WO2024178239A1

WO2024178239A1 - Engineered cell lines and uses thereof

Info

Publication number: WO2024178239A1
Application number: PCT/US2024/016927
Authority: WO
Inventors: Taylor Schreiber; Abhinav Shukla; Vishruti Makani
Original assignee: Shattuck Labs Inc
Current assignee: Shattuck Labs Inc
Priority date: 2023-02-22
Filing date: 2024-02-22
Publication date: 2024-08-29
Anticipated expiration: 2025-08-22

Abstract

The present disclosure relates, inter alia, to improved host cells for the production of Fab and/or Fc domain-containing recombinant protein at high productivity and low levels of contaminants, methods of making the improved host cells, methods of culturing the improved host cells, and preparing proteins (without limitation, e.g., therapeutic proteins including monoclonal antibodies and Fc domain-containing fusion proteins) the improved host cells.

Description

ENGINEERED CELL LINES AND USES THEREOF

FIELD OF THE DISCLOSURE

The present disclosure relates to, in part, improved host cells for the production of recombinant proteins at high productivity and low levels of contaminants. The present disclosure also relates, in part, to methods of making such host cells, to culturing such host cells, and preparing proteins (without limitation, e.g., therapeutic proteins including monoclonal antibodies) using such host cells.

PRIORITY

This application claims the benefit of, and priority to, U.S. Provisional Application No. 63/486,401 , filed February 22, 2023, the contents of which are hereby incorporated by reference in their entirety.

BACKGROUND

Purified recombinant proteins find use in the laboratory and in the clinic. Recombinant proteins are produced by transfected cells, either mammalian cells or bacterial cells. When purifying recombinant proteins, the quantity and purity of the protein must be sufficient for experimental and/or clinical uses. Low yields and the presence of contaminants are two problems that occur during purification of such recombinant proteins. The contaminants may be derived from the cells used to produce the recombinant protein (including, but not limited to: Chinese hamster ovary cells, human embryonic kidney fibroblasts, Escherichia coli, etc.), and include host cell proteins (HCPs), nucleic acids, lipids, and other cellular material that may be released into the culture media along with the desired recombinant protein. The presence of HCPs in biotherapeutic recombinant proteins destined for the clinic may present a risk to the patient if not mitigated. The risk includes potential immunogenicity (including hypersensitivity to HCPs), which is often unpredictable, side effects caused by the HCPs (e.g., when HCPs have their own biological activities), and decrease in stability or activity of the biotherapeutic (e.g., when HCPs comprise proteases or other enzymes that modify the biotherapeutic). For the proteins used for experimental uses, the risk include data artifacts (e.g. caused by biological activities of HCPs). Some HCPs co-purify with product proteins by the virtue of their known associations, including some known interactions, with product proteins. Removing the HCPs from product proteins requires the use of additional downstream processing steps and results in increased costs and low yields.

Thus, methods for improving recombinant protein purification for experimental and/or clinical uses are needed. SUMMARY

Accordingly, the present disclosure relates to an engineered cell line that is adapted for high-level production of recombinant protein. In some aspects the engineered cell line disclosed herein improves recombinant protein yields, decreases production costs of recombinant protein, and/or reduces time and complexity of the process required to purify recombinant proteins.

In some aspects, the present disclosure relates to an engineered cell line adapted for production of a Fab and/or Fc domain-containing recombinant protein at high levels, e.g., as compared to the cell lines that are currently used. In some aspects, the engineered cell line comprising one or more mutation and/or a nucleic acid that causes a decrease in the amount or activity of clusterin or other host cell proteins. In embodiments, the modification is a hypomorphic mutation in clusterin gene or a regulatory element thereof. In embodiments, the modification is a clusterin null mutation. In embodiments, the modification is selected from an amino acid substitution mutation, insertion, deletion, frame-shift mutation, and non-sense mutation.

In some aspects, the present disclosure relates to an engineered cell line adapted for production of a Fab and/or Fc domain-containing recombinant protein, the engineered cell line comprising a modification that causes a decrease in the amount or activity of a host cell protein. In embodiments, the host cell protein is clusterin. In embodiments, the modification is a hypomorphic mutation in clusterin gene or a regulatory element thereof. In embodiments, the modification is a clusterin null mutation. In embodiments, the modification is selected from an amino acid substitution mutation, insertion, deletion, frame-shift mutation, and non-sense mutation. In embodiments, the modification is a conditional mutation (e.g., temperature sensitive mutation or an inducible deletion), a degron or an epigenetic alteration.

In embodiments, the modification is a genomic modification in the engineered cell line. In embodiments, the genomic modification is a heterozygous modification. In embodiments, the genomic modification is a homozygous modification.

In embodiments, modification is expression of a nucleic acid that causes the decrease in the amount or activity of clusterin. In embodiments, the nucleic acid is inserted on chromosome and/or located on an extrachromosomal element. In embodiments, the nucleic acid is selected from an antisense polynucleotide, an shRNA, an siRNA and an miRNA that causes the decrease in the amount or activity of clusterin.

In embodiments, the growth rate of the engineered cell line is at least about 30%, or at least about 40%, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 125%, or at least about 150%, or more compared to the growth rate of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin. In embodiments, the cell productivity of the engineered cell line in a culture is at least about 30%, or at least about 40%, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 125%, or at least about 150%, or more compared to the cell productivity of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin. In embodiments, the growth rate and/or the cell productivity of the engineered cell line in a culture is at least about 5%, or at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, greater compared to the cell productivity of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin.

In embodiments, the culture is selected from a fed-batch culture, a batch culture and a continuous culture.

In embodiments, the engineered cell line is a clusterin mutant derivative of a cell line selected from Chinese hamster ovary (CHO) cells, NS0 murine myeloma cells, PER.C6 human cells, baby hamster kidney (BHK21) cells, murine myeloma Sp2/0 cells, human embryonic kidney 293 (HEK293) cells, HT-1080 cells, Hela cells, CAP cells, HKB-11 cells, HuH-7 cells, Sf9 cells, and a derivative thereof. In embodiments, the engineered cell line is a clusterin mutant derivative of a cell line selected from CHO DUXB11 , CHO DG44, CHOK1 , ExpiCHO and Expi293.

In embodiments, the Fab and/or Fc domain-containing recombinant protein comprises a mammalian Fc domain. In embodiments, the Fc domain is selected from a human Fc domain, a mouse Fc domain, a rat Fc domain, a sheep Fc domain, a goat Fc domain, a donkey Fc domain, a feline Fc domain a hamster Fc domain, a guinea pig Fc domain, a horse Fc domain, a rabbit Fc domain, a pig Fc domain, a dog Fc domain, and a cow Fc domain. In embodiments, the Fc domain comprises a human Fc domain. In embodiments, the Fc domain comprises a Fc domain selected from an IgG Fc domain, an IgA Fc domain an IgM Fc domain, an IgE Fc domain and an IgD Fc domain. In embodiments, the IgG Fc domain is selected from an lgG1 Fc domain, an I gG2 Fc domain, an I gG3 Fc domain, and an I gG4 Fc domain. In embodiments, the IgA is selected from an lgA1 and an lgA2.

In embodiments, the fusion protein comprises the formula: (i) X-Fc, wherein X is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof; (ii) Fc-Y, wherein Y is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof; or (iii) X-Fc-Y, wherein X and Y are independently selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof. In embodiments, the X and/or Y is an antigen or a fragment thereof. In embodiments, the antigen is derived from a pathogen. In embodiments, the pathogen is selected from a virus, a bacterium, a protozoan, a parasite, and a fungus. In embodiments, the antigen is a cancer antigen. In embodiments, the cancer antigen is a neoantigen.

In embodiments, the X and/or Y is a mammalian intracellular protein or a fragment thereof. In embodiments, the X and/or Y is a mammalian secreted protein or a biologically active fragment thereof. In embodiments, the mammalian secreted protein is a coagulation factor, a cytokine or a serum protein. In embodiments, the X and/ or Y is independently a mammalian membrane protein, or a fragment thereof. In embodiments, the X is a Type I membrane protein, or a fragment thereof. In embodiments, the Type I membrane protein is selected from SIRPo/CD172a, TIM-3, BTLA, PD-1, CTLA-4, LAG-3, CD244, CSF1 R, CD160, TIGIT, 2B4, VISTA, VSIG8, LAG3, CD200, B7-H3, BTNL3, BTNL8, BTN2A1 , BTN3A1 , BTN3A2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H, and TMIGD2, or a fragment thereof. In embodiments, the Type I membrane protein is the extracellular domain or the ligand binding portion thereof. In embodiments, the Y is a mammalian membrane protein is a Type II membrane protein, or a fragment thereof. In embodiments, the Type II membrane protein is selected from CD40 ligand (CD40L), OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), 4-1 BB Ligand (4-1 BBL), CD70, CD30 ligand (CD30L), a C-type lectin domain (CLEC) family member, CD137 ligand, RANKL, TRAIL, FasL, TL1A, CD80, CD86, CD58, PD-1, SLAMF6, SIRPo and TGFBR2, or a fragment thereof. In embodiments, fragment of the Type II membrane protein is the extracellular domain or a ligand binding portion thereof.

In embodiments, the fusion protein is capable of modulating an immune response. In embodiments, the fusion protein is a vaccine.

In embodiments, the engineered cell line comprises additional one or more mutations that decrease the amount or activity of another host cell protein (HCP) that remains associated with the Fab and/or Fc domaincontaining recombinant protein after purification, optionally wherein the another HCP is selected from thrombospondin and lipoprotein lipase.

In embodiments, the engineered cell line comprises a nucleic acid encoding the Fab and/or Fc domaincontaining recombinant protein. In some aspects, the present disclosure relates to a cell bank comprising the engineered cell line any of the embodiments disclosed herein.

In some aspects, the present disclosure relates to a method of making a cell bank for production of a Fab and/or Fc domain-containing recombinant protein, comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, (b) obtaining a plurality of cells harboring a nucleic acid encoding the Fab and/or Fc domain-containing recombinant protein by introducing (e.g., transfecting) the nucleic acid in the engineered cell line, (c) optionally obtaining a clone of the cell, (d) optionally expanding the plurality of cells and/or the clone, (e) optionally aliquoting the plurality of cells and/or the clone, and (f) preparing the cell bank. In embodiments, the method optionally further comprises freezing the cell bank. In embodiments, the method optionally further comprises storing the cell bank at a temperature below about -20°C, or below about -80°C, optionally in a freezer, in liquid nitrogen or on dry ice. In embodiments, the method optionally further comprising culturing the plurality of cells and/or the clone, or an aliquot thereof.

In some aspects, the present disclosure relates to a Fab and/or Fc domain-containing recombinant protein prepared by using a cell bank comprising the engineered cell line of any of the embodiments disclosed herein.

In some aspects, the present disclosure relates to a method of culturing the clusterin mutant cells, comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, wherein the engineered cell line optionally harbors a nucleic acid encoding the Fab and/or Fc domain-containing recombinant protein, (b) contacting the engineered cell line with a medium that supports growth of the engineered cell line, (c) incubating at a temperature and for a time that supports growth of the engineered cell line, optionally wherein the temperature is in the range of about 25°C to about 55°C, optionally wherein CO2 is provided during incubation at a level that supports growth of the engineered cell line, optionally wherein the level is in the range of about 2% and about 20%. In embodiments, the culturing is selected from a fed-batch culturing, a batch culturing and a continuous culturing. In embodiments, the culture conditions are selected from fed- batch, batch and continuous culture.

In some aspects, the present disclosure relates to a Fab and/or Fc domain-containing recombinant protein prepared by culturing the engineered cell line of any of the embodiments disclosed herein, optionally wherein the culturing is performed using any of the methods disclosed herein.

In some aspects, the present disclosure relates to a method for isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein, the method comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, wherein the engineered cell line is capable of expressing the Fab and/or Fc domain-containing recombinant protein, (b) culturing the engineered cell line in a growth medium, at a temperature and for a time that supports growth of the engineered cell line, (c) obtaining a cell pellet and/or a culture supernatant; and (d) isolating and/or purifying the Fab and/or Fc domain-containing recombinant protein from the cell pellet and/or the culture supernatant. In embodiments, the temperature is in the range of about 25°C to about 45°C In embodiments, the time is at least about 4 hours, or at least about 12 hours, or at least about 1 day, or at least about 2 days, or at least about 4 days, or at least about 7 days, or at least about 10 days, or at least about 14 days, or at least about 20 days, or at least about 30 days. In embodiments, the method further comprises providing CO2 during culturing, optionally at a level that supports growth of the engineered cell line, optionally wherein the level is in the range of about 2% and about 20%.

In embodiments, the culture supernatant is a clarified supernatant. In embodiments, the culture supernatant or the clarified supernatant is obtained by centrifugation and/or filtration of the culture. In embodiments, isolating and/or purifying comprises a purification step optionally selected from a chromatography step, a precipitation step, and extraction step

In some aspects, the present disclosure relates to a Fab and/or Fc domain-containing recombinant protein prepared according to the method of any of the embodiments disclosed herein. In embodiments, the Fab and/or Fc domain-containing recombinant protein is isolated and/or purified. In embodiments, the Fab and/or Fc domain-containing recombinant protein is at least about 90%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 99.2%, or at least about 99.4%, or at least about 99.5% pure. In embodiments, the Fab and/or Fc domain-containing recombinant protein comprises reduced amount of at least one host cell protein (HCP) compared to the Fab and/or Fc domain-containing recombinant protein prepared using a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin. In embodiments, the HCP is clusterin. In embodiments, the Fab and/or Fc domain-containing recombinant protein is substantially free of clusterin.

In some aspects, the present disclosure relates to a composition comprising the Fab and/or Fc domaincontaining recombinant protein of any of the embodiments disclosed herein. In some aspects, the present disclosure relates to a composition comprising an isolated and/or purified Fab and/or Fc domain-containing recombinant protein prepared according to the method of any of the embodiments disclosed herein. In some aspects, the present disclosure relates to a composition comprising an isolated and/or purified Fab and/or Fc domain-containing recombinant protein prepared using the engineered cell line of any of the embodiments disclosed herein.

Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates that a preparation of an Fc domain-containing chimeric protein prepared using conventional methods comprises various individual CHO host cell proteins (HCPs). HCP profiling of harvest material from a satellite run shows that clusterin is the most abundant HCP.

FIG. 2 shows genomic organization of the clusterin gene, which is located on chromosome 1 of Chinese hamster. Clusterin gene contains 11 exons. Clusterin is a secreted glycoprotein consisting of two chains, o- clusterin and |3-cl usterin . Secretory clusterin, which works as a chaperone facilitating clearance of misfolded proteins, has a molecular weight of 75-80 kDa.

FIG. 3 diagrammatically illustrates the generation of a knockout cell line using a knock-in technology.

FIG. 4 shows the a gRNA selection scheme for the generation of a knockout cell line.

FIG. 5 shows the analysis of CHOCKO (CHO Clusterin KO) stable pools for the detection of intracellular clusterin (left panel) and secreted clusterin (right panel) by western blotting.

FIG. 6 shows the growth curve (solid lines) and viability (dotted lines) of a CHOCKO stable pools in comparison with a clusterin-*- control clone and a clone that received Cas9 but not gRNA.

FIG. 7 shows a clusterin dot blot of CHOCKO clones.

FIG. 8 shows the analysis of five CHOCKO clones for secreted clusterin by western blotting. Culture supernatant of five CHOCKO clones were analyzed by Western blot using non-reduced (lane “N”) or reduced (lane “R”) conditions. A clustering control clones (indicated as “Con”) was used as a positive control.

FIG. 9A and FIG. 9B show the culture profile of three CHOCKO clone in a 14 day fed batch. FIG. 9A shows a growth curve and FIG. 9B shows the viability as a function of time.

FIG. 10 is a bar graph showing relative expression of clusterin mRNA in top 11 clusterin-deletant clones as determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). FIG. 11 A and FIG. 11 B show the western blot analysis of top 7 clusterin-deletant clones under non-reduced (FIG. 11 A) or reduced (FIG. 11 B) conditions.

FIG. 12 is a bar graph showing the population doubling time of the DG44-CHOCKO cell line in comparison with the parental CHO DG44 parent cell line.

FIG. 13 is a bar graph illustrating the expression of monoclonal antibody molecules in the DG44-CHOCKO cell line.

DETAILED DESCRIPTION

The present disclosure is based, in part, on the discovery and construction of a clusterin-modulated cell line, which is well-adapted for high-level production of Fab and/or Fc domain-containing recombinant protein. This engineered cell line provides improved yields, decreased production costs, and reduced time required to purify recombinant proteins, e.g., in comparison with a cell line without clusterin modulation.

Previous studies have shown that a small number of HCPs that are not related to each other form major contaminants. Singh et al., Understanding the mechanism of copurification of “difficult to remove” host cell proteins in rituximab biosimilar products, Biotechnology Progress 36:1-12 (2020); Levy et al., Identification and characterization of host cell protein product-associated impurities in monoclonal antibody bioprocessing, Biotechnol Bioeng 111 (5):904-12 (2014); Zhang et al., Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry, MAbs. 2014;6(3):659-70; Zhang et al., Characterization of the co-elution of host cell proteins with monoclonal antibodies during protein A purification, Biotechnol Prog 32(3)708-17 (2016); Gilgunn et al., Identification and tracking of problematic host cell proteins removed by a synthetic, highly functionalized nonwoven media in downstream bioprocessing of monoclonal antibodies, Journal of Chromatography A 1595: 28-38 (2019); Esser-Skala et al., Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins, Analytical and Bioanalytical Chemistry 412: 6583-6593 (2020); Molden et al., Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development, Mabs, 13 (1): e1955811 (2021); Wilson et al., Identification and classification of host cell proteins during biopharmaceutical process development, Biotechnol Progress. 38:e3224 (2022).

Some HCPs co-purify with the product proteins by the virtue of their known to associations, including some known interactions, with the product proteins. For example, clusterin is an extracellular chaperone that binds to a large number of proteins. Clusterin has been shown to bind to a variety of molecules with high affinity including lipids, peptides, and proteins. Bailey et al., Clusterin, a Binding Protein with a Molten Globule-like Region, Biochemistry 40(39) 11828-11840 (2001). Clusterin is a major contaminant of Fc domain-containing chimeric proteins made from commonly used cells lines such as CHO and its derivative, possibly because it binds the Fc and Fab regions of IgG. Wilson and Easterbrook-Smith, Biochim Biophys Acta 1159(3):319-26 (1992).

Traditional techniques use harsh conditions for HCP removal. These harsh conditions include low pH, very low or very high salt concentration, washes with buffers that include protein denaturants (e.g., urea and guanidine hydrochloride) chemicals like arginine, sodium caprylate, organic solvents (e.g., ethanol, acetone and methanol), and extra chromatography steps. Aboulaich et al., A Novel Approach to Monitor Clearance of Host Cell Proteins Associated With Monoclonal Antibodies, Biotechnol Prog. 30(5): 1114-1124 (2014); Tscheliessnig et al., Ethanol Precipitation for Purification of Recombinant Antibodies, Journal of Biotechnology 188: 17-28 (2014); Jeon et al., Optimization for Simultaneous Removal of Product/Process- Related Impurities of Peptide Fc-Fusion Protein Using Cation Exchange Chromatography, Processes 10(11), 2359 (2022). As a consequence, the high cost of manufacturing Fc- and Fab-containing proteins (e.g., Fc fusion proteins and monoclonal antibodies) derives mainly from the purification process, which contributes to 50%— 80% of the total manufacturing cost. See Tuameh et al., Methods for Addressing Host Cell Protein Impurities in Biopharmaceutical Product Development, Biotechnology Journal 18(3):e2200115 (2023).

Accordingly, in various aspects the present disclosure relates to clusterin mutant cells, methods of making cell banks of the clusterin mutant cells for producing a protein of interest, methods of culturing the clusterin mutant cells, methods of amplifying the cell banks, and methods of preparing proteins using clusterin mutant cells. In embodiments, the culture is selected from a fed-batch culture, a batch culture and a continuous culture. In various aspects and embodiments, the clusterin knockout cell lines disclosed herein efficiently produce various Fc- and Fab-containing proteins. In various aspects and embodiments, allow a streamlined downstream process (e.g., that does not require harsh chromatography conditions required to remove HCPs) and still yield at least about 30% higher overall purified product with significant improvement in product quality. Accordingly, in various aspects and embodiments, the clusterin knockout cell lines disclosed herein produce Fc- and Fab-containing proteins (e.g., Fc fusion proteins and monoclonal antibodies) with high yield, low production cost, higher overall purified product yield with significantly improved product quality. Clusterin Mutant Cell Lines and Cell Banks

In some aspects, the present disclosure relates to an engineered cell line adapted for high-level production of a Fab and/or Fc domain-containing recombinant protein, the engineered cell line comprising one or more mutation and/or a nucleic acid that causes a decrease in the amount or activity of clusterin. In embodiments, the modification is a hypomorphic mutation in clusterin gene or a regulatory element thereof. In embodiments, the modification is a clusterin null mutation. In embodiments, the modification is selected from an amino acid substitution mutation, insertion, deletion, frame-shift mutation, and non-sense mutation.

In some aspects, the present disclosure relates to an engineered cell line adapted for production of a Fab and/or Fc domain-containing recombinant protein, the engineered cell line comprising a modification that causes a decrease in the amount or activity of clusterin. In embodiments, the modification is a hypomorphic mutation in clusterin gene or a regulatory element thereof. In embodiments, the modification is a clusterin null mutation. In embodiments, the modification is selected from an amino acid substitution mutation, insertion, deletion, frame-shift mutation, and non-sense mutation. In embodiments, the modification is a conditional mutation (e.g., temperature sensitive mutation or an inducible deletion), a degron or an epigenetic alteration.

In embodiments, modification is expression of a nucleic acid that causes the decrease in the amount or activity of clusterin. In embodiments, the nucleic acid is inserted on chromosome and/or located on an extrachromosomal element. In embodiments, the nucleic acid is selected from an antisense polynucleotide, an shRNA, an siRNA and an miRNA that causes the decrease in the amount or activity of clusterin. In embodiments, the growth rate of the engineered cell line is at least about 30%, or at least about 40%, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 125%, or at least about 150%, or more compared to the growth rate of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin. In embodiments, the cell productivity of the engineered cell line in a culture is at least about 30%, or at least about 40%, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 125%, or at least about 150%, or more compared to the cell productivity of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin. In embodiments, the growth rate of the engineered cell line in a culture is at least about 5%, or at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, greater compared to the cell productivity of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin. In embodiments, the cell productivity of the engineered cell line in a culture is at least about 5%, or at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, greater compared to the cell productivity of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin.

In embodiments, the engineered cell line is derived by knocking out clusterin from any cell line choice. In embodiments, the engineered cell line is derived using a CRISPR-Cas9 system. In embodiments, the engineered cell line is derived via an insertion or deletion induced by a single guide RNA (gRNA). In embodiments, the engineered cell line is derived by conventional mutagenesis using a mutagen. In an illustrative embodiment, the mutagen is methylnitronitrosoguanidine (MNNG). In embodiments, the engineered cell line is derived via transposon mutagenesis. In embodiments, the engineered cell line is derived from a clusterin mutant animal. In embodiments, the engineered cell line is derived from a clusterin knock-out animal.

In embodiments, the engineered cell line is derived from a cell line any origin. In illustrative embodiments, the engineered cell line is a derivative of a Chinese hamster cell line, human cell line, African green monkey cell line, mouse cell line, etc. In another illustrative embodiment, the engineered cell line is a derivative of a Chinese hamster cell line. In embodiments, the engineered cell line is a derivative of a cell line selected from Chinese hamster ovary (CHO) cells, NS0 murine myeloma cells, PER.C6 human cells, baby hamster kidney (BHK21) cells, murine myeloma Sp2/0 cells, human embryonic kidney 293 (HEK293) cells, HT-1080 cells, Hela cells, CAP cells, HKB-11 cells, HuH-7 cells, Sf9 cells, and a derivative thereof. In embodiments, the engineered cell line is a clusterin mutant derivative of a cell line selected from CHO DUXB11, CHO DG44, CHOK1 , ExpiCHO and Expi293.

In embodiments, the engineered cell line disclosed herein comprises a hypomorphic mutation in clusterin gene or a regulatory element thereof. In embodiments, the modification is a clusterin null mutation. In embodiments, the modification is selected from an amino acid substitution mutation, insertion, deletion, frame-shift mutation, and non-sense mutation. In embodiments, the Fab and/or Fc domain-containing recombinant protein that may be purified using the engineered cell line disclosed herein comprises a clusterin knockout. In embodiments, the engineered cell line disclosed herein comprises a homozygous clusterin knockout. In embodiments, the engineered cell line disclosed herein is derived from a homozygous clusterin knockout animal. Mclaughlin et al., Apolipoprotein J/clusterin limits the severity of murine autoimmune myocarditis, J. Clin. Invest. 106: 1105-1113 (2000); Han et al., Clusterin contributes to caspase-3- independent brain injury following neonatal hypoxia-ischemia, Nat. Med. 7: 338-343 (2001).

In embodiments, the engineered cell line comprises additional one or more mutations that decrease the amount or activity of another host cell protein (HCP) that remains associated with the Fab and/or Fc domaincontaining recombinant protein after purification, optionally wherein the another HCP is selected from thrombospondin and lipoprotein lipase. In embodiments, the engineered cell line comprises at least one, or at least about two, or at least about three, or more mutations that decrease the amount or activity of at least one, or at least two or at least about three host cell proteins (HCPs) that associate with the Fab and/or Fc domain-containing recombinant proteins during purification. In embodiments, the HCPs are selected from clusterin, thrombospondin and lipoprotein lipase.

In embodiments, the engineered cell line comprises a nucleic acid encoding the Fab and/or Fc domaincontaining recombinant protein.

In some aspects, the present disclosure relates to a cell bank comprising the engineered cell line any of the embodiments disclosed herein.

In some aspects, the present disclosure relates to a Fab and/or Fc domain-containing recombinant protein prepared using a cell bank comprising the engineered cell line of any of the embodiments disclosed herein. In some aspects, the present disclosure relates to a Fab and/or Fc domain-containing recombinant protein prepared by culturing plurality of cells and/or the clone of the engineered cell line of any of the embodiments disclosed herein, or an aliquot thereof. In some aspects, the present disclosure relates to a Fab and/or Fc domain-containing recombinant protein prepared by culturing a cell bank comprising the engineered cell line of any of the embodiments disclosed herein.

In some aspects, the present disclosure relates to a method of culturing the clusterin mutant cells, comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, wherein the engineered cell line optionally harbors a nucleic acid encoding the Fab and/or Fc domain-containing recombinant protein, (b) contacting the engineered cell line with a medium that supports growth of the engineered cell line, (c) incubating at a temperature and for a time that supports growth of the engineered cell line, optionally wherein the temperature is in the range of about 25°C to about 55°C, optionally wherein CO2 is provided during incubation at a level that supports growth of the engineered cell line, optionally wherein the level is in the range of about 2% and about 20%. In embodiments, the culturing is selected from a fed-batch culturing, a batch culturing and a continuous culturing. In embodiments, the culture conditions are selected from fed- batch, batch and continuous culture

Protein Purification using Clusterin Mutant Cell Lines

In some aspects, the present disclosure relates to purification of a recombinant protein (without limitation, e.g., a Fab and/or Fc domain-containing recombinant protein) expressed by an engineered cell of any of the embodiments disclosed herein. In embodiments, purification comprises culturing the engineered cell, obtaining cell-free extract and/or culture supernatant, optionally clarifying the cell-free extract and/or culture supernatant, and subjecting the optionally clarified cell-free extract and/or culture supernatant to a chromatography step, a precipitation step, and/or an extraction step. In embodiments, at least one purification step is liquid chromatography.

In one aspect, the present disclosure provides a method for isolating and/or purifying a chimeric protein from a solution, wherein the chimeric protein has the structure selected from: X-Linker and Linker-Y wherein the X or the Y is selected from a mammalian secreted protein, mammalian intracellular protein, a mammalian membrane protein, an antigen, or a fragment thereof, and the linker comprises an Fc domain, the method comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, (b) introducing a nucleic acid encoding the chimeric protein in the engineered cell line, (c) culturing the engineered cell line harboring the nucleic acid encoding the chimeric protein, (d) obtaining a solution comprising the chimeric protein (without limitation, e.g., a culture supernatant, a clarified culture supernatant and a cell-free extract), and (e) contacting the solution comprising the chimeric protein with a matrix capable of binding the chimeric protein (without limitation, e.g., a suitable chromatography column or membrane).

In one aspect, the present disclosure provides a method for isolating and/or purifying a chimeric protein from a solution, wherein the chimeric protein has the structure selected from: X-Linker and Linker-Y wherein the X or the Y is selected from a mammalian secreted protein, mammalian intracellular protein, a mammalian membrane protein, an antigen, or a fragment thereof, and the linker comprises an Fc domain, the method comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, wherein the engineered cell line is capable of expressing the chimeric protein, (b) culturing the engineered cell line in a growth medium, at a temperature and for a time that supports growth of the engineered cell line, (c) obtaining a cell pellet and/or a culture supernatant; and (d) isolating and/or purifying the chimeric protein from the cell pellet and/or the culture supernatant. In embodiments, the temperature is in the range of about 25°C to about 45°C In embodiments, the time is at least about 4 hours, or at least about 12 hours, or at least about 1 day, or at least about 2 days, or at least about 4 days, or at least about 7 days, or at least about 10 days, or at least about 14 days, or at least about 20 days, or at least about 30 days. In embodiments, the method further comprises providing CO2 during culturing, optionally at a level that supports growth of the engineered cell line, optionally wherein the level is in the range of about 2% and about 20%.

In one aspect, the present disclosure provides a method for isolating and/or purifying a chimeric protein from a solution, wherein the chimeric protein has the structure: X-Linker-Y wherein the X and the Y are independently absent or selected from a mammalian secreted protein, mammalian intracellular protein, a mammalian membrane protein, an antigen, or a fragment thereof, and the linker comprises an Fc domain, the method comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, (b) introducing a nucleic acid encoding the chimeric protein in the engineered cell line, (c) culturing the engineered cell line harboring the nucleic acid encoding the chimeric protein, (d) obtaining a solution comprising the chimeric protein (without limitation, e.g., a culture supernatant, a clarified culture supernatant and a cell-free extract), and (e) contacting the solution comprising the chimeric protein with a matrix capable of binding the chimeric protein (without limitation, e.g., a suitable chromatography column or membrane). In embodiments, the chimeric protein is selected from PD1-Fc-OX40L, PD1-Fc-GITRL, SIRPa/CD172a-Fc- CD40L, CD172a-Fc-OX40L, PD1-Fc-TL1A, BTLA-Fc-OX40L, TMIGD2-Fc-OX40L, TIM3-Fc-OX40L, TIM3- Fc-CD40L, PD1 -Fc-4-1 BBL, CD172a-Fc-LIGHT, VSIG8-Fc-4-1 BBL, VSIG8-Fc-CD30L, VSIG8-Fc-CD40L, VSIG8-Fc-FasL, VSIG8- Fc-GITRL, VSIG8-FC-LIGHT, VSIG8-Fc-TL1A, and VSIG8-Fc-TRAIL, CSF1 R-Fc- CD40L, TIGIT-Fc-4-1 BBL, TIGIT-Fc-GITRL, TIGIT- Fc-LIGHT, TIGIT-Fc-OX40L, TIGIT-Fc-TL1 A, PD-1 -Fc- LIGHT, CD86-FC-NKG2A, CD80-Fc-NKG2A, CD48-Fc-NKG2A, PD-1-FC-NKG2A, SLAMF6-FC-NKG2A, SIRPo-Fc-NKG2A, TGFBR2-Fc-NKG2A, TGFBR2-Fc-4-1 BBL, BTNL2A1/BTNL3A1-Fc-scFv, BTNL3A1/BTNL3A2-Fc-scFv, BTNL3A1/BTNL3A3-Fc-scFv, BTNL3 /BTNL8-Fc-scFv, IL-35-Fc-IL-6R (a heterodimer of IL12a-Fc-IL6RA/Gp130 and IL27 -Fc-Gp130/IL6RA), IL-6R-Fc-IL-35 (a heterodimer of IL6RA-Fc-IL12o/IL27p and Gp130-Fc- IL27p/IL12o), IL-35-Fc-IFNyR (a heterodimer of IL12a-Fc- IFNgR/IFNGR2 and IL27p-Fc- IFNGR2/IFNgR), IFNyR-Fc-IL-35 (a heterodimer of IFNgR-Fc-IL12o/IL27p and IFNGR2-Fc-IL27p/IL12a), IL-35-Fc-IL-21 R (a heterodimer of IL12a-Fc-IL-21 r/IL-2rg and IL27p-Fc-IL-2rg /IL-21 r), IL-21 R-Fc-IL-35 (a heterodimer of IL-21 r-Fc-IL12a/IL270 and IL-2rg-Fc- IL27p/IL12a), IL-35-Fc- integrin a4 7 (a heterodimer of IL12a-Fc-integrin a4/integrin 07 and IL270-Fc-integrin 07/integrin a4), integrin a407-Fc-IL-35 (a heterodimer of integrin a4-Fc-IL12a/IL270 and integrin 07-Fc- IL270/IL12a), IL-35-Fc- MAdCAM (a heterodimer of IL12a-Fc-MAdCAM and IL270-Fc-MAdCAM), MAdCAM-Fc-IL-35 (a heterodimer of MAdCAM-Fc-IL12a and MAdCAM-Fc-IL270), IL-35-Fc-DcR3 (a heterodimer of I L12a-Fc-DcR3 and IL270- Fc-DcR3), and IL-6R-Fc-IL-35 (a heterodimer of DcR3-Fc-IL12a and DcR3-Fc- IL270).

In one aspect, the present disclosure provides a method for isolating and/or purifying a chimeric protein from a solution, wherein the chimeric protein has the structure: X-Linker-Y wherein the X and the Y are independently absent or selected from a mammalian secreted protein, mammalian intracellular protein, a mammalian membrane protein, an antigen, or a fragment thereof, and the linker comprises an Fc domain, the method comprising: (a) providing the engineered cell line any of the embodiments disclosed herein, wherein the engineered cell line is capable of expressing the chimeric protein, (b) culturing the engineered cell line in a growth medium, at a temperature and for a time that supports growth of the engineered cell line, (c) obtaining a cell pellet and/or a culture supernatant; and (d) isolating and/or purifying the chimeric protein from the cell pellet and/or the culture supernatant. In embodiments, the temperature is in the range of about 25°C to about 45°C In embodiments, the time is at least about 4 hours, or at least about 12 hours, or at least about 1 day, or at least about 2 days, or at least about 4 days, or at least about 7 days, or at least about 10 days, or at least about 14 days, or at least about 20 days, or at least about 30 days. In embodiments, the method further comprises providing CO2 during culturing, optionally at a level that supports growth of the engineered cell line, optionally wherein the level is in the range of about 2% and about 20%. In embodiments, the chimeric protein is selected from PD1-Fc-OX40L, PD1 -Fc-GITRL, SIRPa/CD172a-Fc-CD40L, CD172a- FC-OX40L, PD1-FC-TL1A, BTLA-Fc-OX40L, TMIGD2-Fc-OX40L, TIM3-Fc-OX40L, TIM3-Fc-CD40L, PD1- Fc-4-1 BBL, CD172a-Fc-LIGHT, VSIG8-Fc-4-1 BBL, VSIG8-Fc-CD30L, VSIG8-Fc-CD40L, VSIG8-Fc-FasL, VSIG8- Fc-GITRL, VSIG8-FC-LIGHT, VSIG8-Fc-TL1A, and VSIG8-Fc-TRAIL, CSF1 R-Fc-CD40L, TIGIT-Fc- 4-1 BBL, TIGIT-Fc-GITRL, TIGIT- Fc-LIGHT, TIGIT-FC-OX40L, TIGIT-Fc-TLI A, PD-1 -Fc-LIGHT, CD86-Fc- NKG2A, CD80-Fc-NKG2A, CD48-Fc-NKG2A, PD-1-Fc-NKG2A, SLAMF6-Fc-NKG2A, SIRPa-Fc-NKG2A, TGFBR2-FC-NKG2A, TGFBR2-Fc-4-1 BBL, BTNL2A1/BTNL3A1-Fc-scFv, BTNL3A1/BTNL3A2-Fc-scFv, BTNL3A1/BTNL3A3-Fc-scFv, BTNL3 /BTNL8-Fc-scFv, IL-35-Fc-IL-6R (a heterodimer of IL12a-Fc- IL6RA/Gp130 and IL270-Fc-Gp130/IL6RA), IL-6R-Fc-IL-35 (a heterodimer of IL6RA-Fc-IL12a/IL27p and Gp130-Fc- IL27p/IL12a), IL-35-Fc-IFNyR (a heterodimer of IL12a-Fc-IFNgR/IFNGR2 and IL27p-Fc- IFNGR2/IFNgR), IFNyR-Fc-IL-35 (a heterodimer of IFNgR-Fc-IL12a/IL27p and IFNGR2-Fc-IL27p/IL12o), IL- 35-FC-IL-21 R (a heterodimer of IL12a-Fc-IL-21r/IL-2rg and IL27p-Fc-IL-2rg /IL-21r), IL-21 R-Fc-IL-35 (a heterodimer of IL-21 r-Fc-IL12a/IL27 and IL-2rg-Fc- IL27 /IL12o), IL-35-Fc-integrin a4 7 (a heterodimer of IL12a-Fc-integrin a4/integrin 07 and IL27p-Fc-integrin p7/integrin a4), integrin a4p7-Fc-l L-35 (a heterodimer of integrin a4-Fc-IL12o/IL27p and integrin P7-Fc- IL27p/IL12a), IL-35-Fc-MAdCAM (a heterodimer of IL12a- Fc-MAdCAM and IL27p-Fc-MAdCAM), MAdCAM-Fc-IL-35 (a heterodimer of MAdCAM-Fc-IL12o and MAdCAM-Fc-IL27P), IL-35-Fc-DcR3 (a heterodimer of IL12o-Fc-DcR3 and IL27p-Fc-DcR3), and IL-6R-Fc- IL-35 (a heterodimer of DcR3-Fc-IL12o and DcR3-Fc- IL27P).

In embodiments, the culture supernatant is a clarified supernatant. In embodiments, the culture supernatant or the clarified supernatant is obtained by centrifugation and/or filtration of the culture. In embodiments, isolating and/or purifying comprises a purification step optionally selected from a chromatography step, a precipitation step, and extraction step.

In embodiments, the method further comprises at least one, or at least two or at least three purification steps. In embodiments, the at least one, or at least two or at least three purification steps are or comprise liquid chromatography.

In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein is more streamlined compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein includes lesser number of purification steps compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein includes lesser number of chromatography steps compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein does not require a harsh chromatography conditions that are required to remove HCPs. In embodiments, the harsh chromatography conditions are selected from low pH, very low or very high salt concentration, protein denaturants, detergents, protein precipitants. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein does not use high salt buffers (e.g., buffers comprising at least about 1 M, or at least about 1 .5 M, or at least about 2 M, or at least about 2.5 M or more NaCI). In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein does not use low salt buffers (e.g., buffers comprising less than about 100 mM, or less than about 50 mM, or less than about 25 mM, or less than about 10 M or less NaCI). In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein uses lower amounts of protein denaturants (e.g., guanidine hydrochloride, urea, and sodium caprylate) compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein does not use protein denaturants (e.g., guanidine hydrochloride, urea, and sodium caprylate). In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein does not use detergents (e.g., sodium caprylate) or uses lower amounts of detergents compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein does not use organic solvents (e.g., ethanol, methanol and acetone) or uses lower amounts of organic solvents compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein does not use low pH buffers (e.g., having pH of less than about 4.5, or less than about 4, or less than about 3.5, or less than about 3, or less than about 2.5, or less). In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein yields at least about 10%, at least about 20%, at least about 30%, or at least about 50%, at least about 75%, or at least about 100%, or at least about 150%, or more isolated and/or purified Fab and/or Fc domain-containing recombinant protein compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein comprises at least about 10%, at least about 20%, at least about 30%, or at least about 40%, at least about 50%, or more or more decrease in amount of at least one HCPs other than clusterin compared to methods that do not use a clusterin mutant cell line. In embodiments, the method of isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein disclosed herein yields isolated and/or purified Fab and/or Fc domain-containing recombinant protein that comprises at least about 10%, at least about 20%, at least about 30%, or at least about 40%, at least about 50%, or more or more reduction in amount of at least one HCPs other than clusterin compared to methods that do not use a clusterin mutant cell line.

Proteins that maybe Purified using Clusterin Mutant Cell Lines Disclosed Herein

Clusterin is an extracellular chaperone that binds to a large number of proteins. Clusterin has been shown to bind to a variety of molecules with high affinity including lipids, peptides, and proteins. Bailey et al., Clusterin, a Binding Protein with a Molten Globule-like Region, Biochemistry 40(39) 11828-11840 (2001). For example, clusterin binds the Fc and Fab regions of IgG. Wilson and Easterbrook-Smith, Biochim Biophys Acta 1159(3):319-26 (1992). Accordingly, in embodiments, the engineered cell line disclosed herein may be used for purifying any protein. In embodiments, any proteins produced in mammalian cell systems may be purified using the engineered cell line disclosed herein. In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) may be purified using the engineered cell line disclosed herein.

In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) comprises a mammalian Fc domain. In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domaincontaining fusion protein) comprises a Fc domain selected from a human Fc domain, a mouse Fc domain, a rat Fc domain, a sheep Fc domain, a goat Fc domain, a donkey Fc domain, a feline Fc domain a hamster Fc domain, a guinea pig Fc domain, a horse Fc domain, a rabbit Fc domain, a pig Fc domain, a dog Fc domain, and a cow Fc domain. In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) comprises a human Fc domain, a human Fab domain and/or a humanized Fab domain.

In embodiments, the Fab and/or Fc domain-containing recombinant protein that may be purified using the engineered cell line disclosed herein comprises a mammalian Fc domain. In embodiments, the Fc domain is selected from a human Fc domain, a mouse Fc domain, a rat Fc domain, a sheep Fc domain, a goat Fc domain, a donkey Fc domain, a feline Fc domain a hamster Fc domain, a guinea pig Fc domain, a horse Fc domain, a rabbit Fc domain, a pig Fc domain, a dog Fc domain, and a cow Fc domain. In embodiments, the Fc domain comprises a human Fc domain. In embodiments, the Fc domain comprises a Fc domain selected from an IgG Fc domain, an IgA Fc domain an IgM Fc domain, an IgE Fc domain and an IgD Fc domain. In embodiments, the IgG Fc domain is selected from an lgG1 Fc domain, an lgG2 Fc domain, an lgG3 Fc domain, and an lgG4 Fc domain. In embodiments, the IgA is selected from an lgA1 and an I gA2. In embodiments, the Fab and/or Fc domain-containing recombinant protein is an antibody, an antibody-like molecule, or a derivative thereof. In embodiments, the Fab and/or Fc domain-containing recombinant protein is a monoclonal antibody. In embodiments, the Fab and/or Fc domain-containing recombinant protein is a monoclonal antibody selected from lgG1 , lgG2, lgG3, lgG4, lgA1, lgA2, IgM and IgE antibody. In embodiments, the Fab and/or Fc domain-containing recombinant protein is an antibody selected from lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2, IgM and IgE antibody. In embodiments, the derivative of the antibody is selected from Fab, Fd, F(ab')2, Fab', and Fv, or a binding fragment thereof. In embodiments, the derivative of the antibody-like molecule is selected from a single-domain antibody, a heavy-chain-only antibody (VHH), a single-chain antibody (scFv), scFv, ScFv-Fc, a diabody, a ScFv-CH, a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody or a binding fragment thereof.

In embodiments, the Fab and/or Fc domain-containing recombinant protein is a fusion protein. In embodiments, the fusion protein is bispecific or tri specific. In embodiments, the fusion protein is selected from Fab-scFv, Fab-L-scFv, Fab-H-scFv, tribody, Fab-(scFv)2, a TriFab, a Fab-Fab fusion protein, a scFv-Fc- Fab fusion protein, a bispecific T-cell engager (BiTE), a Fab-Fv fusion protein, and a Fab-dsFv fusion protein. In embodiments, the fusion protein is an Fc fusion protein. In embodiments, the Fc fusion protein comprises a fusion partner selected from single-chain Fv (scFv), single-chain diabody (scDb), Fv, Fab, and F(ab')2.

In embodiments, the fusion protein comprises the formula: (i) X-Fc, wherein X is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof; (ii) Fc-Y, wherein Y is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof; or (iii) X-Fc-Y, wherein X and Y are independently selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof. In embodiments, the X and/or Y is an antigen or a fragment thereof. In embodiments, the antigen is derived from a pathogen. In embodiments, the pathogen is selected from a virus, a bacterium, a protozoan, a parasite, and a fungus. In embodiments, the antigen is a cancer antigen. In embodiments, the cancer antigen is a neoantigen. In embodiments, the X and/or Y is a mammalian intracellular protein or a fragment thereof. In embodiments, the X and/or Y is a mammalian secreted protein or a biologically active fragment thereof. In embodiments, the mammalian secreted protein is a coagulation factor, a cytokine or a serum protein. In embodiments, the cytokine is selected from IFN-a, IFN-p, IFN-E, IFN-K, IFN-W IFN-y, IL-1a, IL-1 p, IL-1 , IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-35, TNF, leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte colony-stimulating factor (G-CSF), TNF-a, TNF-p, lymphotoxin (LT)-p, LIGHT, Fas ligand (FasL)/CD178, TGF-p1, TGF-p2, TGF-p3, XCL1 , XCL2, CCL1 , CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11 , CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1 , CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CX3CL1 , Epo, Tpo, SCF, and FLT- 3L.

In embodiments, the X and/or Y is a mammalian membrane protein selected from SLAMF4, IL-2 R a, 4- 1 BB/TNFRSF9, IL-2 R p, ALCAM, B7-1 , IL-4 R, B7-H3, BLAME/SLAMF4, CEACAM1, IL-6 R, IL-7 Ra, IL- 10R a, IL-I 0 R p, IL-12 R p 1 , IL-12 R p 2, CD2, IL-13 R a 1 , IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, Integrin a 4/CD49d, CDS, Integrin a E/CD103, CD6, Integrin a M/CD 11 b, CDS, Integrin a X/CD11c, Integrin p 2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, Leukotriene B4-R1 , CD84/SLAMF5, NCAM-L1 , CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common y Chain/IL-2 R y, Osteopontin, CRACC/SLAMF7, PD-1 , CRTAM, PSGL-1 , CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1 , L-Selectin, SIRP p1 , SLAM, TCCR/WSX-1 , DNAM-1 , Thymopoietin, EMMPRIN/CD147, TIM-1 , EphB6, TIM- 2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fey RIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1 B, TRAIL RI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM- 2/CD102, TRAILR3/TNFRSF10C,IFN-yR1, TRAILR4/TNFRSF10D, IFN-y R2, TSLP, IL-1 R1 and TSLP R, or a fragment thereof.

In embodiments, the X and/ or Y is independently a mammalian membrane protein, or a fragment thereof. In embodiments, the X is a Type I membrane protein, or a fragment thereof. In embodiments, the Type I membrane protein is selected from SIRPa/CD172a, TIM-3, BTLA, PD-1 , CTLA-4, LAG-3, CD244, CSF1 R, CD160, TIGIT, 2B4, VISTA, VSIG8, LAG3, CD200, B7-H3, BTNL3, BTNL8, BTN2A1 , BTN3A1, BTN3A2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H, and TMIGD2, or a fragment thereof. In embodiments, the Type I membrane protein is the extracellular domain or the ligand binding portion thereof. In embodiments, the Y is a mammalian membrane protein is a Type II membrane protein, or a fragment thereof. In embodiments, the Type II membrane protein is selected from CD40 ligand (CD40L), OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), 4-1 BB Ligand (4-1 BBL), CD70, CD30 ligand (CD30L), a C- type lectin domain (CLEC) family member, CD137 ligand, RANKL, TRAIL, FasL, TL1A, CD80, CD86, CD58, PD-1 , SLAMF6, SIRPo and TGFBR2, or a fragment thereof. In embodiments, fragment of the Type II membrane protein is the extracellular domain or a ligand binding portion thereof.

In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) comprises a Fc domain selected from an IgG Fc domain, an IgA Fc domain an IgM Fc domain, an IgE Fc domain and an IgD Fc domain. In embodiments, the IgG Fc domain is selected from an I gG1 Fc domain, an I gG2 Fc domain, an I gG3 Fc domain, and an I gG4 Fc domain. In embodiments, the IgA is selected from an I gA1 and an lgA2.

In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) is an immunoglobulin. In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) is an antibody, an antibody-like molecule, or a derivative thereof. In embodiments, the derivative of the antibody is selected from Fab, Fd, F(ab')2, Fab', and Fv, or a binding fragment thereof. In embodiments, the derivative of the antibody-like molecule is selected from a single-domain antibody, a heavy-chain-only antibody (VHH), a single-chain antibody (scFv), scFv, ScFv-Fc, a diabody, a ScFv-CH, a shark heavy-chain- only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody or a binding fragment thereof. These types of binding agents are disclosed In US Patent Nos. or Patent Publication Nos. US 7,417,130, US 2004/132094, US 5,831,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794,144, US 2010/239633, US 7,803,907, US 2010/119446, and/or US 7,166,697, the contents of which are hereby incorporated by reference in their entireties. See also, Storz MAbs. 2011 May-Jun; 3(3): 310-317. In embodiments, the Fc domain is a mammalian Fc domain. In embodiments, the chimeric protein comprises a Fc domain selected from a human Fc domain, a mouse Fc domain, a rat Fc domain, a sheep Fc domain, a goat Fc domain, a donkey Fc domain, a feline Fc domain a hamster Fc domain, a guinea pig Fc domain, a horse Fc domain, a rabbit Fc domain, a pig Fc domain, a dog Fc domain, and a cow Fc domain. In embodiments, the chimeric protein comprises a human Fc domain. In embodiments, the chimeric protein comprises a Fc domain selected from an IgG Fc domain, an IgA Fc domain, an IgM Fc domain, an IgE Fc domain and an IgD Fc domain. In embodiments, the IgG Fc domain is selected from an lgG1 Fc domain, an lgG2 Fc domain, an lgG3 Fc domain, and an I gG4 Fc domain. In embodiments, the IgA is selected from an lgA1 and an lgA2.

In embodiments, the X and/or Y is independently an antigen, a mammalian intracellular protein, a mammalian secreted protein, a mammalian membrane protein, or a fragment thereof. In embodiments, the X and/or Y is an antigen, wherein the antigen is derived from a pathogen. In embodiments, the pathogen is selected from a virus, a bacterium, a protozoan, a parasite, and a fungus. In embodiments, the antigen is a cancer antigen. In embodiments, the cancer antigen is a neoantigen.

In embodiments, the X and/or Y is a mammalian intracellular protein, or a fragment thereof.

In embodiments, the X and/or Y is a mammalian secreted protein, or a fragment thereof. In embodiments, the secreted protein is a cytokine. In embodiments, the cytokine is selected from I FN-a, I FN- , IFN-s, IFN-K, IFN-w IFN-y, IL-1a, IL-1 p, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-15, IL-16, IL- 17, IL-18, IL-19, IL-20, IL-35, TNF, leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte colonystimulating factor (G-CSF), TNF-a, TNF-p, lymphotoxin (LT)-p, LIGHT, Fas ligand (FasL)/CD178, TGF-01 , TGF-p2, TGF-p3, XCL1 , XCL2, CCL1 , CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11 , CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1 , CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CX3CL1, Epo, Tpo, SCF, and FLT-3L.

In embodiments, the X and/or Y is a mammalian membrane protein, or a fragment thereof. In embodiments, the mammalian membrane protein is selected from SLAMF4, IL-2 R a, 4-1 BB/TNFRSF9, IL-2 R , ALCAM, B7-1, IL-4 R, B7-H3, BLAME/SLAMF4, CEACAM1 , IL-6 R, IL-7 Ra, IL-1 OR a, IL-1 0 R p, IL-12 R p 1, IL-12 R p 2, CD2, IL-13 R a 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, Integrin a 4/CD49d, CDS, Integrin a E/CD103, CD6, Integrin a M/CD 11 b, CDS, Integrin a X/CD11 c, Integrin 2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1 , KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, Leukotriene B4-R1 , CD84/SLAMF5, NCAM-L1 , CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F- 10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common y Chain/IL-2 R y, Osteopontin, CRACC/SLAMF7, PD- 1 , CRTAM, PSGL-1 , CTLA-4, RANK/TNFRSF11A, CX3CR1 , CX3CL1, L-Selectin, SIRP 1, SLAM, TCCR/WSX-1 , DNAM-1 , Thymopoietin, EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fey RIII/CD16, TIM-6, TNFR1/TNFRSF1 A, Granulysin, TNF RIII/TNFRSF1 B, TRAIL RI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAILR3/TNFRSF10C,IFN-yR1 , TRAILR4/TNFRSF10D, IFN-y R2, TSLP, IL-1 R1 and TSLP R, or a fragment thereof.

In embodiments, the X is a mammalian membrane protein is a Type I membrane protein, or a fragment thereof. In embodiments, the Type I membrane protein is selected from SIRPa/CD172a, TIM-3, BTLA, PD- 1 , CTLA-4, LAG-3, CD244, CSF1 R, CD160, TIGIT, 2B4, VISTA, VSIG8, LAG3, CD200, B7-H3, BTNL3, BTNL8, BTN2A1 , BTN3A1 , BTN3A2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H, and TMIGD2, or a fragment thereof. In embodiments, the fragment of the Type I membrane protein is the extracellular domain or the ligand binding portion thereof.

In embodiments, the Y is a mammalian membrane protein is a Type II membrane protein, or a fragment thereof. In embodiments, the Type II membrane protein is selected from CD40 ligand (CD40L), OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), 4-1 BB Ligand (4-1 BBL), CD70, CD30 ligand (CD30L), a C- type lectin domain (CLEC) family member, CD137 ligand, RANKL, TRAIL, FasL, TL1A, CD80, CD86, CD58, PD-1 , SLAMF6, SIRPa and TGFBR2, or a fragment thereof. In embodiments, the fragment of the Type II membrane protein is the extracellular domain thereof. In embodiments, the Type II membrane protein is the ligand binding portion thereof.

In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) is a fusion protein. In embodiments, the fusion protein is a Fab fusion protein. In embodiments, the Fab fusion protein is bispecific or tri specific. In embodiments, the Fab fusion protein is selected from Fab-scFv, Fab-L-scFv, Fab-H-scFv, tri body, Fab-(scFv)2, a Tri Fab, a Fab- Fab fusion protein, a scFv-Fc-Fab fusion protein, a bispecific T-cell engager (BiTE), a Fab-Fv fusion protein, and a Fab-dsFv fusion protein.

In embodiments, the fusion protein is an Fc fusion protein. In embodiments, the Fc fusion protein comprises a fusion partner selected from single-chain Fv (scFv), single-chain diabody (scDb), Fv, Fab, and F(ab')2. In embodiments, the fusion protein comprises the formula: (i) X-Fc, wherein X is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof; (ii) Fc-Y, wherein Y is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof; or (iii) X-Fc-Y, wherein X and Y are independently selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof.

In embodiments, the X and/or the Y are an antigen or a fragment thereof. In embodiments, the antigen is derived from a pathogen. In embodiments, the pathogen is selected from a virus, a bacterium, a protozoan, a parasite, and a fungus. In embodiments, the antigen is a cancer antigen. In embodiments, the cancer antigen is a neoantigen.

In embodiments, the X and/or the Y are a mammalian intracellular protein or a fragment thereof.

In embodiments, a mammalian secreted protein or a biologically active fragment thereof. In embodiments, the mammalian secreted protein is a coagulation factor, a cytokine or a serum protein. In embodiments, the cytokine is selected from IFN-a, IFN-0, IFN-e, IFN-K, IFN-W IFN-y, IL-1a, IL-1 , IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-35, TNF, leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte colony-stimulating factor (G-CSF), TNF-a, TNF-0, lymphotoxin (LT)-P, LIGHT, Fas ligand (FasL)/CD178, TGF-p1, TGF-02, TGF-03, XCL1 , XCL2, CCL1 , CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11 , CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1 , CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CX3CL1 , Epo, Tpo, SCF, and FLT- 3L.

In embodiments, the X and/or the Y are a mammalian membrane protein, or a fragment thereof. In embodiments, the mammalian membrane protein is selected from SLAMF4, IL-2 R a, 4-1 BB/TNFRSF9, IL- 2 R 0, ALCAM, B7-1 , IL-4 R, B7-H3, BLAME/SLAMF4, CEACAM1, IL-6 R, IL-7 Ra, IL-1 OR a, IL-I 0 R P, IL- 12 R p 1, IL-12 R p 2, CD2, IL-13 R a 1 , IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, Integrin a 4/CD49d, CDS, Integrin a E/CD103, CD6, Integrin a M/CD 11 b, CDS, Integrin a X/CDUc, Integrin p 2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1 , CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1 , KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1 , CD45, LAIR2, CDS3, Leukotriene B4-R1, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common y Chain/IL-2 R y, Osteopontin, CRACC/SLAMF7, PD-1 , CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, SIRP 1, SLAM, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1 , EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fey RIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1 B, TRAIL RI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAILR3/TNFRSF10C,IFN-yR1 , TRAILR4/TNFRSF10D, IFN-y R2, TSLP, IL-1 R1 and TSLP R, or a fragment thereof.

In embodiments, the X and/or Y is independently a mammalian membrane protein, or a fragment thereof. In embodiments, the X is a Type I membrane protein, or a fragment thereof. In embodiments, the Type I membrane protein is selected from SIRPa/CD172a, TIM-3, BTLA, PD-1 , CTLA-4, LAG-3, CD244, CSF1 R, CD160, TIGIT, 2B4, VISTA, VSIG8, LAG3, CD200, B7-H3, BTNL3, BTNL8, BTN2A1 , BTN3A1, BTN3A2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H, and TMIGD2, or a fragment thereof. In embodiments, the fragment of the Type I membrane protein is the extracellular domain or the ligand binding portion thereof. In embodiments, the Y is a mammalian membrane protein is a Type II membrane protein, or a fragment thereof. In embodiments, the Type II membrane protein is selected from CD40 ligand (CD40L), OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), 4-1 BB Ligand (4-1 BBL), CD70, CD30 ligand (CD30L), a C-type lectin domain (CLEC) family member, CD137 ligand, RANKL, TRAIL, FasL, TL1A, CD80, CD86, CD58, PD-1 , SLAMF6, SIRPa and TGFBR2, or a fragment thereof.

In embodiments, the Type I transmembrane protein is selected from PD 1 , TIM 3, CD172a(SIRPo), TIGIT, CD115 (CSF1R), BTLA, TMIGD2, and VSIG8, or a variant thereof. In embodiments, the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4 1 BBL (CD137L), GITRL, TL1A, CD30L, LIGHT, and CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is selected from PD 1 , TIM 3, CD172a(SIRPa), TIGIT, CD115 (CSF1 R), BTLA, TMIGD2, and VSIG8, ora variant thereof; and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4 1 BBL (CD137L), GITRL, TL1A, CD30L, LIGHT, and CD70, or a variant thereof.

In embodiments, the Type I transmembrane protein is PD 1 , or a variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4 1 BBL (CD137L), GITRL, TL1A, CD30L, and CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is TIM 3, or a variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4- 1 BBL (CD137L), GITRL, TL1A, CD30L, LIGHT, or CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is CD172a(SIRPo), or a variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4 1 BBL (CD137L), TL1A, CD30L, or CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is TIGIT, or a variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4-1 BBL (CD137L), GITRL, TL1A, CD30L, LIGHT, or CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is CD115 (CSF1R), ora variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4 1 BBL (CD137L), TL1A, CD30L, or CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is BTLA, or a variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4-1 BBL (CD137L), GITRL, TL1A, CD30L, LIGHT, or CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is TMIGD2, or a variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4-1 BBL (CD137L), GITRL, TL1A, CD30L, LIGHT, or CD70, or a variant thereof. In embodiments, the Type I transmembrane protein is VSIG8, or a variant thereof, and the Type II transmembrane protein is selected from OX40L, CD40L, LIGHT (CD258), 4-1 BBL (CD137L), GITRL, TL1 A, CD30L, LIGHT, or CD70, or a variant thereof.

In embodiments, the Type I transmembrane protein is PD-1 and the Type II transmembrane protein is OX40L; the Type I transmembrane protein is PD-1 and the Type II transmembrane protein is GITRL; the Type I transmembrane protein is PD-1 and the Type II transmembrane protein is 4-1 BBL; the Type I transmembrane protein is PD-1 and the Type II transmembrane protein is LIGHT; the Type I transmembrane protein is PD-1 and the Type II transmembrane protein is TL1A; the Type I transmembrane protein is SIRPa/CD172a and the Type II transmembrane protein is CD40L; the Type I transmembrane protein is SIRPa/CD172a and the Type II transmembrane protein is OX40L; the Type I transmembrane protein is SIRPo/CD172a and the Type II transmembrane protein is LIGHT; the Type I transmembrane protein is BTLA and the Type II transmembrane protein is OX40L; the Type I transmembrane protein is TMIGD2 and the Type II transmembrane protein is OX40L; the Type I transmembrane protein is TIM3 and the Type II transmembrane protein is OX40L; the Type I transmembrane protein is TIM3 and the Type II transmembrane protein is CD40L; the Type I transmembrane protein is VSIG8 and the Type II transmembrane protein is OX40L; the

Type I transmembrane protein is VSIG8 and the Type II transmembrane protein is 4-1 BBL; the Type I transmembrane protein is VSIG8 and the Type transmembrane protein is CD30L; the Type I transmembrane protein is CSF1R and the Type

transmembrane protein is CD40L; the Type I transmembrane protein is CSF1R and the Type

transmembrane protein is CD40L; the Type I transmembrane protein is TIGIT and the Type II transmembrane protein is 4-1 BBL; the Type I transmembrane protein is TIGIT and the Type II transmembrane protein is GITRL; the Type I transmembrane protein is TIGIT and the Type II transmembrane protein is LIGHT; the Type I transmembrane protein is TIGIT and the Type II transmembrane protein is OX40L; or the Type I transmembrane protein is TIGIT and the Type II transmembrane protein is TL1 A.

In embodiments, the Type I transmembrane protein is selected from CD86, CD80, CD48, PD-1 , SIRPa, SLAMF6, and TGFBR; and wherein the Type II transmembrane protein is NKG2A. In embodiments, the Type I transmembrane protein is TGFBR2, and the Type II transmembrane protein is selected from 4-1 BB Ligand (4-1 BBL), CD30 Ligand (CD30L) and an NKG2 receptor. In embodiments, the Type I transmembrane protein is FLT3L, and the Type II transmembrane protein is selected from CD40L, 4-1 BBL, OX40L, and GITRL.

In embodiments, the fragment of the Type II membrane protein is the extracellular domain or a ligand binding portion thereof. In embodiments, the fusion protein is selected from PD1-Fc-OX40L, PD1-Fc-GITRL, SIRPd/CD172a-Fc-CD40L, CD172a-Fc-OX40L, PD1-Fc-TL1A, BTLA-Fc-OX40L, TMIGD2-Fc-OX40L, TIM3- FC-OX40L, TIM3-Fc-CD40L, PD1 -Fc-4-1 BBL, CD172a-Fc-LIGHT, VSIG8-Fc-4-1 BBL, VSIG8-Fc-CD30L, VSIG8-Fc-CD40L, VSIG8-Fc-FasL, VSIG8- Fc-GITRL, VSIG8-FC-LIGHT, VSIG8-Fc-TL1A, and VSIG8-Fc- TRAIL, CSF1 R-Fc-CD40L, TIGIT-Fc-4-1 BBL, TIGIT-Fc-GITRL, TIGIT- Fc-LIGHT, TIGIT-FC-OX40L, TIGIT- Fc-TLIA, PD-1 -Fc-LIGHT, CD86-Fc-NKG2A, CD80-Fc-NKG2A, CD48-Fc-NKG2A, PD-1-Fc-NKG2A, SLAMF6-FC-NKG2A, SIRPa-Fc-NKG2A, TGFBR2-Fc-NKG2A, TGFBR2-Fc-4-1 BBL, BTNL2A1/BTNL3A1- Fc-scFv, BTNL3A1/BTNL3A2-Fc-scFv, BTNL3A1/BTNL3A3-Fc-scFv, BTNL3 /BTNL8-Fc-scFv, IL-35-Fc-IL- 6R (a heterodimer of IL12o-Fc-IL6RA/Gp130 and IL27 -Fc-Gp130/IL6RA), IL-6R-Fc-IL-35 (a heterodimer of IL6RA-Fc-IL12a/IL27p and Gp130-Fc- IL270/IL12a), IL-35-Fc-IFNyR (a heterodimer of IL12a-Fc- IFNgR/IFNGR2 and IL270-FC- IFNGR2/IFNgR), IFNyR-Fc-IL-35 (a heterodimer of IFNgR-Fc-IL12a/IL270 and IFNGR2-FC-IL270/IL12a), IL-35-Fc-IL-21 R (a heterodimer of IL12a-Fc-IL-21 r/IL-2rg and IL27p-Fc-IL-2rg /IL-21 r), IL-21 R-Fc-IL-35 (a heterodimer of IL-21 r-Fc-IL12a/IL27 and IL-2rg-Fc- IL27p/IL12a), IL-35-Fc- integrin a4p7 (a heterodimer of IL12a-Fc-integrin a4/integrin P7 and IL27p-Fc-integrin p7/integrin a4), integrin a4p7-Fc-IL-35 (a heterodimer of integrin a4-Fc-IL12a/IL27p and integrin P7-Fc- IL27p/IL12a), IL-35-Fc- MAdCAM (a heterodimer of IL12a-Fc-MAdCAM and IL27p-Fc-MAdCAM), MAdCAM-Fc-IL-35 (a heterodimer of MAdCAM-Fc-IL12a and MAdCAM-Fc-IL27p), IL-35-Fc-DcR3 (a heterodimer of I L12a-Fc-DcR3 and IL27p- Fc-DcR3), and IL-6R-Fc-IL-35 (a heterodimer of DcR3-Fc-IL12a and DcR3-Fc- IL27P).

In embodiments, the chimeric protein comprises an extracellular domain from BTLA and an extracellular domain from OX40L, e.g., BTLA-FC-OX40L.

In embodiments, the chimeric protein comprises an extracellular domain from CSF1 R and an extracellular domain from CD40L, e.g., CSF1 R-Fc-CD40L. In embodiments, the chimeric protein comprises an extracellular domain from PD-1 and an extracellular domain from CD40L, e.g., PD-1-Fc-CD40L.

In embodiments, the chimeric protein comprises an extracellular domain from PD1 and an extracellular domain from LIGHT, e.g., PD1-Fc-LIGHT.

In embodiments, the chimeric protein comprises an extracellular domain from PD-1 and an extracellular domain from 4-1 BBL, e.g., PD-1 -Fc-4-1 BBL.

In embodiments, the chimeric protein comprises an extracellular domain from PD-1 and an extracellular domain from GITRL, e.g., PD-1-Fc-GITRL.

In embodiments, the chimeric protein comprises an extracellular domain from PD-1 and an extracellular domain from OX40L, e.g., PD-1-Fc-OX40L.

In embodiments, the chimeric protein comprises an extracellular domain from PD-1 and an extracellular domain from TL1 A, e.g., PD-1-Fc-TL1A.

In embodiments, the chimeric protein comprises an extracellular domain from SIRPo and an extracellular domain from LIGHT, e.g., SIRP1 a-Fc-LIGHT.

In embodiments, the chimeric protein comprises an extracellular domain from SIRPo and an extracellular domain from CD40L, e.g., SIRPo-Fc-CD40L.

In embodiments, the chimeric protein comprises an extracellular domain from SIRPo and an extracellular domain from OX40L, e.g., SIRPo-Fc-OX40L.

In embodiments, the chimeric protein comprises an extracellular domain from TIGIT and an extracellular domain from LIGHT, e.g., TIGIT-Fc-LIGHT.

In embodiments, the chimeric protein comprises an extracellular domain from TIGIT and an extracellular domain from OX40L, e.g., TIGIT-Fc-OX40L.

In embodiments, the chimeric protein comprises an extracellular domain from TIM-3 and an extracellular domain from CD40L, e.g., TIM-3-Fc-CD40L.

In embodiments, the chimeric protein comprises an extracellular domain from TIM3 and an extracellular domain from OX40L, e.g., TIM3-Fc-OX40L. In embodiments, the chimeric protein comprises an extracellular domain from TMIGD2 and an extracellular domain from OX40L, e.g., TMIGD2-Fc-OX40L.

In embodiments, the chimeric protein comprises an extracellular domain from VSIG8 and an extracellular domain from OX40L, e.g., VSIG8-Fc-OX40L. In embodiments, a chimeric protein is capable of binding murine ligand(s)/receptor(s).

In embodiments, a chimeric protein is capable of binding human ligand(s)/receptor(s).

Exemplary proteins that may be purified using the engineered cell line disclosed herein are disclosed in PCT International Publication Nos. WO 2017/059168, WO 2018/157163, WO 2018/157164, WO 2018/157165, WO 2018/157162, WO 2019/246508, WO 2020/047325, WO 2020/047327, WO 2020/047328, WO 2020/047329, WO 2020/047319, WO 2020/047322, WO 2020/146393, WO 2020/176718, WO 2020/232365, the contents of each of which are hereby incorporated by reference in their entireties.

In embodiments, each X domain and Y domain of the chimeric protein is independently capable of binding to its cognate receptor or ligand with a KD of about 1 nM to about 5 nM, for example, about 1 nM, about 1 .5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM. In embodiments, the chimeric protein binds to a cognate receptor or ligand with a KD of about 5 nM to about 15 nM, for example, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, about 10 nM, about 10.5 nM, about 11 nM, about 11.5 nM, about 12 nM, about 12.5 nM, about 13 nM, about 13.5 nM, about 14 nM, about 14.5 nM, or about 15 nM.

In embodiments, each X domain and Y domain of the chimeric protein is independently capable of binding to its cognate receptor or ligand with a KD of less than about 1 pM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 150 nM, about 130 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM, or about 1 nM (as measured, for example, by surface plasmon resonance or biolayer interferometry). In embodiments, the chimeric protein binds to human cognate receptor or ligand with a KD of less than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (as measured, for example, by surface plasmon resonance or biolayer interferometry).

Cells and Nucleic Acids

Aspects of the present invention include a clusterin mutant host cell of any of the embodiments disclosed herein comprising an expression vector that encodes a Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an Fc domain-containing protein). In embodiments, the Fab and/or Fc domaincontaining recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) (without limitation, e.g., an Fc domain-containing protein) is translated as a single unit. In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domaincontaining fusion protein) (without limitation, e.g., an Fc domain-containing protein) is producible as a secretable and fully functional single protein chain. In embodiments, the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) refers to a Fab and/or Fc domain-containing recombinant protein of multiple protein chains, e.g., multiple extracellular domains disclosed herein, that are combined (via covalent or no-covalent bonding) to yield a single unit, e.g., in vitro (e.g., with one or more synthetic linkers disclosed herein).

In embodiments, the expression vector comprises a nucleic acid encoding the Fab and/or Fc domaincontaining recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) (without limitation, e.g., an Fc domain-containing protein) disclosed herein. In embodiments, the expression vector comprises DNA or RNA. In embodiments, the expression vector is a mammalian expression vector.

Both prokaryotic and eukaryotic vectors can be used for expression of the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) (without limitation, e.g., an Fc domain-containing protein). Prokaryotic vectors include constructs based on E. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 and APL. Nonlimiting examples of prokaryotic expression vectors may include the Agt vector series such as Agt11 (Huynh et al., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89). Prokaryotic hostvectorsystems cannot perform much of the post-translational processing of mammalian cells, however. Thus, eukaryotic host- vector systems may be particularly useful. A variety of regulatory regions can be used for expression of the Fc domain-containing proteins (without limitation, e.g., an Fc domain-containing protein) in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the p-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) in the clusterin mutant host cell of any of the embodiments disclosed herein.

Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function. As used herein, the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).

Expression systems functional in human cells are well known in the art, and include viral systems. A promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3’ terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.

There are varieties of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some situations, it is desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).

Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacterio!., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667- 678, 2004), sleeping beauty, transposases of the mariner family (Plasterk et al, supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al, Ann. Rev. Pharm. Toxicol, 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.

In embodiments, the expression vectors for the expression of the Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) are viral vectors. Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21 : 1 17, 122, 2003. Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and a viruses, though other viral vectors may also be used. For in vivo uses, viral vectors that do not integrate into the host genome are suitable for use, such as a viruses and adenoviruses. Illustrative types of a viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and Antiviruses. In embodiments, the invention provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention.

Aspects of the present invention include the clusterin mutant host cell of any of the embodiments disclosed herein comprising the expression vector which comprises the Fab and/or Fc domain-containing recombinant protein (without limitation, e.g., an antibody or Fc domain-containing fusion protein) (without limitation, e.g., an Fc domain-containing protein) disclosed herein.

Expression vectors can be introduced into the clusterin mutant host cell of any of the embodiments disclosed herein for producing the present Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein). Cells may be cultured in vitro or genetically engineered, for example. Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler in “Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.). These include monkey kidney cell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651 ); human embryonic kidney lines e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells (e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells, (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51). Illustrative cancer cell types for expressing the Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) disclosed herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, EG7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.

Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection (ATCC), or from commercial suppliers.

The clusterin mutant host cells that can be used for production of Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) in vitro, ex vivo, and/or in vivo include, without limitation, clusterin mutant epithelial cells, endothelial cells, kerati nocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells. The choice of cell type depends on the type of tumor or infectious disease being treated or prevented, and can be determined by one of skill in the art.

According to the present invention, Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) may be purified using the clusterin mutant host cell of any of the embodiments disclosed herein using specific solid substrates/solid supports, e.g., beads and chromatography resins, or using chromatography methods that do not depend upon Protein A capture. In embodiments, the Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) may be purified in an oligomeric state, or in multiple oligomeric states, and enriched for a specific oligomeric state using specific methods. Without being bound by theory, these methods could include treatment with specific buffers including specified salt concentrations, pH and additive compositions. In other examples, such methods could include treatments that favor one oligomeric state over another. The Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domaincontaining protein) obtained herein may be additionally ‘polished’ using methods that are specified in the art. In embodiments, the Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) are highly stable and able to tolerate a wide range of pH exposure (between pH 3-12), are able to tolerate a large number of freeze/thaw stresses (greater than 3 freeze/thaw cycles) and are able to tolerate extended incubation at elevated temperatures (longer than 2 weeks at 40 degrees C). In embodiments, the Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) are shown to remain intact, without evidence of degradation, deamidation, etc. under such stress conditions. Isolated/purified Fab and/or Fc domain-containing recombinant proteins (without limitation, e.g., an Fc domain-containing protein) may be stored in liquid form for some period of time, frozen for extended periods of time or in some cases lyophilized.

Subjects and/or Animals

In embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In embodiments, the subject and/or animal may comprise fluorescently tagged cells (with e.g. GFP). In embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell.

In embodiments, the subject and/or animal is a human. In embodiments, the human is a pediatric human. In embodiments, the human is an adult human. In embodiments, the human is a geriatric human. In embodiments, the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.

In embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.

EXAMPLES

The examples herein are provided to illustrate advantages and benefits of the present disclosure and to further assist a person of ordinary skill in the art with preparing a clusterin mutant cell and using the clusterin mutant cell for the production of proteins (without limitation, e.g., Fc domain-containing proteins). The examples herein are also presented in order to more fully illustrate the preferred aspects of the present disclosure. The examples should in no way be construed as limiting the scope of the present disclosure, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects or embodiments of the present disclosure described above. The variations, aspects or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present disclosure.

Example 1: The Individual CHO Host Cell Proteins (HCPs) that Form Contaminants in Preparations of an Fc Domain-Containing Protein Purified Using Conventional Methods

Purification of Fc domain-containing proteins (e.g., antibodies and Fc fusion proteins) from mammalian cell expression systems typically involves growth of transfected mammalian cells (e.g., Chinese hamster ovary (CHO) cells and derivatives) in large bioreactors. The cells are typically harvested by centrifugation and/or filtration to yield a clarified supernatant. The protein is then substantially enriched with an affinity chromatography resin, such as protein A or FcXL chromatography and additional processing steps to efficiently purify the protein from other host cell impurities. See, e.g., McCue et al., Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein, Biologicals 43(4): 213-219 (2015).

To purify a chimeric protein (the SIRPa-Fc-CD40L chimeric protein, which is a homomultimeric -500 kDa protein), a conventional scheme was developed. In brief, CHO cells were transfected with a DNA construct encoding the chimeric protein having an N-terminal secretion signal. Cells were grown in a bioreactor. A clarified harvest was generated from the culture supernatant by filtration of intact cells, organelles and other large protein complexes, which was used as the starting material for the purification of SIRPo-Fc-CD40L chimeric protein. The clarified harvest was subjected to affinity chromatography using FcXL resin and additional processing steps. The FcXL resin captures Fc domain of the protein and thereby purifies the chimeric protein. See, e.g., McCue et al., Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein, Biologicals 43(4): 213-219 (2015). The resultant purified preparation was highly enriched for the chimeric protein but it contained elevated level of contaminating proteins. A systematic identification of individual CHO host cell proteins that contaminate the protein was undertaken. Mass spectrometry was performed after purification according to the above scheme. As shown in FIG. 1, clusterin was highly abundant in the purified sample of the protein containing an Fc domain. Clusterin was found to be a main protein that was consistently found to be a contaminant. These data suggest, inter alia, that due to the larger size of the chimeric protein (-500 kDa), several host cell proteins associate tightly with the protein molecule. A small number of product-specific host cell proteins, specifically clusterin, were believed to be bound to the chimeric protein.

Example 2: Construction of a Clusterin Mutant Cells

A clusterin knock-out mutant engineered cell line was generated using a CRISPR-based knock-in technology.

FIG. 2 shows genomic organization of the clusterin gene, which is located on chromosome 1 of Chinese hamster. Clusterin gene contains 11 exons. Clusterin is a secreted glycoprotein consisting of two chains, a- clusterin and -cl usterin . Secretory clusterin, which works as a chaperone facilitating clearance of misfolded proteins, has a molecular weight of 75-80 kDa.

FIG. 3 diagrammatically illustrates the generation of a knockout engineered cell line using a knock-in technology. FIG. 4 shows the a gRNA selection scheme for the generation of a knockout cell line. Briefly, two neighboring single strand breaks were generated on the two opposite strands of the clusterin gene using Cas9, which were repaired by the cells using one of two possible pathways. A repair by non-homologous end joining (NHEJ) in the context of sgRNA can create a loss of function mutation (including a null mutation and a hypomorphic mutation). The mutations generated by NHEJ point mutations, including substation, small insertions or deletions, sometimes causing frameshift). A repair using homologous recombination the using a donor DNA as template can create any desired mutation such as an insertion, deletion of point mutation, as is designed on the template DNA. These mutations can also be hypomorphic mutations (such as amino acid substitutions) or null mutations (such as amino acid substitution, insertion, deletion, frame-shift mutation, non-sense mutation, etc.).

CHO DG44 cells expressing a chimeric protein (“the parental clonal cell line” expressing the SIRPa-Fc- CD40L chimeric protein, which is also referred to herein as “ARC”) were stably electroporated with pSpCas9- BB-2A-Puro vector (Genscript) with or without clusterin sgRNA and HiUGE DisrupTag-2A-Puromycin vectors (CasTag Biosciences) to knockout clusterin from the parental clonal cell line. The SIRPa-Fc-CD40L clonal cell line was transfected only with pSpCas9-BB-2A-Puro vector were used as Cas9 control cell line. Following transfection, stably transfected cells were selected using different concentrations of puromycin to generate CHO Clusterin KO (CHOCKO) stable pools and Cas9 control pools.

CHOCKO stable pools were evaluated for cell growth, viability and cell productivity. Briefly, CHOCKO pools were passaged for 4-5 passages using Excell-Advanced CHO Fed-batch medium in the presence of puromycin prior to inoculation for 14 day shake flask fed batch. CHOCKO stable pools and Cas9 control pools were seeded at 0.5 million cells/mL using 500 mL shake flask at a working volume of 120 mL. The shake flask cultures reached peak viable cell density (VCD) of 14-16 million cells/mL. Day 14 fed batch harvest samples were assessed for both intracellular and secreted clusterin by western blotting. Cell culture supernatants and cell lysates from respective fed batch harvest samples were exposed to reducing (lane “R” in FIG. 5) and non-reducing conditions (lane “N” in FIG. 5), followed by running them on SDS-PAGE. Purified recombinant clusterin (left lanes) and the Cas9 control pool (indicated as “ARC-control” clones) were used as positive controls. The gels were probed using a biotin-conjugated anti-CHO clusterin-5 antibody and detected using IRDye 680-conjugated streptavidin. As shown in FIG. 5, the non-reduced heterodimeric form of clusterin has a molecular mass of 75-80 kDa, whereas reduced clusterin monomer migrates at ~37 kDa. The western blotting data clearly shows >90% decrease in levels of intracellular and secreted clusterin in CHOCKO pools compared to Cas9 control pools (FIG. 5).

These results indicate, inter alia, that a clusterin mutant cell line was generated.

Example 3: Characterization of the Clusterin Mutant Cells Expressing an Fc domain-Containing Protein

CHOCKO stable pools were evaluated for cell growth, viability and cell productivity. Briefly, CHOCKO pools were passaged for 4-5 passages using Excell-Advanced CHO Fed-batch medium in the presence of puromycin prior to inoculation for 14 day shake flask fed batch. CHOCKO stable pools and Cas9 control pools were seeded at 0.5 million cells/mL using 500 mL shake flask at a working volume of 120 mL. FIG. 6 shows the growth curve (solid lines) and viability (dotted lines). The shake flask cultures reached peak viable cell density (VCD) of 14-16 million cells/mL. The growth trends were remarkably similar between CHOCKO pools (indicated by “1 ” in FIG. 6) and Cas9 control pools (indicated by “2” in FIG. 6) with harvest day (day 14) viability above 95%, suggesting clusterin knockout did not adversely affect CHO cell growth and viability. As shown in FIG. 6, CHOCKO stable pools and CAS9 control showed higher peak VCD compared to ARC parent clone (the parental clonal cell line expressing the SIRPa-Fc-CD40L chimeric protein, indicated by “3” in FIG. 6). However, 14 day fed batch titer was comparable, suggesting clusterin KO did not affect cell productivity (FIG. 6).

These results indicate, inter alia, that the clusterin mutant cell lines disclosed herein grow as well as clusterin+ cell lines.

Moreover, day 14 harvest samples were evaluated for expression of the chimeric protein in CHOCKO and Cas9 control pools by FcXL HPLC method. Both CHOCKO and Cas9 control pools showed comparable day 14 harvest titer, suggesting, inter alia, that clusterin knockout did not affect the productivity of the chimeric protein by the parental clonal cell line.

To generate sufficient material for downstream processing and further confirm the comparability of CHOCKO pool growth profile to that of Cas9 control and the parental clonal cell line (“ARC parent clone” expressing SIRPo-Fc-CD40L chimeric protein or), CHOCKO and Cas9 control stable pools along with the chimeric protein parent clone were inoculated in 3L production bioreactors for 14 day fed-batch. The growth profiles and peak VCDs for CHOCKO and Cas9 control pools trended slightly higher than the ARC parent clone, suggesting that clusterin knockout did not adversely affect CHO cell growth and viability (FIG. 6). On day 14, all bioreactors were harvested and clarified harvest samples were processed for downstream purification of the chimeric protein.

A 3L bioreactor harvest was subjected to downstream purification. The purification of harvest of the parental clonal cell line was performed as follows: A clarified harvest was generated from the culture supernatant by filtration of intact cells, organelles and other large protein complexes. The clarified harvest was used as the starting material for the purification of the chimeric protein. The clarified harvest was subjected to affinity chromatography using FcXL resin. This resin captures Fc domain of the protein and thereby purifies the chimeric protein. The protein eluate of the FcXL chromatography was subjected to additional processing. The resultant the chimeric protein purified using this process contained < 50 ppm HCPs.

The purification of harvest of the CHOCKO stable pools expressing the chimeric protein was performed as follows: A clarified harvest was generated from the culture supernatant by filtration of intact cells, organelles and other large protein complexes, which was used as the starting material for the purification of the chimeric protein. The clarified harvest was subjected to affinity chromatography using FcXL resin. This resin captures Fc domain of the protein and thereby purifies the chimeric protein. The protein eluate of the FcXL chromatography was subjected to a streamlined processing process than above. The resultant the chimeric protein purified using this streamlined process contained < 50 ppm HCPs. Comparison of the purification showed improvement in overall process yield by ~30 % when the CHOCKO stable pool was used compared to the process when the parental clonal cell line was used. Moreover the streamlined process provided a cost-effective and faster process.

These results indicate, inter alia, that the clusterin mutant cells disclosed herein allows a streamlined downstream process and still yielding 30% higher overall purified product with significant improvement in product quality. Accordingly, these results, suggest, inter alia, that the clusterin mutant cells disclosed herein provide for high-level production of recombinant proteins with increased yields, decreased production costs, and reduced time required to purify recombinant proteins.

Example 4: Generation and Characterization of the Clusterin Mutant Cell Line Clones Expressing an Fc domain-Containing Protein

Single cell clones of CHOCKO stable pool were generated to identify clonal clusterin knockout cell line expressing a chimeric protein molecule (the SIRPa-Fc-CD40L chimeric protein). CHOCKO stable pool was single cell cloned using Solentim VIPS cell sorter. After monoclonality verification, CHOCKO single cell clones were scaled up through static stages (96 well plate, 24 well plate, and 6 well plate). The top 60 clones at 6 well plate stage was evaluated for the secreted levels of clusterin using clusterin dot blot and the chimeric protein titer. Briefly, 2 L sample was loaded to each grid section. The following controls were used: 1) the parental clonal cell line, 2) CHOCKO stable pool expressing the Fc domain-containing protein, and 3) purified recombinant clusterin protein. The SIRPa-Fc-CD40L clonal cell line and the purified recombinant clusterin protein were used as positive controls for clusterin expression and the CHOCKO pool was used as a negative control. The dot blot was probed using a biotin-conjugated anti-CHO clusterin-5 antibody and detected using IRDye 680-conjugated streptavidin. Parallel dot blots were probed using an anti-GAPDH as a control, which showed expression of GAPDH by all 60 clones. FIG. 7 shows a clusterin dot blot of CHOCKO clones. As shown in FIG. 7, most of the clones showed no clusterin signal, indicating, inter alia, creation of a clusterin null mutation.

The top 24 clones with no detectable clusterin and highest protein titer were scaled up from 6 well plates to shake flasks. After passaging top clones through 4-5 passages, all 24 clones were inoculated into 125 mL shake flask for 14 day fed batch study. On day 14, cell culture harvest samples were evaluated for the chimeric protein titer and clusterin levels. Western blot data with six of the clusterin knock-out clones is shown in FIG. 8. Briefly, intracellular clusterin and secreted clusterin were analyzed by western blotting. Whole cell extract and culture supernatant of CHOCKO clones were analyzed by Western blot using non-reduced (lane “N” in FIG. 8) or reduced (lane “R” in FIG. 8) conditions. Clusterin+ control clone (indicated as “Con” clones) was used as positive control. The gels were probed using a biotin-conjugated anti-CHO clusterin-5 antibody and detected using IRDye 680-conjugated streptavidin. As shown in FIG. 8, compared to the clusterin+ control clone, all tested CHOCKO clones had no detectable levels of secreted clusterin.

Several CHOCKO clones were grown in bioreactors and their growth rate and viability were measured. Growth data of top 3 clones based on the chimeric protein titer is shown in FIG. 9A and FIG. 9B. FIG. 9A shows a growth curve and FIG. 9B shows the viability as a function of time. The three clones are indicated in FIG. 9A and FIG. 9B by numbers “1 ,” “2,” and “3." All tested CHOCKO clones had a comparable growth profile to CHOCKO stable pool and ARC parent clone. All tested CHOCKO clones had a comparable 14-day fed-batch titer, suggesting clusterin mutation did not affect cell productivity. These results indicate, inter alia, that a complete knockout of clusterin did not have any adverse effect on CHO cell growth and productivity.

This example shows, inter alia, that the clusterin mutant cell lines disclosed herein allows high-level production of recombinant protein with improved yields, decreased production costs, and reduced time required to purify recombinant proteins. These results also indicate, e.g., that the clusterin mutant cell lines disclosed herein grow as well as clusterin+ cell lines.

Example 5: Construction of Another Clusterin Mutant Cell Line

CHO DG44 host cells were stably electroporated with the pSpCas9-BB-2A-Puro vector (GENSCRIPT) with or without clusterin sgRNA and HiUGE DisrupTag-2A-Puromycin vectors (CASTAG BIOSCIENCES) to knockout (KO) clusterin from CHO DG44 host cells. Following transfection, stably transfected cells were selected using different concentrations of puromycin to generate a second set of CHO Clusterin KO (CHOCKO) stable pools.

DG44 CHOCKO stable host pools were evaluated for clusterin knockout efficiency, followed by single cell cloning to generate clonal CHOCKO cell line. Briefly, a DG44 CHOCKO stable host pool was single cell cloned using Solentim VIPS cell sorter. After the verification of monoclonality, CHOCKO host single cell clones were scaled up through static stages (96 well plate, 24 well plate, and 6 well plate). The top clones at 6 well plate stage was evaluated for the secreted levels of clusterin using clusterin dot blot. The top 11 CHOCKO cell line clones with no detectable clusterin were scaled up from 6 well plates to shake flasks.

The top 11 CHOCKO cell line clones were evaluated for clusterin mRNA level using RT-qPCR. Briefly, top 11 CHOCKO cell lines and the parental CHO DG44 cells were cultured in 6 well plates and RNA was extracted from the cells using standard techniques. The extracted RNA was subjected to cDNA synthesis. Quantitative polymerase chain reaction (RT-PCR) was performed to examine the relative expression of clusterin mRNA in top 11 CHOCKO cell lines and the parental CHO DG44 cells. GAPDH as a housekeeping gene control. The levels of clusterin mRNA expression were normalized based on the GAPDH mRNA expression. As shown in FIG. 10, clones clone 01 , clone 04, clone 05, clone 08, clone 11 , clone 12, clone 13, and clone 14 showed only background levels of clusterin mRNA. The observed expression of clusterin mRNA in Clone 07 and Clone 10 may be an artifact (e.g., an incomplete clusterin gene sequence left over during cell line construction) since of clusterin protein expression was not observed by western blot analysis (also see FIG. 11 A and FIG. 11 B).

Based on clusterin mRNA and population doubling time of the top 11 clones, top 7 clones were selected for further analysis. The top seven clones were clone 01 , clone 05, clone 08, clone 10, clone 13, clone 14, and clone 15. These clones were also analyzed by western blot analysis. Briefly, the top 11 CHOCKO cell lines and the parental CHO DG44 cells were cultured and culture supernatants were analyzed by western blotting. In one experiment, untreated denatured culture supernatant samples (i.e., boiled in the presence of SDS, without a treatment with a reducing agent) were prepared and resolved using denaturing gels. The gels were probed using a biotin-conjugated anti-CHO clusterin-5 antibody and detected using IRDye 680-conjugated streptavidin. The results are shown in FIG. 11 A. The samples are respectively culture supernatants of clone 15 (lane 1 ), clone 14 (lane 2), clone 13 (lane 3), clone 10 (lane 4), clone 08 (lane 5), clone 05 (lane 6), clone 01 (lane 7), CHO DG44 control (lane 8), a clusterin standard (lane 9), and a molecular weight ladder (lane 10). As shown in FIG. 11 A, compared to the CHO DG44 control, all tested CHOCKO clones had no detectable levels of clusterin.

In another experiment, reduced culture supernatants (i.e., treated only with p-mercaptoethanol, and boiled in the presence of SDS) were prepared and resolved using denaturing gels. The gels were probed using a biotin-conjugated anti-CHO clusterin-5 antibody and detected using IRDye 680-conjugated streptavidin. The results are shown in FIG. 11 B. The samples are respectively a molecular weight ladder (lane 1), a clusterin standard (lane 2), and culture supernatants of CHO DG44 control (lane 3), clone 01 (lane 4), clone 05 (lane 5), clone 08 (lane 6), clone 10 (lane 7), clone 13 (lane 8), clone 14 (lane 9), and clone 15 (lane 10). As shown in FIG. 11 B, compared to the CHO DG44 control, all tested CHOCKO clones had no detectable levels of clusterin.

The western blot analysis results indicated, inter alia, that none of the 7 clones had any detectable levels of a secreted clusterin.

Based on clusterin mRNA levels, protein levels, and population doubling time of a top performing clone (clone 8) was selected for further analysis. Growth characteristics of this clone was analyzed in comparison with the CHO DG44 parental cells. Briefly, CHOCKO clone 8 and CHO DG44 parental cells were repeatedly passaged and growth was measured based on viable cell counts. Population doubling time was calculated and plotted. As shown in FIG. 12, the population doubling time of the DG44-CHOCKO cell line was comparable, if not better, compared to the parental CHO DG44 parent cell line. These results indicated, inter alia, that clusterin knockout did not affect cell viability or growth characteristics of the cells.

Example 6: Expression of Monoclonal Antibody Molecules in DG44 CHOCKO Clone 08

DG44 CHOCKO clone 08 was used to express three different monoclonal antibody (mAb) molecules. Briefly, DG44 CHOCKO cells were stably transfected with a vector encoding antibody heavy and light chain transgenes to produce three different mAb molecules. Next day, the primary transfectants were seeded as mini-pools (50000 cells/well) in 24 well plates under different concentrations (50 and 100 nM) of methotrexate selection. Then, wells in which cell growth was observed were screened for mAb production. Mini-pools with high mAb concentrations were expanded in 125 mL shake flasks. Top mini-pools for each mAb molecule were evaluated in a 14-day fed-batch culture for antibody production. The titer measurement of harvest material on day 14 of fed-batch showed that DG44 CHOCKO host cell line can be used for expression of mAb molecules (FIG. 13). The non-optimized levels of antibody production observed here compare favorably with those published in literature. See, e.g., Reinhart et al., Benchmarking of commercially available CHO cell culture media for antibody production, Appl Microbiol Biotechnol. 2015; 99(11): 4645-4657.

Collectively, these results indicated, inter alia, that clusterin knockout cell lines disclosed herein have growth characteristic that are very similar to commercially available cells. These results further indicated, inter alia, that clusterin knockout cell lines disclosed herein efficiently produce various Fc- and Fab-containing proteins and allow a streamlined downstream process (that does not require harsh chromatography conditions required to remove HCPs) and still yield at least 30% higher overall purified product with significant improvement in product quality.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. EQUIVALENTS

While the disclosure has been disclosed in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments disclosed specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

CLAIMS What is claimed is:

1. An engineered cell line adapted for production of a Fab and/or Fc domain-containing recombinant protein, the engineered cell line comprising a modification that causes a decrease in the amount or activity of a host cell protein (HCP).

2. The engineered cell line of claim 1 , wherein the HCP is a secreted protein.

3. The engineered cell line of claim 1 , wherein the HCP is clusterin.

4. The engineered cell line of claim 3, wherein the modification is a hypomorphic mutation in clusterin gene or a regulatory element thereof.

5. The engineered cell line of claim 3 or claim 4, wherein the modification is a clusterin null mutation.

6. The engineered cell line of any one of claims 3 to 5, wherein the modification is selected from an amino acid substitution mutation, insertion, deletion, frame-shift mutation, and non-sense mutation.

7. The engineered cell line of any one of claims 3 to 6, wherein the modification is a conditional mutation (e.g., temperature sensitive mutation or an inducible deletion), a degron or an epigenetic alteration.

8. The engineered cell line of any one of claims 3 to 7, wherein the modification is a genomic modification in the engineered cell line.

9. The engineered cell line of claim 8, wherein the genomic modification is a heterozygous modification.

10. The engineered cell line of claim 9, wherein the genomic modification is a homozygous modification.

11. The engineered cell line of any one of claims 3 to 5, wherein the modification is expression of a nucleic acid that causes the decrease in the amount or activity of clusterin.

12. The engineered cell line of claim 11 , wherein the nucleic acid is inserted on chromosome.

13. The engineered cell line of claim 11 or claim 12, wherein the nucleic acid is located on an extrachromosomal element.

14. The engineered cell line of any one of claims 11 to 13, wherein the nucleic acid is selected from an antisense polynucleotide, an shRNA, an siRNA and an miRNA that causes the decrease in the amount or activity of clusterin.

15. The engineered cell line of any one of claims 1 to 14, wherein the growth rate of the engineered cell line is at least about 30%, or at least about 40%, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 125%, or at least about 150%, or more compared to the growth rate of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin.

16. The engineered cell line of any one of claims 1 to 15, wherein the cell productivity of the engineered cell line in a culture is at least about 30%, or at least about 40%, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 125%, or at least about 150%, or more compared to the cell productivity of a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin.

17. The engineered cell line of claim 16, wherein the culture is selected from a fed-batch culture, a batch culture and a continuous culture.

18. The engineered cell line of any one of claims 1 to 17, wherein the engineered cell line is a clusterin mutant derivative of a cell line selected from Chinese hamster ovary (CHO) cells, NS0 murine myeloma cells, PER.C6 human cells, baby hamster kidney (BHK21) cells, murine myeloma Sp2/0 cells, human embryonic kidney 293 (HEK293) cells, HT-1080 cells, Hela cells, CAP cells, HKB-11 cells, HuH-7 cells, Sf9 cells, and a derivative thereof.

19. The engineered cell line of any one of claims 1 to 18, wherein the engineered cell line is a clusterin mutant derivative of a cell line selected from CHO DUXB11 , CHO DG44, CHOK1 , ExpiCHO and Expi293.

20. The engineered cell line of any one of claims 1 to 19, wherein the Fab and/or Fc domain-containing recombinant protein has a molecular weight of at least about 20 kDa, or at least about 30 kDa, or at least about 50 kDa, or at least about 100 kDa, or at least about 150 kDa, or at least about 200 kDa, or at least about 250 kDa, or at least about 300 kDa, or at least about 350 kDa, or at least about 400 kDa, or at least about 500 kDa.

21 . The engineered cell line of any one of claims 1 to 20, wherein the Fab and/or Fc domain-containing recombinant protein has a molecular weight of less than about 30 kDa, or less than about 50 kDa, or less than about 100 kDa, or less than about 150 kDa, or less than about 200 kDa, or less than about 250 kDa, or less than about 300 kDa, or less than about 350 kDa, or less than about 400 kDa, or less than about 500 kDa.

22. The engineered cell line of claim 21 , wherein the molecular weight is a native molecular weight of the Fab and/or Fc domain-containing recombinant protein.

23. The engineered cell line of claim 21 , wherein the molecular weight is a molecular weight of one or more monomers of the Fab and/or Fc domain-containing recombinant protein.

24. The engineered cell line of any one of claims 21 to 23, wherein the molecular weight includes the molecular weight of a post-translational processing.

25. The engineered cell line of any one of claims 21 to 23, wherein the post-translational modification includes one or more of glycosylation, acylation, phosphorylation, and proteolytic processing.

26. The engineered cell line of any one of claims 1 to 25, wherein the Fab and/or Fc domain-containing recombinant protein is a monomeric protein.

27. The engineered cell line of any one of claims 1 to 25, wherein the Fab and/or Fc domain-containing recombinant protein is a multimeric protein.

28. The engineered cell line of claim 26, wherein the multimeric protein is a dimeric protein, trimeric protein, a tetrameric protein, a pentameric protein, a hexameric protein or a protein comprising more than six monomers.

29. The engineered cell line of claim 27 or claim 28, wherein the multimeric protein is a homomultimeric protein or a heteromultimeric protein.

30. The engineered cell line of claim 29, wherein the heteromultimeric protein comprises two or three or four, or more distinct monomers.

31 . The engineered cell line of any one of claims 1 to 20, wherein the Fab and/or Fc domain-containing recombinant protein comprises a mammalian Fc domain.

32. The engineered cell line of claim 31 , wherein the Fc domain is selected from a human Fc domain, a mouse Fc domain, a rat Fc domain, a sheep Fc domain, a goat Fc domain, a donkey Fc domain, a feline Fc domain a hamster Fc domain, a guinea pig Fc domain, a horse Fc domain, a rabbit Fc domain, a pig Fc domain, a dog Fc domain, and a cow Fc domain.

33. The cell line of claim 31 or claim 32, wherein the Fc domain comprises a human Fc domain.

34. The engineered cell line of any one of claims 31 to 33, wherein the Fc domain comprises a Fc domain selected from an IgG Fc domain, an IgA Fc domain an IgM Fc domain, an IgE Fc domain and an IgD Fc domain.

35. The engineered cell line of claim 34, wherein the IgG Fc domain is selected from an lgG1 Fc domain, an lgG2 Fc domain, an lgG3 Fc domain, and an lgG4 Fc domain; and/or the IgA is selected from an I gA1 and an lgA2.

36. The engineered cell line of claim 1 to 35, wherein the Fab and/or Fc domain-containing recombinant protein is an antibody, an antibody-like molecule, or a derivative thereof.

37. The engineered cell line of claim 36, wherein the derivative of the antibody is selected from Fab, Fd, F(ab')2, Fab', and Fv, or a binding fragment thereof.

38. The engineered cell line of claim 37, wherein the derivative of the antibody-like molecule is selected from a single-domain antibody, a heavy-chain-only antibody (VHH), a single-chain antibody (scFv), scFv, ScFv-Fc, a diabody, a ScFv-CH, a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody or a binding fragment thereof.

39. The engineered cell line of any one of claims 1 to 18, wherein the Fab and/or Fc domain-containing recombinant protein is a fusion protein.

40. The engineered cell line of claim 39, wherein the fusion protein is bispecific or tri specific.

41. The engineered cell line of claim 39, wherein the fusion protein is selected from Fab-scFv, Fab-L- scFv, Fab-H-scFv, tribody, Fab-(scFv)2, a TriFab, a Fab-Fab fusion protein, a scFv-Fc-Fab fusion protein, a bispecific T-cell engager (BiTE), a Fab-Fv fusion protein, and a Fab-dsFv fusion protein.

42. The engineered cell line of claim 39, wherein the fusion protein is an Fc fusion protein.

43. The engineered cell line of claim 42, wherein the Fc fusion protein comprises a fusion partner selected from single-chain Fv (scFv), single-chain diabody (scDb), Fv, Fab, and F(ab')2.

44. The engineered cell line of claim 39, wherein the fusion protein comprises the formula: (i) X-Fc, wherein X is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof;

(ii) Fc-Y, wherein Y is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof; or

(iii) X-Fc-Y, wherein X and Y are independently selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof.

45. The engineered cell line of claim 44, wherein the X and/or Y is an antigen or a fragment thereof.

46. The engineered cell line of claim 45, wherein the antigen is derived from a pathogen.

47. The engineered cell line of claim 46, wherein the pathogen is selected from a virus, a bacterium, a protozoan, a parasite, and a fungus.

48. The engineered cell line of claim 47, wherein the antigen is a cancer antigen.

49. The engineered cell line of claim 48, wherein the cancer antigen is a neoantigen.

50. The engineered cell line of claim 44, wherein the X and/or Y is a mammalian intracellular protein or a fragment thereof.

51. The engineered cell line of claim 44, wherein the X and/or Y is a mammalian secreted protein or a biologically active fragment thereof.

52. The engineered cell line of claim 51 , wherein the mammalian secreted protein is a coagulation factor, a cytokine or a serum protein.

53. The engineered cell line of claim 52, wherein the cytokine is selected from IFN-a, IFN- , 1 FN-e, IFN- K, IFN-w IFN-y, IL-1a, IL-1 p, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-35, TNF, leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte colony-stimulating factor (G-CSF), TNF-a, TNF-p, lymphotoxin (LT)-p, LIGHT, Fas ligand (FasL)/CD178, TGF-p1 , TGF-p2, TGF-p3, XCL1 , XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11 , CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1 , CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CX3CL1 , Epo, Tpo, SCF, and FLT-3L.

54. The engineered cell line of claim 53, wherein the X and/or Y is a mammalian membrane protein selected from SLAMF4, IL-2 R a, 4-1 BB/TNFRSF9, IL-2 R p, ALCAM, B7-1 , IL-4 R, B7-H3, BLAME/SLAMF4, CEACAM1, IL-6 R, IL-7 Ra, IL-1 OR a, IL-1 0 R p, IL-12 R p 1 , IL-12 R p 2, CD2, IL-13 R a 1 , IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, Integrin a 4/CD49d, CDS, Integrin a E/CD103, CD6, Integrin a M/CD 11 b, CDS, Integrin a X/CD11c, Integrin p 2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1 , CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1 , KIR2DS4, CD4O Ligand/TNFSF5, LAG-3, CD43, LAIR1 , CD45, LAIR2, CDS3, Leukotriene B4-R1, CD84/SLAMF5, NCAM-L1 , CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common y Chain/IL-2 R y, Osteopontin, CRACC/SLAMF7, PD-1 , CRTAM, PSGL-1 , CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1 , L-Selectin, SIRP p1 , SLAM, TCCR/WSX-1 , DNAM-1 , Thymopoietin, EMMPRIN/CD147, TIM-1 , EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fey RIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1 B, TRAIL RI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF1 OB, ICAM-2/CD102, TRAILR3/TNFRSF10C, I FN-yR1 , TRAILR4/TNFRSF10D, IFN-y R2, TSLP, IL-1 R1 and TSLP R, or a fragment thereof.

55. The engineered cell line of claim 54, wherein the X and/ or Y is independently a mammalian membrane protein, or a fragment thereof.

56. The engineered cell line of claim 55, wherein the X is a Type I membrane protein, or a fragment thereof.

57. The engineered cell line of claim 56, wherein the Type I membrane protein is selected from SIRPo/CD172a, TIM-3, BTLA, PD-1 , CTLA-4, LAG-3, CD244, CSF1 R, CD160, TIGIT, 2B4, VISTA, VSIG8, LAG3, CD200, B7-H3, BTNL3, BTNL8, BTN2A1 , BTN3A1 , BTN3A2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H, and TMIGD2, or a fragment thereof.

58. The engineered cell line of claim 57, wherein the fragment of the Type I membrane protein is the extracellular domain or the ligand binding portion thereof.

59. The engineered cell line of claim 56, wherein the Y is a mammalian membrane protein is a Type II membrane protein, or a fragment thereof.

60. The engineered cell line of claim 59, wherein the Type II membrane protein is selected from CD40 ligand (CD40L), OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), 4-1 BB Ligand (4-1 BBL), CD70, CD30 ligand (CD30L), a C-type lectin domain (CLEC) family member, CD137 ligand, RANKL, TRAIL, FasL, TL1A, CD80, CD86, CD58, PD-1 , SLAMF6, SIRPo and TGFBR2, or a fragment thereof.

61. The engineered cell line of claim 60, wherein the fragment of the Type II membrane protein is the extracellular domain or a ligand binding portion thereof.

62. The engineered cell line of any one of claims 44 to 48, wherein the fusion protein is a vaccine.

63. The engineered cell line one of claims 44 to 62, wherein the fusion protein is capable of modulating an immune response.

64. The engineered cell line of any one of claims 1 to 63, wherein the engineered cell line comprises additional one or more mutations that decrease the amount or activity of another host cell protein (HCP) that remains associated with the Fab and/or Fc domain-containing recombinant protein after purification, optionally wherein the another HCP is selected from thrombospondin and lipoprotein lipase.

65. The engineered cell line of any one of claims 1 to 64, wherein the engineered cell line comprises a nucleic acid encoding the Fab and/or Fc domain-containing recombinant protein.

66. A cell bank comprising a plurality of engineered cells of the engineered cell line of any one of claims 1 to 65.

67. A method of making a cell bank for production of a Fab and/or Fc domain-containing recombinant protein, comprising:

(a) providing the engineered cell line of any one of claims 1 to 65,

(b) obtaining a plurality of cells harboring a nucleic acid encoding the Fab and/or Fc domaincontaining recombinant protein by introducing (e.g., transfecting) the nucleic acid in the engineered cell line,

(c) optionally obtaining a clone of the cell,

(d) optionally expanding the plurality of cells and/or the clone,

(e) optionally aliquoting the plurality of cells and/or the clone, and

(f) preparing the cell bank.

68. The method of claim 67, further comprising freezing the cell bank.

69. The method of claim 67 or claim 68, further comprising storing the cell bank at a temperature below about -20°C, optionally in a freezer, below about -80°C, in liquid nitrogen or on dry ice.

70. The method of any one of claims 67 to 69, further comprising culturing the plurality of cells and/or the clone, or an aliquot thereof.

71. A method of culturing the clusterin mutant cells, comprising: (a) providing the engineered cell line of any one of claims 1 to 65, wherein the engineered cell line optionally harbors a nucleic acid encoding the Fab and/or Fc domain-containing recombinant protein,

(b) contacting the engineered cell line with a medium that supports growth of the engineered cell line,

(c) incubating at a temperature and for a time that supports growth of the engineered cell line, optionally wherein the temperature is in the range of about 25°C to about 55°C, optionally wherein CO2 is provided during incubation at a level that supports growth of the engineered cell line, optionally wherein the level is in the range of about 2% and about 20%.

72. The method of claim 71 , wherein the culturing is selected from a fed-batch culturing, a batch culturing and a continuous culturing.

73. A method for isolating and/or purifying a Fab and/or Fc domain-containing recombinant protein, the method comprising:

(a) providing the engineered cell line of any one of claims 1 to 65, wherein the engineered cell line is capable of expressing the Fab and/or Fc domain-containing recombinant protein,

(b) culturing the engineered cell line in a growth medium, at a temperature and for a time that supports growth of the engineered cell line,

(c) obtaining a cell pellet and/or a culture supernatant; and

(d) isolating and/or purifying the Fab and/or Fc domain-containing recombinant protein from the cell pellet and/or the culture supernatant.

74. The method of claim 73, wherein the temperature is in the range of about 25°C to 45°C

75. The method of claim 73 or claim 74, wherein the time is at least about 4 hours, or at least about 12 hours, or at least about 1 day, or at least about 2 days, or at least about 4 days, or at least about 7 days, or at least about 10 days, or at least about 14 days, or at least about 20 days, or at least about 30 days.

76. The method of any one of claims 73 to 75, further comprising providing CO2 during culturing, optionally at a level that supports growth of the engineered cell line, optionally wherein the level is in the range of about 2% and about 20%.

77. The method any one of claims 73 to 76, wherein the culture supernatant is a clarified supernatant.

78. The method any one of claims 73 to 77, wherein the culture supernatant or the clarified supernatant is obtained by centrifugation and/or filtration of the culture.

79. The method of any one of claims 73 to 78, wherein isolating and/or purifying comprises a purification step optionally selected from a chromatography step, a precipitation step, and extraction step.

80. The method of any one of claims 73 to 79, wherein the Fab and/or Fc domain-containing recombinant protein comprises a mammalian Fc domain.

81. The method of claim 80, wherein the Fc domain is selected from a human Fc domain, a mouse Fc domain, a rat Fc domain, a sheep Fc domain, a goat Fc domain, a donkey Fc domain, a feline Fc domain a hamster Fc domain, a guinea pig Fc domain, a horse Fc domain, a rabbit Fc domain, a pig Fc domain, a dog Fc domain, and a cow Fc domain.

82. The method of claim 80 or claim 81 , wherein the Fc domain comprises a human Fc domain.

83. The method of any one of claims 80 to 82, wherein the Fc domain comprises a Fc domain selected from an IgG Fc domain, an IgA Fc domain an IgM Fc domain, an IgE Fc domain and an IgD Fc domain.

84. The method of claim 83, wherein the IgG Fc domain is selected from an lgG1 Fc domain, an lgG2 Fc domain, an I gG3 Fc domain, and an lgG4 Fc domain.

85. The method of claim 84, wherein the IgA is selected from an I gA1 and an lgA2.

86. The method of claim 83 to 85, wherein the Fab and/or Fc domain-containing recombinant protein is an antibody, an antibody-like molecule, or a derivative thereof.

87. The method of claim 86, wherein the derivative of the antibody is selected from Fab, Fd, F(ab')2, Fab', and Fv, or a binding fragment thereof.

88. The method of claim 87, wherein the derivative of the antibody-like molecule is selected from a single-domain antibody, a heavy-chain-only antibody (VHH), a single-chain antibody (scFv), scFv, ScFv-Fc, a diabody, a ScFv-CH, a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody or a binding fragment thereof.

89. The method of any one of claims 83 to 88, wherein the Fab and/or Fc domain-containing recombinant protein is a fusion protein.

90. The method of claim 89, wherein the fusion protein is bispecific or tri specific.

91. The method of claim 89, wherein the fusion protein is selected from Fab-scFv, Fab-L-scFv, Fab-H- scFv, tribody, Fab-(scFv)2, a TriFab, a Fab-Fab fusion protein, a scFv-Fc-Fab fusion protein, a bispecific T- cell engager (BiTE), a Fab-Fv fusion protein, and a Fab-dsFv fusion protein.

92. The method of claim 89, wherein the fusion protein is an Fc fusion protein.

93. The method of claim 92, wherein the Fc fusion protein comprises a fusion partner selected from single-chain Fv (scFv), single-chain diabody (scDb), Fv, Fab, and F(ab')2.

94. The method of claim 89, wherein the fusion protein comprises the formula:

(i) X-Fc, wherein X is selected from an antigen, a mammalian intracellular protein, a mammalian membrane protein, a mammalian secreted protein or a fragment thereof;

95. The method of claim 94, wherein the X and/or Y is an antigen or a fragment thereof.

96. The method of claim 95, wherein the antigen is derived from a pathogen.

97. The method of claim 96, wherein the pathogen is selected from a virus, a bacterium, a protozoan, a parasite, and a fungus.

98. The method of claim 97, wherein the antigen is a cancer antigen.

99. The method of claim 98, wherein the cancer antigen is a neoantigen.

100. The method of claim 94, wherein the X and/or Y is a mammalian intracellular protein or a fragment thereof.

101 . The method of claim 94, wherein the X and/or Y is a mammalian secreted protein or a biologically active fragment thereof.

102. The method of claim 101, wherein the mammalian secreted protein is a coagulation factor, a cytokine or a serum protein.

103. The method of claim 102, wherein the cytokine is selected from IFN-a, I FN-0, IFN-e, IFN-K, IFN-CO IFN-y, IL-1a, IL-1 p, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL- 18, IL-19, IL-20, IL-35, TNF, leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte colony- stimulating factor (G-CSF), TNF-a, TNF-p, lymphotoxin (LT)-p, LIGHT, Fas ligand (FasL)/CD178, TGF- 1 , TGF-p2, TGF-p3, XCL1 , XCL2, CCL1 , CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11 , CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CXCL1 , CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CX3CL1, Epo, Tpo, SCF, and FLT-3L.

104. The method of claim 94, wherein the X and/or Y is a mammalian membrane protein selected from SLAMF4, IL-2 R a, 4-1 BB/TNFRSF9, IL-2 R p, ALCAM, B7-1 , IL-4 R, B7-H3, BLAME/SLAMF4, CEACAM1 , IL-6 R, IL-7 Ra, IL-1 OR a, IL-1 0 R p, IL-12 R p 1 , IL-12 R p 2, CD2, IL-13 R a 1 , IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, I LT4/CDS5d, ILT5/CDS5a, Integrin a 4/CD49d, CDS, Integrin a E/CD103, CD6, Integrin a M/CD 11 b, CDS, Integrin a X/CD11c, Integrin p 2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1 , CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, Leukotriene B4-R1 , CD84/SLAMF5, NCAM-L1 , CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common y Chain/IL-2 R y, Osteopontin, CRACC/SLAMF7, PD-1 , CRTAM, PSGL-1 , CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1 , L-Selectin, SIRP p1 , SLAM, TCCR/WSX-1 , DNAM-1 , Thymopoietin, EMMPRIN/CD147, TIM-1 , EphB6, TIM- 2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fey RIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1 B, TRAIL RI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM- 2/CD102, TRAILR3/TNFRSF10C,IFN-yR1, TRAILR4/TNFRSF10D, IFN-y R2, TSLP, IL-1 R1 and TSLP R, or a fragment thereof.

105. The method of claim 94, wherein the X and/ or Y is independently a mammalian membrane protein, or a fragment thereof.

106. The method of claim 105, wherein the X is a Type I membrane protein, or a fragment thereof.

107. The method of claim 106, wherein the Type I membrane protein is selected from SI RPa/CD172a, TIM-3, BTLA, PD-1 , CTLA-4, LAG-3, CD244, CSF1 R, CD160, TIGIT, 2B4, VISTA, VSIG8, LAG3, CD200, B7-H3, BTNL3, BTNL8, BTN2A1, BTN3A1, BTN3A2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG2H, and TMIGD2, or a fragment thereof.

108. The method of claim 107, wherein the fragment of the Type I membrane protein is the extracellular domain or the ligand binding portion thereof.

109. The method of claim 106, wherein the Y is a mammalian membrane protein is a Type II membrane protein, or a fragment thereof.

110. The method of claim 109, wherein the Type II membrane protein is selected from CD40 ligand (CD40L), OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), 4-1 BB Ligand (4-1 BBL), CD70, CD30 ligand (CD30L), a C-type lectin domain (CLEC) family member, CD137 ligand, RANKL, TRAIL, FasL, TL1A, CD80, CD86, CD58, PD-1, SLAMF6, SIRPa and TGFBR2, or a fragment thereof.

111. The method of any one of claims 79 to 110, wherein the chromatography step is or comprises liquid chromatography conducted using a chromatography column and/or a membrane.

112. A Fab and/or Fc domain-containing recombinant protein prepared according to the method of any one of claims 73 to 111.

113. The Fab and/or Fc domain-containing recombinant protein of claim 112, wherein the Fab and/or Fc domain-containing recombinant protein is isolated and/or purified.

114. The Fab and/or Fc domain-containing recombinant protein of claim 112 or claim 113, wherein the Fab and/or Fc domain-containing recombinant protein is at least about 90%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 99.2%, or at least about 99.4%, or at least about 99.5% pure.

115. The Fab and/or Fc domain-containing recombinant protein of any one of claims 112 to 114, wherein the Fab and/or Fc domain-containing recombinant protein comprises reduced amount of at least one host cell protein (HCP) compared to the Fab and/or Fc domain-containing recombinant protein prepared using a clusterin positive cell line or a cell line not having a mutation that causes a decrease in the amount or activity of clusterin.

116. The Fab and/or Fc domain-containing recombinant protein of any one of claims 112 to 115, wherein the HCP is clusterin.

117. The Fab and/or Fc domain-containing recombinant protein of any one of claims 112 to 116, wherein the Fab and/or Fc domain-containing recombinant protein is substantially free of clusterin.

118. A composition comprising an isolated and/or purified Fab and/or Fc domain-containing recombinant protein prepared using the engineered cell line of any one of claims 1 to 65.

119. A composition comprising an isolated and/or purified Fab and/or Fc domain-containing recombinant protein prepared according to the method of any one of claims 73 to 111 .

120. A composition comprising the Fab and/or Fc domain-containing recombinant protein of any one of claims 112 to 117.