HK40055956A - Methods of producing multimeric proteins in eukaryotic host cells - Google Patents

Description

Method for producing multimeric proteins in eukaryotic host cells

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application serial No. 62/796,014 filed on 23/1/2019, which is incorporated herein by reference in its entirety.

Submitting sequence Listing in ASCII text files

The contents of the ASCII text files submitted below are incorporated herein by reference in their entirety: computer Readable Format (CRF) of sequence Listing (filename: 146392045240SEQLIST. TXT, recording date: 1 month 22 days 2020, size: 5 KB).

Technical Field

The present disclosure relates to methods of producing multimeric polypeptides in eukaryotic (e.g., mammalian) host cells, and cells, methods, and kits or articles of manufacture related thereto.

Background

Therapeutic antibodies represent the most successful biopharmaceuticals. Over the last 30 years, over 40 therapeutic antibodies have been approved for various indications, including cancer, autoimmunity, infection, and vascular disease. Thus, therapeutic antibodies represent one of the fastest growing areas of the pharmaceutical industry, the clinical impact of which is significant.

Due to the fact that most diseases involve multiple parallel signaling pathways, multiple inhibition of receptors and ligands may lead to better therapeutic results. Thus, bispecific antibodies (bsAb) with the ability to bind two different epitopes have been expected to be applied in a number of disease treatments, such as inflammatory and autoimmune diseases (Chan AC, Carter PJ. nat Rev Immunol.2010; 10(5):301-16), Cancer (Kou G, Shi J, Chen L, Zhang D, Hou S, ZHao L, et al Cancer Lett.2010; 299(2): 130-6; Dong J, Sereno A, Aivazian D, Langley E, Miller BR, Snyder WB, et al MAbs.2011; 3) (273-88) and infectious diseases (Denn Bernard F, Liu H, O' MahoR, La Valle R, Bartollino S, Dini S, et al J Infect.195; Acfect 1-149, Lancek J.2011H, Sal J.7, Inc.: 19, Saurur.7, et al, (Janu J. Saurc J.04, et al; Sanwich R.7, Sagnan J.32, Saurc E, et al, (Konu J. K.32, K.7, et al; Janu J. K. E, K. K U, Kontermann RE. MAbs.2017; 9(2) 182 and 212; carter PJ, Lazar ga. nat Rev Drug discov.2018; 17(3):197-223). Over the past decades, genetic engineering has produced over 60 different bispecific antibody formats that have improved the immunogenicity, pharmacokinetic properties and distribution of bsabs (Spiess C, Zhai Q, Carter pj. mol immunol. 2015; 67(2Pt a): 95-106).

Regardless of the application of bsAb, the generation process has been challenging because promiscuous pairing of two different heavy chains and two different light chains produces 16 different combinations, only one of which is bispecific, which makes it difficult to generate them in sufficient quantity, quality, and assembly to support preclinical and clinical development. Antibody engineering has been used to alleviate the chain mismatch problem by heterodimerization of either the heavy or light chains using the mortar-in-mortar and crossMab techniques, respectively (Ridgway JB, Presta LG, Carter P. protein Eng. 1996; 9(7): 617-21; Atwell S, Ridgway JB, Wells JA, Carter P. Jmol biol. 1997; 270(1): 26-35; Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW, Presta LG, et al Nat Biotechnol. 1998; 16(7): 677-81). One manufacturing process solution is to use separate expression of half-antibodies, followed by assembly in vitro to bsAb (Spiess C, Merchant M, Huang a, Zheng Z, Yang NY, Peng J, et al Nat biotechnol.2013; 31(8): 753-8). However, this is associated with a more time consuming and costly manufacturing process, since two different stable cell lines are generated.

Thus, co-expression of both antibodies in a single cell may be more straightforward, but at the same time often requires more extensive optimization of the expression system to direct the light chain to its cognate heavy chain and to obtain the appropriate amount of correctly assembled bsAb, avoiding the presence of unwanted by-products and the need for complex purification processes.

Mammalian cells such as Chinese Hamster Ovary (CHO) cells have been the primary host of the biopharmaceutical industry for the past few decades (Wurm FM. Nat Biotechnol. 2004; 22(11): 1393-8). Bioprocess innovations and cell engineering improved product titers (Ayyar BV, Arora S, Ravi SS. methods.2017; 116: 51-62; Kelley B, Kiss R, Laird M.adv Biochem Eng Biotechnol.2018.Epub 2018/05/04); however, uncharacterized cellular processes and gene regulatory mechanisms still hinder cell growth, specific productivity (specific productivity) and protein quality. Currently, there are several systematic approaches to precisely control the level of translation of recombinant proteins in mammalian cells.

Despite advances in the engineering of complex antibody formats (such as bispecific antibodies), manufacturability of these antibody formats often shows low potency in a single mammalian expression system. Low titer and insufficient product quality are two factors that lead to difficulty in stable production of bsAb. Therefore, there is an urgent need to identify limiting steps in production systems and to provide for the production of multimeric polypeptides that allow for improved correct assembly, e.g., in eukaryotic or mammalian host cell systems.

All references cited herein, including patent applications, patent publications, non-patent documents, and UniProtKB/Swiss-Prot accession numbers, are incorporated by reference in their entirety as if each reference were specifically and individually indicated to be incorporated by reference.

Disclosure of Invention

To meet these and other needs, provided herein are methods of producing multimeric polypeptides in eukaryotic (e.g., mammalian) host cells. These multimeric polypeptides comprise two or more subunits, each subunit comprising two or more polypeptide chains (e.g., as with multispecific or bispecific antibodies). Advantageously, these methods provide more precise manipulation of the translation level of each polypeptide chain in the multimeric polypeptide, allowing for improved production of correctly assembled multimeric polypeptides.

Certain aspects of the present disclosure relate to methods of producing multimeric polypeptides in eukaryotic host cells. In some embodiments, the multimeric polypeptide comprises a first subunit comprising a first polypeptide chain and a second polypeptide chain, and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain. In some embodiments, the method comprises: providing a eukaryotic host cell; culturing a eukaryotic host cell under conditions suitable for expression of a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein upon expression the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain form a multimeric polypeptide; and recovering the multimeric polypeptide produced by the eukaryotic host cell. In some embodiments, the eukaryotic host cell comprises a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding a first polypeptide chain, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding a second polypeptide chain, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a third polypeptide chain, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a fourth polypeptide chain. In some embodiments, when each subunit is expressed individually in a eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit, and one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence. In some embodiments, when all subunits are expressed together in the same eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit, and one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence. In some embodiments, the multimeric polypeptide is a bispecific antibody. In some embodiments, the multimeric polypeptide is a bispecific antibody, the first and third polypeptide chains are antibody heavy chains, the second and fourth polypeptide chains are antibody light chains, the first subunit is a first half-antibody that binds a first antigen, and the second subunit is a second half-antibody that binds a second antigen.

Other aspects of the disclosure relate to methods of producing a bispecific antibody in a eukaryotic host cell, wherein the bispecific antibody comprises a first half antibody comprising a first antibody heavy chain and a first antibody light chain and a second half antibody chain comprising a second antibody heavy chain and a second antibody light chain, the method comprising: (a) providing a eukaryotic host cell, wherein the eukaryotic host cell comprises a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding a first antibody heavy chain, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding a first antibody light chain, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a second antibody heavy chain, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a second antibody light chain, wherein when each half-antibody is expressed alone in the eukaryotic host cell, the first half-antibody is expressed at a lower level than the second half-antibody, and wherein one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence; (b) culturing a eukaryotic host cell under conditions suitable for expression of a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain, and a second antibody light chain, wherein the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain, and the second antibody light chain form a bispecific antibody, and wherein the first half-antibody binds a first antigen and the second half-antibody binds a second antigen; and (c) recovering the bispecific antibody produced by the eukaryotic host cell.

In some embodiments, the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the heavy chain of the second antibody comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region; and wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region. In some embodiments, the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the heavy chain of the second antibody comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region; and wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region. In some embodiments, the knob mutation comprises at least one of: T366Y, T366W, T394W and F405W, numbered according to the EU index based on human IgG 1. In some embodiments, the hole mutation comprises at least one of: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbering based on human IgG1 according to the EU index. In some embodiments, the knob mutation comprises T366W, and wherein the hole mutation comprises at least one, at least two, or all three of T366S, L368A, and Y407V, numbered according to the EU index based on human IgG 1. In some embodiments, the first antibody light chain comprises a first mutation, the first antibody heavy chain comprises a second mutation, and the first mutation and the second mutation facilitate selective association of the first antibody light chain with the first antibody heavy chain. In some embodiments, the first mutation comprises an amino acid substitution at V133 and/or the second mutation comprises an amino acid substitution at S183, numbering based on the EU index. In some embodiments, the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and/or the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D. In some embodiments, the second antibody light chain comprises a third mutation, the second antibody heavy chain comprises a fourth mutation, and the third mutation and the fourth mutation facilitate selective association of the second antibody light chain with the second antibody heavy chain. In some embodiments, the third mutation comprises an amino acid substitution at V133 and/or the fourth mutation comprises an amino acid substitution at S183, numbering based on the EU index. In some embodiments, the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and/or the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D. In some embodiments, the amino acid substitution at S183 produces a positively charged residue (e.g., S183K), and the amino acid substitution at V133 produces a negatively charged residue (e.g., V133E). In some embodiments, the amino acid substitution at S183 produces a negatively charged residue (e.g., S183E), and the amino acid substitution at V133 produces a positively charged residue (e.g., V133K).

In some embodiments, the methods result in higher yields of the multimeric polypeptide in the eukaryotic host cell, e.g., as compared to production of an open reading frame in which one or more, two or more, three or more, or four polynucleotides encoding polypeptide chains of the multimeric polypeptide comprise an operable linkage to a native or unmodified translation initiation sequence, or as compared to production of a eukaryotic host cell in which each polynucleotide encoding polypeptide chains of the multimeric polypeptide comprises the same translation initiation sequence. In some embodiments, the methods result in fewer mis-paired by-products in the eukaryotic host cell, e.g., as compared to production of an open reading frame in which one or more, two or more, three or more, or four polynucleotides encoding polypeptide chains of the multimeric polypeptide comprise an operable linkage with a native or unmodified translation initiation sequence, or in which each polynucleotide encoding polypeptide chains of the multimeric polypeptide comprises the same translation initiation sequence. For example, transient or stable transfectants can be cultured, and production of multimeric polypeptides can be assayed. Fed-batch or perfusion cultures are cultured and the product titer in the cell culture medium or on the cell surface can be determined. For surface expression, cells can be stained with antibodies to detect products and analyzed, for example, by flow cytometry. The product quality and/or purity can be analyzed by, for example, electrophoresis and/or mass spectrometry. In some embodiments, when each subunit is expressed individually in a eukaryotic host cell, one or both polypeptide chains of the first subunit are translated at a slower rate than one or both polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed individually in a eukaryotic host cell, one or both polypeptide chains of the first subunit fold more slowly and/or less efficiently than one or both polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed individually in a eukaryotic host cell, the first subunit assembles at a slower rate than the second subunit. In some embodiments, the first translation initiation sequence and/or the second translation initiation sequence is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, 5% to 30%, 5% to 50%, 5% to 75%, 10% to 30%, 10% to 50%, 10% to 75%, 25% to 50%, 25% to 75%, 25% to 100%, 50% to 75%, 50% to 100%, or 75% to 100% weaker than the third translation initiation sequence and/or the fourth translation initiation sequence. In some embodiments, the first translation initiation sequence and/or the second translation initiation sequence is at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, 1.3-fold to 3-fold, 1.5-fold to 3-fold, 2-fold to 10-fold, 2-fold to 5-fold, 3-fold to 10-fold, 5-fold to 10-fold, or 7-fold to 10-fold weaker than the third translation initiation sequence and/or the fourth translation initiation sequence. In some embodiments, the first subunit or first half antibody is expressed at a level at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, 5% to 30%, 5% to 50%, 5% to 75%, 10% to 30%, 10% to 50%, 10% to 75%, 25% to 50%, 25% to 75%, 25% to 100%, 50% to 75%, 50% to 100%, or 75% to 100% lower than the level of expression of the second subunit or second half antibody, e.g., when each subunit or half antibody is expressed separately in a eukaryotic host cell. In some embodiments, the first subunit or first half-antibody is expressed at a level at least 1.3 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, 1.3 fold to 3 fold, 1.5 fold to 3 fold, 2 fold to 10 fold, 2 fold to 5 fold, 3 fold to 10 fold, 5 fold to 10 fold, or 7 fold to 10 fold lower than the level of expression of the second subunit or second half-antibody, e.g., when each subunit or half-antibody is expressed alone in a eukaryotic host cell. In some embodiments, the first subunit or first half-antibody is expressed at a level 0.2-fold to 0.8-fold, less than 0.5-fold, or less than 0.3-fold lower than the expression level of the second subunit or second half-antibody, e.g., when each subunit or half-antibody is expressed alone in a eukaryotic host cell.

Other aspects of the disclosure relate to a plurality or composition of multimeric polypeptides or bispecific antibodies, wherein each multimeric polypeptide or bispecific antibody of the plurality or composition is produced according to the method of any one of the embodiments described herein.

Other aspects of the present disclosure relate to a recombinant eukaryotic host cell for expressing a non-natural multimeric polypeptide comprising a first subunit comprising a first polypeptide chain and a second subunit chain comprising a third polypeptide chain and a fourth polypeptide chain, the host cell comprising: a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the first polypeptide chain; a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the second polypeptide chain; a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the third polypeptide chain; and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the fourth polypeptide chain; wherein when each subunit is expressed individually in the recombinant eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit; and wherein one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence. In some embodiments, the multimeric polypeptide is a bispecific antibody, the first and third polypeptide chains are antibody heavy chains, the second and fourth polypeptide chains are antibody light chains, the first subunit is a first half-antibody that binds a first antigen, and the second subunit is a second half-antibody that binds a second antigen.

Other aspects of the present disclosure relate to a recombinant eukaryotic host cell for expressing a bispecific antibody, wherein the bispecific antibody comprises a first half-antibody comprising a first antibody heavy chain and a first antibody light chain, and a second half-antibody comprising a second antibody heavy chain and a second antibody light chain, the recombinant cell comprising: (a) a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the heavy chain of the first antibody; (b) a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the light chain of the first antibody; (c) a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a heavy chain of the second antibody; and (d) a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the light chain of the second antibody; wherein the first half antibody is expressed at a lower level than the second half antibody when each half antibody is expressed individually in the recombinant eukaryotic host cell, and wherein one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence. In some embodiments, the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain, and the second antibody light chain form a bispecific antibody, and the first half-antibody binds to a first antigen and the second half-antibody binds to a second antigen.

In some embodiments, the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the heavy chain of the second antibody comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region; and wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region. In some embodiments, the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the heavy chain of the second antibody comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region; and wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region. In some embodiments, the knob mutation comprises at least one of: T366Y, T366W, T394W and F405W, numbered according to the EU index based on human IgG 1. In some embodiments, the hole mutation comprises at least one of: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbering based on human IgG1 according to the EU index. In some embodiments, the knob mutation comprises T366W, and wherein the hole mutation comprises at least one, at least two, or all three of T366S, L368A, and Y407V, numbered according to the EU index based on human IgG 1. In some embodiments, the first antibody light chain comprises a first mutation, the first antibody heavy chain comprises a second mutation, and the first mutation and the second mutation facilitate selective association of the first antibody light chain with the first antibody heavy chain. In some embodiments, the first mutation comprises an amino acid substitution at V133 and/or the second mutation comprises an amino acid substitution at S183, numbering based on the EU index. In some embodiments, the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and/or the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D. In some embodiments, the second antibody light chain comprises a third mutation, the second antibody heavy chain comprises a fourth mutation, and the third mutation and the fourth mutation facilitate selective association of the second antibody light chain with the second antibody heavy chain. In some embodiments, the third mutation comprises an amino acid substitution at V133 and/or the fourth mutation comprises an amino acid substitution at S183, numbering based on the EU index. In some embodiments, the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and/or the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D. In some embodiments, the amino acid substitution at S183 produces a positively charged residue (e.g., S183K), and the amino acid substitution at V133 produces a negatively charged residue (e.g., V133E). In some embodiments, the amino acid substitution at S183 produces a negatively charged residue (e.g., S183E), and the amino acid substitution at V133 produces a positively charged residue (e.g., V133K). In some embodiments, the first antibody light chain comprises a V133K mutation, the first antibody heavy chain comprises a S183E mutation, the second antibody light chain comprises a V133E mutation, and the second antibody heavy chain comprises a S183K mutation, numbered based on the EU index. In some embodiments, the first antibody heavy chain further comprises a T366S, L368A, and Y407V mutation, and the second antibody heavy chain further comprises a T366W mutation, numbered according to the EU index based on human IgG 1. In some embodiments, the second antibody light chain comprises a V133K mutation, the second antibody heavy chain comprises a S183E mutation, the first antibody light chain comprises a V133E mutation, and the first antibody heavy chain comprises a S183K mutation, numbered based on the EU index. In some embodiments, the second antibody heavy chain further comprises a T366S, L368A, and Y407V mutation, and the first antibody heavy chain further comprises a T366W mutation, numbered according to the EU index based on human IgG 1.

Other aspects of the disclosure relate to methods of identifying a combination of translation initiation sequences for expression of a multimeric polypeptide in a eukaryotic host cell, wherein the multimeric polypeptide comprises a first subunit comprising a first polypeptide chain and a second polypeptide chain, and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain. In some embodiments, the method comprises: providing a library comprising a plurality of eukaryotic host cells, wherein each eukaryotic host cell in the plurality comprises: a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the first polypeptide chain, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the second polypeptide chain, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the third polypeptide chain, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the fourth polypeptide chain, wherein multiple combinations of first, second, third, and fourth translation initiation sequences are present in a plurality of eukaryotic host cells; culturing the library of eukaryotic host cells under conditions suitable for expression of the multimeric polypeptide by the eukaryotic host cells in the plurality; measuring the amount of multimeric polypeptide expressed by a single eukaryotic host cell of the plurality or a clone of a single eukaryotic host cell of the plurality; and identifying one or more clones of a single eukaryotic host cell in the plurality or individual eukaryotic host cells in the plurality that express the multimeric polypeptide the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence. In some embodiments, one or more clones of a single eukaryotic host cell in a plurality or clones of a single eukaryotic host cell in a plurality that express the multimeric polypeptide are identified based on higher yields of the multimeric polypeptide and/or lower levels of mis-paired by-products, e.g., a first translation initiation sequence, a second translation initiation sequence, a third translation initiation sequence, and a fourth translation initiation sequence, as compared to a reference or different combinations of four translation initiation sequences. In some embodiments, all of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence in each host cell in the plurality comprise a sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1).

Other aspects of the disclosure relate to kits or articles of manufacture comprising a polynucleotide for expression of a multimeric polypeptide, wherein the multimeric polypeptide comprises a first subunit comprising a first polypeptide chain and a second polypeptide chain, and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain. In some embodiments, the kit or article of manufacture comprises a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the first polypeptide chain; a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the second polypeptide chain; a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the third polypeptide chain; and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the fourth polypeptide chain; wherein one or more of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence are not operably linked to their respective open reading frames in that the respective open reading frames are present in a naturally occurring host cell genome. In some embodiments, the multimeric polypeptide is a bispecific antibody, the first and third polypeptide chains are antibody heavy chains, the second and fourth polypeptide chains are antibody light chains, the first subunit is a first half-antibody that binds a first antigen, and the second subunit is a second half-antibody that binds a second antigen.

In some embodiments, when each subunit is produced individually, one or both polypeptide chains of the first subunit are produced at a lower level than one or both polypeptide chains of the second subunit. In some embodiments, when all subunits are produced by the same host cell, one or both polypeptide chains of the first subunit are produced at a lower level than one or both polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed separately in a eukaryotic host cell, the first subunit assembles with more of the incorrectly paired byproducts than the second subunit. In some embodiments, when all subunits are expressed together in the same eukaryotic host cell, the first subunit assembles with more incorrectly paired byproducts than the second subunit.

In some embodiments of any of the embodiments described herein, all of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence comprise a sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1). In some embodiments, one or both of the first translation initiation sequence and the second translation initiation sequence comprises a sequence selected from the group consisting of: 8-10 of SEQ ID NO. In some embodiments, one or both of the third translation initiation sequence and the fourth translation initiation sequence comprises the sequence ACCATGG (SEQ ID NO:3) or GAAGTATGA (SEQ ID NO: 11). In some embodiments, the first translation initiation sequence comprises a sequence selected from the group consisting of: 8-10, the second translation initiation sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 8-10, the third translation initiation sequence comprises the sequence of SEQ ID NO 2, and the fourth translation initiation sequence comprises the sequence of SEQ ID NO 11. In some embodiments, the first translation initiation sequence comprises the sequence of SEQ ID NO 9, the second translation initiation sequence comprises the sequence of SEQ ID NO 9, the third translation initiation sequence comprises the sequence of SEQ ID NO 2, and the fourth translation initiation sequence comprises the sequence of SEQ ID NO 11. In some embodiments, each of the first, second, third, and fourth polynucleotides is operably linked to a promoter. In some embodiments, the first polynucleotide and the second polynucleotide are operably linked to the same promoter, and the third polynucleotide and the fourth polynucleotide are operably linked to the same promoter. In some embodiments, the first translation initiation sequence is weaker than the third translation initiation sequence. In some embodiments, the second translation initiation sequence is weaker than the fourth translation initiation sequence. In some embodiments, the first translation initiation sequence is weaker than the fourth translation initiation sequence. In some embodiments, the second translation initiation sequence is weaker than the third translation initiation sequence. In some embodiments, the first translation initiation sequence is identical to the second translation initiation sequence. In some embodiments, the third translation initiation sequence is identical to the fourth translation initiation sequence. In some of any of the embodiments described herein, the multimeric polypeptide specifically binds to one or more target antigens. In some embodiments, the multimeric polypeptide is a multispecific antigen-binding protein.

Also provided herein are polynucleotides comprising an open reading frame operably linked to a translation initiation sequence selected from the group consisting of: 8-11 of SEQ ID NO. Further provided herein is a set of polynucleotides comprising: a first translation initiation sequence operably linked to a first open reading frame comprising a sequence selected from the group consisting of: 8-10, a second translation initiation sequence operably linked to a second open reading frame comprising a sequence selected from the group consisting of SEQ ID NOs: 8-10, a third translation initiation sequence comprising the sequence of SEQ ID NO 2 operably linked to a third open reading frame, and a fourth translation initiation sequence comprising the sequence of SEQ ID NO 11 operably linked to a fourth open reading frame. In some embodiments, the first translation initiation sequence comprises the sequence of SEQ ID NO 9, the second translation initiation sequence comprises the sequence of SEQ ID NO 9, the third translation initiation sequence comprises the sequence of SEQ ID NO 2, and the fourth translation initiation sequence comprises the sequence of SEQ ID NO 11. Further provided herein is a host cell comprising a polynucleotide or set of polynucleotides according to any one of the embodiments described herein.

In some embodiments of any of the embodiments described herein, the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into one or more chromosomes of the eukaryotic host cell. In some embodiments, the first, second, third, and fourth polynucleotides are integrated into the same chromosomal locus of the eukaryotic host cell. In some embodiments, the first, second, third, and fourth polynucleotides are part of one of a plurality of extrachromosomal polynucleotides in the eukaryotic host cell. In some embodiments, the eukaryotic host cell is a mammalian host cell. In some embodiments, the mammalian host cell is a Chinese Hamster Ovary (CHO) cell.

It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the invention are further described by the following detailed description.

Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

Fig. 1 shows the names and nucleotide sequences of kozak (kz) sequence variants designed and analyzed by transient transfection. The initiation codon is grey and underlined. The sequences shown correspond to SEQ ID NOS: 3-7 (top to bottom).

Figure 2 provides normalized titers of Fc fusion proteins a and B with different Kozak sequence variants, tested by transient transfection followed by homogeneous time-resolved fluorescence (HTRF). Data are presented as mean and error bars represent Standard Deviation (SD) of four independent transfections. Changes in the Kozak consensus sequence modulate the titer of the Fc fusion protein.

FIG. 3 illustrates the process of designing a Kozak library, transforming E.coli cells with the library, sequencing to determine the nucleotide sequence of the variants, screening the variants by transient transfection, sorting and narrowing the variants within the range, and finally selecting variants that cover the entire expression range.

FIG. 4 shows the Wild Type (WT) Kozak sequence in the upper row. The designed Kozak library is shown in the lower row. N is A, T, G or C. The initiation codon is grey and underlined. Positions-3 and +4 of the start codon are indicated. The sequences shown correspond (from top to bottom) to SEQ ID NO 23 and 1, respectively.

Fig. 5 shows the normalized antibody titers of 111 Kozak sequence variants transiently transfected into CHO cells. Data are presented as mean values and error bars represent Standard Deviation (SD) of two independent transfections. An almost continuous translation range of 0.05 to 1.54 units of normalized titer was observed (1.0 corresponds to the titer produced using the Wt Kozak sequence).

FIG. 6 provides the nucleotide distribution of 108 colonies of the Kozak library. The percentage of each nucleotide at each position is shown. The nucleotide distribution of the analyzed variants was random and diverse at each position, indicating no bias in library screening.

FIG. 7 shows the screening process to reduce the number of Kozak variants by transient transfection in CHO cells. Criteria for selection of variants in rounds are reproducibility between transient transfections, stability between DNA preparations and preservation of nucleotide diversity.

Fig. 8A and 8B provide normalized titers and sequences for the last set of Kozak sequence variants. Fig. 8A shows the normalized antibody titers for the designated Kozak sequence variants transiently transfected into CHO cells. Data are presented as mean values and error bars represent standard deviation of eleven independent transfections. The 11 Kozak sequence variants represent a 0.2 to 1.3 fold expression range of Wt Kozak, enabling high precision titer adjustment. Asterisks indicate Kozak sequence variants selected as a representative group of five variants (including Wt Kozak) because they cover the broad expression levels observed in transient transfections. Fig. 8B provides the sequences of the last set of Kozak sequence variants. Wt consensus Kozak sequence is boxed in one box.

FIG. 9 shows that higher percentages of correctly assembled BsAb can be obtained by manipulating the ratio of light chains (LC1: LC 2). For example, modification of the ratio of LC1: LC2 from 1:2.5 to 1.5:1 resulted in a 40% increase in correctly paired light chains.

FIG. 10 provides the translational strength of the listed Kozak variants relative to Wt Kozak (1X). The sequences of the named Kozak variants are provided on the right. These Kozak sequence variants were selected as a representative group of five variants (including Wt Kozak) because they cover the broad expression levels observed in transient transfections. The sequences correspond to SEQ ID NOS: 8, 9, 10, 2 and 11 (top to bottom).

Fig. 11 provides a combination of Kozak variants used to develop 25 integrated expression vectors for each group of Ab1/Ab2 BsAb. A mixture of 50 plasmids was transfected into CHO cells. The diversity generated by this approach achieves a potential of 625 different combinations of chain ratios. Only the combination of Ab1 is shown, but the same combination was used to generate Ab2 group vectors.

FIG. 12 shows transfection efficiency and number of transfections for a given Kozak mix pool. Transfection efficiency was monitored by cell surface staining analysis with Allophycocyanin (APC) -labeled anti-hu IgG antibodies and measurement of antibody-APC expression by flow cytometry. Empty hosts were used to set gates for Ab expression negative and GFP positive cells. The X-axis shows GFP expression and the Y-axis shows APC antibody expression. Similar transfection efficiencies were observed for Kozak mixes and Wt Kozak pools.

Fig. 13A and 13B show the stabilization pool recovery time process. Two pools for each condition are shown. The addition of the selection drug to establish a stable pool is indicated by the arrow. The Y-axis shows the percent viability and the X-axis shows the days after addition of the selected drug.

Fig. 14A and 14B provide the absolute titers of 704 individual clones of either Kozak mixed clones or Wt Kozak clones selected from each transfection and condition and cloned as single cells. Titers were measured by HTRF. Presented in pairs according to the number of transfections indicated.

15A and 15B provide the viability of the 14 day shake flask fed-batch generation of the Kozak mixed clone and the Wt-Kozak clone. The X-axis shows days and the Y-axis shows percent viability. The number of transfections is indicated. All clones studied showed comparable viability throughout the production assay. The final viability at day 14 under both conditions was about 80-95%, except for one Kozak mixed clone which was 74%.

16A and 16B show viable cell counts generated by a 14 day shake flask fed-batch for both the Kozak mixed clone and the Wt-Kozak clone. X-axis shows days and Y-axis shows 10 per ml⁶Number of living cells in (1). The number of transfections is indicated. An exponential growth rate was observed until day 7, after which all individual clones reached plateau.

FIG. 17 shows the general absolute titers of the first 11 Kozak mixed clones and the Wt Kozak clone for each transfection. Titers were measured on day 14 of shake flask fed-batch production. In both transfections, Wt Kozak clones alone showed higher general absolute antibody titers than Kozak mixed clones alone. Note that titers represent correctly assembled bispecific antibodies as well as half-antibodies and other unwanted side products.

Fig. 18 provides an assessment of the quality of assembled antibody for each transfected Kozak mix and the first 11 clones of Wt Kozak. Samples were analyzed by non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) to distinguish and quantify the two antibody forms: major peaks equal to full antibody in the form of correctly and incorrectly assembled BsAb (full-ab), and the sum of prepeaks representing other species of different molecular weights. The number of transfections is indicated. Among the first 22 clones, there were fewer Kozak mixed clones with a high percentage of all-ab form than Wt Kozak clones.

FIGS. 19A and 19B show the quality and assembly efficiency of moderate (labeled with M) and low (labeled with L) HCCF titer Kozak mixed clones. FIG. 19A shows the general absolute titers of 3 medium and 4 low Kozak mixed clones measured by the shake flask fed-batch generation assay on day 14. The resulting titers of these clones showed similar behavior to that previously observed in the primary screen. Fig. 19B provides an assessment of the quality of assembled antibodies for medium and low Kozak mixed clones. Samples were analyzed by non-reducing CE-SDS to distinguish and quantify the main peak equal to the incorrectly paired by-product and correctly assembled full antibody in BsAb form (full-ab), as well as the sum of pre-peaks representing other species of different molecular weights. There is no direct correlation between the typical titer and the highest level of whole antibody. For example, some Kozak mixed clones with more moderate titers showed similar percent all-ab, approximately 50-60%, compared to the first 11 clones.

FIGS. 20A and 20B show the quality and effective BsAb titer of indicated Wt Kozak and Kozak mixed clones. Fig. 20A provides a quantification of the species detected by mass spectrometry of eight Kozak mixed clones and eight Wt Kozak clones. The structure of each species is indicated in the legend. Correctly assembled BsAb is highlighted in white. The best individual Kozak mixed clone showed about 40% of correctly assembled BsAb, which is more than twice (about 18%) that of the best individual Wt Kozak clone. Figure 20B provides estimated titers of bispecific antibody and various mispaired by-products and half antibody species. It is calculated as the percentage of each form multiplied by the general titer. Bispecific effective titers are highlighted in white. Due to the fact that some Kozak mixed clones had higher BsAb assembly than Wt Kozak clones, some Kozak mixed clones overcome the deficiencies of the general titer shown previously in fig. 17.

FIGS. 21A-21C show the combination of Kozak sequence variants and the translational strengths of the indicated Kozak mixed clones. Fig. 21A shows Kozak sequence variant combinations for Kozak mixed clones. Grey highlighted is unavailable sequencing data. The name and translational strength of each Kozak sequence variant are shown on the right. Fig. 21B shows the combination of Kozak sequence variants for the best antibody-producing Kozak mixed clones. The name and transfection number of the Kozak mixed clone are indicated. Grey highlighted is unavailable sequencing data. Fig. 21C provides Kozak sequence variant combinations for 3 medium (labeled M) and 4 low Kozak mixed clones (labeled L) on the left. On the right, the name and translational strength of each Kozak sequence variant is provided. Grey highlighted is unavailable sequencing data.

Figure 22A shows the quantification of bispecific antibody, mis-paired by-products and half antibody species by mass spectrometry for WT Kozak clone 50 and Kozak mixed clones 69 and 17M. Correctly assembled bispecific antibodies are highlighted in white.

Figure 22B shows the effective titers of bispecific antibody, incorrectly paired by-products, and half antibody species, calculated as the percentage of each species multiplied by the general titer, shown on the right. Correctly assembled bispecific antibodies are highlighted in white.

The top panel, fig. 22C, shows the productivity of clone 17M. The CE-SDS data of this clone showed a product mass of about 48% for full-ab and about 52% for half-ab. Mass spectral data indicate that in approximately 48% of whole ab, all of these represent correctly assembled bispecific antibodies. On the other hand, almost all the semi-Ab species were knob 1/2Ab2, consistent with the fact that the heavy and light chains of Ab2 were under Wt Kozak and the strongest Kozak sequence variant #228, respectively. At the bottom, the name and translational strength of each Kozak sequence variant are shown. Clone 17M carried both the heavy and light chains of Ab1 under the weaker Kozak sequence variant.

Figure 22D provides quantification of bispecific antibody, incorrectly paired by-products and half-antibody formats by mass spectrometry of indicated medium low Kozak mixed clones. The percent of BsAb assembly was less than 10%, except for Kozak mixed clone 17. The absolute abundance of each species correlates with the intensity of expression of each chain.

Detailed Description

I. Definition of

Before the present disclosure is described in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a", "an", "the" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes a combination of two or more such molecules, and the like.

The term "about" as used herein refers to the usual range of error for the corresponding value as readily known to those of skill in the art. References herein to "about" a value or parameter include (and describe) embodiments that refer to the value or parameter itself.

It is understood that the aspects and embodiments of the disclosure described herein include aspects and embodiments that are referred to as "comprising," consisting of, "and" consisting essentially of.

The terms "polypeptide" or "protein" are used interchangeably herein to refer to an amino acid polymer of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component or toxin. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically include antibodies and antigen binding proteins.

As used herein, a "multimeric" polypeptide or protein may refer to any complex comprising more than one polypeptide or protein monomer (e.g., polypeptide chain). For example, a multimeric polypeptide or protein may refer to a complex comprising two or more subunits, each subunit comprising one, two, or more different polypeptide chains. The term "multimeric" polypeptide or protein specifically includes antibodies and antigen binding proteins. In certain embodiments, the multimeric polypeptide is a bispecific antibody. In certain embodiments, the bispecific antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain. In certain embodiments, the bispecific antibody is an IgG1, IgG2, or IgG4 isotype. In certain embodiments, the bispecific antibody is an IgG1 or IgG4 isotype. In certain embodiments, the bispecific antibody is an IgG1 isotype.

As used herein, the term "subunit," when used to refer to a multimeric polypeptide or a component of a protein, is intended to refer to any polypeptide comprising more than one different polypeptide chain. In some embodiments, a subunit may comprise a macromolecular complex of two or more polypeptides linked together by one or more intermolecular bonds, including but not limited to one or more disulfide bonds. In certain embodiments, the subunits comprise immunoglobulin heavy and light chains. In certain embodiments, the subunit comprises a half-antibody. In certain embodiments, the immunoglobulin heavy and light chains or half-antibodies are of the IgG1, IgG2, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy and light chains or half-antibodies are of the IgG1 or IgG4 isotype. In certain embodiments, the immunoglobulin heavy and light chains or half-antibodies are of the IgG1 isotype.

As used herein, "incorrectly paired by-products" refers to assembly of one or more polypeptide chain components of a multimeric polypeptide in an undesirable arrangement. For example, if the multimeric polypeptide is a bispecific antibody comprising subunit light chain a/heavy chain a and subunit light chain B/heavy chain B, incorrectly paired by-products result from incorrect pairing during the production of heavy chain a dimers, heavy chain B dimers, and/or incorrect pairing of one or more light chain a/heavy chain a and light chain B/heavy chain B subunits, and include any product other than a correctly assembled bispecific antibody. Specific examples of incorrectly paired by-products are referenced and shown in fig. 20A, 20B, and 22A-22D. Other methods for assessing protein folding, such as differential scanning fluorescence, MS, or using computer tools to predict the outcome may also be used.

As used herein, the term "translation initiation sequence" refers to a polynucleotide sequence comprising a start codon and adjacent bases of a polynucleotide comprising an open reading frame. According to the translational scanning model, the ribosome pre-initiation complex binds to the 5 'end of the polynucleotide and proceeds linearly in the 3' direction to look for a stop codon. Work on Marilyn Kozak based on a scanning model describing translation initiation in detail (see, e.g., Kozak M. nucleic Acids Res.1981; 9(20): 5233-52; Kozak M. nucleic Acids Res.1987; 15(20): 8125-48; Kozak M. EMBO J.1997; 16(9): 2482-92; Kozak M. Jmol biol.1987; 196(4): 947-50; Hamilton R, Watanabe CK, de Boer HA. nucleic Acids Res.1987; 15(8): 81-93; Cavener DR. nucleic Acids Res.1987; 15(4): 1353-61; Kozak M. nucleic Acids Res.283; 3512. DR. nucleic Acids Res.1987; 15(4): 1353-61; Kozak M. Res. 1984; Kiven DR. 8512-857; Kizak J.857; Kozak M. Biozak sequence of Kozak M. J.102, Kozak M. SJ.32; Kozak M. JK.15, SJ.10, J.15, 15, 35, 120, 15, 3-61; Kozak, III.

As used herein, the terms "native" and "non-native" refer to one or more genetic elements (e.g., encoding polypeptides, promoters, translation units, or combinations thereof), meaning the genomic context in which the genetic element in the host cell chromosome is found in nature. For example, a polypeptide (e.g., a multimeric polypeptide) is "native" to a host cell or host cell chromosome when the polynucleotide encoding the polypeptide naturally occurs in the genome of the host cell, and is "non-native" when the polynucleotide encoding the polypeptide does not naturally occur in the genome of the host cell. A translation initiation sequence or open reading frame is "native" to a host cell or host cell chromosome when the translation initiation sequence or open reading frame is naturally present in the genome of the host cell, and is "non-native" when the translation initiation sequence or open reading frame is not naturally present in the genome of the host cell. An operable combination of a translation initiation sequence and an open reading frame is "non-natural" when the translation initiation sequence is not naturally present in the genome of the host cell in the same operable linkage as the open reading frame, or vice versa. For example, the translation initiation sequence: when one or both of the translation initiation sequence and the open reading frame are not naturally occurring in the genome of the host cell, when the translation initiation sequence is present in the genome of the host cell in operable linkage with an open reading frame that is not operably combinable in the naturally occurring host genome (even if the same translation initiation sequence is naturally occurring elsewhere in the genome of the host cell), or when the open reading frame is present in the genome of the host cell in operable linkage with a translation initiation sequence that is not operably combinable in the naturally occurring host genome (even if the same open reading frame sequence is naturally occurring elsewhere in the genome of the host cell), the translation initiation sequence: the open reading frame combination is "non-native" to the host cell or host cell chromosome.

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication and episomal mammalian vectors). After introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell and thereby replicated together with the host genome. In addition, certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector.

"operably linked" refers to the juxtaposition of two or more components wherein the components are in a relationship permitting them to function in their intended manner. Typically, but not necessarily, DNA sequences that are "operably linked" are contiguous and, where necessary to join two protein coding regions, or, in the case of a secretory leader, contiguous and in reading frame. However, while an operably linked promoter is typically located upstream of a coding sequence or translational unit, it need not be contiguous therewith. Operably linked enhancers can be located upstream, within, or downstream of the coding sequence/translational unit, and at considerable distance from the promoter. Ligation is accomplished by recombinant methods known in the art, for example using PCR methods, by annealing or by ligation at convenient restriction sites. If suitable restriction sites are not present, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional specifications.

"promoter" refers to a polynucleotide sequence that controls the transcription of a gene or sequence to which it is operably linked. Promoters include signals for RNA polymerase binding and transcription initiation. The promoter used will function in the cell type of the host cell in which the selected sequence is expected to be expressed. A large number of promoters, including constitutive, inducible and repressible promoters from a variety of different sources, are well known in the art (and identified in databases such as GenBank).

As used herein, the term "host cell" (or "recombinant host cell") means a cell that has been genetically altered or can be genetically altered by the introduction of an exogenous or non-native polynucleotide, e.g., a recombinant plasmid or vector. It is understood that these terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in the progeny due to mutation or environmental impact, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "antigen" is used herein in the broadest sense and encompasses various forms of both polypeptide and non-polypeptide antigens, including but not limited to small peptide antigens, full-length protein antigens, carbohydrate antigens, lipid antigens, and nucleic acid antigens.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as the antibody fragment exhibits the desired antigen-binding activity. The term "immunoglobulin" (Ig) is used interchangeably herein with antibody.

An "isolated" antibody is an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are materials that would interfere with antibody research, diagnostic or therapeutic uses, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight of the antibody (e.g., as determined by the Lowry method), in some embodiments, greater than 99% by weight; (2) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence (e.g., by using a rotary cup sequencer), or (3) homogenization (SDS-PAGE under reducing or non-reducing conditions, using, for example, coomassie blue or silver staining). Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment will not be present. Typically, however, the isolated antibody will be prepared by at least one purification step.

The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin (i.e., the variable domain, which comprises the antigen binding site). Constant Domain comprising heavy chain C_H1、C_H2 and C_HDomain 3 (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "V_H". The variable domain of the light chain may be referred to as "V_L. These domains are usually the most variable part of the antibody and contain the antigen binding site.

The term "variable" refers to the fact that: certain portions of the variable domains vary widely in sequence between antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed among the variable domains of the antibody. It is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, predominantly in the beta sheet structure, connected by three HVRs, which form loops connecting and in some cases forming part of the beta sheet structure. The HVRs in each chain are held tightly together by the FR region and, together with the HVRs in the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, U.S. department of health and public service, national institute of health, Bessesda, Maryland (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but have respective effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

The "light chain" of an antibody (immunoglobulin) from any mammalian species can be assigned to one of two distinctly different classes, termed kappa ("κ") and lambda ("λ"), respectively, based on the amino acid sequence of its constant domain.

As used herein, the term IgG "isotype" or "subclass" refers to any subclass of immunoglobulin defined by the chemical and antigenic characteristics of the constant regions of the immunoglobulin.

Antibodies (immunoglobulins) can be classified into different classes according to the amino acid sequence of their heavy chain constant domains. Immunoglobulins are largely divided into five classes: IgA, IgD, IgE, IgG and IgM, and some of them may be further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described in, for example, the following documents: abbas et al, Cellular and molecular immunology, 4 th edition (w.b. saunders, co., 2000). The antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as defined below. The term particularly refers to antibodies having a heavy chain comprising an Fc region.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, an antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site and a residual "Fc" fragment, the name reflecting its ability to crystallize readily. F (ab') produced by pepsin treatment₂The fragment has two antigen binding sites and is still capable of cross-linking with antigen.

"Fv" is the smallest antibody fragment that contains the complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light chain variable domain in tight and non-covalent association. In the single chain Fv (scfv) class, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker such that the light and heavy chains may associate into a "dimer" structure similar to that in the two chain Fv class. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity on the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

Fab fragments contain a heavy chain variable domain and a light chain variable domain and also contain the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab 'fragments differ from Fab fragments in that the Fab' fragments have added to the carboxy terminus of the heavy chain CH1 domain residues including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domains carry a free thiol group. F (ab')₂Antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, allowing the scFv to form the desired antigen binding structure. For reviews on scFv, see for example Pluckthun, Pharmacology of Monoclonal Antibodies (The Pharmacology of Monoclonal Antibodies), Vol.113, eds, Rosenburg and Moore, (Springer-Verlag, New York,1994), p.269-315.

The term "diabodies" refers to antibody fragments having two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain and create two antigen binding sites. Diabodies can be bivalent antibodies or bispecific antibodies. Diabodies are more fully described, for example, in: EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Trisomal and tetrasomal antibodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible minor mutations, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be to select a unique clone from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to increase affinity for the target, to humanize the target binding sequence, to increase its production in cell culture, to decrease its immunogenicity in vivo, to produce a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the disclosure. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibody preparations are also advantageous in that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, Monoclonal Antibodies used in accordance with the present disclosure may be prepared by a variety of techniques including, for example, expression in prokaryotic host cells, Hybridoma methods (e.g., Kohler and Milstein, Nature,256:495-97 (1975); Hongo et al, Hybridoma,14(3):253-260(1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd edition 1988); Hammerling et al, Monoclonal Antibodies and T-Cell Hybridoma 563-681(Elsevier, N.Y.,1981)), recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), phage display technology 2004 (see, for example, Clackson et al, Nature 352, 624: 628 (1991); Mardks et al, Mardhks et al, 12442. J12431; Meldhl et al, 134: 96-340 (Ledhe et al), Nature, Ledhk et al, 134-5, 134-35; Ledhk et al, 134-35; Nature, Ser. 32-35; Ledhk et al, 1981); Biodhe et al, Biol 5, Ser-32, WO 32,338, 76, methods 284(1-2):119-132(2004)) and techniques for producing human antibodies or human-like antibodies in animals having part or all of a human immunoglobulin locus or gene encoding a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; jakobovits et al, Proc.Natl.Acad.Sci.USA 90:2551 (1993); jakobovits et al, Nature 362:255-258 (1993); bruggemann et al, Yeast in Immunol.7:33 (1993); U.S. Pat. nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016; marks et al, Bio/Technology 10:779-783 (1992); lonberg et al, Nature 368:856-859 (1994); morrison, Nature 368: 812-; fishwild et al, Nature Biotechnol.14: 845-; neuberger, Nature Biotechnol.14:826(1996) and Lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995)).

The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of an antibody variable domain which is hypervariable in sequence and/or forms structurally defined loops. Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 showed the most diversity among six HVRs, and in particular H3 was thought to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al, Immunity 13:37-45 (2000); johnson and Wu, Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camelid antibodies consisting of only heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996).

Many HVR descriptions are used and are included herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, department of public and health services, national institutes of health, Besserda, Md. (1991)). In contrast, Chothia refers to the position of the structural loop (Chothia and Lesk J.mol.biol.196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and Chothia structural loops and was adopted by the AbM antibody modeling software of Oxford Molecular (Oxford Molecular). The "contact" HVRs are based on available analysis results of complex crystal structures. The residues of each of these HVRs are described below.

TABLE 1a. hypervariable regions of antibodies

The HVRs can include the following "extended HVRs": 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97 or 89-96(L3) in VL, and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable domain residues are numbered according to the method of Kabat et al, supra.

"framework" or "FR" residues are those variable domain residues other than the HVR residues as defined herein.

The term "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof refers to the numbering system proposed in the Kabat et al reference above for either the heavy chain variable domain or the light chain variable domain. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to a shortening or insertion of the FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat numbering) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat numbering, etc.) after heavy chain FR residue 82. The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequences to regions of homology of "standard" Kabat numbered sequences.

When referring to residues in the variable domain (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al, Sequences of Immunological interest, 5 th edition, department of U.S. department of health and public service, national institutes of health, betesday, maryland (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" (e.g., the EU index reported by Kabat et al, supra) is typically used.

The expression "linear antibody" refers to the antibody described by Zapata et al (1995Protein Eng,8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandemly connected Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions with a complementary light chain polypeptide. Linear antibodies may be bispecific or monospecific.

Multimeric polypeptide production and screening methods

Provided herein are methods of producing multimeric polypeptides in eukaryotic host cells (e.g., mammalian host cells). In some embodiments, the method comprises: providing a eukaryotic host cell comprising a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding a first polypeptide chain of a multimeric polypeptide, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding a second polypeptide chain of a multimeric polypeptide, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a third polypeptide chain of a multimeric polypeptide, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a fourth polypeptide chain of a multimeric polypeptide; culturing a eukaryotic host cell under conditions suitable for expression of the first, second, third, and fourth polypeptide chains, wherein the first, second, third, and fourth polypeptide chains form a multimeric polypeptide; and recovering the multimeric polypeptide produced by the eukaryotic host cell.

In some embodiments, when each polypeptide chain is produced alone in a eukaryotic host cell or when two polypeptide chains are produced together by the same eukaryotic host cell, the first subunit is produced at a lower level than the second subunit, and one or both of the first and second translation initiation sequences are weaker than one or both of the third and fourth translation initiation sequences. In some embodiments, each subunit comprises a half-antibody comprising an antibody heavy chain and an antibody light chain, and the multimeric polypeptide is a bispecific antibody.

The production methods described herein are based, at least in part, on the demonstration herein that adjusting translational strength using different translation initiation sequences can improve multimeric polypeptide (e.g., bispecific antibody) assembly and reduce product-related impurities, such as incorrectly paired by-products. It has been surprisingly found that a reduction in translation of selected polypeptide chains relative to the level of translation of other polypeptide chains of a multimeric polypeptide can increase the efficiency of multimeric polypeptide assembly and increase overall product quality by limiting the accumulation of incorrectly paired by-products. Without wishing to be bound by theory, it is believed that the production of some polypeptide chains at the strongest translation initiation sequence actually drives the production of more incorrectly paired by-products comprising the chain, rather than more correctly assembled multimeric polypeptides. Thus, down-regulating translation of one or more chains of a multimeric polypeptide (e.g., a bispecific antibody) may slow the assembly of the multimeric polypeptide (e.g., a bispecific antibody), which may actually positively impact assembly efficiency and reduce unwanted mis-paired by-products. Thus, the methods described herein not only allow for higher levels of multimeric polypeptide expression, but also reduce the production of impurities, such as incorrectly paired by-products, which carry a burden, such as requiring expensive purification methods to remove them.

In some embodiments, when each subunit is produced individually in a eukaryotic host cell, or when all subunits are produced together in the same eukaryotic host cell, one or both polypeptide chains of the first subunit are translated at a lower level than one or both polypeptide chains of the second subunit. In some embodiments, when each subunit is produced individually in a eukaryotic host cell, or when all subunits are produced together in the same eukaryotic host cell, the first subunit assembles with more incorrectly paired byproducts than the second subunit. In some embodiments, the use of a weaker translation initiation sequence operably linked to one or more (e.g., one or two) polypeptide chains of a first subunit (e.g., a weaker or more difficult to express subunit in a host cell) results in higher yields and/or fewer mis-paired by-products of the multimeric polypeptide compared to the translation initiation sequence of one or more (e.g., one or two) polypeptide chains linked to a second subunit. In some embodiments, when each subunit is expressed individually in a eukaryotic host cell, one or both polypeptide chains of the first subunit are translated at a slower rate than one or both polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed individually in a eukaryotic host cell, one or both polypeptide chains of the first subunit fold more slowly and/or less efficiently than one or both polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed individually in a eukaryotic host cell, the first subunit assembles at a slower rate than the second subunit. Assays for measuring protein translation rates are known in the art and include, but are not limited to, 35S methionine labeling and ribosome profiling (see, e.g., Ingolia, N. (2016) Cell 165: 22-33).

As described herein, in some embodiments, a multimeric polypeptide of the present disclosure comprises two subunits. In some embodiments, a subunit of the disclosure comprises two or more polypeptide chains. In some embodiments, a subunit of the disclosure comprises two polypeptide chains. In some embodiments, each polynucleotide encoding a polypeptide chain of the multimeric polypeptide comprises a translation initiation sequence operably linked to an open reading frame. In some embodiments, the multimeric polypeptides of the present disclosure comprise two subunits, each subunit comprising two polypeptide chains, a first subunit of the multimeric polypeptide being expressed at a lower level than a second subunit when each subunit is expressed alone in a eukaryotic host cell or when all subunits are expressed together in the same eukaryotic host cell, and one or both of the first translation initiation sequence (operably linked to the first open reading frame) and the second translation initiation sequence (operably linked to the second open reading frame) being weaker than one or both of the third translation initiation sequence (operably linked to the third open reading frame) and the fourth translation initiation sequence (operably linked to the fourth open reading frame). In some embodiments, the multimeric polypeptide is a bispecific antibody.

In some embodiments, a subunit or half-antibody of the present disclosure is expressed at a lower level than another subunit or half-antibody, e.g., when each subunit or half-antibody is expressed separately in a eukaryotic host cell. In some embodiments, a subunit or half-antibody of the disclosure is expressed at a level that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, 5% to 30%, 5% to 50%, 5% to 75%, 10% to 30%, 10% to 50%, 10% to 75%, 25% to 50%, 25% to 75%, 25% to 100%, 50% to 75%, 50% to 100%, or 75% to 100% lower than another subunit or half-antibody, e.g., when each subunit or half-antibody is expressed separately in a eukaryotic host cell. In some embodiments, a subunit or half-antibody of the disclosure is expressed at a level at least 1.3 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, 1.3 fold to 3 fold, 1.5 fold to 3 fold, 2 fold to 10 fold, 2 fold to 5 fold, 3 fold to 10 fold, 5 fold to 10 fold, or 7 fold to 10 fold lower than another subunit or half-antibody, e.g., when each subunit or half-antibody is expressed alone in a eukaryotic host cell. In some embodiments, a subunit or half-antibody of the disclosure is expressed at a level 0.2-fold to 0.8-fold, less than 0.5-fold, or less than 0.3-fold lower than another subunit or half-antibody, e.g., when each subunit or half-antibody is expressed alone in a eukaryotic host cell.

In some embodiments, the method comprises: providing a eukaryotic host cell comprising a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame of a first antibody heavy chain encoding a bispecific antibody, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame of a first antibody light chain encoding a bispecific antibody, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame of a second antibody heavy chain encoding a bispecific antibody, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame of a second antibody light chain encoding a bispecific antibody; culturing a eukaryotic host cell under conditions suitable for expression of the first, second, third, and fourth polypeptide chains, wherein upon expression the first, second, third, and fourth polypeptide chains form a bispecific antibody; recovering said bispecific antibody from the eukaryotic host cell. In some embodiments, the bispecific antibody specifically binds to two antigens (e.g., a first antibody heavy and light chain forms an antigen binding domain that binds a first antigen, and a second antibody heavy and light chain forms an antigen binding domain that binds a second antigen).

In some embodiments, a first translation initiation sequence is said to be weaker than another translation initiation sequence when the first translation initiation sequence results in lower translation efficiency and/or expression of the open reading frame to which it is operably linked than the reference or efficiency/expression of the same open reading frame to which the other translation initiation sequence is operably linked. Suitable methods for comparing the strength of various translation initiation sequences in eukaryotic host cells are described and illustrated herein. For example, transient or stable transfectants can be cultured, and production of multimeric polypeptides can be assayed. Fed-batch or perfusion cultures are cultured and the product titer in the cell culture medium or on the cell surface can be determined. For surface expression, cells can be stained with antibodies to detect products and analyzed, for example, by flow cytometry. The product quality and/or purity can be analyzed by, for example, electrophoresis and/or mass spectrometry.

In some embodiments, a translation initiation sequence is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, 5% to 30%, 5% to 50%, 5% to 75%, 10% to 30%, 10% to 50%, 10% to 75%, 25% to 50%, 25% to 75%, 25% to 100%, 50% to 75%, 50% to 100%, or 75% to 100% weaker than another translation initiation sequence. In some embodiments, when one translation initiation sequence results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, 5% to 30%, 5% to 50%, 5% to 75%, 10% to 30%, 10% to 50%, 10% to 75%, 25% to 100%, 50% to 75%, 50% to 100%, or 75% to 100% less efficient translation of the same open reading frame to which it is operably linked as compared to a reference or efficiency of the same open reading frame to which another translation initiation sequence is operably linked (e.g., when measured as described above), the translation initiation sequence is said to be weaker than the other translation initiation sequence. In some embodiments, when one translation initiation sequence results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, 5% to 30%, 5% to 50%, 5% to 75%, 10% to 30%, 10% to 50%, 10% to 75%, 25% to 100%, 50% to 75%, 50% to 100%, or 75% to 100% less expression of an open reading frame operably linked to another translation initiation sequence than a reference or level of expression of the same open reading frame operably linked to another translation initiation sequence (e.g., when measured as described above), the translation initiation sequence is said to be weaker than the other translation initiation sequence.

In some embodiments, one translation initiation sequence and is at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, 1.3-fold to 3-fold, 1.5-fold to 3-fold, 2-fold to 10-fold, 2-fold to 5-fold, 3-fold to 10-fold, 5-fold to 10-fold, or 7-fold to 10-fold weaker than the other translation initiation sequence. In some embodiments, a translation initiation sequence is said to be weaker than another translation initiation sequence when the translation initiation sequence results in at least 1.3-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, 1.3-fold to 3-fold, 1.5-fold to 3-fold, 2-fold to 10-fold, 2-fold to 5-fold, 3-fold to 10-fold, 5-fold to 10-fold, or 7-fold to 10-fold less efficient translation of the same open reading frame operably linked to the other translation initiation sequence, as compared to a reference or efficiency of the same open reading frame operably linked to the other translation initiation sequence (e.g., when measured as described above). In some embodiments, a translation initiation sequence is said to be weaker than another translation initiation sequence when the translation initiation sequence results in at least 1.3 fold, at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, 1.3 fold to 3 fold, 1.5 fold to 3 fold, 2 fold to 10 fold, 2 fold to 5 fold, 3 fold to 10 fold, 5 fold to 10 fold, or 7 fold to 10 fold less expression of the same open reading frame operably linked thereto as compared to a reference or level of expression of the same open reading frame operably linked to another translation initiation sequence (e.g., when measured as described above). In some embodiments, a translation initiation sequence is said to be weaker than another translation initiation sequence when the one translation initiation sequence results in expression that is 0.2-fold to 0.8-fold, less than 0.5-fold, or less than 0.3-fold greater than the expression of the same open reading frame to which it is operably linked, as compared to a reference or expression level of the same open reading frame to which the other translation initiation sequence is operably linked (e.g., when measured as described above).

In some embodiments, the first translation initiation sequence is weaker than the third translation initiation sequence. In some embodiments, the second translation initiation sequence is weaker than the fourth translation initiation sequence. In some embodiments, the first translation initiation sequence is weaker than the fourth translation initiation sequence. In some embodiments, the second translation initiation sequence is weaker than the third translation initiation sequence. In some embodiments, the first translation initiation sequence is identical to the second translation initiation sequence. In some embodiments, the first translation initiation sequence is different from the second translation initiation sequence. In some embodiments, the third translation initiation sequence is identical to the fourth translation initiation sequence. In some embodiments, the third translation initiation sequence is different from the fourth translation initiation sequence.

In some embodiments, for example, the methods of the present disclosure result in higher yields of multimeric polypeptides in eukaryotic host cells as compared to producing multimeric polypeptides using existing methods. In some embodiments, for example, the methods of the present disclosure result in a higher yield of multimeric polypeptide (e.g., a higher level of correctly assembled multimeric polypeptide, such as a bispecific antibody) in a eukaryotic host cell of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to the production of multimeric polypeptide using existing methods. For example, expression can be compared to expression in which one or more, two or more, three or more, or four polynucleotides encoding polypeptide chains of the multimeric polypeptide comprise an open reading frame operably linked to a native or unmodified translation initiation sequence, or in which each polynucleotide encoding polypeptide chains of the multimeric polypeptide comprises the same translation initiation sequence.

In some embodiments, for example, the methods of the present disclosure result in fewer mis-paired by-products of the multimeric polypeptide in the eukaryotic host cell as compared to producing the multimeric polypeptide using existing methods. In some embodiments, for example, the methods of the present disclosure result in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% fewer mis-paired byproducts of the multimeric polypeptide in the eukaryotic host cell as compared to producing the multimeric polypeptide using existing methods. The methods of the present disclosure facilitate downstream purification in manufacturing due to fewer impurities (e.g., incorrectly ventilated by-products) in the cell culture supernatant. In some embodiments, the yield can be compared to a yield in which one or more, two or more, three or more, or four polynucleotides encoding polypeptide chains of the multimeric polypeptide comprise an open reading frame operably linked to a native or unmodified translation initiation sequence, or in which each polynucleotide encoding polypeptide chains of the multimeric polypeptide comprises the same translation initiation sequence. In some embodiments, the amount of mis-paired byproducts refers to the amount of one or more particular mis-paired byproducts. In some embodiments, the amount of mis-paired byproducts refers to the total amount of all mis-paired byproducts.

In some embodiments, the methods of the present disclosure can be used to express four subunits of a multimeric polypeptide of the present disclosure at a chain ratio of 1:1:1: 1. However, the methods described herein can also be used to modulate the expression of the four subunits of the multimeric polypeptides of the present disclosure at other desired chain ratios, e.g., to improve the expression and/or assembly of correctly paired multimeric polypeptides. For example, it is well established that antibody light chains can serve as chaperones for antibody heavy chains (see, e.g., Lee, Y.K, et al (1999) mol. biol. cell 10:2209-2219), and that it may be an advantage to have higher light chain expression during production. Thus, in some embodiments, the methods of the present disclosure are used to increase the expression of one or both light chains of a bispecific antibody relative to the expression of a heavy chain. Furthermore, the data provided herein indicate that in some cases, a higher proportion of correctly assembled bispecific antibody can be obtained by manipulating the ratio of light chain 1: light chain 2. Thus, in some embodiments, the methods of the present disclosure are used to increase the expression of one light chain of a bispecific antibody relative to the expression of another light chain. In some embodiments, the methods of the present disclosure can be used to express the four polypeptide chains of a bispecific antibody in a 1:1:1:1 chain ratio or another chain ratio, depending on the optimal method of production of the bispecific antibody, which can be determined as described herein.

In some embodiments, one or more of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence comprises a sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1). In some embodiments, all of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence comprise the sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1). In some embodiments, one or both translation initiation sequences operably linked to an open reading frame encoding a polypeptide chain of a first subunit (e.g., a less expressed subunit of a multimeric polypeptide) comprises a sequence selected from the group consisting of: 8-10 of SEQ ID NO. In some embodiments, one or both translation initiation sequences operably linked to an open reading frame of a polypeptide chain encoding a second subunit (e.g., a more strongly expressed subunit of a multimeric polypeptide) comprises a sequence selected from the group consisting of: 2, 3 and 11.

Exemplary translation initiation sequences are provided in table 2. The relative strengths of exemplary translation initiation sequences are provided in fig. 10.

TABLE 2 translation initiation sequence.

In some embodiments, the translation efficiency (i.e., strength) of the translation initiation sequence is compared to a reference. In some embodiments, the reference is a translation initiation sequence comprising a wild-type Kozak sequence in the host cell. In some embodiments, the reference sequence comprises the polynucleotide sequence of SEQ ID NO. 2 or 3. Exemplary assays suitable for determining the relative strength of translation initiation sequences are described and illustrated herein (see, e.g., examples 1-3). In some embodiments, the strength of the translation initiation sequence is determined by expressing (e.g., after transient transfection) a translation initiation sequence comprising an open reading frame (e.g., encoding a polynucleotide, subunit, or multimeric polypeptide of the disclosure, such as an antibody or half-antibody) operably linked to a polypeptide in a host cell and measuring the yield of the polypeptide product. In some embodiments, the level of production of the polypeptide product (e.g., product titer) is compared to a reference, e.g., the level of production of the same product in the same host cell type, wherein the open reading frame is operably linked to a reference translation initiation sequence, e.g., a wild-type Kozak sequence that is active in the host cell. The relative strengths of exemplary translation initiation sequences are provided in fig. 10. For example, transient or stable transfectants can be cultured, and production of multimeric polypeptides can be assayed. Fed-batch or perfusion cultures are cultured and the product titer in the cell culture medium or on the cell surface can be determined. For surface expression, cells can be stained with antibodies to detect products and analyzed, for example, by flow cytometry. The product quality and/or purity can be analyzed by, for example, electrophoresis and/or mass spectrometry.

In some embodiments, the open reading frame of the first polypeptide chain of the first subunit (e.g., the less expressed subunit) of the multimeric protein is operably linked to a translation initiation sequence comprising SEQ ID NO: 9. In some embodiments, the open reading frame of the second polypeptide chain of the first subunit (e.g., the less expressed subunit) of the multimeric protein is operably linked to a translation initiation sequence comprising SEQ ID NO: 9. In some embodiments, the open reading frame of the first polypeptide chain of the second subunit (e.g., the more strongly expressed subunit) of the multimeric protein is operably linked to a translation initiation sequence comprising SEQ ID NO:2 or 11. In some embodiments, the open reading frame of the second polypeptide chain of the second subunit (e.g., the less expressed subunit) of the multimeric protein is operably linked to a translation initiation sequence comprising SEQ ID NOs 2 or 11. In some embodiments, the open reading frame of the first polypeptide chain of the first subunit (e.g., the subunit expressing the weaker) is operably linked to the translation initiation sequence comprising SEQ ID NO:9, the open reading frame of the second polypeptide chain of the polypeptide chains of the first subunit (e.g., the subunit expressing the weaker) is operably linked to the translation initiation sequence comprising SEQ ID NO:9, the open reading frame of the first polypeptide chain of the second subunit (e.g., the subunit expressing the stronger) is operably linked to the translation initiation sequence comprising SEQ ID NO:2 or 11, and the open reading frame of the second polypeptide chain of the second subunit (e.g., the subunit expressing the stronger) is operably linked to the translation initiation sequence comprising SEQ ID NO:2 or 11. In some embodiments, the multimeric protein is a bispecific antibody. In some embodiments, the first subunit and the second subunit comprise half-antibodies. In some embodiments, the two polypeptide chains of each subunit represent an antibody heavy chain and an antibody light chain, respectively. In some embodiments, the open reading frame encoding the heavy chain of the first half antibody is operably linked to a translation initiation sequence comprising SEQ ID No. 9, the open reading frame encoding the light chain of the first half antibody is operably linked to a translation initiation sequence comprising SEQ ID No. 9, the open reading frame encoding the heavy chain of the second half antibody is operably linked to a translation initiation sequence comprising SEQ ID No. 3, and the open reading frame encoding the light chain of the second half antibody is operably linked to a translation initiation sequence comprising SEQ ID No. 11.

Promoters

In some embodiments, the translation initiation sequences and/or open reading frames of the present disclosure are operably linked to a promoter. In some embodiments, each of the first, second, third, and fourth polynucleotides is operably linked to a promoter. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron and can regulate its expression. Prokaryotic promoters are generally classified into two classes, inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the level of transcription of a cistron under its control in response to a change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. A variety of promoters suitable for use in eukaryotic host cells are known in the art. In some embodiments, the polynucleotides encoding the first and second polypeptide chains of a subunit are operably linked to the same promoter. In some embodiments, the polynucleotides encoding the first and second polypeptide chains of a subunit are operably linked to different promoters.

Numerous promoters recognized by a variety of potential host cells are well known. The selected promoter may be operably linked to cistron DNA encoding, for example, a light or heavy chain, by removing the promoter from the source DNA by restriction endonuclease digestion and inserting the isolated promoter sequence into the vector of the invention. Both native promoter sequences and many heterologous promoters can be used to direct amplification and/or expression of a target gene. In some embodiments, heterologous promoters are used because they generally allow for greater transcription and higher yields of expressed target genes as compared to the native target polypeptide promoter.

Expression and cloning vectors typically contain a promoter that is recognized by the host organism and operably linked to the desired Fc-containing polypeptide (e.g., antibody) nucleic acid. Promoter sequences for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream from the transcription start site. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. The 3 'end of most eukaryotic genes is the AATAAA sequence, which may be a signal to add a poly A tail to the 3' end of the coding sequence. All these sequences are suitably inserted into eukaryotic expression vectors.

For the production of Fc-containing polypeptides (e.g., antibodies), antibody transcription of the vector in mammalian host cells is controlled, for example, by: promoters obtained from the genome of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, for example, the actin promoter or immunoglobulin promoter, or from heat shock promoters, provided that these promoters are compatible with the host cell system. In some embodiments, the promoter is a CMV promoter.

The early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments, which also contain the replication origin of the SV40 virus. The immediate early promoter of human cytomegalovirus is conveniently obtained as a Hind 111E restriction fragment. U.S. Pat. No. 4,419,446 discloses a system for expressing DNA in a mammalian host using bovine papilloma virus as a vector. Modifications of this system are described in U.S. Pat. No. 4,601,978. See also Reyes et al, Nature 297:598-601(1982) for the expression of human interferon-beta cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, Rous Sarcoma Virus (Rous Sarcoma Virus) long terminal repeat can be used as a promoter.

Transcription of DNA encoding antigen-binding polypeptides (e.g., antibodies) by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin genes). Similarly, an enhancer from a eukaryotic cell virus may be used. Examples include the SV40 enhancer on the posterior side of the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the origin of replication, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) for a description of elements that enhance activation of eukaryotic promoters. Enhancers may be spliced into the vector at the 5' or 3' position of the antibody polypeptide coding sequence, provided that enhancement is achieved, but are typically located at the 5' site of the promoter.

Multispecific multimeric polypeptides

Certain aspects of the disclosure, for example, relate to multimeric polypeptides comprising two or more subunits, each subunit comprising two or more polypeptide chains. In some embodiments, one or more polypeptide chains, one or more subunits, or one or more multimeric polypeptides of the disclosure are non-native to the host cell.

In some embodiments, the subunits of the present disclosure are monomers of heterodimers. As used herein, a heterodimer may refer to any polypeptide complex comprising two distinct polypeptides or polypeptide complexes operably linked. A non-limiting example of a heterodimer is a bispecific or bivalent antibody composed of two distinct antibody monomers (i.e., in an operably linked light-heavy chain pair). In this example, folding and assembly of a first heavy chain-light chain pair that recognizes a first antigen produces a first antibody monomer. The folding and assembly of a second heavy chain-light chain pair recognizing a second antigen produces a second antibody monomer. These monomers can be assembled by any means known in the art (described in more detail below for bispecific antibodies) to form heterodimers. For more details on an illustrative example of heterodimeric antibody formation, see Ridgway JBB et al 1996Protein Eng.9(7): 617-621.

In some embodiments, the multimeric polypeptide or subunit of the disclosure is a secreted protein. As used herein, a secreted protein may refer to any protein secreted by a host cell into the host cell periplasm or extracellular environment. The secreted protein may be a protein that is endogenously secreted by the host cell, or the secreted protein may be a protein that is not endogenously secreted by the host cell but is modified in a manner that promotes its secretion. For example, the presence of a signal sequence, typically found at the N-terminus of a polypeptide, can direct the polypeptide to the secretory pathway for secretion. Many signal sequences are known in the art and can be used to promote secretion of secreted proteins or to allow secretion of proteins not naturally secreted by the host cell, see, e.g., Picken et al, infection.Immun.42: 269-275 (1983); simmons and Yansura, Nature Biotechnology 14:629-634 (1996); and Humphreys DP et al 2000Protein Expr. Purif.20(2): 252. One non-limiting example of a signal sequence is the heat stable enterotoxin ii (stii) signal sequence.

In some embodiments, the polypeptide chains of the subunits of the present disclosure are linked to each other by at least one disulfide bond. In some embodiments, the subunits of the multimeric polypeptides of the present disclosure are linked to each other by at least one disulfide bond. A disulfide bond may refer to any covalent bond linking two thiol groups. Disulfide bonds in polypeptides are typically formed between thiol groups of cysteine residues. Polypeptide disulfide bonds are known in the art to be important for folding and assembly of many polypeptides, such as the two-chain proteins of the present disclosure. Polypeptide disulfide bonds may include disulfide bonds (i.e., intramolecular or intrachain disulfide bonds) between cysteine residues in a single polypeptide chain. Polypeptide disulfide bonds may also include disulfide bonds between cysteine residues found on separate polypeptide chains (i.e., intermolecular or interchain disulfide bonds).

Disulfide bonds are known in the art to be important for the folding and assembly of antibodies and antibody fragments. Different antibody isotopes and different subclasses within isotopes are known to have different disulfide bond patterns. For example, depending on the particular IgG subclass, an IgG antibody May contain 12 intrachain disulfide bonds, one interchain disulfide bond between each light chain and its corresponding heavy chain, and 2 to 11 interchain disulfide bonds between heavy chains (see e Liu H and May K2012 mabs.4(1):17 for a more detailed description). IgM (see, e.g., Wiersma EJ and Shulman MJ 1995J. Immunol.154(10):5265), IgE (see, e.g., Helm BA et al 1991Eur. J. Immunol.21(6):1543), IgA (see, e.g., Chintalacharuvu KR et al 2002J. Immunol.169(9):5072), and IgD (see, e.g., Shin SU et al 1992hum. antibodies hybrids 3(2):65) are also known to form disulfide bonds during folding and assembly.

In some embodiments, the multimeric polypeptide of the present disclosure comprises a multispecific antigen-binding protein. In some embodiments, the multispecific antigen-binding protein binds to two or more epitopes of one, two or more polypeptides or other antigens. Multispecific antibodies have binding specificities for at least two different epitopes, wherein the epitopes are typically derived from different antigens. Although such molecules normally bind only two different epitopes (i.e. bispecific antibodies, BsAb), when used herein, this expression encompasses antibodies with other specificities, e.g. trispecific antibodies. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

In some embodiments, the multimeric polypeptide of the present disclosure is an antibody. In some embodiments, the subunit of the disclosure is a half-antibody. In some embodiments, the antibodies provided herein are chimeric, human, or humanized antibodies. Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments. Antibodies are prepared using techniques available in the art for producing antibodies, as described below, exemplary methods of which are described in more detail in the following sections. One skilled in the art will recognize that many of the methods described below can be applied to multimeric polypeptides other than antibodies.

In some embodiments, the subunit of the disclosure is a monovalent antibody, wherein the first chain and the second chain represent an immunoglobulin heavy chain and an immunoglobulin light chain. As used herein, a monovalent antibody may refer to any polypeptide complex consisting of an antibody heavy chain and an antibody light chain operably linked together to form a heavy chain-light chain pair, wherein the heavy chain-light chain pair is not operably linked to a second heavy chain-light chain pair. The term "half antibody (hAb)" is used interchangeably herein.

In some embodiments, the multimeric polypeptide specifically binds to one or more target antigens. In some embodiments, the multimeric polypeptide or subunit of the disclosure is capable of specifically binding an antigen. As used herein, the terms "bind," "specifically binds," or "specifically" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds that target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the antigen, e.g., as measured by Radioimmunoassay (RIA). In certain embodiments, the antibody that specifically binds to the target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved between proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding. In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 150nM, less than or equal to 100nM, less than or equal to 50nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM or less than or equal to 0.001nM (e.g., 10 nM)^-8M or less,E.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) with the Fab form of the antibody of interest and its antigen as described in the assay below. By using the minimum concentration in the presence of a series of unlabeled antigen titrations (¹²⁵I) The solution binding affinity of Fab for antigen was measured by equilibration of Fab with labeled antigen and subsequent capture of the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.mol.biol.293:865 881 (1999)). To determine the conditions for the assay, capture anti-Fab antibodies (Cappel Labs) were coated with 5. mu.g/ml in 50mM sodium carbonate (pH 9.6)The well plate (Thermo Scientific) was left overnight and then blocked with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23 ℃) for two to five hours. In the non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Mixing the antigen with serial dilutions of the Fab of interest. Then incubating the target Fab overnight; however, incubation may be continued for a longer period of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution was then removed and used with 0.1% polysorbate 20 in PBSThe plate was washed eight times. When the plates had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TM(ii) a Packard) and in TOPCOUNT^TMThe gamma counter (Packard) counts the plate for tens of minutes. The concentration of each Fab that gives less than or equal to 20% maximal binding is selected for use in a competitive binding assay.

According to another example, at 25 ℃, using immobilized antigen CM5 chips at about 10 Response Units (RU)-2000 or3000(BIAcore, Inc., Piscataway, NJ), Kd measured by surface plasmon resonance assay. Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μ g/ml (about 0.2 μ M) with 10mM sodium acetate pH 4.8 before injection at a rate of 5 μ L/min to obtain approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, injection containing 0.05% polysorbate 20 (TWEEN-20) was performed at 25 ℃ at a flow rate of about 25. mu.l/min^TM) Two-fold serial dilutions (0.78nM to 500nM) of Fab in PBS of surfactant (PBST). By fitting both association and dissociation sensorgrams simultaneously, using a simple one-to-one Langmuir binding model: (Evaluation software version 3.2) calculate association rate (kon) and dissociation rate (koff). The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, Chen, Y, et al, J.mol.biol.293:865-881 (1999). If the association rate exceeds 106M-1s-1 as determined by surface plasmon resonance as described above, the association rate can be determined by using a fluorescence quenching technique, e.g., in a spectrometer such as an Aviv Instruments equipped with a flow stopping device or a 8000 series SLM-AMINCO^TMThe increase or decrease in fluorescence emission intensity (excitation 295 nM; emission 340nM, 16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 ℃ was measured in a spectrophotometer (ThermoSpectronic) with a stirred cuvette in the presence of increasing concentrations of antigen.

In some embodiments, the multimeric polypeptide of the present disclosure comprises a bispecific antibody. In some embodiments, the first and third polypeptide chains are antibody heavy chains and the second and fourth polypeptide chains are antibody light chains. In some embodiments, the first subunit is a first half-antibody that binds a first antigen and the second subunit is a second half-antibody that binds a second antigen. In some embodiments, the first antigen and the second antigen are different. In some embodiments, the first antigen and the second antigen are different epitopes of the same target. Other antibodies, such as trispecific antibodies or tetravalent antibodies are also contemplated.

Methods of making bispecific antibodies are known in the art. The traditional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature,305:537-539 (1983)). Due to the random diversity of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome and the product yield is low. Similar procedures are disclosed in WO 93/08829 and Traunecker et al, EMBO J.,10:3655-3659 (1991).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In certain embodiments, the disclosure contemplates antibody variants with some, but not all, effector functions, which make them desirable candidates for use where the half-life of the antibody in vivo is important and certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells NK cells mediating ADCC express only Fc (RIII, whereas monocytes express Fc (RI, Fc (RII and FcR on hematopoietic cells are summarized in Table 3 on page 464 of ravech and Kinet, Kinet, Annu. Rev. Immunol.9:457-492 (1991). in vitro assays for assessing ADCC activity of molecules of interestOther non-limiting examples are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al Proc. nat' l Acad. Sci. USA 83: 7059-; 5,821,337 (see Bruggemann, M. et al, J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox)Non-radioactive cytotoxicity assays (Madison, WI) useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells alternatively or additionally ADCC activity of the molecule of interest can be assessed in vivo, e.g. in animal models such as disclosed in Clynes et al, proc. nat' l acad. sci. usa 95:652-656(1998) CDC 1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and hence lacks CDC activity see, e.g., C1q and C3C binding elisa in WO 2006/029879 and WO 2005/100402 CDC assays can be performed to assess complement activation (see, e.g., Gazzano-Santoro et al, j.methods.202: 163) and r.g, 104m.1996, r.s. 104101 g. 1052-1052, M.S. and M.J.Glennie, Blood 103: 2738-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

Antibodies with reduced effector function include those with substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acids 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al, J.biol.chem 9(2):6591-6604 (2001))

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In one exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A and K334A.

In some embodiments, alterations that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC) are made in the Fc region, for example, as described in U.S. Pat. Nos. 6,194,551, WO 99/51642, and Idusogene et al J.Immunol.164: 4178-.

Antibodies with extended half-life and improved neonatal Fc receptor (FcRn) binding, responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587 (1976); and Kim et al, J.Immunol.24:249(1994)) are described in US2005/0014934A1(Hinton et al). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 in the Fc region (U.S. patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351.

The antibodies of the present disclosure can be further modified to include additional non-protein moieties known in the art and readily available. In certain embodiments, suitable moieties for antibody derivatization are water soluble polymers.

Pestle-in-mortar method

One method known in the art for making bispecific antibodies is the "knob-to-hole" or "bulge-to-cavity" method (see, e.g., U.S. Pat. No. 5,731,168). In this method, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise an interface. The interface of one immunoglobulin polypeptide interacts with the corresponding interface of another immunoglobulin polypeptide, thereby associating the two immunoglobulin polypeptides. These interfaces can be engineered such that a "knob" or "protrusion" (which terms are used interchangeably herein) located on one immunoglobulin polypeptide interface corresponds to a "hole" or "cavity" (which terms are used interchangeably herein) located on another immunoglobulin polypeptide interface. In some embodiments, the hole has the same or similar dimensions as the pestle, and is suitably positioned such that when two interfaces interact, the pestle of one interface can be positioned in the corresponding hole of the other interface. Without wishing to be bound by theory, it is believed that this stabilizes the heteromultimer and favors the formation of the heteromultimer over other species (e.g., homomultimers). In some embodiments, the methods can be used to facilitate heteromultimerization of two different immunoglobulin polypeptides, resulting in a bispecific antibody comprising two immunoglobulin polypeptides having binding specificity for different epitopes.

In some embodiments, the knob may be constructed by replacing the small amino acid side chain with a larger side chain. In some embodiments, the socket can be constructed by replacing the large amino acid side chain with a smaller side chain. The pestle or mortar may be present in the original interface or may be synthetically introduced. For example, the knob or hole can be recombinantly introduced by altering the nucleic acid sequence encoding the interface to replace at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for altering nucleic acid sequences may include standard molecular biology techniques well known in the art. The following table shows the side chain volumes of the various amino acid residues. In some embodiments, the original residue has a small side chain volume (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and the input residues that form the knob are naturally occurring amino acids and can include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and the import residue for forming the socket is a naturally occurring amino acid, and can include alanine, serine, threonine, and valine.

In some embodiments, the original residues used to form the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining three-dimensional structures may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these examples, human IgG₁The CH3/CH3 interface of (a) involves sixteen residues on each domain located on four antiparallel beta strands. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel beta strands to minimize the risk of the knob being held by the surrounding solvent rather than the compensating hole in the partner CH3 domain. In some embodiments, the mutations that form the corresponding knob and hole in the two immunoglobulin polypeptides correspond to one or more of the pairs provided in the table below.

In certain embodiments, the CH3 and/or CH2 domain of an antibody of the disclosure is from the IgG4 subtype. In some embodiments, the IgG4 CH3 and/or CH2 domain of the antibodies of the present disclosure may comprise one or more additional mutations, including but not limited to the S228P mutation (EU numbering).

TABLE 1b Properties of amino acid residues

^aThe molecular weight of the amino acid minus the molecular weight of water. From Handbook of Chemistry and Physics,43^rdValues of ed.cleveland, Chemical Rubber Publishing co., 1961.

^bValues from A.A Zamyytnin, prog.Biophys.mol.biol.24:107-123, 1972.

^cValues from C.Chothia, J.mol.biol.105:1-14,1975. The accessible surface areas are defined in fig. 6-20 of this reference.

In some embodiments, the polypeptide chain of one subunit comprises at least one hole mutation and the polypeptide chain of another subunit comprises at least one knob mutation. For example, the first subunit comprises an antibody Fc region comprising at least one hole mutation and the second subunit comprises an antibody Fc region comprising at least one knob mutation, or the first subunit comprises an antibody Fc region comprising at least one knob mutation and the second subunit comprises an antibody Fc region comprising at least one hole mutation.

In some embodiments, the CH3 domain of an antibody of the disclosure is from an IgG (e.g., an IgG1 subtype, an IgG2 subtype, an IgG2A subtype, an IgG2B subtype, an IgG3 subtype, or an IgG4 subtype). In some embodiments, the CH3 domain of an antibody of the present disclosure may comprise one or more knob or hole mutations, such as those described in table 3 below.

TABLE 3 exemplary corresponding set of point and hole mutations

CH3 of the first immunoglobulin	CH3 of a second immunoglobulin
		T366Y	Y407T
T366W	Y407A
		T366W	T366S：L368A:Y407V
F405A	T394W
		Y407T	T366Y
T366Y:F405A	T394W:Y407T
		T366W:F405W	T394S:Y407A
F405W:Y407A	T366W:T394S
		F405W	T394S

In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain, the CH3 domain comprising one or more amino acid substitutions listed in table 2 above. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more amino acid substitutions listed in the left column of table 3 and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more corresponding amino acid substitutions listed in the right column of table 3. In some embodiments, one subunit of a multimeric polypeptide of the present disclosure comprises a mutation listed in the left column of the row in table 3, and another subunit of the multimeric polypeptide comprises a mutation listed in the right column of the same row in table 3. As a non-limiting example of a knob and hole pair, in some embodiments, a bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising a T366W mutation and a second immunoglobulin polypeptide comprising a CH3 domain comprising a T366S, L368A, and Y407V mutation. In some embodiments, the at least one knob mutation is selected from the group consisting of: T366Y, T366W, T394W and F405W, numbered according to the EU index based on human IgG 1. In some embodiments, the at least one hole mutation is selected from the group consisting of: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbering based on human IgG1 according to the EU index. In certain embodiments, the knob mutation comprises a T366W substitution and the hole mutation comprises T366S, L368A, and Y407V substitutions, numbered according to the EU index based on human IgG 1. Other descriptions of knob and hole mutations that can be used in the methods and cells described herein can be found, for example, in Ridgway JBB et al 1996Protein Eng.9(7): 617. sup. 621and www.imgt.org/IMGTbiotechnology/Knobs-into-holes _ IgG.html.

Following DNA mutation as described above, polynucleotides encoding modified immunoglobulin polypeptides having one or more corresponding knob or hole mutations can be expressed and purified using standard recombinant techniques and cell systems known in the art. For example, all four polypeptide chains of a bispecific antibody can be produced and assembled in a single host cell, e.g., as described and exemplified in the examples below.

Bispecific antibodies have been generated using leucine zippers. Kostelny et al, J.Immunol.,148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. The method can also be used for the production of antibody homodimers. The "diabody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-. The fragments comprise a light chain variable domain (V) linked by a linker_H) Heavy chain variable domain of (V)_L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V of a segment_HAnd V_LThe domains are forced to complement the V of another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, J.Immunol.,152:5368 (1994).

Preparation of bispecificAnother technique for antibody fragments is "bispecific T cell adaptors" orMethods (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO 2008/119567). The method utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain includes two single chain fv (scFv) fragments, each having a variable heavy chain (V) separated by a polypeptide linker_H) And variable light chain (V)_L) A domain, said linker being of sufficient length to allow intramolecular association between the two domains. The single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, such that when each scFv is conjugated to its cognate epitope, cells of the two different cell types will be in close proximity or bound together. One particular embodiment of the method includes identifying a scFv for a cell surface antigen expressed by an immune cell, such as a CD3 polypeptide on a T cell, linked to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

Antibodies having more than two valencies are contemplated. For example, trispecific antibodies may be prepared. Tuft et al J.Immunol.147:60 (1991).

In some embodiments, the antibody described herein is a single domain antibody. A single domain antibody is a single polypeptide chain comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516B 1). In one embodiment, the single domain antibody consists of all or part of the heavy chain variable domain of an antibody.

Mutations that promote selective light-heavy chain pairing

In some embodiments, the multimeric polypeptides of the present disclosure comprise one or more mutations that facilitate selective association of an antibody light chain with an antibody heavy chain. Exemplary amino acid substitutions that can be used in the methods and cells described herein to promote selective association between an antibody heavy chain and an antibody light chain can be found in WO 2016/172485. In some embodiments, the antibody heavy chains of the subunits comprise one or more mutations that promote selective association, and the antibody light chains of the subunits comprise one or more mutations that promote selective association in combination with the heavy chains. In some embodiments, both half-antibodies of a bispecific antibody comprise one or more mutations that facilitate selective association of an antibody light chain with an antibody heavy chain (e.g., one heavy chain has a positively charged mutation and its corresponding light chain has a negatively charged mutation, while the other heavy chain has a negatively charged mutation and its corresponding light chain has a positively charged mutation, or vice versa). In some embodiments, only one half-antibody of a bispecific antibody comprises one or more mutations that facilitate selective association of an antibody light chain with an antibody heavy chain (e.g., one heavy chain has a positively charged mutation and its corresponding light chain has a negatively charged mutation, or vice versa).

In some embodiments, the antibody heavy chain (e.g., CH1 domain) comprises an amino acid substitution at S183 and the antibody light chain (e.g., CL domain) comprises an amino acid substitution at V133 (numbering based on the EU index). In some embodiments, the S183 substitution is selected from the group consisting of: S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R and S183K. In some embodiments, the V133 substitutes for a sequence selected from the group consisting of: V133E, V133S, V133L, V133W, V133K, V133R and V133D. In some embodiments, the amino acid substitution at S183 produces a positively charged residue, and wherein the amino acid substitution at V133 produces a negatively charged residue. In some embodiments, the amino acid substitution at S183 produces a negatively charged residue, and wherein the amino acid substitution at V133 produces a positively charged residue.

Mutations that promote selective association of heavy/light chain pairs can be combined with mutations that promote selective binding of heavy chains (e.g., knob and hole mutations). In some embodiments, a multimeric polypeptide (e.g., a bispecific antibody) can include a set of knob and hole mutations, and one or more mutations that facilitate selective association of an antibody light chain with an antibody heavy chain. Advantageously, this facilitates proper heavy/light chain association in one or both half-antibodies, as well as proper assembly of the bispecific antibody (e.g., as opposed to forming dimers for each half-antibody), resulting in fewer mis-paired side products. In some embodiments, a polypeptide chain of the present disclosure (e.g., an antibody heavy chain of a bispecific antibody or half-antibody) comprises one or more knob or hole mutations, and one or more mutations that facilitate selective association of an antibody light chain with an antibody heavy chain. For example, in some embodiments, a first antibody heavy chain of a bispecific antibody comprises a negative charge producing mutation (e.g., the S183E mutation) and a hole mutation, a first antibody light chain comprises a positive charge producing mutation (e.g., the V133K mutation), a second anti-heavy chain of the bispecific antibody comprises a positive charge producing mutation (e.g., the S183K mutation) and a knob mutation, and a second anti-light chain comprises a negative charge producing mutation (e.g., the V133E mutation).

Multiple multimeric polypeptides and compositions of multimeric polypeptides

Further provided herein are multimeric polypeptides produced according to the methods described herein, as well as various multimeric polypeptides or compositions of multimeric polypeptides. Advantageously, the present disclosure demonstrates that the methods described herein allow for improved production of multimeric polypeptides with higher yields of desired multimeric polypeptides and/or lower amounts of impurities, such as incorrectly paired by-products. Thus, in some embodiments, a plurality of multimeric polypeptides and multimeric polypeptide compositions produced by the methods of the present disclosure comprise fewer incorrectly paired by-products than a plurality and compositions produced by the prior art (e.g., as compared to an expression system in which one or more, two or more, three or more, or four polynucleotides encoding polypeptide chains of the multimeric polypeptide comprise an open reading frame operably linked to a native or unmodified translation initiation sequence, or as compared to expression in which each polynucleotide encoding polypeptide chains of the multimeric polypeptide comprises the same translation initiation sequence). In some embodiments, a plurality of multimeric polypeptides and multimeric polypeptide compositions produced by the methods of the present disclosure comprise a higher proportion of multimeric polypeptides and incorrectly paired by-products than those produced by the prior art (e.g., as compared to an expression system in which one or more, two or more, three or more, or four polynucleotides encoding polypeptide chains of the multimeric polypeptides comprise an open reading frame operably linked to a native or unmodified translation initiation sequence, or as compared to expression in which each polynucleotide encoding polypeptide chains of the multimeric polypeptides comprises the same translation initiation sequence). In some embodiments, all polypeptide chains of the multimeric polypeptide are produced by a single eukaryotic (e.g., mammalian) cell. In some embodiments, the multimeric polypeptide is a bispecific antibody.

Multimeric protein production

Host cells (e.g., as described in section III below) are transformed with one or more polynucleotides or vectors (e.g., expression vectors) and cultured in conventional nutrient media, suitably modified to induce promoters, select transformants, or amplify genes encoding the desired sequences.

Host cells for producing a desired multimeric polypeptide of the present disclosure or a subunit thereof can be cultured in a variety of media. Commercially available media such as Ham's F10(Sigma), minimal essential medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's modified eagle's medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, in Ham et al, meth.Enz.58:44(1979), Barnes et al, anal. biochem.102:255(1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469; WO 90/03430; any of the media described in WO 87/00195 or U.S. reissue patent 30,985 may be used as the medium for the host cells. Any of these media may be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN)^TMDrugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Or may be determined by one skilled in the artThe appropriate concentrations known to the practitioner include any other necessary supplements. Culture conditions such as temperature, pH, etc., are conditions previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

Described herein are methods for recovering the multimeric polypeptides of the disclosure. When recombinant techniques are used, the multimeric polypeptides of the disclosure or subunits thereof can be produced intracellularly, or directly secreted into the culture medium. If the polypeptide is produced intracellularly, as a first step, particulate debris of the host cells or of the lysed fragments is removed, for example by centrifugation or ultrafiltration. In the case of polypeptides secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

Polypeptide compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein a as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J.Immunol. meth.62:1-13 (1983)). Protein G is recommended for all mouse isoforms and human gamma 3(Guss et al, EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is attached is mostly agarose, but other matrices may be used. Mechanically stable matrices, such as controlled pore glass or poly (styrene divinyl) have faster flow rates and shorter processing times than agarose. When the antibody comprises a CH3 domain, Bakerbond ABX^TMResins (j.t.baker, phillips burg, NJ) can be used for purification. Other protein purification techniques may also be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica gel, chromatography on heparin, chromatography on anion or cation exchange resins (such as polyaspartic acid columns)SEPHAROSE of a line^TMChromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation, depending on the antibody to be recovered.

After any preliminary purification step, the mixture comprising the polypeptide of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., about 0-0.25M salt). The production of the antigen-binding polypeptide may alternatively or additionally (for any of the foregoing particular methods) comprise dialyzing a solution comprising the mixture of polypeptides.

In one embodiment, the multimeric polypeptide of the present disclosure, or a subunit thereof produced herein, is further purified to obtain a substantially homogeneous preparation for further assay and use. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractional distillation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica gel or on cation exchange resins (e.g., DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, for example using Sephadex G-75.

Purification of the multimeric polypeptide can be performed using known chromatographic techniques, including, for example, protein a or protein G column chromatography. In one embodiment, the target multimeric polypeptide may be recovered from the solid phase of the column by elution into a solution containing a chaotropic agent or mild detergent. Exemplary chaotropic agents and mild detergents include, but are not limited to, guanidine hydrochloride, urea, lithium perchlorate, arginine, histidine, SDS (sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which are commercially available.

In one embodiment, for example, protein a immobilized on a solid phase is used for immunoaffinity purification of the antigen binding polypeptides of the invention. Protein a is a 41kD cell wall protein from staphylococcus aureus that binds with high affinity to the Fc region of an antigen binding polypeptide. Lindmark et al (1983) J.Immunol.meth.62: 1-13. The solid phase for immobilizing protein A is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, to prevent non-specific adhesion of contaminants.

As a first step of purification, a preparation derived from the above cell culture is applied to a protein a immobilized solid phase to specifically bind the antigen binding polypeptide of interest to protein a. The solid phase is then washed to remove contaminants non-specifically bound to the solid phase. The antigen binding polypeptide (e.g., antibody) is recovered from the solid phase by elution.

Screening method

Further provided herein are methods of identifying a combination of translation initiation sequences for expression of a multimeric polypeptide in a eukaryotic host cell of the present disclosure. In some embodiments, the method comprises providing a library comprising a plurality of eukaryotic host cells, culturing the library of eukaryotic host cells under conditions suitable for expression of the multimeric polypeptide by the plurality of eukaryotic host cells, measuring the amount of the multimeric polypeptide produced by a single eukaryotic host cell in the plurality or a clone of a single eukaryotic host cell in the plurality, and identifying a first translation initiation sequence, a second translation initiation sequence, a third translation initiation sequence, and a fourth translation initiation sequence of a clone of a single eukaryotic host cell in the one or more pluralities that produces the multimeric polypeptide or a single eukaryotic host cell in the plurality. Exemplary such methods are described and illustrated below.

In some embodiments, a library of the present disclosure comprises a plurality of host cells of the present disclosure. In some embodiments, two or more or all of the plurality of host cells comprise the set of polynucleotides necessary to encode each polypeptide chain of a subunit or multimeric polypeptide as described herein. For example, in some embodiments, a plurality or all of the host cells in the library comprise a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding a first polypeptide chain of a multimeric polypeptide of the present disclosure, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding a second polypeptide chain of a multimeric polypeptide of the present disclosure, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a third polypeptide chain of a multimeric polypeptide of the present disclosure, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a fourth polypeptide chain of a multimeric polypeptide of the present disclosure. In some embodiments, the plurality of host cells or libraries comprise a plurality of combinations of a first translation initiation sequence, a second translation initiation sequence, a third translation initiation sequence, and a fourth translation initiation sequence. Thus, screening libraries can be used to identify combinations of translation initiation sequences that produce a property of interest (e.g., expression of multimeric polypeptides, e.g., high level expression or low level of incorrectly paired by-products). The present disclosure demonstrates that expression of multimeric polypeptides can be improved by modulating the strength of a translation initiation sequence operably linked to a polynucleotide encoding one or more constituent polypeptide chains.

In some embodiments, the one or more translation initiation sequences operably linked to the open reading frame in each of the plurality of host cells comprises the sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1).

Various methods described herein can be used to measure the amount of multimeric polypeptide produced by one or more host cells or clonal populations of host cells of the present disclosure. For example, transient or stable transfectants can be cultured, and production of multimeric polypeptides can be assayed. Fed-batch or perfusion cultures are cultured and the product titer in the cell culture medium or on the cell surface can be determined. For surface expression, cells can be stained with antibodies to detect products and analyzed, for example, by flow cytometry. The product quality and/or purity can be analyzed by, for example, electrophoresis and/or mass spectrometry.

Antibodies of the disclosure can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics, such as the method described in example 3. Additional Methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178:1-37(O' Brien et al, eds., Human Press, Totowa, NJ,2001) and further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in Molecular Biology 248:161-175(Lo, ed., Human Press, Totowa, NJ, 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5): 1073-; fellouse, proc.natl.acad.sci.usa 101 (34); 12467-12472 (2004); and Lee et al, J.Immunol.methods 284(1-2):119-132 (2004).

In some phage display methods, the repertoire of VH and VL genes are individually cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library from which antigen-binding phage can then be selected, as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments as single chain fv (scfv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, all natural components (e.g., all natural components from humans) can be cloned to provide a single source of antibodies to a wide range of non-self and self-antigens without any immunization as described by Griffiths et al, EMBO J,12: 725-. Finally, natural libraries can also be made by cloning unrearranged V gene segments from stem cells; and the use of PCR primers containing random sequences to encode highly variable CDR3 regions and to accomplish in vitro rearrangement as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. publication nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

III. cells

Also provided herein are recombinant eukaryotic host cells that can be used to produce multimeric polypeptides, e.g., according to the methods described in section II. In some embodiments, the host cell described herein comprises a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding a first polypeptide chain of a multimeric polypeptide of the present disclosure, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding a second polypeptide chain of a multimeric polypeptide of the present disclosure, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a third polypeptide chain of a multimeric polypeptide of the present disclosure, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a fourth polypeptide chain of a multimeric polypeptide of the present disclosure. In some embodiments, when each subunit is expressed individually in a recombinant eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit, and one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence. In some embodiments, the multimeric polypeptide is non-native to the recombinant eukaryotic host cell. In some embodiments, the recombinant eukaryotic host cell is an isolated recombinant eukaryotic host cell.

In some embodiments, the host cell of the present disclosure is a eukaryotic cell. In some embodiments, the host cell of the present disclosure is a mammalian cell.

Suitable host cells include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine process. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse support cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human liver cancer cell line (Hep G2).

In some embodiments, the host cell of the present disclosure is a Chinese Hamster Ovary (CHO) cell or cell line. In some embodiments, the host cell of the present disclosure is the CHO-K1 cell line (see, e.g., ATCC accession number CCL-61)^TMAnd Lewis, N.E. et al (2013) nat. Biotechnol.31: 759-. In some embodiments, the host cells of the present disclosure are CHO cells deficient in dihydrofolate reductase (DHFR) activity (see, e.g., Urlaub, G. and Chasin, L.A. (1980) Proc. Natl. Acad. Sci.77: 4216-.

In some embodiments, all polypeptide chains of the multimeric polypeptide are produced by a single eukaryotic (e.g., mammalian) cell. Advantageously, this eliminates the need for post-production assembly of multimeric polypeptides (e.g., bispecific antibodies), as is the case when two subunits (e.g., half-antibodies) are assembled in vitro after production.

Any promoter and/or translation initiation sequence described herein can be used in the host cells of the present disclosure.

In some embodiments, one or more polynucleotides encoding a polypeptide chain of a subunit of a multimeric polypeptide of the present disclosure are present on a chromosome of a host cell of the present disclosure (e.g., by integration). In some embodiments, each polynucleotide encoding a polypeptide chain of a subunit of a multimeric polypeptide of the present disclosure is present on a chromosome of a host cell of the present disclosure (e.g., by integration). In some embodiments, each polynucleotide encoding a polypeptide chain of a subunit of a multimeric polypeptide of the present disclosure is present in the same chromosome or chromosomal locus of a host cell of the present disclosure (e.g., by integration).

The stable integration or stable transfection of a polynucleotide of the present disclosure onto a host cell chromosome can provide a stable production cell line for the production of the multimeric polypeptides of the present disclosure. Various methods suitable for integrating polynucleotides into the host cell genome are known in the art, including random integration or site-specific integration (e.g., the "landing pad" method); see, e.g., Zhao, M. et al (2018) appl. Microbiol. Biotechnol.102: 6105-6117; lee, j.s. et al (2015) sci.rep.5: 8572; and Gaidukov, L.et al (2018) Nucleic Acids Res.46: 4072-.

In some embodiments, one or more polynucleotides encoding a polypeptide chain of a subunit of a multimeric polypeptide of the disclosure are maintained as extrachromosomal polynucleotides in a host cell. In some embodiments, each of the polynucleotides encoding a polypeptide chain of a subunit of a multimeric polypeptide of the disclosure is maintained as an extrachromosomal polynucleotide in a host cell. In some embodiments, one or more polynucleotides encoding a polypeptide chain of a subunit of a multimeric polypeptide of the disclosure are present in a vector (e.g., an expression vector). In some embodiments, each of the polynucleotides encoding a polypeptide chain of a subunit of a multimeric polypeptide of the disclosure is present in one or more vectors (e.g., expression vectors).

Carrier components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence.

Vectors for eukaryotic host cells may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. Preferably, the heterologous signal sequence of choice is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences are available as well as viral secretory leaders, e.g., the herpes simplex gD signal. The DNA of such precursor region is linked in reading frame to DNA encoding the desired antigen-binding polypeptide (e.g., an antibody).

Typically, mammalian expression vectors do not require an origin of replication component. For example, an SV40 source can generally be used, but only because it contains an early promoter.

Any promoter and/or translation initiation sequence described herein can be used in the vectors of the present disclosure.

Expression and cloning vectors may comprise a selection gene, also referred to as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) complement auxotrophic deficiencies when relevant, or (c) supply key nutrients not available from complex media.

One example of a selection scheme utilizes a drug to retard the growth of the host cell. Those cells successfully transformed with the heterologous gene produce a protein conferring drug resistance and thus survive the selection protocol. Examples of such dominant selection use neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those markers that can identify cells capable of uptake of antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein-I and metallothionein-II (preferably primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like).

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in medium containing methotrexate (Mtx), a DHFR competitive antagonist. When wild-type DHFR is used, a suitable host cell is a Chinese Hamster Ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts comprising endogenous DHFR) transformed or co-transformed with DNA sequences encoding a polypeptide chain of the disclosure, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3' -phosphotransferase (APH), can be selected by cell growth in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See, for example, U.S. Pat. No. 4,965,199.

Expression vectors used in eukaryotic host cells also typically contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically obtained from the 5 '(and sometimes 3') untranslated region of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See W094/11026 and the expression vectors disclosed therein.

Host cells are transformed with the above expression or cloning vectors to produce the desired polypeptide (e.g., multimeric polypeptide) and cultured in conventional nutrient media, suitably modified to induce promoters, select transformants, or amplify genes encoding the desired sequences.

Kits and articles of manufacture

Further provided herein are useful kits or articles of manufacture, e.g., for expression of multimeric polypeptides.

In some embodiments, the kit comprises a set of polynucleotides encoding each polypeptide chain and/or subunit of the multimeric polypeptide. For example, in some embodiments, a kit described herein comprises a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding a first polypeptide chain of a multimeric polypeptide of the present disclosure, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding a second polypeptide chain of a multimeric polypeptide of the present disclosure, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a third polypeptide chain of a multimeric polypeptide of the present disclosure, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a fourth polypeptide chain of a multimeric polypeptide of the present disclosure. In some embodiments, one or more of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence are not operably linked to their respective open reading frames because the respective open reading frames are present in a naturally occurring host cell genome.

In some embodiments, each polynucleotide in a kit of the present disclosure is part of one or more expression vectors of the present disclosure.

In some embodiments, each polynucleotide in a kit of the present disclosure is operably linked to a promoter of the present disclosure. For example, in some embodiments, each polynucleotide is operably linked to a different promoter. In some embodiments, two or more polynucleotides are operably linked to the same promoter. For example, if the multimeric polypeptide is a bispecific antibody, polynucleotides encoding the open reading frames of the heavy and light chains of each half-antibody may be operably linked to the same promoter. In some embodiments, "the same promoter" refers to the same physical polynucleotide. In some embodiments, "identical promoter" refers to physically different polynucleotides that share the same promoter sequence.

In some embodiments, the kit or article of manufacture further comprises instructions for using the polynucleotide set to produce a subunit or multimeric polypeptide of the disclosure, e.g., according to any of the methods described in section II above or using any of the host cells described in section III above.

Examples of the invention

The present disclosure will be more fully understood with reference to the following examples. However, the examples should not be construed as limiting the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: use of Kozak sequence variants to modulate protein production in transient transfection of mammalian cells

Currently, there are several systematic approaches to precisely control the level of translation of recombinant proteins in mammalian cells. The region in which translation initiates polypeptide synthesis is called the Translation Initiation Site (TIS). The Translation Initiation Site (TIS) consists of the initiation codon and its adjacent bases. In eukaryotes, translation initiation generally follows a model of the scanning mechanism (Kozak M.cell.1978; 15(4):1109-23) which assumes that the ribosome pre-initiation complex, consisting of small 40S ribosomal subunits, Met-tRNA, eIF2-GTP, eIF1, eIF1A, eIF3 and eIF5, binds at the 5 'end of the mRNA and proceeds linearly in the 3' direction looking for the initiation codon. After the small 40S ribosomal subunit is located in the initiation codon, the initiation factor dissociates, the large subunit binds to the small 40S ribosomal subunit to form a ribosomal complex, translation begins (Nanda JS, Saini AK, Munoz AM, Hinnebucch AG, Lorsch JR.J Biol chem.2013; 288(8): 5316-29; Pestova TV, Kolupaeva VGgenes Dev.2002; 16(22): 2906-22). Initiation is not always limited to the initiation codon (AUG) closest to the 5' end. If the first AUG codon occurs in an optimal environment, the ribosome complex initiates translation, but if the Translation Initiation Site (TIS) around the first AUG triplet is suboptimal, some 40S subunits bypass this site and initiate further downstream. Thus, cells can control the level of protein translation by modulating TIS, making sequences around the initiation codon play a major role in the initiation and subsequent enhancement of translational efficiency (Kozak M.J Biol chem.1991; 266(30): 19867-70; Kozak M.JCelbiol.1991; 115(4): 887-.

Kozak has reported CCRCCAUGG (purine, R ═ A or G; the start codon is underlined) is a highly potent mammalian TIS (Kozak M. nucleic Acids Res.1981; 9(20): 5233-52). In this sequence, purines at position-3 (3 nucleotides upstream of the AUG codon) are most conserved in vertebrate messenger RNA (Kozak M. nucleic Acids Res.1987; 15(20): 8125-48). Point mutation studies provide evidence that A or G in position-3 and G in position +4 (immediately after the AUG codon) are critical for optimal translation efficiency (Kozak M. EMBO J. 1997; 16(9): 2482-92). The Kozak consensus sequence differs in length and nucleotide composition between species, but is conserved for most genes within a species. Not surprisingly, point mutations in the Kozak sequence affect more advanced (Kozak M.cell.1986; 44(2):283-92) and lower (Dvir S, Velten L, Sharon E, Zeevi D, Carey LB, Weinberger A, et al, Proc Natl Acad Sci U S A.2013; 110(30): E2792-801) eukaryotes and are associated with the development of human diseases, including cancer and metabolic disorders (Sonenberg N, Hinnebucch AG. cell.2009; 136(4): 731-45; Mohan RA, van Engelen K, Stefanovic S, Barnett P, Ilgun A, Baars MJ 2014, et al, Am J Genet A.A.11; 2732-8).

To investigate whether variants of the Kozak sequence could modulate protein production in industrially generated cell lines, the combination of-3 and-3, -2 and-1 positions upstream of the start codon of the ORF encoding the Fc fusion protein was altered.

Method

Cell lines and cell cultures

The CHO cell line was used for transient transfection assays in plates and for cell lines that produce constitutive antibodies by target integration. CHO cells were cultured in DMEM/F12 based medium, placed in 125mL shake flask vessel at 150rpm, 37 ℃ and 5% CO₂And (6) oscillating. Every 3-4 days with 3x10⁵The cells were passaged at a seeding density of individual cells/ml.

Transient transfection

CHO cells were transfected with transient transfection plasmids using lipofectamine 2000CD according to the manufacturer's recommendations (Invitrogen, Carlsbad, ca). As an internal control, DNA was also carried which showed good expression of the antibody and poor expression of the antibody. Briefly, DNA vector (2. mu.g) and 10. mu.l lipofectamine were incubated in 500. mu.l medium at room temperature for 30min to facilitate complex formation. The transfection complexes were transferred to 96-well plates (falconR) containing exponentially growing CHO cells in DMEM/F12-based medium and 5% DFBS in a total working volume of approximately 2.5 ml. Transfected cells were incubated at 37 ℃ with 5% CO₂And incubation at 80% humidity. 24h after transfection, the cell culture medium was changed to production medium, and then the cells were cultured at 33 ℃. Each transfection was performed in biological replicates. For antibody concentration determination, supernatant samples were collected from the cultures at 48h post-transfection and repeated by HTRF (homogeneous time-resolved FRET) assay. Homogeneous time-resolved FRET (HTRF) assay

For HTRF determination, see Degorce, f., et al (2009). Curr Chem Genomics 3: 22-32.

Design and construction of Kozak sequence variants

Variants with lower and medium frequencies at these positions were designed based on the nucleotide frequencies around the vertebrate translation initiation site as determined by Kozak (Kozak M. nucleic Acids Res. 1987; 15(20):8125-48), as shown in FIG. 1. DNA for Kozak sequence variants of Fc fusion proteins a or B was synthesized by Genewiz, inc. The variants were cloned into transient transfection vectors of the gene tack under the transcriptional control of the CMV promoter and ampicillin resistance marker. The sequence was verified using universal forward and reverse primers.

DNA library design

Fc fusion protein B was used as the backbone for a synthetic DNA library of Kozak sequence. The positions of diversification are-5, -4, -3, -2, -1 and +4 of the start codon of any of the four nucleotides. The library was synthesized by Genewiz, inc. The library was cloned into a transient transfection vector under the transcriptional control of the CMV promoter and an ampicillin resistance marker. Transfection of the library into MaxDH5 α competent cells (Invitrogen, Carlsbad, california) and performed multiple dilutions to optimize selection of individual colonies. DNA preparation of individual colonies was carried out according to the manufacturer's recommendations (QIAGEN, Hilden, Germany). Sequencing was performed using universal forward and reverse primers.

Construction of a Stable vector

Two integrated antibody expression vectors were used to develop bispecific stable cell lines. The expression vector contains two independent Cytomegalovirus (CMV) promoters to direct transcription of the Heavy Chain (HC) and Light Chain (LC) as two independent units. Streptomyces niger puromycin-N-acetyltransferase (PUR) (Vara JA, Portela A, Ortin J, Jimenez A. nucleic Acids Res.1986; 14(11):4617-24) gene was used as a selection marker in a plasmid. The gene encoding a fusion protein containing the positive and negative selection marker HyTK conferring hygromycin (Hyg R) resistance was used as a selectable marker in another plasmid. Each set of 25 expression vectors was generated by cloning a combination of 5 heavy chains and 5 light chains under different Kozak sequence variants to generate a Kozak mixed pool. Two expression plasmids were used to develop the Wt Kozak library, one for each antibody, with heavy and light chains under the Wt Kozak sequence.

Development of stable cell lines

CHO cells were transfected using the MaxCyte STX transfection system according to the manufacturer's recommendations (MaxCyte, Gaithersburg, Md.). Transfected cells were pooled into two separate pools and selected with selective medium containing puromycin 5 μ g/ml and FIAU 0.5 μ M. After recovery, one well was Single Cell Cloned (SCC) using a Wellmate microplate dispenser (Thermo Matrix) with an Integra viafil sterile 8-channel tube (Integra Λ) with a pore size of 0.5mm with 1 cell/well limiting dilution to 384-well clear, flat-bottom, tissue culture processing plates (corning Inc, corning, new york). The plates were incubated at 37 ℃ and 5% CO₂And (4) incubating. Three to four weeks after inoculation, 704 individual colonies were picked into 96-well plates (corning Inc, corning, new york) and antibody production was assessed after about 2 days using homogeneous time-resolved fret (htrf). The first 48 and then the first 24 clones expressing the antibody were analyzed by HTRF. The first 11 single cell clones were adapted to suspension growth and evaluated in the production assay. Seven additional clones showing neutralized low HCCF titers compared to the top-ranked clone were also selected for further analysis.

Shake flask fed-batch production assay

On days 7 and 10, fed-batch production cultures were performed in shake flasks (corning Inc, corning, new york) using chemically defined basal medium and fast feed. At 1.0x10⁶Cells/ml seeded cells. A temperature shift from 37 ℃ to 35 ℃ was performed on day 3. Titers at day 14 were determined using protein a affinity chromatography with UV detection. Percent viability and viable Cell count were determined using a Vi-Cell XR instrument (Beckman Coulter) on days 0,3, 7, 10 and 14. Glucose and lactate concentrations were monitored on day 7 and day 14 using a Bioprofile 400 analyzer (Nova Biomedical),

antibody surface staining protocol

For antibody surface staining, approximately 200 million cells were pelleted and washed twice with PBS buffer. Cells were then resuspended at 1:100 dilution in 0.5mL PBS containing anti-human IgG (H + L) Allophycocyanin (APC) -conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA) and incubated for 20min with shaking at 37 ℃. Unstained control samples were resuspended in PBS without antibody. Empty hosts were also stained to set gating. After incubation with staining antibody for 20min, cells were washed once with PBS and resuspended in 400 μ l PBS. Then, the cells were analyzed in a FACscan flow cytometer (Attune NxT flow cytometer, Life Technologies). For each analysis 20000 events were recorded.

Product quality analysis

Standard product quality analysis was performed by subjecting total protein A purified antibody to non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS). The TECAN Evo200 system was used to automate sample preparation. All samples were diluted to 1mg/ml to ensure consistent liquid handling and to maintain the optimal dye to protein ratio. The prepared samples were immediately analyzed by the labChip gxi system and the data were processed using Chromeleon software. Signal intensity, peak profile and relative peak area distribution were evaluated.

Mass spectrometry

To identify and quantify variants associated with protein products, qualitative analysis and mass determination were performed on an Agilent 6230 time-of-flight mass spectrometer using HPLC-chip separation and ion source. Data deconvolution was performed using MassHunter software. We determined the ion abundance of the species with the bispecific and incorrect LC and HC stoichiometries.

Genomic DNA extraction, PCR amplification and sequencing analysis

DNeasy Blood was used&Extraction of genomic DNA was carried out with Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's manual. Genomic DNA was quantified using a simplina microspectrophotometer (GE life sciences). Use ofHot Start High-Fidelity 2X Master Mix (Biotech, N.Y.) was subjected to PCR amplification. Mu.g of gDNA was used as template. And optimizing PCR conditions. An annealing temperature of 64 ℃ and 30 cycles were used. Specific annealing primers for each chain variable region were designed to distinguish identical chains between molecules. The following forward (F) and reverse (R) primers were used to amplify Ab1 heavy chain, Ab1 light chain, Ab2 heavy chain, and Ab2 light chain: HC-Ab 1: F-5'-GATACCAGCACCAGCACCGCCT-3' (S)EQ ID NO:12) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO: 13); LC-Ab 1: F-5'-CTGAACAGCCGCACCCGCAA-3' (SEQ ID NO:14) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO: 15); HC-Ab 2: 5'-GTGATTTGGCGCGGCGGCA-3' (SEQ ID NO:16) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO: 17); and LC-Ab 2: 5'-GTGCGCAACCTGGTGGTGTGG-3' (SEQ ID NO:18) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO: 19). The PCR products were cleaned using a PCR purification kit (QIAGEN) according to the manufacturer's instructions and analyzed for correct size by 12-well 2% precast agarose gel (seimer feishell technology). The following primers were used for sequencing: SEQ 1: 5'-AACGGTGCATTGGAACGCGG-3' (SEQ ID NO:20) and SEQ 2: 5'-TGGCTTCGTTAGAACGCAGC-3' (SEQ ID NO: 21). The Kozak sequence variants for each strand in each clone were confirmed at least twice. Sequencing revealed a Kozak mixed clone lacking one of the strands. These are Kozak mixed clones 3, 11, 29, 60 and 21L for Ab2-LC, Ab2-HC, Ab1-LC, Ab2-HC and Ab1-LC, respectively. Kozak mixed clone 24 had no HC and LC for Ab1 (FIGS. 23A-23C).

Results

To investigate whether changes in the consensus Kozak sequence could modulate protein production in industrially generated cell lines, the combination of-3 and-3, -2 and-1 positions upstream of the start codon of the ORF encoding the Fc fusion protein was changed (FIG. 1). Kozak sequence variants were tested by transient transfection in CHO-producing cell lines and Fc fusion protein production was assessed 48 hours post-transfection by HTRF (homogeneous time-resolved fluorescence) assay. Well-expressed antibodies and poorly-expressed antibodies were also included as internal assay controls.

As shown in fig. 2, changes in the consensus Kozak sequence modulated Fc fusion protein a titers. The results confirmed that the presence of a purine at position-3 (GCCATGG and ACCATGG, SEQ ID NO: 4 and 3, respectively) produced high levels of Fc fusion protein A titers. The weakest Kozak sequence variant evaluated, TTTATGG (SEQ ID NO:7), adjusted the titer to a level of half of the Kozak consensus sequence, hereinafter referred to as wild-type (Wt) Kozak.

To determine whether Kozak sequence variants are able to modulate protein production regardless of the molecule, these combinations in different Fc fusion proteins were tested: fc fusion 'B' (fig. 2). Similar to cells for Fc fusion a transfected with Kozak sequence variants, cells for Fc fusion B transfected with variants of Kozak sequence showed a change in titer compared to Wt Kozak, and a similar pattern was observed for variants of Fc fusion B. These results indicate that changes in Wt Kozak sequence can be used to adjust antibody titers.

Example 2: design and screening of Kozak sequence library to expand the expression level range

Other Kozak sequence variants were tested to achieve a comprehensive range of expression levels from 0.1-fold to 1.0-fold for Wt Kozak. As shown in the detailed workflow provided in fig. 3, a library of Kozak sequence variants using ORF encoding Fc fusion protein B (representative of well behaved molecules) was designed and screened. The library included randomization of five bases upstream (-5, -4, -3, -2, and-1) and one base downstream (+ 4) of the initiation codon into any of four nucleotides (FIG. 4). The expected diversity of this construction is about 4100 variants of the Kozak sequence. When this position is occupied by adenine (A) or cytosine (C), randomization of position +4 results in an amino acid change from glycine to arginine present at position 2 of the signal sequence. The library was transformed into competent E.coli cells and individual variants were sequenced to exclude those showing non-design mutations, deletions, and those containing a stop codon as the second amino acid of the signal sequence, which occurred when position +4 was occupied by thymine (T).

Results

Initial screens for transient transfections in CHO cells were performed with 111 correct variants, including Wt Kozak and Wt Kozak with C or A at position +4, as controls for the second amino acid change in the signal sequence. As shown in fig. 5, Fc fusion protein titers measured 48 hours after transient transfection by HTRF assay showed broad expression levels. An almost continuous translation range of normalized titers from 0.05 to 1.54 units was observed, where 1.0 corresponds to Wt Kozak. These variants meet the target of the desired expression range, thus stopping further variant screening. Generally, the presence of a or C in position +4 increases the titer of Wt Kozak regardless of the Kozak sequence upstream of the start codon. The screen was repeated and data for two independent transient transfections are presented (figure 5).

As shown in fig. 6, the nucleotide distribution of the analyzed variants was random, indicating no bias in library screening. Next, the variants were divided into five groups according to their potency and performance. Group ranges are: group I, 0.05-0.3; group II, 0.31-0.6; group III, 0.61-0.8; group IV, 0.81-1.00; and group V, 1.01-1.34. No other correlation between antibody production and Kozak sequence variants was detected, except for the weakest group prefers adenine and thymine at most positions, and arginine as the second amino acid of the strongest group of signal sequences. Several rounds of transient transfection were performed to reduce the number of variants (FIG. 7). Criteria for selection of variants in rounds are reproducibility between transient transfections, stability between different DNA preparations and preservation of nucleotide diversity. As shown in fig. 8A-8B, the screen established 11 Kozak sequence variants, representing a 0.2 to 1.3 fold expression range of Wt Kozak, enabling high precision titer adjustment.

Example 3: generation of bispecific stable clones with mixture of Kozak sequence variants

Despite advances in the engineering of complex antibody formats (such as bispecific antibodies), manufacturability of these antibody formats often shows low potency in a single mammalian expression system. Low titer and insufficient product quality are two factors that make stable production of bispecific antibodies (BsAb) difficult. Therefore, there is a strong need to identify the restriction steps in the production system. One bottleneck that limits the efficiency of assembly of bispecific and other multi-stranded formats in a single cell is limited control of the individual strand level. Designing a vector that can adjust the individual strand ratios in a multi-stranded fashion can improve assembly efficiency as previously demonstrated in E.coli with a Translation Initiation Region (TIR) variant (Simmons LC, Yansura DG. nat Biotechnol. 1996; 14(5): 629-34). For example, as shown in FIG. 9, the ratio of light chains (LC1: LC2) that make up the BsAb can be manipulated to achieve a higher percentage of correctly assembled BsAbs.

Because of the need to identify unique ratios of parent antibodies combined into bispecific forms to generate bispecific antibodies, Kozak sequence variants were tested for their ability to produce higher BsAb assembly and production in single cells. In the establishment of stable CHO host cell lines, the successful application of Kozak sequence variants as a technology to regulate protein expression was evaluated. To do this, a representative group of five variants, including Wt Kozak, was selected (fig. 10 and 8A). For Kozak sequence variants #3, #135, #148, Wt, and #228, the relative protein production intensity of these variants was about 0.3-fold, about 0.5-fold, about 0.8-fold, about 1.0-fold, and about 1.3-fold, respectively, that of the Wt Kozak sequence. Thus, this set of variants covers the broad expression levels observed in transient transfections (fig. 10).

Bispecific antibody Ab1/Ab2 was selected as a model to evaluate the effect of Kozak sequence variation on regulatory chain ratios based on the following criteria. First, for Ab1/Ab2, a well-characterized process development platform can be used that facilitates analysis and characterization of the product. Second, Ab1 is a poorly expressed molecule with a bottleneck downstream of the transcription process, while Ab2 represents an example of a well-behaved molecule. Thus, this is a suitable scenario to examine the ability of Kozak sequence variants to create successful strand pairings.

To preferentially promote heavy chain heterodimerization, the Heavy Chains (HC) of Ab2 and Ab1 carry knob and hole mutations, respectively. Point mutations are used to reduce Light Chain (LC) mismatches (Dillon M, Yin Y, Zhou J, McCarty L, Ellerman D, Slaga D, et al MAbs.2017; 9(2): 213-30). The previously described combinations of heavy and light chains under the five Kozak sequence variant groups were used to develop 25 integrated expression vectors per group (fig. 11). A mixture of 50 plasmids was transfected into CHO cells (hereinafter referred to as Kozak mix). The diversity generated by this approach achieves a potential of 625 different combinations of chain ratios. As a reference value, 4 strands of BsAb were cloned under Wt Kozak sequence, transfected into CHO cells, and carried in parallel (hereinafter abbreviated as Wt Kozak). Two different transfections were performed for each condition.

Results

Transfection efficiency was monitored by cell surface staining for total IgG and measured by FACS analysis. FIG. 12 shows that similar transfection efficiencies were observed for Kozak mixing and Wt Kozak cells. After transfection of the expression plasmid, a stable cell pool was established by drug selection. As shown in fig. 13A and 13B, similar cell viability was observed for both Kozak mixing and Wt Kozak cells after addition of selection drug (indicated by black arrows). One stable pool per transfection per condition was then selected and single cell cloning was performed. After recovery, plates were analyzed for clone recovery, and 704 clones recovered per well and condition were picked into 96-well plates. Preliminary screening based on the titer of Harvested Cell Culture Fluid (HCCF) showed differences in profile and absolute titer between different conditions. The Kozak mixed clones showed lower absolute titers and more significant titer reduction compared to the Wt Kozak clones (fig. 14A and 14B). The results matched the planned diversity of the chain ratio combinations in the Kozak mixed clones, while the profile of Wt Kozak clones was consistent with that expected for clones carrying only one chain ratio combination. Several rounds of screening based on HCCF titer, suspension adaptation and scale-up were performed to determine the first 11 clones per transfection pool and per condition, which resulted in a total of 22 clones per condition. Since each Kozak mixed clone is likely to carry a different combination of Kozak sequence variants, and to see the differences between the combinations, seven more clones were also selected for further analysis that exhibited moderately low HCCF titers compared to the top-ranked clones.

Example 4: shake flask performance of bispecific stable clones derived from mixtures of Kozak sequence variants

A 14-day shake flask fed-batch production was used to evaluate the productivity and bispecific assembly of each individual clone selected from example 3 above. All clones studied showed comparable viability throughout the production assay. The final viability at day 14 under both conditions was about 80-95%, except that one Kozak mixed clone had a final viability of 74% (fig. 15A and 15B). Similarly, viable cell counts showed an exponential rate of increase until day 7 for all individual clones, followed by a plateau (fig. 16A and 16B). For the general titer of each clone, mass production quality and assembly efficiency were measured at day 14.

As shown in fig. 17, Wt Kozak clones alone showed higher general absolute antibody titers than Kozak mixed clones alone in both transfections. Note that in this titer, properly assembled bispecific antibody, half antibody and other unwanted side products are all indicated. To assess product quality, samples were analyzed by non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) to provide information about molecular weight forms and other impurities. This technique allows two forms to be distinguished and quantified: major peaks equal to full antibody in the form of correctly and incorrectly assembled BsAb (full-ab), and the sum of prepeaks representing other species of different molecular weights. Generally, fewer Kozak mixed clones with a high percentage of all-ab formation were observed in the first 22 clones compared to Wt Kozak clones (fig. 18). This result is consistent with the fact that, despite the differences in the chain ratio combinations between conditions (Wt Kozak clone and Kozak mixed clone 1and 625, respectively), each condition screened and selected the same number of individual clones, which facilitated finding a greater number of Wt Kozak clones with high product quality. The percentage of whole antibody varies from clone to clone. The range of four individual Kozak mixed clones for transfection 2 was approximately 60% of the total antibody, and then the percentage was reduced to 30%.

All individual Kozak mixed clones transfected with 1 showed approximately 30% of the main peak (fig. 18). This result is consistent with the fact that among the diversity of the chain ratio combinations of Kozak mixed pools, some of them were weak in all-ab production. In addition, not all clones producing the highest antibodies (typical titers) were associated with the highest content of all-ab in Kozak mixed clones.

Generally, the best single Wt Kozak clone ranged between 80-90% of the full-ab yield. Similar to the Kozak mixed clones, Wt Kozak clones alone showed variability depending on the percentage of the main peak of the clone. In addition, a correlation between high general titres and high main peak content was observed in these clones (fig. 18).

In parallel, an evaluation of the production performance of seven additional clones with low HCCF titers compared to the top-ranked clone was performed. The resulting titers of these clones showed similar behavior to that previously observed in the primary screen (fig. 19A). CE-SDS data indicate that product quality varies from clone to clone. Also, there is no direct correlation between the general titer and the highest level of whole antibody. In this way, some Kozak mixed clones with more moderate titers showed similar percent all-ab, about 50-60%, compared to the first 11 clones (fig. 19B).

Example 5: bispecific assembly in stable clones with enhanced Kozak sequence variants

The first four clones from the CE-SDS analysis were analyzed for each condition and transfection to identify and quantify the mismatch between HC and LC. IgG was recovered from the cell culture medium using protein a affinity chromatography and the resulting mixture was analyzed by complete liquid chromatography mass spectrometry (LC-MS). After deconvolution of the data, several product-related variants were identified, including half-antibody (HC + LC), homodimer (two identical HCs), and mismatched LC-HC species. Using the calculations reported in the methods section, LC-MS data were used to orthogonally weight the CE-SDS results to report the percentage of species that were analyzed for overlap on the CE-SDS due to mass similarity.

Results

Seven different bispecific antibody formats and three half-antibody formats were distinguished and quantified (fig. 20A). The best individual Kozak mixed clone showed about 40% of correctly assembled BsAb, which is more than twice (about 18%) the best observed individual Wt Kozak clone (fig. 20A). Since each single Kozak mixed clone may carry a different combination of Kozak sequence variants, each clone shows a different percentage of BsAb assembly, while Wt Kozak clones carrying the same combination show a similar number of BsAb assemblies. Notably, incorrectly assembled bispecific version Ab1-HC + Ab2-HC +2x Ab2-LC with light chain mismatches accounted for 41% to 64% of the whole antibody produced by Wt Kozak clones (fig. 20A). These data point to the importance of adjusting the light chain ratio to achieve BsAb assembly when produced using single cells. Mass spectrometry of Kozak mixed clones with the lowest full-ab formation yield as measured by CE-SDS confirmed that most of the production material of those clones was half-antibodies. The forms are Ab1-HC + Ab2-LC and mortar half-Ab 1. These clones provided BsAb forms that were mainly incorrectly assembled BsAb 2x Ab1-HC +2x Ab2-LC and at a lower percentage 2xAb2-HC + Ab1-LC + Ab2-LC (fig. 20A).

Knowing the percentage of each form, effective bispecific titers were calculated (fig. 20B). Due to the fact that some Kozak mixed clones had higher BsAb assembly than Wt Kozak clones, Kozak mixed clones overcome the deficiencies of the general titer previously shown (fig. 17 and fig. 20A-20B). The effective bispecific titers of the best clones were similar under each condition: the best Wt Kozak and Kozak mixed clones were 0.7g/L and 0.6g/L, respectively. Since each Kozak mixed clone likely carried a different combination of Kozak variants, each clone showed a different percentage of bsAb assembly, while WT Kozak clones carrying the same combination showed a narrower bsAb assembly range (fig. 20A). Notably, the incorrectly assembled bispecific version Ab2 HC + Ab1HC +2x Ab2 LC with light chain mismatches accounted for 41-64% of the all-Ab produced by the Wt Kozak clone (fig. 20A). These data indicate the importance of adjusting the level of light chain translation to achieve correct bsAb assembly when produced using single cells.

However, as shown in fig. 20B, Kozak clones provided a smaller amount of impurities that had to be removed during purification than Wt Kozak clones. Due to the fact that some Kozak mixed clones had higher BsAb assembly than Wt Kozak clones, some Kozak mixed clones overcome the deficiencies of the general titer shown previously in fig. 17. Importantly, the Kozak mixed clones also exhibited less product related impurities than the WT Kozak clones (fig. 20B). Purification of correctly assembled bsabs from large amounts of nearly identical potential byproducts is challenging. Since some by-products are very similar to the target bsAb, their removal downstream is usually at the expense of yield or product quality. New analytical methods are required to distinguish the target product from these unique impurities, but they require costly manufacturing and complex techniques. The Kozak mixed clones minimized the formation of impurities and product-related variants (fig. 20B and 22B), demonstrating the benefit of this approach to bsAb production in single cells.

These data indicate that adjusting translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells.

Example 6: regulation of strand ratio using Kozak sequence variants yields high BsAb yields

The heterogeneity and diversity observed between Kozak mixed clones in terms of BsAb assembly efficiency and product quality indicates that each clone has a chain expressed under a different Kozak sequence variant. To identify the Kozak combinations that each clone has and determine the best combinations, 29 Kozak mixed clones were sequenced.

Results

The data show that Kozak mixed clones with low product yield expressed the heavy chain of Ab2 under one of the weakest Kozak sequence variants kz.135 and kz.3, with the remaining chains having Wt or the strongest Kozak sequence variant (fig. 21A-21B). As a result, the amount of heavy chain of Ab2 that may be produced is insufficient, and therefore the final product is preferably half-Ab and mortar half-Ab 2 in the form of Ab1-HC + Ab2-LC (fig. 20A). In other words, there is a range that can reduce heavy chain expression before disrupting whole ab assembly.

Otherwise, when the combination of Wt Kozak and the strongest Kozak sequence variant regulated the expression of four chains, the Kozak mixed clone showed high BsAb assembly with high titers (fig. 21A). On the other hand, when the combination of WT Kozak and the strongest Kozak sequence variant (kz.228; relative strength of 1.3) regulated the expression of four chains, the Kozak mixed clones showed high bsAb assembly and effective titers (fig. 20A and 21A, Kozak mixed clones 69, 61 and 88). Interestingly, Kozak hybrid clone 69 and Kozak hybrid clone 61 showed a slight light chain ratio of 1.3-fold Ab1 light chain to 1.0-fold Ab2 light chain, respectively, or vice versa, significantly reducing the Ab2 light chain mismatch observed in WT Kozak clones (FIG. 20A; Ab2 HC + Ab1HC +2x Ab2 LC). These data indicate that reduced translation of selected strands relative to the level of translation of other strands can improve bsAb assembly efficiency and improve overall product quality by limiting the accumulation of subspecies. However, under the strongest Kozak sequence variant, expression of either light chain prompted the production of more daughter products consisting of that chain rather than the more correctly assembled BsAb. For example, Kozak clone 69 with light chain Ab1 under the strongest Kozak sequence showed approximately 30% of the product as hole 1/2Ab1 (fig. 20A). This observation is consistent with the fact that the manufacture of BsAb requires stoichiometric chain pairing to merge into a bispecific format. In this regard, saturation of the protein production system can lead to the accumulation of subspecies.

In view of this, and without wishing to be bound by theory, it is believed that the reduction in translation may prevent the accumulation of species and result in an increase in yield. Consistent with this conclusion, one combination, performed at half the titer of the top-ranked Kozak clone and carrying Ab1 heavy and light chains under the weaker Kozak sequence variant #135, showed the highest bispecific assembly detected (fig. 22D). The CE-SDS data of this clone showed a product mass of about 48% for full-ab and about 52% for half-ab. MS data indicated that 100% of all-ab corresponds to the bispecific format only, which is about 50% of the correctly assembled BsAb. On the other hand, almost all the semi-Ab species were pestles 1/2Ab2 (fig. 22A-22B), consistent with the fact that the heavy and light chains of Ab2 were under Wt Kozak and the strongest Kozak sequence variant #228, respectively. Thus, in certain areas, down-regulation of the expression of both strands of a molecule, which may present a bottleneck downstream of translation during production, has a positive impact on assembly efficiency.

Clone 17M was one of the additional clones that advanced, although the primary HCCF titer was moderate (fig. 19A). Although the effective titer of this clone was only about half that of the top Kozak clone, it showed the highest bispecific assembly, about 48%. MS data indicated that 100% of all-ab corresponded only to the correct bispecific format, and that the half-ab species were almost exclusively restricted to pestle 1/2mAb2 (fig. 22A and 22B).

These data can be interpreted from the sequencing results (FIG. 22C). Both the heavy and light chains of aAb1 are under the weaker Kozak variant (kz.135), and Ab1 is a difficult to express molecule, as previously described. Downregulation of both chains, as is the case with kz.135, with a relative strength of about 0.5, can alleviate translational downstream bottlenecks, such as protein folding, and thus positively impact assembly efficiency. With respect to Ab2, the heavy and light chains were under stronger Kozak sequence, WT Kozak and the strongest Kozak variant (kz.228), respectively (fig. 22C).

This combination, in combination with the weaker Kozak sequence of aAb1, would result in accumulation of aAb2 half-ab while limiting any significant accumulation of other species (fig. 22A and 22B).

In the same manner, seven additional clones with low HCCF titers compared to the top-ranked clones were sequenced. In general, it was observed that expression of most of the chains of both molecules was controlled by the weakest Kozak sequence variant in these clones (fig. 21C). This result supports the low performance of these clones compared to the top-ranked clones.

To elucidate the Ab species and BsAb format, 5 of these Kozak mixed clones carrying the combination of Kozak sequence variants that are most different from the first 11 clones were analyzed by mass spectrometry. The percent of BsAb assembly was less than 10%, except for Kozak mixed clone 17. The absolute abundance of each species correlated with the expression intensity of each chain (fig. 22D).

Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the illustration and example should not be construed to limit the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

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<400> 16

gtgatttggc gcggcggca 19

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 17

atgggcggta ggcgtgtacg g 21

<210> 18

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 18

gtgcgcaacc tggtggtgtg g 21

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 19

atgggcggta ggcgtgtacg g 21

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 20

aacggtgcat tggaacgcgg 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 21

tggcttcgtt agaacgcagc 20

<210> 22

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 22

ccaccatggg 10

<210> 23

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 23

ccaccatggg a 11

Claims (97)

1. A method of producing a multimeric polypeptide in a eukaryotic host cell, wherein the multimeric polypeptide comprises a first subunit comprising a first polypeptide chain and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain, the method comprising:

(a) providing a eukaryotic host cell as described herein,

wherein said eukaryotic host cell comprises a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding said first polypeptide chain, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding said second polypeptide chain, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding said third polypeptide chain, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding said fourth polypeptide chain,

wherein when each subunit is expressed individually in the eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit, and

wherein one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence;

(b) culturing the eukaryotic host cell under conditions suitable for expression of the first, second, third, and fourth polypeptide chains, wherein the multimeric polypeptide is formed upon expression of the first, second, third, and fourth polypeptide chains; and

(c) recovering the multimeric polypeptide produced by the eukaryotic host cell.

2. The method of claim 1, wherein one or two polypeptide chains of the first subunit are expressed at a lower level than one or two polypeptide chains of the second subunit when all subunits are expressed in the same host cell.

3. The method of claim 1 or claim 2, wherein all of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence comprise the sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1).

4. The method of any one of claims 1-3, wherein one or both of the first translation initiation sequence and the second translation initiation sequence comprises a sequence selected from the group consisting of SEQ ID NOs 8-10.

5. The method of any one of claims 1-4, wherein one or both of the third translation initiation sequence and the fourth translation initiation sequence comprises the sequence ACCATGG (SEQ ID NO:3) or GAAGTATGA (SEQ ID NO: 11).

6. The method of claim 1 or claim 2, wherein the first translation initiation sequence comprises the sequence of SEQ ID No. 9, wherein the second translation initiation sequence comprises the sequence of SEQ ID No. 9, wherein the third translation initiation sequence comprises the sequence of SEQ ID No. 2, and wherein the fourth translation initiation sequence comprises the sequence of SEQ ID No. 11.

7. The method of any one of claims 1-6, wherein each of the first, second, third, and fourth polynucleotides is operably linked to a promoter.

8. The method of claim 7, wherein the first polynucleotide and the second polynucleotide are operably linked to the same promoter, and wherein the third polynucleotide and the fourth polynucleotide are operably linked to the same promoter.

9. The method of any one of claims 1-8, wherein the first translation initiation sequence is weaker than the third translation initiation sequence.

10. The method of any one of claims 1-9, wherein the second translation initiation sequence is weaker than the fourth translation initiation sequence.

11. The method of any one of claims 1-10, wherein the first translation initiation sequence is weaker than the fourth translation initiation sequence.

12. The method of any one of claims 1-11, wherein the second translation initiation sequence is weaker than the third translation initiation sequence.

13. The method of any one of claims 1-12, wherein the first translation initiation sequence is the same as the second translation initiation sequence.

14. The method of any one of claims 1-13, wherein the third translation initiation sequence is the same as the fourth translation initiation sequence.

15. The method of any one of claims 1-14, wherein the multimeric polypeptide specifically binds to one or more target antigens.

16. The method of any one of claims 1-15, wherein the multimeric polypeptide is a multispecific antigen-binding protein.

17. A method of producing a bispecific antibody in a eukaryotic host cell, wherein the bispecific antibody comprises a first half antibody comprising a first antibody heavy chain and a first antibody light chain and a second half antibody comprising a second antibody heavy chain and a second antibody light chain, the method comprising:

(a) providing a eukaryotic host cell as described herein,

wherein the eukaryotic host cell comprises a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the heavy chain of a first antibody, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the light chain of a first antibody, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the heavy chain of a second antibody, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the light chain of a second antibody,

wherein when each half-antibody is expressed individually in the eukaryotic host cell, the first half-antibody is expressed at a lower level than the second half-antibody, and

wherein one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence;

(b) culturing the eukaryotic host cell under conditions suitable for expression of the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain, and the second antibody light chain, wherein the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain, and the second antibody light chain form the bispecific antibody, and wherein the first half-antibody binds a first antigen and the second half-antibody binds a second antigen; and

(c) recovering the bispecific antibody produced by the eukaryotic host cell.

18. The method of claim 17, wherein the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region; and wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region.

19. The method of claim 17, wherein the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region; and wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region.

20. The method of claim 18 or claim 19, wherein the knob mutation comprises at least one of: T366Y, T366W, T394W and F405W, numbered according to the EU index based on human IgG 1.

21. The method according to any one of claims 18-20, wherein the hole mutation comprises at least one of: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbering based on human IgG1 according to the EU index.

22. The method of any one of claims 18-21, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises at least one, at least two, or all three of T366S, L368A, and Y407V, numbered according to the EU index based on human IgG 1.

23. The method of any one of claims 17-22, wherein the first antibody light chain comprises a first mutation, wherein the first antibody heavy chain comprises a second mutation, and wherein the first mutation and the second mutation facilitate selective association of the first antibody light chain with the first antibody heavy chain.

24. The method of claim 23, wherein the first mutation comprises an amino acid substitution at V133, and wherein the second mutation comprises an amino acid substitution at S183, numbering based on the EU index.

25. The method of claim 24, wherein the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and wherein the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D.

26. The method of claim 24 or claim 25, wherein the amino acid substitution at S183 produces a positively charged residue and the amino acid substitution at V133 produces a negatively charged residue, or the amino acid substitution at S183 produces a negatively charged residue and the amino acid substitution at V133 produces a positively charged residue.

27. The method of any one of claims 17-22, wherein the second antibody light chain comprises a third mutation, wherein the second antibody heavy chain comprises a fourth mutation, and wherein the third mutation and the fourth mutation facilitate selective association of the second antibody light chain with the second antibody heavy chain.

28. The method of claim 27, wherein the third mutation comprises an amino acid substitution at V133, and wherein the fourth mutation comprises an amino acid substitution at S183, numbering based on the EU index.

29. The method of claim 28, wherein the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and wherein the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D.

30. The method of claim 28 or claim 29, wherein the amino acid substitution at S183 produces a positively charged residue and the amino acid substitution at V133 produces a negatively charged residue, or the amino acid substitution at S183 produces a negatively charged residue and the amino acid substitution at V133 produces a positively charged residue.

31. The method of any one of claims 17-22, wherein the first antibody light chain comprises a V133K mutation, the first antibody heavy chain comprises a S183E mutation, the second antibody light chain comprises a V133E mutation, and the second antibody heavy chain comprises a S183K mutation, numbered based on the EU index.

32. The method of claim 31, wherein the first antibody heavy chain further comprises T366S, L368A, and Y407V mutations, and the second antibody heavy chain further comprises a T366W mutation, numbered according to the EU index based on human IgG 1.

33. The method of any one of claims 17-22, wherein the second antibody light chain comprises a V133K mutation, the second antibody heavy chain comprises a S183E mutation, the first antibody light chain comprises a V133E mutation, and the first antibody heavy chain comprises a S183K mutation, numbered based on the EU index.

34. The method of claim 33, wherein the second antibody heavy chain further comprises T366S, L368A, and Y407V mutations, and the first antibody heavy chain further comprises a T366W mutation, numbered according to the EU index based on human IgG 1.

35. The method of any one of claims 1-34, wherein the first, second, third, and fourth polynucleotides are integrated into one or more chromosomes of the eukaryotic host cell.

36. The method of claim 35, wherein the first, second, third, and fourth polynucleotides are integrated into the same chromosomal locus of the eukaryotic host cell.

37. The method of any one of claims 1-34, wherein the first, second, third, and fourth polynucleotides are part of one of a plurality of extrachromosomal polynucleotides in the eukaryotic host cell.

38. The method of any one of claims 1-37, wherein the eukaryotic host cell is a mammalian host cell.

39. The method of claim 38, wherein the mammalian host cell is a Chinese Hamster Ovary (CHO) cell.

40. The method of any one of claims 1-39, wherein the first translation initiation sequence and/or the second translation initiation sequence is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% weaker than the third translation initiation sequence and/or the fourth translation initiation sequence.

41. The method of any one of claims 1-39, wherein when each subunit or half-antibody is expressed individually in said eukaryotic host cell, said first subunit or said first half-antibody is expressed at a level that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% lower than the level of expression of said second subunit or said second half-antibody.

42. A plurality of multimeric polypeptides, wherein each multimeric polypeptide of the plurality of multimeric polypeptides is produced according to the method of any one of claims 1-41.

43. A recombinant eukaryotic host cell for expression of a non-natural multimeric polypeptide, wherein the non-natural multimeric polypeptide comprises a first subunit comprising a first polypeptide chain and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain, the recombinant cell comprising:

(a) a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the first polypeptide chain;

(b) a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the second polypeptide chain;

(c) a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the third polypeptide chain; and

(d) a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the fourth polypeptide chain;

wherein when each subunit is expressed individually in the recombinant eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit; and is

Wherein one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence.

44. The cell of claim 43, wherein one or two polypeptide chains of the first subunit are expressed at a lower level than one or two polypeptide chains of the second subunit when all subunits are expressed in the same host cell.

45. The cell of claim 43 or claim 44, wherein all of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence comprise a sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1).

46. The cell of any one of claims 43-45, wherein one or both of the first translation initiation sequence and the second translation initiation sequence comprises a sequence selected from the group consisting of SEQ ID NOs 8-10.

47. The cell of any one of claims 43-46, wherein one or both of the third translation initiation sequence and the fourth translation initiation sequence comprises the sequence ACCATGG (SEQ ID NO:3) or GAAGTATGA (SEQ ID NO: 11).

48. The cell of any one of claims 43-45, wherein the first translation initiation sequence comprises the sequence of SEQ ID NO 9, wherein the second translation initiation sequence comprises the sequence of SEQ ID NO 9, wherein the third translation initiation sequence comprises the sequence of SEQ ID NO 2, and wherein the fourth translation initiation sequence comprises the sequence of SEQ ID NO 11.

49. The cell of any one of claims 43-48, wherein each of the first, second, third, and fourth polynucleotides is operably linked to a promoter.

50. The cell of claim 49, wherein the first polynucleotide and the second polynucleotide are operably linked to the same promoter, and wherein the third polynucleotide and the fourth polynucleotide are operably linked to the same promoter.

51. The cell of any one of claims 43-50, wherein the first translation initiation sequence is weaker than the third translation initiation sequence.

52. The cell of any one of claims 43-51, wherein the second translation initiation sequence is weaker than the fourth translation initiation sequence.

53. The cell of any one of claims 43-52, wherein the first translation initiation sequence is weaker than the fourth translation initiation sequence.

54. The cell of any one of claims 43-53, wherein the second translation initiation sequence is weaker than the third translation initiation sequence.

55. The cell of any one of claims 43-54, wherein the first translation initiation sequence is identical to the second translation initiation sequence.

56. The cell of any one of claims 43-55, wherein the third translation initiation sequence is identical to the fourth translation initiation sequence.

57. The cell of any one of claims 43-56, wherein the folded and assembled multimeric polypeptide specifically binds to one or more target antigens.

58. The cell of any one of claims 43-57, wherein the multimeric polypeptide is a multispecific antigen-binding protein.

59. A recombinant eukaryotic host cell for expression of a bispecific antibody comprising a first half-antibody comprising a first antibody heavy chain and a first antibody light chain and a second half-antibody comprising a second antibody heavy chain and a second antibody light chain, the recombinant cell comprising:

(a) a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the heavy chain of the first antibody;

(b) a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the light chain of the first antibody;

(c) a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a heavy chain of the second antibody; and

(d) a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a light chain of the second antibody;

wherein when each half-antibody is expressed individually in the recombinant eukaryotic host cell, the first half-antibody is expressed at a lower level than the second half-antibody; and is

Wherein one or both of the first translation initiation sequence and the second translation initiation sequence is weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence.

60. The cell of claim 59, wherein the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region; and wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region.

61. The cell of claim 59, wherein the first antibody heavy chain comprises a first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the CH3 domain of the second antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with amino acid residues having a smaller side chain volume, thereby creating a hole on the surface of the CH3 domain of the second antibody Fc region that interacts with the CH3 domain of the first antibody Fc region; and wherein the CH3 domain of the first antibody Fc region is altered such that within the CH3/CH3 interface one or more amino acid residues are substituted with an amino acid residue having a larger side chain volume, thereby creating a knob on the surface of the CH3 domain of the first antibody Fc region that interacts with the CH3 domain of the second antibody Fc region.

62. The cell of claim 60 or claim 61, wherein the knob mutation comprises at least one of: T366Y, T366W, T394W and F405W, numbered according to the EU index based on human IgG 1.

63. The cell of any one of claims 60-62, wherein the hole mutation comprises at least one of: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbering based on human IgG1 according to the EU index.

64. The cell of any one of claims 60-63, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises at least one, at least two, or all three of T366S, L368A, and Y407V, numbered according to the EU index based on human IgG 1.

65. The cell of any one of claims 59-64, wherein the first antibody light chain comprises a first mutation, wherein the first antibody heavy chain comprises a second mutation, and wherein the first mutation and the second mutation facilitate selective association of the first antibody light chain with the first antibody heavy chain.

66. The cell of claim 65, wherein the first mutation comprises an amino acid substitution at V133, and wherein the second mutation comprises an amino acid substitution at S183, numbering based on the EU index.

67. The cell of claim 66, wherein the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and wherein the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D.

68. The cell of claim 66 or claim 67, wherein the amino acid substitution at S183 produces a positively charged residue and the amino acid substitution at V133 produces a negatively charged residue, or the amino acid substitution at S183 produces a negatively charged residue and the amino acid substitution at V133 produces a positively charged residue.

69. The cell of any one of claims 59-64, wherein the second antibody light chain comprises a third mutation, wherein the second antibody heavy chain comprises a fourth mutation, and wherein the third mutation and the fourth mutation facilitate selective association of the second antibody light chain with the second antibody heavy chain.

70. The cell of claim 69, wherein the third mutation comprises an amino acid substitution at V133, and wherein the fourth mutation comprises an amino acid substitution at S183, numbering based on the EU index.

71. The cell of claim 70, wherein the S183 substitution is selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R, and S183K; and wherein the V133 substitution is selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R, and V133D.

72. The cell of claim 70 or claim 71, wherein the amino acid substitution at S183 produces a positively charged residue and the amino acid substitution at V133 produces a negatively charged residue, or the amino acid substitution at S183 produces a negatively charged residue and the amino acid substitution at V133 produces a positively charged residue.

73. The cell of any one of claims 59-64, wherein the first antibody light chain comprises a V133K mutation, the first antibody heavy chain comprises a S183E mutation, the second antibody light chain comprises a V133E mutation, and the second antibody heavy chain comprises a S183K mutation, numbered based on the EU index.

74. The cell of claim 73, wherein the first antibody heavy chain further comprises T366S, L368A, and Y407V mutations, and the second antibody heavy chain further comprises a T366W mutation, numbered according to the EU index based on human IgG 1.

75. The cell of any one of claims 59-64, wherein the second antibody light chain comprises a V133K mutation, the second antibody heavy chain comprises a S183E mutation, the first antibody light chain comprises a V133E mutation, and the first antibody heavy chain comprises a S183K mutation, numbered based on the EU index.

76. The cell of claim 75, wherein the second antibody heavy chain further comprises T366S, L368A, and Y407V mutations, and the first antibody heavy chain further comprises a T366W mutation, numbered according to the EU index based on human IgG 1.

77. The cell of any one of claims 43-76, wherein the first, second, third, and fourth polynucleotides are integrated into one or more chromosomes of the eukaryotic host cell.

78. The cell of claim 77, wherein the first, second, third, and fourth polynucleotides are integrated into the same chromosomal locus of the eukaryotic host cell.

79. The cell of any one of claims 43-76, wherein the first, second, third, and fourth polynucleotides are part of one of a plurality of extrachromosomal polynucleotides in the eukaryotic host cell.

80. The cell of any one of claims 43-79, wherein the eukaryotic host cell is a mammalian host cell.

81. The cell of claim 80, wherein the method results in higher production of the multimeric polypeptide in the eukaryotic host cell.

82. A method of identifying a combination of translation initiation sequences for expression of a multimeric polypeptide in a eukaryotic host cell, wherein the multimeric polypeptide comprises a first subunit comprising a first polypeptide chain and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain, the method comprising:

(a) providing a library comprising a plurality of eukaryotic host cells, wherein each eukaryotic host cell in the plurality comprises:

a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the first polypeptide chain,

a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the second polypeptide chain,

a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the third polypeptide chain, and

a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding said fourth polypeptide chain,

wherein a plurality of combinations of a first translation initiation sequence, a second translation initiation sequence, a third translation initiation sequence, and a fourth translation initiation sequence are present in the plurality of eukaryotic host cells;

(b) culturing a library of said eukaryotic host cells under conditions suitable for expression of said multimeric polypeptides by eukaryotic host cells in said plurality;

(c) measuring the amount of the multimeric polypeptide expressed by a single eukaryotic host cell of the plurality or a clone of a single eukaryotic host cell of the plurality; and

(d) identifying the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence of one or more single eukaryotic host cells of the plurality or clones of single eukaryotic host cells of the plurality that express the multimeric polypeptide.

83. The method of claim 82, wherein all of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence in each host cell in the plurality each comprise the sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1).

84. The method of claim 82 or claim 83, wherein the multimeric polypeptide specifically binds to one or more target antigens.

85. The method according to any one of claims 82-84, wherein the multimeric polypeptide is a multispecific antigen-binding protein.

86. The method of any one of claims 82-84, wherein the multimeric polypeptide is a bispecific antibody, wherein the first polypeptide chain and the third polypeptide chain are antibody heavy chains, wherein the second polypeptide chain and the fourth polypeptide chain are antibody light chains, wherein the first subunit is a first half-antibody that binds a first antigen, and wherein the second subunit is a second half-antibody that binds a second antigen.

87. The method of any one of claims 82-86, wherein the first, second, third, and fourth polynucleotides are integrated into one or more chromosomes of each eukaryotic host cell in the plurality.

88. The method of claim 87, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the same chromosomal locus of each eukaryotic host cell in the plurality.

89. The method of any one of claims 82-86, wherein said first polynucleotide, said second polynucleotide, said third polynucleotide, and said fourth polynucleotide are part of one of a plurality of extrachromosomal polynucleotides in each eukaryotic host cell in said plurality.

90. The method of any one of claims 82-89, wherein the eukaryotic host cell is a mammalian host cell.

91. The method of claim 90, wherein the mammalian host cell is a Chinese Hamster Ovary (CHO) cell.

92. A polynucleotide kit for expression of a multimeric polypeptide comprising a first subunit comprising a first polypeptide chain and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain, the kit comprising:

(a) a first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the first polypeptide chain;

(b) a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the second polypeptide chain;

(c) a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the third polypeptide chain; and

(d) a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the fourth polypeptide chain;

wherein one or more of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence are not operably linked to their respective open reading frames in that the respective open reading frames are present in a naturally occurring host cell genome.

93. The kit of claim 92, wherein the first, second, third, and fourth polynucleotides are part of one or more expression vectors.

94. The kit of claim 92 or claim 93, wherein all of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence comprise the sequence (from 5 'to 3') NNNNNATGNGA, wherein N is C, G, A or T/U (SEQ ID NO: 1).

95. The kit of any one of claims 92-94, wherein the first translation initiation sequence comprises the sequence of SEQ ID No. 9, wherein the second translation initiation sequence comprises the sequence of SEQ ID No. 9, wherein the third translation initiation sequence comprises the sequence of SEQ ID No. 2, and wherein the fourth translation initiation sequence comprises the sequence of SEQ ID No. 11.

96. The kit of any one of claims 92-95, wherein each of the first, second, third, and fourth polynucleotides is operably linked to a promoter.

97. The kit of claim 96, wherein the first polynucleotide and the second polynucleotide are operably linked to the same promoter, and wherein the third polynucleotide and the fourth polynucleotide are operably linked to the same promoter.