Flow cytometry attenuated reporter expression (FLARE) multiple reporter system and methods of use thereof

Abstract

Compositions and methods are provided for expressing multiple target peptides at a high level in mammalian host cells. Each target peptide is encoded on a polynucleotide comprising a nucleotide sequence encoding a unique cell surface marker peptide and a nucleotide sequence encoding a unique target peptide, wherein both the nucleotide sequence encoding the unique cell surface marker peptide and the nucleotide sequence encoding the unique target peptide are transcribed on the same mRNA. Each mammalian host cell contains multiple different polynucleotides, enabling the cell to express multiple unique target peptides and multiple unique cell surface marker peptides. In some embodiments, the multiple unique cell surface marker peptides are variants of CD52. The compositions and methods can be used to express multimeric target proteins, such as cross-crossed dual variable domain (CODV) triantibodies.

Description

Technical Field

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 116,094, filed November 19, 2020, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on November 2, 2021, is named 723336_SA9-271PC_ST25.txt and has a size of 16,727 bytes.

[0005] This disclosure generally relates to high-throughput systems and methods for identifying high-yielding strains that can be used to produce therapeutic and diagnostic proteins. Prior Technology

[0006] Commercial production of therapeutic proteins requires stable, high-performing recombinant cell lines. Existing methods typically rely on dihydrofolate reductase-deficient (DHFR) Chinese hamster ovary (CHO) cell lines to generate selected cell lines capable of producing one or more of these proteins. High-yielding cell lines are obtained by selectively repeating the amplification of transfected cell pools using agents such as methotrexate (MTX). This process is time-consuming, labor-intensive, and fails to specifically identify cell lines that will produce high levels of protein.

[0007] Flow cytometry, including fluorescence-activated cell sorting (FACS), has been used to improve the development and selection of high-yielding CHO cell lines. This technology enables rapid identification and isolation of high-yielding lines from heterogeneous transfected cell populations, reducing the labor and time associated with random selection methods. It allows for the identification of desired cells without requiring repeated MTX amplification of the pool.

[0008] However, after isolating single-celled colonies via FACS-based sorting methods, screening a sufficient number of colonies to isolate stable, high-yielding cell lines remains a time-consuming process. Therefore, efficient and accurate screening methods are highly advantageous at this stage of cell line development. Furthermore, screening at the 96-well plate stage of colony development is preferable, as it focuses on further amplification of those colonies with high productivity. Analysis of cell culture medium harvests is often used to identify colonies secreting high levels of therapeutic proteins. However, this method is not optimal because it does not account for differences in cell density or culture medium volume between wells. Therefore, it may not accurately predict colonies with high specific productivity and high titers. Moreover, considerable effort must often be devoted to developing and optimizing new assays for each new target therapeutic protein.

[0009] Commercially valuable proteins typically consist of two or more covalently and / or non-covalently associated polypeptide chains. IgG antibodies and their derivatives, as heterodimeric tetramers, are examples of such proteins. The generation of multi-chain proteins usually involves the co-expression of individual polypeptide chains as well as the isolation of the intact protein.

[0010] WO 2008 / 036255 discloses a FACS-based and reporter protein-based system for high-throughput development of therapeutic proteins, which is used to identify, select, and generate populations of recombinant eukaryotic host cells that produce stable and highly expressive target peptides.

[0011] WO 2017 / 062722 discloses a fluorescence-activated cell sorting (FACS) method for bulk selection of production cells expressing target peptides.

[0012] There remains a need in the art for compositions and methods for screening colonies to select recombinant cell lines that stably produce high levels of target multi-chain proteins. This invention addresses this need and provides related advantages. Summary of the Invention

[0013] This article discloses methods and compositions suitable for the identification, selection, and generation of recombinant mammalian host cells that stably express a target multimeric polypeptide (“target polypeptide”) at a high level, and for the development and large-scale production of said target polypeptide. These methods and compositions will be used, for example, for the development and large-scale production of engineered antibodies comprising cross-cross dual variable domain (CODV) Ig-like proteins. Steinmetz A. et al. (2016) MAbs8(5): 867-878.

[0014] One aspect of this disclosure is a method for representing multiple target peptides at a high level, the method comprising: (a) Culture multiple mammalian host cells, each containing multiple recombinant polynucleotides, among which Each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA. The culture was conducted under conditions that allowed each unique cell surface marker peptide to be expressed on the surface of mammalian host cells, as well as the expression of each unique target peptide. (b) Contact the cultured mammalian host cells of step (a) with a variety of detectable agents, each of which is capable of uniquely binding to one or more of the unique cell surface marker peptides expressed on the surface of the mammalian host cells; (c) Perform at least one round of fluorescence-activated cell sorting on the contacted cells from step (b) to select one or more mammalian host cells that are uniquely bound to at least one of the plurality of detectable agents; (d) Prepare one or more colony populations of the mammalian host cells selected in step (c); (e) Analyze one or more colonies from step (d) by detecting the performance levels of at least two unique cell surface marker peptides in each colony population; (f) Select one or more colonies exhibiting high levels of performance with at least two unique cell surface marker peptides; and (g) Cultivate one or more colonies selected in step (f) under conditions that allow for high-level expression of the various target peptides.

[0015] In some embodiments, step (c) includes performing a single-round fluorescence-activated cell sorting on cells from step (b) that have been in contact with at least the first detectable agent and the second detectable agent, thereby selecting one or more mammalian host cells that are uniquely bound by at least both the first and second detectable agents.

[0016] In some embodiments, step (c) includes (c1) Performing a first round of fluorescence-activated cell sorting on at least a first cell surface-labeled polypeptide to select one or more mammalian host cells bound to at least a first detectable agent; and (c2) Perform a second round of fluorescence-activated cell sorting on the cells selected in step (c1) for at least a second cell surface labeled peptide, thereby selecting one or more mammalian host cells that are bound to at least the first detectable agent and the second detectable agent.

[0017] In some embodiments, step (e) is performed 7 to 28 days after step (d).

[0018] In some embodiments, the analysis in step (e) includes flow cytometry.

[0019] In some embodiments, at least one of the various unique cell surface marker peptides in step (f) exhibits a higher performance level than the corresponding performance level of at least 70% of the colony population analyzed in step (e).

[0020] In some embodiments, each of the multiple unique cell surface marker peptides in step (f) exhibits a higher performance level than the corresponding performance level of at least 70% of the colony population analyzed in step (e).

[0021] In some embodiments, at least the first of the multiple unique cell surface peptides in step (f) exhibits a higher performance level than at least the second of the multiple unique cell surface peptides in step (f).

[0022] In some embodiments, the first of the multiple unique cell surface peptides in step (f) exhibits a higher performance level than the second of the multiple unique cell surface peptides in step (f).

[0023] In some embodiments, the method further includes: (h) Isolate at least the first unique target polypeptide and the second unique target polypeptide expressed in step (g) from the one or more selected colonies or from the cell culture medium in which the one or more selected colonies are cultured.

[0024] In some embodiments, the plurality of unique target peptides comprises 2 to 8 unique target peptides.

[0025] In some embodiments, the plurality of unique target peptides comprises 2 to 4 unique target peptides.

[0026] In some embodiments, the plurality of unique target peptides includes two unique target peptides.

[0027] In some embodiments, the plurality of unique target peptides includes three unique target peptides.

[0028] In some embodiments, the plurality of unique target peptides includes four unique target peptides.

[0029] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique cell surface marker polypeptide, and a nucleotide sequence encoding the unique target polypeptide from the 5' end to the 3' end.

[0030] In some embodiments, each recombinant polynucleotide comprises 1 to 4 promoters, 1 to 4 nucleotide sequences each encoding a unique cell surface marker polypeptide, and 1 to 4 nucleotide sequences each encoding a unique target polypeptide, wherein one of the nucleotide sequences encoding the unique cell surface marker polypeptide and one of the nucleotide sequences encoding the unique target polypeptide are transcribed on the same mRNA.

[0031] In some embodiments, each recombinant polynucleotide is a bicistronic polynucleotide comprising two promoters and two nucleotide sequences each encoding a unique target polypeptide, and also comprising one or two nucleotide sequences each encoding a unique cell surface marker polypeptide.

[0032] In some embodiments, each bicistronic polynucleotide contains a single nucleotide sequence encoding a unique cell surface marker polypeptide.

[0033] In some embodiments, each bicistronic polynucleotide comprises two nucleotide sequences that encode a unique cell surface marker polypeptide, and each of the nucleotide sequences encoding the unique cell surface marker polypeptide is transcribed on the same mRNA as one of the nucleotide sequences encoding a unique target polypeptide.

[0034] In some embodiments, each recombinant polynucleotide comprises, from its 5' to 3' end, one of one to four promoters, one of one to four nucleotide sequences encoding a unique cell surface marker polypeptide, and one of one to four nucleotide sequences encoding a unique target polypeptide. In some embodiments, each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA.

[0035] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique cell surface marker polypeptide and at the 5' end of the nucleotide sequence encoding the unique target polypeptide.

[0036] In some embodiments, each recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the unique target polypeptide.

[0037] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG.

[0038] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of GTG and TTG.

[0039] In some embodiments, the nucleotide sequence encoding the unique cell surface marker polypeptide lacks any ATG triplet.

[0040] In some embodiments, at least the first recombinant polynucleotide comprises, from the 5' end to the 3' end, a first promoter, a nucleotide sequence encoding a first unique cell surface marker polypeptide, and a nucleotide sequence encoding a first unique target polypeptide; and at least the second recombinant polynucleotide comprises, from the 5' end to the 3' end, a second promoter, a nucleotide sequence encoding a second unique target polypeptide, and a nucleotide sequence encoding a second unique cell surface marker polypeptide.

[0041] In some embodiments, the first recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the first unique cell surface marker polypeptide and the 5' end of the nucleotide sequence encoding the first unique target polypeptide.

[0042] In some embodiments, the first recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the first unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the first unique target polypeptide.

[0043] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG.

[0044] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of GTG and TTG.

[0045] In some embodiments, the nucleotide sequence encoding the first unique cell surface marker polypeptide lacks any ATG triplet.

[0046] In some embodiments, the second recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the second unique target polypeptide and the 5' end of the nucleotide sequence encoding the second unique cell surface marker polypeptide.

[0047] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique target polypeptide, and a nucleotide sequence encoding the unique cell surface marker polypeptide from the 5' end to the 3' end.

[0048] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique target polypeptide and the 5' end of the nucleotide sequence encoding the unique cell surface marker polypeptide.

[0049] In some embodiments, at least one promoter is the β-actin promoter.

[0050] In some embodiments, each promoter is a β-actin promoter.

[0051] In some embodiments, at least one promoter is the hamster β-actin promoter.

[0052] In some embodiments, each promoter is a hamster β-actin promoter.

[0053] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0054] In some embodiments, at least the second unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0055] In some embodiments, each unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0056] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of human CD52 and its variants.

[0057] In some embodiments, at least the second unique cell surface marker peptide is selected from the group consisting of human CD52 and its variants.

[0058] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of human CD52 and its variants.

[0059] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52 and CD59.

[0060] In some embodiments, at least the second unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0061] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0062] In some embodiments, at least the first unique cell surface marker peptide is human CD52.

[0063] In some embodiments, at least the second unique cell surface marker peptide is human CD52.

[0064] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X2 is A.

[0065] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X4 is A.

[0066] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein X7 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X7 is A.

[0067] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 22 (GQNDTSQX 8SSPS), wherein X 8 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X 8 is A.

[0068] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 23 (GQNDTSQX 8X 9SPS), wherein each of X 8 and X 9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8 and X 9 is A.

[0069] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 24 (GQNDTSQX 8SX 10PS), wherein each of X8 and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X8 and X10 is A.

[0070] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 25 (GQNDTSQX 8X 9X 10PS), wherein each of X 8, X 9, and X 10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8, X 9, and X 10 is A.

[0071] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 26 (GX 2NDTSQX 8X 9SPS), wherein each of X2, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X9 is A.

[0072] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS), wherein each of X2, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X10 is A.

[0073] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X9 is A.

[0074] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X10 is A.

[0075] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V.

[0076] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V.

[0077] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0078] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0079] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0080] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0081] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0082] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0083] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0084] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 23 (GX 2NDTSQX 8X 9SPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X4, X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V.

[0085] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X4, X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V.

[0086] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 15 (GANDTSQAASPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 17 (GQNDTSAAASPS).

[0087] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 16 (GANDTSQASAPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 18 (GQNDTSAASAPS).

[0088] In some embodiments, at least one unique target polypeptide comprises a therapeutic polypeptide or a therapeutic protein.

[0089] In some embodiments, at least one unique target polypeptide is a polypeptide of a multi-chain protein.

[0090] In some embodiments, each unique target polypeptide is a polypeptide of a multi-chain protein.

[0091] In some embodiments, at least one unique target polypeptide is an antibody polypeptide.

[0092] In some embodiments, each unique target polypeptide is a polypeptide of an antibody.

[0093] In some embodiments, at least one unique target polypeptide is a polypeptide of a cross-cross dual variable domain (CODV) Ig-like protein.

[0094] In some embodiments, each unique target polypeptide is a polypeptide of a cross-cross dual variable domain (CODV) Ig-like protein.

[0095] In some embodiments, at least one unique target polypeptide is a polypeptide of a CODV triantibody.

[0096] In some embodiments, each unique target polypeptide is a polypeptide of a CODV triantibody.

[0097] In some embodiments, the recombinant mammalian host cell is a CHO cell.

[0098] Another aspect of this disclosure is an engineered mammalian host cell comprising a variety of recombinant polynucleotides, wherein each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are both transcribed on the same mRNA.

[0099] In some embodiments, the plurality of unique target peptides comprises 2 to 8 unique target peptides.

[0100] In some embodiments, the plurality of unique target peptides comprises 2 to 4 unique target peptides.

[0101] In some embodiments, the plurality of unique target peptides includes two unique target peptides.

[0102] In some embodiments, the plurality of unique target peptides includes three unique target peptides.

[0103] In some embodiments, the plurality of unique target peptides includes four unique target peptides.

[0104] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique cell surface marker polypeptide, and a nucleotide sequence encoding the unique target polypeptide from the 5' end to the 3' end.

[0105] In some embodiments, each recombinant polynucleotide comprises 1 to 4 promoters, 1 to 4 nucleotide sequences each encoding a unique cell surface marker polypeptide, and 1 to 4 nucleotide sequences each encoding a unique target polypeptide, wherein one of the nucleotide sequences encoding the unique cell surface marker polypeptide and one of the nucleotide sequences encoding the unique target polypeptide are transcribed on the same mRNA.

[0106] In some embodiments, each recombinant polynucleotide is a bicistronic polynucleotide comprising two promoters and two nucleotide sequences each encoding a unique target polypeptide, and also comprising one or two nucleotide sequences each encoding a unique cell surface marker polypeptide.

[0107] In some embodiments, each bicistronic polynucleotide contains a single nucleotide sequence encoding a unique cell surface marker polypeptide.

[0108] In some embodiments, each bicistronic polynucleotide comprises two nucleotide sequences that encode a unique cell surface marker polypeptide, and each of the nucleotide sequences encoding the unique cell surface marker polypeptide is transcribed on the same mRNA as one of the nucleotide sequences encoding a unique target polypeptide.

[0109] In some embodiments, each recombinant polynucleotide comprises, from its 5' to 3' end, one of one to four promoters, one of one to four nucleotide sequences encoding a unique cell surface marker polypeptide, and one of one to four nucleotide sequences encoding a unique target polypeptide. In some embodiments, each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA.

[0110] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique cell surface marker polypeptide and at the 5' end of the nucleotide sequence encoding the unique target polypeptide.

[0111] In some embodiments, each recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the unique target polypeptide.

[0112] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG.

[0113] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of GTG and TTG.

[0114] In some embodiments, the nucleotide sequence encoding the unique cell surface marker polypeptide lacks any ATG triplet.

[0115] In some embodiments, at least the first recombinant polynucleotide comprises, from the 5' end to the 3' end, a first promoter, a nucleotide sequence encoding a first unique cell surface marker polypeptide, and a nucleotide sequence encoding a first unique target polypeptide; and at least the second recombinant polynucleotide comprises, from the 5' end to the 3' end, a second promoter, a nucleotide sequence encoding a second unique target polypeptide, and a nucleotide sequence encoding a second unique cell surface marker polypeptide.

[0116] In some embodiments, the first recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the first unique cell surface marker polypeptide and the 5' end of the nucleotide sequence encoding the first unique target polypeptide.

[0117] In some embodiments, the first recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the first unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the first unique target polypeptide.

[0118] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG.

[0119] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of GTG and TTG.

[0120] In some embodiments, the nucleotide sequence encoding the first unique cell surface marker polypeptide lacks any ATG triplet.

[0121] In some embodiments, the second recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the second unique target polypeptide and the 5' end of the nucleotide sequence encoding the second unique cell surface marker polypeptide.

[0122] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique target polypeptide, and a nucleotide sequence encoding the unique cell surface marker polypeptide from the 5' end to the 3' end.

[0123] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique target polypeptide and the 5' end of the nucleotide sequence encoding the unique cell surface marker polypeptide.

[0124] In some embodiments, at least one promoter is the β-actin promoter.

[0125] In some embodiments, each promoter is a β-actin promoter.

[0126] In some embodiments, at least one promoter is the hamster β-actin promoter.

[0127] In some embodiments, each promoter is a hamster β-actin promoter.

[0128] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0129] In some embodiments, at least the second unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0130] In some embodiments, each unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0131] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of human CD52 and its variants.

[0132] In some embodiments, at least the second unique cell surface marker peptide is selected from the group consisting of human CD52 and its variants.

[0133] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of human CD52 and its variants.

[0134] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52 and CD59.

[0135] In some embodiments, at least the second unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0136] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0137] In some embodiments, at least the first unique cell surface marker peptide is human CD52.

[0138] In some embodiments, at least the second unique cell surface marker peptide is human CD52.

[0139] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X2 is A.

[0140] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X4 is A.

[0141] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein X7 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X7 is A.

[0142] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 22 (GQNDTSQX 8SSPS), wherein X 8 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X 8 is A.

[0143] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 23 (GQNDTSQX 8X 9SPS), wherein each of X 8 and X 9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8 and X 9 is A.

[0144] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 24 (GQNDTSQX 8SX 10PS), wherein each of X8 and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X8 and X10 is A.

[0145] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 25 (GQNDTSQX 8X 9X 10PS), wherein each of X 8, X 9, and X 10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8, X 9, and X 10 is A.

[0146] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 26 (GX 2NDTSQX 8X 9SPS), wherein each of X2, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X9 is A.

[0147] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS), wherein each of X2, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X10 is A.

[0148] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X9 is A.

[0149] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X10 is A.

[0150] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V.

[0151] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V.

[0152] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0153] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0154] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0155] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0156] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0157] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0158] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0159] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 23 (GX 2NDTSQX 8X 9SPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X4, X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V.

[0160] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X4, X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V.

[0161] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 15 (GANDTSQAASPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 17 (GQNDTSAAASPS).

[0162] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 16 (GANDTSQASAPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 18 (GQNDTSAASAPS).

[0163] In some embodiments, at least one unique target polypeptide comprises a therapeutic polypeptide or a therapeutic protein.

[0164] In some embodiments, at least one unique target polypeptide is a polypeptide of a multi-chain protein.

[0165] In some embodiments, each unique target polypeptide is a polypeptide of a multi-chain protein.

[0166] In some embodiments, at least one unique target polypeptide is an antibody polypeptide.

[0167] In some embodiments, each unique target polypeptide is a polypeptide of an antibody.

[0168] In some embodiments, at least one unique target polypeptide is a polypeptide of a cross-cross dual variable domain (CODV) Ig-like protein.

[0169] In some embodiments, each unique target polypeptide is a polypeptide of a cross-cross dual variable domain (CODV) Ig-like protein.

[0170] In some embodiments, at least one unique target polypeptide is a polypeptide of a CODV triantibody.

[0171] In some embodiments, each unique target polypeptide is a polypeptide of a CODV triantibody.

[0172] In some embodiments, the recombinant mammalian host cell is a CHO cell.

[0173] Another aspect of this disclosure is an isolated human CD52 variant comprising the amino acid sequence of SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X2 is A.

[0174] Another aspect of this disclosure is an isolated human CD52 variant comprising the amino acid sequence of SEQ ID NO: 20 (GQNX 4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X4 is A.

[0175] Another aspect of this disclosure is an isolated human CD52 variant comprising the amino acid sequence of SEQ ID NO: 22 (GQNDTSQX 8SSPS), wherein X 8 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X 8 is A. Simple Explanation of the Diagram

[0176] Figure 1 is a schematic depiction of the recombinant polynucleotides and corresponding mRNA transcripts according to this disclosure. Only a single representative recombinant polynucleotide is shown in this depiction. The nucleotide sequence encoding a unique cell surface marker polypeptide (reporter) has a TTG start codon, and the nucleotide sequence encoding a unique target polypeptide (therapeutic protein) has an ATG start codon.

[0177] Figure 2A depicts the binding of mAb 4B10 and 7F11 to CHO cells expressing wild-type hCD52 (WT) and each of 10 hCD52 single-point mutants, with each mutant replacing the indicator amino acid of WT with alanine (A). Binding with untransfected CHO cells (CHO parent) is also shown.

[0178] Figure 2B depicts the binding of mAb CF1D12, 5F7, 3G7, 4G7, 9D9, and 11C11 to CHO cells expressing wild-type hCD52 (WT) and each of 10 hCD52 single-point mutants, with each mutant replacing the indicator amino acid of WT with alanine (A). Binding with untransfected CHO cells (CHO parent) is also shown.

[0179] Figure 2C depicts the binding of mAb Campath-1H, 2C3, 12G6, and 23E6 to CHO cells expressing wild-type hCD52 (WT) and each of 10 hCD52 single-point mutants, with each mutant replacing the indicator amino acid of WT with alanine (A). Binding with untransfected CHO cells (CHO parent) is also shown.

[0180] Figure 3 depicts the sequence alignment of natural (wild-type) hCD52 (SEQ ID NO: 30) and the engineered mutant hCD52 (Mut4; SEQ ID NO: 31), the latter including a non-AUG (alternative) start codon with optimal Kozak context, mutations in all internal ATG codons, eliminated splice sites, and a single-point mutation at amino acid 4 in hCD52 where aspartic acid is replaced by alanine. Mut4 is bound by Camppath-1H (C-1H) but not by mAb 9D9. The partial amino acid sequence shown corresponds to SEQ ID NO: 4.

[0181] Figure 4 depicts the sequence alignments of natural (wild-type) hCD52 (SEQ ID NO: 30) and the engineered mutant hCD52 (Mut8; SEQ ID NO: 32), the latter including a non-AUG (substitutional) start codon with optimal Kozak background, mutations in all internal ATG codons, eliminated splice sites, and a single-point mutation at amino acid 8 in hCD52 where alanine replaces threonine. Mut8 is bound by mAb 9D9 but not by Camppath-1H (C-1H). The partial amino acid sequence shown corresponds to SEQ ID NO: 8.

[0182] Figure 5 depicts the sequence alignment of natural (wild-type) hCD52 (SEQ ID NO: 30) and the engineered mutant hCD52 (Mut8 / 10; SEQ ID NO: 33), the latter including a non-AUG (substitution) start codon with optimal Kozak background, mutations in all internal ATG codons, eliminated splice sites, and two point mutations: alanine substitution for threonine at amino acid 8 and alanine substitution for serine at amino acid 10 in hCD52. Mut8 / 10 is bound by mAb 9D9 but not by Camppath-1H (C-1H). The partial amino acid sequence shown corresponds to SEQ ID NO: 13.

[0183] Figure 6 shows four flow cytometry bars depicting the binding of Campath-1H or 9D9 to indicated hCD52 mutants, where the polynucleotide encoding each hCD52 mutant has the ATG start codon.

[0184] Figure 7 shows four flow cytometry bar graphs depicting the binding of Campath-1H or 9D9 to indicated hCD52 mutants, where the polynucleotide encoding each hCD52 mutant has the ATG start codon.

[0185] Figure 8 shows four flow cytometry bar graphs depicting the binding of Camphor-1H or 9D9 to indicated hCD52 mutants, where the polynucleotide encoding each hCD52 mutant has an indicated start codon (ATG, CTG, GTG, or TTG).

[0186] Figure 9 is a pair of bar charts depicting the relative binding of Camppath-1H or 9D9 with Mut4 (left) and Mut8 (right), where each hCD52 mutant is encoded by a polynucleotide with an indicated start codon (ATG, CTG, GTG, or TTG).

[0187] Figure 10 is a flow cytometry bar graph depicting the relative levels of reporter and target gene (GOI) expression, depending on the choice of the reporter's start codon (ATG / AUG, CTG / CUG, GTG, or TTG). The depicted partial sequences correspond to SEQ ID NO: 35 to 38.

[0188] Figure 11 is a set of bar charts depicting the protocol of a three-reporter system using indicated hCD52 mutants (Mut4, Mut2 / 8 / 9, and Mut7 / 8 / 9) and indicated antibodies (7F11, 9D9, and Camppath-1H).

[0189] Figure 12 is a set of bar charts depicting the protocol of a three-reporter system using indicated hCD52 mutants (Mut4, Mut2 / 8 / 10, and Mut7 / 8 / 10) and indicated antibodies (7F11, 9D9, and Camppath-1H).

[0190] Figure 13A is a schematic diagram depicting four vectors encoding the CODV triantibody. Two reporter substances are present in this system.

[0191] Figure 13B is a schematic diagram depicting two bicistronic vectors encoding a CODV triantibody. Two reporter molecules are present in this system. Implementation

[0192] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by way of identification. The disclosures of these publications, patents, and published patent specifications are hereby incorporated into this disclosure by reference to provide a more comprehensive description of the prior art in the field to which this invention pertains.

[0193] As used herein, specific terms have the meanings defined below.

[0194] [I.] [definition]

[0195] Unless otherwise indicated, the practice of this invention will employ conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols in Molecular Biology (edited by FM Ausubel et al., (1987)); series: Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (edited by MJ MacPherson, BD Hames, and GR Taylor, (1995)), Harlow and Lane (edited by Harlow and Lane, (1988); Antibodies, A Laboratory Manual; and Animal Cell Culture (edited by RI Freshney, (1987)).

[0196] Unless the context clearly indicates otherwise, as used in the specification and claims, the singular forms "a / an" and "the" include plural indicators. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

[0197] As used herein, the term "comprising" is intended to mean that a composition and method includes the listed elements, but does not exclude other elements. When used to define compositions and methods, "consisting substantially of" should mean excluding other elements that are of any significant importance to the composition. Thus, a composition consisting substantially of elements as defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers (such as phosphate-buffered saline), preservatives, etc. "Containing" will mean excluding elements that are more than trace amounts of other components as well as the substantial method steps for administering the compositions of the invention. Examples defined by each of these transitional terms are within the scope of the invention.

[0198] As used herein, the term "polynucleotide" or "nucleotide sequence" refers to any form of nucleotide polymer, examples of which include, but are not limited to, genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, complementary DNA (cDNA), recombinant polynucleotides, branched polynucleotides, plastids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs.

[0199] As used herein, the term "peptide" refers to a polymeric form of an amino acid or amino acid analog of any length, including, but not limited to, full-length single-chain polypeptides, full-length single-chain proteins, fragments of full-length single-chain polypeptides, fragments of full-length single-chain proteins; full-length multi-chain polypeptides, full-length multi-chain proteins, fragments of full-length multi-chain polypeptides, fragments of full-length multi-chain proteins; and full-length single-chain multi-chain polypeptides, full-length single-chain multi-chain proteins, fragments of single-chain multi-chain polypeptides, and fragments of single-chain multi-chain proteins.

[0200] As used herein, "multi-chain polypeptide" or "multi-chain protein" refers to any polypeptide or protein composed of two or more single-chain polypeptides. The two or more single-chain polypeptides may associate via any combination of covalent bonds (e.g., disulfide bonds) and / or non-covalent bonds (e.g., ionic bonds or hydrogen bonds). For example, a conventional IgG antibody consists of two heavy chains linked by one or more disulfide bonds in a hinge region, and two light chains, each light chain linked to a single heavy chain by one or more disulfide bonds between its respective variable light chain (VL) and variable heavy chain (VH) domains. As another example, a CODV triantibody consists of two distinct heavy chains (a CODV heavy chain and a Fab heavy chain, having a pestle and mortar relationship) and two distinct light chains (a CODV light chain and a Fab light chain).

[0201] Other examples of multi-chain proteins (also known as multimeric proteins) include homomeric and heteromeric multimers, including but not limited to certain enzymes, ion channels, receptors, cell adhesion molecules, voltage-gated potassium channels, and therapeutic proteins (e.g., insulin and α-galactosidase). Many soluble proteins and membrane proteins form homomeric complexes in cells, and most proteins in protein databases are homomeric. Approximately 65% ​​of proteins in prokaryotes and 55% of proteins in eukaryotes exist as dimers or higher-order complexes (excluding very high-order structures such as the cytoskeleton, ribosomes, etc.). In complexes, homomers are about four times more frequent than heteromers in unicellular species, while the two types are equally frequent in vertebrates. Thus, heteromers constitute about 10% of proteins in unicellular species and nearly 30% in vertebrates. Lynch M, Mol Biol Evol.29(5): 1353-1366 (2012). Homo-oligomers are responsible for the diversity and specificity of many pathways, and they may mediate and regulate gene expression, enzyme activity, ion channels, receptors, and cell adhesion processes. Voltage-gated potassium channels in the plasma membrane of neurons are heteropolymeric proteins composed of four of the forty known α subunits.

[0202] As used herein, the term "IRES" or "polynucleotide with IRES bioactivity" is intended to include any molecule, such as a polynucleotide or its reverse transcript, capable of initiating the translation of a polynucleotide operatively linked to an IRES without the benefit of a cap site in a eukaryotic cell. IRES or polynucleotides with IRES bioactivity may be identical to sequences found in nature, such as microRNA viruses IRES, or they may be non-naturally occurring or unnatural sequences that perform the same function when introduced into a suitable host cell.

[0203] As used herein, the term "alternative start codon" is intended to include any non-ATG polynucleotide (typically a triplet) that serves as a start site for translation initiation and has reduced efficiency relative to the ATG start codon. The use of naturally occurring alternative start codons is known in the art and described, for example, in Kozak (1991) J Cell Biol. 115(4): 887-903; Mehdi et al. (1990) Gene91: 173-178; Kozak (1989) Mol Cell Biol. 9(11): 5073-5080. Generally, alternative start codons have reduced translation efficiency compared to ATG; for example, the alternative start codon GTG may have 3% to 5% translation efficiency compared to the ATG translation efficiency (100%). The translation efficiency of alternative start codons may also be affected by their sequence background: for example, it has been reported that the optimal Kozak common sequence has a positive impact on the translation initiation of alternative start codons. Mehdi et al. (1990) Gene91:173-178; Kozak (1989) Mol Cell Biol.9(11):5073-5080. The complete Kozak common sequence is GCCRCC. [ATG]G (SEQ ID NO: 34), where the start codon ATG is in bold, and the A in the ATG start codon is specified as... The "R" at position [+]1 and position -3 is a purine (A or G). These two most conserved positions are purines, preferably A at -3 and G at +4 (Kozak (1991) J Cell Biol. 115(4): 887-903). Alternative start codons for selecting markers with reduced performance are described in U.S. Patent Publication No. 2006 / 0172382 and U.S. Patent Publication No. 2006 / 0141577. Those skilled in the art will recognize that sequences described herein as DNA will have corresponding sequences as RNA molecules; for example, the DNA sequence ATG will correspond to the RNA sequence AUG.

[0204] As used in this article, "high performance level" means a performance level that is higher than at least 50%, 70%, 80%, 90%, 95%, or 99% of the total cells analyzed.

[0205] As used herein, "CD20" refers to a four-transmembrane protein expressed on the surface of B cells, beginning in the pre-B cell stage and also expressed on mature B cells in the bone marrow and periphery. CD20 presents three surface-available antigenic regions. The cDNA and amino acid sequences of human and mouse CD20 are available from GenBank, e.g., accessions NM_152866, NM_021950, and NM_152867 (human); NM_007641 (mouse); NP_068769, NP_690605, and NP_690606 (human); and NP_031667 (mouse). The full-length human CD20 is 297 amino acids long, and the full-length mouse CD20 is 291 amino acids long.

[0206] CD52 (also known as the Camphor-1H antigen) is a short glycoprotein expressed on mature lymphocytes; although its function is not fully understood, it is thought to inhibit cell adhesion. The cDNA and amino acid sequences of human and mouse CD52 are available from GenBank, for example, accession numbers BC000644 (human); NM_013706 (mouse); AAH00644 (human); and EDL30035 (mouse). The full-length human CD52 is 61 amino acids long, including a 42-amino acid message sequence, while the full-length mouse CD52 is 74 amino acids long, including a 23-amino acid message sequence.

[0207] CD59 (also known as the membrane attack complex (MAC) inhibitor and protectin) is a cell surface glycoprotein that inhibits the polymerization of complement component C9 and the formation of the membrane attack complex. The cDNA and amino acid sequences of human and mouse CD59 are available from GenBank, for example, accessions NM_203331, NM_0006111, NM_001127223, NM_001127225, and NM_001127226 (human); NM_181858 and NM_001368215 (mouse); NP_000602, NP_001120695, NP_001120697, NP_001120698, and NP_001120699 (human); and NP_862906 and NP_001355144 (mouse). The full-length human CD59 is 128 amino acids long, including a message sequence of 25 amino acids, while the full-length mouse CD59 is 129 amino acids long, including a message sequence of 23 amino acids.

[0208] As used herein, "therapeutic peptide" or "therapeutic protein" means any protein or peptide that can be produced in a host cell and in the aspects illustrated herein, selected for its potential as a therapeutic agent or drug, such as antibodies, antibody fragments, antibody-like molecules (e.g., cross-crossed dual variable domain (CODV) Ig-like proteins) or enzymes.

[0209] [II.] [method]

[0210] One aspect of this disclosure is a method for representing multiple target peptides at a high level, the method comprising: (a) Culture multiple mammalian host cells, each containing multiple recombinant polynucleotides, among which Each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA. The culture was conducted under conditions that allowed each unique cell surface marker peptide to be expressed on the surface of mammalian host cells, as well as the expression of each unique target peptide. (b) Contact the cultured mammalian host cells of step (a) with a variety of detectable agents, each of which is capable of uniquely binding to one or more of the unique cell surface marker peptides expressed on the surface of the mammalian host cells; (c) Perform at least one round of fluorescence-activated cell sorting on the contacted cells from step (b) to select one or more mammalian host cells that are uniquely bound to at least one of the plurality of detectable agents; (d) Prepare one or more colony populations of the mammalian host cells selected in step (c); (e) Analyze one or more colonies from step (d) by detecting the performance levels of at least two unique cell surface marker peptides in each colony population; (f) Select one or more colonies exhibiting high levels of performance with at least two unique cell surface marker peptides; and (g) Cultivate one or more colonies selected in step (f) under conditions that allow for high-level expression of the various target peptides.

[0211] The plurality of mammalian host cells may be a population of mammalian host cells, such as a population of mammalian cell lines. In step (a), the plurality of cells or cell populations typically include 100 or more, 500 or more, 1,000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, or 10⁶ or more cells. The cells are typically all of one type, such as CHO cells, HEK-293 cells, BHK-21 cells, HepG2 cells, BAE-1 cells, SH-SY5Y cells, myeloma cells, hybridoma cells, HeLa cells, Vero cells, NIH3T3 cells, WEHI231 cells, YAC cells, Jurkat cells and their derivatives, such as CHO-K1 cells and CHO / DHFR- cells.

[0212] Cultures that allow each unique cell surface marker peptide to be expressed on the surface of mammalian host cells, and for each unique target peptide to be expressed, may include, for example, typical conditions for mammalian cells in tissue culture, such as at 37ºC in humidified air supplemented with 5% CO2 in a suitable medium (e.g., DMEM, Ham's F12, or serum-free medium, optionally supplemented with bovine fetal serum (FCS) or fetal bovine serum (FBS), L-glutamic acid, and penicillin / streptomycin). The culture can be performed in any suitable manner, such as multi-well plate culture, petri dish culture, mass culture, or bioreactor culture. The culture allows time for the expression of cell surface marker peptides, and also time for cell population expansion.

[0213] The plurality of recombinant polynucleotides includes two or more different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 2 different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 3 different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 4 different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 5 different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 6 different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 7 different recombinant polynucleotides. In some embodiments, the plurality of recombinant polynucleotides includes 8 different recombinant polynucleotides.

[0214] Of course, each mammalian host cell can contain more than a single copy of each recombinant polynucleotide. For example, each mammalian host cell can contain approximately 10¹ to approximately 10⁶ copies of each recombinant polynucleotide.

[0215] Additionally, each mammalian host cell may contain approximately the same or different numbers of copies of each recombinant polynucleotide. In some embodiments, it is preferred that each mammalian host cell contains approximately the same number of copies of at least two different recombinant polynucleotides. In some embodiments, it is preferred that each mammalian host cell contains approximately the same number of copies of each recombinant polynucleotide. In some other embodiments, it is preferred that each mammalian host cell contains different numbers of copies of at least two different recombinant polynucleotides. In some other embodiments, it is preferred that each mammalian host cell contains different numbers of copies of each recombinant polynucleotide. The number of copies of each different recombinant polynucleotide may vary and be selected based on the conditions used to introduce the different recombinant polynucleotides into the host mammalian cell.

[0216] Each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA.

[0217] Because each recombinant polynucleotide contains a nucleotide sequence encoding a unique cell surface marker polypeptide, and because both the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA, each mammalian host cell can express as many unique (i.e., different) cell surface marker polypeptides as the number of different recombinant polynucleotides. For example, mammalian host cells transfected with two different recombinant polynucleotides can express two unique (i.e., different) cell surface marker polypeptides. Similarly, mammalian host cells transfected with three different recombinant polynucleotides can express three unique (i.e., different) cell surface marker polypeptides, and so on. Regardless of the number of nucleotide sequences encoding unique (i.e., different) cell surface marker polypeptides, the expression of each such cell surface marker polypeptide will be associated with the expression of the corresponding unique (i.e., different) target polypeptide.

[0218] Similarly, because each recombinant polynucleotide contains a nucleotide sequence encoding a unique cell surface marker polypeptide, and because both the nucleotide sequence encoding the unique target polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA, each mammalian host cell can express as many unique (i.e., different) target polypeptides as different recombinant polynucleotides. For example, mammalian host cells transfected with two different recombinant polynucleotides can express two unique (i.e., different) target polypeptides. Similarly, mammalian host cells transfected with three different recombinant polynucleotides can express three unique (i.e., different) target polypeptides, and so on. Regardless of the number of nucleotide sequences encoding unique (i.e., different) target polypeptides, the expression of each such target polypeptide will be associated with the expression of the corresponding unique (i.e., different) cell surface marker polypeptide.

[0219] Step (b) involves contacting the cultured mammalian host cells of step (a) with a plurality of detectable agents, each detectable agent capable of uniquely binding to one or more of the unique cell surface marker peptides expressed on the surface of the mammalian host cells. In some embodiments, at least one unique detectable agent can bind to only one unique cell surface marker peptide. In some embodiments, each unique detectable agent can bind to only one unique cell surface marker peptide. In some embodiments, at least one unique detectable agent can bind to more than one unique cell surface marker peptide; for example, such a unique detectable agent may be capable of binding to two different (i.e., unique) cell surface marker peptides.

[0220] Step (c) involves performing at least one round of fluorescence-activated cell sorting (FACS) on the contacted cells from step (b) to select one or more mammalian host cells that are uniquely bound to at least one of the plurality of detectable agents. In some embodiments, step (c) includes performing a single round of FACS on the cells from step (b) that have been contacted with at least a first detectable agent and a second detectable agent to select one or more mammalian host cells that are uniquely bound to both the first and second detectable agents. In some embodiments, step (c) includes performing a single round of FACS on the cells from step (b) that have been contacted with three or more detectable agents to select one or more mammalian host cells that are uniquely bound to the three or more detectable agents. In some embodiments, step (c) includes performing a single round of FACS on the cells from step (b) that have been contacted with three detectable agents to select one or more mammalian host cells that are uniquely bound to the three detectable agents.

[0221] In some embodiments, step (c) includes performing a single round of FACS on cells from step (b) that have been contacted with four detectable agents, thereby selecting one or more mammalian host cells that uniquely bind to the four detectable agents. In some embodiments, step (c) includes performing a single round of FACS on cells from step (b) that have been contacted with five detectable agents, thereby selecting one or more mammalian host cells that uniquely bind to the five detectable agents. In some embodiments, step (c) includes performing a single round of FACS on cells from step (b) that have been contacted with six detectable agents, thereby selecting one or more mammalian host cells that uniquely bind to the six detectable agents. In some embodiments, step (c) includes performing a single round of FACS on cells from step (b) that have been contacted with seven detectable agents, thereby selecting one or more mammalian host cells that uniquely bind to the seven detectable agents. In some embodiments, step (c) includes performing a single round of FACS on cells from step (b) that have been in contact with the eight detectable agents, thereby selecting one or more mammalian host cells that uniquely bind to the eight detectable agents.

[0222] In some embodiments, step (c) includes (c1) Performing a first round of fluorescence-activated cell sorting on at least a first cell surface-labeled polypeptide to select one or more mammalian host cells bound to at least a first detectable agent; and (c2) Perform a second round of fluorescence-activated cell sorting on the cells selected in step (c1) for at least a second cell surface labeled peptide, thereby selecting one or more mammalian host cells that are bound to at least the first detectable agent and the second detectable agent.

[0223] Of course, in some embodiments, several additional rounds of FACS are performed to select one or more mammalian host cells that are bound to any number of specific detectable agents. For example, in some embodiments, step (c) further includes... (c3) Perform a third round of FACS on the cells selected in step (c2) against at least a third cell surface marker peptide, thereby selecting one or more mammalian host cells that are bound to at least the first, second and third detectable agents.

[0224] Similarly, in some embodiments, step (c) further includes (c4) Perform a fourth round of FACS on the cells selected in step (c3) against at least a fourth cell surface marker peptide, thereby selecting one or more mammalian host cells that are bound by at least the first, second, third and fourth detectable agents.

[0225] In some embodiments, step (e) is performed 7 to 28 days after step (d). In some embodiments, step (e) is performed 7 to 21 days after step (d). In some embodiments, step (e) is performed 7 to 14 days after step (d). In some embodiments, step (e) is performed 7 to 13 days after step (d). In some embodiments, step (e) is performed 7 to 12 days after step (d). In some embodiments, step (e) is performed 7 to 11 days after step (d). In some embodiments, step (e) is performed 7 to 10 days after step (d). In some embodiments, step (e) is performed 7 to 9 days after step (d). In some embodiments, step (e) is performed 7 to 8 days after step (d). In some embodiments, step (e) is performed 7 to 9 days after step (d). In some embodiments, step (e) is performed 7 days after step (d).

[0226] In some embodiments, step (e) is performed 8 to 28 days after step (d). In some embodiments, step (e) is performed 8 to 21 days after step (d). In some embodiments, step (e) is performed 8 to 14 days after step (d). In some embodiments, step (e) is performed 8 to 13 days after step (d). In some embodiments, step (e) is performed 8 to 12 days after step (d). In some embodiments, step (e) is performed 8 to 11 days after step (d). In some embodiments, step (e) is performed 8 to 10 days after step (d). In some embodiments, step (e) is performed 8 to 9 days after step (d). In some embodiments, step (e) is performed 8 days after step (d).

[0227] In some embodiments, step (e) is performed 9 to 28 days after step (d). In some embodiments, step (e) is performed 9 to 21 days after step (d). In some embodiments, step (e) is performed 9 to 14 days after step (d). In some embodiments, step (e) is performed 9 to 13 days after step (d). In some embodiments, step (e) is performed 9 to 12 days after step (d). In some embodiments, step (e) is performed 9 to 11 days after step (d). In some embodiments, step (e) is performed 9 to 10 days after step (d). In some embodiments, step (e) is performed 9 days after step (d).

[0228] In some embodiments, step (e) is performed 10 to 28 days after step (d). In some embodiments, step (e) is performed 10 to 21 days after step (d). In some embodiments, step (e) is performed 10 to 14 days after step (d). In some embodiments, step (e) is performed 10 to 13 days after step (d). In some embodiments, step (e) is performed 10 to 12 days after step (d). In some embodiments, step (e) is performed 10 to 11 days after step (d). In some embodiments, step (e) is performed 10 days after step (d).

[0229] In some embodiments, step (e) is performed 11 to 28 days after step (d). In some embodiments, step (e) is performed 11 to 21 days after step (d). In some embodiments, step (e) is performed 11 to 14 days after step (d). In some embodiments, step (e) is performed 11 to 13 days after step (d). In some embodiments, step (e) is performed 11 to 12 days after step (d). In some embodiments, step (e) is performed 11 days after step (d).

[0230] In some embodiments, step (e) is performed 12 to 28 days after step (d). In some embodiments, step (e) is performed 12 to 21 days after step (d). In some embodiments, step (e) is performed 12 to 14 days after step (d). In some embodiments, step (e) is performed 12 to 13 days after step (d). In some embodiments, step (e) is performed 12 days after step (d).

[0231] In some embodiments, step (e) is performed 13 to 28 days after step (d). In some embodiments, step (e) is performed 13 to 21 days after step (d). In some embodiments, step (e) is performed 13 to 14 days after step (d). In some embodiments, step (e) is performed 13 days after step (d).

[0232] In some embodiments, step (e) is performed 14 to 28 days after step (d). In some embodiments, step (e) is performed 14 to 21 days after step (d). In some embodiments, step (e) is performed 14 days after step (d).

[0233] In some embodiments, step (e) is performed 15 to 28 days after step (d). In some embodiments, step (e) is performed 15 to 21 days after step (d). In some embodiments, step (e) is performed 15 days after step (d).

[0234] In some embodiments, step (e) is performed 16 to 28 days after step (d). In some embodiments, step (e) is performed 16 to 21 days after step (d). In some embodiments, step (e) is performed 16 days after step (d).

[0235] In some embodiments, step (e) is performed 17 to 28 days after step (d). In some embodiments, step (e) is performed 17 to 21 days after step (d). In some embodiments, step (e) is performed 17 days after step (d).

[0236] In some embodiments, step (e) is performed 18 to 28 days after step (d). In some embodiments, step (e) is performed 18 to 21 days after step (d). In some embodiments, step (e) is performed 18 days after step (d).

[0237] In some embodiments, step (e) is performed 19 to 28 days after step (d). In some embodiments, step (e) is performed 19 to 21 days after step (d). In some embodiments, step (e) is performed 19 days after step (d).

[0238] In some embodiments, step (e) is performed 20 to 28 days after step (d). In some embodiments, step (e) is performed 20 to 21 days after step (d). In some embodiments, step (e) is performed 20 days after step (d).

[0239] In some embodiments, step (e) is performed 21 to 28 days after step (d). In some embodiments, step (e) is performed 21 days after step (d).

[0240] In some embodiments, step (e) is performed 22 to 28 days after step (d). In some embodiments, step (e) is performed 22 days after step (d).

[0241] In some embodiments, step (e) is performed 23 to 28 days after step (d).

[0242] In some embodiments, step (e) is performed 24 to 28 days after step (d). In some embodiments, step (e) is performed 24 days after step (d).

[0243] In some embodiments, step (e) is performed 25 to 28 days after step (d). In some embodiments, step (e) is performed 25 days after step (d).

[0244] In some embodiments, step (e) is performed 26 to 28 days after step (d). In some embodiments, step (e) is performed 26 days after step (d).

[0245] In some embodiments, step (e) is performed 27 to 28 days after step (d).

[0246] In some embodiments, step (e) is performed 28 days after step (d).

[0247] In some embodiments, the analysis in step (e) includes flow cytometry. In some embodiments, the flow cytometry analysis in step (e) includes cell sorting. In some other embodiments, the flow cytometry analysis in step (e) does not include cell sorting.

[0248] In some embodiments, at least one of the multiple unique cell surface marker peptides in step (f) exhibits a higher performance level than the corresponding performance level of at least 70% of the colony population analyzed in step (e). For example, if one of the multiple unique cell surface marker peptides is CD52, then the performance level of CD52 in step (f) may be higher than the CD52 performance level of at least 70% of the colony population analyzed in step (e). In some embodiments, at least one of the multiple unique cell surface marker peptides in step (f) is a single unique cell surface marker. In some other embodiments, at least one of the multiple unique cell surface marker peptides in step (f) is two unique cell surface marker peptides. In some other embodiments, at least one of the multiple unique cell surface marker peptides in step (f) is three unique cell surface marker peptides, four unique cell surface marker peptides, etc. The same principle applies to any number of unique cell surface marker peptides.

[0249] In some embodiments, each of the multiple unique cell surface marker peptides in step (f) exhibits a higher performance level than the corresponding performance level of at least 70% of the colony population analyzed in step (e).

[0250] In some embodiments, at least a first of the multiple unique cell surface marker peptides in step (f) exhibits a higher performance level than at least a second of the multiple unique cell surface marker peptides in step (f). For example, if one of the multiple unique cell surface marker peptides is a first CD52 variant and another of the multiple unique cell surface marker peptides is a second CD52 variant, then the performance level of at least the first CD52 variant in step (f) may be higher than the performance level of at least the second CD52 variant in step (f). Similarly, if one of the multiple unique cell surface marker peptides is a first CD52 variant, another of the multiple unique cell surface marker peptides is a second CD52 variant, and yet another of the multiple unique cell surface marker peptides is a third CD52 variant, then the performance level of at least the first CD52 variant in step (f) may be higher than the performance levels of both the second CD52 variant and the third CD52 variant in step (f). As another example, if one of the plurality of unique cell surface marker peptides is a first CD52 variant, another of the plurality of unique cell surface marker peptides is a second CD52 variant, and yet another of the plurality of unique cell surface marker peptides is a third CD52 variant, then in step (f) at least the performance level of the first CD52 variant and the performance level of the second CD52 variant can be higher than the performance level of the third CD52 variant in step (f). The same principle applies to any number of unique cell surface marker peptides.

[0251] In some embodiments, the first of the multiple unique cell surface peptides in step (f) exhibits a higher performance level than the second of the multiple unique cell surface peptides in step (f).

[0252] In some embodiments, the method further includes:

[0253] (h) Isolate at least the first and second unique target peptides exhibited in step (g) from one or more selected colonies or from the cell culture medium in which the one or more selected colonies are cultured. Any suitable method for isolating or purifying the target peptides exhibiting the desired performance can be used. For example, in some embodiments, the isolation may involve affinity chromatography, gel filtration / particle size exclusion, hydrophobic interaction chromatography, immunoprecipitation, ion exchange chromatography, or any combination thereof. For secreted target peptides, the isolation or purification may be performed directly from the cell culture medium or from dialysate and / or from its concentrate. For other target peptides, the isolation or purification may include a cell lysis step or a membrane disruption step. Such general isolation and purification methods are well known to those skilled in the art.

[0254] In some embodiments, the plurality of unique target peptides comprises 2 to 8 unique target peptides.

[0255] In some embodiments, the plurality of unique target peptides comprises 2 to 4 unique target peptides.

[0256] In some embodiments, the plurality of unique target peptides includes two unique target peptides.

[0257] In some embodiments, the plurality of unique target peptides includes three unique target peptides.

[0258] In some embodiments, the plurality of unique target peptides includes four unique target peptides.

[0259] In some embodiments, the plurality of unique target peptides includes five unique target peptides.

[0260] In some embodiments, the plurality of unique target peptides includes six unique target peptides.

[0261] In some embodiments, the plurality of unique target peptides includes seven unique target peptides.

[0262] In some embodiments, the plurality of unique target peptides comprises eight unique target peptides.

[0263] In some embodiments, the plurality of unique target peptides comprises eight or more unique target peptides.

[0264] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique cell surface marker polypeptide, and a nucleotide sequence encoding the unique target polypeptide from the 5' end to the 3' end.

[0265] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique cell surface marker polypeptide and at the 5' end of the nucleotide sequence encoding the unique target polypeptide.

[0266] In some embodiments, each recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the unique target polypeptide.

[0267] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG. That is, the alternative (non-ATG) start codons may be the same or different; they may be partially or completely the same, or they may be partially or completely different.

[0268] In some embodiments, at least one alternative start codon is CTG.

[0269] In some embodiments, at least one alternative start codon is GTG.

[0270] In some embodiments, at least one alternative start codon is TTG.

[0271] In some embodiments, at least one alternative start codon is ATT.

[0272] In some embodiments, at least one alternative start codon is ATA.

[0273] In some embodiments, at least one alternative start codon is ACG.

[0274] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of GTG and TTG. In some embodiments, at least one alternative start codon is GTG. In some embodiments, at least one alternative start codon is TTG. In some embodiments, each alternative (non-ATG) start codon is GTG. In some other embodiments, each alternative (non-ATG) start codon is TTG.

[0275] In some embodiments, the nucleotide sequence encoding the unique cell surface marker polypeptide lacks any ATG triplet.

[0276] In some embodiments, at least a first recombinant polynucleotide comprises, from its 5' end to its 3' end, a first promoter, a nucleotide sequence encoding a first unique cell surface marker polypeptide, and a nucleotide sequence encoding a first unique target polypeptide; and at least a second recombinant polynucleotide comprises, from its 5' end to its 3' end, a second promoter, a nucleotide sequence encoding a second unique target polypeptide, and a nucleotide sequence encoding a second unique cell surface marker polypeptide. As described above, this disclosure also contemplates embodiments that may include, but are not limited to, at least a third recombinant polynucleotide having the corresponding element, at least a fourth recombinant polynucleotide having the corresponding element, at least a fifth recombinant polynucleotide having the corresponding element, at least a sixth recombinant polynucleotide having the corresponding element, at least a seventh recombinant polynucleotide having the corresponding element, and at least an eighth recombinant polynucleotide having the corresponding element.

[0277] In some embodiments, the first recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the first unique cell surface marker polypeptide and the 5' end of the nucleotide sequence encoding the first unique target polypeptide.

[0278] In some embodiments, the first recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the first unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the first unique target polypeptide.

[0279] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG.

[0280] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of GTG and TTG.

[0281] In some embodiments, the alternative (non-ATG) start codon is CTG.

[0282] In some embodiments, the alternative (non-ATG) start codon is GTG.

[0283] In some embodiments, the alternative (non-ATG) start codon is TTG.

[0284] In some embodiments, the alternative (non-ATG) start codon is ATT.

[0285] In some embodiments, the alternative (non-ATG) start codon is ATA.

[0286] In some embodiments, the alternative (non-ATG) start codon is ACG.

[0287] In some embodiments, the nucleotide sequence encoding the first unique cell surface marker polypeptide lacks any ATG triplet.

[0288] In some embodiments, the second recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the second unique target polypeptide and the 5' end of the nucleotide sequence encoding the second unique cell surface marker polypeptide.

[0289] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique target polypeptide, and a nucleotide sequence encoding the unique cell surface marker polypeptide from the 5' end to the 3' end.

[0290] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique target polypeptide and the 5' end of the nucleotide sequence encoding the unique cell surface marker polypeptide.

[0291] The selection of each promoter is independent of any other promoter. In some embodiments, no two promoters are the same. In some embodiments, at least two promoters are the same. In some embodiments, all promoters are the same.

[0292] In some embodiments, at least one promoter is the β-actin promoter.

[0293] In some embodiments, each promoter is a β-actin promoter.

[0294] In some embodiments, at least one promoter is the hamster β-actin promoter.

[0295] In some embodiments, each promoter is a hamster β-actin promoter.

[0296] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0297] As used herein, unless otherwise stated, CD20, CD52, and CD59 include human and non-human forms of CD20, CD52, and CD59. In some embodiments, CD20 is human CD20 (hCD20). In some embodiments, CD52 is human CD52 (hCD52). In some embodiments, CD59 is human CD59 (hCD59). Non-human forms of CD20, CD52, and CD59 include, but are not limited to, mouse, rat, and non-human primate forms of CD20, CD52, and CD59.

[0298] As used herein, a "variant" refers to a mutated form of a specified molecule in which at least one amino acid residue differs from that of the wild-type molecule. For example, a "variant" may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 point mutations compared to a wild-type polypeptide, provided that the variant is expressible on the surface of a host mammalian cell. For a variant having two or more point mutations, the mutations may be adjacent to each other or not. In some embodiments, at least two point mutations are adjacent to each other. In some other embodiments, no two point mutations are adjacent to each other. In some embodiments, some point mutations are adjacent to each other, and other point mutations are not adjacent to each other.

[0299] In some embodiments, the first unique cell surface marker polypeptide is CD20 or a variant thereof.

[0300] In some embodiments, the first unique cell surface marker polypeptide is CD20.

[0301] In some embodiments, the first unique cell surface marker polypeptide is a variant of CD20.

[0302] In some embodiments, the first unique cell surface marker polypeptide is CD52 or a variant thereof.

[0303] In some embodiments, the first unique cell surface marker peptide is CD52.

[0304] In some embodiments, the first unique cell surface marker polypeptide is a variant of CD52.

[0305] In some embodiments, the first unique cell surface marker polypeptide is CD59 or a variant thereof.

[0306] In some embodiments, the first unique cell surface marker polypeptide is CD59.

[0307] In some embodiments, the first unique cell surface marker polypeptide is a variant of CD59.

[0308] In some embodiments, at least the second unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59, and their variants. In such embodiments, the first and second unique cell surface marker polypeptides are different from each other. For example, the first unique cell surface marker polypeptide may be wild-type CD52, and the second unique cell surface marker polypeptide may be a variant of CD52. As another example, the first unique cell surface marker polypeptide may be a first variant of CD52, and the second unique cell surface marker polypeptide may be a second variant of CD52, wherein the first and second variant CD52 molecules are different.

[0309] In some embodiments, each unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0310] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of human CD52 and its variants.

[0311] In some embodiments, at least the second unique cell surface marker polypeptide is selected from the group consisting of human CD52 and its variants. In such embodiments, the first and second unique cell surface marker polypeptides are different from each other. For example, the first unique cell surface marker polypeptide may be wild-type human CD52, and the second unique cell surface marker polypeptide may be a human CD52 variant. As another example, the first unique cell surface marker polypeptide may be a first human CD52 variant, and the second unique cell surface marker polypeptide may be a second human CD52 variant, wherein the first and second human CD52 variant molecules are different.

[0312] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of human CD52 and its variants.

[0313] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52 and CD59.

[0314] In some embodiments, at least the second unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0315] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0316] In some embodiments, at least the first unique cell surface marker peptide is human CD52.

[0317] In some embodiments, at least the second unique cell surface marker peptide is human CD52.

[0318] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X2 is A.

[0319] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X4 is A.

[0320] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein X7 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X7 is A.

[0321] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 22 (GQNDTSQX 8SSPS), wherein X 8 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X 8 is A.

[0322] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 23 (GQNDTSQX 8X 9SPS), wherein each of X 8 and X 9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8 and X 9 is A.

[0323] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 24 (GQNDTSQX 8SX 10PS), wherein each of X8 and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X8 and X10 is A.

[0324] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 25 (GQNDTSQX 8X 9X 10PS), wherein each of X 8, X 9, and X 10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8, X 9, and X 10 is A.

[0325] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 26 (GX 2NDTSQX 8X 9SPS), wherein each of X2, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X9 is A.

[0326] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS), wherein each of X2, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X10 is A.

[0327] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X9 is A.

[0328] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X10 is A.

[0329] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V.

[0330] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V.

[0331] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0332] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0333] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0334] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0335] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0336] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0337] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0338] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 23 (GX 2NDTSQX 8X 9SPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X4, X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V.

[0339] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X4, X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V.

[0340] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 15 (GANDTSQAASPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 17 (GQNDTSAAASPS).

[0341] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 16 (GANDTSQASAPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 18 (GQNDTSAASAPS).

[0342] In some embodiments, at least one unique target polypeptide comprises a therapeutic polypeptide or a therapeutic protein.

[0343] In some embodiments, at least one unique target polypeptide is a polypeptide of a multi-chain protein.

[0344] In some embodiments, each unique target polypeptide is a polypeptide of a multi-chain protein.

[0345] In some embodiments, at least one unique target polypeptide is an antibody polypeptide.

[0346] In some embodiments, each unique target polypeptide is a polypeptide of an antibody.

[0347] In some embodiments, at least one unique target polypeptide is a polypeptide of a cross-cutting dual variable domain (CODV) Ig-like protein. In some embodiments, at least one unique target polypeptide is a polypeptide of a cross-cutting dual variable domain (CODV) Ig-like protein triantibody.

[0348] In some embodiments, each unique target polypeptide is a polypeptide of a cross-cutting dual variable domain (CODV) Ig-like protein. In some embodiments, each unique target polypeptide is a polypeptide of a cross-cutting dual variable domain (CODV) Ig-like protein triantibody.

[0349] The recombinant mammalian host cell can be any mammalian cell or cell line capable of expressing one or more of the target peptides according to the methods and / or compositions disclosed herein. In some embodiments, the recombinant mammalian host cell is selected from the group consisting of CHO cells, HEK-293 cells, BHK-21 cells, HepG2 cells, BAE-1 cells, SH-SY5Y cells, myeloma cells (e.g., Sp2 / O-Ag14), hybridoma cells, HeLa cells, Vero cells, NIH3T3 cells, WEHI231 cells, YAC cells, Jurkat cells, and derivatives thereof, such as CHO-K1 cells and CHO / DHFR- cells. In some embodiments, the recombinant mammalian host cell is a CHO cell.

[0350] [III.] [Composition]

[0351] Another aspect of this disclosure is an engineered mammalian host cell comprising a variety of recombinant polynucleotides, wherein each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are both transcribed on the same mRNA.

[0352] In some embodiments, the plurality of unique target peptides comprises 2 to 8 unique target peptides.

[0353] In some embodiments, the plurality of unique target peptides comprises 2 to 4 unique target peptides.

[0354] In some embodiments, the plurality of unique target peptides includes two unique target peptides.

[0355] In some embodiments, the plurality of unique target peptides includes three unique target peptides.

[0356] In some embodiments, the plurality of unique target peptides includes four unique target peptides.

[0357] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique cell surface marker polypeptide, and a nucleotide sequence encoding the unique target polypeptide from the 5' end to the 3' end.

[0358] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique cell surface marker polypeptide and at the 5' end of the nucleotide sequence encoding the unique target polypeptide.

[0359] In some embodiments, each recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the unique target polypeptide.

[0360] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG.

[0361] In some embodiments, each alternative (non-ATG) start codon is independently selected from the group consisting of GTG and TTG.

[0362] In some embodiments, the nucleotide sequence encoding the unique cell surface marker polypeptide lacks any ATG triplet.

[0363] In some embodiments, at least the first recombinant polynucleotide comprises, from the 5' end to the 3' end, a first promoter, a nucleotide sequence encoding a first unique cell surface marker polypeptide, and a nucleotide sequence encoding a first unique target polypeptide; and at least the second recombinant polynucleotide comprises, from the 5' end to the 3' end, a second promoter, a nucleotide sequence encoding a second unique target polypeptide, and a nucleotide sequence encoding a second unique cell surface marker polypeptide.

[0364] In some embodiments, the first recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the first unique cell surface marker polypeptide and the 5' end of the nucleotide sequence encoding the first unique target polypeptide.

[0365] In some embodiments, the first recombinant polynucleotide further comprises an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the first unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the first unique target polypeptide.

[0366] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of CTG, GTG, TTG, ATT, ATA, and ACG.

[0367] In some embodiments, the alternative (non-ATG) start codon is selected from the group consisting of GTG and TTG.

[0368] In some embodiments, the nucleotide sequence encoding the first unique cell surface marker polypeptide lacks any ATG triplet.

[0369] In some embodiments, the second recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the second unique target polypeptide and the 5' end of the nucleotide sequence encoding the second unique cell surface marker polypeptide.

[0370] In some embodiments, each recombinant polynucleotide includes a promoter, a nucleotide sequence encoding the unique target polypeptide, and a nucleotide sequence encoding the unique cell surface marker polypeptide from the 5' end to the 3' end.

[0371] In some embodiments, each recombinant polynucleotide further includes an internal ribosome entry site (IRES) located at the 3' end of the nucleotide sequence encoding the unique target polypeptide and the 5' end of the nucleotide sequence encoding the unique cell surface marker polypeptide.

[0372] In some embodiments, at least one promoter is the β-actin promoter.

[0373] In some embodiments, each promoter is a β-actin promoter.

[0374] In some embodiments, at least one promoter is the hamster β-actin promoter.

[0375] In some embodiments, each promoter is a hamster β-actin promoter.

[0376] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0377] In some embodiments, at least the second unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0378] In some embodiments, each unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52, CD59 and their variants.

[0379] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of human CD52 and its variants.

[0380] In some embodiments, at least the second unique cell surface marker peptide is selected from the group consisting of human CD52 and its variants.

[0381] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of human CD52 and its variants.

[0382] In some embodiments, at least the first unique cell surface marker polypeptide is selected from the group consisting of CD20, CD52 and CD59.

[0383] In some embodiments, at least the second unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0384] In some embodiments, each unique cell surface marker peptide is selected from the group consisting of CD20, CD52 and CD59.

[0385] In some embodiments, at least the first unique cell surface marker peptide is human CD52.

[0386] In some embodiments, at least the second unique cell surface marker peptide is human CD52.

[0387] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X2 is A.

[0388] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X4 is A.

[0389] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein X7 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X7 is A.

[0390] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 22 (GQNDTSQX 8SSPS), wherein X 8 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X 8 is A.

[0391] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 23 (GQNDTSQX 8X 9SPS), wherein each of X 8 and X 9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8 and X 9 is A.

[0392] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 24 (GQNDTSQX 8SX 10PS), wherein each of X8 and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X8 and X10 is A.

[0393] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 25 (GQNDTSQX 8X 9X 10PS), wherein each of X 8, X 9, and X 10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X 8, X 9, and X 10 is A.

[0394] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 26 (GX 2NDTSQX 8X 9SPS), wherein each of X2, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X9 is A.

[0395] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS), wherein each of X2, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X2, X8, and X10 is A.

[0396] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X9 is A.

[0397] In some embodiments, at least one unique cell surface marker polypeptide is a human CD52 variant comprising the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V. In some embodiments, each of X7, X8, and X10 is A.

[0398] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L and V; and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence selected from SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L and V.

[0399] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX 4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX 7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V.

[0400] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0401] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS).

[0402] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0403] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS).

[0404] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0405] In some embodiments, at least a first unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant, which is composed of the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0406] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS).

[0407] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 23 (GX 2NDTSQX 8X 9SPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX 7X 8X 9SPS), wherein each of X4, X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V.

[0408] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 20 (GQNX 4TSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 27 (GX 2NDTSQX 8SX 10PS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX 7X 8SX 10PS), wherein each of X4, X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V.

[0409] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 15 (GANDTSQAASPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 17 (GQNDTSAAASPS).

[0410] In some embodiments, at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 16 (GANDTSQASAPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 18 (GQNDTSAASAPS).

[0411] In some embodiments, at least one unique target polypeptide comprises a therapeutic polypeptide or a therapeutic protein.

[0412] In some embodiments, at least one unique target polypeptide is a polypeptide of a multi-chain protein.

[0413] In some embodiments, each unique target polypeptide is a polypeptide of a multi-chain protein.

[0414] In some embodiments, at least one unique target polypeptide is an antibody polypeptide.

[0415] In some embodiments, each unique target polypeptide is a polypeptide of an antibody.

[0416] In some embodiments, at least one unique target polypeptide is a polypeptide of a cross-cross dual variable domain (CODV) Ig-like protein.

[0417] In some embodiments, each unique target polypeptide is a polypeptide of a cross-cross dual variable domain (CODV) Ig-like protein.

[0418] In some embodiments, the recombinant mammalian host cell is a CHO cell.

[0419] Another aspect of this disclosure is an isolated human CD52 variant comprising the amino acid sequence of SEQ ID NO: 19 (GX 2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X2 is A.

[0420] Another aspect of this disclosure is an isolated human CD52 variant comprising the amino acid sequence of SEQ ID NO: 20 (GQNX 4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X4 is A.

[0421] Another aspect of this disclosure is an isolated human CD52 variant comprising the amino acid sequence of SEQ ID NO: 21 (GQNDTSQX 8SSPS), wherein X 8 is selected from the group consisting of A, G, I, L, and V. In some embodiments, X 8 is A.

[0422] Example

[0423] [Example] [1.] Generation and characterization of novel anti-hCD52 monoclonal antibodies

[0424] Mice were immunized with human CD52 (hCD52), and 11 seed strains expressing mouse anti-hCD52 monoclonal antibodies were prepared from the conjugated antibodies using standard methods. These 11 monoclonal antibodies were then converted into chimeric monoclonal antibodies containing mouse variable regions and human constant regions using standard methods again. Affinity of the 11 seed strains to hCD52 peptide mimetopes was determined. These 11 seed strains are shown in Table 1.

[0425] [surface] [1] [:] HCD52-resistant strains mAb Primitive (mouse) isotype Transformed (human) homotype Kd (nM)* 7F11 mIgG 1 hIgG 1 ND 4B10 mIgG 2A hIgG 1 ND 2C3 mIgG 3 hIgG 1 4.0 12G6 mIgG 3 hIgG 1 4.2 23E6 mIgG 3 hIgG 1 6.1 9D9 mIgG 3 hIgG 1 43 11C11 mIgG 3 hIgG 1 70 3G7 mIgG 2B hIgG 1 79 4G7 mIgG 3 hIgG 1 101 8G3 mIgG 3 hIgG 1 127 5F7 mIgG 3 hIgG 1 260 ND, not performed * Campath-1H has a measurement capacity of 68 nM Kd.

[0426] Next, the strains were characterized based on ELISA-based binding maps of truncated hCD52 peptide epitopes and alanine scan series. Comparisons were made with Camppath-1H (C-1H). The results are shown in Tables 2 and 3. Table 2 shows that C-1H primarily binds to the C-terminal region of hCD52, while 9D9, 11C11, 3G7, and CF1D12 primarily bind to the N-terminal region of hCD52. These results are generally consistent with the data shown in Table 3, namely that alanine scans of the C-terminal region of hCD52 eliminated binding to C-1H, while alanine scans of the N-terminal region of hCD52 eliminated binding to 9D9, 11C11, and 3G7.

[0427] Some of these strains (antibodies) were also characterized based on their FACS-based binding maps. In short, hCD52 and individual alanine scan variants of hCD52 corresponding to the peptide mimic epitopes in Table 3 were individually represented by CHO cells and then analyzed by FACS using the antibodies selected in this example (4B10, 7F11, CF1D12, 5F7, 3G7, 4G7, 9D9, 11C11, Camppath-1H, 2C3, 12G8, and 23E6). The amino acid sequences of the test antigens are as follows: Mut1 AQNDTSQTSSPS SEQ ID NO: 1 Mut2 GANDTSQTSSPS SEQ ID NO: 2 Mut3 GQADTSQTSSPS SEQ ID NO: 3 Mut4 GQNATSQTSSPS SEQ ID NO: 4 Mut5 GQNDASQTSSPS SEQ ID NO: 5 Mut6 GQNDTAQTSSPS SEQ ID NO: 6 Mut7 GQNDTSATSSPS SEQ ID NO: 7 Mut8 GQNDTSQASSPS SEQ ID NO: 8 Mut9 GQNDTSQTASPS SEQ ID NO: 9 Mut10 GQNDTSQTSAPS SEQ ID NO: 10 WT GQNDTSQTSSPS SEQ ID NO: 11

[0428] The results are shown in [picture] [2A] [To the image] [2C], which showed that there was a clear difference between the hCD52 alanine mutant 4 (GQNATSQTSSPS; SEQ ID NO: 4) and 8 (GQNDTSQASSPS; SEQ ID NO: 8) on the cell surface, Campath-1H and strain 9D9.

[0429] The results of these studies provide proof of principle for the development of systems designed to monitor the co-expression of multiple different target peptides in individual cells by evaluating the expression of corresponding unique cell surface marker peptides.

[0430] [surface] [2]: ELISA-based binding maps of truncated series of hCD52 peptide mimic epitopes. peptides SEQ ID NO: 3G7 4G7 9D9 11C 11 8G3 7F 11 4B 10 12 G6 2C3 twenty three E6 5F7 C- 1H CF1 D12 GQNDTSQTSSPSAD 39 [+] [+] [+] [+] [+] - - [+] [+] [+] [+] [+] [+] QNDTSQTSSPSAD 40 - [+] - - - - - [+] [+] [+] - [+] - NDTSQTSSPSAD 41 - [+] - - - - - [+] [+] [+] - [+] - DTSQTSSPSAD 42 - [+] - - - - - [+] [+] [+] - [+] - TSQTSSPSAD 43 - [+] - - - - - [+] [+] [+] - [+] - SQTSSPSAD 44 - [+] - - - - - [+] [+] [+] - [+] - QTSSPSAD 45 - [+] - - - - - [+] [+] [+] - [+] - TSSPSAD 46 - [+] - - - - - - - - - - [+] GQNDTSQTSSPAD 47 [+] [+] [+] [+] [+] - - [+] [+] [+] [+] - [+] GQNDTSQTSSAD 48 [+] [+] [+] [+] - - - - - - - - [+] GQNDTSQTSAD 49 [+] [+] [+] [+] - - - - - - - - [+] GQNDTSQTAD 50 [+] - [+] [+] - - - - - - - - [+] GQNDTSQAD 51 [+] [+] [+] [+] - - - - - - [+] - [+] GQNDTSAD 52 - [+] [+] [+] - - - - - - - - [+] GQNDTAD 53 - - - - - - - - - - - - [+]

[0431] Table 3: ELISA-based binding maps of alanine-scanned series of hCD52 peptide mimic epitopes. peptides SEQ ID NO: 3G7 4G7 9D9 11C 11 8G3 7F 11 4B 10 12 G6 2C3 twenty three E6 5F7 C- 1H CF1 D12 GQNDTSQTSSPSAD 39 [+] [+] [+] [+] [+] - - [+] [+] [+] [+] [+] [+] [A]QNDTSQTSSPSAD 54 - [+] - - - - - [+] [+] [+] - [+] [+] G [A]NDTSQTSSPSAD 55 [+] [+] [+] [+] [+] - - [+] [+] [+] [+] [+] [+] GQ [A]DTSQTSSPSAD 56 - [+] - - - - - [+] [+] [+] - [+] [+] GQN [A]TSQTSSPSAD 57 - - - - - - - [+] [+] [+] - [+] - GQND [A]SQTSSPSAD 58 - - - - - - - [+] [+] [+] - [+] [+] GQNDT [A]QTSSPSAD 59 - - - - - - - [+] [+] [+] - [+] [+] GQNDTS [A]TSSPSAD 60 - [+] - [+] - - - - - - - [+] [+] GQNDTSQ [A]SSPSAD 61 [+] [+] [+] [+] [+] - - [+] [+] [+] - [+] [+] GQNDTSQT [A]SPSAD 62 [+] [+] [+] [+] [+] - - [+] [+] [+] [+] - [+] GQNDTSQTS [A]PSAD 63 [+] [+] [+] [+] [+] - - [+] [+] [+] [+] - [+] GQNDTSQTSS [A]SAD 64 [+] [+] [+] [+] - - - [+] [+] [+] - - [+] GQNDTSQTSSP [A]AD 65 [+] [+] [+] [+] [+] - - [+] [+] [+] [+] - [+] [, , ]

[0432] [Example] [2.] Exemplary hCD52 mutants encoding peptides that can be used as unique cell surface markers are multinucleotide constructs.

[0433] Various mutant forms of the polynucleotide encoding hCD52 were prepared, including non-AUG (alternative) start codons with optimal Kozak background, mutations in all internal ATG codons, eliminated splice sites, and single-point mutations in which selected amino acids of hCD52 were replaced with alanine.

[0434] [picture] [3] Sequence alignments of natural (wild-type) hCD52 (SEQ ID NO: 30) and engineered mutant hCD52 (Mut4; SEQ ID NO: 31) were depicted, the latter including a non-AUG (substitution) start codon with optimal Kozak background, mutations in all internal ATG codons, eliminated splice sites, and a single-point mutation at amino acid 4 of hCD52 replacing aspartic acid with alanine. Mut4 is bound by Camppath-1H (C-1H) but not by mAb 9D9.

[0435] [picture] [4] Sequence alignments of natural (wild-type) hCD52 (SEQ ID NO: 30) and engineered mutant hCD52 (Mut8; SEQ ID NO: 32) were depicted, the latter including a non-AUG (substitution) start codon with optimal Kozak background, mutations in all internal ATG codons, eliminated splice sites, and a single-point mutation at amino acid 8 of hCD52 where threonine is replaced by alanine. Mut8 is bound by mAb 9D9 but not by Camppath-1H (C-1H).

[0436] Figure 5 depicts the sequence alignment of natural (wild-type) hCD52 (SEQ ID NO: 30) and engineered double mutant hCD52 (Mut8 / 10; SEQ ID NO: 33), the latter including a non-AUG (substitution) start codon with optimal Kozak background, mutations in all internal ATG codons, eliminated splice sites, and two point mutations: alanine substitution for threonine at amino acid 8 and alanine substitution for serine at amino acid 10 in hCD52. Similar to Mut8, Mut8 / 10 is bound by mAb 9D9 but not by Camppath-1H (C-1H).

[0437] [Example] [3.] Additional polynucleotide constructs encoding exemplary hCD52 mutants that can be used as unique cell surface marker peptides.

[0438] CHO cells were transfected with polynucleotides encoding the following hCD52 mutants: Mut4, Mut8, Mut8 / 9, Mut8 / 10, and Mut8 / 9 / 10. All mutants possessed the ATG start codon. Cells were analyzed by FACS using fluorophore-labeled Camppath-1H and 9D9 antibodies. Results are shown in [Figure / Table / Insert Figure / Text ... [picture] [6] and [picture] [7]

[0439] [picture] [6] are four flow cytometry bar graphs depicting the binding of Camppath-1H or 9D9 to cells expressing hCD52, specifically Mut4 (SEQ ID NO: 4) or Mut8 / 10 (SEQ ID NO: 13), where the polynucleotide encoding each hCD52 mutant has an ATG start codon. The graphs indicate that Mut4 mutants can be easily distinguished from Mut8 / 10 mutants using Camppath-1H and 9D9.

[0440] [picture] [7] are four flow cytometry bar graphs depicting the binding of Camppath-1H or 9D9 to hCD52-expressing Mut4 (SEQ ID NO: 4), Mut8 (SEQ ID NO: 8), Mut8 / 9 (SEQ ID NO: 12), Mut8 / 10 (SEQ ID NO: 13), or Mut8 / 9 / 10 (SEQ ID NO: 14), where the polynucleotide encoding each hCD52 mutant has an ATG start codon. The graphs indicate that the Mut4 mutant can be easily distinguished from all four Mut8-containing mutants using Camppath-1H and 9D9.

[0441] [Example] [4.] The impact of using non-ATG start codons

[0442] CHO cells were transfected with polynucleotides encoding the hCD52 mutants Mut4 and Mut8, each polynucleotide containing an ATG, CTG, GTG, or TTG start codon. Cells were analyzed by FACS using fluorophore-labeled Camppath-1H and 9D9 antibodies. Results are shown in [Figure / Table / Insert Figure / Text ... [picture] [8] and [picture] [9]

[0443] [picture] [8] are four flow cytometry bar graphs that individually depict the binding of Camppath-1H or 9D9 to cells expressing Mut4 (SEQ ID NO: 4) or Mut8 (SEQ ID NO: 8) based on the start codon. The graphs show that the expression of each of Mut4 and Mut8 follows the pattern ATG > CTG > GTG > TTG > mimic.

[0444] [picture] [9] is a bar chart representation of the data from Figure 8, which shows that Mut4 and Mut8 mutants can be easily distinguished using Campath-1H and 9D9, especially mutants with ATG, CTG or GTG start codons.

[0445] [Example] [5.] The effect of cell surface marker start codons on the expression of target peptides

[0446] Stable transfected CHO pools co-expressing alternative initiator reporters and soluble receptor-Fc fusion proteins were analyzed using flow cytometry. Each reporter was expressed using UUG, GUG, CUG, or AUG as the start codon. Bar overlays show cell surface expression levels of CD52 or CD59, with all AUG mutations at the 3' end of the start codon. Cairns et al. (2011) Biotechnol. Bioeng. 108: 2611-22. Representative results are shown in... [picture]

[10] . The figure shows an inverse relationship between reporter (cell surface marker) performance and target gene (GOI; target polypeptide) performance, which varies depending on the start codon in the polynucleotide encoding the reporter. The results indicate that TTG showed the lowest detectable level of CD52 performance, which would allow for better ribosome read-through to the GOI. Since the performance of the target protein is ultimately more important than that of the reporter, the figure shows that choosing a suboptimal non-ATG start codon such as TTG is sufficient and preferred.

[0447] [Example] [6.] A combination of hCD52 alanine mutants of three reporter agents can be enabled.

[0448] As further evidence for the concept, [picture]

[11] illustrates how specific combinations of certain hCD52 alanine mutants can enable three unique reporter agents. For example... [picture]

[11] As shown, hCD52 mutant 4 (GQNATSQTSSPS; SEQ ID NO: 4) was identified using Camppath-1H (C-1H) instead of 9D9 or 7F11. hCD52 mutant Mut2 / 8 / 9 (GANDTSQAASPS; SEQ ID NO: 15) was identified by binding to 9D9 but not to C1H or 7F11. hCD52 mutant Mut7 / 8 / 9 (GQNDTSAAASPS; SEQ ID NO: 17) was identified by binding to 7F11 but not to C1H or 9D9.

[0449] [Example] [7.] Additional hCD52 alanine mutant combinations of the three reporter can be enabled.

[0450] As further evidence of the concept, [picture]

[12] illustrates how specific combinations of certain hCD52 alanine mutants can enable three unique reporter agents. For example... [picture]

[12] As shown, hCD52 mutant 4 (GQNATSQTSSPS; SEQ ID NO: 4) was identified using Campath-1H (C-1H) instead of 9D9 or 7F11. hCD52 mutant Mut2 / 8 / 10 (GANDTSQASAPS; SEQ ID NO: 16) was identified by binding to 9D9 but not to C1H or 7F11. hCD52 mutant Mut7 / 8 / 9 (GQNDTSAASAPS; SEQ ID NO: 18) was identified by binding to 7F11 but not to C1H or 9D9.

[0451] [Example] [8.] Dual reporter system for CODV triple antibody expression

[0452] Although CODV triantibodies have been described, their production is extremely challenging. These constructs have four distinct polypeptide chains: two distinct heavy chains (the CODV heavy chain and the Fab heavy chain, with mutual mortar and pestle structures) and two distinct light chains (the CODV light chain and the Fab light chain). Balancing the expression of these four individual polypeptide chains is difficult, and excessive free heavy chains can be toxic to the cells expressing them.

[0453] In one embodiment, each heavy chain is represented by its own separate expression vector along with a unique cell surface marker polypeptide encoded on a single mRNA, and each light chain is represented by its own separate expression vector without any unique cell surface marker polypeptide. The corresponding heavy and light chains (CODV heavy and light chains, and Fab heavy and light chains) self-associate to form the desired CODV triantibody. See also [picture] [13A].

[0454] It is worth noting that this method provides the ability to independently isolate and screen the strains of each arm, and it can provide ideal screening for product quality (i.e., homogeneous trispecific performance).

[0455] In another embodiment, a similar approach can be used, the difference being that the corresponding heavy and light chains are encoded on a single bicistronic vector. See also [picture] [13B].

[0456] none

[0457]

Claims

1. A method for expressing multiple target peptides at a high level, the method comprising: (a) Culturing multiple mammalian host cells, each containing multiple recombinant polynucleotides, wherein each recombinant polynucleotide contains a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA, wherein the culture is performed under conditions that allow each unique cell surface marker polypeptide to be expressed on the surface of the mammalian host cell and each unique target polypeptide to be expressed; (b) Contacting the cultured mammalian host cells of step (a) with multiple detectable agents, each detectable agent being capable of uniquely binding to one or more of the unique cell surface marker polypeptides expressed on the surface of the mammalian host cell; (c) [The text abruptly ends here, likely due to an incomplete sentence or missing information.] (b) performing at least one round of fluorescence-activated cell sorting on the contacted cells to select one or more mammalian host cells that are uniquely bound to at least one of the plurality of detectable agents; (d) preparing one or more colonies of the mammalian host cells selected in step (c); (e) analyzing one or more colonies from step (d) by detecting the performance levels of at least two unique cell surface marker peptides on each colony; (f) selecting one or more colonies having high performance levels of the at least two unique cell surface marker peptides; and (g) culturing one or more colonies selected in step (f) under conditions that allow for high-level performance of the plurality of target peptides, wherein the at least two unique cell surface marker peptides are variants of CD52, the variants comprising: (a) SEQ (a) The amino acid sequence shown in SEQ ID NO: 19 (GX2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; (b) The amino acid sequence shown in SEQ ID NO: 20 (GQNX4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V; (c) The amino acid sequence shown in SEQ ID NO: 21 (GQNDTSX7TSSPS), wherein X7 is selected from the group consisting of A, G, I, L, and V; (d) The amino acid sequence shown in SEQ ID NO: 22 (GQNDTSQX8SSPS), wherein X8 is selected from the group consisting of A, G, I, L, and V; (e) The amino acid sequence shown in SEQ ID NO: 23 (GQNDTSQX8X9SPS), wherein each of X8 and X9 is independently selected from the group consisting of A, G, I, L, and V; (f) SEQ ID The amino acid sequence shown in NO: 24 (GQNDTSQX8SX10PS), wherein each of X8 and X10 is independently selected from the group consisting of A, G, I, L and V;(g) The amino acid sequence shown in SEQ ID NO: 25 (GQNDTSQX8X9X10PS), wherein each of X8, X9, and X10 is independently selected from the group consisting of A, G, I, L, and V; (h) The amino acid sequence shown in SEQ ID NO: 26 (GX2NDTSQX8X9SPS), wherein each of X2, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V; (i) The amino acid sequence shown in SEQ ID NO: 27 (GX2NDTSQX8SX10PS), wherein each of X2, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V; (j) The amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX7X8X9SPS), wherein each of X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V; or (k) SEQ ID The amino acid sequence shown in NO: 29 (GQNDTSX7X8SX10PS), wherein each of X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V, and wherein each recombinant polynucleotide also includes an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the unique target polypeptide.

2. The method as described in claim 1, wherein step (c) comprises: (i) Perform a single round of fluorescence-activated cell sorting on cells from step (b) that have been in contact with at least the first detectable agent and the second detectable agent, thereby selecting one or more mammalian host cells that are uniquely bound by at least the first detectable agent and the second detectable agent; or (ii) (c1) Perform a first round of fluorescence-activated cell sorting on at least the first cell surface labeled peptide, thereby selecting one or more mammalian host cells that are bound by at least the first detectable agent; and (c2) Perform a second round of fluorescence-activated cell sorting on the cells selected in step (c1) for at least the second cell surface labeled peptide, thereby selecting one or more mammalian host cells that are bound by at least the first detectable agent and the second detectable agent.

3. The method as described in request item 1, wherein: (i) Step (e) is performed 7 to 28 days after step (d); and / or (ii) the analysis in step (e) includes flow cytometry; and / or (iii) at least one or each of the multiple unique cell surface marker peptides in step (f) has a higher performance level than the corresponding performance level of at least 70% of the colony population analyzed in step (e); or (iv) at least the first of the multiple unique cell surface peptides in step (f) has a higher performance level than at least the second of the multiple unique cell surface peptides in step (f).

4. The method of any one of claims 1 to 3, wherein the plurality of unique target peptides comprises 2 to 8 unique target peptides, 2 to 4 unique target peptides, 2 unique target peptides, 3 unique target peptides, or 4 unique target peptides.

5. The method as described in any one of claims 1 to 3, wherein: (i) at least one promoter is a β-actin promoter; (ii) each promoter is a β-actin promoter; (iii) at least one promoter is a hamster β-actin promoter; or (iv) each promoter is a hamster β-actin promoter.

6. The method of any one of claims 1 to 3, wherein each recombinant polynucleotide comprises, from the 5' end to the 3' end, a promoter, a nucleotide sequence encoding the unique cell surface marker polypeptide, and a nucleotide sequence encoding the unique target polypeptide.

7. The method of any one of claims 1 to 3, wherein each recombinant polynucleotide comprises 1 to 4 promoters, 1 to 4 nucleotide sequences each encoding a unique cell surface marker polypeptide and 1 to 4 nucleotide sequences each encoding a unique target polypeptide, wherein one of the nucleotide sequences encoding the unique cell surface marker polypeptide and one of the nucleotide sequences encoding the unique target polypeptide are transcribed on the same mRNA.

8. The method of any one of claims 1 to 3, wherein each recombinant polynucleotide comprises 1 to 4 promoters, 1 to 4 nucleotide sequences each encoding a unique cell surface marker polypeptide and 1 to 4 nucleotide sequences each encoding a unique target polypeptide, wherein one of the nucleotide sequences encoding the unique cell surface marker polypeptide and one of the nucleotide sequences encoding the unique target polypeptide are transcribed on the same mRNA; wherein each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA.

9. The method of claim 7, wherein each recombinant polynucleotide comprises, from the 5' end to the 3' end, one of one to four promoters, one of one to four nucleotide sequences encoding a unique cell surface marker polypeptide, and one of one to four nucleotide sequences encoding a unique target polypeptide.

10. The method of any one of claims 1 to 3, wherein (i) each alternative (non-ATG) start codon is independently selected from the group consisting of CTG, GTG, TTG, ATT, ATA and ACG, or each alternative (non-ATG) start codon is independently selected from the group consisting of GTG and TTG and / or (ii) the nucleotide sequence encoding the unique cell surface marker polypeptide lacks any ATG triplet.

11. The method as described in any one of claims 1 to 3, wherein: (a) At least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 19 (GX2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V; (b) at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (GX2NDTSQTSSPS). NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS); (c) at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 2 (GQNDTSATSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS); (d) at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GQNDTSATSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GQNDTSATSSPS). (e) a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a first unique cell surface marker polypeptide consisting of a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide consisting of a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and (f) at least a first unique cell surface marker polypeptide comprising a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS);And at least the second unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); (g) at least the first unique cell surface marker peptide is composed of a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least the second unique cell surface marker peptide is composed of a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); (h) at least the first unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least the second unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); (i) at least the first unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); The human CD52 variant contains the amino acid sequence shown in SEQ ID NO: 20 (GQNX4TSQTSSPS); at least the second unique cell surface marker polypeptide contains the human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 23 (GX2NDTSQX8X9SPS); and at least the third unique cell surface marker polypeptide contains the human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX7X8X9SPS), wherein each of X4, X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V; (j) at least the first unique cell surface marker polypeptide contains the amino acid sequence shown in SEQ ID NO: 20 (GQNX4TSQTSSPS); at least the second unique cell surface marker polypeptide contains the human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 27 (GX2NDTSQX8SX10PS); and at least the third unique cell surface marker polypeptide contains the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX7X8X9SPS). (k) A human CD52 variant containing the amino acid sequence shown in NO: 29 (GQNDTSX7X8SX10PS), wherein each of X4, X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V; and at least a first unique cell surface marker polypeptide contains the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a second unique cell surface marker polypeptide contains a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 15 (GANDTSQAASPS).And at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 17 (GQNDTSAAASPS); or (l) at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 16 (GANDTSQASAPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 18 (GQNDTSAASAPS).

12. The method as described in any one of claims 1 to 3, wherein: (i) at least one unique target polypeptide comprises a therapeutic polypeptide or a therapeutic protein; (ii) at least one unique target polypeptide is a polypeptide of a multi-chain protein; (iv) at least one unique target polypeptide is a polypeptide of an antibody; (vi) at least one unique target polypeptide is a polypeptide of a crossover dual-variable domain (CODV) Ig-like protein; or (vii) at least one unique target polypeptide is a polypeptide of a CODV triantibody.

13. An engineered mammalian host cell comprising multiple recombinant polynucleotides, wherein each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide, and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA, wherein at least two of the unique cell surface marker polypeptides are variants of CD52, the variants comprising: (a) The amino acid sequence shown in SEQ ID NO: 19 (GX2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; (b) The amino acid sequence shown in SEQ ID NO: 20 (GQNX4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L, and V; (c) The amino acid sequence shown in SEQ ID NO: 21 (GQNDTSX7TSSPS), wherein X7 is selected from the group consisting of A, G, I, L, and V; (d) The amino acid sequence shown in SEQ ID NO: 22 (GQNDTSQX8SSPS), wherein X8 is selected from the group consisting of A, G, I, L, and V; (e) The amino acid sequence shown in SEQ ID NO: 23 (GQNDTSQX8X9SPS), wherein each of X8 and X9 is independently selected from the group consisting of A, G, I, L, and V; (f) SEQ ID (g) The amino acid sequence shown in SEQ ID NO: 24 (GQNDTSQX8SX10PS), wherein each of X8 and X10 is independently selected from the group consisting of A, G, I, L and V; (h) The amino acid sequence shown in SEQ ID NO: 25 (GQNDTSQX8X9X10PS), wherein each of X8, X9 and X10 is independently selected from the group consisting of A, G, I, L and V; (h) The amino acid sequence shown in SEQ ID NO: 26 (GX2NDTSQX8X9SPS), wherein each of X2, X8 and X9 is independently selected from the group consisting of A, G, I, L and V; (i) The amino acid sequence shown in SEQ ID NO: 27 (GX2NDTSQX8SX10PS), wherein each of X2, X8 and X10 is independently selected from the group consisting of A, G, I, L and V; (j) SEQ ID The amino acid sequence shown in NO: 28 (GQNDTSX7X8X9SPS), wherein each of X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V; or (k) the amino acid sequence shown in SEQ ID NO: 29 (GQNDTSX7X8SX10PS), wherein each of X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V, and wherein each recombinant polynucleotide further includes an alternative (non-ATG) start codon for translation initiation of the nucleotide sequence encoding the unique cell surface marker polypeptide and an ATG start codon for translation initiation of the nucleotide sequence encoding the unique target polypeptide.

14. The engineered mammal as claimed in claim 13, wherein each recombinant polynucleotide comprises, from the 5' end to the 3' end, a promoter, a nucleotide sequence encoding the unique cell surface marker polypeptide, and a nucleotide sequence encoding the unique target polypeptide.

15. The engineered mammal of claim 13, wherein each recombinant polynucleotide comprises 1 to 4 promoters, 1 to 4 nucleotide sequences encoding a unique cell surface marker polypeptide, and 1 to 4 nucleotide sequences encoding a unique target polypeptide, wherein one of the nucleotide sequences encoding the unique cell surface marker polypeptide and one of the nucleotide sequences encoding the unique target polypeptide are transcribed on the same mRNA.

16. The engineered mammal of claim 13, wherein each recombinant polynucleotide comprises 1 to 4 promoters, 1 to 4 nucleotide sequences encoding a unique cell surface marker polypeptide and 1 to 4 nucleotide sequences encoding a unique target polypeptide, wherein one of the nucleotide sequences encoding the unique cell surface marker polypeptide and one of the nucleotide sequences encoding the unique target polypeptide are transcribed on the same mRNA, wherein each recombinant polynucleotide comprises a promoter, a nucleotide sequence encoding a unique cell surface marker polypeptide and a nucleotide sequence encoding a unique target polypeptide, wherein the nucleotide sequence encoding the unique cell surface marker polypeptide and the nucleotide sequence encoding the unique target polypeptide are transcribed on the same mRNA.

17. The engineered mammal as claimed in claim 15, wherein each recombinant polynucleotide comprises, from the 5' end to the 3' end, one of one to four promoters, one of one to four nucleotide sequences encoding a unique cell surface marker polypeptide, and one of one to four nucleotide sequences encoding a unique target polypeptide.

18. The engineered mammal as claimed in claim 13, wherein (i) each alternative (non-ATG) start codon is independently selected from the group consisting of CTG, GTG, TTG, ATT, ATA and ACG, or each alternative (non-ATG) start codon is independently selected from the group consisting of GTG and TTG and / or (ii) the nucleotide sequence encoding the unique cell surface marker polypeptide lacks any ATG triplet.

19. The engineered mammalian host cell as described in claim 13, wherein: (a) At least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 19 (GX2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L, and V; and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (GQNX4TSQTSSPS) and SEQ ID NO: 21 (GQNDTSX7TSSPS), wherein each of X4 and X7 is independently selected from the group consisting of A, G, I, L, and V; (b) At least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (GX2NDTSQTSSPS). NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS); (c) at least a first unique cell surface marker polypeptide is composed of a human CD52 variant consisting of the amino acid sequence shown in SEQ ID NO: 2 (GQNDTSATSSPS); and at least a second unique cell surface marker polypeptide is composed of a human CD52 variant consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 (GQNATSQTSSPS) and SEQ ID NO: 7 (GQNDTSATSSPS); (d) at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GQNDTSATSSPS); and at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GQNDTSATSSPS). (e) a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a first unique cell surface marker polypeptide consisting of a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least a second unique cell surface marker polypeptide consisting of a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and (f) at least a first unique cell surface marker polypeptide comprising a human CD52 variant having the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS);And at least the second unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); (g) at least the first unique cell surface marker peptide is composed of a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 2 (GANDTSQTSSPS); and at least the second unique cell surface marker peptide is composed of a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); (h) at least the first unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least the second unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); (i) at least the first unique cell surface marker peptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 7 (GQNDTSATSSPS); The human CD52 variant contains the amino acid sequence shown in SEQ ID NO: 20 (GQNX4TSQTSSPS); at least the second unique cell surface marker polypeptide contains the human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 23 (GX2NDTSQX8X9SPS); and at least the third unique cell surface marker polypeptide contains the human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX7X8X9SPS), wherein each of X4, X7, X8, and X9 is independently selected from the group consisting of A, G, I, L, and V; (j) at least the first unique cell surface marker polypeptide contains the amino acid sequence shown in SEQ ID NO: 20 (GQNX4TSQTSSPS); at least the second unique cell surface marker polypeptide contains the human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 27 (GX2NDTSQX8SX10PS); and at least the third unique cell surface marker polypeptide contains the amino acid sequence shown in SEQ ID NO: 28 (GQNDTSX7X8X9SPS). (k) A human CD52 variant containing the amino acid sequence shown in NO: 29 (GQNDTSX7X8SX10PS), wherein each of X4, X7, X8, and X10 is independently selected from the group consisting of A, G, I, L, and V; and at least a first unique cell surface marker polypeptide contains the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); and at least a second unique cell surface marker polypeptide contains a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 15 (GANDTSQAASPS).And at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 17 (GQNDTSAAASPS); or (l) at least a first unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 4 (GQNATSQTSSPS); at least a second unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 16 (GANDTSQASAPS); and at least a third unique cell surface marker polypeptide comprises a human CD52 variant containing the amino acid sequence shown in SEQ ID NO: 18 (GQNDTSAASAPS).

20. The engineered mammalian host cell as described in claim 13, wherein: (i) at least one unique target polypeptide comprises a therapeutic polypeptide or a therapeutic protein; (ii) at least one unique target polypeptide is a polypeptide of a multi-chain protein; (iv) at least one unique target polypeptide is a polypeptide of an antibody; (vi) at least one unique target polypeptide is a polypeptide of a cross-crossed dual variable domain (CODV) Ig-like protein; or (vii) at least one unique target polypeptide is a polypeptide of a CODV triantibody.

21. An isolated human CD52 variant comprising an amino acid sequence consisting of: (i) SEQ ID NO: 19 (GX2NDTSQTSSPS), wherein X2 is selected from the group consisting of A, G, I, L and V; (ii) SEQ ID NO: 20 (GQNX4TSQTSSPS), wherein X4 is selected from the group consisting of A, G, I, L and V; or (iii) SEQ ID NO: 22 (GQNDTSQX8SSPS), wherein X8 is selected from the group consisting of A, G, I, L and V.

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