Methods for producing multimeric proteins in eukaryotic host cells

Abstract

The present disclosure relates to producing a multimeric polypeptide (e.g., comprising two or more subunits, each subunit comprising two or more polypeptide chains, such as a multispecific or bispecific antibody) in a eukaryotic host cell. In some embodiments, provided herein is a method for producing a multimeric polypeptide in a eukaryotic host cell using a host cell comprising a polynucleotide, the polynucleotide comprising a translation initiation sequence operably connected to an open reading frame of each polypeptide encoding the multimeric polypeptide. Advantageously, the present disclosure demonstrates that regulating the intensity of the translation initiation sequence connected to each open reading frame allows for a higher yield of multimeric polypeptides with fewer misassembled by-products. The present disclosure further provides cells, screening methods, and kits associated therewith.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 796,014, filed on January 23, 2019, which is incorporated herein by reference in its entirety.

[0003] Submit sequence listing as ASCII text file

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Computer Readable Format (CRF) of a Sequence Listing (File Name: 146392045240SEQLIST.TXT, Record Date: January 22, 2020, Size: 5KB). Technical Field

[0005] The present disclosure relates to methods of producing multimeric polypeptides in eukaryotic (eg, mammalian) host cells, and cells, methods, and kits or articles of manufacture related thereto. Background Art

[0006] Therapeutic antibodies represent the most successful biological drugs. Over the past 30 years, more than 40 therapeutic antibodies have been approved for a variety of indications, including cancer, autoimmunity, infection, and vascular disease. As such, therapeutic antibodies represent one of the fastest growing areas of the pharmaceutical industry, and their clinical impact is significant.

[0007] Due to the fact that most diseases involve multiple parallel signaling pathways, multiple inhibition of receptors and ligands may achieve better therapeutic effects. Therefore, bispecific antibodies (bsAbs) with the ability to bind to two different epitopes have been shown to be promising for the treatment of a variety of diseases, 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(3):273-88) and infectious diseases (De Bernardis F, Liu H, O'Mahony R, La Valle R, Bartollino S, Sandini S, et al. J Infect Dis. 2007; 195(1):149-57; Laventie BJ, Rademaker HJ, Saleh M, de Boer E, Janssens R, Bourcier T, et al. Proc Natl Acad Sci US A. 2011; 108(39): 16404-9) etc. (Kontermann RE. MAbs. 2012; 4(2): 182-97; Brinkmann U, Kontermann RE. MAbs. 2017; 9(2): 182-212; Carter PJ, Lazar GA. Nat Rev Drug Discov. 2018; 17(3): 197-223). In the past few decades, genetic engineering has produced more than 60 different bispecific antibody formats, which have improved the immunogenicity, pharmacokinetic properties and distribution of bsAb (Spiess C, Zhai Q, Carter PJ. Mol Immunol. 2015; 67(2Pt A): 95-106).

[0008] Regardless of the application of bsAbs, the production process has been challenging due to the promiscuous pairing of two different heavy chains and two different light chains to produce 16 different combinations, only one of which is bispecific, which makes it difficult to produce them in sufficient quantity, quality and assembly to support preclinical and clinical development. Antibody engineering has been used to alleviate the chain mispairing problem by heterodimerization of heavy or light chains using knobs-in-holes and crossMab technologies, respectively (Ridgway JB, Presta LG, Carter P. Protein Eng. 1996; 9(7): 617-21; Atwell S, Ridgway JB, Wells JA, Carter P. J Mol 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 in vitro assembly into bsAbs (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 due to the production of two different stable cell lines.

[0009] Therefore, co-expression of two antibodies in a single cell may be more straightforward, but it often requires more extensive optimization of the expression system to direct the light chain to its cognate heavy chain and obtain appropriate amounts of correctly assembled bsAbs, avoiding the presence of unwanted byproducts and the need for complex purification processes.

[0010] In the past few decades, mammalian cells such as Chinese hamster ovary (CHO) cells have become the main host in the biopharmaceutical industry (Wurm FM. Nat Biotechnol. 2004; 22 (11): 1393-8). Bioprocess innovation and cell engineering have 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 cell processes and gene regulation mechanisms still hinder cell growth, specific productivity and protein quality. Currently, there are some systematic methods to precisely control the translation level of recombinant proteins in mammalian cells.

[0011] Although progress has been made in the engineering of complex antibody formats (such as bispecific antibodies), the manufacturability of these antibody formats in a single mammalian expression system generally exhibits low efficiency. Low titer and insufficient product quality are two factors that lead to difficulty in stably producing bsAbs. Therefore, there is an urgent need to identify the limiting steps in the production system and provide the production of multimeric polypeptides that allow improvement in correct assembly, for example, in eukaryotic or mammalian host cell systems.

[0012] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety to the same extent as if each reference was specifically and individually indicated to be incorporated by reference. Summary of the invention

[0013] To meet these and other needs, methods for producing multimeric polypeptides in eukaryotic (e.g., mammalian) host cells are provided herein. These multimeric polypeptides comprise two or more subunits, each of which comprises 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.

[0014] Certain aspects of the present disclosure relate to methods for 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 the eukaryotic host cell under conditions suitable for the expression of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain form a multimeric polypeptide upon expression; and recovering the multimeric polypeptide produced by the eukaryotic host cell. In some embodiments, the eukaryotic host cell comprises a first polynucleotide, a second polynucleotide, a third nucleotide, and a fourth polynucleotide, the first polynucleotide comprising a first translation initiation sequence operably connected to a first open reading frame encoding a first polypeptide chain, the second polynucleotide comprising a second translation initiation sequence operably connected to a second open reading frame encoding a second polypeptide chain, the third polynucleotide comprising a third translation initiation sequence operably connected to a third open reading frame encoding a third polypeptide chain, and the fourth polynucleotide comprising a fourth translation initiation sequence operably connected to a fourth open reading frame encoding a fourth polypeptide chain. In some embodiments, when each subunit is expressed separately in a eukaryotic host cell, the first subunit is expressed at a level lower than the second subunit, and one or both of the first translation initiation sequence and the second translation initiation sequence are 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 level lower than the second subunit, and one or both of the first translation initiation sequence and the second translation initiation sequence are 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 polypeptide chain and the third polypeptide chain are antibody heavy chains, the second polypeptide chain and the fourth polypeptide chain are antibody light chains, the first subunit is a first half antibody that binds to a first antigen, and the second subunit is a second half antibody that binds to a second antigen.

[0015] Other aspects of the present disclosure relate to methods for 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, a second polynucleotide, a third nucleotide, and a fourth polynucleotide, the first polynucleotide comprising a first translation initiation sequence operably linked to a first open reading frame encoding the first antibody heavy chain, the second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the first antibody light chain, the third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the second antibody heavy chain, and the fourth polynucleotide comprising a first polynucleotide, a second polynucleotide, a third nucleotide, 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 the first half antibody is expressed at a lower level than the second half antibody when each half antibody is expressed separately in a eukaryotic host cell, and wherein one or both of the first translation initiation sequence and the second translation initiation sequence are 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 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.

[0016] In some embodiments, 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 amino acid residues having a smaller side chain volume, thereby generating a hole on the surface of the CH3 domain of the Fc region of the first antibody that interacts with the CH3 domain of the Fc region of the second antibody; 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 amino acid residues having a larger side chain volume, thereby generating a knob on the surface of the CH3 domain of the Fc region of the second antibody that interacts with the CH3 domain of the Fc region of the first antibody. In some embodiments, 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 generating 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 amino acid residues having a larger side chain volume, thereby generating 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 the following: T366Y, T366W, T394W, and F405W, numbered according to the EU index based on human IgG1. In some embodiments, the hole mutation comprises at least one of the following: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbered according to the EU index based on human IgG1. 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 IgG1. 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 promote the 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, numbered according to the EU index.In some embodiments, S183 substitutions are selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R and S183K; and / or V133 substitutions are 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 promote the selective association of the second antibody light chain with the second antibody heavy chain. In some embodiments, the third mutation includes an amino acid substitution at V133 and / or the fourth mutation includes an amino acid substitution at S183, based on EU index numbering. In some embodiments, S183 substitutions are selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R and S183K; and / or V133 substitutions are selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R and V133D. In some embodiments, the amino acid substitutions at S183 produce positively charged residues (e.g., S183K), and the amino acid substitutions at V133 produce negatively charged residues (e.g., V133E). In some embodiments, the amino acid substitutions at S183 produce negatively charged residues (e.g., S183E), and the amino acid substitutions at V133 produce positively charged residues (e.g., V133K).

[0017] In some embodiments, the method results in a higher yield of a multimeric polypeptide in a eukaryotic host cell, for example, compared to the production of an open reading frame operably connected to a natural or unmodified translation initiation sequence, or compared to the production of each polynucleotide of a polypeptide chain encoding a multimeric polypeptide. In some embodiments, the method results in fewer mispaired byproducts in a eukaryotic host cell, for example, compared to the production of an open reading frame operably connected to a natural or unmodified translation initiation sequence, or compared to the production of each polynucleotide of a polypeptide chain encoding a multimeric polypeptide, wherein one or more, two or more, three or more or four polynucleotides of a polypeptide chain encoding a multimeric polypeptide comprise a natural or unmodified translation initiation sequence. For example, transient or stable transfectants can be cultured, and the production of multimeric polypeptides can be determined. Culture feed batches or perfusion cultures can determine the product titer in a cell culture medium or on a cell surface. For surface expression, cells can be stained with antibodies to detect the product and analyzed, for example, by flow cytometry. Product quality and / or purity can be analyzed by, for example, electrophoresis and / or mass spectrometry. In some embodiments, when each subunit is expressed alone in a eukaryotic host cell, one or two polypeptide chains of the first subunit are translated at a slower rate than one or two polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed alone in a eukaryotic host cell, one or two polypeptide chains of the first subunit fold slower and / or less efficiently than one or two polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed alone in a eukaryotic host cell, the first subunit assembles at a slower rate than the second subunit. In some embodiments, the first translation start sequence and / or the second translation start 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 start sequence and / or the fourth translation start 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 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 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 the expression level of the second subunit or second half antibody, e.g., when each subunit or half antibody is expressed individually in a eukaryotic host cell. In some embodiments, the first subunit or the first half antibody is expressed at a level that 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 5-fold, 3-fold to 10-fold, 5-fold to 10-fold, or 7-fold to 10-fold lower than the expression level of the second subunit or the second half antibody, for example, when each subunit or half antibody is expressed alone in a eukaryotic host cell. In some embodiments, the first subunit or the first half antibody is expressed at a level that is 0.2-fold to 0.8-fold, less than 0.8-fold, less than 0.5-fold, or less than 0.3-fold lower than the expression level of the second subunit or the second half antibody, for example, when each subunit or half antibody is expressed alone in a eukaryotic host cell.

[0018] Other aspects of the present 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.

[0019] 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 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 separately in the recombinant eukaryotic host cell, the expression level of the first subunit is lower than that of the second subunit; and wherein one or both of the first translation initiation sequence and the second translation initiation sequence are 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 polypeptide chain and the third polypeptide chain are antibody heavy chains, the second polypeptide chain and the fourth polypeptide chain 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.

[0020] 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 first antibody heavy chain; (b) a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the first antibody light chain; (c) a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the second antibody heavy chain; and (d) a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the second antibody light chain; wherein the first half antibody is expressed at a lower level than the second half antibody when each half antibody is expressed separately in the recombinant eukaryotic host cell, and wherein one or both of the first translation initiation sequence and the second translation initiation sequence are 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 a first antigen and the second half antibody binds a second antigen.

[0021] In some embodiments, 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 amino acid residues having a smaller side chain volume, thereby generating a hole on the surface of the CH3 domain of the Fc region of the first antibody that interacts with the CH3 domain of the Fc region of the second antibody; 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 amino acid residues having a larger side chain volume, thereby generating a knob on the surface of the CH3 domain of the Fc region of the second antibody that interacts with the CH3 domain of the Fc region of the first antibody. In some embodiments, 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 generating 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 amino acid residues having a larger side chain volume, thereby generating 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 the following: T366Y, T366W, T394W, and F405W, numbered according to the EU index based on human IgG1. In some embodiments, the hole mutation comprises at least one of the following: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbered according to the EU index based on human IgG1. 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 IgG1. 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 promote the 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, numbered according to the EU index.In some embodiments, S183 substitutions are selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R and S183K; and / or V133 substitutions are 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 promote the selective association of the second antibody light chain with the second antibody heavy chain. In some embodiments, the third mutation includes an amino acid substitution at V133 and / or the fourth mutation includes an amino acid substitution at S183, based on EU index numbering. In some embodiments, S183 substitutions are selected from the group consisting of S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R and S183K; and / or V133 substitutions are selected from the group consisting of V133E, V133S, V133L, V133W, V133K, V133R and V133D. In some embodiments, the amino acid substitutions at S183 produce positively charged residues (e.g., S183K), and the amino acid substitutions at V133 produce negatively charged residues (e.g., V133E). In some embodiments, the amino acid substitutions at S183 produce negatively charged residues (e.g., S183E), and the amino acid substitutions at V133 produce positively charged residues (e.g., V133K). In some embodiments, the first antibody light chain comprises a V133K mutation, the first antibody heavy chain comprises an S183E mutation, the second antibody light chain comprises a V133E mutation, and the second antibody heavy chain comprises an S183K mutation, based on the EU index numbering. In some embodiments, the first antibody heavy chain further comprises T366S, L368A and Y407V mutations, and the second antibody heavy chain further comprises a T366W mutation, based on the EU index numbering based on human IgG1. In some embodiments, the second antibody light chain comprises a V133K mutation, the second antibody heavy chain comprises an S183E mutation, the first antibody light chain comprises a V133E mutation, and the first antibody heavy chain comprises an S183K mutation, based on the EU index numbering. In some embodiments, the second antibody heavy chain further comprises T366S, L368A and Y407V mutations, and the first antibody heavy chain further comprises a T366W mutation, based on the EU index numbering based on human IgG1.

[0022] Other aspects of the present disclosure relate to methods for identifying a combination of translation initiation sequences for expressing 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 A plurality of combinations of the first translation initiation sequence, the second translation initiation sequence, the third translation initiation sequence, and the fourth translation initiation sequence are present in a plurality of eukaryotic host cells; the eukaryotic host cell library is cultured under conditions suitable for expressing the multimeric polypeptide by the eukaryotic host cells in the plurality; the amount of the multimeric polypeptide expressed by a single eukaryotic host cell in the plurality or a clone of a single eukaryotic host cell in the plurality is measured; and 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 clones of a single eukaryotic host cell in the plurality or a single eukaryotic host cell in the plurality expressing the multimeric polypeptide are identified. In some embodiments, based on a higher yield of multimeric polypeptides and / or a lower level of mispaired byproducts, for example, compared to a reference or different combinations of four translation initiation sequences, 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 clones of a single eukaryotic host cell in the plurality expressing the multimeric polypeptide or a single eukaryotic host cell in the plurality are identified. In some embodiments, all of the first translation start sequence, the second translation start sequence, the third translation start sequence, and the fourth translation start sequence in each of the plurality of host cells comprise the sequence (from 5' to 3') NNNNNATGNGA, wherein N is C, G, A, or T / U (SEQ ID NO: 1).

[0023] Other aspects of the present disclosure relate to a kit or article comprising a polynucleotide for the 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 comprises a first polynucleotide comprising a first translation initiation sequence operably connected to a first open reading frame encoding the first polypeptide chain; a second polynucleotide comprising a second translation initiation sequence operably connected to a second open reading frame encoding the second polypeptide chain; a third polynucleotide comprising a third translation initiation sequence operably connected to a third open reading frame encoding the third polypeptide chain; and a fourth polynucleotide comprising a fourth translation initiation sequence operably connected 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 connected to their respective open reading frames, because the respective open reading frames are present in the naturally occurring host cell genome. In some embodiments, the multimeric polypeptide is a bispecific antibody, the first polypeptide chain and the third polypeptide chain are antibody heavy chains, the second polypeptide chain and the fourth polypeptide chain 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.

[0024] In some embodiments, when each subunit is produced separately, one or two polypeptide chains of the first subunit are produced at a lower level than one or two polypeptide chains of the second subunit. In some embodiments, when all subunits are produced by the same host cell, one or two polypeptide chains of the first subunit are produced at a lower level than one or two 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 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.

[0025] 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 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 of the first translation initiation sequence and the second translation initiation sequence comprise a sequence selected from the group consisting of: SEQ ID NO: 8-10. In some embodiments, one or both of the third translation initiation sequence and the fourth translation initiation sequence comprise 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: SEQ ID NO: 8-10, the second translation initiation sequence comprises a sequence selected from the group consisting of: SEQ ID NO: 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 a sequence of SEQ ID NO: 9, the second translation initiation sequence comprises a sequence of SEQ ID NO: 9, the third translation initiation sequence comprises a sequence of SEQ ID NO: 2, and the fourth translation initiation sequence comprises a sequence of SEQ ID NO: 11. In some embodiments, each of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide 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 second translation initiation sequence is weaker than the third translation initiation sequence. In some embodiments, the first translation initiation sequence is the same as the second translation initiation sequence. In some embodiments, the third translation initiation sequence is the same as the fourth translation initiation sequence. In some embodiments 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.

[0026] Also provided herein are polynucleotides comprising an open reading frame operably linked to a translation initiation sequence, the translation initiation sequence being selected from the group consisting of: SEQ ID NO: 8-11. Further provided herein are 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: SEQ ID NO: 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 No: 8-10, a third translation initiation sequence operably linked to a third open reading frame, comprising a sequence of SEQ ID NO: 2, and a fourth translation initiation sequence operably linked to a fourth open reading frame, comprising a sequence of SEQ ID NO: 11. In some embodiments, the first translation initiation sequence comprises a sequence of SEQ ID NO: 9, the second translation initiation sequence comprises a sequence of SEQ ID NO: 9, the third translation initiation sequence comprises a sequence of SEQ ID NO: 2, and the fourth translation initiation sequence comprises a sequence of SEQ ID NO: 11. Further provided herein are host cells comprising a polynucleotide or a set of polynucleotides according to any one embodiment described herein.

[0027] In some embodiments of any embodiment 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 certain embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide and the fourth polynucleotide are integrated into the same chromosome locus of the eukaryotic host cell. In certain embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide and the fourth polynucleotide are part of one of the multiple extrachromosomal polynucleotides in the eukaryotic host cell. In certain embodiments, the eukaryotic host cell is a mammalian host cell. In certain embodiments, the mammalian host cell is a Chinese hamster ovary (CHO) cell.

[0028] It should 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 present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0030] Figure 1 The names and nucleotide sequences of Kozak (Kz) sequence variants designed and analyzed by transient transfection are shown. The start codon is gray and underlined. The sequences shown correspond to SEQ ID NOs: 3-7 (from top to bottom).

[0031] Figure 2 Provided are the normalized titers of Fc fusion proteins A and B with different Kozak sequence variants, which were tested by transient transfection followed by homogeneous time-resolved fluorescence (HTRF). Data are presented as mean values, with error bars representing the standard deviation (SD) of four independent transfections. Changes in the Kozak consensus sequence modulate the titer of the Fc fusion protein.

[0032] Figure 3 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 is described.

[0033] Figure 4 The wild-type (WT) Kozak sequence in the upper row is shown. The designed Kozak library is shown in the lower row. N is A, T, G or C. The start codon is gray and underlined. The -3 and +4 positions of the start codon are indicated. The sequences shown correspond to (from top to bottom) SEQ ID NOs: 23 and 1, respectively.

[0034] Figure 5 Shown are the normalized antibody titers of 111 Kozak sequence variants transiently transfected into CHO cells. Data are expressed as mean values, and error bars represent the standard deviation (SD) of two independent transfections. An almost continuous translation range of normalized titers of 0.05 to 1.54 units was observed (1.0 corresponds to the titer produced using the Wt Kozak sequence).

[0035] Figure 6 The nucleotide distribution of 108 colonies of the Kozak library is provided. The percentage of each nucleotide at each position is shown. The nucleotide distribution of the analyzed variants is random and diverse at each position, indicating that the library screening is not biased.

[0036] Figure 7 The screening process to reduce the number of Kozak variants by transient transfection in CHO cells is shown. The criteria for selecting variants between rounds were reproducibility between transient transfections, stability between DNA preparations, and preservation of nucleotide diversity.

[0037] Fig. 8A and 8BNormalized titers and sequences for the final set of Kozak sequence variants are provided. Fig. 8A Shown are normalized antibody titers of specified Kozak sequence variants transiently transfected into CHO cells. Data are expressed as mean values, and error bars represent standard deviations of eleven independent transfections. 11 Kozak sequence variants represent an expression range of 0.2 to 1.3 times that of Wt Kozak, thereby enabling high precision adjustment of titers. Asterisks indicate Kozak sequence variants selected as a representative group of five variants (including Wt Kozak) because they cover a wide range of expression levels observed in transient transfections. Figure 8B The sequences of the last set of Kozak sequence variants are provided. The wt consensus Kozak sequence is framed in a box.

[0038] Fig. 9 It was shown that a higher percentage of correctly assembled BsAbs can be obtained by manipulating the ratio of light chains (LC1:LC2). For example, modification of the LC1:LC2 ratio from 1:2.5 to 1.5:1 resulted in a 40% increase in correctly paired light chains.

[0039] Fig.10 The translation strength of the listed Kozak variants relative to Wt Kozak (1x) is provided. 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 a wide range of expression levels observed in transient transfections. The sequences correspond to SEQ ID NOs: 8, 9, 10, 2, and 11 (from top to bottom).

[0040] Fig.11 A combination of Kozak variants for developing 25 integrated expression vectors for each group of Ab1 / Ab2 BsAbs is provided. A mixture of 50 plasmids is transfected into CHO cells. The diversity generated by this method reaches a potential 625 different chain ratio combinations. Only the combination of Ab1 is shown, but the same combination is used to generate the Ab2 group vectors.

[0041] Fig.12 The transfection efficiency and transfection number of the designated Kozak mixed pools are shown. The transfection efficiency was monitored by cell surface staining analysis with anti-hu IgG antibodies labeled with allophycocyanin (APC) and by measuring antibody-APC expression by flow cytometry. Gates were set for Ab expression negative and GFP positive cells using an empty host. The X-axis shows GFP expression and the Y-axis shows APC antibody expression. Similar transfection efficiencies were observed for the Kozak mixed and Wt Kozak pools.

[0042] Fig.13A and 13BThe stable pool recovery time course is shown. Two pools for each condition are shown. The addition of selection drugs to establish stable pools is indicated by arrows. The Y axis shows the percentage of viability, and the X axis shows the number of days after the addition of the selection drug.

[0043] Fig.14A and 14B The absolute titers of 704 individual clones of Kozak pools or Wt Kozak clones selected and single-cell cloned from each transfection and condition are presented. Titers were measured by HTRF. Presented in pairs according to the number of transfections indicated.

[0044] 15A and 15B provide the viability of 14-day shake flask fed batch production of Kozak hybrid clones and Wt-Kozak clones. The X-axis shows the number of days and the Y-axis shows the percentage of viability. The number of transfections is indicated. All clones studied showed comparable viability throughout the production assay. The final viability on day 14 under both conditions was approximately 80-95%, except for one Kozak hybrid clone with a final viability of 74%.

[0045] 16A and 16B show the viable cell counts of a 14-day shake flask fed-batch of a Kozak hybrid clone and a Wt-Kozak clone. The X-axis shows days, and the Y-axis shows cells per ml per 10 6 The number of viable cells in . The number of transfections is indicated. An exponential growth rate was observed until day 7, after which all individual clones reached a plateau phase.

[0046] Fig.17 The general absolute titers of the first 11 Kozak pool clones and the Wt Kozak clones for each transfection are shown. Titers were measured on day 14 of the shake flask fed-batch production. In both transfections, the Wt Kozak clones alone showed higher general absolute antibody titers than the Kozak pool clones alone. Note that the titers represent correctly assembled bispecific antibodies as well as half antibodies and other unwanted byproducts.

[0047] Fig.18 Provided is an assessment of the quality of assembled antibodies for the first 11 clones of Kozak hybrid and Wt Kozak for each transfection. Samples were analyzed by non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) to distinguish and quantify two antibody forms: a main peak equal to the full antibody (full-ab) of the BsAb form assembled correctly and incorrectly, and the sum of the front peaks of other species representing different molecular weights. The number of transfections is indicated. Among the first 22 clones, Kozak hybrid clones with a high percentage of full-ab forms were less than Wt Kozak clones.

[0048] Fig.19A and 19BThe quality and assembly efficiency of medium (marked with M) and low (marked with L) HCCF titer Kozak pool clones are shown. Fig.19A Shown are typical absolute titers of 3 medium and 4 low Kozak pool clones measured by shake flask fed-batch production assay on day 14. The production titers of these clones showed similar behavior to that observed previously in the primary screen. Fig.19B Provides an assessment of the quality of assembled antibodies of low and medium Kozak hybrid clones. Samples were analyzed by non-reducing CE-SDS to distinguish and quantify the main peaks equal to the incorrectly paired byproducts and the whole antibody (whole-ab) in the form of correctly assembled BsAbs, as well as the sum of the front peaks of other species representing different molecular weights. There is no direct correlation between the general titer and the highest content of the whole antibody. For example, compared with the first 11 clones, some Kozak hybrid clones with more moderate titers showed similar whole-ab percentages of about 50-60%.

[0049] Fig. 20A and 20B The quality and effective BsAb titers of the indicated Wt Kozak and Kozak mixed clones are shown. Fig. 20A Provided are quantifications of species detected by mass spectrometry of eight Kozak hybrid clones and eight Wt Kozak clones. The structure of each species is indicated in the legend. Correctly assembled BsAbs are highlighted in white. The best individual Kozak hybrid clone showed approximately 40% correctly assembled BsAbs, which is more than twice (approximately 18%) of the best individual Wt Kozak clones. Fig. 20B The estimated titers of the bispecific antibodies and various mispaired byproducts and half-antibody species are provided. It is calculated as the percentage of each form multiplied by the general titer. The bispecific effective titer is highlighted in white. Some Kozak hybrid clones overcome the previous Fig.17 The general titer shown in the figure is insufficient.

[0050] Figures 21A-21C The translation intensities of Kozak sequence variant combinations and the indicated Kozak hybrid clones are shown. Fig.21A A combination of Kozak sequence variants from a Kozak hybrid clone is shown. Sequencing data was not available for this sequence. The name and translation strength of each Kozak sequence variant is shown on the right. Fig.21B The Kozak sequence variant combinations of the best antibody-producing Kozak pool clones are shown. The Kozak pool clone name and transfection number are indicated. Sequencing data were not available for the gray highlight. Fig. 21CKozak sequence variant combinations of 3 medium (marked with M) and 4 low Kozak hybrid clones (marked with L) on the left are provided. On the right, the name and translation strength of each Kozak sequence variant are provided. Grey highlights indicate that sequencing data was not available.

[0051] Fig.22A Quantification of bispecific antibodies, mispaired byproducts and half-antibody species by mass spectrometry is shown for WT Kozak clone 50 and Kozak hybrid clones 69 and 17M. Correctly assembled bispecific antibodies are highlighted in white.

[0052] Fig. 22B The effective titers of bispecific antibodies, improperly paired byproducts, and half-antibody species, calculated as the percentage of each species multiplied by the general titer, are shown on the right. Correctly assembled bispecific antibodies are highlighted in white.

[0053] Top Fig. 22C The production performance of clone 17M is shown. The CE-SDS data of this clone showed that the product mass was about 48% of the full-ab and about 52% of the half-ab. The mass spectrometry data showed that of the approximately 48% full-abs, all of these represented correctly assembled bispecific antibodies. On the other hand, almost all of the half-ab species were knob 1 / 2Ab2, which is consistent with the fact that the heavy and light chains of Ab2 are respectively under Wt Kozak and the strongest Kozak sequence variant #228. At the bottom, the name and translation strength of each Kozak sequence variant are shown. Clone 17M carries both the heavy and light chains of Ab1 under the weaker Kozak sequence variant.

[0054] Fig.22D Quantification of bispecific antibodies, improperly paired byproducts, and half-antibody forms by mass spectra of the indicated low-medium Kozak hybrid clones is provided. The percentage of BsAb assembly was less than 10%, except for Kozak hybrid clone 17. The absolute abundance of each species is related to the expression intensity of each chain. DETAILED DESCRIPTION

[0055] I. Definitions

[0056] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to specific compositions or biological systems, which can of course vary. In addition, it should be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting.

[0057] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" 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.

[0058] The term "about" as used herein refers to the common error range of the corresponding value that is readily known to those skilled in the art. References herein to "about" values ​​or parameters include (and describe) embodiments involving the value or parameter itself.

[0059] It should be understood that the disclosed aspects and embodiments described herein include aspects and embodiments referred to as "comprising," "consisting of," and "consisting essentially of."

[0060] The terms "polypeptide" or "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with 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 with a labeling component or toxin. Also included within the definition are, for example, one or more analogs containing amino acids (including, for example, non-natural amino acids, etc.) and other modified polypeptides known in the art. As used herein, the terms "polypeptide" and "protein" specifically include antibodies and antigen binding proteins.

[0061] As used herein, a "multimer" polypeptide or protein may refer to any complex comprising more than one polypeptide or protein monomer (e.g., polypeptide chain). For example, a multimer 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 "multimer" polypeptide or protein specifically includes antibodies and antigen-binding proteins. In certain embodiments, the multimer 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.

[0062] As used herein, the term "subunit" when used to refer to a component of a multimeric polypeptide or protein is intended to refer to any polypeptide comprising more than one different polypeptide chain. In some embodiments, a subunit may include 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, a subunit comprises an immunoglobulin heavy chain and a light chain. In certain embodiments, a subunit comprises a half antibody. In certain embodiments, the immunoglobulin heavy chain and light chain or a half antibody is an IgG1, IgG2, or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain and light chain or a half antibody is an IgG1 or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain and light chain or a half antibody is an IgG1 or IgG4 isotype. In certain embodiments, the immunoglobulin heavy chain and light chain or a half antibody is an IgG1 isotype.

[0063] As used herein, "incorrectly paired byproducts" refers to the 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 subunits light chain A / heavy chain A and subunits light chain B / heavy chain B, incorrectly paired byproducts arise 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 byproducts are referenced and shown in Fig. 20A , 20B and 22A-22D. Other methods for assessing protein folding, such as differential scanning fluorimetry, MS, or using computer tools to predict the outcome can also be used.

[0064] 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 translation scanning model, a ribosome pre-initiation complex binds to the 5' end of a polynucleotide and proceeds linearly in the 3' direction to search for a stop codon. Based on the work of Marilyn Kozak, who described in detail the scanning model of translation initiation (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. J Mol Biol. 1987; 196(4):947-50; Hamilton R, Watanabe CK, de Boer HA. Nucleic Acids Res. 1987; 15(8):3581-93; Cavener DR. Nucleic Acids Res. 1987; 15(4):1353-61; Kozak M. Nucleic Acids Res.1984;12(2):857-72; Gupta P, Rangan L, Ramesh TV, Gupta M. J Theor Biol.2016;404:303-11; Grzegorski SJ, Chiari EF, Robbins A, Kish PE, Kahana A. PLoS One.2014;9(9):e108475; and Kozak M. Cell.1986;44(2):283-92), the translation initiation sequence may include a "Kozak" sequence, as well as the variant Kozak sequences described herein.

[0065] As used herein, the terms "natural" and "non-natural" refer to one or more genetic elements (e.g., encoding polypeptides, promoters, translation units, or combinations thereof), meaning the genomic context when the genetic elements in the host cell chromosomes are present in nature. For example, when the polynucleotide encoding the polypeptide is naturally present in the genome of the host cell, the polypeptide (e.g., a multimeric polypeptide) is "natural" for the host cell or the host cell chromosome, and when the polynucleotide encoding the polypeptide is not naturally present in the genome of the host cell, it is "non-natural". When the translation initiation sequence or open reading frame is naturally present in the genome of the host cell, the translation initiation sequence or open reading frame is "natural" for the host cell or the host cell chromosome, and when the translation initiation sequence or open reading frame is not naturally present in the genome of the host cell, it is "non-natural". When the translation initiation sequence is not naturally present in the genome of the host cell in the same operably connected as the open reading frame, the operable combination of the translation initiation sequence and the open reading frame is "non-natural", or vice versa. For example, a translation initiation sequence:open reading frame combination is "non-native" to the host cell or host cell chromosome when one or both of the translation initiation sequence and open reading frame do not naturally occur in the host cell genome, when the translation initiation sequence is present in the host cell genome in operable linkage with an open reading frame that is not operable combined in a naturally occurring host genome (even if the same translation initiation sequence is naturally present elsewhere in the host cell genome), or when the open reading frame is present in the host cell genome in operable linkage with a translation initiation sequence that is not operable combined in a naturally occurring host genome (even if the same open reading frame sequence naturally occurs elsewhere in the host cell genome).

[0066] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell (e.g., a vector with a replication origin and an episomal mammalian vector) into which they are introduced. After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of the nucleic acid operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms.

[0067] "Operably linked" refers to the juxtaposition of two or more components, wherein the components are in a relationship that allows them to function in their intended manner. Typically, but not necessarily, the DNA sequence "operably linked" is continuous and, where two protein coding regions need to be connected, or in the case of a secretory leader, continuous and in reading frame. However, although the promoter operably linked is usually located upstream of the coding sequence or translation unit, it is not necessarily continuous with it. The enhancer operably linked can be located upstream, within or downstream of the coding sequence / translation unit and is quite far from the promoter. Connection is completed by recombinant methods known in the art, such as using PCR methods, by annealing or by connection at a convenient restriction site. If a suitable restriction site does not exist, then a synthetic oligonucleotide adapter or a linker is used according to conventional specifications.

[0068] "Promoter" refers to a polynucleotide sequence that controls the transcription of a gene or sequence to which it is operably linked. A promoter includes 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).

[0069] As used herein, the term "host cell" (or "recombinant host cell") means a cell that has been genetically altered or is capable of being genetically altered by the introduction of exogenous or non-natural polynucleotides, such as a recombinant plasmid or vector. It should be understood that these terms refer not only to the specific subject cell, but also to the progeny of the cell. Because certain modifications may occur in the progeny due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0070] The term "antigen" herein is used 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.

[0071] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as the antibody fragment exhibits the desired antigen-binding activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0072] An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that interfere with the research, diagnostic, or therapeutic use of the antibody, and may include enzymes, hormones, and other protein or non-protein 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 a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence (e.g., by using a spinning cup sequencer), or (3) homogeneous (SDS-PAGE performed under reducing or non-reducing conditions, using, for example, Coomassie blue or silver stain). Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, typically, isolated antibodies will be prepared by at least one purification step.

[0073] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than another portion of the immunoglobulin (i.e., the variable domain, which contains the antigen binding site). The constant domain contains the C H 1. C H 2 and C H The three domains (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

[0074] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as the "V H The variable domain of the light chain may be referred to as the "V L ” These domains are generally the most variable parts of an antibody and contain the antigen-binding site.

[0075] The term "variable" refers to the fact that some parts of the variable domains have very different sequences between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed in the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) in the light chain and heavy chain variable domains. The more conservative part of the variable domain is called the framework region (FR). The variable domains of natural heavy and light chains each include four FR regions, which mainly adopt a β-folded structure and are connected by three HVRs, which form a loop connecting the β-folded structure and form a part of the β-folded structure in some cases. The HVRs in each chain are closely held 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 Human Services, National Institutes of Health, Bethesda, Maryland (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but have respective effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0076] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0077] As used herein, the term IgG "isotype" or "subclass" refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of the immunoglobulin constant region.

[0078] According to the amino acid sequence of its heavy chain constant domain, antibodies (immunoglobulins) can be divided into different categories.Immunoglobulins are mainly divided into five categories: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.The heavy chain constant domains corresponding to different classes of immunoglobulins are respectively referred to as α, γ, ε, γ and μ.The subunit structure and three-dimensional configuration of different types of immunoglobulins are well known and generally described in, for example, the following documents: Abbas et al., Cellular and Mol. Immunology, 4th edition (WB Saunders, Co., 2000).Antibodies can be part of a larger fusion molecule, which is formed by the covalent or non-covalent association of antibodies with one or more other proteins or peptides.

[0079] 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 specifically refers to an antibody having heavy chains that include an Fc region.

[0080] "Antibody fragments" include a portion of an intact antibody, preferably including its antigen binding region. In some embodiments, the antibody fragments described herein are antigen binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0081] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0082] "Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, a double-chain Fv species is composed of a dimer of a heavy chain and a light chain variable domain that are tightly and non-covalently associated. In a single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can be associated into a "dimer" structure similar to that in a double-chain Fv species. With this configuration, the three HVRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Six HVRs jointly confer antigen binding specificity to antibodies. However, even a single variable domain (or only half of an Fv comprising three HVRs that are specific to an antigen) also has the ability to recognize and bind antigens, although its affinity is lower than that of a complete binding site.

[0083] The Fab fragment contains the heavy chain variable domain and the light chain variable domain and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that the Fab' fragment adds some residues at the carboxyl terminus of the heavy chain CH1 domain, which residues include one or more cysteines from the antibody hinge region. Fab'-SH is the nomenclature herein for Fab' in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0084] "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 a desired antigen binding structure. For a review of scFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0085] The term "diabody" refers to an antibody fragment with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) connected 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 another chain and produce two antigen binding sites. Diabodies can be bivalent antibodies or bispecific antibodies. Diabodies are more fully described in, for example: EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0086] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody group, for example, except for a small amount of mutations that may exist, such as naturally occurring mutations, the individual antibodies contained in the antibody group are identical. Therefore, the modifier "monoclonal" indicates that the characteristic of the antibody is not a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody generally includes an antibody containing a polypeptide sequence that binds a target, wherein the target binding polypeptide sequence is obtained by including a process of 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 plurality of clones, such as a collection of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the target binding sequence of selection can be further changed, for example, to improve affinity to the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, produce multi-specific antibodies, etc., and the antibody comprising the changed target binding sequence is also a monoclonal antibody of the present disclosure. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibody preparations is that they are generally free of contamination by other immunoglobulins.

[0087] The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can 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, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. 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., Year in Immunol.7:33 (1993); U.S. Patent 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 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0088] The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the most diversity among the six HVRs, and H3 in particular is considered to play a unique role in conferring fine specificity to antibodies. 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, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0089] Many HVR descriptions are used and are included herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland (1991)). In contrast, Chothia refers to the position of the structural loop (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops, and is adopted by the AbM antibody modeling software of Oxford Molecular. "Contact" HVR is based on the analysis results of available complex crystal structures. The residues of each of these HVRs are described below.

[0090] Table 1a. Antibody hypervariable regions

[0091]

[0092] HVRs may 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., above.

[0093] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0094] The term "variable domain residue numbering as described in Kabat" or "amino acid position numbering as described in Kabat" and variations thereof refer to the numbering system for heavy chain variable domains or light chain variable domains proposed in the above-mentioned document by Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion fragment after residue 52 of H2 (residue 52a according to Kabat numbering) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat numbering). The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence with a region of homology to a "standard" Kabat numbering sequence.

[0095] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed., U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, Maryland (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported by Kabat et al., supra).

[0096] The expression "linear antibody" refers to the antibodies described by Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). In short, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0097] II. Multimeric polypeptide production and screening methods

[0098] Provided herein is a method for producing a multimeric polypeptide in a eukaryotic host cell (e.g., a mammalian host cell). In some embodiments, the method includes: providing a eukaryotic host cell, the eukaryotic host cell comprising a first polynucleotide comprising a first translation initiation sequence operably connected 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 connected 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 connected 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 connected to a fourth open reading frame encoding a fourth polypeptide chain of a multimeric polypeptide; culturing the eukaryotic host cell under conditions suitable for expression of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain, wherein 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.

[0099] In some embodiments, when each polypeptide chain is produced separately in a eukaryotic host cell or when the 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 translation initiation sequence and the second translation initiation sequence are weaker than one or both of the third translation initiation sequence and the fourth translation initiation sequence. 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.

[0100] The production method described herein is based at least in part on the proof of this article, that is, using different translation initiation sequences to adjust the translation strength can improve the assembly of multimeric polypeptides (e.g., bispecific antibodies) and reduce impurities associated with the product, such as incorrectly paired by-products. Surprisingly, it was found that the reduction of the selected polypeptide chain translation relative to the translation level of other polypeptide chains of the multimeric polypeptide can improve the multimeric polypeptide assembly efficiency and improve the 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 under the strongest translation initiation sequence actually promotes the production of more incorrectly paired by-products including the chain, rather than more correctly assembled multimeric polypeptides. Therefore, down-regulating the translation of one or more chains of a multimeric polypeptide (e.g., a bispecific antibody), which may slow down the assembly of a multimeric polypeptide (e.g., a bispecific antibody), may actually have a positive impact on assembly efficiency and reducing unwanted mispaired by-products. Therefore, the method described herein not only allows a higher level of multimeric polypeptide expression, but also reduces the generation of impurities, such as incorrectly paired by-products, which are burdensome, such as requiring expensive purification methods to remove them.

[0101] In some embodiments, when each subunit is produced separately in a eukaryotic host cell, or when all subunits are produced together in the same eukaryotic host cell, one or two polypeptide chains of the first subunit are translated at a lower level than one or two polypeptide chains of the second subunit. In some embodiments, when each subunit is produced separately in a eukaryotic host cell, or when all subunits are produced together in the same eukaryotic host cell, the first subunit is assembled with more incorrectly paired byproducts than the second subunit. In some embodiments, using a weaker translation initiation sequence operably linked to one or more (e.g., one or two) polypeptide chains of the first subunit (e.g., a subunit that is weaker or more difficult to express in the host cell) results in a higher yield of multimeric polypeptides and / or fewer incorrectly paired byproducts. In some embodiments, when each subunit is expressed separately in a eukaryotic host cell, one or two polypeptide chains of the first subunit are translated at a slower rate than one or two polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed separately in a eukaryotic host cell, one or two polypeptide chains of the first subunit fold slower and / or less efficiently than one or two polypeptide chains of the second subunit. In some embodiments, when each subunit is expressed separately 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 mapping (see, e.g., Ingolia, N. (2016) Cell 165: 22-33).

[0102] As described herein, in some embodiments, the multimeric polypeptide of the present disclosure comprises two subunits. In some embodiments, the subunit of the present disclosure comprises two or more polypeptide chains. In some embodiments, the subunit of the present disclosure comprises two polypeptide chains. In some embodiments, each polynucleotide encoding the polypeptide chain of the multimeric polypeptide comprises a translation initiation sequence operably connected to an open reading frame. In some embodiments, the multimeric polypeptide of the present disclosure comprises two subunits, each subunit comprises two polypeptide chains, and when each subunit is expressed alone in a eukaryotic host cell or when all subunits are expressed together in the same eukaryotic host cell, the first subunit of the multimeric polypeptide is expressed at a level lower than that of the second subunit, and one or both of the first translation initiation sequence (operably connected to the first open reading frame) and the second translation initiation sequence (operably connected to the second open reading frame) are weaker than the third translation initiation sequence (operably connected to the third open reading frame) and the fourth translation initiation sequence (operably connected to the fourth open reading frame) One or both. In some embodiments, the multimeric polypeptide is a bispecific antibody.

[0103] In some embodiments, a subunit or half antibody of the disclosure is expressed at a lower level than another subunit or half antibody, for example, when each subunit or half antibody is expressed separately in a eukaryotic host cell. In some embodiments, a subunit or half antibody of the present 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 individually in a eukaryotic host cell. In some embodiments, a subunit or half antibody of the present disclosure is expressed at a level that 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 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 present disclosure is expressed at a level that is 0.2-fold to 0.8-fold, less than 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.

[0104] 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 antibody heavy chain of a bispecific antibody, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding a first antibody light chain of the bispecific antibody, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding a second antibody heavy chain of the bispecific antibody, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding a second antibody light chain of the bispecific antibody; culturing the eukaryotic host cell under conditions suitable for expression of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the 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 bispecific antibody; and recovering the bispecific antibody from the eukaryotic host cell. In some embodiments, a bispecific antibody specifically binds to two antigens (e.g., a first antibody heavy chain and light chain form an antigen binding domain that binds a first antigen, and a second antibody heavy chain and light chain form an antigen binding domain that binds a second antigen).

[0105] In certain embodiments, when the first translation initiation sequence causes a lower translation efficiency and / or expression of an open reading frame operably connected to another translation initiation sequence, compared to a reference or efficiency / expression of the same open reading frame operably connected to another translation initiation sequence, the first translation initiation sequence is referred to as being weaker than another translation initiation sequence. This paper describes and illustrates a suitable method for comparing various translation initiation sequence intensities in eukaryotic host cells. For example, transient or stable transfectants can be cultivated, and the production of multimeric polypeptides can be measured. Cultivate fed-batch or perfuse cultures, and the product titer in the cell culture medium or on the cell surface can be measured. For surface expression, cells can be stained with antibodies to detect the product and analyzed, for example, by flow cytometry. Product quality and / or purity can be analyzed by, for example, electrophoresis and / or mass spectrometry.

[0106] 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 a translation initiation sequence causes the translation of an open reading frame to which it is operably linked to be 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% less efficient than a reference or the efficiency of the same open reading frame operably linked to another translation initiation sequence (e.g., when measured as described above), A translation initiation sequence is said to be weaker than another translation initiation sequence if the sequence is at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, between 5% and 30%, between 5% and 50%, between 5% and 75%, between 10% and 30%, between 10% and 50%, between 10% and 75%, between 25% and 50%, between 25% and 75%, between 25% and 100%, between 50% and 75%, between 50% and 100%, or between 75% and 100%. In some embodiments, when a translation initiation sequence causes the expression of an open reading frame to which it is operably linked to be 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% lower than a reference or expression level of the same open reading frame operably linked to another translation initiation sequence (e.g., when measured as described above), the expression of an open reading frame to which it is operably linked 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% lower than a reference or expression level of the same open reading frame operably linked to another translation initiation sequence (e.g., when measured as described above). A translation initiation sequence is said to be weaker than another translation initiation sequence if the sequence is at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, between 5% and 30%, between 5% and 50%, between 5% and 75%, between 10% and 30%, between 10% and 50%, between 10% and 75%, between 25% and 50%, between 25% and 75%, between 25% and 100%, between 50% and 75%, between 50% and 100%, or between 75% and 100%.

[0107] In some embodiments, one 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 5-fold, 3-fold to 10-fold, 5-fold to 10-fold, or 7-fold to 10-fold weaker than another translation initiation sequence. In some embodiments, a translation initiation sequence is said to be weaker than another translation initiation sequence when it causes the translation of an open reading frame to which it is operably linked to be 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 5-fold, 3-fold to 10-fold, 5-fold to 10-fold, or 7-fold to 10-fold less efficient than a reference or the efficiency of the same open reading frame to which it is operably linked (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 it causes expression of an open reading frame to which it is operably linked to be 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 5-fold, 3-fold to 10-fold, 5-fold to 10-fold, or 7-fold to 10-fold lower than a reference or expression level of the same open reading frame to which it is operably linked (e.g., when measured as described above). In some embodiments, a translation initiation sequence is referred to as being weaker than another translation initiation sequence when it results in 0.2-fold to 0.8-fold, less than 0.8-fold, less than 0.5-fold, or less than 0.3-fold expression of an open reading frame to which it is operably linked, compared to a reference or expression level of the same open reading frame to which it is operably linked (e.g., when measured as described above).

[0108] 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.

[0109] In some embodiments, for example, compared to using existing methods to produce multimeric polypeptides, the methods of the present disclosure result in a higher yield of multimeric polypeptides in eukaryotic host cells. In some embodiments, for example, compared to using existing methods to produce multimeric polypeptides, the methods of the present disclosure result in a higher yield of multimeric polypeptides in eukaryotic host cells 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% (e.g., correctly assembled multimeric polypeptides, such as higher levels of bispecific antibodies). For example, expression can be compared to expression in which one or more, two or more, three or more, or four polynucleotides encoding a polypeptide chain of a multimeric polypeptide comprise an open reading frame operably linked to a native or unmodified translation start sequence, or in which each polynucleotide encoding a polypeptide chain of a multimeric polypeptide comprises the same translation start sequence.

[0110] In some embodiments, for example, compared to using existing methods to produce multimeric polypeptides, the method of the present disclosure results in fewer mispaired by-products of multimeric polypeptides in eukaryotic host cells. In some embodiments, for example, compared to using existing methods to produce multimeric polypeptides, the method of the present disclosure 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% or at least 100% less mispaired by-products of multimeric polypeptides in eukaryotic host cells. Due to fewer impurities (e.g., by-products of incorrect ventilation) in the cell culture supernatant, the method of the present disclosure is conducive to downstream purification in manufacturing. In some embodiments, the yield can be compared to the yield in which one or more, two or more, three or more or four polynucleotides of the polypeptide chain encoding the multimeric polypeptide comprise an open reading frame operably connected to a natural or unmodified translation initiation sequence, or the yield in which each polynucleotide of the polypeptide chain encoding the multimeric polypeptide comprises the same translation initiation sequence. In some embodiments, the amount of the by-product of mispairing refers to the amount of one or more specific by-products of mispairing. In some embodiments, the amount of the by-product of mispairing refers to the total amount of all by-products of mispairing.

[0111] In some embodiments, the methods of the present disclosure can be used to express the four subunits of the multimeric polypeptide of the present disclosure with a chain ratio of 1:1:1:1. However, the methods described herein can also be used to regulate the expression of the four subunits of the multimeric polypeptide of the present disclosure with other desired chain ratios, for example, to improve the expression and / or assembly of correctly paired multimeric polypeptides. For example, it has been well demonstrated that antibody light chains can serve as molecular chaperones for antibody heavy chains (see, for example, Lee, YK, et al. (1999) Mol. Biol. Cell 10: 2209-2219), and having higher light chain expression during production may be an advantage. Therefore, in some embodiments, the methods of the present disclosure are used to increase the expression of one or two light chains of a bispecific antibody relative to the expression of the heavy chain. In addition, the data provided herein show that, in some cases, a higher proportion of correctly assembled bispecific antibodies can be obtained by manipulating the ratio of light chain 1: light chain 2. Therefore, 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 at a chain ratio of 1:1:1:1, or another chain ratio, depending on the optimal method for production of the bispecific antibody, which can be determined as described herein.

[0112] 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 comprise the 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 the open reading frame encoding the polypeptide chain of the first subunit (e.g., the more weakly expressed subunit of the multimeric polypeptide) comprise a sequence selected from the group consisting of: SEQ ID NO: 8-10. In some embodiments, one or both translation initiation sequences operably linked to the open reading frame encoding the polypeptide chain of the second subunit (e.g., the more strongly expressed subunit of the multimeric polypeptide) comprise a sequence selected from the group consisting of: SEQ ID NO: 2, 3, and 11.

[0113] Exemplary translation initiation sequences are provided in Table 2. The relative strengths of the exemplary translation initiation sequences are Fig.10 Available in.

[0114] Table 2. Translation start sequences.

[0115]

[0116] 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 a host cell. In some embodiments, the reference sequence comprises a polynucleotide sequence of SEQ ID NO: 2 or 3. Exemplary assays suitable for determining the relative strength of a translation initiation sequence are described and illustrated herein (see, e.g., Examples 1-3). In some embodiments, the strength of a translation initiation sequence is determined by expressing (e.g., after transient transfection) a translation initiation sequence operably linked to an open reading frame (e.g., encoding a polynucleotide, subunit or multimeric polypeptide of the present disclosure, such as an antibody or half antibody) in a host cell, and measuring the yield of the polypeptide product. In some embodiments, the production level (e.g., product titer) of the polypeptide product is compared to a reference, such as the production level of the same product in the same host cell type, wherein the open reading frame is operably linked to a reference translation initiation sequence, such as a wild-type Kozak sequence active in the host cell. The relative strength of an exemplary translation initiation sequence is measured in Fig.10For example, transient or stable transfectants can be cultured and the production of multimeric polypeptides can be determined. Fed-batch or perfusion cultures can be cultured and product titers can be determined in the cell culture medium or on the cell surface. For surface expression, cells can be stained with antibodies to detect the product and analyzed, for example, by flow cytometry. Product quality and / or purity can be analyzed, for example, by electrophoresis and / or mass spectrometry.

[0117] In some embodiments, the open reading frame of the first polypeptide chain of the first subunit (e.g., the more weakly 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 more weakly 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 more weakly 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 first polypeptide chain of the first subunit (e.g., the more weakly expressed subunit) is operably linked to a translation initiation sequence comprising SEQ ID NO: 9, the open reading frame of the second polypeptide chain of the polypeptide chain of the first subunit (e.g., the more weakly expressed subunit) is operably linked to a translation initiation sequence comprising SEQ ID NO: 9, the open reading frame of the first polypeptide chain of the second subunit (e.g., the more strongly expressed subunit) is operably linked to a 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 more strongly expressed subunit) is operably linked to a 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 a half antibody. 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 first half antibody heavy chain is operably linked to a translation start sequence comprising SEQ ID NO:9, the open reading frame encoding the first half antibody light chain is operably linked to a translation start sequence comprising SEQ ID NO:9, the open reading frame encoding the second half antibody heavy chain is operably linked to a translation start sequence comprising SEQ ID NO:3, and the open reading frame encoding the second half antibody light chain is operably linked to a translation start sequence comprising SEQ ID NO:11.

[0118] Promoter

[0119] In certain embodiments, the translation initiation sequence and / or open reading frame of the present disclosure are operably connected to a promoter. In certain embodiments, each of the first polynucleotide, the second polynucleotide, the third polynucleotide and the fourth polynucleotide is operably connected to a promoter. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that can regulate its expression. Prokaryotic promoters are generally divided into two categories, inducible and constitutive. An inducible promoter is a promoter that responds to changes in culture conditions, such as the presence or absence of nutrients or changes in temperature, and initiates an increase in the transcription level of a cistron under its control.

[0120] 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 polypeptide chain and the second polypeptide chain of the subunit are operably connected to the same promoter. In some embodiments, the polynucleotides encoding the first polypeptide chain and the second polypeptide chain of the subunit are operably connected to different promoters.

[0121] A large number of promoters recognized by various potential host cells are well known. Remove the promoter from the source DNA by restriction endonuclease digestion and insert the separated promoter sequence into the vector of the present invention, and the promoter selected can be operably connected with the cistron DNA of encoding for example light chain or heavy chain. Natural promoter sequence and many heterologous promoters can be used for guiding the amplification and / or expression of target gene. In certain embodiments, heterologous promoters are used because they usually allow larger transcription and higher yield of the target gene expressed compared with the natural target polypeptide promoter.

[0122] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and operably connected to the desired Fc polypeptide (e.g., antibody) nucleic acid. The promoter sequence of eukaryotic organisms is known. In fact, all eukaryotic genes have an AT-rich region, located at the position of about 25 to 30 bases upstream of the transcription start site. Another sequence of 70 to 80 bases found upstream of many gene transcriptions is the CNCAAT region, in which N can be any nucleotide. The 3' end of most eukaryotic genes is an AATAAA sequence, which may be a signal that the poly A tail is added to the 3' end of the coding sequence. All these sequences are suitably inserted into the eukaryotic expression vector.

[0123] For the production of Fc-containing polypeptides (e.g., antibodies), antibody transcription from a vector in a mammalian host cell is controlled, for example, by a promoter obtained from a viral genome, 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 a heterologous mammalian promoter, e.g., an actin promoter or an immunoglobulin promoter, or from a heat shock promoter, provided that such promoters are compatible with the host cell system. In some embodiments, the promoter is a CMV promoter.

[0124] The early and late promoters of SV40 virus are conveniently available in the form of SV40 restriction fragments, which also contain the replication origin of SV40 virus. The immediate early promoter of human cytomegalovirus can be conveniently obtained as Hind 111E restriction fragment. U.S. Patent 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. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) about the expression of human beta-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, Rous Sarcoma Virus long terminal repeats can be used as promoters.

[0125] By inserting enhancer sequences into vectors, the transcription of DNA encoding antigen-binding polypeptides (e.g., antibodies) can be increased in higher eukaryotes. It is now known that many enhancer sequences come from mammalian genes (e.g., globulin, elastase, albumin, alpha-fetoprotein, and insulin genes). Similarly, enhancers from eukaryotic cell viruses can be used. Examples include SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers on the rear side of the replication origin (bp 100-270). See also Yaniv, Nature 297: 17-18 (1982) for a description of elements that enhance eukaryotic promoter activation. Enhancers can be spliced ​​into vectors at the 5' or 3' position of the antibody polypeptide coding sequence, provided that enhancement is achieved, but are generally located at the 5' site of the promoter.

[0126] Multispecific multimeric peptides

[0127] Certain aspects of the present disclosure, for example, relate to a multimeric polypeptide 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 present disclosure are non-native to a host cell.

[0128] In certain embodiments, the subunit of the present disclosure is a monomer of a heterodimer. As used herein, a heterodimer may refer to any polypeptide complex comprising two distinct polypeptides or polypeptide complexes that are operably connected. A non-limiting example of a heterodimer is a bispecific or bivalent antibody consisting of two distinct antibody monomers (i.e., a light chain-heavy chain pair that is operably connected). In this example, the folding and assembly of the first heavy chain-light chain pair that recognizes the first antigen produces a first antibody monomer. The folding and assembly of the second heavy chain-light chain pair that recognizes the 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 a heterodimer. For more detailed information on illustrative examples of heterodimeric antibody formation, see Ridgway JBB et al. 1996 Protein Eng. 9 (7): 617-621.

[0129] In some embodiments, the multimeric polypeptides or subunits disclosed herein are secretory proteins. As used herein, secretory proteins may refer to any protein secreted by a host cell into the periplasm of a host cell or into the extracellular environment. A secretory protein may be a protein endogenously secreted by a host cell, or a secretory protein may be a protein that is not endogenously secreted by a 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 may direct the polypeptide to a secretory pathway for secretion. Many signal sequences are known in the art and may be used to promote the secretion of secretory proteins or to allow secretion of proteins that are not naturally secreted by host cells, see, e.g., Picken et al., Infect. Immun. 42: 269-275 (1983); Simmons and Yansura, Nature Biotechnology 14: 629-634 (1996); and Humphreys DP et al. 2000 Protein Expr. Purif. 20 (2): 252. A non-limiting example of a signal sequence is the heat stable enterotoxin II (STII) signal sequence.

[0130] In some embodiments, the polypeptide chains of the subunits of the present disclosure are connected to each other by at least one disulfide bond. In some embodiments, the subunits of the multimeric polypeptides of the present disclosure are connected to each other by at least one disulfide bond. A disulfide bond may refer to any covalent bond connecting two thiol groups. Disulfide bonds in polypeptides are usually formed between thiol groups of cysteine ​​residues. It is known in the art that polypeptide disulfide bonds are important for the folding and assembly of many polypeptides such as double-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 (i.e., intermolecular or interchain disulfide bonds) between cysteine ​​residues found on separate polypeptide chains.

[0131] It is known in the art that disulfide bonds are 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 specific 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 (for a more detailed description, see Liu H and May K 2012 MAbs. 4 (1): 17). It is also known that IgM (see, e.g., Wiersma EJ and Shulman MJ 1995 J. Immunol. 154(10):5265), IgE (see, e.g., Helm BA et al. 1991 Eur. J. Immunol. 21(6):1543), IgA (see, e.g., Chintalacharuvu KR et al. 2002 J. Immunol. 169(9):5072) and IgD (see, e.g., Shin SU et al. 1992 Hum. Antibodies Hybridomas 3(2):65) form disulfide bonds during folding and assembly.

[0132] In some embodiments, the multimeric polypeptides of the present disclosure include multispecific antigen-binding proteins. In some embodiments, multispecific antigen-binding proteins bind to two or more epitopes of one, two or more polypeptides or other antigens. Multispecific antibodies have binding specificity to at least two different epitopes, wherein these epitopes are generally from different antigens. Although such molecules normally only bind to two different epitopes (i.e., bispecific antibodies, BsAbs), when used herein, this statement encompasses antibodies with other specificities, such as trispecific antibodies. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0133] In some embodiments, the multimeric polypeptides of the present disclosure are antibodies. In some embodiments, the subunits of the present disclosure are half antibodies. In some embodiments, the antibodies provided herein are chimeric antibodies, human antibodies or humanized antibodies. Antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments herein. As described below, antibodies are prepared using techniques that can be used to produce antibodies in the art, and exemplary methods thereof are described in more detail in the following sections. Those skilled in the art will recognize that many of the methods described below can be applied to multimeric polypeptides other than antibodies.

[0134] In some embodiments, the subunit of the present 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 that are 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.

[0135] In some embodiments, the multimeric polypeptide specifically binds to one or more target antigens. In some embodiments, the multimeric polypeptide or subunit of the present disclosure can specifically bind to an antigen. As used herein, the term "binding", "specifically binding" or "having specificity" refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which determines the presence of a 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 to the target with greater affinity, avidity, easier and / or longer duration than it binds to other targets. In one embodiment, the degree of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to an antigen, for example, as measured by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM or ≤0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In another embodiment, specific binding may include but does not require exclusive binding. In certain embodiments, the dissociation constant (Kd) of the antibodies provided herein is ≤1 μM, ≤150 nM, ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0136] In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) with the Fab form of the antibody of interest and its antigen as described in the following assay. The Kd is measured by using a minimum concentration ( 125 The solution binding affinity of Fab for antigen is measured by equilibrating Fab with 1) labeled antigen and then capturing bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, 5 μg / ml capture anti-Fab antibody (Cappel Labs) was used to coat the plate in 50 mM sodium carbonate (pH 9.6). Multiwell plates (Thermo Scientific) were blocked overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with a serial dilution of the target Fab. The target Fab is then incubated overnight; however, the incubation can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate to incubate at room temperature (e.g., one hour). The solution is then removed and incubated with 0.1% polysorbate in PBS The plates were washed eight times. When the plates were dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and in TOPCOUNT TM The plates were counted for tens of minutes on a gamma counter (Packard).The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for use in the competitive binding assay.

[0137] According to another embodiment, at 25°C, using an immobilized antigen CM5 chip, at approximately 10 response units (RU), -2000 or -3000 (BIAcore, Inc., Piscataway, NJ), Kd was 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. Antigens were diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of coupled protein. After injection of antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, 5% paraformaldehyde (PVA) in a solution containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant (PBST) in PBS. A simple one-to-one Langmuir binding model ( The association rate (kon) and dissociation rate (koff) are calculated using the ELISA software (Aviv Instruments, Inc., New York, NY) version 3.2. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y. et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 106 M-1 s-1 as determined by surface plasmon resonance as described above, the association rate can be determined by using a fluorescence quenching technique, i.e., in a spectrometer such as a spectrophotometer equipped with a stop flow device (Aviv Instruments) or a 8000 series SLM-AMINCO TM The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C was measured in a spectrophotometer (ThermoSpectronic) using a stirred cuvette in the presence of increasing concentrations of antigen.

[0138] In some embodiments, the multimeric polypeptides of the present disclosure comprise bispecific antibodies. In some embodiments, the first polypeptide chain and the third polypeptide chain are antibody heavy chains, and the second polypeptide chain and the fourth polypeptide chain are antibody light chains. In some embodiments, the first subunit is a first half antibody that binds to a first antigen, and the second subunit is a second half antibody that binds to 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 represent different epitopes of the same target. Other antibodies, such as trispecific antibodies or tetravalent antibodies, are also contemplated.

[0139] Methods for preparing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, in which 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 usually completed by an affinity chromatography step is quite cumbersome, and the product yield is low. Similar processes are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0140] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0141] In certain embodiments, the present disclosure contemplates antibody variants that have some but not all effector functions, making them desirable candidates for applications 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 can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells that mediate ADCC NK cells express only Fc(RIII), while monocytes express Fc(RI, Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Other non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)). 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA; and Nonradioactive cytotoxicity assays (Promega, 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 target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0142] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0143] Certain antibody variants with improved or decreased binding to FcRs 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).)

[0144] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) in the Fc region. In an exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

[0145] In some embodiments, changes are made in the Fc region that result in altered (ie, improved or reduced) CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0146] 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 US 2005 / 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 with 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, such as substitutions at Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature 322: 738-40 (1988) for other examples of Fc region variants; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351.

[0147] The antibodies of the present disclosure may be further modified to include additional non-proteinaceous moieties that are known in the art and readily available.In certain embodiments, the moiety suitable for derivatization of the antibody is a water soluble polymer.

[0148] Pestle-in-Mortar Method

[0149] One method known in the art for preparing bispecific antibodies is the "knobs-into-holes" or "protuberance-into-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 the other immunoglobulin polypeptide, thereby associating the two immunoglobulin polypeptides. These interfaces can be engineered so that a "knob" or "protrusion" (these terms are used interchangeably herein) located on the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms are used interchangeably herein) located on the interface of the other immunoglobulin polypeptide. In some embodiments, the hole has the same or similar dimensions as the knob and is appropriately positioned so that when the two interfaces interact, the knob 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 heteromultimers and facilitates the formation of heteromultimers more than other species (e.g., homomultimers). In some embodiments, the method can be used to promote heteromultimerization of two different immunoglobulin polypeptides to generate a bispecific antibody comprising two immunoglobulin polypeptides with binding specificities for different epitopes.

[0150] In some embodiments, a knob can be constructed by replacing a small amino acid side chain with a larger side chain. In some embodiments, a hole can be constructed by replacing a large amino acid side chain with a smaller side chain. The knob or hole may be present in the original interface, or may be introduced synthetically. For example, a knob or hole may be recombinantly introduced by changing 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 changing nucleic acid sequences may include standard molecular biology techniques well known in the art. The following table shows the side chain volumes of 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 import residue that forms the knob is a naturally occurring amino acid, and may 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 used to form the hole is a naturally occurring amino acid, and may include alanine, serine, threonine, and valine.

[0151] In some embodiments, the original residues for forming the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining the three-dimensional structure may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these embodiments, the CH3 / CH3 interface of human IgG1 involves sixteen residues on each domain located on four antiparallel β strands. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel β strands to minimize the risk of the knob being accommodated by the surrounding solvent rather than the compensating hole in the partner CH3 domain. In some embodiments, the mutations that form the corresponding knobs and holes in two immunoglobulin polypeptides correspond to one or more pairs provided in the following table.

[0152] In certain embodiments, the CH3 and / or CH2 domains of the antibodies of the present disclosure are from the IgG4 subtype. In some embodiments, the IgG4 CH3 and / or CH2 domains of the antibodies of the present disclosure may comprise one or more additional mutations, including but not limited to the S228P mutation (EU numbering).

[0153] Table 1b. Properties of amino acid residues

[0154]

[0155] a The molecular weight of the amino acid minus the molecular weight of water. From Handbook of Chemistry and Physics, 43 rd ed.Cleveland,Chemical Rubber Publishing Co.,1961.

[0156] b Values ​​from AA. Values ​​from Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107-123, 1972.

[0157] c Values ​​from C. Chothia, J. Mol. Biol. 105: 1-14, 1975. Figure 6-2 0 defines the accessible surface area.

[0158] In some embodiments, the polypeptide chain of one subunit comprises at least one hole mutation and the polypeptide chain of the other 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.

[0159] In some embodiments, the CH3 domain of an antibody of the present disclosure is from IgG (e.g., IgG1 subtype, IgG2 subtype, IgG2A subtype, IgG2B subtype, IgG3 subtype, or 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.

[0160] Table 3. Exemplary sets of corresponding knob and hole mutations

[0161] CH3 of the first immunoglobulin CH3 of the 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

[0162] In some embodiments, the immunoglobulin polypeptide comprises a 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 the second immunoglobulin polypeptide comprises 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 a row of Table 3, and another subunit of the multimeric polypeptide comprises a mutation listed in the right column of the same row of Table 3. As a non-limiting example of a knob and hole pair, in some embodiments, the 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 T366S, L368A, and Y407V mutations. In some embodiments, 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 IgG1. In some embodiments, at least one hole mutation is selected from the group consisting of: F405A, Y407T, Y407A, T366S, L368A, Y407V, and T394S, numbered according to the EU index based on human IgG1. In certain embodiments, the knob mutation comprises a T366W substitution, and the hole mutation comprises a T366S, L368A, and Y407V substitution, numbered according to the EU index based on human IgG1. Additional 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. 1996 Protein Eng. 9(7): 617-621 and www.imgt.org / IMGTbiotechnology / Knobs-into-holes_IgG.html.

[0163] 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.

[0164] Bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). By gene fusion, leucine zipper peptides from Fos protein and Jun protein are connected to the Fab' portion of two different antibodies. Antibody homodimers are reduced to form monomers in the hinge region and then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. The fragments contain a light chain variable domain (V H )'s heavy chain variable domain (V L ), the linker is too short to allow pairing between the two domains on the same chain. H and V L The domain is forced to mate with the complementary V L and V H The domains pair to form two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152: 5368 (1994).

[0165] Another technique for making bispecific antibody fragments is “bispecific T cell engagers” or Methods (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 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) domains, the length of the linker being sufficient to allow intramolecular association between the two domains. The single polypeptide further comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes can be specific for different cell types, so that when each scFv engages with its cognate epitope, cells of two different cell types are brought into close proximity or bound together. A specific embodiment of the method includes an scFv that recognizes a cell surface antigen expressed by an immune cell, such as a CD3 polypeptide on a T cell, which is linked to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0166] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147:60 (1991).

[0167] In some embodiments, the antibodies described herein are single domain antibodies. A single domain antibody is a single polypeptide chain comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516B1). In one embodiment, a single domain antibody consists of all or part of the heavy chain variable domain of an antibody.

[0168] Mutations that promote alternative light-heavy chain pairing

[0169] In some embodiments, the multimeric polypeptide of the present disclosure includes one or more mutations that promote the selective association of antibody light chain with antibody heavy chain. Exemplary amino acid substitutions that can be used in the methods and cells described herein to promote the selective association between antibody heavy chain and antibody light chain can be found in WO2016 / 172485. In some embodiments, the antibody heavy chain of the subunit includes one or more mutations that promote selective association, and the antibody light chain of the subunit includes one or more mutations that promote the selective association of heavy chain combination. In some embodiments, both half antibodies of the bispecific antibody include one or more mutations that promote the selective association of antibody light chain with antibody heavy chain (for example, a heavy chain has a positively charged mutation, and its corresponding light chain has a negatively charged mutation, and the other heavy chain has a negatively charged mutation, and its corresponding light chain has a positively charged mutation, and vice versa). In some embodiments, only one half antibody of the bispecific antibody includes one or more mutations that promote the selective association of antibody light chain with antibody heavy chain (for example, a heavy chain has a positively charged mutation, and its corresponding light chain has a negatively charged mutation, and vice versa).

[0170] 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 (based on EU index numbering). In some embodiments, S183 substitutions are selected from the group consisting of the following items: S183A, S183T, S183V, S183Y, S183F, S183H, S183N, S183D, S183E, S183R and S183K. In some embodiments, the V133 substitutions are selected from the sequence of the group consisting of the following items: 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 results in a negatively charged residue, and wherein the amino acid substitution at V133 results in a positively charged residue.

[0171] Mutations that promote selective association of heavy chain / 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) may include a set of knob and hole mutations, and one or more mutations that promote selective association of an antibody light chain with an antibody heavy chain. Advantageously, this promotes correct heavy chain / light chain association in one or both half antibodies, as well as correct assembly of the bispecific antibody (e.g., as opposed to forming dimers of each half antibody), resulting in fewer mispaired byproducts. In some embodiments, a polypeptide chain of the present disclosure (e.g., an antibody heavy chain of a bispecific antibody or half antibody) includes one or more knob or hole mutations, and one or more mutations that promote selective association of an antibody light chain with an antibody heavy chain. For example, in some embodiments, the first antibody heavy chain of the bispecific antibody comprises a mutation that generates a negative charge (e.g., an S183E mutation) and a hole mutation, the first antibody light chain comprises a mutation that generates a positive charge (e.g., a V133K mutation), the second anti-heavy chain of the bispecific antibody comprises a mutation that generates a positive charge (e.g., an S183K mutation) and a knob mutation, and the second anti-light chain comprises a mutation that generates a negative charge (e.g., a V133E mutation).

[0172] Multiple multimeric polypeptides and combinations of multimeric polypeptides

[0173] Further provided herein are multimeric polypeptides produced according to the methods described herein, and compositions of various multimeric polypeptides or multimeric polypeptides. Advantageously, the disclosure proves that the methods described herein allow improved production of desired multimeric polypeptides with higher yields and / or lower amounts of impurities, such as the production of multimeric polypeptides of incorrectly paired byproducts. Therefore, in some embodiments, compared with various and compositions produced by prior art (e.g., compared with an expression system in which one or more, two or more, three or more or four polynucleotides of a polypeptide chain encoding a multimeric polypeptide include an open reading frame operably connected to a natural or unmodified translation initiation sequence, or compared with the expression in which each polynucleotide of a polypeptide chain encoding a multimeric polypeptide includes the same translation initiation sequence), various multimeric polypeptides and multimeric polypeptide compositions produced by the methods disclosed herein include fewer incorrectly paired byproducts. In some embodiments, a plurality of multimeric polypeptides and multimeric polypeptide compositions produced by the methods of the present disclosure comprise a higher ratio of multimeric polypeptides and improperly paired byproducts than a plurality and compositions produced by prior art techniques (e.g., compared to an expression system in which one or more, two or more, three or more, or four polynucleotides encoding a polypeptide chain of a multimeric polypeptide comprise an open reading frame operably linked to a native or unmodified translation initiation sequence, or compared to expression in which each polynucleotide encoding a polypeptide chain of a multimeric polypeptide comprises the same translation initiation sequence). In some embodiments, all polypeptide chains of a multimeric polypeptide are produced by a single eukaryotic (e.g., mammalian) cell. In some embodiments, the multimeric polypeptide is a bispecific antibody.

[0174] Multimeric protein production

[0175] 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 modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0176] Host cells for producing the desired multimeric polypeptides or subunits thereof of the present disclosure can be cultured in a variety of culture media. Commercially available culture 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 host cells. In addition, any culture medium described 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; WO 87 / 00195 or U.S. Reissued Patent 30,985 can be used as a culture medium for 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 TM Drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, and the like are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.

[0177] Described herein is the method for reclaiming multimeric polypeptide of the present disclosure.When using recombinant technology, multimeric polypeptide of the present disclosure or its subunit can be produced in cell, or directly secreted in culture medium.If polypeptide is produced in cell, then as the first step, the particulate debris of host cell or cleavage fragment is removed, for example, by centrifugation or ultrafiltration.When polypeptide is secreted in culture medium, usually first use commercially available protein concentration filter, for example Amicon or MilliporePellicon ultrafiltration unit, concentrate the supernatant from this type of expression system.Protease inhibitors such as PMSF can be included in the aforementioned any step to inhibit proteolysis and antibiotics can be included to prevent the growth of foreign contaminants.

[0178] The polypeptide composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is 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 γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is mostly agarose, but other matrices may also 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 TM Resins (JT Baker, Phillipsburg, 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, SEPHAROSE on anion or cation exchange resins such as polyaspartic acid columns, TM Chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody to be recovered.

[0179] After any preliminary purification step, the mixture comprising the target polypeptide and contaminants can 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 0-0.25 M salt). The production of antigen-binding polypeptides may alternatively or additionally (for any of the aforementioned specific methods) comprise dialysis of a solution comprising the polypeptide mixture.

[0180] In one embodiment, the multimeric polypeptide of the present disclosure or its subunit produced in this article is further purified to obtain a substantially homogeneous preparation for further determination and use. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on immunoaffinity column or ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica gel or on cation exchange resin (such as DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration, for example, using Sephadex G-75.

[0181] 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 can be recovered from the solid phase of the column by eluting into a solution containing a chaotropic agent or a 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.

[0182] In one embodiment, for example, protein A immobilized on a solid phase is used for immunoaffinity purification of the antigen-binding polypeptides of the present invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureus that binds with high affinity to the Fc region of the antigen-binding polypeptide. Lindmark et al. (1983) J. Immunol. Meth. 62: 1-13. The solid phase to which protein A is immobilized 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 nonspecific adhesion of contaminants.

[0183] As the first step of purification, the preparation derived from the above cell culture is applied to a protein A-immobilized solid phase to allow the target antigen-binding polypeptide to specifically bind to protein A. The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. The antigen-binding polypeptide (e.g., antibody) is recovered from the solid phase by elution.

[0184] Screening methods

[0185] Further provided herein is a method for identifying a combination of translation initiation sequences for expressing multimeric polypeptides in eukaryotic host cells of the present disclosure. In certain embodiments, the method includes providing a library comprising a variety of eukaryotic host cells, cultivating a eukaryotic host cell library under conditions suitable for expression of a variety of eukaryotic host cell multimeric polypeptides, measuring the amount of multimeric polypeptides produced by a clone of a single eukaryotic host cell in a variety or a single eukaryotic host cell in a variety, and identifying one or more clones of a single eukaryotic host cell in a variety or a single eukaryotic host cell in a variety that produce multimeric polypeptides, a first translation initiation sequence, a second translation initiation sequence, a third translation initiation sequence, and a fourth translation initiation sequence. Exemplary such methods are described and illustrated below.

[0186] In some embodiments, the 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 a polynucleotide set necessary for encoding each polypeptide chain of a subunit or multimeric polypeptide as described herein. For example, in some embodiments, a plurality of or all host cells in the library comprise a first polynucleotide comprising a first translation initiation sequence operably connected 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 connected 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 connected 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 connected to a fourth open reading frame encoding a fourth polypeptide chain of a multimeric polypeptide of the present disclosure. In some embodiments, a plurality of host cells or libraries comprise a combination of a plurality of first translation initiation sequences, a second translation initiation sequence, a third translation initiation sequence, and a fourth translation initiation sequence. Therefore, screening libraries can be used to identify combinations of translation initiation sequences that produce a desired property (e.g., expression of a multimeric polypeptide, such as high level expression or low level of incorrectly paired byproducts). The present disclosure demonstrates that expression of a multimeric polypeptide can be improved by modulating the strength of a translation initiation sequence operably linked to a polynucleotide encoding one or more constituent polypeptide chains.

[0187] 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).

[0188] The various methods described herein can be used to measure the amount of multimeric polypeptides produced by one or more host cells or clonal populations of host cells disclosed herein. For example, transient or stable transfectants can be cultured, and the production of multimeric polypeptides can be determined. Culture feed batch or perfusion cultures can determine the product titer in the cell culture medium or on the cell surface. For surface expression, cells can be stained with antibodies to detect the product and analyzed, for example, by flow cytometry. Product quality and / or purity can be analyzed by, for example, electrophoresis and / or mass spectrometry.

[0189] Antibodies of the present 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 to obtain antibodies having desired binding characteristics, such as the methods described in Example 3. Additional methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described in, for example, McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); 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):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0190] In certain phage display methods, the repertoire of VH and VL genes is cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, 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 immunogens without the need to construct hybridomas. Alternatively, all natural repertoires (e.g., all natural repertoires from humans) can be cloned to provide a single source of antibodies to a wide range of non-self-antigens and self-antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared by cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode highly variable CDR3 regions and perform 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. Patent 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.

[0191] III. Cells

[0192] Also provided herein are recombinant eukaryotic host cells that can be used to produce multimeric polypeptides, for example, according to the methods described in Part II. In some embodiments, the host cell described herein comprises a first polynucleotide comprising a first translation initiation sequence operably connected 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 connected 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 connected 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 connected 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 separately in a recombinant eukaryotic host cell, the first subunit is expressed at a level lower than that of the second subunit, and one or both of the first translation initiation sequence and the second translation initiation sequence are 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-natural to a recombinant eukaryotic host cell. In some embodiments, the recombinant eukaryotic host cell is an isolated recombinant eukaryotic host cell.

[0193] In some embodiments, the host cells of the present disclosure are eukaryotic cells. In some embodiments, the host cells of the present disclosure are mammalian cells.

[0194] Suitable host cells include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (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 supporting cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).

[0195] 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 a CHO-K1 cell line (see, e.g., ATCC catalog number CCL-61 TM and Lewis, NE et al. (2013) Nat. Biotechnol. 31:759-765). 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, LA (1980) Proc. Natl. Acad. Sci. 77:4216-4220).

[0196] 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 the multimeric polypeptide (e.g., bispecific antibody), as in the case of post-production assembly of two subunits (e.g., half antibodies).

[0197] Any of the promoters and / or translation initiation sequences described herein may be used in the host cells of the present disclosure.

[0198] In some embodiments, one or more polynucleotides encoding a polypeptide chain of a subunit of a multimeric polypeptide of the present invention are present on a chromosome of a host cell of the present invention (e.g., by integration). In some embodiments, each polynucleotide encoding a polypeptide chain of a subunit of a multimeric polypeptide of the present invention is present on a chromosome of a host cell of the present invention (e.g., by integration). In some embodiments, each polynucleotide encoding a polypeptide chain of a subunit of a multimeric polypeptide of the present invention is present on the same chromosome or chromosomal locus of a host cell of the present invention (e.g., by integration).

[0199] The polynucleotides of the present disclosure are stably integrated or stably transfected into the host cell chromosome to provide a stable production cell line for producing 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., "landing pad" method); see, e.g., Zhao, M. et al. (2018) Appl. Microbiol. Biotechnol. 102: 6105-6117; Lee, JS et al. (2015) Sci. Rep. 5: 8572; and Gaidukov, L. et al. (2018) Nucleic Acids Res. 46: 4072-4086.

[0200] In some embodiments, one or more polynucleotides encoding the polypeptide chains of the subunits of the multimeric polypeptides of the present invention are maintained as extrachromosomal polynucleotides in the host cell. In some embodiments, each of the polynucleotides encoding the polypeptide chains of the subunits of the multimeric polypeptides of the present invention is maintained as an extrachromosomal polynucleotide in the host cell. In some embodiments, one or more polynucleotides encoding the polypeptide chains of the subunits of the multimeric polypeptides of the present invention are present in a vector (e.g., an expression vector). In some embodiments, each of the polynucleotides encoding the polypeptide chains of the subunits of the multimeric polypeptides of the present invention is present in one or more vectors (e.g., an expression vector).

[0201] Vector components generally 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.

[0202] The vector for eukaryotic host cells may also include a signal sequence or other polypeptides with a specific cleavage site at the N-terminus of the target mature protein or polypeptide. Preferably, the selected heterologous signal sequence is a heterologous signal sequence that is recognized and processed (i.e., cut by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences and viral secretory leader sequences, such as herpes simplex gD signals, may be obtained. The DNA in this precursor region is connected in frame to a DNA encoding a desired antigen-binding polypeptide (e.g., an antibody).

[0203] Typically, mammalian expression vectors do not require an origin of replication component. For example, the SV40 origin can often be used, but only because it contains the early promoter.

[0204] Any promoter and / or translation initiation sequence described herein can be used in the vectors of the present disclosure.

[0205] Expression and cloning vectors may contain selection genes, also called selectable markers. 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 where relevant, or (c) supply critical nutrients that are not available in complex media.

[0206] An example of a selection scheme utilizes a drug to retard the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and thus survive the selection scheme. Examples of this type of dominant selection use neomycin, mycophenolic acid, and hygromycin.

[0207] Another example of suitable selectable markers for mammalian cells are those markers that can identify cells capable of taking up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and metallothionein-II (preferably primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, etc.).

[0208] For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line (e.g., ATCC CRL-9096) deficient in DHFR activity.

[0209] Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding polypeptide chains of the disclosure, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by growing the cells in a medium containing a selection agent for the selectable marker (such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418). See, e.g., U.S. Pat. No. 4,965,199.

[0210] The expression vectors used in eukaryotic host cells also typically contain sequences necessary for terminating transcription and stabilizing mRNA. Such sequences are typically available from the 5' (sometimes 3') untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0211] Host cells are transformed with the above-described expression or cloning vectors to produce the desired polypeptide (eg, multimeric polypeptide), and cultured in a conventional nutrient medium modified appropriately to induce promoters, select transformants, or amplify genes encoding the desired sequences.

[0212] IV. Kits and Articles

[0213] Further provided herein are useful kits or articles of manufacture, eg, for expression of multimeric polypeptides.

[0214] In some embodiments, the kit comprises a set of polynucleotides encoding each polypeptide chain and / or subunit of a multimeric polypeptide. For example, in some embodiments, the kit described herein comprises a first polynucleotide comprising a first translation initiation sequence operably connected 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 connected 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 connected 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 connected 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 connected to their respective open reading frames because their respective open reading frames are present in the naturally occurring host cell genome.

[0215] In some embodiments, each polynucleotide in the kits of the present disclosure is part of one or more expression vectors of the present disclosure.

[0216] In some embodiments, each polynucleotide in the kit of the present disclosure is operably connected to a promoter of the present disclosure. For example, in some embodiments, each polynucleotide is operably connected to different promoters. In some embodiments, two or more polynucleotides are operably connected to the same promoter. For example, if the multimeric polypeptide is a bispecific antibody, the polynucleotides encoding the open reading frames of the heavy chain and light chain of each half antibody can be operably connected to the same promoter. In some embodiments, "the same promoter" refers to the same physical polynucleotide. In some embodiments, "the same promoter" refers to physically different polynucleotides that share the same promoter sequence.

[0217] 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.

[0218] Examples

[0219] The present disclosure will be more fully understood with reference to the following examples. However, examples should not be construed as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are only for illustrative purposes, and various modifications or changes thereof will be suggested to those skilled in the art, and will be included in the spirit and limits of the present application and within the scope of the appended claims.

[0220] Example 1: Modulation of protein production using Kozak sequence variants in transient transfection of mammalian cells

[0221] At present, there are some systematic methods to accurately control the translation level of recombinant proteins in mammalian cells. The region where translation initiates polypeptide synthesis is called the translation initiation site (TIS). The translation initiation site (TIS) consists of a start codon and its adjacent bases. In eukaryotes, translation initiation generally follows a scanning mechanism model (Kozak M. Cell. 1978; 15 (4): 1109-23), which assumes that the ribosome pre-initiation complex composed of a small 40S ribosomal subunit, Met-tRNA, eIF2-GTP, eIF1, eIF1A, eIF3 and eIF5 binds to the 5' end of the mRNA and moves linearly in the 3' direction to find the start codon. After positioning the small 40S ribosomal subunit in the start codon, the initiation factors dissociate, the large subunit combines with the small 40S ribosomal subunit to form a ribosome complex, and translation begins (Nanda JS, Saini AK, Munoz AM, Hinnebusch AG, Lorsch JR. J Biol Chem. 2013; 288 (8): 5316-29; Pestova TV, Kolupaeva VG Genes Dev. 2002; 16 (22): 2906-22). Initiation is not always limited to the start codon (AUG) closest to the 5' end. If the first AUG codon appears in the 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. Therefore, cells can control the level of protein translation by regulating TIS so that the sequence around the start codon plays a major role in the initiation and subsequent enhancement of translation efficiency (Kozak MJ Biol Chem. 1991; 266(30): 19867-70; Kozak M. J Cell Biol. 1991; 115(4): 887-903; Sonenberg N, Hinnebusch AG. Cell. 2009; 136(4): 731-45; Ivanov IP, Loughran G, Sachs MS, Atkins JF. Proc Natl Acad Sci US A. 2010; 107(42): 18056-60).

[0222] Kozak has reported CCRCC AUGG (purine, R = A or G; start codon is underlined) is an efficient mammalian TIS (Kozak M. Nucleic Acids Res. 1981; 9(20): 5233-52). In this sequence, the purine at position -3 (3 nucleotides upstream of the AUG codon) is the 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 essential for optimal translation efficiency (Kozak M. EMBO J. 1997; 16(9): 2482-92). The Kozak consensus sequence varies 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 both higher (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 US A. 2013; 110(30): E2792-801) eukaryotes and are associated with the development of human diseases, including cancer and metabolic disorders (Sonenberg N, Hinnebusch AG. Cell. 2009; 136(4): 731-45; Mohan RA, van Engelen K, Stefanovic S, Barnett P, Ilgun A, Baars MJ, et al. Am J Med Genet A. 2014; 164A(11): 2732-8).

[0223] To investigate whether variants of the Kozak sequence could modulate protein production in industrial production cell lines, position -3 and the combination of positions -3, -2 and -1 upstream of the start codon of the ORF encoding the Fc fusion protein were altered.

[0224] method

[0225] Cell lines and cell culture

[0226] CHO cell lines were used for transient transfection analysis in plates and for cell lines producing constitutive antibodies by target integration. CHO cells were cultured in DMEM / F12-based medium in 125 mL shake flasks at 150 rpm, 37°C, and 5% CO2. 3x 10 5 The cells were passaged at a seeding density of 10 cells / ml.

[0227] Transient transfection

[0228] According to the manufacturer's suggestion (Invitrogen, Carlsbad, California), lipofectamine2000CD was used to transfect CHO cells with transient transfection plasmids. As an internal control, DNA of well-expressed antibodies and poorly expressed antibodies was also carried. In brief, DNA carrier (2 μg) and 10 μl lipofectamine were incubated at room temperature for 30 min in 500 μl culture medium to promote complex formation. The transfection complex was transferred to a 96-well plate (FalconR), which contained exponentially growing CHO cells in a culture medium based on DMEM / F12 and 5% DFBS, and the total working volume was about 2.5 ml. The transfected cells were cultured at 37 ° C, 5% CO2 and 80% humidity. After transfection for 24 h, the cell culture medium was replaced with a production culture medium, and then the cells were cultured at 33 ° C. Each transfection was performed with biological repetition. For antibody concentration determination, supernatant samples were collected from the culture 48 h after transfection, and repeated determination was performed by HTRF (homogeneous time-resolved FRET) determination. Homogeneous Time-Resolved FRET (HTRF) Assay

[0229] For HTRF assay, see Degorce, F., et al. (2009). Curr Chem Genomics 3:22-32.

[0230] Design and construction of Kozak sequence variants

[0231] Based on the nucleotide frequencies around the translation start site of vertebrates determined by Kozak (Kozak M. Nucleic Acids Res. 1987; 15(20):8125-48), variants with lower and intermediate frequencies at these positions were designed, such as Figure 1 As shown. DNA of Kozak sequence variants of Fc fusion protein A or B was synthesized by Genewiz, Inc. The variants were cloned into Genentech's transient transfection vector under the transcriptional control of the CMV promoter and ampicillin resistance marker. The sequences were verified using universal forward and reverse primers.

[0232] DNA library design

[0233] Fc fusion protein B was used as the backbone of a synthetic DNA library of Kozak sequences. The diversified positions were -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. The library was transfected into Max DH5α competent cells (Invitrogen, Carlsbad, California) were used and multiple dilutions were performed to optimize the selection of single colonies. DNA preparations were performed for individual colonies according to the manufacturer's recommendations (QIAGEN, Hilden, Germany). Sequencing was performed using universal forward and reverse primers.

[0234] Stable vector construction

[0235] Two integrated antibody expression vectors are used to develop bispecific stable cell lines. The expression vector contains two independent cytomegalovirus (CMV) promoters to guide the transcription of heavy chain (HC) and light chain (LC) as two independent units. The Streptomyces nigricans puromycin-N-acetyltransferase (PUR) (Vara JA, Portela A, Ortin J, Jimenez A. Nucleic Acids Res. 1986; 14 (11): 4617-24) gene is used as a selection marker in one plasmid. The gene encoding the fusion protein containing the positive and negative selection marker HyTK that confers hygromycin (Hyg R) resistance is used as a selectable marker in another plasmid. By cloning a combination of 5 heavy chains and 5 light chains under different Kozak sequence variants to generate a Kozak mixed pool, 25 expression vectors per group are generated. Two expression plasmids are used to develop the Wt Kozak library, one for each antibody, wherein the heavy chain and light chain are under the Wt Kozak sequence.

[0236] Stable cell line development

[0237] According to the manufacturer's advice (MaxCyte, Gaithersburg, MD), MaxCyte STX transfection system was used to transfect CHO cells. Transfected cells were merged into two separate pools and selected with a selective culture medium containing puromycin 5 μg / ml and FIAU 0.5 μM. After recovery, the Wellmate microplate dispenser (Thermo Matrix) of the sterile 8-channel tube (IntegrΛ) of Integra Viafill with a 0.5mm aperture was used to limit dilution to 384-well transparent, flat-bottomed, tissue culture treated plates (Coming Inc, Coming, New York), and a pool was subjected to single cell cloning (SCC). Plates were incubated at 37 ° C and 5% CO2. After three to four weeks of inoculation, 704 separate colonies were selected into 96-well plates (Coming Inc, Coming, New York), and homogeneous time-resolved FRET (HTRF) was used to assess antibody production after approximately 2 days. The clones of the first 48 and the first 24 subsequently expressing antibodies were analyzed by HTRF. The top 11 single cell clones were adapted to growth in suspension and evaluated in a production assay. Seven additional clones that exhibited intermediate and low HCCF titers compared to the top ranked clones were also selected for further analysis.

[0238] Shake flask fed-batch production assay

[0239] Fed-batch production cultures were performed in shake flasks (Corning Inc, Corning, NY) using chemically defined basal medium with rapid feed on days 7 and 10. 6 Cells were seeded at 10 cells / ml. A temperature shift from 37°C to 35°C was performed on day 3. Titers on day 14 were determined using protein A affinity chromatography with UV detection. Percent viability and viable cell counts were determined on days 0, 3, 7, 10, and 14 using a Vi-Cell XR instrument (Beckman Coulter). Glucose and lactate concentrations were monitored on days 7 and 14 using a Bioprofile 400 analyzer (Nova Biomedical).

[0240] Antibody surface staining protocol

[0241] For antibody surface staining, about 2 million cells were precipitated and washed twice with PBS buffer. The cells were then resuspended in 0.5 mL PBS containing anti-human IgG (H+L) allophycocyanin (APC) conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) at a dilution of 1:100 and incubated for 20 min at 37 ° C with shaking. Unstained control samples were resuspended in PBS without antibodies. Empty hosts were also stained to set gates. After incubation with staining antibodies for 20 minutes, cells were washed once with PBS and resuspended in 400 μl of PBS. Then, cells were analyzed in a FACscan flow cytometer (Attune NxT flow cytometer, Life Technologies). For each analysis, 20,000 events were recorded.

[0242] Product quality analysis

[0243] Standard product quality analysis was performed by subjecting the purified antibodies from total protein A to non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS). The TECAN Evo200 system was used for automated sample preparation. All samples were diluted to 1 mg / ml to ensure consistent liquid handling and maintain an optimal dye to protein ratio. The prepared samples were immediately analyzed by the labChip GXII system and the data were processed using Chromeleon software. Signal intensity, peak curves, and relative peak area distributions were evaluated.

[0244] Mass spectrometry

[0245] To identify and quantify variants associated with the protein product, 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 bispecifics as well as species with incorrect LC and HC stoichiometries.

[0246] Genomic DNA extraction, PCR amplification, and sequencing analysis

[0247] Genomic DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA was quantified using a SimpliNano micro-volume spectrophotometer (GE Life Sciences). Hot Start High-Fidelity 2X Master Mix (New England Biotechnology) was used for PCR amplification. 1 μg of gDNA was used as template. PCR conditions were optimized. An annealing temperature of 64°C and 30 cycles were used. Specific annealing primers for the variable region of each chain were designed to distinguish the same chain between molecules. The following forward (F) and reverse (R) primers were used to amplify the Abl heavy chain, Abl light chain, Ab2 heavy chain, and Ab2 light chain: HC-Abl: F-5'-GATACCAGCACCAGCACCGCCT-3' (SEQ ID NO: 12) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO: 13); LC-Abl: F-5'-CTGAACAGCCGCACCCGCAA-3' (SEQ ID NO: 14) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO: 15); HC-Ab2: 5'-GTGATTTGGCGCGGCGGCA-3' (SEQ ID NO: 16) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO: 17); NO:17); and LC-Ab2: 5'-GTGCGCAACCTGGTGGTGTGG-3' (SEQ ID NO:18) and R-5'-ATGGGCGGTAGGCGTGTACGG-3' (SEQ ID NO:19). PCR products were cleaned up using a PCR purification kit (QIAGEN) according to the manufacturer's instructions and analyzed for correct size by 12-well 2% precast agarose gel (Thermo Fisher Scientific). Sequencing was performed using the following primers: SEQ1: 5'-AACGGTGCATTGGAACGCGG-3' (SEQ ID NO:20) and SEQ2: 5'-TGGCTTCGTTAGAACGCAGC-3' (SEQ ID NO:21). Kozak sequence variants for each chain in each clone were confirmed at least twice. Sequencing showed Kozak hybrid clones lacking one of the chains. These are Kozak hybrid clones 3, 11, 29, 60, and 21L, for Ab2-LC, Ab2-HC, Ab1-LC, Ab2-HC, and Ab1-LC, respectively. Kozak hybrid clone 24 lacks the HC and LC of Ab1 ( Figures 21A-21C ).

[0248] result

[0249] To investigate whether changes in the consensus Kozak sequence could modulate protein production in industrial production cell lines, the -3 position and the combination of -3, -2, and -1 positions upstream of the start codon of the ORF encoding the Fc fusion protein were altered ( Figure 1 ). Kozak sequence variants were tested by transient transfection in a CHO production cell line and the production of Fc fusion proteins was assessed by HTRF (homogeneous time-resolved fluorescence) assay 48 hours after transfection. Well-expressing antibodies and poorly expressing antibodies were also included as internal assay controls.

[0250] like Figure 2 As shown, changes in the consensus Kozak sequence modulate Fc fusion protein A titers. The results demonstrate that the presence of a purine at position -3 (GCCATGG and ACCATGG, SEQ ID NOs: 4 and 3, respectively) produces high levels of Fc fusion protein A titers. The weakest Kozak sequence variant evaluated, TTTATGG (SEQ ID NO: 7), modulates titers to half the level of the Kozak consensus sequence, hereinafter referred to as wild-type (Wt) Kozak.

[0251] To determine whether Kozak sequence variants could modulate protein production regardless of molecule, these combinations in different Fc fusion proteins were tested: Fc fusion 'B' ( Figure 2 ). Similar to cells transfected with Kozak sequence variants for Fc fusion A, cells transfected with variants of the Kozak sequence for Fc fusion B showed titer changes compared to Wt Kozak, and a similar pattern was observed for variants of Fc fusion B. These results suggest that variations of the Wt Kozak sequence can be used to adjust antibody titers.

[0252] Example 2: Design and screening of Kozak sequence libraries to expand the range of expression levels

[0253] Other Kozak sequence variants were tested to achieve a full range of expression levels from 0.1-fold to 1.0-fold that of Wt Kozak. Figure 3 As shown in the detailed workflow provided in , a Kozak sequence variant library using the ORF encoding Fc fusion protein B (a representative of well-performing molecules) was designed and screened. The library included randomization of five bases upstream of the start codon (-5, -4, -3, -2, and -1) and one base downstream (+4) to any of four nucleotides ( Figure 4). The expected diversity of this construction is about 4100 variants of the Kozak sequence. Randomization of the +4 position generates an amino acid change from glycine, which is present at position 2 of the signal sequence, to arginine, when this position is occupied by adenine (A) or cytosine (C). The library is transformed into competent E. coli cells and individual variants are sequenced to exclude those that display undesigned mutations, deletions, and those that contain a stop codon as the second amino acid of the signal sequence, which occurs when position +4 is occupied by thymine (T).

[0254] result

[0255] Initial screening in transient transfections in CHO cells was performed with 111 correct variants, including Wt Kozak and Wt Kozak with a C or A at position +4 as a control for the second amino acid change in the signal sequence. Figure 5 As shown, Fc fusion protein titers measured by HTRF at 48 hours after transient transfection showed a wide range of expression levels. An almost continuous translation range of standardized titers from 0.05 to 1.54 units was observed, with 1.0 corresponding to Wt Kozak. These variants met the target of the desired expression range, thus stopping further variant screening. In general, regardless of the Kozak sequence upstream of the start codon, the presence of A or C in the +4 position increased the titer of Wt Kozak. The screening was repeated and represents data from two independent transient transfections ( Figure 5 ).

[0256] like Figure 6 As shown, the nucleotide distribution of the analyzed variants was random, indicating that the library screening was unbiased. Next, the variants were divided into five groups based on their potency and performance. The group ranges were: 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. Except for the preference of adenine and thymine at most positions in the weakest group, and arginine as the second amino acid of the signal sequence of the strongest group, no other correlation between antibody production and Kozak sequence variants was detected. Several rounds of transient transfection were performed to reduce the number of variants ( Figure 7 ). Criteria for selection of variants between rounds were reproducibility between transient transfections, stability between different DNA preparations, and preservation of nucleotide diversity. Figures 8A-8B As shown, this screen established 11 Kozak sequence variants representing an expression range of 0.2 to 1.3-fold that of Wt Kozak, enabling titer modulation with high precision.

[0257] Example 3: Generation of bispecific stable clones with a mixture of Kozak sequence variants

[0258] Despite progress in the engineering of complex antibody formats such as bispecific antibodies, the manufacturability of these antibody formats in single mammalian expression systems generally exhibits low efficiency. Low titer and insufficient product quality are two factors that make bispecific antibodies (BsAbs) difficult to stably produce. Therefore, there is an urgent need to identify limiting steps in production systems. One bottleneck that limits the assembly efficiency of bispecific and other multi-chain formats in a single cell is the limited control of the individual chain level. Designing vectors that can adjust the individual chain ratio in multi-chain formats can improve assembly efficiency, as previously demonstrated in Escherichia coli with translation initiation region (TIR) ​​variants (Simmons LC, Yansura DG. Nat Biotechnol. 1996; 14 (5): 629-34). For example, Fig. 9 As shown, the ratio of light chains (LC1:LC2) that make up a BsAb can be manipulated to achieve a higher percentage of correctly assembled BsAbs.

[0259] Since it is necessary to identify unique ratios of parent antibodies that are combined into a bispecific format to generate bispecific antibodies, Kozak sequence variants were tested for their ability to produce higher BsAb assembly and production in a single cell. The successful application of Kozak sequence variants as a technology for regulating protein expression was evaluated in the establishment of a stable CHO host cell line. To do this, a representative group of five variants including Wt Kozak was selected ( Fig.10 and Fig. 8A For Kozak sequence variants #3, #135, #148, Wt, and #228, the relative protein production intensities of these variants were about 0.3, about 0.5, about 0.8, about 1.0, and about 1.3 times that of the Wt Kozak sequence, respectively. Thus, this set of variants covers a wide range of expression levels observed in transient transfections ( Fig.10 ).

[0260] The bispecific antibodies Ab1 / Ab2 were chosen as a model to evaluate the effects of Kozak sequence variations on regulating chain ratios based on the following criteria. First, for Ab1 / Ab2, a well-characterized process development platform was available that facilitated analysis and characterization of the products. Second, Ab1 was a poorly expressed molecule with a bottleneck downstream in the transcription process, whereas Ab2 represented an example of a well-performing molecule. Therefore, this was an appropriate scenario to examine the ability of Kozak sequence variants to create successful chain pairing.

[0261] 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 alleviate light chain (LC) mispairing (Dillon M, Yin Y, Zhou J, McCarty L, Ellerman D, Slaga D, et al. MAbs. 2017; 9(2): 213-30). Combinations of heavy and light chains under the five Kozak sequence variant groups described previously 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 method reached a potential 625 different chain ratio combinations. As a reference value, the 4 chains of the BsAb were cloned under the Wt Kozak sequence, transfected into CHO cells, and carried in parallel (hereinafter referred to as WtKozak). Two different transfections were performed for each condition.

[0262] result

[0263] Transfection efficiency was monitored by cell surface staining of total IgG and measured by FACS analysis. Fig.12 It is shown that similar transfection efficiencies were observed for the Kozak mix and the Wt Kozak pool. After transfection of the expression plasmid, stable cell pools were established by drug selection. Fig.13A and 13B As shown, similar cell viability was observed for both the Kozak mix and the Wt Kozak pools after addition of the selection drug (indicated by the black arrow). Then, one stable pool per transfection per condition was selected and single cell cloned. After recovery, the plates were analyzed for clone recovery and 704 clones recovered per pool and condition were picked into 96-well plates. Preliminary screening based on harvest cell culture fluid (HCCF) titers showed differences in profiles and absolute titers between conditions. Kozak mix clones showed lower absolute titers and more significant titer reductions compared to Wt Kozak clones ( Fig.14A and 14B ). This result matches the planned diversity of chain ratio combinations in the Kozak pool clones, while the profile of the Wt Kozak clones is consistent with expectations for clones carrying only one chain ratio combination. Several rounds of screening based on HCCF titer, suspension adaptation, and amplification were completed to determine the top 11 clones for each transfection pool and each condition, which resulted in a total of 22 clones per condition. Since each Kozak pool clone has the potential to carry a different combination of Kozak sequence variants, and in order to understand the differences between the combinations, an additional seven clones that exhibited low HCCF titers compared to the top-ranked clones were also selected for further analysis.

[0264] Example 4: Shake flask performance of bispecific stable clones derived from a mix of Kozak sequence variants

[0265] The productivity and bispecific assembly of each individual clone selected from Example 3 above were evaluated using 14-day shake flask fed-batch production. All clones studied showed comparable viability throughout the production assay. The final viability at day 14 was approximately 80-95% under both conditions, except for one Kozak pool clone with a final viability of 74% ( Fig.15A and 15B Similarly, for all individual clones, viable cell counts showed an exponential growth rate until day 7, then reached a plateau ( Fig.16A and 16B ). For each clone's typical titer, product quality and assembly efficiency were measured on day 14.

[0266] like Fig.17 As shown, in both transfections, the individual Wt Kozak clones showed higher general absolute antibody titers than the individual Kozak mixed clones. Note that in this titer, correctly assembled bispecific antibodies, half antibodies, and other unwanted side products are represented. 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 the distinction and quantification of two forms: a main peak equivalent to the whole antibody (all-ab) in correctly and incorrectly assembled BsAb forms, and the sum of the pre-peaks representing other species of different molecular weights. In general, fewer Kozak mixed clones with a high percentage of full-ab formation were observed among the top 22 clones compared to the Wt Kozak clones ( Fig.18 ). This result is consistent with the fact that, despite the different chain ratio combinations between conditions (1 and 625 for Wt Kozak clones and Kozak mixed clones, respectively), the same number of individual clones were screened and selected for each condition, which favored finding a greater number of Wt Kozak clones with high product quality. The percentage of full antibody varied between clones. The four individual Kozak mixed clones for transfection 2 ranged from approximately 60% of full antibody, and then the percentage dropped to 30%.

[0267] All individual Kozak pool clones transfected with 1 showed a main peak of approximately 30% ( Fig.18 ). This result is consistent with the fact that among the diversity of chain ratio combinations in the Kozak pool, some of them are weak in full-AB production. In addition, not all clones producing the highest antibodies (general titer) are associated with the highest content of full-AB in the Kozak pool.

[0268] Typically, the best individual Wt Kozak clones ranged between 80-90% of full-ab production. Similar to Kozak pool clones, individual Wt Kozak clones showed variability in the percentage of the main peak depending on the clone. In addition, a correlation between high general titer and high main peak content was observed in these clones ( Fig.18 ).

[0269] In parallel, the production performance of seven additional clones with low to medium HCCF titers compared to the top-ranked clones was evaluated. The production titers of these clones showed similar behavior to that previously observed in the primary screen ( Fig.19A ). CE-SDS data indicated that product quality varied from clone to clone. Again, there was no direct correlation between general titer and maximum full-Ab content. In this way, some Kozak pool clones with more modest titers showed similar full-Ab percentages of approximately 50-60% ( Fig.19B ).

[0270] Example 5: Bispecific assembly in stable clones enhanced by Kozak sequence variants

[0271] The first four clones from CE-SDS analysis in each condition and transfection were analyzed to identify and quantify the mispairing of 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 data deconvolution, several product-related variants were identified, including half antibodies (HC+LC), homodimers (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 overlapped on CE-SDS analysis due to similar mass.

[0272] result

[0273] Distinguish and quantify seven different bispecific antibody formats and three half-antibody formats ( Fig. 20A The best individual Kozak pool clones showed approximately 40% correctly assembled BsAbs, which was more than double that observed for the best individual Wt Kozak clone (approximately 18%) ( Fig. 20A). Since each single Kozak hybrid clone may carry a different combination of Kozak sequence variants, each clone showed a different percentage of bsAb assembly, while Wt Kozak clones carrying the same combination showed similar numbers of bsAb assembly. Notably, the improperly assembled bispecific format Ab1-HC+Ab2-HC+2x Ab2-LC with light chain mismatch accounted for 41% to 64% of the full antibodies produced by the Wt Kozak clones ( Fig. 20A ). These data point out the importance of adjusting the light chain ratio to achieve BsAb assembly when using a single cell to produce. The mass spectrometry of the Kozak mixed clones with the lowest full-ab formation yield measured by CE-SDS confirms that most of the production materials of those clones are half antibodies. The form is Ab1-HC+Ab2-LC and half-Ab1. The BsAb forms provided by these clones are mainly BsAb 2x Ab1-HC+2x Ab2-LC that is not properly assembled, and the BsAb form 2xAb2-HC+Ab1-LC+Ab2-LC ( Fig. 20A ).

[0274] Knowing the percentage of each form, calculate the effective bispecific titer ( Fig. 20B Due to the fact that some Kozak pool clones have higher BsAb assembly than the Wt Kozak clone, the Kozak pool clones overcome the general titer deficiency shown previously ( Fig.17 and Figures 20A-20B ). The effective bispecific titers of the best clones in each condition were similar: 0.7 g / L and 0.6 g / L for the best WT Kozak and Kozak hybrid clones, respectively. Since each Kozak hybrid clone may carry 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 range of bsAb assembly ( Fig. 20A ). Notably, the improperly assembled bispecific format Ab2 HC+Ab1 HC+2x Ab2 LC with light chain mispairing accounted for 41-64% of the full-ab produced by the Wt Kozak clone ( Fig. 20A ). These data point to the importance of tuning light chain translation levels to achieve correct bsAb assembly when using single cell production.

[0275] However, if Fig. 20B As shown, the Kozak clones provide a smaller amount of impurities that must be removed during purification compared to the Wt Kozak clones. Some Kozak hybrid clones overcome the previous Fig.17Importantly, the Kozak pool clones also exhibited less product-related impurities than the WT Kozak clones ( Fig. 20B ). Purification of correctly assembled bsAbs from a large number of nearly identical potential byproducts is challenging. Because some byproducts are very similar to the target bsAb, their removal downstream often comes at the expense of yield or product quality. New analytical methods are needed to distinguish the target product from these unique impurities, but they require high-cost manufacturing and complex technologies. Kozak hybrid cloning minimizes the formation of impurities and product-related variants ( Fig. 20B and 22B ), demonstrating the benefit of this approach to produce bsAbs in single cells.

[0276] These data suggest that tuning translation strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells.

[0277] Example 6: Adjusting chain ratios using Kozak sequence variants to produce high BsAb yields

[0278] The heterogeneity and diversity observed between Kozak pooled clones in terms of BsAb assembly efficiency and product quality indicated that each clone had chains expressed under different Kozak sequence variants. To identify the Kozak combination possessed by each clone and determine the best combination, 29 Kozak pooled clones were sequenced.

[0279] result

[0280] The data showed that the Kozak pooled clones with low product yields expressed the heavy chain of Ab2 under one of the weakest Kozak sequence variants, Kz.135 and Kz.3, and the remaining chains had either Wt or the strongest Kozak sequence variant ( Figures 21A-21B As a result, insufficient amounts of heavy chains of Ab2 may be generated, and thus the final products are preferably half-Ab and half-Ab2 in the form of Ab1-HC+Ab2-LC ( Fig. 20A ). In other words, there is a range in which heavy chain expression can be reduced before full ab assembly is disrupted.

[0281] Otherwise, when the combination of Wt Kozak and the strongest Kozak sequence variant regulated the expression of the four chains, the Kozak mixed clone showed high BsAb assembly with high titer ( Fig.21A On the other hand, when the combination of WT Kozak and the strongest Kozak sequence variant (Kz.228; relative intensity of 1.3) modulated the expression of the four chains, the Kozak hybrid clone showed high bsAb assembly and effective titer ( Fig. 20A and21A Interestingly, Kozak pooled clones 69, 61, and 88 exhibited a minor light chain ratio of 1.3-fold Ab1 light chain to 1.0-fold Ab2 light chain, or vice versa, respectively, significantly reducing the Ab2 light chain mispairing observed in the WT Kozak clones ( Fig. 20A ; Ab2 HC + Ab1 HC + 2x Ab2 LC). These data suggest that reduced translation of a selected chain relative to the translation levels of the other chains 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 drove the production of more daughter products composed of that chain rather than a more correctly assembled BsAb. For example, Kozak clone 69 with light chain Ab1 under the strongest Kozak sequence showed approximately 30% of the product to be 1 / 2Ab1 ( Fig. 20A ). This observation is consistent with the fact that the manufacture of BsAbs 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.

[0282] With this in mind, and not wishing to be bound by theory, it is believed that the reduction in translation may prevent the accumulation of species and lead to an increase in yield. Consistent with this conclusion, a combination of Abl heavy and light chains carried at half the titer of the top Kozak clone and under the weaker Kozak sequence variant #135 exhibited the highest bispecific assembly detected ( Fig.22D ). CE-SDS data of this clone showed that the product mass was about 48% of the full-ab and about 52% of the half-ab. MS data indicated that 100% of the full-ab corresponded only to the bispecific form, which was about 50% of the correctly assembled BsAb. On the other hand, almost all of the half-ab species were knob 1 / 2 Ab2 ( Figures 22A-22B ), which is consistent with the fact that the heavy and light chains of Ab2 are under the Wt Kozak and strongest Kozak sequence variant #228, respectively. Therefore, within a certain range, the downregulation of the expression of the two chains of the molecule, which may present a bottleneck downstream of translation during the production process, has a positive impact on the assembly efficiency.

[0283] Clone 17M was one of the additional clones that moved forward, despite a modest primary HCCF titer ( Fig.19A ). Although this clone had only about half the effective titer of the top Kozak clone, it showed the highest bispecific assembly at about 48%. MS data indicated that 100% of the full-abs corresponded only to the correct bispecific format, and that the half-ab species were almost exclusively limited to the knob 1 / 2 mAb2 ( Fig.22A and 22B ).

[0284] These data can be obtained from the sequencing results ( Fig. 22C ). Both the heavy and light chains of aAb1 are under a weaker Kozak variant (Kz.135), and as mentioned before, Ab1 is a difficult molecule to express. Downregulation of both chains, as in the case of using Kz.135 with a relative strength of about 0.5, can relieve bottlenecks downstream of translation, such as protein folding, thus having a positive impact on assembly efficiency. Regarding Ab2, the heavy and light chains are under a stronger Kozak sequence, WT Kozak, and the strongest Kozak variant (Kz.228), respectively ( Fig. 22C ).

[0285] This combination, combined with the weaker Kozak sequence of aAb1, will result in accumulation of the aAb2 half-ab while limiting any significant accumulation of the other species ( Fig.22A and 22B ).

[0286] In the same way, seven additional clones with medium to low HCCF titers compared to the top-ranked clones were sequenced. In general, it was observed that the expression of most 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.

[0287] To elucidate Ab species and BsAb forms, five of these Kozak hybrid clones carrying combinations of Kozak sequence variants that were most distinct from the first 11 clones were analyzed by mass spectrometry. The percentage of BsAb assembly was less than 10%, except for Kozak hybrid clone 17. The absolute abundance of each species was correlated with the expression intensity of each chain ( Fig.22D ).

[0288] 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 as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety. Sequence Listing <110> Hoffmann-La Roche GmbH <120> 14639-20452.40 <130> Methods for producing multimeric proteins in eukaryotic host cells <140> Not yet specified <141> Also attach <150> US 62 / 796,014 <151> 2019-01-23 <160> twenty three <170> FastSEQ for Windows, Version 4.0 <210> 1 <211> 11 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> misc_feature <222> 1, 2, 3, 4, 5, 9 <223> n = A, T, C or G <400> 1 nnnnnatgng a 11 <210> 2 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 2 ccaccatgg 9 <210> 3 <211> 7 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 3 accatgg 7 <210> 4 <211> 7 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 gccatgg 7 <210> 5 <211> 7 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 5 tccatgg 7 <210> 6 <211> 7 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 6 tagatgg 7 <210> 7 <211> 7 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 7 tttatgg 7 <210> 8 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 8 tttttatgg 9 <210> 9 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 9 ggctcatgc 9 <210> 10 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 10 cctagatgc 9 <210> 11 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 11 gaagtatga 9 <210> 12 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 12 gataccagca ccagcaccgc ct 22 <210> 13 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 13 atgggcggta ggcgtgtacg g 21 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 14 ctgaacagcc gcacccgcaa 20 <210> 15 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 15 atgggcggta ggcgtgtacg g 21 <210> 16 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 16 gtgatttggc gcggcggca 19 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 17 atgggcggta ggcgtgtacg g 21 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 18 gtgcgcaacc tggtggtgtg g 21 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 19 atgggcggta ggcgtgtacg g 21 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 20 aacggtgcat tggaacgcgg 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty one tggcttcgtt agaacgcagc 20 <210> twenty two <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty two ccaccatggg 10 <210> twenty three <211> 11 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty three ccaccatggg a 11

Claims

1. A method for 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 polypeptide chain and a second subunit comprising a third polypeptide chain and a fourth polypeptide chain, the method comprising: (a) providing the eukaryotic host cell, wherein the eukaryotic host cell comprises a first polynucleotide, a second polynucleotide, a third nucleotide and a fourth polynucleotide, wherein the first polynucleotide comprises a first translation initiation sequence operably linked to a first open reading frame encoding the first polypeptide chain, the second polynucleotide comprises a second translation initiation sequence operably linked to a second open reading frame encoding the second polypeptide chain, the third polynucleotide comprises a third translation initiation sequence operably linked to a third open reading frame encoding the third polypeptide chain, and the fourth polynucleotide comprises a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the fourth polypeptide chain, wherein when each subunit is expressed separately in the eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit, and wherein (1) the first translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the second translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the third translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11); or, (2) the first translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), the second translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), the third translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11); (b) culturing the eukaryotic host cell under conditions suitable for expression of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain, wherein upon expression the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain form the multimeric polypeptide; and (c) recovering the multimeric polypeptide produced by the eukaryotic host cell, 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 to a first antigen, and wherein the second subunit is a second half antibody that binds to a second antigen. 2 . The method of claim 1 , wherein each of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide is operably linked to a promoter.

3. The method of claim 2, 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.

4. A method for 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 the 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 the first antibody heavy chain, a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the first antibody light chain, a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the second antibody heavy chain, and a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the second antibody light chain, wherein when each half antibody is expressed separately in the eukaryotic host cell, the first half antibody is expressed at a lower level than the second half antibody, and wherein (1) the first translation start sequence comprises the sequence GGCTCATGC (SEQ ID NO:9), the second translation start sequence comprises the sequence GGCTCATGC (SEQ ID NO:9), the third translation start sequence comprises the sequence CCACCATGG (SEQ ID NO:2), and the fourth translation start sequence comprises the sequence GAAGTATGA (SEQ ID NO:11), or, (2) the first translation start sequence comprises the sequence GAAGTATGA (SEQ ID NO:11), the second translation start sequence comprises the sequence CCACCATGG (SEQ ID NO:2), the third translation start sequence comprises the sequence GAAGTATGA (SEQ ID NO:11), and the fourth translation start sequence comprises the sequence GAAGTATGA (SEQ ID NO:11); (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.

5. The method of claim 4, wherein the first antibody heavy chain comprises a first antibody Fc region, the first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region, the 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 amino acid residues having a smaller side chain volume, thereby generating 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 amino acid residues having a larger side chain volume, thereby generating 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.

6. The method of claim 4, wherein the first antibody heavy chain comprises a first antibody Fc region, the first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region, the 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 generating 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 amino acid residues having a larger side chain volume, thereby generating 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.

7. The method of claim 5 or claim 6, wherein the knob mutation comprises at least one of the following: T366Y, T366W, T394W and F405W, numbered according to the EU index based on human IgG1.

8. The method of any one of claims 5-7, wherein the hole mutation comprises at least one of the following: F405A, Y407T, Y407A, T366S, L368A, Y407V and T394S, numbered according to the EU index based on human IgG1.

9. The method of any one of claims 5-8, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises at least one of T366S, L368A, and Y407V, numbered based on human IgG1 according to the EU index.

10. The method of any one of claims 5-8, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises at least two of T366S, L368A, and Y407V, numbered based on human IgG1 according to the EU index.

11. The method of any one of claims 5-8, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises all three of T366S, L368A, and Y407V, numbered based on human IgG1 according to the EU index.

12. The method of any one of claims 4-11, 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 promote selective association of the first antibody light chain with the first antibody heavy chain.

13. The method of claim 12, wherein the first mutation comprises an amino acid substitution at V133, and wherein the second mutation comprises an amino acid substitution at S183, based on EU index numbering.

14. The method of claim 13, 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.

15. The method of claim 13 or claim 14, wherein an amino acid substitution at S183 results in a positively charged residue and an amino acid substitution at V133 results in a negatively charged residue, or an amino acid substitution at S183 results in a negatively charged residue and an amino acid substitution at V133 results in a positively charged residue.

16. The method of any one of claims 4-11, 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 promote selective association of the second antibody light chain with the second antibody heavy chain.

17. The method of claim 16, wherein the third mutation comprises an amino acid substitution at V133, and wherein the fourth mutation comprises an amino acid substitution at S183, based on EU index numbering.

18. The method of claim 17, 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.

19. The method of claim 17 or claim 18, wherein an amino acid substitution at S183 results in a positively charged residue and an amino acid substitution at V133 results in a negatively charged residue, or an amino acid substitution at S183 results in a negatively charged residue and an amino acid substitution at V133 results in a positively charged residue.

20. The method of any one of claims 4-11, 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, based on EU index numbering.

21. The method of claim 20, 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 IgG1.

22. The method of any one of claims 4-11, 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, based on EU index numbering.

23. The method of claim 22, 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 IgG1.

24. The method of any one of claims 1-23, wherein 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.

25. The method of claim 24, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the same chromosomal locus of the eukaryotic host cell.

26. The method of any one of claims 1-23, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are part of one of a plurality of extrachromosomal polynucleotides in the eukaryotic host cell.

27. The method of any one of claims 1-26, wherein the eukaryotic host cell is a mammalian host cell.

28. The method of claim 27, wherein the mammalian host cell is a Chinese Hamster Ovary (CHO) cell.

29. 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 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 said third polypeptide chain; and (d) 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 separately in the recombinant eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit; and in, (1) the first translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the second translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the third translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), or, (2) the first translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), the second translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), the third translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), wherein the non-natural 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.

30. The cell of claim 29, wherein each of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide is operably linked to a promoter.

31. The cell of claim 30, 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.

32. A recombinant eukaryotic host cell for expression of a bispecific antibody, the 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 first antibody heavy chain; (b) a second polynucleotide comprising a second translation initiation sequence operably linked to a second open reading frame encoding the first antibody light chain; (c) a third polynucleotide comprising a third translation initiation sequence operably linked to a third open reading frame encoding the second antibody heavy chain; and (d) a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding the second antibody light chain; wherein when each half antibody is expressed separately in the recombinant eukaryotic host cell, the first half antibody is expressed at a lower level than the second half antibody; and in, (1) the first translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the second translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the third translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), or, (2) the first translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), the second translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), the third translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11).

33. The cell of claim 32, wherein the first antibody heavy chain comprises a first antibody Fc region, the first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region, the 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 amino acid residues having a smaller side chain volume, thereby generating 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 amino acid residues having a larger side chain volume, thereby generating 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.

34. The cell of claim 32, wherein the first antibody heavy chain comprises a first antibody Fc region, the first antibody Fc region comprising a CH2 domain and a CH3 domain; wherein the second antibody heavy chain comprises a second antibody Fc region, the 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 generating 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 amino acid residues having a larger side chain volume, thereby generating 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.

35. A cell according to claim 33 or claim 34, wherein the knob mutation comprises at least one of the following: T366Y, T366W, T394W and F405W, numbered according to the EU index based on human IgGl.

36. The cell of any one of claims 33-35, wherein the hole mutations comprise at least one of the following: F405A, Y407T, Y407A, T366S, L368A, Y407V, and T394S, numbered according to the EU index based on human IgG1.

37. The cell of any one of claims 33-36, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises at least one of T366S, L368A, and Y407V, numbered based on human IgG1 according to the EU index.

38. The cell of any one of claims 33-36, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises at least two of T366S, L368A, and Y407V, numbered according to the EU index based on human IgG1.

39. The cell of any one of claims 33-36, wherein the knob mutation comprises T366W, and wherein the hole mutation comprises all three of T366S, L368A, and Y407V, numbered based on human IgG1 according to the EU index.

40. The cell of any one of claims 32-39, 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 promote selective association of the first antibody light chain with the first antibody heavy chain.

41. The cell of claim 40, wherein the first mutation comprises an amino acid substitution at V133, and wherein the second mutation comprises an amino acid substitution at S183, based on EU index numbering.

42. The cell of claim 41, 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.

43. A cell according to claim 41 or claim 42, wherein an amino acid substitution at S183 results in a positively charged residue and an amino acid substitution at V133 results in a negatively charged residue, or an amino acid substitution at S183 results in a negatively charged residue and an amino acid substitution at V133 results in a positively charged residue.

44. The cell of any one of claims 32-39, 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 promote selective association of the second antibody light chain with the second antibody heavy chain.

45. The cell of claim 44, wherein the third mutation comprises an amino acid substitution at V133, and wherein the fourth mutation comprises an amino acid substitution at S183, based on EU index numbering.

46. ​​The cell of claim 45, 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.

47. A cell according to claim 45 or claim 46, wherein an amino acid substitution at S183 results in a positively charged residue and an amino acid substitution at V133 results in a negatively charged residue, or an amino acid substitution at S183 results in a negatively charged residue and an amino acid substitution at V133 results in a positively charged residue.

48. The cell of any one of claims 32-39, 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, based on EU index numbering.

49. The cell of claim 48, 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 IgG1.

50. The cell of any one of claims 32-39, 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, based on EU index numbering.

51. The cell of claim 50, 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 IgG1.

52. The cell of any one of claims 29-51, wherein 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.

53. The cell of claim 52, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the same chromosomal locus of the eukaryotic host cell.

54. The cell of any one of claims 29-51, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are part of one of a plurality of extrachromosomal polynucleotides in the eukaryotic host cell.

55. The cell of any one of claims 29-54, wherein the eukaryotic host cell is a mammalian host cell.

56. A polynucleotide kit for expression of a multimeric polypeptide comprising 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, 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 said third polypeptide chain; and (d) a fourth polynucleotide comprising a fourth translation initiation sequence operably linked to a fourth open reading frame encoding said 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 present in the genome of a naturally occurring host cell, wherein when each subunit is expressed separately in a eukaryotic host cell, the first subunit is expressed at a lower level than the second subunit, and in, (1) the first translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the second translation initiation sequence comprises the sequence GGCTCATGC (SEQ ID NO: 9), the third translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), or, (2) the first translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), the second translation initiation sequence comprises the sequence CCACCATGG (SEQ ID NO: 2), the third translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), and the fourth translation initiation sequence comprises the sequence GAAGTATGA (SEQ ID NO: 11), 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.

57. The kit of claim 56, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are part of one or more expression vectors.

58. The kit of claim 56 or claim 57, wherein each of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide is operably linked to a promoter.

59. The kit of claim 58, 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.

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