS. NIH, NIH Publication no. 91-3242, 1991.

[0254] In some embodiments, the RPP comprises or consists of humanized antibodies. A humanized antibody has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and / or additions, such that the humanized antibody is less likely to induce an immune response, and / or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and / or light chains of the non- human species antibody are mutated to produce the humanized antibody. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5.886,152 and 5,877,293.

[0255] Fragments or analogs of antibodies can be readily prepared by those of ordinary' skill in the art follo ving the teachings of this specification and using techniques well-known in the art. Preferred amino- and carboxy -termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of thenucleotide and / or amino acid sequence data to public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. See, e.g., Bowie et al., 1991, Science 253:164.

[0256] An ABP of an RPP can also be any synthetic 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, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (scFv proteins).

[0257] Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units”, or “hypervariable region”) can be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. CDRs can be obtained by constructing polynucleotides that encode the CDR of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody producing cells as a template (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).

[0258] The variable region domains of ABPs can be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain that has been created using recombinant DNA engineering techniques. Such engineered versions include those created, for example, from a specific antibody variable region by insertions, deletions, or changes in or to the amino acid sequences of the specific antibody. Particular examples include engineered variable region domains containing 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.

[0259] The variable region domain may be covalently attached at a C terminal amino acid to at least one other antibody domain or a fragment thereof. Thus, for example, a Vn domain that is present in the variable region domain may be linked to an immunoglobulin CHI domain, or a fragment thereof. Similarly, a VL domain may be linked to a CK domain or a fragment thereof. In this way, for example, the antibody may be a Fab fragment wherein the antigen binding domain contains associated VH and VL domains covalently linked at their C termini to a CHI and CK domain, respectively. The CHI domain may be extended with further amino acids, for example to provide a hinge region or a portion of a hinge region domain as found in a Fab’ fragment, or to provide further domains, such as antibody CH2 and CH3 domains.

[0260] The RPP can include ABPs comprising, e.g., the cognate pairs of heavy and light chain CDR3 sequence disclosed herein. For example, CDRs may be incorporated into known antibody framework regions (IgGl, IgG2, etc.), or conjugated to a suitable vehicle to enhance the half-life thereof. 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 may be in monomeric, dimeric, tetrameric, or other form. In one embodiment, one or more water-soluble polymer is bonded at one or more specific position, for example at the amino terminus, of a binding agent.

[0261] In certain embodiments, the ABP comprises one or more water soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., 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, a derivative binding agent comprises one or more of monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidonej-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide / ethylene oxide co-polymer, polyoxy ethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of such polymers. In certain embodiments, one or more water-soluble polymer is randomly attached to one or more side chains. In certain embodiments, PEG can act to improve the therapeutic capacity for a binding agent, such as an antibody. Certain such methods are discussed, for example, in U.S. Pat. No. 6,133,426, which is hereby incorporated by reference for any purpose.

[0262] An ABP of an RPP can have, for example, the structure of a naturally occurring immunoglobulin. An “immunoglobulin” is a tetrameric molecule. In a naturally occurringimmunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. 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’s isotype as IgM, IgD. IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology7Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

[0263] Different ABPs may bind to different domains of disease targets or act by different mechanisms of action. As indicated herein inter alia, the domain regions are designated such as to be inclusive of the group, unless otherwise indicated. For example, amino acids 4-12 refers to nine amino acids: amino acids at positions 4, and 12, as well as the seven intervening amino acids in the sequence. Other examples include antigen binding proteins that inhibit binding of a pathogen to its target cell, i.e., neutralizing activity. An antigen binding protein need not completely inhibit a binding to target cell to find use in the present invention.

[0264] The ABPs described herein can include an Fc region, e.g., a dimer Fc polypeptide. One suitable Fc polypeptide, described in PCT application WO 93 / 10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgGl antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and in Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93 / 10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity' for Fc receptors.

[0265] Antigen-binding fragments of ABPs of the invention can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab and F(ab’)2fragments. Antibody fragments and derivatives produced by genetic engineering techniques also are contemplated.

[0266] Additional embodiments include chimeric antibodies, e.g., humanized versions of non-human (e.g., murine) monoclonal antibodies. Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity' when the antibodies are administered to humans. In one embodiment, a humanized antibody comprises the variable domain of a murine antibody (or all or part of the antigen binding site thereof) and a constant domain derived from a human antibody. Alternatively, a 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 l. , 1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDR grafted antibody. Techniques for humanizing antibodies are discussed in, e.g., U.S. Pat. No.s 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.

[0267] Procedures have been developed for generating human or partially human antibodies in non-human animals. For example, mice in which one or more endogenous immunoglobulin genes have been inactivated by various means have been prepared. Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. Antibodies produced in the animal incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. 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 pare generated in the animal.

[0268] Examples of techniques for production and use of transgenic animals for the production of human or partially human antibodies are described in U.S. Patents 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, Cur r 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 ’I 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 'I Immunol. 6: 579-91, Tomizuka et al., 1997, Nature Genetics 16: 133-43, Tomizuka et al., 2000, Pro. Nat’lAcad. Sci. USA 97 : 722-27, Tuaillon et al., 1993, Pro.Nat 'lAcad.Sci. USA 90 : 3720-24, and Tuaillon et al., 1994, J.Immunol. 152 : 2912-20.

[0269] ABPs of the invention can comprise any constant region known in the art. The light chain constant region can be, for example, a kappa- or lambda-ty pe light chain constant region, e.g, a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.

[0270] Techniques are known for deriving an antibody of a different subclass or isotype from an antibody of interest, i.e., subclass switching. Thus, IgG antibodies may be derived from an IgM antibody, for example, and vice versa. Such techniques allow' the preparation of new' antibodies that possess the antigen-binding properties of a given antibody (the 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 employed. Cloned DNA encoding particular antibody polypeptides may be employed in such procedures, e.g, DNA encoding the constant domain of an antibody of the desired isotype. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.

[0271] Single chain antibodies (scFv) may be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker, e.g, a synthetic sequence of amino acid residues), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (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). By combining different VL and Vn-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., 2001. Biomol. Eng. 18:31-40). Techniques developed for the production of single chain antibodies 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.

[0272] An ABP according to the invention may have a binding affinity for antigen target of less than or equal to 5 x 10'7M, less than or equal to 1 x 10'7M, less than or equal to 0.5 x 10’7M, less than or equal to 1 x 10'8M, less than or equal to 1 x 10’9M, less than or equal to 1 x 10’10M, less than or equal to 1 x 10-11M, or less than or equal to 1 x 10’12M.

[0273] The affinity of an ABP, as well as the extent to which the ABP inhibits binding, can be determined by one of ordinary skill in the art using conventional techniques, for example those described by Scatchard et al. (Ann. N. Y. Acad. Sci. 51:660-672 (1949)) or by surface plasmon resonance (SPR; BIAcore, Biosensor, Piscataway. NJ). For surface plasmon resonance, target molecules are immobilized on a solid phase and exposed to ligands in a mobile phase running along a flow cell. If ligand binding to the immobilized target occurs, the local refractive index changes, leading to a change in SPR angle, which can be monitored in real time by detecting changes in the intensity’ of the reflected light. The rates of change of the SPR signal can be analyzed to yield 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, e.g., Wolff et al., Cancer Res. 53:2560-65 (1993)).RPP size

[0274] In some embodiments, the RPP comprises at least about any of 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. 50. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62. 63. 64. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74. 75. 76. 77. 78. 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more ABPs.

[0275] In some embodiments, the RPP comprises at least 100, at least 500, at least 1000, at least 2000. at least 3000. at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 15,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, at least 500,000, or at least 1000,000 unique ABPs.Nucleic acids

[0276] In one aspect, the present invention provides isolated nucleic acid molecules. The nucleic acids comprise, for example, polynucleotides that encode all or part of an RPP, for example, one or both chains of an antibody of the invention, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes. PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing. The nucleic acids can be any length. They 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 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be part of a larger nucleic acid, for example, a vector. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids).

[0277] Polynucleotides encoding antibody polypeptides (e.g., heavy or light chain, variable domain only, CDRs only, or full length) can be isolated from B cells, plasma cells, or plasmablasts of a subject that has been exposed to an antigen, e g., by being infected by virus or immunized with a vaccine. The nucleic acid can be isolated by conventional procedures such as polymerase chain reaction (PCR) or methods described herein (e.g., single cell OE- RT-PCR).

[0278] Polypeptide sequences of the CDR3 from the variable regions of the heavy and light chain variable regions are shown herein. The skilled artisan will appreciate that, due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein isencoded by a large number of other nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding each RPP of the invention.

[0279] Methods for hybridizing nucleic acids are well-known in the art. See, e.g., Curr. Prot. in Mol. Biol., John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, a moderately stringent hybridization condition uses a prewashing solution containing 5X sodium chloride / sodium citrate (SSC), 0.5% SDS. 1.0 rnM EDTA (pH 8.0), hybridization buffer of about 50% formamide. 6X SSC. and a hybridization temperature of 55° C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42° C), and washing conditions of 60° C, in 0.5X SSC, 0. 1% SDS. A stringent hybridization condition hybridizes in 6X SSC at 45° C, followed by one or more washes in 0. IX SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% identical to each other typically remain hybridized to each other. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by. for example. 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 can be readily determined by those having ordinary skill in the art based on, for example, the length and / or base composition of the DNA.

[0280] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g.. an RPP) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However, it is made, a mutant polypeptide can be expressed and screened for a desired property (e.g., binding to a virus).

[0281] In another aspect, the present invention provides nucleic acid molecules that are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences of the invention. A nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide of the invention, for example, afragment that can be used as a probe or primer or a fragment encoding an active portion (e.g. , a virus binding portion) of a polypeptide of the invention.

[0282] Probes based on the sequence of a nucleic acid of the invention can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of the invention. The probe can comprise a label group, e.g. , a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide

[0283] In another aspect, the present invention provides libraries of nucleic acids that encode for RPP or a variant or derivative thereof, derived from B cells, plasmablasts, and plasma cells. These libraries of nucleic acids are generated by isolating plasmablasts and plasma cells into single-cell reaction containers, wherein they are lysed and antibody-specific nucleic acids are purified or captured, for example on solid supports such as beads. The present invention provides methods for performing capture of transcripts from millions of single cells in parallel. Capture of transcripts is followed by amplification of nucleic acids that encode heavy and light chain immunoglobulins, and subsequent linkage of said nucleic acids into libraries of fused constructs that encode both heavy and light chain immunoglobulins. In such libraries the native pairing of heavy and light chain immunoglobulins, as originally found in the input B cells, plasmablasts, and plasma cells, is maintained. Such methods are performed in parallel on millions of single cells, such that the resulting library of fused heavy and light chain immunoglobulin nucleic acids comprises natively paired sequences for millions of single cells. Such methods are described elsewhere (Adler et al., Mabs 9, 1282-1996, 2017; WO2020 / 223573 which are incorporated by reference in its entirety herein).Vectors and host cells

[0284] The present invention provides vectors, each vector comprising a nucleic acid encoding a polypeptide of the invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors.

[0285] In another aspect of the present invention, expression vectors containing the nucleic acid molecules and polynucleotides of the present invention are also provided, and host cells transformed with such vectors, and methods of producing the polypeptides are also provided. The term “expression vector” refers to a plasmid, phage, virus or vector for expressing a polypeptide from a polynucleotide sequence. Vectors for the expression of the polypeptidescontain at a minimum sequence required for vector propagation and for expression of the cloned insert. An expression vector comprises a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a sequence that encodes polypeptides and proteins to be transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. These sequences may further include a selection 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 which function in specific cells or tissues, and viral vectors for the expression of polypeptides in targeted human or animal cells.

[0286] The recombinant expression vectors of the invention can comprise a nucleic acid 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 on the basis of the host cells to be used for expression, which is 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. SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter), those that direct expression of the nucleotide sequence only in certain host cells (e g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11 :287, Maniatis et al., 1987, Science 236: 1237. incorporated by reference herein in their entireties), and those that direct inducible expression of a nucleotide sequence in response to particular treatment or condition (e.g., the metallothionin promoter in mammalian cells and the tet-responsive and / or streptomycin responsive promoter in both prokaryotic and eukaryotic systems (see id.). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[0287] The invention further provides methods of making polypeptides, e.g., RPP. A variety of other expression / host systems may be utilized. Vector DNA can be introduced into prokaryotic or eukaryotic systems via conventional transformation or transfection techniques. These systems include but are not limited to microorganisms such as bacteria (for example, E. coli) transformed with recombinant bacteriophage, plasmid or cosmid DNA expressionvectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems 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 systems. Mammalian cells useful in recombinant protein production include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient in 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 lines, the 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 embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants. HL-60. U937, HaK or Jurkat cells. Mammalian expression allows for the production of secreted or soluble polypeptides which may be recovered from the growth medium.

[0288] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g, for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Once such cells are transformed with vectors that contain selectable markers as well as the desired expression cassette, the cells can be allowed to grow in an enriched media before they are switched to selective media, for example. The selectable marker is designed to allow' growth and recovery of cells that successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell hne employed. An overview' of expression of recombinant proteins is found in Methods of Enzymology, v. 185, Goeddell, D.V., ed., Academic Press (1990). Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g, cellsthat have incorporated the selectable marker gene will survive, while the other cells die), among other methods.

[0289] The transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification procedures (as defined above).

[0290] In some cases, such as in expression using prokaryotic systems, the expressed polypeptides of this invention may need to be “refolded” and oxidized into a proper tertiary structure and disulfide linkages generated in order to be biologically active. Refolding can be accomplished using a number of procedures well known in the art. Such methods include, for example, exposing the solubilized polypeptide to a pH usually above 7 in the presence of a chaotropic agent. The selection of chaotrope is similar to the choices used for inclusion body solubilization; however, a chaotrope is typically used at a lower concentration. Exemplary7chaotropic agents are guanidine and urea. In most cases, the refolding / oxidation solution will also contain a reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential which allows for disulfide shuffling to occur for the formation of cysteine bridges. Some commonly used redox couples include cysteine / cystamine, glutathione / dithiobisGSH, cupric chloride, dithiothreitol DTT / dithiane DTT, and 2- mercaptoethanol (bME) / dithio-bME. In many instances, a co-solvent may be used to increase the efficiency of the refolding. Commonly used cosolvents include glycerol, polyethylene glycol of various molecular weights, and arginine.

[0291] In addition, the polypeptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d.Ed., Pierce Chemical Co. (1984); Tam et al., J Am Chem Soc, 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).

[0292] The polypeptides and proteins of the present invention can be purified according to protein purification techniques well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the proteinaceous and non-proteinaceous fractions. Having separated the peptide polypeptides from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretictechniques 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, wherein the polypeptide is purified to any degree relative to its naturally -obtainable state. A purified polypeptide therefore also refers to a polypeptide that is free from the environment in which it may naturally occur. Generally, “purified” will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, such as constituting about 50 %, about 60 %, about 70 %, about 80 %, about 85 %, or about 90 % or more of the proteins in the composition.

[0293] Various techniques suitable for use in purification are well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies (immunoprecipitation) and the like or by heat denaturation, followed by centrifugation; chromatography such as affinity chromatography (Protein-A columns), 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, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide. Exemplary purification steps are provided in the Examples below.

[0294] Various methods for quantifying the degree of purification of polypeptide are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific binding activity of an active fraction, or assessing the amount of peptide or polypeptide within a 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, to compare it to the binding activity of the initial extract, and to thus calculate the degree of purification, herein assessed by a “-fold purification number.” The actual units used to represent the amount of binding activity' are, of course, be dependent upon the particular assay technique chosen to follow' the purification and whether or not the polypeptide or peptide exhibits a detectable binding activity.

[0295] In some aspects, the present invention includes libraries of antibody-encoding nucleic acid vectors for site-directed integration into mammalian genomes. Such vectors include plasmids, retroviruses, and lentivirus. These libraries of vectors encode libraries of antibody sequences, which are then be used to engineer mammalian cells for production of RPPs. The libraries of nucleic acid vectors may include 10, 100, 1,000, 10,000, or more than 100,000 different antibody-encoding sequences. The sequences are derived from plasmablasts and plasma cells. These libraries of nucleic acids are generated by isolating plasmablasts and plasma cells into single-cell reaction containers, wherein they are lysed and antibody-specific nucleic acids are purified or captured, for example on solid supports such as beads. The present invention provides methods for performing capture of transcripts from millions of single cells in parallel. Capture of transcripts is followed by amplification of nucleic acids that encode heavy and light chain immunoglobulins, and subsequent linkage of said nucleic acids into libranes of fused constructs that encode both heavy and light chain immunoglobulins. In such libraries the native pairing of heavy and light chain immunoglobulins, as originally found in the input plasmablasts and plasma cells, is maintained. Such methods are performed in parallel on millions of single cells, such that the resulting library of fused heavy and light chain immunoglobulin nucleic acids comprises natively paired sequences for millions of single cells. These paired fused amplicons are then engineered into full-length antibody constructs using Gibson Assembly, restriction endonucleases, or other recombinant DNA techniques.

[0296] Engineering into full-length antibody constructs is performed on the full library en masse, such that the antibody sequence content and antibody sequence counts of the library are essentially maintained throughout the process. In some aspects, the library' of expression vectors is engineered in two steps, such that the scFv amplicon is subcloned into an intermediate vector, and then a second round of Gibson Assembly, restriction digestion, or other recombinant technique is used to engineer additional domains of the antibody into the linker of the scFv. The method is described in US Patent No. 9,422,547, which is incorporated by reference in its entirety herein. The native pairing of heavy and light chain immunoglobulins is essentially maintained throughout the process of engineering into full- length expression vector libraries. The vectors are designed in various orientations, for example, two separate promoters drive expression of heavy and light chain immunoglobulins, or one promoter drives expression of both heavy' and light chain immunoglobulins, and a translational skip motif is used to separately translate the heavy and light chainimmunoglobulins into separate polypeptides. In some embodiments, the expression vectors comprise sequences for site-directed integration into mammalian production cells, for example, CRISPR-Cas9. Flp-In, Cre / Lox, or zinc finger recombination methods. Site- directed integration ensures that each mammalian production cell encodes a single antibody sequence, and decreases variability in expression levels between single production cells.

[0297] In another aspect, the present disclosure provides a host cell or a library of host cells, each comprising a polynucleotide encoding the RPP described herein. In some embodiments, the host cell comprises a vector or comprises a polynucleotide stably integrated into its genome. In some embodiments, the host cell comprises a polynucleotide encoding a RPP stably integrated into the genome using a Flp recombinase recognition target (FRT) landing pad or a similar method known in the art. In some embodiments, the host cell is a mammalian or prolaryotic cell. In some embodiments, the host cell is a human cell or a yeast cell. In some embodiments, the host cell is CHO cell.

[0298] In some embodiments, the host cell comprises an expression vector or integrated polynucleotide for production of RPPs. In some embodiments, a library of host cells can be used produce a RPP described herein. In some embodiments, a library of host cells comprise cells selected based on their production yields or neutralization or binding titers of RPP produced by them.Methods of generating a custom RPP

[0299] In one aspect, the present disclosure provides a method of generating a recombinant polyclonal protein (RPP) specific to a target molecule or complex of target molecules. In some embodiments, the method comprises: (1) obtaining an input antigen binding protein (ABP) library dataset including an ABP profile for each of a plurality of ABPs and (2) generating a filtered ABP library dataset corresponding to a subset of the plurality of ABPs and comprising a reference to each of the subset of the plurality of ABPs. In some embodiments, the filtered ABP library dataset is provided for generation of a composition comprising the ABPs. In some embodiments, the method further comprises the step of (3) generating a composition comprising ABPs corresponding to the ABP references in the filtered ABP library dataset, thereby generating the RPP.

[0300] In some embodiments, the filtered ABP library dataset corresponds to a subset of the plurality of ABPs having at least one of a plurality of characteristic descriptors having a value within a predetermined range for the characteristic. In some embodiments, the filtered ABPlibrary dataset corresponds to a subset of the plurality of ABPs having one or more preferred library properties. In some embodiments, the filtered ABP library dataset corresponds to a subset of the plurality of ABPs having at least one of the pluralities of characteristic descriptors having a value within a predetermined range for the characteristic and having have one or more preferred library properties.

[0301] In some embodiments, the one or more preferred library properties are selected from:(i) the set of heavy chain CDR3 sequences contained in the subset of the plurality of ABPs comprises at least about 10, 20, 50, 100, 200, or 1000 unique sequences;(ii) the subset of the plurality of ABPs specifically bind to at least two unique epitopes associated with the target molecule or complex;(iii) the subset of the plurality of ABPs is capable of modulating at least two target antigen variants;(iv) the set of heavy chain V genes represented in the subset of the plurality of ABPs comprises at least two unique V genes;(v) the set of light chain V genes represented in the subset of the plurality of ABPs comprises at least two unique V genes;(vi) the set of heavy chain J genes represented in the subset of the plurality of ABPs comprises at least two unique J genes;(vii) the set of light chain J genes represented in the subset of the plurality of ABPs comprises at least two unique J genes;(viii) the average percent germline identity of heavy chain V genes represented in the subset of the plurality of ABPs is between about 50% and about 100%;(ix) the average percent germline identity of light chain V genes represented in the subset of the plurality of ABPs is between about 50% and about 100%;(x) the average percent germline identity of heavy chain J genes represented in the subset of the plurality of ABPs is between about 50% and about 100%; and(xi) the average percent germline identity of light chain J genes represented in the subset of the plurality of ABPs is between about 50% and about 100%.

[0302] In some embodiments, the at least one of a plurality of characteristic descriptors are selected from:(i) a binding affinity of the respective ABP for the respective target antigen;(ii) an effector activity of the respective ABP against the target molecule or complex;(iii) a solubility score of the respective ABP;(iv) an aggregation score of the respective ABP;(v) a hydrophobicity score of the respective ABP;(vi) an isoelectric point of the respective ABP;(vii) a stability7score of the respective ABP;(viii) a molecular weight of the respective ABP;(ix) a number of unpaired cysteine residues in the respective ABP;(x) an abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen;(xi) a fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, as compared to the abundance frequency prior to enrichment;(xii) a partial or full sequence of the respective ABP;(xiii) a number of non-canonical glycosylation sites in the respective ABP;(xiv) a number of cleavage sites in the respective ABP;(xv) a number of deamidation sites in the respective ABP;(xvi) a number of isomerization sites in the respective ABP;(xvii) a number of oxidation sites in the respective ABP;(xviii) CDR3H length of the respective ABP; and (xix) binding specificity of the respective ABP.

[0303] In some embodiments, the plurality7of characteristic descriptors for ABPs are obtained experimentally, by in silico method, or from other sources. Any method known in the art for obtaining the relevant information can be adopted in various embodiments.

[0304] In some embodiments, the plurality7of characteristic descriptors comprises the binding affinity of the respective ABP for the respective target antigen. In some embodiments, the binding affinity7is expressed in KD, and the predetermined range for the binding affinity is less than about 10 pM, 1 pM, 100 nM, 10 nM, 1 nM, or lower, optionally wherein the binding affinity7is determined by surface plasmon resonance (SPR) or biolayer interferometry (BLI). In some embodiments, the ABP has a KD less than about 100 pM (such as less than about any of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM. 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM. 8 pM, 7 pM. 6 pM, 5 pM. 4 pM, 3 pM. 2 pM, 1 pM. 900 nM, 800 nM, 700 nM. 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 1 nM, 900 pM, 800 pM, 700pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 1 pM, or lower). In some embodiments, the ABP has a KD from about 100 pM to about 1 pM (such as about any of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM. 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM. 5 pM, 4 pM. 3 pM, 2 pM. 1 pM, 900 nM, 800 nM. 700 nM, 600 nM, 500 nM. 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or 1 pM, including any ranges between any of these values). In some embodiments, the binding affinity is determined by a PolyMap assay comprising the steps of: providing a library of target-decorated cells, wherein each of the target-decorated cells presents the target molecule or complex on the membrane; contacting the library of target-decorated cells with a pl urality of ABP-ribosome- mRNA (ARM) complexes corresponding to the one or more of the pluralities of ABPs, thereby inducing binding between the target-decorated cells and the ARM complexes; generating a plurality of monodisperse or poly disperse emulsion microdroplets, wherein each microdroplet contains a single cell out of the target-decorated cells, one or more ARM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; capturing RNA released from the single cell on a solid surface or within a semi- permeable shell; generating a library of hybrid polynucleic acids that comprise a sequence from a transcript of the single cell and / or a sequence from the mRNA of the ARM complex; sequencing the library of hybrid polynucleic acids; and determining a presence or absence of binding of each of the one or more of the pluralities of ABPs to their respective target antigen.

[0305] In some embodiments, the predetermined range for the binding affinity is a presence of binding of the respective ABP to their respective target antigen in the PolyMap assay.

[0306] In some embodiments, the plurality of characteristic descriptors comprises the effector activity of the respective ABP against the target molecule or target molecule complex. In some embodiments, the target molecule or target molecule complex comprises a virus, and the effector activity is a neutralization activity determined by a pseudovirusneutralization assay or a live virus neutralization assay. In some embodiments, the neutralization activity7is determined by a pseudovirus neutralization assay and corresponds to an IC50 from about 1 ng / mL to about 500 mg / mL (such as about any of 1 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL. 50 ng / mL, 60 ng / mL, 70 ng / mL. 80 ng / mL. 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1 pg / mL, 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / rnL, 50 pg / mL, 60 pg / mL, 70 pg / mL. 80 pg / mL, 90 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, 1 mg / mL. 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, or 500 mg / mL, including any ranges between any of these values). In some embodiments, the neutralization activity is determined by a live virus neutralization assay and corresponds to an IC50 from about 1 ng / mL to about 500 mg / mL (such as about any of 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1 pg / mL, 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL. 80 pg / mL. 90 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, 1 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL. 200 mg / mL, 300 mg / mL, 400 mg / mL, or 500 mg / mL, including any ranges between any of these values). In some embodiments, the predetermined range for the target molecule or target molecule complex neutralization activity7corresponds to (a) an IC50 from about 0.08 pg / mL to about 900 pg / mL when determined by7pseudovirus neutralization assay or (b) an IC50 from about 0.003 pg / mL to about 1350 pg / mL when determined by live virus neutralization assay.

[0307] In some embodiments, the target molecule or target molecule complex comprises a bacterium, and the effector activity7is a bactericidal activity determined by a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPKA). In some embodiments, the target molecule or target molecule complex comprises baculoviral particles. In some embodiments, the bactericidal activity corresponds to a concentration where 50% bactericidal activity7is observed from about 1 ng / ml to about 500 mg / ml (such as about any of 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL. 9 ng / mL. 10 ng / mL, 20 ng / mL, 30 ng / mL. 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL. 80ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1 pg / mL, 10 pg / mL. 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 gg / mL, 60 gg / mL, 70 gg / mL, 80 gg / mL. 90 gg / mL, 100 gg / mL, 200 gg / mL, 300 gg / mL, 400 gg / mL, 500 gg / mL. 600 gg / mL, 700 gg / mL, 800 gg / mL. 900 gg / mL, 1 mg / mL. 10 mg / rnL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, or 500 mg / mL, including any ranges between any of these values). In some embodiments, the predetermined range for the bactericidal activity corresponds to a concentration where 50% bactericidal activity is observed from about 0.08 gg / ml to about 3600 gg / ml.

[0308] In some embodiments, the plurality of characteristic descriptors comprises the solubility score, optionally wherein the solubility score is determined using SKADE. In some embodiments, the solubility score is greater than about 0.5 (such as greater than about any of 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78. 0.79, 0.8, 0.81, 0.82. 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96. 0.97, 0.98, or 0.99). In some embodiments, the solubility score is from about 0.5 to about 0.8 (such as about any of 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, or 0.8, including any ranges between any of these values). In some embodiments, the predetermined range for the solubility score is greater than about 0.5, or between 0.5 and 0.8.

[0309] In some embodiments, the plurality7of characteristic descriptors comprises the aggregation score, optionally wherein the aggregation score corresponds to the number of residues predicted to have a propensity to aggregate and is determined by a method comprising the steps of: determining a 3D structure of the ABP, optionally wherein the 3D structure is determined using ABodyBuilder2; and determining the aggregation score based on the 3D structure, optionally wherein the aggregation score is determined using Aggrescan3D. In some embodiments, the aggregation score is fewer than 50 (such as fewer than any of 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) aggregation-prone sites. In some embodiments, the predetermined range for the aggregation score is fewer than 20, 15, or lower aggregation-prone sites.

[0310] In some embodiments, the plurality of characteristic descriptors comprises the hydrophobicity score, optionally wherein the hydrophobic score is determined as the grand average of hydropathy (GRAVY), optionally wherein the hydropathy value of each amino acid is calculated using the Eisenberg scale. In some embodiments, the hydrophobicity score is less than about 0.1 (such as less than about any of 0.1, 0.095, 0.09, 0.085, 0.08, 0.075, 0.07, 0.065, 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, 0.01, 0.005, or lower).In some embodiments, the predetermined range for the hydrophobicity score is less than 0.03, less than 0.02, or less than 0.015.

[0311] In some embodiments, the plurality7of characteristic descriptors comprises the isoelectric point, optionally wherein the isoelectric point is determined as EMBOSS pK values. In some embodiments, the isoelectric point is between about 6.5 and about 9.5 (such as about any of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5, including any ranges between any of these values). In some embodiments, the predetermined range for the isoelectric point is between about 7.0 and about 9.0 or between about 8.0 and about 8.5.

[0312] In some embodiments, the plurality of characteristic descriptors comprises the stability' score, optionally wherein the stability score is determined by a method comprising the steps of calculating an aliphatic index by determining the relative volume of A, V. L, and I residues, wherein the stability score corresponds to the aliphatic index. In some embodiments, the stability score is from about 60 to about 80 (such as about any of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80, including any ranges between any of these values). In some embodiments, the predetermined range for the stability score is from about 65 to about 73.

[0313] In some embodiments, the plurality of characteristic descriptors comprises the molecular yveight and the predetermined range for the molecular weight is less than about 250 kDa (such as less than about any of 250 kDa, 245 kDa, 240 kDa, 235 kDa, 230 kDa, 225 kDa, 220 kDa, 215 kDa, 210 kDa, 205 kDa, 200 kDa, 195 kDa, 190 kDa, 185 kDa, 180 kDa, 175 kDa, 170 kDa, 165 kDa, 160 kDa, 155 kDa, 150 kDa, 145 kDa, 140 kDa, 135 kDa, 130 kDa, 125 kDa, 120 kDa, 115 kDa, 110 kDa, 105 kDa, 100 kDa, or less). In some embodiments, the predetermined range for the molecular weight is less than about 170 kDa, less than about 160 kDa, less than about 150 kDa, less than about 140 kDa, less than about 130 kDa, less than about 120 kDa, or lower.

[0314] In some embodiments, the plurality of characteristic descriptors comprises a number of unpaired cysteine residues and the predetermined range for the number of unpaired cysteine residues is less than 5, 4, 3, 2, or 1.

[0315] In some embodiments, the plurality of characteristic descriptors comprises an abundance frequency or fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, optionally wherein the sorting process is fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) sorting, further optionally wherein the sorting is carried by yeast display.

[0316] In some embodiments, the predetermined range for the post-sort abundance frequency is greater than about 0.01% (such as greater than about any of 0.010%, 0.012%, 0.013%, 0.014%, 0.015%, 0.016%, 0.017%, 0.018%, 0.019%, 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.030%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%. 0.039%, 0.040%, 0.045%.0.026%, 0.047%, 0.048%, 0.049%, 0.050%, 0.055%, 0.026%, 0.057%, 0.058%, 0.059%, 0.060%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.070%, 0.075%, 0.076%, 0.077%, 0.078%, 0.079%, 0.080%, 0.085%, 0.086%, 0.087%, 0.088%, 0.089%, 0.090%, 0.095%, 0.096%, 0.097%, 0.098%, 0.099%, 0.100%, 1%, 2%, 5%, 10%, 15%, 16%, 17%, 18%, 19%, 20%. 25%. 30%. 35%. 40%. 45%. 50%. 55%. or 60%, or greater) within a pool of ABPs obtained after the sorting process. In some embodiments, the predetermined range for the post-sort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 1%, 2%, 5%, 10%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% or greater within a pool of ABPs obtained after the sorting process. In some embodiments, the predetermined range for the post-sort fold-change in abundance frequency is greater than about any of 0.5 (such as greater than about any of 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, or greater). In some embodiments, the predetermined range for the post-sort fold-change is greater than about any of 1, 1.5, 2, 2.5, 3, 4, or greater. In some embodiments, the enrichment is performed by FACS or MACS.

[0317] In some embodiments, the plurality of characteristic descriptors comprises the number of non-canonical glycosylation sites, optionally wherein the predetermined range for the number of non-canonical glycosylation sites is less than 5, 4, 3, 2, or 1.

[0318] In some embodiments, the plurality of characteristic descriptors comprises the number of cleavage sites, optionally wherein the predetermined range for the number of cleavage sites is less than 5, 4. 3, 2, or 1, further optionally wherein the cleavage site is a DP motif in the variable heavy or variable light chain region of the respective ABP.

[0319] In some embodiments, the plurality' of characteristic descriptors comprises the number of deamidation sites, optionally wherein the predetermined range for the number of deamidation sites is less than 5, 4. 3. 2, or 1. further optionally wherein the deamidation site is an NG, NS, or NA motif in CDR2H or CDR1L of the respective ABP.

[0320] In some embodiments, the plurality of characteristic descriptors comprises the number of isomerization sites, optionally wherein the predetermined range for the number of isomerization sites is less than 5, 4, 3, 2, or 1, further optionally wherein the isomerization site is a DG or DS motif in CDR2H, CDR3H, or CDR1L of the respective ABP.

[0321] In some embodiments, the plurality of characteristic descriptors comprises the number of oxidation sites, optionally wherein the predetermined range for the number of oxidation sites is less than 5, 4, 3, 2, or 1, further optionally wherein the oxidation site is a W or M residue in the CDRHs or CDRLs of the respective ABP.

[0322] In some embodiments, the plurality of characteristic descriptors comprises the CDR3H length, optionally wherein the predetermined range for the CDR3H length is from about 10 to about 14 amino acids.

[0323] In some embodiments, the plurality of characteristic descriptors comprises the binding specificity, optionally wherein the binding specificity corresponds to the number of variants of a target antigen capable of being targeted by the respective ABP and the predetermined range for the binding specificity is capability of binding to at least 2 (such as at least any of 3, 4, 5, 6, 7, 8, 9, 10, or more) variants of the target antigen, further optionally wherein the binding specificity is determined by a Poly Map assay.

[0324] In some embodiments, the plurality of characteristic descriptors comprises the binding specificity, optionally wherein the binding specificity corresponds to the number of variants of a target antigen capable of being targeted by the respective ABP and the predetermined range for the binding specificity is capability of binding to at least 2 (such as at least any of 3, 4, 5, 6, 7. 8, 9, 10, or more) variants of the target antigen, further optionally wherein the binding specificity is determined by a Poly Map assay and / or a polyreactivity assay. In some embodiments, the polyreactivity assay is a binding assay. In some embodiments, thepolyreactivity assay is a FACS-based assay. In some embodiments, the polyreactivity assay is a plate-based assay with purified ABPs. Non-limiting examples of binding assays to determine polyreactivity or polyspecificity can be found in Jain et al. (Biophysical properties of the clinical-stage antibody landscape. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):944- 949); Makowski et al. (Highly sensitive detection of antibody nonspecific interactions using flow cytometry. mAbs, 2021 13( 1 )); Kelly et al. (Reduction of nonspecificity motifs in synthetic antibody libraries, Journal of Molecular Biology; Volume 430, Issue 1, 5 January' 2018, Pages 119-130); and Chen et al. (Human antibody polyreactivity is governed primarily by the heavy-chain complementarity-determining regions. Cell Reports. Volume 43, Issue 10, 22 October 2024, 114801), each of which is incorporated by reference in its entirety.

[0325] In some embodiments, the polyreactivity’ assay allows for isolating antibodies with high and low poly reactivity. In some embodiments, the plurality of characteristic descriptors comprises, at least in part, ABP features that result in polyreactivity. In some embodiments, the plurality' of characteristic descriptors comprises, at least in part, an oligoclonality assay.

[0326] In some embodiments, the method of the present disclosure comprises generating a dataset for a filtered ABP library' comprising selected ABPs, wherein the dataset comprises a reference to each of the selected ABPs and: (a) the filtered ABP library comprises at least 10 selected ABPs which is a subset of the at least 100 candidate ABPs; and (b) each selected ABP has at least one of the plurality of characteristic descriptors meets a preferred criteria. In some embodiments, the method further comprises providing the dataset for the filtered ABP library' for generation of a composition comprising selected ABPs, thereby generating the RPP. In some embodiments, the method further comprises generating a composition comprising selected ABPs using the dataset.

[0327] In some embodiments, the preferred criteria is a binding affinity to the respective target antigen is ranked at least top 25% among all the candidate ABPs in the input ABP library. In some embodiments, the preferred criteria is a binding affinity to the respective target antigen is ranked at least top 10%, at least top 20%, at least top 30%, top 35%, top 40%, top 45%, or top 50% among all the candidate ABPs in the input ABP library.

[0328] In some embodiments, the preferred criteria is an effector activity against the target molecule or complex is ranked at least top 25% among all the candidate ABPs in the input ABP library. In some embodiments, the preferred criteria is an effector activity' against thetarget molecule or complex is ranked at least top 10%, top 20%, top 30%, top 35%, top 40%, top 45%, or top 50% among all the candidate ABPs in the input ABP library7.

[0329] In some embodiments, the preferred criteria is a binding pattern for the respective target antigen and its variants, where the binding pattern is shared with less than 30% of other candidate ABPs in the input ABP library7. In some embodiments, the binding pattern corresponds to a subset of the respective target antigen and its variants capable of binding to the respective ABP. In some embodiments, the preferred criteria is a binding pattern for the respective target antigen and its variants, where the binding pattern is shared with less than 20% of other candidate ABPs in the input ABP library. In some embodiments, the preferred criteria is a binding pattern for the respective target antigen and its variants, where the binding pattern is shared with less than 40%, less than 30%, less than 20%. less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of other candidate ABPs in the input ABP library. In some embodiments, the preferred criteria is that the binding pattern of the respective ABP is shared with at least 5. at least 6, at least 7, at least 8, at least 9. or at least 10 other candidate ABPs in the input ABP library. In some embodiments, the preferred criterion is that the binding pattern of the respective ABP is shared with less than 10, less than 9, less than 8, less than 7, less than 5, less than 4, less than 3, or less than 2 other candidate ABPs in the input ABP library7.

[0330] In some embodiments, the preferred criteria is an abundance frequency of the ABP following sorting the input ABP library7or a subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, the preferred criteria is an abundance frequency following sorting the input ABP library or a subset thereof is ranked at least top 15%, at least top 20%, at least top 25%, at least top 30%, at least top 35%, at least top 40%, or at least top 50% among all the candidate ABPs in the input ABP library7or the subset thereof.

[0331] In some embodiments, the preferred is a fold-change of the increase in the abundance frequency following sorting the input ABP or the subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof. In some embodiments, the preferred is a fold-change of the increase in the abundance frequency following sorting the input ABP or the subset thereof is ranked at least top 15%, at least top 20%, at least top 25%, at least top 30%, at least top 35%, at least top 40%, or at least top 50%among all the candidate ABPs in the input ABP library or the subset thereof. Fold change can be defined as the ratio of the ABPs post-enrichment frequency to its pre-enrichment frequency.Experimental method

[0332] Various experimental method known in the art can be adopted and used for determination and prediction of characteristics of ABPs. For example, binding affinity of an ABP can be determined by surface plasmon resonance (SPR) or biolayer interferometry (BLI), but other method can be also used.PolyMap assay

[0333] In some embodiments, Poly Map assay is used to determine binding of an ABP to a target antigen. Poly Map assay is the method described in PCT / US2024 / 012238 filed Jan 19, 2024 which is incorporated by reference in its entirety.

[0334] In short, PolyMap assay is a method for high-throughput analysis of antibodies. Specifically, the method can comprise the steps of (i) providing a library of target-decorated cells, wherein each of the target-decorated cells presents a target of interest on the membrane; (ii) contacting the library of target-decorated cells with a plurality of TBP-ribosome-mRNA (TRM) complexes, thereby inducing binding between the target-decorated cells and the TRM complexes: (iii) generating a plurality of emulsion microdroplets, wherein each microdroplet contains a single cell out of the target-decorated cells, one or more TRM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; (iv) capturing RNA released from the single cell on a solid surface or within a semi-permeable shell; and (v) generating a library' of hybrid polynucleic acids that comprise a sequence from a transcript of the single cell and / or a sequence from the mRNA of the TRM complex.

[0335] The library' of hybrid polynucleic acids can be analyzed or sequenced to provide information related to binding between the target of interest and the TRM complex. For example, based on the sequence, a TBP that binds to the target of interest and / or their binding affinity or specificity can be studied. Accordingly, the method can further comprise the step of identifying a target-TBP pair based on the sequencing of the library of hybrid polynucleic acids. In some embodiments, a plurality of target-TBP pairs are identified by sequencing the library of hybrid polynucleic acids. In some embodiments, more than two, three, four, five, six, seven, eight, nine, ten, twenty or more pairs of target-TBP pairs are identified. In some embodiments, the method further comprises the step of identifying a target binding proteinspecific to the target of interest. In some embodiments, the method further comprises the step of identifying binding affinity or specificity of a target binding protein specific to the target of interest.

[0336] In some embodiments, the PolyMap assay comprises the steps of: providing a library of target-decorated cells, wherein each of the target-decorated cells presents the target molecule or complex on the membrane; contacting the library of target-decorated cells with a plurality of ABP-ribosome-mRNA (ARM) complexes corresponding to the one or more of the plurality of ABPs, thereby inducing binding between the target-decorated cells and the ARM complexes; generating a plurality of monodisperse or poly disperse emulsion microdroplets, wherein each microdroplet contains a single cell out of the target-decorated cells, one or more ARM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; capturing RNA released from the single cell on a solid surface or within a semi-permeable shell; generating a library of hybrid polynucleic acids that comprise a sequence from a transcript of the single cell and / or a sequence from the mRNA of the ARM complex; sequencing the library of hybrid polynucleic acids; and determining a presence or absence of binding of each of the one or more of the plurality of ABPs to their respective target antigen.In silico method

[0337] In some embodiments, at least one characteristic descriptor of the plurality of characteristic descriptors is determined using an in silico method. In some embodiments, the in silico method obtains the respective characteristic based on the sequence of the respective ABP, or a nucleic acid encoding the ABP, as an input. In some embodiments, the in silico method obtains the respective characteristic based on the protein structure prediction, paratope-epitope prediction and antibody-antigen docking.

[0338] In some embodiments, one or more in silico methods are used to determine the solubility score, aggregation score, hydrophobicity score, isoelectric point, stability score, number of cysteine residues, number of glycosylation sites, number of cleavage sites, number of deamidation sites, number of isomerization sites, and / or number of oxidation sites of one or more of the ABPs referenced in the input ABP library dataset.

[0339] In some embodiments, the in silico methods are used to determine binding affinity, effector activity (e.g., neutralization activity or killing activity), solubility, aggregation, hydrophobicity, isoelectric point, stability, molecular weight, number of cysteine residues.abundance frequency and fold-change in abundance following sorting of a library of ABPs, number of glycosylation sites, number of cleavage sites, number of deamidation sites, number of isomerization sites, number of oxidation sites, CDR3H length, binding specificity, and full or partial amino acid sequence. In some embodiments, an in silico method is used for epitope mapping, affinity maturation, and humanization while ensuring compatibility for therapeutic use. In some embodiments, the in silico method is used for assessment of antibody developability', including aggregation, solubility, viscosity, and excipient formulation. In some embodiments, an in silico method is used for binding predicting, neutralization prediction, ACE2 competition prediction and / or epitope prediction.

[0340] In some embodiments, an in silico method is used for sequence analysis of a group of ABPs, to determine V and J gene usage and / or V and J gene sequence percent identity to germline sequences. In some embodiments, an in silico method is used for functional analysis of a group of ABPs e.g., binding profile and / or effector activity (e.g., neutralization activity or killing activity ) profile of the group of ABPs.

[0341] In some embodiments, the in silico method is used to determine one, two, three, four, five, six, seven or more of the characteristic descriptors.

[0342] In some embodiments, the functional characteristics are experimentally generated. In some embodiments, the functional characteristics are computationally calculated. In some embodiments, the functional characteristics are obtained from a database.

[0343] In some embodiments, each antibody sequence is paired with its known functional properties, such as binding affinity, effector activity (e.g., neutralization activity or killing activity ), solubility7, aggregation, hydrophobicity, isoelectric point, stability7, abundance frequency and fold-change in abundance following sorting of a library7of ABPs and / or binding specificity of the antibodies. In some embodiments, various preprocessing steps are employed, such as sequence alignment, normalization, and removal of redundant or erroneous data.

[0344] In some embodiments, the model is trained to predict binding affinity, effector activity (e.g., neutralization activity or killing activity), solubility, aggregation, hydrophobicity7, isoelectric point, stability, abundance frequency and fold-change in abundance following sorting of a library7of ABPs and / or binding specificity7of the antibodies. In some embodiments, the model predicts interaction with an antibody with other antibodiesin a library. In some embodiments, the model predicts suitability of an antibody for a polyclonal antibody.

[0345] In some embodiments, the model is trained using a dataset divided into training, validation, and test subsets. In some embodiments, the model is cross validated to fine tune hyperparameters or to select the best-performing model configuration. In some embodiments, data augmentation strategies, such as random perturbations and synthetic sequence generation, are implemented to increase the diversity of the training data. In some embodiments, the performance of the model is assessed using various metrics, including accuracy, precision, recall, and the area under the receiver operating characteristic (ROC) curve.

[0346] In some embodiments, the model generates an antibody sequence or variation thereof that has preferred functional characteristics and suitable for generation of a polyclonal antibody.Methods of producing an RPP

[0347] One aspect of the present disclosures relates to production of an RPP comprising ABPs disclosed herein. In some embodiments, an RPP is generated by using the sequences of ABPs selected by the method disclosed herein. For example, the RPP can be generated from host cells, each comprising a polynucleotide encoding an ABP. In some embodiments, an RPP is generated by using dataset for the filtered ABP library comprising selected ABPs, wherein each selected ABP has at least one of the pluralities of characteristic descriptors meets a preferred criteria described herein.

[0348] An ABP can be generated and purified from host cells that comprise nucleic acid encoding the ABP using conventional methods known in the art.

[0349] ABPs can be prepared, and screened for desired properties, by any of conventional techniques. Certain techniques involve isolating a nucleic acid encoding a polypeptide chain (or portion thereof) of an ABP of interest, and manipulating the nucleic acid through recombinant DNA technology. The nucleic acid may be fused to another nucleic acid of interest, or altered (e.g, by mutagenesis or other conventional techniques) to add, delete, or substitute one or more amino acid residues, for example. Furthermore, the antigen binding proteins may be purified from cells that naturally express them (<?.g, an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems, using any technique known in the art. See. for example, Monoclonal Antibodies, Hybridomas:A New Dimension in Biological Analyses, Kennet el 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).

[0350] Once synthesized, the DNA encoding an ABP can be propagated and expressed according to any of a variety of w ell-know n procedures for nucleic acid excision, ligation, transformation, and transfection using any number of known expression vectors. Thus, in certain embodiments expression of an antibody fragment may be preferred in a prokaryotic host, such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol.178:497-515). In certain other embodiments, expression of the antibody or a fragment thereof may be preferred in a eukaryotic host cell, 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 (such as a mouse NSO line), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, com, soybean, and rice cells.

[0351] Replicable expression vectors containing DNA encoding an antibody variable and / or constant region may be prepared and used to transform an appropriate cell line, for example, anon-producing myeloma cell line, such as a mouse NSO line or bacteria, such as E. coli, in which production of the antibody will occur. To obtain efficient transcription and translation, the DNA sequence in each vector should include appropriate regulatory sequences, particularly a promoter and leader sequence operatively linked to the variable domain sequence. Particular methods for producing antibodies in this way are generally well-known and routinely used. For example, basic molecular biology procedures are described by Maniatis et al. (Molecular Cloning, A Laboratory Manual, 2nd ed.. Cold Spring Harbor Laboratory, New' York, 1989; see also Maniatis et al, 3rd ed., Cold Spring Harbor Laboratory7, New' York, (2001)). DNA sequencing can be performed as described in Sanger et al. (PNAS 74:5463, (1977)) and the Amersham International pic sequencing handbook, and site directed mutagenesis 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). Additionally, numerous publications describe techniques suitable for the preparation of antibodies by manipulation of DNA, creation of expression vectors, and 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).

[0352] Any expression system known in the art can be used to make the recombinant polypeptides of the invention. In general, host cells are transformed with a recombinant expression vector that comprises DNA encoding a desired polypeptide. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram-positive organisms, for example E. coli or Bacilli. 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 lines, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. {Cloning Vectors: A Laboratory Manual. Elsevier, New Y ork, 1985).

[0353] Production cell lines for monoclonal antibodies (mAbs) are ty pically produced by randomly inserting expression constructs into a mammalian production cell genome, for example, a CHO genome (Rita Costa et al., 2010). However, this canonical method produces cell lines with multiple copies of mAb inserted into the CHO genome. If the polyclonal antibody construct libraries were randomly inserted into the CHO genome, many clones would express multiple antibodies, which would result in frequent non-native pairing between heavy and light chain Ig. Additionally, different genome locations have different transcriptional activity levels (Kito et al., 2002), which could result in heterogeneous, inconsistent and / or unstable bioproduction. Thus, in some aspects the current invention provides a CHO cell line with a Flp recombinase recognition target (FRT) landing pad stably engineered into the genome. Such site-directed genome integration cell lines are then used for stable expression of RPP.

[0354] It will be understood by one skilled in the art that some proteins, such as antibodies, may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the protein as well as the culture conditions. Such modifications may include variations in glycosylation, methionineoxidation, diketopiperizine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy -terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, R.J. Journal of Chromatography 705: 129- 134, 1995 ).Pharmaceutical compositions

[0355] Pharmaceutical compositions containing one or more of the RPPs of the present disclosure are also provided. Such compositions comprise a therapeutically or prophylactically effective amount of the polypeptide or protein in a mixture with pharmaceutically acceptable materials.

[0356] In some embodiments, the pharmaceutical composition comprises about 10, 100, 500, 1,000, 5,000, 10,000, 50,000 or more than 100,000 distinct ABPs, each having a unique sequence. In some embodiments, the pharmaceutical composition comprises at least 10, 100, 500, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000 or more than 100,000 distinct ABPs, each having a unique sequence.

[0357] The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.

[0358] Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspendingagents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, ty loxapal); stability’ enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. Neutral buffered saline or saline mixed with conspecific serum albumin are examples of appropriate diluents. In accordance with appropriate industry standards, preservatives such as benzyl alcohol may also be added. The composition may be formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Suitable components are nontoxic to recipients at the dosages and concentrations employed. Further examples of components that may be employed in pharmaceutical formulations are presented in Remington’s Pharmaceutical Sciences, 16thEd. (1980) and 20thEd. (2000), Mack Publishing Company. Easton, PA.

[0359] Optionally, the composition additionally comprises one or more physiologically active agents, for example, an anti-viral agent, plasma IVIg, etc. In various embodiments, the composition comprises one, two, three, four, five, or six physiologically active agents in addition to an RPP.

[0360] In another embodiment of the invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically -acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

[0361] The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also beincorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.

[0362] The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery' format, and desired dosage. See for example, Remington’s Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the polypeptide. For example, suitable compositions may be water for injection, physiological saline solution for parenteral administration.Content of pharmaceutically active ingredients

[0363] In typical embodiments, the active ingredient (i.e., the proteins and polypeptides (e.g., RPP) of the present invention) is present in the pharmaceutical composition at a concentration of at least O.Olmg / ml. at least O. lmg / ml, at least 0.5mg / ml. or at least Img / 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. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 100 mg / ml, 250 mg / ml, 500 mg / ml, 750 mg / ml, Ig / ml, 5g / ml, lOg / ml, or 50 g / ml.Formulation Generally

[0364] The pharmaceutical composition can be in any form appropriate for human or veterinary medicine, including a liquid, an oil, an emulsion, a gel, a colloid, an aerosol or a solid.

[0365] The pharmaceutical composition can be formulated for administration by any route of administration appropriate for human or veterinary medicine, including enteral and parenteral routes of administration.

[0366] In some embodiments, the pharmaceutical composition is formulated for intravenous, intraperitoneal, intramuscular, or subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection or intravenous infusion.

[0367] In some embodiments, the pharmaceutical composition is formulated for intrathecal or intracerebroventricular administration.

[0368] In some embodiments, the pharmaceutical composition is formulated for topical administration.Pharmacological compositions adapted for injection

[0369] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stabi 1 ity. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives can be included, as required.

[0370] In various embodiments, the unit dosage form is a vial, ampule, bottle, or pre-filled syringe. In some embodiments, the unit dosage form contains 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 of the pharmaceutical composition. In some embodiments, the unit dosage form contains 125 mg, 150 mg, 175 mg, or 200 mg of the pharmaceutical composition. In some embodiments, the unit dosage form contains at least 250 mg, 1g, 10g, 20g, 30g, 40g, 50g, 60g, 70g, 80g, 90g, or 100g of the pharmaceutical composition. In some embodiments, the unit dosage form contains about 250 mg, 1g, 10g, 20g, 30g, 40g, 50g, 60g, 70g, 80g, 90g, or 100g of the pharmaceutical composition.

[0371] In typical embodiments, the pharmaceutical composition in the unit dosage form is in liquid form. In various embodiments, the unit dosage form contains between 0. 1 mL and 50 ml of the pharmaceutical composition. In some embodiments, the unit dosage form contains 1 ml, 2.5 ml, 5 ml, 7.5 ml, 10 ml, 25 ml, or 50 ml of pharmaceutical composition.

[0372] In particular embodiments, the unit dosage form is a vial containing 1 ml of the pharmaceutical composition containing an active ingredient (e.g., RPP) at a concentration of 0.01 mg / ml, 0. 1 mg / ml, 0.5 mg / ml, or Img / ml. In particular embodiments, the unit dosage form is a vial containing 1 ml of the pharmaceutical composition containing an active ingredient (e.g., RPP) at a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, Img / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml or 50 mg / ml, 100 mg / ml, 250 mg / ml, 500 mg / ml, 750 mg / ml, Ig / ml, 5g / ml, lOg / ml, or 50 g / ml. In some embodiments, the unit dosage form is a vial containing 2 ml of the pharmaceutical composition containing an active ingredient at aconcentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, Img / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml or 50 mg / ml, 100 mg / ml, 250 mg / ml, 500 mg / ml, 750 mg / ml. Ig / ml, 5g / ml, lOg / ml, or 50 g / ml.

[0373] In some embodiments, the pharmaceutical composition is formulated for injection of an active ingredient at a single dose or multiple doses of between 0.010 and 5 g / kg body weight. In some embodiments, the pharmaceutical composition is formulated for injection of an active ingredient at a single dose of 0.010 g / kg body weight. In some embodiments, the pharmaceutical composition is formulated for injection at a single dose of 0.01, 0.05, 0. 15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60. 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 0. 1 g / kg body weight.

[0374] In some embodiments, the unit dose contains at least 0.1g, 0.5g, 1g, 1.5g, 2g, 2.5g, 3g, 3.5g, 4g, 5g, 10g, 20g, 30g, 40g, or 50g of the active ingredient (e.g., RPP). In some embodiments, the unit dose contains about 0. 1g, 0.5g. 1g. 1.5g. 2g, 2.5g, 3g, 3.5g, 4g, 5g, 10g, 20g, 30g, 40g, or 50g of the active ingredient (e.g., RPP).

[0375] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

[0376] The pharmaceutical compositions may conveniently be presented in unit dosage form.

[0377] The unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition.

[0378] In some embodiments, the pharmaceutical composition in the unit dosage form is in solid form, such as a lyophilate, suitable for solubilization.

[0379] In some embodiments, the unit dosage form is suitable for subcutaneous, intradermal, or intramuscular administration include preloaded syringes, auto-injectors, and autoinject pens, each containing a predetermined amount of the pharmaceutical composition described hereinabove.

[0380] In various embodiments, the unit dosage form is a preloaded syringe, compnsing a syringe and a predetermined amount of the pharmaceutical composition. In certain preloaded syringe embodiments, the syringe is adapted for subcutaneous administration. In certain embodiments, the syringe is suitable for self-administration. In particular embodiments, the preloaded syringe is a single use syringe.

[0381] In certain embodiments, the unit dosage form is an autoinject pen. The autoinject pen comprises an autoinject pen containing a pharmaceutical composition as described herein. In some embodiments, the autoinject pen delivers a predetermined volume of pharmaceutical composition. In other embodiments, the autoinject pen is configured to deliver a volume of pharmaceutical composition set by the user.Mixtures of plasma IVIg with recombinant hyperimmunes

[0382] In some embodiments, a recombinant hyperimmune is spiked into conventional plasma IVIg to increase the anti-pathogen titer of IVIg. In some embodiments, several antipathogen recombinant hyperimmunes are spiked into conventional plasma IVIg. Any number of spike-ins can be mixed with plasma IVIg to generate increased titers against any number of pathogens.

[0383] In some embodiments, the spike-in recombinant hyperimmunes are mixed with plasma IVIg by the pharmacist. In some embodiments, the spike-in recombinant hyperimmunes are mixed with plasma IVIg by the manufacturer.Methods of treating a disease responsive to an RPP

[0384] In another aspect, methods are presented for treating a subject having a disease responsive to an RPP. In some embodiments, the disease is associated with a target molecule or complex of target molecules of the ABPs.

[0385] The disease can be a viral infection. In some embodiments, the method comprises administering the pharmaceutical composition, the RPP disclosed herein to a patient infected with coronavirus or having a symptom of COVID-19. The disease can be a bacterial infection.

[0386] In vivo and / or in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0387] The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery', the method of administration andother factors know n to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0388] In some embodiments, the pharmaceutical composition is administered by injection, infusion, or by topical application. In some embodiments, the pharmaceutical composition is administered by intravenous infusion.

[0389] In some embodiments, the subject is treated with the pharmaceutical composition in combination with another therapeutic agent. In some embodiments, the pharmaceutical composition and another therapeutic agent is administered concurrently or individually. In some embodiments, the anther therapeutic agent is IVIg.

[0390] In some embodiments, the pharmaceutical composition is administered at a dose of 0.01, 0.05, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45. 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85. 0.90. 0.95, or 0.1 g active ingredient (RPP) / kg body weight. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.01, 0.05, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 0.1 g active ingredient (RPP) / kg body weight. In some embodiments, the pharmaceutical composition is administered at a dose of more than 0.01, 0.05, 0.15, 0.20, 0.25, 0.30. 0.35. 0.40. 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 0.1 g active ingredient (RPP) / kg body weight.

[0391] 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.

[0392] In some embodiments, the pharmaceutical composition is administered once, twice, three times, four times, five times, or more. In some embodiments, the pharmaceutical composition is administered once a day for one, two, three, four, five, or more days. In some embodiments, the pharmaceutical composition is administered until the desired outcome is observed.

[0393] In some embodiments, the pharmaceutical composition is administered w ith plasma IVIg.6. NON-LIMITING ASPECTS AND EMBODIMENTS OF THE DISCLOSURE

[0394] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments.Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-110 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0395] Aspect 1. A method of generating a recombinant polyclonal protein library (RPP) specific for a target molecule or complex of target molecules, comprising:(1) obtaining an input antigen binding protein (ABP) library dataset including an ABP profile for each of a plurality of ABPs, wherein(a) each ABP of the plurality of ABPs specifically binds a target antigen associated with the target molecule or complex; and(b) each ABP profile comprises a reference to the respective ABP and a plurality of characteristic descriptors for the respective ABP selected from:(i) a binding affinity of the respective ABP for the respective target antigen;(ii) an effector activity of the respective ABP against the target molecule or complex;(iii) a solubility score of the respective ABP;(iv) an aggregation score of the respective ABP;(v) a hy drophobicity score of the respective ABP;(vi) an isoelectric point of the respective ABP;(vii) a stability score of the respective ABP;(viii) a molecular weight of the respective ABP;(ix) a number of unpaired cysteine residues in the respective ABP;(x) an abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen;(xi) a fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, as compared to the abundance frequency prior to enrichment;(xii) a partial or full sequence of the respective ABP;(xiii) a number of non-canonical glycosylation sites in the respective ABP;(xiv) a number of cleavage sites in the respective ABP;(xv) a number of deamidation sites in the respective ABP;(xvi) a number of isomerization sites in the respective ABP;(xvii) a number of oxidation sites in the respective ABP;(xviii) CDR3H length of the respective ABP; and(xix) binding specificity of the respective ABP;(2) generating a filtered ABP library dataset corresponding to a subset of the plurality of ABPs and comprising a reference to each of the subset of the plurality of ABPs, wherein(a) each ABP in the subset of the plurality of ABPs has at least one of the plurality of characteristic descriptors having a value within a predetermined range for the characteristic; and(b) the subset of the plurality of ABPs have one or more preferred library properties selected from:(i) the set of heavy chain CDR3 sequences contained in the subset of the plurality of ABPs comprises at least about 10. 20. 50. 100, 200. or 1000 unique sequences;(ii) the subset of the plurality of ABPs specifically bind to at least two unique epitopes associated with the target molecule or complex;(iii) the subset of the plurality of ABPs is capable of modulating at least two target antigen variants;(iv) the set of heavy chain V genes represented in the subset of the plurality of ABPs comprises at least two unique V genes;(v) the set of light chain V genes represented in the subset of the plurality of ABPs comprises at least two unique V genes;(vi) the set of heavy chain J genes represented in the subset of the plurality7of ABPs comprises at least two unique J genes;(vii) the set of light chain J genes represented in the subset of the plurality7of ABPs comprises at least two unique J genes;(viii) the average percent germline identity of heavy chain V genes represented in the subset of the plurality7of ABPs is between about 50% and about 100%;(ix) the average percent germline identity of light chain V genes represented in the subset of the plurality of ABPs is between about 50% and about 100%;(x) the average percent germline identity of heavy chain J genes represented in the subset of the plurality of ABPs is between about 50% and about 100%; and(xi) the average percent germline identity of light chain J genes represented in the subset of the plurality of ABPs is between about 50% and about 100%; and(3) providing the fdtered ABP library dataset for generation of a composition comprising ABPs corresponding to the ABP references in the fdtered ABP library dataset, thereby generating the RPP.

[0396] Aspect 2. The method of aspect 1, wherein the plurality' of characteristic descriptors in (2)(a) comprises the binding affinity’ of the respective ABP for the respective target antigen.

[0397] Aspect 3. The method of aspect 1, yvherein the binding affinity is expressed in KD. and the predetermined range for the binding affinity’ is less than about 10 pM, 1 pM. 100 nM, 10 nM, 1 nM, or lower, optionally wherein the binding affinity7is determined by surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0398] Aspect 4. The method of aspect 1, yvherein the binding affinity is determined by a PolyMap assay comprising the steps of: providing a library of target-decorated cells, wherein each of the target-decorated cells presents the target molecule or complex on the membrane; contacting the library’ of target-decorated cells yvith a plurality’ of ABP-ribosome- mRNA (ARM) complexes corresponding to the one or more of the plurality' of ABPs, thereby inducing binding between the target-decorated cells and the ARM complexes; generating a plurality’ of monodisperse or polydisperse emulsion microdroplets, wherein each microdroplet contains a single cell out of the target-decorated cells, one or more ARM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; capturing RNA released from the single cell on a solid surface or yvithin a semi- permeable shell; generating a library' of hybrid polynucleic acids that comprise a sequence from a transcript of the single cell and / or a sequence from the mRNA of the ARM complex; sequencing the library of hybrid polynucleic acids; anddetermining a presence or absence of binding of each of the one or more of the plurality of ABPs to their respective target antigen.

[0399] Aspect 5. The method of aspect 1, wherein the predetermined range for the binding affinity is a presence of binding of the respective ABP to their respective target antigen in the Poly Map assay.

[0400] Aspect 6. The method of any one of aspects 1-5, wherein the plurality of characteristic descriptors in (2)(a) comprises the effector activity of the respective ABP against the target molecule or target molecule complex.

[0401] Aspect 7. The method of aspect 1-6, wherein the target molecule or target molecule complex comprises a virus, and the effector activity is a neutralization activity determined by a pseudovirus neutralization assay or a live virus neutralization assay.

[0402] Aspect 8. The method of aspect 7, wherein the predetermined range for the neutralization activity corresponds to (a) an ICso from about 0.08 pg / mL to about 900 pg / mL when determined by pseudovirus neutralization assay or (b) an ICso from about 0.003 pg / mL to about 1350 pg / mL when determined by live virus neutralization assay.

[0403] Aspect 9. The method of aspect Error! Reference source not found., wherein the target molecule or target molecule complex comprises a bacterium, and the effector activity is a bactericidal activity determined by a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPKA).

[0404] Aspect 10. The method of aspect 8, wherein the predetermined range for the bactericidal activity corresponds to a concentration where 50% bactericidal activity is observed from about 0.08 pg / ml to about 3600 pg / ml.

[0405] Aspect 11. The method of any one of aspects 1-10 wherein the plurality of characteristic descriptors in (2)(a) comprises the solubility score, optionally wherein the solubility score is determined using SKADE.

[0406] Aspect 12. The method of aspect 12, wherein the predetermined range for the solubility score is greater than about 0.5, or between 0.5 and 0.8.

[0407] Aspect 13. The method of any one of aspects 1-12, wherein the plurality of characteristic descriptors in (2)(a) comprises the aggregation score, optionally wherein the aggregation score corresponds to the number of residues predicted to have a propensity to aggregate and is determined by a method comprising the steps of:determining a 3D structure of the ABP, optionally wherein the 3D structure is determined using ABodyBuilder2; and determining the aggregation score based on the 3D structure, optionally wherein the aggregation score is determined using Aggrescan3D.

[0408] Aspect 14. The method of aspect 13. wherein the predetermined range for the aggregation score is fewer than 20, 15, or lower aggregation-prone sites.

[0409] Aspect 15. The method of any one of aspects 1-14, wherein the plurality' of characteristic descriptors in (2)(a) comprises the hydrophobicity score, optionally wherein the hydrophobic score is determined as the grand average of hydropathy (GRAVY), optionally wherein the hydropathy value of each amino acid is calculated using the Eisenberg scale.

[0410] Aspect 16. The method of aspect 15. wherein the predetermined range for the hydrophobicity7score is less than 0.03, less than 0.02, or less than 0.015.

[0411] Aspect 17. The method of any one of aspects 1-16, yvherein the plurality' of characteristic descriptors in (2)(a) comprises the isoelectric point, optionally wherein the isoelectric point is determined as EMBOSS pK values.

[0412] Aspect 18. The method of aspect 17, wherein the predetermined range for the isoelectric point is betyveen 7.0 and 9.0 or between 8.0 and 8.5.

[0413] Aspect 19. The method of any one of aspects 1-18, wherein the plurality' of characteristic descriptors in (2)(a) comprises the stability score, optionally wherein the stability score is determined by a method comprising the steps of: calculating an aliphatic index by determining the relative volume of A, V, L, and I residues, wherein the stability score corresponds to the aliphatic index.

[0414] Aspect 20. The method of aspect 19, wherein the predetermined range for the stability' score is from about 65 to about 73.

[0415] Aspect 21. The method of any one of aspects 1-20, yvherein the plurality of characteristic descriptors in (2)(a) comprises the molecular weight and the predetermined range for the molecular yveight is less than 170 kDa, less than 160 kDa, less than 150 kDa, less than 140 kDa, less than 130 kDa, less than 120 kDa, or lower.

[0416] Aspect 22. The method of any one of aspects 1-21, yvherein the plurality of characteristic descriptors in (2)(a) comprises a number of unpaired cysteine residues and the predetermined range for the number of unpaired cysteine residues is less than 5, 4, 3. 2, or 1.

[0417] Aspect 23. The method of any one of aspects 1-22, wherein the plurality of characteristic descriptors in (2)(a) comprises an abundance frequency or fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, optionally wherein the sorting process is fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) sorting, further optionally wherein the sorting is carried by yeast display.

[0418] Aspect 24. The method of aspect 23. wherein the predetermined range for the post-sort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 1%, 2%, 5%, 10%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, or greater within a pool of ABPs obtained after the sorting process.

[0419] Aspect 25. The method of aspect 22 or 23, wherein the predetermined range for the post-sort fold-change is greater than about any of 1, 1.5, 2, 2.5, 3, 4, or greater.

[0420] Aspect 26. The method of any one of aspects 1-25 wherein the enrichment is performed by FACS or MACS.

[0421] Aspect 27. The method of any one of aspects 1-26, wherein the plurality’ of characteristic descriptors in (2)(a) comprises the number of non-canonical glycosylation sites, optionally wherein the predetermined range for the number of non-canonical glycosylation sites is less than 5, 4, 3, 2, or 1.

[0422] Aspect 28. The method of any one of aspects 1-27, wherein the plurality of characteristic descriptors in (2)(a) comprises the number of cleavage sites, optionally wherein the predetermined range for the number of cleavage sites is less than 5, 4, 3, 2, or 1, further optionally wherein the cleavage site is a DP motif in the variable heavy or variable light chain region of the respective ABP.

[0423] Aspect 29. The method of any one of aspects 1-28, wherein the plurality' of characteristic descriptors in (2)(a) comprises the number of deamidation sites, optionally wherein the predetermined range for the number of deamidation sites is less than 5, 4, 3, 2. or 1, further optionally wherein the deamidation site is an NG, NS, or NA motif in CDR2H or CDR1L of the respective ABP.

[0424] Aspect 30. The method of any one of aspects 1-29, wherein the plurality of characteristic descriptors in (2)(a) comprises the number of isomerization sites, optionally wherein the predetermined range for the number of isomerization sites is less than 5, 4, 3, 2,or 1, further optionally wherein the isomerization site is a DG or DS motif in CDR2H, CDR3H, or CDR1L of the respective ABP.

[0425] Aspect 31. The method of any one of aspects 1-30, wherein the plurality of characteristic descriptors in (2)(a) comprises the number of oxidation sites, optionally wherein the predetermined range for the number of oxidation sites is less than 5, 4, 3, 2, or 1, further optionally wherein the oxidation site is a W or M residue in the CDRHs or CDRLs of the respective ABP.

[0426] Aspect 32. The method of any one of aspects 1-31, wherein the plurality' of characteristic descriptors in (2)(a) comprises the CDR3H length, optionally wherein the predetermined range for the CDR3H length is from about 10 to about 14 amino acids.

[0427] Aspect 33. The method of any one of aspects 1-32, wherein the plurality' of characteristic descriptors in (2)(a) comprises the binding specificity, optionally wherein the binding specificity corresponds to the number of variants of a target antigen capable of being targeted by the respective ABP and the predetermined range for the binding specificity is capability of binding to at least 2 (such as at least any of 3, 4, 5, 6, 7, 8, 9, 10, or more) variants of the target antigen, further optionally wherein the binding specificity is determined by a PolyMap assay.

[0428] Aspect 34. The method of any one of aspects 1-33, wherein the plurality of characteristic descriptors comprises at least any of 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 different characteristic descriptors, selected from: (1 )(b)(i) to (l)(b)(xix).

[0429] Aspect 35. The method of any one of aspects 1-34, wherein the partial or full sequence in (l)(b)(xii) comprises CDR sequences of the respective ABP.

[0430] Aspect 36. The method of any one of aspects 1-35, wherein at least one characteristic descriptor of the plurality of characteristic descriptors is determined using an in silico model for the respective characteristic with the sequence of the respective ABP, or a nucleic acid encoding the ABP, as an input.

[0431] Aspect 37. The method of aspect 36, yvherein one or more in silico models are used to determine the solubility score, aggregation score, hydrophobicity score, isoelectric point, stability score, number of cysteine residues, number of glycosylation sites, number of cleavage sites, number of deamidation sites, number of isomerization sites, and / or number of oxidation sites of one or more of the ABPs referenced in the input ABP library' dataset.

[0432] Aspect 38. The method of any one of aspects 1-37, wherein the one or more of preferred library properties comprises at least about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 different preferred library properties, selected from (2)(b)(i) to (2)(b)(xi).

[0433] Aspect 39. The method of any one of aspects 1-38, further comprising calculating, based on the partial or full sequences of the respective ABPs referenced in the input ABP library dataset, the heavy chain and light chain V and J gene usage and the average percent germline identity of the heavy chain and light chain V and J genes.

[0434] Aspect 40. The method of any one of aspects 1-39, wherein each ABP of the plurality of ABPs comprises a cognate pair of heavy chain and light chain variable regions from a single cell out of a blood sample from at least one donor previously exposed to the target molecule or complex.

[0435] Aspect 41. The method of aspect40, wherein the at least one donor comprises at least two, three, four, five. six. seven, eight, nine, ten. eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more donors.

[0436] Aspect 42. The method of aspect 41, wherein the at least one donor has been previously vaccinated with a vaccine denved from the target molecule or complex.

[0437] Aspect 43. The method of any one of aspects 40-42, wherein the blood sample comprises cells purified from peripheral blood mononuclear cells (PBMCs) of the donor.

[0438] Aspect 44. The method of any one of aspects 40-43, wherein the single cell is a B cell, plasma cell, or plasmablast.

[0439] Aspect 45. The method of any one of 1-44, wherein the target molecule or complex is associated with a pathogen.

[0440] Aspect 46. The method of aspect 45 wherein the pathogen is a virus, bacteria, or toxin.

[0441] Aspect 47. The method of any one of aspects 1-46, wherein the plurality' of ABPs comprises at least about any of 10. 20. 30. 40. 50, 100, or 1000 ABPs.

[0442] Aspect 48. The method of any one of aspects 1-47, wherein each of the plurality7of ABPs comprises full-length antibodies or functional fragments thereof.

[0443] Aspect 49. The method of aspect 48, w herein the full-length antibodies or functional fragments thereof are of the human IgGl, IgG2, IgG3, or IgG4 subtype.

[0444] Aspect 50. The method of any one of aspects 1-49, further comprising generating the composition comprising ABPs corresponding to the ABP references in the filtered ABP library dataset.

[0445] Aspect 51. An RPP generated by the method of any one of aspects 1-50.

[0446] Aspect 52. A recombinant polyclonal protein library (RPP) comprising at least 10 unique antigen binding proteins (ABPs) specific for a target molecule or complex, wherein each ABP of the at least 10 unique ABPs has at least one of a plurality of characteristics having a value within a predetermined range for the characteristic, wherein the plurality of characteristics comprises:(i) a binding affinity of a respective ABP for the respective target antigen;(ii) an effector activity of the respective ABP against the target molecule or complex;(iii) a solubility score of the respective ABP;(iv) an aggregation score of the respective ABP;(v) a hydrophobicity score of the respective ABP;(vi) an isoelectric point of the respective ABP;(vii) a stability score of the respective ABP;(viii) a molecular weight of the respective ABP;(ix) a number of unpaired cysteine residues in the respective ABP;(x) an abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen;(xi) a fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, as compared to the abundance frequency prior to enrichment:(xii) a partial or full sequence of the respective ABP;(xiii) a number of non-canonical glycosylation sites in the respective ABP;(xiv) a number of cleavage sites in the respective ABP;(xv) a number of deamidation sites in the respective ABP;(xvi) a number of isomerization sites in the respective ABP;(xvii) a number of oxidation sites in the respective ABP;(xviii) CDR3H length of the respective ABP; and(xix) binding specificity of the respective ABP; and wherein the at least 10 unique ABPs in the RPP have one or more preferred library properties selected from:(i) the set of heavy chain CDR3 sequences contained in the at least 10 unique ABPs comprises at least about 10, 20, 50, 100. 200, or 1000 unique sequences;(ii) the at least 10 unique ABPs specifically bind to at least two unique epitopes associated with the target molecule or complex;(iii) the at least 10 unique ABPs are capable of modulating at least two target antigen variants;(iv) the set of heavy chain V genes represented in the at least 10 unique ABPs comprises at least two unique V genes;(v) the set of light chain V genes represented in the at least 10 unique ABPs comprises at least two unique V genes;(vi) the set of heavy chain J genes represented in the at least 10 unique ABPs comprises at least two unique J genes;(vii) the set of light chain J genes represented in the at least 10 unique ABPs comprises at least two unique J genes;(viii) the average percent germline identity of heavy chain V genes represented in the at least 10 unique ABPs is between about 50% and about 100%;(ix) the average percent germline identity of light chain V genes represented in the at least 10 unique ABPs is between about 50% and about 100%;(x) the average percent germline identity of heavy chain J genes represented in the at least 10 unique ABPs is between about 50% and about 100%; and(xi) the average percent germline identity7of light chain J genes represented in the at least 10 unique ABPs is between about 50% and about 100%.

[0447] Aspect 53. The RPP of aspect 52, wherein the plurality of characteristics comprises the binding affinity of the respective ABP for the respective target antigen.

[0448] Aspect 54. The RPP of aspect 53, wherein the binding affinity is expressed in KD, and the predetermined range for the binding affinity7is less than about 10 pM, 1 pM, 100 nM, 10 nM, 1 nM, or lower, optionally wherein the binding affinity7is determined by surface plasmon resonance (SPR) or biolayer interferometry' (BLI).

[0449] Aspect 55. The RPP of aspect 53, wherein the binding affinity is determined by a Poly Map assay comprising the steps of: providing a library of target-decorated cells, wherein each of the target-decorated cells presents the target molecule or complex on the membrane; contacting the library of target-decorated cells with a plurality of ABP-ribosome- mRNA (ARM) complexes corresponding to the one or more of the plurality of ABPs, thereby inducing binding between the target-decorated cells and the ARM complexes; generating a plurality of monodisperse or poly disperse emulsion microdroplets, wherein each microdroplet contains a single cell out of the target-decorated cells, one or more ARM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; capturing RNA released from the single cell on a solid surface or within a semi- permeable shell; generating a library' of hybrid polynucleic acids that comprise a sequence from a transcript of the single cell and / or a sequence from the mRNA of the ARM complex; sequencing the library of hybrid polynucleic acids; and determining a presence or absence of binding of each of the one or more of the plurality of ABPs to their respective target antigen.

[0450] Aspect 56. The RPP of aspect 55, wherein the predetermined range for the binding affinity is a presence of binding of the respective ABP to their respective target antigen in the Poly Map assay.

[0451] Aspect 57. The RPP of aspect 52, wherein the plurality’ of characteristics comprises the effector activity of the respective ABP against the target molecule or target molecule complex.

[0452] Aspect 58. The RPP of aspect 57, wherein the target molecule or target molecule complex comprises a virus, and the effector activity is a neutralization activity determined by a pseudovirus neutralization assay or a live virus neutralization assay.

[0453] Aspect 59. The RPP of aspect 58, wherein the predetermined range for the target molecule or target molecule complex neutralization activity corresponds to (a) an ICso from about 0.08 pg / mL to about 900 pg / mL when determined by7pseudovirus neutralization assay or (b) an ICso from about 0.003 pg / mL to about 1350 pg / mL when determined by live virus neutralization assay.

[0454] Aspect 60. The RPP of aspect 59, wherein the target molecule or target molecule complex comprises a bacterium, and the effector activity7is a bactericidal activity determined by a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPKA).

[0455] Aspect 61. The RPP of aspect 60, wherein the predetermined range for the bactericidal activity7corresponds to a concentration where 50% bactericidal activity7is observed from about 0.08 pg / ml to about 3600 pg / ml.

[0456] Aspect 62. The RPP of any one of aspects 52-61, wherein the plurality of characteristics comprises the solubility7score, optionally wherein the solubility score is determined using SKADE.

[0457] Aspect 63. The RPP of aspect 63, wherein the predetermined range for the solubility score is greater than about 0.5, or between 0.5 and 0.8.

[0458] Aspect 64. The RPP of any one of aspects 52-63, wherein the plurality of characteristics comprises the aggregation score, optionally wherein the aggregation score corresponds to the number of regions predicted to have a propensity7to aggregate and is determined by a method comprising the steps of determining a 3D structure of the ABP, optionally wherein the 3D structure is determined using ABodyBuilder2; and determining the aggregation score based on the 3D structure, optionally wherein the aggregation score is determined using Aggrescan3D.

[0459] Aspect 65. The RPP of aspect 64, wherein the predetermined range for the aggregation score is fewer than 20, 15, or lower aggregation-prone sites.

[0460] Aspect 66. The RPP of any one of aspects 52-66, wherein the plurality7of characteristics comprises the hydrophobicity7score, optionally wherein the hydrophobic score is determined as the grand average of hydropathy (GRAVY), optionally wherein the hydropathy value of each amino acid is calculated using the Eisenberg scale.

[0461] Aspect 67. The RPP of aspect 66, wherein the predetermined range for the hydrophobicity7score is less than 0.03, less than 0.02, or less than 0.015.

[0462] Aspect 68. The RPP of any one of aspects 52-67, wherein the plurality7of characteristics comprises the isoelectric point, optionally wherein the isoelectric point is determined as EMBOSS pK values.

[0463] Aspect 69. The RPP of aspect 68, wherein the predetermined range for the isoelectric point is between 7.0 and 9.0 or between 8.0 and 8.5.

[0464] Aspect 70. The RPP of any one of aspects 52-69, wherein the plurality of characteristics comprises the stability score, optionally wherein the stability score is determined by a method comprising the steps of: calculating an aliphatic index by determining the relative volume of A, V, L, and I residues, wherein the stability score corresponds to the aliphatic index.

[0465] Aspect 71. The RPP of aspect 70, wherein the predetermined range for the stability score is from about 65 to about 73.

[0466] Aspect 72. The RPP of any one of aspects 52-71, wherein the plurality of characteristics comprises the molecular weight and the predetermined range for the molecular weight is less than 150 kDa, less than 140 kDa, less than 130 kDa, less than 120 kDa, less than 110 kDa, less than 100 kDa, or lower.

[0467] Aspect 73. The RPP of any one of aspects 52-72, wherein the plurality of characteristics comprises a number of unpaired cysteine residues and the predetermined range for the number of unpaired cysteine residues is less than 5, 4, 3, 2, or 1.

[0468] Aspect 74. The RPP of any one of aspects 52-73, wherein the plurality of characteristics comprises an abundance frequency or fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, optionally wherein the sorting process is fluorescence- activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) sorting, further optionally wherein the sorting is carried by yeast display.

[0469] Aspect 75. The RPP of aspect 74, wherein the predetermined range for the postsort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 1%, 2%, 5%. 10%. 15%. 16%. 17%. 18%. 19%. 20%. 25%. 30%. 35%. 40%. 45%, 50%. 55%, or 60%, or greater within a pool of ABPs obtained after the sorting process.

[0470] Aspect 76. The RPP of aspect 74 or 75, wherein the predetermined range for the post-sort fold-change is greater than about any of 1. 1.5, 2, 2.5. 3. 4, 5, 6. 7, 8, 9. 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or greater.

[0471] Aspect 77. The RPP of any one of aspects 2-76, wherein the enrichment is performed experimentally by FACS or MACS.

[0472] Aspect 78. The RPP of any one of aspects 52-77, wherein the plurality of characteristics comprises the number of non-canonical glycosylation sites, optionally wherein the predetermined range for the number of non-canonical glycosylation sites is less than 5, 4, 3, 2, or 1.

[0473] Aspect 79. The RPP of any one of aspects 52-78, wherein the plurality' of characteristics comprises the number of cleavage sites, optionally wherein the predetermined range for the number of cleavage sites is less than 5, 4, 3. 2. or 1. further optionally wherein the cleavage site is a DP motif in the variable heavy or variable light chain region of the respective ABP.

[0474] Aspect 80. The RPP of any one of aspects 52-78, wherein the plurality of characteristics comprises the number of deamidation sites, optionally wherein the predetermined range for the number of deamidation sites is less than 5, 4, 3, 2, or 1, further optionally wherein the deamidation site is an NG, NS, or NA motif in CDR2H or CDR1L of the respective ABP.

[0475] Aspect 81. The RPP of any one of aspects 52-80, wherein the plurality7of characteristics comprises the number of isomerization sites, optionally wherein the predetermined range for the number of isomerization sites is less than 5, 4, 3. 2, or 1. further optionally wherein the isomerization site is a DG or DS motif in CDR2H, CDR3H, or CDR1L of the respective ABP.

[0476] Aspect 82. The RPP of any one of aspects 52-81, wherein the plurality of characteristics comprises the number of oxidation sites, optionally wherein the predetermined range for the number of oxidation sites is less than 5, 4, 3, 2, or 1, further optionally yvherein the oxidation site is a W or M residue in the CDRHs or CDRLs of the respective ABP.

[0477] Aspect 83. The RPP of any one of aspects 52-82, wherein the plurality of characteristics comprises the CDR3H length, optionally yvherein the predetermined range for the CDR3H length is from about 10 to about 14 amino acids.

[0478] Aspect 84. The RPP of any one of aspects 52-83, wherein the plurality of characteristics comprises the binding specificity, optionally yvherein the binding specificity' corresponds to the number of variants of a target antigen capable of being targeted by the respective ABP and the predetermined range for the binding specificity is capability of binding to at least 2 (such as at least any of 3, 4, 5, 6, 7, 8, 9, 10, or more) variants of thetarget antigen, further optionally wherein the binding specificity is determined by a Poly Map assay.

[0479] Aspect 85. The RPP of any one of aspects 52-84, wherein the plurality of characteristics comprises at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 different characteristics.

[0480] Aspect 86. The RPP of any one of aspects 52-85, wherein the partial or full sequence in (l)(b)(xii) comprises CDR sequences of the respective ABP.

[0481] Aspect 87. The RPP of any one of aspects 52-86, wherein at least one characteristic of the plurality of characteristics is determined using an in silico model for the respective characteristic with the sequence of the respective ABP, or a nucleic acid encoding the ABP, as an input.

[0482] Aspect 88. The RPP of aspect 87, wherein one or more in silico models are used to determine the solubility score, aggregation score, hydrophobicity score, isoelectric point, stability score, number of cysteine residues, number of glycosylation sites, number of cleavage sites, number of deamidation sites, number of isomerization sites, and / or number of oxidation sites of one or more of the at least 10 unique ABPs.

[0483] Aspect 89. The RPP of any one of aspects 52-88, wherein the one or more of preferred library properties comprises at least about any of 2, 3. 4, 5, 6, 7, 8, 9, 10, or 11 different preferred library properties.

[0484] Aspect 90. The RPP of any one of aspects 52-87, wherein the target molecule or complex is a pathogen or a group of pathogens.

[0485] Aspect 91. The RPP of aspect 90, wherein the RPP is capable of neutralizing or killing one or more variants of the pathogen or the group of pathogens, optionally wherein the RPP is capable of neutralizing or killing at least any of two. three, four, five, ten, fifteen, or more different variants of the pathogen or the group of pathogens.

[0486] Aspect 92. A plurality of isolated polynucleotides, wherein each of the isolated polynucleotides encodes one of the ABPs of the RPP of any one of aspects 52-90.

[0487] Aspect 93. A plurality of polynucleotide constructs, comprising the isolated polynucleotides of aspect 92 cloned into an expression vector.

[0488] Aspect 94. A plurality of host cells comprising the plurality of isolated polynucleotides of aspect 92, or the plurality of polynucleotide constructs of aspect 93.

[0489] Aspect 95. A method of producing an RPP, the method comprising culturing the plurality7of host cells of aspect 94under conditions for expression of the ABPs and isolating the ABPs.

[0490] Aspect 96. A pharmaceutical composition comprising the RPP of any one of aspects 52-91 and a pharmaceutically acceptable excipient.

[0491] Aspect 97. A method of treating a patient in need thereof by administering an effective amount of the pharmaceutical composition of aspect 96.

[0492] Aspect 98. The method of aspect 97. wherein the patient has been exposed to the target molecule or complex or a variant thereof.

[0493] Aspect 99. The method of aspect 97, wherein the patient has a disease associated with the target molecule or complex or a variant thereof.

[0494] Aspect 100. The method of any one of aspects 97-99, wherein the pharmaceutical composition is administered intramuscularly, subcutaneously, intravenously, intradermally, orally, or through inhalation.

[0495] Aspect 101. The method of any one of aspects 97-100, wherein the effective amount is sufficient to treat the disease associated with the target molecule or complex or a variant thereof.7. EXAMPLES

[0496] Below are examples of specific embodiments for carry ing out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g, amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0497] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., 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 3rdEd. (Plenum Press) Vols A and B(1992).Example 1: FACS-enrichment of antibody clones in COVID antibody libraries

[0498] Eight COVID-19 related anti-SARS-CoV-2 antibody libraries (COV-1 to COV-8) were generated using samples from sixteen donors (each library’ from two donor samples) and their antibody sequences were obtained. A superpool was also generated by pooling all eight libraries. The antibodies in each of the eight libraries were sorted by FACS using the receptor-binding domain (RBD) and the superpool was sorted using the Omicron / BA2 variant of the RBD or the full-length SARS-CoV-2 spike protein.

[0499] FIG. 2 is a heatmap where the column represents each library or the superpool before or after sorting and the row represents each antibody clone. When post- vs. pre-sort fold change >= 1.8 and post-sort frequency >= 0.1%, the clones are defined to be FACS-enriched. From the experiment, total 194 clones were enriched by FACS (binders to SARS-CoV-2)Example 2: COVID antibody V / J gene usage and percent identity to germline sequences

[0500] V(D)J recombination of antibody gene segments contributes to the vast combinatorial diversity of antibodies. The sequences of COVID antibody libraries were analyzed for their V heavy chain and J heavy chain gene usages and the gene usages are summarized in the heatmap provided in FIG. 3. The heatmap shows the combinatorial diversity of the COVID antibodies. The information related to V / J gene usage can be used to design a custom polyclonal antibody. For example, a custom polyclonal can be designed to have more diverse functionalities by including antibodies with diverse V / J gene usage.

[0501] V heavy and J heavy chain sequences in the COVID antibody libraries were further compared against their germline sequences. The % identities from the comparison are provided in FIG. 4. “All” depicts the % identities for all antibodies in the COVID antibody libraries, while “Enriched” depicts the % identities for the FACS-enriched antibodies. During antibody affinity maturation, somatic hypermutation introduces point mutations into antibody V and J genes. Thus, antibodies with lower % identity to germline can be considered to be more affinity matured.Example 3: COVID antibody library PolyMap profileI l l

[0502] Binding of antibodies in the COVID antibody library (columns) to the surface antigen of different coronavirus variants (CoV-2 WT, EU1, Beta, Alpha, Gamma, etc.) was tested by PolyMap using the method described in PCT / US2024 / 012238 filed on Jan 19, 2024.

[0503] In short, antibodies in the COVID antibody library were expressed in a ribosomedisplay format, which uses a tethered mRNA to provide genotype-phenotype linkage of the soluble protein. Antigens were expressed at a high level on the surface of a mammalian cell, which can then be bound by the soluble ARM complex.

[0504] For Drop-seq microfluidic analysis of the larger libraries, cells expressing each CoV-2 S sequence variant were pooled together and then stained with antibody-ribosome-mRNA (ARM) complexes of the anti-CoV-2 antibody library. After identifying antigen and antibody sequences with Drop-seq cell barcodes, a merged list of every antibody and antigen associated with each cell barcode was generated. As each antibody has equal opportunity to bind each antigen, the total antibody reads for each clone across all cell lines were counted, then the percentage of reads for each antibody was normalized by the number of associated cells. When examining the antibodies based on the total number of antibodies reads, differential binding patterns were observed across the antibody sequences (FIG. 5). Many of the antibody clones showed minimal enrichment to the Omicron spike variants (2 IK, 22 A / D, 23B), which was expected as this library was generated from donors convalescent from the original ancestral WT strain in early 2020.Example 4: COVID antibody developability profiles

[0505] Antibody developability refers to the set of characteristics that influence its ability to be successfully developed as a therapeutic drug. Antibodies need to remain structurally intact and active under various conditions (stability), including temperature and pH changes, to maintain their efficacy during manufacturing, storage, and administration. Further, antibodies need to be soluble with minimal aggregation to allow for efficient manufacturing and formulation.

[0506] These developability profiles were computationally generated using antibody sequences from the COVID or Jain database and provided in FIG. 6. The antibodies investigated in Jain et al. (Jain, Tushar, et al. "Biophysical properties of the clinical-stage antibody landscape." Proceedings of the National Academy of Sciences 114.5 (2017): 944- 949) reached advanced clinical stages and are used here to determine upper and lower bounds of each developability property7. Amber lines indicate upper and lower 5% thresholds of thedistribution for each property and the red lines indicate the minimum and maximum of the distribution for each property'. The COVID antibodies have distributions similar to those of the clinical antibodies, suggesting that most (but not all) antibodies in this dataset have promising developability profiles.Example 5: Design and generation of custom RPPs

[0507] A comprehensive list of antibody sequences was curated, specifically focusing on the amino acid sequences of the variable light (VL) and variable heavy (VH) chains. Next, relevant parameters for each sequence were calculated, such as frequency, enrichment, hydrophobicity, solubility, etc. of each clone, to inform the selection process. Based on these calculations, appropriate clone pools were selected that meet the desired criteria.

[0508] For each identified clone, the amino acid sequences of the variable light (VL) and variable heavy (VH) chains were determined and then codon-optimized to enhance expression in the chosen host system. These optimized sequences were ordered with specific overlapping regions to facilitate Gibson assembly. Both the plasmid backbone and the insert fragments were prepared using PCR to ensure compatibility- for assembly. The Gibson assembly reaction was then set up in a plate format, allowing for efficient and parallel processing of multiple constructs. Following assembly, a heat shock step yvas performed in the plate to transform the assembled plasmids into competent cells. The resulting transformed cells were pooled, plated on selective media, and individual colonies were picked and sequenced to confirm the correct assembly and integrity of the constructs. The antibody proteins from the stable cell lines were purified and assays were conducted to evaluate their functionality and other key characteristics.

[0509] The process allo ved successful cloning of 1 10 antibody sequences from the GIGA- 2050 library for expression studies. Approximately 85% of these sequences yvere successfully assembled, indicating a high efficiency in the assembly process. When sequencing at twice the input level, we were able to recover and confirm 50-75% of the clones, while increasing the sequencing depth to three times the input alloyved us to recover up to 80% of the clones.

[0510] The anti-SARS-CoV-2 antibodies w ere pooled in a variety- of libraries as summarized in the beloyv table.

[0511] As shown in the table above, there were 8 sub-libraries that were pooled to generate GIGA-2050. Each clone had a frequency in its sub-library', and a frequency once mixed together. The "most frequent (sub library )" takes the clones at highest % in any sub library, whereas the "2050 top" takes the top clones by % after pooling. These libraries (1-16) were specifically designed to answer the questions, such as (i) is frequency or enrichment more correlated with function? (ii) can rational selection (e.g., using Polymap) improve function?(iii) do developability' metrics help address manufacturability? Should we set the threshold?(iv) can we capture GIGA2050 efficacy with fewer clones? Can we improve GIGA2050 manufacturability?

[0512] Specifically, GIGA2050 is a pool of 8 sub-libraries. The ‘‘Most frequent clones (sublibrary)’' refers to a library of the clones at highest % in any sub library, whereas the “Most frequent clones (GIGA2050)” refers to a library of the top clones by % after pooling. The “Polymap ‘top’ binders” is a library of clones selected as the most frequence clones in GIGA-2050 and then further selected based on the Polymap profiles. In particular, the clones having unique Poly Map patterns were selected and the ones having repeat patterns were excluded from the library’. In other words, the library includes the top clones from GIGA- 2050 with any duplicate PolyMap profiles removed. The "Polymap ‘rare’ binders” is a library of clones selected purely on Poly Map profiles, aiming to include 10 clones with unique profiles. The clone with the best Poly Map score was picked for 10 antigen pools: (e.g., WT / EU1, alpha, beta / gamma, delta / epsilon, eta / iota, kappa, lambda, mu, omicron, covl).

[0513] Each of the library was experimentally tested for various functions such as binding (e.g., ELISA), neutralization activity and developability (e.g., titer, purification, recovery', CEX-HPLC and SEC-HPLC profiles, Tm, stability). The results showed antibody characteristics preferred for generation of a custom polyclonal antibody. Specifically, the results showed whether abundance frequency and fold-change in abundance of individual antibodies following sorting of a library correlates with its function, whether combination of diverse binders improve function, whether developability metrics of each antibody addresses manufacturability and whether there is a threshold in the developability metrics for developability and how many clones of antibodies are needed to replicate the function of naturally occurring or other recombinant polyclonal antibodies.Example 6: Characterization of custom RPPs (sequence analysis)

[0514] For generation of the libraries described in Example 5, plasmids encoding the selected antibody proteins were pooled in equal amounts and transfected into the landing pad CHOZN cell line, followed by selection for successful integration using the glutamine synthetase gene as a marker. Genomic DNA (gDNA) and RNA were then isolated from the resulting cell lines. Both gDNA and RNA samples were subjected to sequencing. For gDNA analysis, PCR was performed using primer pools that cover the VH gene region, incorporating Illumina adaptors for compatibility’ with next-generation sequencing. Similarly, for RNA analysis, RT- PCR was conducted using primer pools targeting the VH gene, also with Illumina adaptors, to enable comprehensive sequencing of the antibody genes. For smaller libraries, sequencing results demonstrated high recovery rates, with genomic DNA (gDNA) analysis returning 97- 100% of the clones and RNA analysis yielding 87-100% of the clones. For the larger library’, the recovery rates were somewhat reduced; gDNA sequencing returned 87-97% of the clones, while RNA sequencing showed a broader range and lower recovery, returning only 33-80%of the clones. These findings highlight the impact of library size on the efficiency of clone recovery from both gDNA and RNA, with smaller libraries providing more comprehensive representation in both types of analyses. The results are provided in Table 5 below.

[0515] Plasmids corresponding to the top 10, 25, and 50 clones were pooled in equal amounts and subsequently used to transfect cells, with the goal of generating representative cell lines. After transfection, the resulting cell lines were sequenced to assess the distribution of clones.Interestingly, as provided in FIG. 7, the sequencing data for top 10 / 25 / 50 clones revealed that the transfection process introduced an unexpected skew in clone representation, suggesting that certain clones were preferentially incorporated or expressed in the cells despite the initial equal pooling of plasmids. This observation indicates that factors beyond plasmid input, such as transfection efficiency or cellular selection, may influence the final composition of the cell lines.

[0516] Despite pooling of plasmids by mass, analysis revealed that the input mix was already skewed, indicating that equal mass does not necessarily translate to equal representation of each clone. Furthermore, there was a lack of correlation between the proportion of input plasmid and the percentage of genomic DNA (gDNA) reads observed after transfection, suggesting that factors other than initial plasmid quantity are influencing clone abundance. When comparing the observed read frequencies to the theoretical expectations based on the input mix, some clones consistently appeared at lower frequencies. This discrepancy does not seem to be due to issues with DNA concentration, which was verified to be within acceptable ranges. These findings raise the possibility’ of amplification bias during library preparation or sequencing, or perhaps biological factors such as the impact of specific protein sequences on cell survival, which could lead to selective loss or underrepresentation of certain clones in the final cell population.Example 7: Characterization of custom RPPs (protein purification and developability testing)

[0517] A production run was carried out in which a protein mix was successfully purified using protein A affinity chromatography. Following purification, developability parameters for each antibody were calculated based on their amino acid sequences, allowing for an assessment of their potential suitability for further development. To facilitate comparison, pools of ten antibodies each were selected and categorized as either "good" or "bad" based on these developability metrics, and both pools were produced and purified under the same conditions. The resulting data were visualized in FIG. 8, with the distribution of the tested antibodies shown in grey, alongside a reference distribution of clinical antibodies as reported by Jain et al., (Biophysical properties of the clinical-stage antibody landscape. Proc Natl Acad Sci U S A. 2017 Jan 31 ; 114(5):944-949) providing context for how the experimental antibodies compare to those that have advanced to clinical use.

[0518] A 50 mL production run was conducted, during which the observed titers ranged from 0.6 to 1.6 g / L, which is somewhat lower than typically expected for this process. Upon review, it was determined that the lower titers were likely due to seeding the cultures at a lower cell density than is standard in process development (PD) runs. Importantly, as provided in FIG. 9, the selection of antibodies based on developability criteria did not appear to have a significant effect on the titers achieved.

[0519] Samples categorized as either "good" or "bad" based on these developability metrics were further analyzed using SEC-MALS following high-throughput Protein A (HTP ProA) purification. The percentage of monomers was determined by considering all chrom...

Claims

CLAIMS1. A method of generating a recombinant polyclonal protein library7(RPP) specific to a target molecule or complex of target molecules, comprising:(1) obtaining a dataset for an input antigen binding protein (ABP) library comprising at least 100 candidate ABPs, wherein the input ABP library dataset comprises an ABP profile for each of the at least 100 candidate ABPs, wherein(a) each candidate ABP of the input ABP library is capable of specific binding to a target antigen associated with the target molecule or complex; and(b) each ABP profile comprises a reference to a respective candidate ABP and a plurality7of characteristic descriptors for the respective candidate ABP selected from:(i) a binding affinity7of the respective candidate ABP to the respective target antigen;(ii) an effector activity of the respective candidate ABP against the target molecule or complex;(iii) a binding pattern of the respective candidate ABP to the respective target antigen and its variants;(iv) an abundance frequency of the respective candidate ABP following sorting the input ABP library7or a subset thereof to enrich for binding to the respective target antigen; and(v) a fold-change of the increase in the abundance frequency of the respective candidate ABP following sorting the input ABP library or a subset thereof to enrich for binding to the respective target antigen, as compared to the abundance frequency prior to enrichment;(2) generating a dataset for a filtered ABP library comprising selected ABPs, wherein the dataset comprises a reference to each of the selected ABPs and(a) the filtered ABP library comprises at least 10 selected ABPs which is a subset of the at least 100 candidate ABPs; and(b) each selected ABP has at least one of the plurality of characteristic descriptors meets a preferred criteria selected from:(i) a binding affinity to the respective target antigen is ranked at least top 25% among all the candidate ABPs in the input ABP library7;(ii) an effector activity against the target molecule or complex is ranked at least top 25% among all the candidate ABPs in the input ABP library;(iii) a binding pattern for the respective target antigen and its variants is shared among less than 20% of candidate ABPs in the input ABP library;(iv) an abundance frequency of the selected ABP following sorting the input ABP library' or a subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof; and(v) a fold-change of the increase in the abundance frequency of the selected ABP following sorting the input ABP or the subset thereof is ranked at least top 25% among all the candidate ABPs in the input ABP library or the subset thereof; and(3) providing the dataset for the filtered ABP library for generation of a composition comprising selected ABPs, thereby generating the RPP.

2. The method of claim 1, wherein the plurality of characteristic descriptors in (2)(b) comprises the binding affinity to the respective target antigen.

3. The method of claim 1, wherein the binding affinity7is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% among all the candidate ABPs in the input ABP library7, optionally wherein the binding affinity is determined by surface plasmon resonance (SPR) or biolayer interferometry (BLI).

4. The method of any one of claims 1-3, wherein the plurality7of characteristic descriptors in (2)(b) comprises the effector activity against the target molecule or target molecule complex.

5. The method of claim 4, wherein the preferred criteria is that the effector activity7against the target molecule or complex is ranked at least top 20%. at least top 15%, at least top 10%, or at least top 5% among all the candidate ABPs in the input ABP library.

6. The method of claim 4 or 5, wherein the target molecule or target molecule complex comprises a virus, and the effector activity is a neutralization activity determined by a pseudovirus neutralization assay or a live virus neutralization assay.

7. The method of any one of claims 4-6, wherein the preferred criteria for the effector activity7is neutralization activity corresponding to (a) an IC50 from about 0.08 pg / mL to about 900 ug / mL when determined by pseudovirus neutralization assay or (b) an ICso from about 0.003 pg / mL to about 1350 pg / mL when determined by live virus neutralization assay.

8. The method of any one of claims 4-7, wherein the target molecule or target molecule complex comprises a bacterium, and the effector activity is a bactericidal activity determined by a serum bactencidal assay (SBA) or an opsonophagocytic killing assay (OPKA).

9. The method of claim 8, wherein the preferred criteria for the bactericidal activity corresponds to a concentration where 50% bactericidal activity is observed from about 0.08 pg / ml to about 3600 pg / ml.

10. The method of any one of claims 1-9, wherein the plurality of characteristic descriptors in (2)(b) comprises an abundance frequency or fold-change of the increase in the abundance frequency of the respective ABP following a sorting process to enrich for binding to the respective target antigen, optionally wherein the sorting process is fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) sorting, further optionally wherein the sorting is carried by yeast display.

11. The method of claim 10, wherein the preferred criteria for the post-sort abundance frequency is greater than about any of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% within a pool of ABPs obtained after the sorting process.

12. The method of claim 11, wherein the preferred criteria for the post-sort abundance frequency is between 0.01% and 50%, between 1% and 50%, between 5% and 45%, between 10% and 40%, between 15% and 35%, or between 20% and 30%.

13. The method of claim 1 1 or 12, wherein the preferred criteria for the post-sort foldchange is greater than about any of 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700 or greater.

14. The method of claim 6, wherein the preferred criteria for the post-sort fold-change ranges from 1.5 to 1000, from 10 to 500, from 10 to 400, from 10 to 1000, from 100 to 1000, from 200 to 1000, from 300 to 1000, from 400 to 1000, or from 500 to 1000.

15. The method of any one of claims 1-14, wherein the enrichment is performed by FACS or MACS.

16. The method of any one of claims 10-13, wherein the preferred criteria for the postsort abundance frequency or the post-sort fold-change is that the corresponding ABP is ranked at least top 20%, at least top 15%, at least top 10%, or at least top 5% within the input ABP library.

17. The method of any one of claims 1-14, wherein the plurality’ of characteristic descriptors in (2)(b) comprises the binding pattern, optionally wherein the binding pattern corresponds to a subset of the respective target antigen and its variants capable of binding to the respective ABP, further optionally wherein the binding pattern is determined by a Poly Map assay.

18. The method of claim 17, wherein the preferred criteria is that the binding pattern of the respective ABP is shared among less than 15%, less than 10%, less than 5%, or less than 3% candidate ABPs in the input ABP library.

19. The method of claim 17, wherein the preferred criteria is that the binding pattern of the respective ABP is shared with less than 100, less than 50, less than 10 or less than 5 other candidate ABPs in the input ABP library .

20. The method of any one of claims 1-19, wherein the selected ABPs in the filtered ABP library as a group meet 1, 2, 3, 4, 5 or 6 different preferred criteria, selected from: (2)(b)(i) to (2)(b)(v).

21. The method of any one of claims 1-19, wherein the selected ABPs in the filtered ABP library meet one or more additional preferred criteria related to: a solubility score of the respective ABP; an aggregation score of the respective ABP; a hydrophobicity score of the respective ABP; an isoelectric point of the respective ABP; a stability score of the respective ABP; a molecular weight of the respective ABP; a number of unpaired cysteine residues in the respective ABP; a partial or full sequence of the respective ABP; a number of non-canonical glycosylation sites in the respective ABP; a number of cleavage sites in the respective ABP; a number of deamidation sites in the respective ABP; a number of isomerization sites in the respective ABP; a number of oxidation sites in the respective ABP; CDR3H length of the respective ABP; or binding specificity of the respective ABP.

22. The method of any one of claims 1-21 , wherein the filtered ABP library meets one or more preferred properties selected from: the set of heavy chain CDR3 sequences contained in the filtered ABP library comprises at least about 10, 20, 50, 100, 200, or 1000 unique sequences; the selected ABPs in the filtered ABP library as a group specifically bind to at least two unique epitopes associated with the target molecule or complex; the selected ABPs in the filtered ABP library’ as a group are capable of modulating at least two target antigen variants; the set of heavy chain V genes represented in the ABPs in the filtered ABP library as a group comprises at least two unique V genes; the set of light chain V genes represented in the ABPs in the filtered ABP library' as a group comprises at least two unique V genes; the set of heavy chain J genes represented in the ABPs in the filtered ABP library’ as a group comprises at least two unique J genes;the set of light chain J genes represented in the ABPs in the filtered ABP library7as a group comprises at least two unique J genes; the average percent germline identity of heavy chain V genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; the average percent germline identity of light chain V genes represented in the ABPs in the filtered ABP library' as a group is between about 50% and about 100%; the average percent germline identity of heavy chain J genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%; and the average percent germline identity of light chain J genes represented in the ABPs in the filtered ABP library as a group is between about 50% and about 100%.

23. The method of any one of claims 1-22, wherein each candidate ABP of the input ABP library comprises a cognate pair of heavy chain and light chain variable regions from a single cell out of a blood sample from at least one donor previously exposed to the target molecule or complex.

24. The method of claim 23, wherein the at least one donor is at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more donors.

25. The method of claim 23 or 24, wherein the at least one donor has been previously vaccinated with a vaccine derived from the target molecule or complex.

26. The method of any' one of claims 23-25, wherein the blood sample comprises cells purified from peripheral blood mononuclear cells (PBMCs) of the donor.

27. The method of any one of claims 23-26. wherein the single cell is a B cell, plasma cell, or plasmablast.

28. The method of any one of claims 1-27, wherein the target molecule or complex is associated with a pathogen, optionally wherein the pathogen is a virus, bacteria, or toxin.

29. The method of any one of claims 1-28, wherein the input ABP library is a pool of ABPs, wherein each of the ABPs have been selected for a selection criteria, wherein the selection criteria is (i) binding or binding affinity to a target antigen or its variant, (ii) activity against the pathogen, or (iii) effector activity.

30. The method of any one of claims 1-29, wherein the input ABP library comprises at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000. at least 1100, at least 1200, at least 1300, at least 1400, at least 1500. at least 1600. at least 1700, at least 1800, at least 1900, at least 2000, at least 3000, at least 4000, at least 5000, at least 10,000, at least 50,000 or more ABPs.

31. The method of any one of claims 1-30, wherein the RPP comprises at least 10, at least 25, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 ABPs.

32. The method of any one of claims 1-30, wherein each ABP of the RPP comprises full- length antibodies or functional fragments thereof.

33. The method of claim 32, wherein the full-length antibodies or functional fragments thereof are of the human IgGl. IgG2, IgG3, or IgG4 subtype.

34. The method of any one of claims 1-33, further comprising generating the composition comprising ABPs corresponding to the ABP references in the fdtered ABP library dataset.

35. The method of any one of claims 1-34, wherein the binding affinity or the binding pattern is determined by a PolyMap assay comprising the steps of:providing a library of target-decorated cells, wherein each of the targetdecorated cells presents the target molecule or complex on the membrane; contacting the library of target-decorated cells with a plurality of ABP- ribosome-mRNA (ARM) complexes corresponding to the one or more of the plurality of ABPs, thereby inducing binding between the target-decorated cells and the ARM complexes; generating a plurality of monodisperse or poly disperse emulsion microdroplets, wherein each microdroplet contains a single cell out of the targetdecorated cells, one or more ARM complexes bound to the single cell, and a lysis reagent inducing lysis of the single cell; capturing RNA released from the single cell on a solid surface or within a semi-permeable shell; generating a library of hybrid polynucleic acids that comprise a sequence from a transcript of the single cell and / or a sequence from the mRNA of the ARM complex; sequencing the library of hybrid polynucleic acids; and determining a presence or absence of binding of each of the one or more of the plurality of ABPs to their respective target antigen.

36. A RPP generated by the method of any one of claims 1-35.

37. A plurality of polynucleotides, wherein each polynucleotide encodes an ABP in the RPP of claim 36.

38. A plurality of polynucleotide constructs, comprising the plurality of polynucleotides of claim 36 cloned into an expression vector.

39. A plurality of host cells comprising the plurality of polynucleotides of claim 34 or the plurality' of polynucleotide constructs of claim 37.

40. A method of producing an RPP, the method comprising culturing the plurality of host cells of claim 39 under conditions for expression of the ABPs and isolating the ABPs.

41. A pharmaceutical composition comprising the RPP of claim 36 and a pharmaceutically acceptable excipient.

42. A method of treating a patient in need thereof by administering an effective amount of the pharmaceutical composition of claim 41.

43. The method of claim 42, wherein the patient has been exposed to the target molecule or complex or a variant thereof.

44. The method of claim 42 or 43, wherein the patient has a disease associated with the target molecule or complex or a variant thereof.

45. The method of any one of claims 42-44. wherein the pharmaceutical composition is administered intramuscularly, subcutaneously, intravenously, intradermally, orally, or through inhalation.

46. The method of any one of claims 42-45. wherein the effective amount is sufficient to treat the disease associated with the target molecule or complex or a variant thereof.

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