Modified pIX vector constructs

Abstract

The present invention provides vector constructs comprising an open reading frame comprising a nucleic acid sequence encoding a modified pIX filamentous bacteriophage coat protein wherein the methionine (M) residue at position 1 of the pIX filamentous bacteriophage coat protein is replaced with a substitution amino acid residue. The invention also provides a bacteriophage particle or a bacteriophage display system containing the vector construct, and a bacteriophage display method and kit.

Description

Technical Field

[0001] The present invention relates to modified pIX filamentous phage coat proteins for phage display and phage particles displaying fusion proteins comprising such modified pIX proteins. More specifically, the present invention relates to vector constructs and nucleic acid molecules encoding the modified pIX phage coat proteins, preferably fused to a protein of interest, so that the protein of interest can be displayed on the surface of the phage as a pIX fusion protein. Background Art

[0002] Over the past three decades, phage display has emerged as a powerful and efficient method for discovering and evolving novel binding proteins. The principle of combinatorial phage display technology is based on a genotype-phenotype linkage, centered on the principle that each virion will display on its surface only the very same proteins encoded by its genome, encapsidated by its protein coat. Phage particles themselves are highly resistant to a wide range of physicochemical conditions; therefore, phage display offers superior versatility in many selection scenarios compared to competing combinatorial technologies. Consequently, despite the existence of competing combinatorial technologies, none demonstrates a high degree of versatility combined with ease of use. Despite this, screening for the desired phenotype following phage panning remains challenging, and identification of the optimal binder is not guaranteed.

[0003] Phage display of heterologous polypeptides has been achieved using all five structural proteins of the filamentous phage coat, but pill has achieved the most widespread application. Pill display has also been described and has been shown to have certain advantages over standard pill display in terms of efficacy in identifying desired target-specific antibody candidates and generating clones with superior biophysical properties ( et al., 2016, Sci. Rep. 6, 39066). Summary of the Invention

[0004] The present inventors have now developed an improved pIX phage display system utilizing an improved pIX expression vector construct. The improvement is reflected in the provision of a vector encoding a modified pIX phage coat protein, wherein, rather than providing a full-length wild-type pIX protein, the vector encodes a modified pIX phage coat protein in which the methionine (M) residue at position 1 is replaced by an alternative amino acid residue.

[0005] Surprisingly, the inventors have demonstrated that such modified pIX carrier is able to cause a significant improvement in phage display when compared to phage display using wild-type pIX protein, either in terms of functional protein display (e.g. in the form of improved antigen or target binding / reactivity or improved fusion protein display or functionality) or in terms of infectious phage particle production (infectious phage titres) or both. Such improvements are highly advantageous for phage display in terms of the ability to successfully select and isolate binders, the quality and / or quantity of binders that can be selected, or in terms of the size of phage display libraries that can be produced. For example, the most effective use of phage display as an engineering and discovery tool requires the combination of the highest possible functional display with the highest possible infectious virion / phage particle production. This will ensure that the largest possible library of heterologous fusion proteins can be screened for the desired variant properties, thereby improving the prospects of identifying proteins with the desired properties. Advantageously, the present invention manages to achieve this combination of properties.

[0006] The advantages of the present invention are further enhanced in a phage display system that is capable of high valency (HV) display, i.e. the advantages of the present invention can be further enhanced in a system designed to maximize the number of pIX fusion proteins displayed on the surface of the phage when compared to the number of wild-type (or non-fusion) pIX proteins displayed on the surface.

[0007] Thus, in one aspect, the present invention provides a vector construct comprising an open reading frame comprising a nucleic acid sequence encoding a modified pIX filamentous bacteriophage coat protein, wherein the methionine (M) residue at position 1 of the pIX filamentous bacteriophage coat protein is replaced by an alternative amino acid residue.

[0008] Viewed from another perspective, the present invention provides a vector construct comprising a nucleic acid sequence encoding a pIX bacteriophage coat protein (modified pIX bacteriophage coat protein), wherein the methionine (M) residue at position 1 is replaced by an alternative amino acid residue.

[0009] The vector of the present invention is an expression vector or expression construct, i.e. generally consisting of nucleic acid sequences capable of expressing (protein synthesis) the desired encoded protein components in a suitable host cell.

[0010] As mentioned above, the present application is in the field of phage display on a pIX phage coat protein. Thus, the vector of the present application can be a phage vector or a phagemid vector (plasmid), the basic structure and components of which are well known to the person skilled in the art and are chosen to enable expression of the phage protein and packaging of the phage particle in a suitable host cell, such that the heterologous or foreign protein (protein of interest, POI) fused to the modified pIX phage coat protein of the present application is displayed on the surface of the phage particle.

[0011] Thus, when the vector of the present application is used to transform a suitable host cell, for example a suitable prokaryotic host cell, for example a suitable E. coli strain, phage particles are produced, which contain the desired POI fused to the modified pIX phage coat protein of the present application and displayed on the surface of the phage particle, the vector sequences or other nucleic acid sequences encoding the various phage components of the phage genome, and also encoding the POI contained within the phage particle.

[0012] In the modified pIX phage coat protein of the present application, the methionine (M) residue at position 1 of the pIX phage coat protein, for example the methionine (M) residue at position 1 of the wild-type pIX phage coat protein or the native pIX phage coat protein, can be replaced or exchanged by any one of the alternative amino acid residues, i.e. any amino acid residue other than methionine (M). In other words, the N-terminal methionine (M) residue of the pIX phage coat protein, for example the N-terminal methionine (M) residue of the wild-type pIX phage coat protein or the native pIX phage coat protein, can be replaced or exchanged by any one of the alternative amino acid residues, i.e. any amino acid residue other than methionine (M). These pIX phage coat proteins used in the present application or encoded by the constructs of the present application are referred to herein as modified pIX phage coat proteins of the present application or non-wild-type pIX phage coat proteins. Thus, in terms of the amino acid sequence, the modified pIX phage coat proteins of the present application do not correspond to the wild-type pIX phage coat protein.

[0013] Since the genetic fusion between the POI and the modified pIX phage coat protein of the present application in a single ORF is advantageous for the present application, conveniently, the suitable one of the alternative amino acids for replacing the M at position 1 of the pIX phage coat protein will be a genetically encoded amino acid, which can then be encoded by a nucleic acid sequence and thus easily be comprised in the vector of the present application.

[0014] In embodiments of the application, the one alternative amino acid residue that replaces methionine (M) is selected from the group consisting of leucine (L), glycine (G), isoleucine (I), phenylalanine (F), tryptophan (W), tyrosine (Y), asparagine (N), glutamine (Q), glutamic acid (E), aspartic acid (D), proline (P), arginine (R), lysine (K), histidine (H), cysteine (C), serine (S), threonine (T), alanine (A), or valine (V).

[0015] In other embodiments of the application, the one alternative amino acid residue is selected from the group consisting of L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T, or A.

[0016] In other embodiments of the application, the one alternative amino acid residue is selected from the group consisting of L, G, I, F, W, Y, N, Q, E, D, P, R, K, or H, or from the group consisting of L, G, I, C, S, T, or A.

[0017] In some preferred embodiments, the one alternative amino acid residue is selected from the group consisting of L, G, I, F, W, Y, N, Q, E, D, P, R, K, or H.

[0018] In other preferred embodiments of the application, the one alternative amino acid residue is selected from the group consisting of L, G, or I, more preferably the one alternative amino acid residue is L or G, most preferably the one alternative amino acid residue is L.

[0019] While any one of the alternative amino acid residues can be used, the choice can also be guided by the desired outcome of the phage display process. For example, in many cases it is desirable to have as high a phage yield as possible combined with as high a functionality of the fusion protein as possible. This will ensure the ability to cover the largest possible functional diversity in any fusion protein library, which should maximize the ability to search and identify the desired new fusion proteins resulting from the library selection. In this case, the results presented herein indicate that replacing methionine with leucine (M1L), glycine (M1G), or isoleucine (M1I) seems to achieve the best combination of these two different but interrelated characteristics.

[0020] However, in cases where for example large fusion protein diversity is less important, for example in cases where a smaller library is sufficient (in which case high phage yield is not required), other M1x identities can be suitable to maximize fusion protein functionality. In this case, the results presented herein indicate that M1E or M1D represent suitable one alternative amino acids that will result in very high target reactivity, and M1F, M1W, M1Y, M1N, or M1Q would be other choices. M1P, M1R, M1K, or M1H would be yet another choice.

[0021] Alternatively, where high phage production has higher importance (but functional fusion protein is a lower priority, e.g., where high affinity cloning is not necessarily required), then other M1x substitutions can be suitable to maximize phage production. In this case, the results presented herein indicate that, in this case, M1C or M1S or M1T or M1A represent suitable alternative amino acids. M1V would be another option.

[0022] In some embodiments, a substitution amino acid residue is not R, K, D, S, A, V, T, C, H, P, E, Q, N, Y, W, F, I, G, or L. For example, in some embodiments, a substitution amino acid is not R. In some embodiments, a substitution amino acid is not K. In some embodiments, a substitution amino acid is not D. In some embodiments, a substitution amino acid is not S. In some embodiments, a substitution amino acid is not A. In some embodiments, a substitution amino acid is not V. In some embodiments, a substitution amino acid is not T. In some embodiments, a substitution amino acid is not C. In some embodiments, a substitution amino acid is not H. In some embodiments, a substitution amino acid is not P. In some embodiments, a substitution amino acid is not E. In some embodiments, a substitution amino acid is not Q. In some embodiments, a substitution amino acid is not N. In some embodiments, a substitution amino acid is not Y. In some embodiments, a substitution amino acid is not W. In some embodiments, a substitution amino acid is not F. In some embodiments, a substitution amino acid is not I. In some embodiments, a substitution amino acid is not G. In some embodiments, a substitution amino acid is not L.

[0023] In some embodiments, position 2 of the modified pIX bacteriophage coat protein of the application is S. In some embodiments, position 3 of the modified pIX bacteriophage coat protein is V. In some embodiments, position 4 of the modified pIX bacteriophage coat protein is L. In some embodiments, one or more, two or more, preferably all of positions 2, 3, and 4 of the modified pIX bacteriophage coat protein are S, V, and L, respectively. In some embodiments, position 2 of the modified pIX bacteriophage coat protein of the application is not S. In some embodiments, position 3 of the modified pIX bacteriophage coat protein is not V. In some embodiments, position 4 of the modified pIX bacteriophage coat protein is not L. In some embodiments, one or more, two or more, preferably all of positions 2, 3, and 4 of the modified pIX bacteriophage coat protein are not S, V, and L, respectively. In some embodiments, position 16 is not C. In some such embodiments, the modified position is not M.

[0024] For the avoidance of doubt, the modified pIX bacteriophage coat protein of the application (or the pIX portion / component of the vector construct or nucleic acid molecule of the application) has (or encodes) an amino acid residue at position 1 of the pIX protein, but the amino acid residue is not methionine (M). In other words, the methionine (M) at position 1 of the pIX protein is replaced or exchanged for an alternative amino acid. Merely deleting or removing the methionine (M) at position 1 is not sufficient. Thus, the present application does not encompass, for example, a modified pIX protein or pIX fragment in which the methionine (M) at position 1 has been deleted or removed (e.g. deleted or removed without being replaced by an alternative amino acid residue). Thus, for example, the present application does not encompass a pIX fragment comprising or consisting of amino acids at and beyond position 2 (e.g. positions 2 to 32 of a full-length pIX protein), or a pIX fragment in which the M at position 1 has been removed. Indeed, it has been demonstrated that a vector construct in which the methionine (M) at position 1 of the pIX protein has been deleted does not display the same advantages and improved properties as the wild-type pIX compared to the vector construct of the application.

[0025] It can be noted that the improvements observed using the vectors and systems of the application as described herein generally refer to improvements relative to equivalent vectors, systems, etc. using wild-type pIX bacteriophage coat proteins or native pIX bacteriophage coat proteins.

[0026] As used herein, the term open reading frame (ORF) has its standard art-recognized meaning. Thus, the term open reading frame (ORF) is used herein to refer to the span of a nucleic acid molecule (typically DNA) between a start codon and a stop codon or between a translation initiation site and a translation termination site. Such ORF typically encodes a polypeptide, in this case a polypeptide comprising a modified pIX bacteriophage coat protein of the application as described herein. A preferred ORF encodes a fusion protein of a POI and a modified pIX bacteriophage coat protein of the application. Suitable start codons are well known to those of skill in the art. A typical start codon and exemplary start codon will be ATG, which encodes methionine. The start codon will be located at a suitable distance upstream of the sequence encoding the modified pIX bacteriophage coat protein in a vector (or nucleic acid molecule) of the application so as to initiate translation of the modified pIX bacteriophage coat protein under suitable conditions. When a POI is also encoded by a vector (or nucleic acid molecule) of the application, the start codon is located at a suitable distance upstream of the sequence encoding the POI-modified pIX fusion, e.g., close to or directly adjacent to the sequence, so as to initiate translation of the POI-modified pIX fusion protein under suitable conditions. Suitable stop codons are well known to those of skill in the art. A typical stop codon and exemplary stop codons are TAA, TGA, or TAG. One or more stop codons can be used. In some embodiments, a vector or nucleic acid molecule of the application contains a single ORF.

[0027] As used herein, the term "pIX phage coat protein" or "pIX protein" or "pIX phage protein" or "pIX coat protein" and the like refers to a pIX protein derived or originating from a filamentous phage, such as a wild-type pIX filamentous phage coat protein sequence or a native pIX filamentous phage coat protein sequence, or a pIX protein having a sequence corresponding to the sequence of such a pIX protein. A preferred filamentous phage from which the pIX protein or the pIX protein corresponding thereto is derived is a M13 phage, fd phage or fl phage. Any suitable pIX protein can be used, provided that the pIX protein has the ability to display a POI as a pIX fusion protein on the surface of a phage particle. Although a wild-type (or native) pIX protein or a wild-type-like pIX protein, such as a wild-type-like pIX protein comprising all of the amino acids of a wild-type (or native) pIX but also comprising one or more additional amino acids, such as a wild-type-like pIX protein that is a conditionally mutated form, is used in some embodiments of the application, such as in various helper phages as described herein, the pIX protein encoded by the vectors of the application, or otherwise used in a fusion protein with a POI so that the POI is displayed on the phage surface, corresponds to a pIX protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced by one alternative amino acid residue. In other words, these pIX proteins are the modified pIX phage coat proteins of the application as described elsewhere herein.

[0028] Preferably, the modified pIX phage coat protein encoded by the vectors of the application comprises or consists of a full-length pIX phage coat protein, provided that the methionine (M) residue at position 1 of the full-length pIX filamentous phage coat protein is replaced by one alternative amino acid residue (e.g. as described elsewhere herein). Such a full-length pIX phage coat protein can typically have 32 amino acids.

[0029] Preferably, the modified pIX phage coat protein encoded by the vectors of the application comprises or consists of the following amino acid sequence corresponding to the wild-type pIX protein from VCS M13 helper phage (Genbank AY598820.1).

[0030] MSVLVYSFASFVLGWCLRSGITYFTRLMETSS

[0031] (SEQ ID NO: 1)

[0032] provided that the methionine (M) residue at position 1 of the pIX filamentous phage coat protein (here: SEQ ID NO: 1) is replaced by one alternative amino acid residue (e.g. as described elsewhere herein).

[0033] An exemplary nucleic acid sequence encoding this sequence for inclusion in a vector of the application is provided elsewhere herein as SEQ ID NO: 2, again provided that the nucleic acid sequence encoding the methionine (M) residue at position 1 of the pIX phage coat protein is replaced with a nucleic acid sequence encoding an alternative amino acid residue (e.g., as described elsewhere herein).

[0034] Thus, yet another embodiment of the application provides a vector construct of the application, wherein the encoded modified pIX phage coat protein corresponds to a pIX coat protein from M13 phage, fd phage, or fl phage, or a variant thereof, provided that the methionine (M) residue at position 1 is replaced with an alternative amino acid residue (e.g., as described elsewhere herein).

[0035] In other embodiments, e.g., when a variant of the modified pIX protein is used, e.g., when a variant of the M13 phage coat protein, fd phage coat protein, or fl pIX phage coat protein is used, the encoded pIX protein comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, or 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% identity, provided that the methionine (M) residue at position 1 of the pIX phage coat protein is replaced with an alternative amino acid residue. In other words, in the modified pIX protein of the application, the position 1 of the pIX protein sequence should not be a methionine (M). Preferred alternative amino acid sequences for inclusion at position 1 are described elsewhere herein, and are capable of leading to improved phage display, e.g., improved production of infectious phage particles (infectious phage titers) and / or improved functional protein display, e.g., improved POI / antibody / fusion protein display, e.g., improved antigen or target binding. Without wishing to be bound by theory, it is believed that replacing the M at position 1 of the pIX protein with an alternative amino acid improves the translation efficiency of the ORF of the vector construct, and in particular, has a positive effect (or improvement) on the amount of POI-modified pIX fusion protein produced.

[0036] Likewise, a nucleic acid molecule encoding a variant of the modified pIX protein of the application can for example comprise or consist of a nucleotide sequence having at least 60%, 65%, 70%, 75%, or 80% sequence identity to the nucleotide sequence of SEQ ID NO: 2, for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, provided that the methionine (M) residue encoded at position 1 of the pIX filamentous phage coat protein is replaced by an alternative amino acid residue (e.g., as described elsewhere herein).

[0037] Accordingly, yet another embodiment of the application provides a vector construct of the application, wherein the encoded modified pIX filamentous phage coat protein comprises SEQ ID NO: 1 (MSVLVYSFASFVLGWCLRSGITYFTRLMETSS) or a sequence having at least 60%, 65%, 70%, 75%, or 80% identity to SEQ ID NO: 1, provided that the methionine (M) residue at position 1 is replaced by an alternative amino acid residue (e.g., as described elsewhere herein).

[0038] Other preferred examples of modified pIX sequences (e.g., variant modified pIX sequences) encoded by a vector or nucleic acid molecule of the application are sequences containing up to 12 altered amino acids in the pIX sequence (e.g., SEQ ID NO: 1), for example up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 altered amino acids in the pIX sequence (e.g., in SEQ ID NO: 1), provided that the methionine (M) residue at position 1 of the pIX filamentous phage coat protein (e.g., SEQ ID NO: 1) is replaced by an alternative amino acid residue (e.g., as described elsewhere herein).

[0039] Accordingly, yet another embodiment of the application provides a vector construct of the application, wherein the encoded modified pIX filamentous phage coat protein comprises SEQ ID NO: 1 (MSVLVYSFASFVLGWCLRSGITYFTRLMETSS) or a sequence having at least 60%, 65%, 70%, 75%, or 80% identity to SEQ ID NO: 1, provided that the methionine (M) residue at position 1 is replaced by an alternative amino acid residue (e.g., as described elsewhere herein).

[0040] These modified pIX sequences or variants thereof, e.g., modified pIX sequences or variant pIX sequences of the application, should retain or have the functional ability to display a POI on the surface of a phage particle as a pIX fusion protein. A functional C-terminal truncation or N-terminal fragment of SEQ ID NO: 1 (or a variant sequence) or other pIX sequence can also be used, provided that the methionine (M) residue at position 1 of the pIX filamentous bacteriophage coat protein is replaced with an alternative amino acid residue (e.g., as described elsewhere herein) and the ability to display a POI as a pIX fusion protein is retained. In other embodiments, a fragment is not used. Thus, alternatively, a full-length pIX protein can be used, e.g., a pIX protein in which all 32 amino acids (or a variant thereof, e.g., as described elsewhere herein) are present, provided that the methionine (M) residue at position 1 of the pIX filamentous bacteriophage coat protein is replaced with an alternative amino acid residue (e.g., as described elsewhere herein).

[0041] In the present application, "sequence identity" is a measure of identity at the amino acid level between proteins and at the nucleotide level between nucleic acids. Protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence when the sequences are aligned. Similarly, nucleic acid sequence identity can be determined by comparing the nucleotide sequence at a given position in each sequence when the sequences are aligned. When reference is made herein to a variant molecule, e.g., by a percent identity value, e.g., at least 60%, 65%, 70%, etc., it will be understood that such variants are typically functional variants or derivatives, i.e., display retained or improved function.

[0042] Methods to determine percent identity of two amino acid sequences or two nucleic acid sequences are well known and described in the art, and any of these methods can be used. For example, to determine percent identity of two amino acid sequences or percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions / # of positions x 100). In some embodiments, the two sequences are of the same length.

[0043] The sequences can be aligned manually and the number of identical amino acids counted. Alternatively, the alignment of the two sequences for the determination of the percentage identity can be done using a mathematical algorithm. Such an algorithm is incorporated in the NBLAST program and the XBLAST program (Altschul et al. 1990). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous (or having a certain % identity) to nucleic acid molecules of the application. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous (or having a certain % identity) to protein molecules of the application. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs can be used. See http: / / www.ncbi.nlm.nih.gov. Alternatively, the sequence identity can be calculated after the sequences have been aligned, for example by the BLAST program in the EMBL database (www.ncbi.nlm.gov / cgi-bin / BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" can be used for the alignment. In the context of the present application, the BLASTN default settings and the PSIBLAST default settings can be advantageous. In the calculation of the percentage identity, only exact matches are counted.

[0044] In preferred vectors of the application, the sequence encoding the modified pIX phage coat protein of the application is linked, e.g. operably linked, to a sequence encoding a protein of interest (POI). Thus, in preferred embodiments, the vector construct or open reading frame (ORF) of the vector construct further comprises a sequence encoding a protein of interest (POI) linked, e.g. operably linked or fused, to the sequence encoding the modified pIX phage coat protein of the application. Thus, preferred vector constructs and nucleic acid molecules of the application encode a POI-modified pIX fusion protein.

[0045] The terms "fusion protein," "fused," and the like are used herein to describe the functional linkage of two or more protein components in the same polypeptide sequence or in the same open reading frame (ORF). Such fusion proteins can also be described as genetic fusions, as these fusion proteins are encoded by the same nucleic acid sequence (sometimes referred to as a "fusion gene" or "fusion nucleotide sequence"). Although the two (or more) protein components (or the encoding nucleic acid sequences) can be directly adjacent to one another in such fusion proteins, these components can likewise be linked by a suitable peptide spacer or linker. As is well known in the art, the spacer or linker is important for allowing each individual protein component to be expressed in a functional manner, e.g., for allowing the individual protein components to form suitable three-dimensional structures to perform or maintain the functions required of the individual protein components.

[0046] Thus, in a fusion protein encoded by a vector or nucleic acid molecule of the present application, a peptide spacer (or linker) is typically included between the protein of interest (POI) and the modified pIX bacteriophage coat protein of the present application. In other embodiments, such a linker or spacer need not be included, or such a linker or spacer can be included only between some of the components. Thus, in a vector or nucleic acid molecule of the present application, the sequence encoding the POI can be fused to the sequence encoding the modified pIX bacteriophage coat protein of the present application, with or without a spacer sequence or linker sequence between the components. All of these possibilities (i.e., fusion proteins or encoding nucleic acids with or without a spacer sequence or linker sequence) are still considered to be direct fusions or direct genetic fusions.

[0047] Although this discussion focuses on the linker or spacer between the modified pIX bacteriophage coat protein of the present application and the POI, linker sequences can be included elsewhere in a vector of the present application as appropriate, e.g., between other components of the vector as discussed herein, e.g., between the VH and VL domains of an antibody POI or other POI involving or comprising two or more individual polypeptide components.

[0048] Thus, the term "pIX fusion protein" refers to a pIX protein (pIX bacteriophage coat protein, pIX filamentous bacteriophage coat protein) fused to an exogenous peptide / polypeptide (e.g., a protein of interest (POI)). Similarly, the term "modified pIX fusion protein" refers to a modified pIX protein (modified pIX bacteriophage coat protein, modified pIX filamentous bacteriophage coat protein) of the present application fused to an exogenous peptide / polypeptide (e.g., a protein of interest (POI)).

[0049] Thus, a preferred vector of the application comprises a sequence (nucleic acid sequence) encoding a modified pIX bacteriophage coat protein of the application fused (genetically fused) to a sequence encoding a POI (sometimes referred to herein as a POI-modified pIX or POI-modified pIX fusion protein). The POI and the modified pIX can be in any suitable order or spacing in the vector, provided that once expressed and packaged into a bacteriophage particle, a functional fusion protein between the POI and the modified pIX is formed, wherein the modified pIX coat protein component of the fusion protein forms part of the bacteriophage coat, and the POI is functionally expressed or displayed on the surface of the bacteriophage particle. Thus, the POI portion of the fusion protein is in frame with the modified pIX coat protein portion of the fusion protein. This means that the POI and the pIX are expressed in the same polypeptide sequence (or as part of the same ORF), or in other words, as a direct fusion. In the vectors of the application, it is generally preferred that the POI component of the fusion protein is N-terminal (or at or near the N-terminus) of the modified pIX component of the fusion protein.

[0050] Another aspect of the application provides a modified pIX filamentous bacteriophage coat protein of the application. In other words, these aspects of the application provide a modified pIX filamentous bacteriophage coat protein, wherein the methionine (M) residue at position 1 of the pIX filamentous bacteriophage coat protein is replaced by a replacement amino acid residue (e.g. as described elsewhere herein). In preferred embodiments, fusion proteins comprising a modified pIX bacteriophage coat protein of the application are provided, e.g. fusion proteins of the application comprising a POI and a modified pIX bacteriophage coat protein. In other words, such aspects of the application provide a POI fused (preferably, N-terminally) to a modified pIX filamentous bacteriophage coat protein, wherein the methionine (M) residue at position 1 of the pIX filamentous bacteriophage coat protein is replaced by a replacement amino acid residue (e.g. as described elsewhere herein). Nucleic acid molecules encoding such modified pIX filamentous bacteriophage coat proteins and fusion proteins are also provided.

[0051] The application extends to nucleic acid sequences or nucleic acid molecules capable of forming part of a vector of the application or comprising a component of a vector of the application. Thus, a further aspect of the application provides a nucleic acid molecule or nucleic acid sequence comprising an open reading frame comprising a nucleic acid sequence encoding a modified pIX filamentous bacteriophage coat protein, wherein the methionine (M) residue at position 1 of the pIX filamentous bacteriophage coat protein is replaced by a replacement amino acid residue (e.g. as described elsewhere herein).

[0052] Furthermore, the present application provides a nucleic acid molecule or nucleic acid sequence encoding a modified pIX bacteriophage coat protein of the present application or a fusion protein of the present application comprising a POI fused to a modified pIX bacteriophage coat protein of the present application.

[0053] In preferred embodiments of the present application, one or more ribosomal (ribosomal) binding sites (RBS) are included in the vector construct. Such components can also be referred to as translation initiation regions (TIR).

[0054] The RBS sequence is located in the vector at a position suitable for the RBS sequence to function. The role of the RBS is to recruit the ribosome during the initiation of protein translation, and is therefore conveniently placed a suitable distance upstream of the start codon of the protein desired to be translated or upstream of the ORF of the protein desired to be translated. Thus, in the vectors of the present application, the RBS sequence is conveniently placed upstream of the sequence encoding the POI-modified pIX fusion protein. In embodiments where the signal peptide is also part of the ORF, e.g. as discussed elsewhere herein, the RBS sequence is also conveniently placed upstream of the sequence encoding the signal peptide (or upstream of the start codon of the ORF comprising the nucleic acid sequence encoding the signal peptide). In embodiments where there is no signal peptide in the ORF, the RBS sequence is then conveniently placed a suitable distance upstream of the sequence encoding the POI-modified pIX fusion protein. The suitable distance will be known or readily determined by the skilled person depending on the RBS selected. An exemplary distance can be seven or eight nucleotides from the ATG (or other) start codon, but this can vary.

[0055] The RBS / TIR sequence modulates the strength of translation (protein expression level) of the sequence located downstream, and different types of RBS are capable of producing different levels of protein expression, e.g. weak or strong expression. Weak RBS / TIR sequences or strong RBS / TIR sequences are well known in the art, and can be readily selected by the skilled person depending on the level of protein expression desired. As would be expected, a strong RBS promotes or induces more translation (strong translation) compared to a weak RBS. Both weak RBS sequences and strong RBS sequences can be used in the vectors of the present application. In some embodiments, a weak RBS is used.

[0056] In particular, in the preferred vectors of the application, the RBS is comprised upstream (or 5' or N-terminal) of the start codon of the sequence encoding the POI-modified pIX fusion protein (or upstream of the start codon of the ORF comprising the nucleic acid sequence encoding the POI-modified pIX fusion protein, etc.). The preferred RBS for use in the application is a Shine Dalgarno (SD) sequence or a SD-based sequence that can be comprised in the vector construct. SD sequences are well known and described in the art, and any of these SD sequences can be used. For example, a core SD sequence is GAGG (SEQ ID NO: 3), and other consensus sequences are GAGGAG (SEQ ID NO: 4) or AGAGGAG (SEQ ID NO: 5) or AGGAGAA (SEQ ID NO: 6), e.g. comprising the sequence AGGAG (SEQ ID NO: 7). Thus, a SD sequence comprising these core sequences or consensus sequences can be used.

[0057] An exemplary structure of a construct of the application having a POI-modified pIX fusion protein is shown in Figure 3 B. As described elsewhere herein, the POI shown in Figure 3 B is a scFv antibody fragment, but this is just an example of a POI or library of POIs that can be used.

[0058] In preferred vectors of the present invention, a sequence encoding a spacer or linker (typically a peptide spacer or linker) is included between the sequence encoding the POI and the sequence encoding the modified pIX phage coat protein. Such sequences are typically synthetic or artificial (e.g., non-natural or unnatural) linker or spacer sequences, for example, sequences that do not encode a functional protein or protein domain. Composite linker sequences can also be used. Such linker or spacer sequences can include tag sequences, such as c-Myc tags or FLAG tags (e.g., DYKDDDDK; SEQ ID NO: 8). Typically, a complete or full-length linker or spacer sequence is used, for example, such sequences are typically not truncated sequences. The inclusion of such a sequence can aid in the folding of the linked protein, in particular, the folding of the N-terminal protein (here, typically the POI), and thus the spacer or linker length can be appropriately adjusted to achieve optimal or satisfactory functional folding of the two components (i.e., the POI and the modified pIX). The appropriate length can be readily determined by one skilled in the art. However, exemplary lengths would be between 5 and 15 amino acids (Weiss et al., 2000, Protein Sci., 9:647-654), for example, 6 to 10 amino acids. A specific linker for use in the present invention is AAAGSKDIR (SEQ ID NO: 12). Alternatively, a linker such as a GS linker, for example, a linker having a certain number of GS repeats (e.g., G4S repeats), can be used.

[0059] If present in a vector or fusion protein of the present invention, such a spacer or linker forms a portion of the vector or fusion protein that is distinct from the modified pIX phage coat protein. In other words, such a spacer or linker is not part of the modified pIX phage coat protein of the present invention; the modified pIX phage coat protein of the present invention is a different component or a separate component, for example, there is a connection between the modified pIX phage coat protein and the upstream portion of the vector. Thus, any of the replacement amino acid residues used as a substitution at position 1 of the modified pIX phage coat protein of the present invention is part of the pIX component (portion) of the vector and is not part of the spacer sequence or linker sequence (or any other portion of the vector); the spacer sequence or linker sequence (or POI sequence) is a different or separate component. Such different portions of the vector construct are typically separated by restriction enzyme sites or site-specific recombination sites. Thus, in some embodiments, a restriction enzyme site or site-specific recombination site is incorporated between the linker component or spacer component (or other portion of the vector) and the modified pIX component.

[0060] As described elsewhere herein, preferred vectors of the application are capable of encoding a protein of interest (POI) or targeting unit fused to a modified pIX phage coat protein. Such embodiments allow for the display of a POI, e.g. a targeting protein, on the modified pIX coat protein of the application. As described elsewhere herein, the use of a modified pIX phage coat protein of the application can result in improved display of a POI.

[0061] Thus, the POI (and indeed any linker sequence or spacer sequence positioned between the POI and the modified pIX phage coat protein) is typically exogenous or heterologous. By exogenous or heterologous protein is meant a protein or peptide which is not originally part of the relevant phage coat protein, e.g. a pIX protein, etc., which is fused to a modified pIX phage coat protein of the application (with or without any linker amino acids or spacer amino acids which are also exogenous or heterologous, and thus not part of or originally part of the relevant phage coat protein), e.g. to the N-terminus of a modified pIX phage coat protein of the application, e.g. to the N-terminal amino acid residue which serves as a replacement for the M residue at position 1 of the pIX phage coat protein.

[0062] Any protein of interest (POI) can be encoded in a vector of the application, provided that any protein of interest (POI) is suitable for display on a phage, in particular as a fusion to a pIX phage coat protein, e.g. a modified pIX phage coat protein of the application. Suitable examples are well known and documented in the art. However, preferred examples will be targeting molecules / targeting units or binding partners / binding proteins which are capable of binding to other entities (targets / target entities, e.g. target proteins). Some preferred examples of POIs will be antibodies or fragments of antibodies (e.g. Fabs, scFv, nanobodies), MHC molecules (class I or class II), T cell receptors (TCRs), or non-Ig derived binding proteins, e.g. DARpins, ankyrin family, fibronectin family, cohesins, anti-calcins, etc. (Hosse et al., 2006, Protein Sci 15: 14-27) and peptides.

[0063] A suitable design of the vector of the application for displaying a selected type of POI on the surface of a phage will be readily determined by the skilled person. For example, if the selected type of POI is in the form of a single polypeptide chain, such as a scFv antibody or a single chain TCR or a single chain MHC molecule, such as a single chain MHC class I or MHC class II, the nucleic acid molecule encoding these polypeptides can simply be positioned in the vector so that a fusion protein is produced with the modified pIX phage coat protein of the application. If the selected type of POI is in the form of two or more polypeptide chains, such as a Fab antibody fragment or a TCR or a MHC molecule with two chains, the nucleic acid molecule encoding one of the polypeptides (one of the chains) can be positioned in the vector so that a fusion protein is produced with the modified pIX phage coat protein of the application, and the other polypeptide chain(s) can be produced separately or independently.

[0064] The vector of the application can be used for classical phage display in order to select binding partners (e.g. antibodies) against a specific target entity (e.g. a target protein or a target antigen). In such applications, a library of POIs can be expressed on phage particles as part of a fusion protein with the modified pIX phage coat protein of the application, and selected for binding to the target entity by standard techniques and art-known techniques.

[0065] Another preferred component of the vector construct of the application is a suitable promoter sequence in order to control the expression of the ORF comprising the modified pIX protein of the application and the expression of the fusion protein comprising the modified pIX protein. Suitable promoter sequences are well known to the skilled person and any one of these sequences can be used. An exemplary promoter sequence can be the lac promoter, which can be induced, for example, with IPTG. Other promoters can include tac, arabB, or psp.

[0066] Optionally, a signal sequence or signal peptide, such as a pelB signal sequence or a pelB signal peptide, can be comprised in the ORF comprising the nucleic acid sequence encoding the modified pIX phage coat protein of the application. Such a signal sequence or signal peptide can thus be present or absent in the vector or nucleic acid molecule of the application. If present, a suitable position will be readily determined. Such a signal sequence is typically located upstream (N-terminal) of the POI-modified pIX fusion protein of the application, but as part of the same ORF. In some embodiments, no signal sequence or signal peptide is used or present. A signal sequence or signal peptide can sometimes also be referred to as a leader sequence or a leader peptide.

[0067] Other optional features that can be present in the vectors (or nucleic acid molecules) of the application are well known to those skilled in the art. For example, these vectors, e.g. bacteriophage vectors or phagemid vectors (which can be collectively referred to as phage display vectors or constructs) can optionally additionally contain other suitable components, such as origins of replication, inducible or non-inducible promoters / operators for initiating transcription, enhancers, termination sequences, antibiotic resistance genes and markers, sequences encoding chaperone proteins (e.g. periplasmic chaperone proteins, such as FkpA), signal sequences, linkers, protease sites, universal tags or reporter molecules, restriction enzymes or site-specific recombination sites to enable cloning and other manipulations, such as for cloning a suitable POI into a suitable position of the vectors of the application to form a fusion protein with the modified pIX bacteriophage coat protein, primer binding sites to enable amplification of the construct by, e.g. PCR, or other desired sequence elements, such as DNA sequences to enable discrimination between different libraries by, e.g. PCR. Suitable sources and positioning of such additional components within the phage display constructs such that the additional components perform the desired function of the additional components will be entirely within the normal practice of those skilled in the art.

[0068] As described elsewhere herein, nucleic acid molecules encoding the modified pIX bacteriophage coat proteins of the application or the fusion proteins of the application comprising the modified pIX bacteriophage coat proteins of the application form a further aspect.

[0069] As described elsewhere herein, the vectors of the application are primarily for use in phage display and can therefore be phagemid vectors or phage vectors. Thus, in yet another embodiment of the application, the vector construct is a phagemid vector or a phage vector.

[0070] Phage display is a well known and described technique in the art. In this respect, in 1985, G.P. Smith established a method to display polypeptides on the surface of filamentous bacteriophage, which is a virus that infects E. coli cells (Smith, G.P., 1985, Science 228, 1315-1317). Since then, so-called phage display has developed into a powerful technology for protein engineering and selection of peptides and proteins that bind to specific targets (Pluckthun, A., 2009, Mol. Biotechnol. 43, 35- 52). and Sandlie, 2012, Methods 58, 40-46). The filamentous bacteriophage M13 is constructed from five different structural proteins. Protein VIII (pVIII) is the major coat protein and one end of the particle is capped by 5 copies of pIII and pVI and the other end is capped by 5 copies of pVII and pIX. The particle infects E. coli with F pilus through pIII and injects its ssDNA into the bacterial cell. Here the bacteriophage DNA is replicated and transcribed and new bacteriophage particles are assembled before being non-lytically secreted into the growth medium.

[0071] In phage display, the gene encoding the protein of interest (POI) is usually placed between the gene encoding the coat protein (usually pIII, but here pIX) and the N-terminal signal sequence of the gene to produce a POI-coat protein fusion, although in some embodiments of the application the signal sequence is not present. The term "phage library" or "library of phage particles" or similar terms refers to a collection of unique phages that differ in the amino acid sequence of the POI and can be made by standard molecular cloning techniques. The library can well comprise >10 10 members and can be used for selection of specific binders.

[0072] The present application therefore also provides a phage or phage particle comprising the vector or nucleic acid molecule of the application and expressing on the surface the modified pIX filamentous bacteriophage coat protein or the modified pIX fusion protein of the application. The phage particle can thus comprise a phage genome or a phagemid, preferably it can comprise a phagemid. Such phage or phage particle can be any filamentous bacteriophage. Preferred examples are enterobacteria phages, such as M13 phage, fd phage or fl phage.

[0073] A further aspect of the application provides a library of phage / phage particles (e.g. filamentous phage) produced using the vector (or nucleic acid molecule) of the application as described herein and thus comprising the vector (or nucleic acid molecule) of the application as described herein. The phage comprises a fusion protein of a POI as described herein with a modified pIX bacteriophage coat protein as described herein. The filamentous phage thus displays a library of POIs or a POI fused to the modified pIX bacteriophage coat protein of the application. As with other phage display libraries, each individual phage particle expresses / display the same POI, but the presence of multiple particles expressing different POIs allows the display of a plurality of (or one library of or multiple) different POIs.

[0074] Thus, in such libraries, a collection of different protein fusions (e.g. different antibody fusions) with different properties are displayed and selected on a desired target in the form of a phage display library (e.g. an antibody phage display library). In phage display for target discovery (e.g. antibody discovery), the library typically consists of a collection of artificially diversified or endogenously diversified target proteins (e.g. antibodies) fused to a phage coat protein (here the modified pIX) and these target proteins (e.g. antibodies) differ in their biophysical, biochemical, and target binding properties. Such libraries are then used to identify those variants with the target properties through a cyclic process called panning, wherein each clone in the library competes against each other to enrich for the favorable variants.

[0075] Thus, another aspect of the application provides a library of phage particles, wherein the phage particles comprise a vector (or nucleic acid molecule) of the application as described herein, and wherein a plurality of different target proteins are expressed on the surface of the phage particles fused to the modified pIX phage coat protein of the application.

[0076] For aspects of the application involving phage display, one can refer to general phage display textbooks for relevant techniques and definitions, such as Phage Display in Biotechnology and Drug Discovery by Sachdev S. Sidhu, 1995, or Phage Display: A Laboratory Manual by Barbas et al., 1994.

[0077] The POI-modified pIX fusion proteins of the present application can be encoded in the context of a complete phage genome by inserting the sequence encoding the POI-modified pIX fusion protein into the phage genome (phage vector display), or on a phagemid (phagemid display). Phagemids are high copy number plasmids that are capable of encoding the POI-modified pIX fusion protein and require superinfection with a helper phage that provides the necessary genetic material for phage production. Thus, in phagemid display, there are typically two sources of coat protein for POI display (here, pIX phage coat protein); the helper phage encodes pIX protein (e.g., pIX protein that is not fused to a POI; non-fusion pIX protein), and the phagemid encodes the POI-pIX phage coat protein fusion (here, the POI-modified pIX fusion protein). The resulting new virions will have a mixture of phagemid-derived POI-modified pIX fusion proteins and helper phage-derived pIX coat proteins (non-fusion pIX coat proteins). Such helper phage-encoded pIX protein / non-fusion pIX protein can be wild-type (or native) or a quasi- wild type pIX protein. Similarly, if a phage genome system is used, it is also typically necessary for non-fusion (e.g., wild-type or quasi- wild type) pIX phage protein to be present, although in some embodiments of the present application, non-fusion (e.g., wild-type or quasi- wild type) forms of pIX coat protein are not present.

[0078] Thus, in the present application, phage particles can be engineered to have one copy or multiple copies of a POI displayed on the modified pIX coat protein.

[0079] In a phage genome system, this can be achieved, for example, by modifying the phage genome to contain a sequence encoding (or an ORF comprising a sequence encoding) a POI-modified pIX fusion protein of the application. If this is the only version / form of pIX phage coat protein in the phage genome, multiple copies of the POI will be displayed on the modified pIX coat protein, and high valency (HV) display should be achieved, as there will be no other version of the pIX phage coat protein competing for display on the surface. On the other hand, if an alternative version of the pIX phage coat protein is provided in the system, such that both versions of the pIX phage coat protein are present in the phage genome, for example by further modifying the phage genome to comprise a sequence encoding (or an ORF comprising a sequence encoding) another pIX protein (non-fusion pIX protein) in addition to the modified pIX phage coat protein of the application, then both versions of the pIX will compete with each other for display on the surface, and a mixture of the POI-modified pIX fusion protein and the non-fusion pIX protein will be present on the surface, thereby achieving low valency (LV) display.

[0080] In a phagemid system, this can be controlled, for example, by the helper phage used, and in a preferred embodiment of the application, phagemid particles are used which have multiple copies of the POI displayed on the modified pIX coat protein. This can be achieved in any suitable way. However, in particular, to increase the level of display and to produce multiple copies of the POI on the surface of the phage, a modified type of helper phage can be used, for example a helper phage known as Delta phage, which allows high valency (HV) display on pIX. Such a modified helper phage is in contrast to the use of a normal helper phage, for example M13K07, VCSM13, R408 or similar normal helper phage, which only allow low valency (LV) display.

[0081] The helper phage reported by Nilssen et al. (Nilssen et al., 2012, Nucleic acids research, 40, e120; WO 2011 / 036555) as Delta phage has at least one (e.g. two) amber mutations, which are inserted into the amber mutation close to the start codon of pIX, i.e. close to the codon for the first methionine (M) residue of the pIX phage coat protein, thereby conditionally inactivating (conditionally repressing) the pIX encoded by the helper phage. Specifically, these amber mutations are placed between the 2ndand 3rdposition of the pIX phage coat protein, i.e. between the residue S and the residue V of the wild type pIX phage coat protein. However, other positions are also possible, provided that the mutant will function to conditionally inactivate (conditionally repress) the pIX encoded by the helper phage.

[0082] If this helper phage is subsequently superinfected into a host cell (e.g. E. coli) transformed with a phagemid encoding a POI-pIX fusion (e.g. a POI-modified pIX fusion protein of the present application), then in a host cell that suppresses the amber mutation, e.g. an amber-suppressing strain of E. coli (e.g. a supE+ strain), a (low) valency display of POI-pIX can be seen, whereas in a host cell that does not suppress the amber mutation, e.g. an amber-non-suppressing strain of E. coli (e.g. a supE- / supE negative strain), a high valency display of POI-pIX can be seen. This is because, in the amber-suppressing host cell strain (e.g. a supE+ E. coli), pIX (non-fused pIX) from the helper phage is produced, which results in an intermediate (low) valency display, as pIX (non-fused pIX) from the helper phage competes with the POI-pIX fusion protein from the phagemid for display, and a mixture of both is displayed. However, in the amber-non-suppressing strain (e.g. a supE- / supE negative strain), the production of pIX (non-fused pIX) from the helper phage is suppressed or blocked, and only the phagemid-encoded POI-pIX fusion, here a POI-modified pIX fusion protein of the present application, should be present, resulting in a high valency display of the fusion protein on the modified pIX.

[0083] Thus, in a preferred embodiment of the application, the vector construct is a phagemid vector encoding a POI-modified pIX fusion protein of the application, and this vector construct is used in combination with a helper phage having a conditional mutation, enabling control of the expression / production of the helper phage encoded pIX (non-fusion pIX) phage protein, which in turn enables control of the number of POI-modified pIX fusion proteins on the surface of the phage. In embodiments where the conditional mutation is not suppressed, for example when using a non-suppressing strain of E. coli, for example using a amber non-suppressing strain, for example a supE / supE negative strain, then the helper phage encoded pIX should not be expressed / produced (will be suppressed), and only the POI-modified pIX should be expressed / produced, resulting in only the POI-modified pIX fusion protein of the application on the surface (high valency, HV, display). In embodiments where the conditional mutation is suppressed, for example when using a suppressing strain of E. coli, for example using an amber suppressing strain, for example a supE+ strain, then the helper phage encoded pIX will be produced / expressed, resulting in a mixture of helper phage pIX (non-fusion pIX) and POI-modified pIX fusion protein on the surface (low valency, LV, display).

[0084] Suitable conditional mutations are well known to the person skilled in the art, and a helper phage vector can easily be designed and a suitable host cell selected, such that the expression of the helper phage encoded pIX is controlled by the conditional mutation. For example, as mentioned above, in a helper phage known as Delta phage, one or more conditional mutations in the form of suppressible stop codons (for example amber mutations / amber stop codons, or other suppressible stop codons, for example ochre mutations / stop codons or opal mutations / stop codons) are used in combination with a suitable host cell to suppress or not suppress the conditional mutation.

[0085] Other means of high valency (HV) display can be achieved with the phagemid (plus helper phage) system. For example, any helper phage in which the pIX phage coat protein is missing (for example has been deleted) or does not produce a functional pIX phage coat protein (for example due to a mutation or truncation) can be combined with a phagemid of the application, i.e. a phagemid comprising a sequence encoding a POI-modified pIX fusion protein of the application (or an ORF comprising a sequence encoding a POI-modified pIX fusion protein of the application), to achieve HV display. Again, such a system is designed such that the POI-modified pIX fusion protein of the application should be the only functional pIX coat protein in the system, and thus should be the only pIX coat protein displayed on the surface of the phage, resulting in HV display.

[0086] In some embodiments of the application, high valency display and systems allowing high valency display are preferred because the modified pIX vectors of the application have proven to be particularly effective and advantageous when combined with high valency display. However, equally, the modified pIX vectors of the application are compatible with low valency display and systems allowing low valency display. Methods and systems to achieve low valency display will be well known to the person skilled in the art. For example, the modified pIX vectors of the application, for example phagemid vectors, can be used with helper phage under inhibitory conditions as described above, for example Delta phage. Alternatively however, the modified pIX vectors of the application can be used with more conventional helper phage encoding pIX (non-fusion pIX) coat proteins, for example where expression of the pIX coat protein is not specifically controlled or inhibited, for example M13K07 or VCSM13, to achieve low valency display on the surface with a mixture of helper phage (non-fusion) pIX and POI-modified pIX fusion proteins. Thus, in some embodiments, low valency display and systems allowing low valency display can be used with the modified pIX vectors of the application.

[0087] HV display has traditionally not been used to identify high affinity binders because it is believed that the avidity effect created by the display of multiple copies of the POI can compromise high affinity selection. Conversely, LV display has generally been used to allow high affinity binders to be identified. However, it has been shown that the modified pIX vectors of the application can be advantageously used in HV display systems to identify high affinity binders. HV display systems have the additional advantage of maximising the functional fraction of phage particles because more of the particles will have POI fusion proteins, which means that there will be a wider functional diversity in the displayed POIs, which in turn means that it is more likely that a binder will be found. Thus, the ability of the HV display systems of the application to combine an improved functional fraction with improved functional properties of the displayed candidates is highly advantageous, for example in successfully identifying binders to a target of interest.

[0088] As used herein, the term high valency (HV) display refers to a phage display system that is designed to maximise the number of copies of a particular phage coat fusion protein, here a modified pIX fusion protein, displayed on the surface of the phage when compared to the number of wild type (or non-fusion) pIX proteins displayed on the surface. Thus, such a system is designed so that all (theoretically all) copies of the particular phage coat protein, here a pIX phage coat protein, displayed on the surface of the phage should be a POI fusion protein, here a POI-modified pIX fusion protein. Thus, in HV display, the system is designed so that it is possible to display 5 copies of a POI-modified pIX fusion protein on each particle.

[0089] As used herein, the term low value (LV) display refers to a phage display system that is designed such that a mixture of coat protein fusion proteins (here, modified pIX fusion proteins) and wild type (or non-fusion) pIX proteins are displayed on the surface of phage. Thus, such systems are designed such that the particular phage coat protein (here, pIX phage coat protein) displayed on the surface of the phage is not all (theoretically not all) copies, i.e., less than 5 copies, e.g., 4, 3, 2, or 1 copy (or non-maximal copies or low copies, e.g., less than 5, 4, 3, 2, or 1 copy) is a POI fusion protein, here a POI-modified pIX fusion protein. Such systems are typically set up to achieve an average of 1 copy or less than 1 copy of POI fusion protein per phage particle (although such systems can be set up to achieve higher averages if desired). Thus, in setting up a system to achieve an average of 1 copy or less than 1 copy of POI fusion protein per phage particle, many phage particles will not display a POI-modified pIX fusion protein at all.

[0090] The application as described herein is designed for prokaryotic systems rather than, for example, eukaryotic systems. Thus, suitable host cells are prokaryotic cells, and in particular, bacterial cells. Suitable bacterial hosts for phage display that can be used to express the vectors and nucleic acid sequences of the application and to package and produce phage particles are well known to those skilled in the art and can be selected accordingly. Preferred bacterial host cells are Gram-negative bacteria, such as E. coli strains. Exemplary E. coli strains include XL-1 blue, TG1, ER2738, AVB100FmkII’, MC1061, SS320, TOP10F’, and K91K. In some embodiments, non-suppressor strains are preferred, such as amber non-suppressor strains, examples of which are SS320, TOP10F’, AVB100FmkII’, MC1061, and K91K. In other embodiments, suppressor strains are used, such as XL-1 Blue, TG1, or ER2738.

[0091] The term “phage”, commonly referred to as bacteriophage, is used herein in its art-recognized form to mean a virus that infects, replicates in, and is secreted from bacteria. A filamentous bacteriophage or filamentous phage is a phage that has a single-stranded DNA genome (ssDNA genome) that is packaged with phage coat proteins. The secreted filamentous phage particles have a filamentous structure in phenotype. Filamentous bacteriophage or filamentous phage is preferably used in the present application.

[0092] As used herein, the term phage or filamentous phage or filamentous bacteriophage encompasses both phage genome-derived virions and phagemid-derived virions.

[0093] The term "phagemid" is a term in the art and refers to a type of cloning vector developed as a hybrid of the filamentous phage Ff and a plasmid to create a vector that can be propagated as a plasmid and also packaged in viral particles as single-stranded DNA. Similar to a plasmid, a phagemid can be used to clone DNA fragments and is introduced into a bacterial host by a range of techniques (e.g. transformation, electroporation). However, infection of the bacterial host containing the phagemid with a "helper" phage (e.g. VCSM13 or M13K07 or the above described Delta phage) provides the essential viral components to enable single-stranded DNA replication and packaging of the phagemid DNA into phage particles.

[0094] The term "helper phage" is a term in the art and refers to a virus that aids the propagation of a separate and unrelated defective virus (e.g. phagemid) by infecting the same host cell already occupied by the defective virus (e.g. phagemid) and by providing proteins missing from the defective virus (e.g. phagemid) and required to complete its life cycle and form viral particles (e.g. containing the phagemid), such defective virus being not a phage genome or functional virus per se, but merely a plasmid containing one or several elements derived from a phage genome (here containing at least one modified pIX protein).

[0095] Preferred helper phages for use in the present application are described elsewhere herein and include M13K07 (Stratagene), Hyperphage (Progen Biotechnik GmbH), R408 (Agilent Technologies), and VCSM13 (Stratagene). In preferred embodiments, the helper phage can be a helper phage having a conditional (or suppressible) mutation as described herein, e.g. a Delta phage helper phage as described herein and in the art, or Phaberge, or Ex-phage.

[0096] Another aspect provided by the present application is a phage display system comprising a vector (or nucleic acid molecule) of the present application. Preferred phage display systems comprise a vector (or nucleic acid molecule) of the present application, e.g., a phagemid vector of the present application, and a helper phage, e.g., a helper phage as described herein, e.g., a helper phage capable of expressing a pIX phage coat protein, e.g., a non-fused pIX phage coat protein. Other preferred phage display systems of the present application comprise a vector (or nucleic acid molecule) of the present application, e.g., a phagemid vector of the present application, and a bacterial host cell, e.g., an E. coli host cell / strain. Suitable host cells / strains are also described elsewhere herein and can be included as components in all phage display systems, kits, methods, and uses described herein. Other preferred phage display systems include a vector (or nucleic acid molecule) of the present application, a helper phage, e.g., a helper phage as described herein, e.g., a helper phage capable of expressing a pIX phage coat protein, e.g., a non-fused pIX phage coat protein, and a bacterial host cell, e.g., a bacterial host cell as described herein, e.g., an E. coli host cell / strain.

[0097] Thus, yet another embodiment of the present application provides a phage display system of the present application as described elsewhere herein, which further comprises a helper phage and / or a bacterial host cell strain, e.g., an E. coli host strain.

[0098] In some embodiments, the pIX phage coat protein encoded by the helper phage is capable of complementing or competing with the modified pIX phage coat protein encoded by the vector construct or nucleic acid molecule of the present application. In such embodiments, the pIX phage coat protein encoded by the helper phage is in fact capable of providing additional copies of pIX phage coat protein, e.g., additional copies of non-fused pIX phage coat protein, e.g., additional copies of functional pIX phage coat protein, which are available for use in forming a phage coat. In some such embodiments, the pIX phage coat protein encoded by the helper phage is produced or expressed under the control of one or more conditional mutations, e.g., one or more suppressible mutations, e.g., as described elsewhere herein. For example, in preferred embodiments, the suppressible mutation is a suppressible stop codon, preferably selected from the group consisting of an amber stop codon, an ochre stop codon, and an opal stop codon, more preferably an amber stop codon. A preferred helper phage for use in such systems is a Delta phage, the details of which are described herein and elsewhere in the art. Suitable and preferred E. coli host strains for use in such embodiments are suppressible strains, preferably amber suppressible strains, more preferably XL-1 Blue, TGI, or ER2738.

[0099] In some embodiments, the pIX phage coat protein encoded by the helper phage is unable to complement or compete with the modified pIX phage coat protein encoded by the vector construct or nucleic acid molecule of the application. This inability to complement or compete can result in any suitable manner. For example, this inability to complement or compete can result from the pIX phage coat protein encoded by the helper phage being non-functional, e.g. due to mutation or truncation, or from the pIX phage coat protein encoded by the helper phage being absent, e.g. due to deletion.

[0100] Alternatively, this inability to complement or compete can result from the pIX phage coat protein encoded by the helper phage not being produced or expressed, e.g. the pIX phage coat protein is produced or expressed under the control of one or more conditional mutations, e.g. one or more repressible mutations, e.g. as described elsewhere herein, and the production or expression is repressed. For example, in preferred embodiments, the repressible mutation is a stop codon that is capable of repression, preferably selected from the group consisting of an amber stop codon, an ochre stop codon, and an opal stop codon, more preferably an amber stop codon, and the production or expression of the pIX phage coat protein is repressed by use of a suitable bacterial host strain. A preferred helper phage for such a system is a Delta phage, details of which are described elsewhere herein. A suitable and preferred E. coli host strain for use in such embodiments is a non-repressible strain that does not allow production or expression of the pIX phage coat protein encoded by the helper phage, preferably an amber non-repressible strain (or ochre non-repressible strain, or opal non-repressible strain), more preferably SS320 or TOP-10F’.

[0101] The vector (or nucleic acid molecule) of the application also has utility in phage display methods, i.e. can be used in phage display methods.

[0102] Accordingly, a further aspect of the application provides a method for producing a phage particle, the method comprising use of the vector construct or nucleic acid molecule of the application or use of the phage display system of the application as described herein. Such phage particles are typically produced by a method comprising the step of introducing the vector construct of the application into a suitable bacterial host cell, along with a suitable helper phage if required, examples of which are described elsewhere herein.

[0103] Accordingly, a further aspect of the application provides a method of phage display, the method comprising the steps of:

[0104] a. providing a bacterial host cell / strain, e.g. an E. coli host strain, comprising a vector construct of the application, wherein the open reading frame further comprises a sequence encoding a protein of interest fused to a sequence encoding a modified pIX phage coat protein of the application, wherein expression of said vector construct leads to the production of a protein of interest-modified pIX fusion protein;

[0105] b. providing a helper phage; and

[0106] c. infecting said bacterial host cell / strain, e.g. said E. coli host strain, with said helper phage under conditions such that said host strain produces phage particles displaying said protein of interest-modified pIX fusion protein.

[0107] In some embodiments, such a method can be used for high- value phage display. Thus, in some embodiments, the method is a method for high-value phage display, wherein the pIX filamentous phage coat protein encoded by the helper phage cannot complement the POI-modified pIX filamentous phage coat protein encoded by the vector construct of the application.

[0108] In particular, the application provides a method for high-value phage display, comprising the steps of:

[0109] a) providing a non-repressor bacterial host cell / strain, e.g. a non-repressor E. coli host strain, comprising a vector construct of the application, wherein the open reading frame further comprises a sequence encoding a protein of interest fused to a sequence encoding a modified pIX phage coat protein of the application, wherein expression of said vector construct leads to the production of a protein of interest-modified pIX fusion protein;

[0110] b) providing a helper phage, wherein expression of the pIX phage coat protein of the helper phage is controlled by one or more repressor mutations;

[0111] c) infecting said non-repressor bacterial host cell / strain, e.g. said E. coli host strain, with said helper phage under conditions such that the pIX phage coat protein encoded by the helper phage is not expressed or produced, such that said non-repressor host cell / strain produces multiple copies of phage particles displaying said protein of interest-modified pIX fusion protein.

[0112] As described elsewhere herein, in some embodiments, such high-value display is preferred.

[0113] In other embodiments, the method of the application can be used for low- cost phage display. Thus, in such embodiments, the method is a method for low-cost phage display, wherein the pIX filamentous phage coat protein encoded by the helper phage is capable of complementing the POI-modified pIX filamentous phage coat protein encoded by the vector construct of the application.

[0114] In particular, the application provides a method for low-cost phage display, the method comprising the steps of:

[0115] a) providing a suppressor bacterial host cell / strain comprising a vector construct of the application, e.g. a suppressor E. coli host strain, wherein the open reading frame further comprises a sequence encoding a protein of interest fused to the sequence encoding the modified pIX phage coat protein of the application, wherein expression of the vector construct results in the production of a protein of interest-modified pIX fusion protein;

[0116] b) providing a helper phage, wherein expression of the pIX phage coat protein of the helper phage is controlled by one or more suppressor mutations;

[0117] c) infecting the suppressor bacterial host cell / strain, e.g. the E. coli host strain, with the helper phage under conditions such that the pIX phage coat protein encoded by the helper phage is expressed or produced, such that the suppressor host cell / strain produces single copy or low copy phage particles displaying the protein of interest-modified pIX fusion protein.

[0118] In any phage display method according to the application and as described herein, e.g. high- cost phage display or low-cost phage display, preferably, a library of the vector constructs of the application encoding a plurality of proteins of interest is used. In other preferred embodiments, the phage display method is used for the selection of proteins that bind to a desired target molecule.

[0119] When one or more proteins (POI) have been selected using the method of the application, these proteins or components, fragments, variants, or derivatives of these proteins can be manufactured or produced, and if desired, formulated together with at least one pharmaceutically acceptable carrier or excipient. Such manufactured molecules or components, fragments, variants, or derivatives of molecules are also encompassed by the application. Alternatively, these molecules can take the form of nucleic acids encoding the proteins, which in turn can be incorporated into suitable expression vectors and / or contained in suitable host cells. Thus, nucleic acid molecules encoding the proteins, or expression vectors containing the nucleic acid molecules, form further aspects of the application.

[0120] Thus, another aspect of the application provides a method of producing or manufacturing a protein (POI), the method comprising the step of selecting a protein according to the method of the application as described herein, manufacturing or producing said protein or a component, fragment, variant, or derivative of the protein, and optionally formulating said manufactured protein with at least one pharmaceutically acceptable carrier or excipient. In other words, said method of the application as described herein, e.g. a method for selecting a protein, can also comprise the step of manufacturing or producing said protein or a component, fragment, variant, or derivative of the protein, and optionally formulating said manufactured or produced antibody with at least one pharmaceutically acceptable carrier or excipient. Said variants or derivatives of the protein can have at least 60%, 70%, 80%, 90%, 95%, or 99% sequence identity with the original polypeptide from which they are derived.

[0121] Another aspect described herein (e.g. for use in the methods of the application) is a kit comprising a vector (or nucleic acid molecule) of the application, or a kit comprising a phage display system of the application as described above, e.g. comprising a phagemid and a helper phage of the application, preferably a helper phage as described herein, e.g. a helper phage wherein the pIX phage coat protein is expressed under the control of a conditional (or suppressible) mutation as described herein, or a kit comprising a vector (or nucleic acid molecule) of the application, e.g. a phagemid vector of the application, and a bacterial host cell, e.g. an E. coli host strain as described herein, e.g. a non-suppressible E. coli host strain. The kit can also include necessary instructions for use. Also provided is a kit comprising a phagemid, a helper phage, and a bacterial host cell of the application as described herein. Preferred vectors, helper phages, and bacterial host cells of the application for use in such kits are as described elsewhere herein.

[0122] Preferred vectors (or nucleic acid molecules) of the application for inclusion in such kits can comprise a modified pIX phage coat protein vector of the application as described herein, further comprising one or more cloning sites (e.g. a multiple cloning site) suitable for cloning a POI, which is then fused to the modified pIX phage coat protein.

[0123] Thus, preferred kits can comprise or consist of a collection of reagents for producing phage particles having a fusion protein of a POI of the application and a modified pIX coat protein. In addition to a vector of the application, the kit can also include one or more components selected from the group consisting of: other phagemids, helper phages, bacterial strains, and instructions. Preferred options for such additional components are as described elsewhere herein.

[0124] Another aspect of the application provides the use of a vector construct, nucleic acid molecule, phage display system or kit of the application to produce phage particles, or for phage display. In other words, the application provides a method for producing phage particles (or a method of phage display), the method comprising the use of a vector construct, nucleic acid molecule, phage display system or kit of the application. Such a method for producing phage particles typically comprises the step of introducing a vector construct or nucleic acid molecule of the application, together with a suitable helper phage if required, into a suitable host cell (e.g. a bacterial host cell), examples of which are described elsewhere herein.

[0125] The phage particles of the application as defined herein can also be used as molecular tools for in vitro applications and assays. The particles can be used in any assay requiring the display of a POI on a pIX phage protein.

[0126] As the preferred phage particles of the application also display a POI, as described elsewhere herein, this POI can be a specific binding partner or targeting unit, e.g. an antibody or the like, which can be used as a member of a specific binding pair or targeting agent, and such phage particles can be used in any assay requiring a specific binding pair member or targeting unit.

[0127] Accordingly, further aspects of the application provide reagents comprising a phage particle of the application as defined herein and the use of such phage particles as molecular tools, e.g. in in vitro assays.

[0128] As used throughout this application, the terms "a" and "one" are intended to denote "at least one", "at least a first", "one or more" or "more than one" of the referenced component or step, unless the context specifically indicates otherwise.

[0129] Further, where the terms "comprising", "including", "containing", or other equivalent terms are used, then in some more specific embodiments these terms include the term "consisting of or "consisting essentially of, or other equivalent terms.

[0130] Where appropriate, a method comprising certain steps also includes a method consisting of those steps. In the methods described herein, the method steps can be performed in any suitable order.

[0131] The terms "increase" or "improve" or "enhance" (or equivalent terms) as described herein include any measurable increase or improvement when compared to a suitable control. Suitable controls can be readily identified by one skilled in the art and can include levels of a particular parameter determined when using wild-type pIX phage coat proteins as compared to the modified pIX phage coat proteins of the application. Preferably, the increase etc. will be significant, e.g. statistically significant, e.g. a probability value of specifically < 0.05, when compared to a suitable control level or value. Methods of determining statistical significance of differences are well known in the art and documented.

[0132] Some of the sequences mentioned herein are summarized in the following table together with the relevant identifiers.

[0133]

[0134]

[0135] According to the conventions of the technical field, all sequences in this table are listed herein 5' to 3' or from N-terminus to C-terminus. BRIEF DESCRIPTION OF DRAWINGS

[0136] The application will be further described with reference to the following non-limiting examples, with reference to the following drawings, in which:

[0137] Figure 1 : Polyclonal phage ELISA and monoclonal screening for OMV reactivity. (A) Normalized phage samples from R0 and R3 outputs were analyzed by ELISA for binding to OMV. Phage displaying an irrelevant specificity (scFv anti-NIP) were included as a control. (B) Random single colonies after R3 were rescued to high value (HV) display for all libraries. Samples were analyzed by ELISA for OMV reactivity and scored positive with a S / B (signal / background) ratio > 3. The percentage of OMV positive clones within each library group is indicated. Supernatant from empty E. coli SS320 was included as a control. (C) Phage libraries after R3 were reformatted by batch cloning for soluble scFv E. coli expression and random single colonies were analyzed by ELISA for OMV binding. et al., 2016).

[0138] Figure 2 : SDS-PAGE / Western blot analysis. Whole human antibody phagemid libraries (R0 and R3) were separated by 4-12% SDS PAGE (A) and Western blot (B) analysis was performed using a polyclonal anti-human Fc antibody. The positions of the molecular weight markers are indicated on the left. (A) Anti-pIX Western blot analysis of normalized amounts of phage (left) and determined anti-phOx scFv phagemid control clone (right) of Katsenelson et al., 2016) probed with polyclonal rabbit anti-pIX serum. M13K07 helper phage was included as a control (C). Both phagemid samples were packaged as low valency (LV rescued with M13K07) or high valency (HV rescued with Delta phage) display. pIXwt and scFv-pIX fusions are indicated.

[0139] Figure 3 (A) Schematic of the pV, pVII, pIX, and pVIII encoding genomic regions of the M13 filamentous phage. The pIX ORF has a start codon internal to the pVII ORF and is expressed as a complete protein without any post-translational processing. (B) Schematic of the scFv-pIX phagemid expression cassette. The heterologous scFv fusion is placed N-terminally to the complete pIX capsid coupled by an artificial linker / spacer as disclosed in Katsenelson et al., 2011). The complete phagemid sequence is available via GenBank accession code HQ528250. Transcription is controlled by the lac promoter (LacPO) through T7 terminator and subsequent mRNA to protein translation of the consecutive open reading frame (ORF) starting at the Met encoding start codon* (C). The starting amino acid sequence (upper region) on the connection of the scFv* and the linker to the native pIX** is shown, wherein the native pIX Met (M) starting residue is indicated in bold underlined**. As indicated (lower panel), site-specific mutagenesis was performed on this residue***.

[0140] Figure 4 : Phage yield after phagemid rescue was determined by infectious titer (cfu ampR / ml) or by total virion content determined by OD using the formula (((A269nm-A320nm) x 6.083 x 10 16 ) / genome size = virions / ml).

[0141] Figure 5M1x anti-phOx scFv phage were produced in E. coli XL1 -blue (A) at LV or in E. coli SS320 (B) at HV, titrated and phage used in phage capture ELISA to assess target binding to phOx-BSA, with similar total amount of virions per sample. In the same graph the level of target binding (left x-axis) is plotted against the corresponding titers (right x-axis). (C) Anti-phOx scFv phage displayed on pIXwt were produced in E. coli XL1 -blue at low value (LV) or in E. coli SS320 at high value (HV), titrated and phage concentration of low titer (LT) 10 8 cfu ampR / ml and high titer (HT) 10 10 cfu ampR / ml were used in phage capture ELISA to assess target binding to phOx-BSA. (D) Anti-pIX protein blot analysis of phage displaying M1x anti-phOx scFv separated by 4-12% SDS PAGE and then probed with polyclonal rabbit anti-pIX serum (upper part). All phagemids were produced in E. coli SS320 at HV, except for the wild type phage, which was also produced in E. coli XL1 -blue at LV. To compare the amount of phage used in the experiment, a protein blot analysis was performed with mouse anti-pIII (lower part).

[0142] Figure 6 : Phage yield after phagemid rescue was determined by infectious titer (cfu ampR / ml) or by total virion content determined from OD using the formula (((A269nm-A320nm) x 6.083 x 10 16 ) / genome size = virions / ml). Each individual phage M1x version was produced and titrated separately. Thereafter, phage samples were grouped according to their biochemical similarity and the average of the average of the combined titers within each group is shown as indicated in the figure.

[0143] Figure 7 : M1x anti-NIP scFv phage were rescued using Delta phage in E. coli XL1 -Blue (A) and E. coli SS320 (B) at LV and HV, respectively, titrated and target binding to NIP-BSA assessed in phage capture ELISA using serial dilutions of each individual M1x variant. Thereafter, variants were grouped according to their biochemical similarity and the average of the average of the combined data of each individual binding curve within each group is shown as indicated in the figure. It is worth noting that some samples produced very little phage and could only be tested at low titers.

[0144] Figure 8 Phage target binding and phage titers were given a score from 1 to 10 based on the relationship between individual values (A). Since target binding did not reach signal saturation for all groups, specific target binding was defined as the titer that produced 2-fold over the baseline value, and the grades were scored accordingly. The M1L_I_G group, representing the best score for the combination between target binding and phage titer (Q4), was separated into individual amino acids (B).

[0145] Figure 9 Comparison of M1L and M1G. M1L phage, M1G phage, and wt anti-phOx and anti-NIP scFv phage were produced in E. coli XL1 -blue or E. coli SS320, respectively, in LV and HV, respectively. Phage were titrated in phage capture ELISA and target binding to phOx-BSA (A, C) or NIP-BSA (B, D) was assessed at serial dilutions.

[0146] Figure 10 Low vs. high display. M1L phage and wt anti-phOx and anti-NIP scFv phage were produced in E. coli XL1 -blue or E. coli SS320, respectively, in LV and HV, respectively. Phage were titrated in phage capture ELISA and target binding to phOx-BSA (A) or NIP-BSA (B) was assessed at serial dilutions. LV phage and HV phage performance was compared to the standard low value protocol using M13K07 rescue and E. coli XL1 -blue (M1L_Standard-LV and wt_Standard-LV).

[0147] Figure 11 Functional binding vs. target concentration. Anti-phOx and anti-NIP scFv displayed on pIXwt and M1L were produced in E. coli SS320 in HV. Phage were titrated using serial phage dilutions and target binding to decreasing amounts of phOx-BSA (A) or NIP-BSA (B) was assessed in phage capture ELISA.

[0148] Figure 12 Evaluation of preferential target-specific enrichment between pIXwt and pIX-M1L in spiked panning II.

[0149] NIP-specific scFv were produced in E. coli SS320 (HV) using Delta phage helper phage and rescued at 1 : 10 7Target-unrelated scFv were included, followed by 3 rounds of immobilized NIP-BSA panning. Then 40 randomly selected single colonies were packaged from each mock library before (RO) and after each round of selection (R1-3) and tested for target reactivity using antigen-specific phage capture ELISA. Clones were considered positive if they exhibited a response at least 3-fold higher than background signal. Results are given as number of positive clones / total number of clones tested as indicated.

[0150] Figure 13 : A previously reported fully human scFv antibody phage library (Krauss et al., 2016) displayed on pIXwt was reformatted to pIX-M1L and two libraries were prepared from E. coli SS320 under standard-LV and HV display using M13K07 helper phage and Delta phage helper phage, respectively. The apparent level of functionally folded scFv on phage was then assessed by binding to the conformation-specific superantigen protein L (pL) in serial dilutions of titrated phage using phage capture ELISA. Non-pL binding scFv control phage were included as a control.

[0151] Figure 14 : Two indicated versions of a fully human scFv antibody phage library were used in separate selections of pHLA specific binders using the same protocol for two unrelated tumor associated antigen (TAA) specific pHLA targets in three consecutive rounds (R1-3) of parallel panning. After panning, equal amounts of polyclonal phage prepared from the R3 output as well as the unselected library (RO) were tested for target specific binding in phage capture ELISA. All samples were tested on both targets as a specificity screen for apparent specificity and as a mutual negative control. Results for each sample are shown as the signal ratio on specific vs. non-specific target as an indirect measure of target specific enrichment. An unrelated scFv control phage was included as a negative control (NC).

[0152] Figure 15 : Random single clones from the R3 output selected against pHLA TAA target 1 were amplified and phage were produced using Delta phage rescue independent of the starting standard LV or HV form used in selection to maximize sensitivity in screening. Phage were individually tested for binding to the matched pHLA target (TAA target 1) and unmatched pHLA targets (TAA target 2 and TAA target 3) in phage capture ELISA. Clones were separated into their displayed capsids (A and B) and the displayed version used in selection (standard-LV or HV). The number of target specific clones in each version is indicated. DETAILED DESCRIPTION​

[0153] Examples

[0154] Improved antibody discovery by modified pIX display

[0155] Example 1: Identification of modified pIX versions that yield improved antibody display

[0156] Materials and methods

[0157] Monoclonal phage expression

[0158] Monoclonals were packaged into 96 deep well plates for screening experiments using 400 μl media or in 50 ml cultures for larger scale expression. Briefly, clones were inoculated into YT-AG and grown ON / 37°C. For 96 deep well expression, 10 μl were transferred into a new plate containing fresh media and grown at 10 9 cfu Delta phage for 3h / 37°C / 600 rpm. For larger scale expression, cultures were re-inoculated into fresh media to an OD 600nm of 0.05 and grown at 37°C with vigorous shaking until an OD 600nm of 0.2 was reached before superinfection at MOI 10 with Delta phage or M13K07. Plates and flasks were further incubated at 37°C / 30 min and further grown for 30 min with vigorous shaking before cells were pelleted and resuspended in 2x YT-AK. Phage were packaged ON / 30°C. 100 μL of clear supernatant from deep well packaging was used for screening in ELISA, while phage from larger scale expression were PEG precipitated before spot titration 1 and used for ELISA analysis and WB analysis.

[0159] Monoclonal phage ELISA using anti-phOx phage and NIP phage

[0160] ELISA plates were coated with a series dilution of phOx-BSA or NIP-BSA in PBS starting at 5 μg / mL, incubated ON / 4°C and blocked for 1 h / RT with 4% skim milk powder in PBST. Phage were added in a series dilution and incubated 1 h / RT. Bound phage particles were detected with anti-M13-HRP (Amersham Biosciences, 1 :5,000). Phage samples and antibodies were diluted in PBST. Plates were developed with TMB solution and read at 450 nm using a microplate reader. Between each step, plates were washed 3 times with PBST.

[0161] SDS-PAGE and Western blotting

[0162] 2 x 10 9 cfu AmpR Normalization of phage samples Phage samples were normalized using BOLT TM LDS sample buffer was heated for 5 min at 95°C and then separated on a 4-12% Tris Plus gel in Bolt MES SDS running buffer (reagents from Invitrogen) at 220 V for 22 min with a wide-range ladder. Protein blots were transferred to Immobilon TM -P membranes (Sigma) using a semi-dry blotting apparatus and membranes were blocked with PBSM. Mouse anti-pIII (MoBiTec, 1 :50 000) and anti-mouse IgG-HRP (1 :10.000) were used for pIII detection. For pIX detection, polyclonal anti-pIX rabbit serum was raised by immunization with a peptide of the C-terminal pIX portion (N-CITYFTRLMETSS-C; SEQ ID NO: 9) (AbMART). Anti-pIX serum was used at 1 :2000 in combination with anti-rabbit IgG-HRP (1 :5000). Protein blots were detected by reading chemiluminescence signals.

[0163] Spiking panning

[0164] Phage displaying NIP-specific scFv on pIXwt or pIX-M1L were rescued with Delta phage from two amber non-suppressor E. coli strains, TOP10F’ or SS320, titered and mixed in approximately 50 / 50 mix, followed by one round of selection on immobilized NIP-BSA. Briefly, ELISA wells (NUNC) were coated with 5 pg / mL phOx-BSA or NIP-BSA in PBS ON / 4°C. Phage were incubated with antigen for 1.5 h at (RT) room temperature with agitation. Wells were washed with 10x PBST and 5x PBS and elution of bound phage was performed by incubation with 0.5 mL 0.5% trypsin for 10 min. Both input phage and eluate were used to infect E. coli and 20 to 24 random single clones from each group were sequenced (Eurofins Genomics).

[0165] Reformatting of pIX libraries

[0166] Reformatting of the original state human scFv-pIX phage library to scFv-pIX_M1L scaffold 2 . Using 10 10 cfu AmpRAs template, 0.25 μΜ each of forward 5'-ATTAAAGAGGAGAAATTAACCATGGCCC-3' (SEQ ID NO: 10) and reverse 5'-TTTTGGATCCAGCGGCCGC-3' (SEQ ID NO: 11), biotinylated primers (Eurofins Genomics) containing Ncol and Notl RE-sites, and 0.05 U / ml Phusion High-Fidelity DNA Polymerase, the scFv cassette was PCR-amplified directly from scFv-pIX library phages. Correct bands were extracted from agarose gels and then subjected to digestion and capture of the biotinylated ends using MyOne Streptavidin Tl magnetic beads (Invitrogen). The scFv cassette was purified and ligated into phosphatase-treated vectors in the presence of polynucleotide kinase at 16°C overnight, followed by purification using Pellet Paint Coprecipitant (Novagen). The ligation mix was used to transform electrocompetent E. coli SS320 (Lucigen) essentially as described before 3 , using 350 μΐ aliquots and an ECM 600 electroporator (BTX). The transformation mix was plated on bioassay dishes (Nunc) and incubated at 30°C overnight (ON). An amount of 8.7 x 1010primary transformants was obtained, which was scraped from the plate and rescued (see phage rescue and PEG / NaCl precipitation section). The size of the library was limited to the size of the scFv-pIX library pool, which was determined to have a diversity of 3 x 1010. 9 8 2

[0167] Phage rescue and PEG / NaCl precipitation

[0168] The scraped material was inoculated in 2x YT supplemented with 30 μg / ml tetracycline, 100 μg / ml ampicillin, and 0.1 M glucose (2x YT-TAG) to an OD 600 ​​nm 0.05 and incubated at 37°C with vigorous shaking until OD reached 0.1 to 0.2 for phagemid rescue. The culture was superinfected with (Delta phage against pIX_M1L library, R1, R2 and R3 or M13K07 against R3 only) at MOI 20 and incubated at 37°C with gentle shaking for 60 min, then vigorous shaking for 30 min, then centrifuged and the medium replaced to 2x YT supplemented with 100 pg / ml ampicillin and 50 pg / ml kanamycin (2x YT-AK) and further incubated at 30°C for 7 h. Phage particles were purified and concentrated by 2x PEG / NaCl precipitation and resuspended in PBS and cfu determined by spot titration. 1

[0169] Protein L ELISA

[0170] ELISA plates were coated with 5 pg / mL protein L (pL) in PBS and incubated ON / 4°C and blocked for 1 h / RT with 2% skim milk powder in PBST. Phage samples were added in serial dilution and incubated for 2 h / RT. Bound phage particles were detected with anti-M13 antibody conjugated with HRP (produced by Norwegian Antibodies with chicken immunized with M13 phage). Phage samples and antibody were diluted in PBST. Plates were developed with TMB solution and read at 450 nm using a microplate reader. Between each step plates were washed 3 times with PBST.

[0171] Phage selection

[0172] Selection was performed using both solid- and solution-based panning. Pre-blocked phage samples were incubated for 1 hour with 100 nM biotinylated HLA-A2 loaded with a panel of irrelevant TAAs captured onto MyOne streptavidin Tl beads. Unbound phage was transferred to a new tube and incubated for 1 hour with 100 nM biotinylated HLA-A2:TAA target 1 pre-captured onto beads (solid-based panning for Rl) or in solution (solution-based panning for R2 and R3) and then captured onto beads. Antigen concentration was reduced to 10thof each round and wash stringency was increased from 8x PBST + 2x PBS in Rl to 13x PBST + 2x PBS in R2 and 18x PBST + 2x PBS in R3. Prior to R2 and R3, phage samples were heat challenged at 65°C for 15 minutes prior to panning. Tubes were briefly vortexed between each wash. 4% (w / v) skimmed milk powder (PBSM) or 2% (w / v) bovine serum albumin (essentially fatty acid free) were used as blocking agents in alternating selection rounds. Elution was performed by incubation with 0.5 ml 0.5% trypsin for 10 minutes and then half of the eluate was used to infect E. coli. Infected colonies were scraped and rescued (see phage rescue and PEG / NaCl precipitation section). A small sample of the infected culture was removed to determine yield.

[0173] Screening of selection output

[0174] Individual clones were packaged into 96 deep well plates as described above. 4 Briefly, clones were inoculated into YT-AG and incubated ON / 37°C / 600 rpm. 10 μΙ_ were transferred to a new plate containing fresh media and 10 9 Growth for 3 hours prior to superinfection with cfu Delta phage. Plates were incubated ON / 37°C / 30 minutes with gentle agitation and for a further 30 minutes with vigorous shaking before cells were pelleted and resuspended in 50 μΙ_ 2x YT-AK and phage packaged ON / 30°C. 100 μΙ_ of clarified supernatant was used for screening in ELISA.

[0175] Monoclonal phage ELISA from phage selection

[0176] ELISA plates were coated with 5 pg / mL NeutrAvidin in PBS, incubated ON / 4°C, and blocked with 5% nonfat dry milk in PBST for 1 h / RT. Biotinylated pHLA variants were captured for 1 h / RT, followed by addition of phage. Bound phage particles were detected with anti-M13-HRP (Amersham Biosciences, 1 :5,000). pHLA, phage samples, and antibody were all diluted in PBST. Plates were developed with TMB solution and read at 450 nm using a microplate reader. Between each step, plates were washed 3 times with PBST.

[0177] References

[0178] 1 G. Kristinsson, S. G. & Sandlie, I. Reliable titration of filamentous bacteriophages independent of pIII fusion moiety and genome size by using trypsin to restore wild-type pIII phenotype. BioTechniques 44, 551-554 (2008).

[0179] 2 L. S. et al. Multivalent pIX phage display selects for distinct and improved antibody properties. Sci. Rep. 6, 39066, doi:10.1038 / srep39066 (2016).

[0180] 3 Tonikian, R., Zhang, Y., Boone, C. & Sidhu, S. S. Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nat Protoc 2, 1368-1386 (2007).

[0181] 4 Frick, R. et al. A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation. Science Immunology 6(62), eabg4925, doi:10.1126 / sciimmunol.abg4925 (2021).

[0182] Results

[0183] We have previously shown that antibody discovery using pIX can improve the efficacy of identifying desired target-specific antibody candidates, as well as generating clones with superior biophysical properties, compared to those found by standard pIII display Figure 1 ) 1 This beneficial property was achieved by the combined use of pIX as an antibody display scaffold and a modified helper phage called Delta phage 2 , which allows multivalent high valency (HV) display of pIX fusion proteins.

[0184] In previous studies, when comparing the use of pIX display and pIII display as an antibody display scaffold, there was a strikingly surprising difference in the actual downstream success rate in target-specific signal sampling bulk output and discovery in target binding assays after phage library selection Figure 1 A vs Figure 1 C). Traditionally, one would expect higher target signals from phage selection output to be an indication of a more successful enrichment of specific clones, but in our case this was not a good correlation. Furthermore, in HV phage display, functional avidity effects (also known as avidity) are expected to impair high affinity selection, in contrast to low valency (LV) display that effectively allows for the identification of higher affinity binders 3 However, this was not the case in our previous studies of two comprehensive antibody library selections, as HV pIX display outperformed LV pIII display and HV pIII display in successful antibody identification Figure 1 C), and identified the strongest target binders consistently 1 There is a strong correlation between phage genome size and virion size 4 As pIX is encoded by a much smaller gene than pIII, virion display in pIX has a smaller virion coat than phage expressing pIII 4 This would lead to a smaller footprint of the target binding assay, e.g.Figure 1 The lower signal shown in A. Moreover, we previously found a higher tendency to form large polyomavirus virions in pIII systems that further amplified this effect, as target-specific detection is based on virion recognition 1,4 .

[0185] To better understand whether the differences in productive scFv antibody display between the use of pIX and pIII as display scaffolds would also influence how to interpret these results, we performed binding experiments of pIX library and pIII library of different antibody pools matched to pL, a superantigen 5 that binds functional antibodies, as well as capsid-specific Western blots to assess the actual display levels. Indeed, both the most successful display approaches, LV pIII and HV pIX, had clearly similar antibody levels in these assays. Moreover, as expected, there was a significant difference in display levels on LV versus HV display.

[0186] Further studies focusing on pIX display using Western blots and subsequent anti-pIX detection did indeed show a clear difference in apparent scFv antibody display between LV and HV formats Figure 2 ).

[0187] However, we also made some additional observations in these assays. First, for unknown reasons, pIX lacking any scFv fusion always produced two different bands of about 12.5 and 18 kDa, which are higher than its estimated MW of about 3.6 kDa. However, deviations in SDS PAGE migration behavior are well known for other M13 coat proteins, such as pIII 6 . Second, although the helper phage system for phage PhiX174 has been designed to completely block pIXwt expression in amber non-suppressor E. coli strains such as TOP10F’ and SS3202, we consistently observed a clear lack of detection of scFv fusion pIX in HV samples Figure 2 . Therefore, in particular, we were interested in the latter observation, as this observation did not immediately point to non-specific degradation, which would typically produce a variety of lower MW intermediates.

[0188] It is well known that pIXwt has a complex and only partially characterized translation initiation that is apparently coupled to the upstream pVII coat due to an inefficient pIX-specific Shine-Dalgarno (SD) site Figure 3 A) 7,8 . Therefore, we re-examined the original scFv-pIX fusion design to consider whether there is another explanation for the apparent defined blend of scFv-pIX and free pIX in HV display samples.Figure 2 ). Our previous design contained only one SD upstream of the scFv ORF, so it should ensure efficient single translation initiation of the complete heterologous fusion protein ORF. However, we used the complete native pIX ORF, so the design also contains two naturally occurring initiation codons encoding a methionine (M) at the N-terminus of the scFv and the native methionine of pIX. We considered the possibility that the hitherto undescribed ribosome wobble or cryptic pIX intrinsic SD-like sequence might lead to independent simultaneous translation of both products Figure 3 B) To our knowledge, such atypical translation, e.g. hopping and translation wobble, has not been described in the context of pIX in filamentous phages9,10.

[0189] To test the hypothesis of independent translation initiated by the native pIX methionine (hereinafter referred to as M1) in our system, we therefore performed a limited M1x targeting design replacing this M1 Figure 3 C) We performed a number of modifications, e.g. removal of side chains (M1A and M1G), introduction of negatively charged residues compatible with the overall negative charge of the virion outer shell (M1D and M1E) 11 and introduction of a small hydrophobic side chain (M1L). In this initial test, we avoided the positively charged R chain, as this might confer virion instability in the close environment of the virion outer shell 12 Various pIX variants were tested in the context of a well-characterized previously described phagemid display of anti-phOx scFv 4 In both the amber-suppressing E. coli strain XL1-Blue and the non-suppressing E. coli strain SS320, phagemid rescue was used to prepare virions using the phage lambda Deltas, presenting LV display and HV display, respectively. First, we assessed whether there are significant differences in virion production between the different M1x versions and the unmodified pIX (referred to as wt) Figure 4 ).

[0190] Here, we determined both the virion content (cfu ampR ) based on infectious particles and the total amount of virions (A 268 ) 2 In normal phage production, about 10% to 50% of the virions are infectious 13 , whereas in the case of multimeric phages forming several genome unit length virions, there is a corresponding difference between infectious virion titers and total virion titers 1 In the case of LV display, where helper phage pIXwt expression is allowed and thus complements the heterologous fusion protein, as expected Figure 4, left panel), we observed uniform and roughly equal virion production as measured by both methods. However, this changed in the case of HV display, such that all pIX M1x variants displayed varying degrees of predominantly reduced infectious titers Figure 4 , right panel). The most pronounced detrimental effects were seen for the M1D variant and the M1E variant, which both exhibited a drop in infectious titers to one percent and no concomitant reduction in total genome counts. On the other hand, the M1A variant, the M1G variant, and the M1L variant only showed a mild reduction in infectious titers compared to the pIXwt variant and a corresponding reduction in total virion titers.

[0191] We also tested the different phage variants in ELISA for target binding capacity Figure 5 ). The normalized amount of virions was set to the limit of detection of the unmodified pIX display response (based on A 268 , corresponding to 8 x 10 7 / ml, Figure 5 C) were used for both LV samples and HV samples to better visualize the M1x response changes and to plot the individual overall undiluted infectious titers together with the target binding responses in the graphs. Corresponding to the results in Figure 4 , the infectious phage input was roughly identical in all LV samples Figure 5 A), while this was significantly different in the HV samples Figure 5 B). Moreover, in the LV samples, there was a clear difference in antigen binding, where all M1x variants bound the scFv to a greater or lesser extent than displayed on unmodified pIX Figure 5 A). Furthermore, two samples stood out clearly in terms of target reactivity and similar levels, namely the M1D variant and the M1L variant. When changing the scFv display mode from LV to HV, the apparent target reactivity was expected to increase due to increased scFv display and due to varying degrees of functional avidity effects caused by multivalent scFv display 2 . In fact, we did observe an improved target reactivity for all M1x variants in the HV format compared to unmodified pIX and the LV counterparts. This effect was pronounced for the M1D variant and the M1E variant, but importantly, these samples also exhibited a large difference between virions and infectious titers. It is noteworthy that the M1L variant again stood out in terms of improved target reactivity and this without severely reducing the infectious titers.

[0192] To gain insight into the actual scFv-pIX display levels, especially on the HV display version, we also performed a pIX-specific Western blot analysis comparing roughly equal amounts of total virions between each sample Figure 5D). This analysis revealed several interesting trends. First, the apparent amounts of scFv were similar between all samples. Second, there was a very clear difference in the amount of free pIX between the different Mlx versions, with unmodified pIX again showing a large fraction of fusions lacking free pIX, in particular this was also the case for the M1A variant. The M1G variant and the M1L variant showed some degree of intermediate phenotype and had significantly less free pIX than unmodified pIX. In sharp contrast to this, no free pIX was observed in the M1D variant and the M1E variant. As mentioned above, it is important to keep in mind here that these HV samples have a strong indication of multibaculovirus formation due to the different discrepancy between total virion counts and infectious titers. Irrespective of the virion length (which varies with the multibaculovirus distribution), the stoichiometry of the tip caps pVII / pIX and pIII / pVI will be similar to the infectious titers 14 Therefore we performed a parallel anti-pIII Western blot to account for the actual virion counts. This analysis indeed confirmed the infectious titers, as the virion counts were particularly low in the two samples (M1D and M1E) that showed the strongest target reactivity. For this reason, we cannot completely rule out that these samples also had traces of free pIX below the detection limit in the assay.

[0193] In summary, the combined results of these analyses of anti-phOx scFv clearly picked out the M1L variant as the most favorable variant, which both maintained overall good virion yields in LV and HV and showed a clear improvement in target reactivity that must be attributed to the significant reduction of scFv lacking the pIX protein component. The results also strongly suggest that it is the native N-terminal pIX methionine that allows the production of this pIX side product and that this effect can be eliminated by changing this particular residue.

[0194] Antibody phage display has major purposes for antibody engineering and discovery, where a collection of different antibody fusions with different properties is displayed and selected in the form of an antibody library 15 These antibodies inevitably have different intrinsic efficiencies in the display and the human anti-phOx scFv represents a rather well-performing unit in this context, as the human anti-phOx scFv was derived from multiple rounds of optimization using phage display engineering 16 Therefore, we extended the analysis of the Mlx effect to also include an anti-NIP scFv derived from a hybridoma that is known and performs poorly in phage display 2,4Since the original analysis clearly showed that the impact on pIX off-target product reduction, virion production, and target reactivity varied depending on which amino acid M1 was exchanged to, we here tested all 20 genetically encoded amino acid variants. Single M1x variants of both the LV (XL1-Blue) and HV (SS320) versions were again prepared using Delta phage and the respective titers were evaluated relative to infectious virion count and total virion count Figure 6 ).

[0195] Overall, the results were similar to those observed with the anti-phOx scFv, since rather uniform and high titers were obtained in the LV version ( Figure 6 , left panel), whereas rather large differences occurred in the HV version when considering infectious virion count ( Figure 6 , right panel). For ease of interpretation, we grouped the data of individual M1x variants that represented similar trends, which also somewhat coincided with the biochemical properties of the amino acids (positive vs. negative charge, hydrophobic vs. hydrophilic, etc.). There were clear differences between some mutant groups in infectious phage production, which was most negatively affected in the M1E and M1D versions, showing a 4 orders of magnitude decrease in infectious titers compared to unmodified (wt) pIX ( Figure 6 , right panel). We also evaluated the antigen-specific reactivity against NIP-BSA in phage capture ELISAs with the same phage preparations ( Figure 7 ).

[0196] Here, we used serial dilutions of all samples, rather than a single point phage concentration that was normalized like for the anti-phOx scFv ( Figure 5 ). Again, we grouped the data of individual M1x variants here that represented similar antigen reactivity. First, there were clear differences in overall reactivity between the LV and HV groups, as expected, likely due to avidity effects associated with HV display ( Figure 7 A vs. B Figure 7B). Notably, this effect is mainly limited to the Mlx variants. Second, the unmodified (wt) pIX version is clearly in the smallest target reactivity group independent of LV display or HV display. Third, the reactivity group curves between the various Mlx variants are slightly more complex depending on LV display or HV display. However, the unmodified pIX variant and the M1V variant are always in the group with the smallest reactivity. As indicated, the M1L variant and the M1I variant are also always in the group with the highest target reactivity in both the LV format and the HV format. As pointed out before, the efficient use of phage display as an engineering and discovery tool requires the highest possible functional display combined with the highest possible infectious virion production. This will ensure the largest possible library of heterologous fusion proteins that can be screened for the desired variant properties.

[0197] Therefore, to integrate these two main properties into the HV display data in the Figure 6 and Figure 7 combination analysis, we score phage production and target binding from 1 to 10 (where 10 is the most favorable) and plot these against each other ( Figure 8 ). Here, it is clear that the group containing M1L, M1I, and M1G is picked out as the most favorable group (Q4) with only small individual differences ( Figure 8 A). Further stratification of this group into their individual amino acids shows that the M1L variant represents the best compromise between improved target binding and preserved phage production ( Figure 8 B). However, the M1G variants also show a comparable performance and can even be slightly better in terms of target binding. Their similar phenotype is also very consistent with the initial analysis on an unrelated anti-phOx scFv showing a strong and similar reduction in free pIX production ( Figure 5 D). Therefore, we focused further evaluation on a side-by-side comparison of these two scFvs using a phage capture ELISA to compare the unmodified pIX to the M1L variant and the M1G variant in terms of their matching sample of target binding capabilities ( Figure 9 ).

[0198] This focused side-by-side comparison further emphasizes the very similar effect of exchanging the M1 position to M1L or M1G with a possible slight improvement in benefit for the M1L version. In the case of the anti-phOx and anti-NIP scFvs, the M1 modification is clearly superior to the unmodified counterpart. In the case of the anti-phOx scFv ( Figure 9 A and Figure 9 C), the effect is greater for the anti-NIP scFv ( Figure 9 B and Figure 9 D), and for both scFvs, the effect is greater in the HV display format compared to the LV display format.

[0199] To confirm and further extend previous analyses, we again used Delta phage with anti-phOx scFv construct and anti-NIP scFv construct for LV rescue and HV rescue, but now also including phagemid rescue with standard M13K07 helper phage in E. coli XL1-Blue 17 It is known that M13K07 (and other equivalent helper phages, such as VCSM13 and R408) yields significantly lower antibody display than the use of Delta phage, which also translates into a reduction of apparent target sensitivity 2 . To test whether this is also the case here, we therefore repeated the corresponding phage capture ELISAs Figure 10 ).

[0200] As seen before, the M1L variant gave the strongest target reactivity with both scFv and always in LV display and HV display. The difference was again largest for the HV version of the anti-NIP scFv. Also, the M13K07 rescued phage were clearly inferior in target reactivity with both scFv compared to the LV version and the HV version. Interestingly, the difference between unmodified pIX and M1L also seemed equal when using M13K07, which suggests the use of Delta phage as a key helper phage reagent to reveal underlying different trends.

[0201] In our previous studies, when comparing LV versions and HV versions as discovery tools 1 , the difference in the degree of influence of functional avidity between the classical pIII display system and our original pIX display system was not obvious, which reduced the ability to distinguish high-affinity binders from low-affinity binders. Since the current M1x modification seems to improve antibody display levels and, in particular, in the HV version, this can indicate the possibility that the modified pIX display approach is also susceptible to functional avidity limitations. To test this hypothesis, we therefore repeated the phage capture ELISAs, in which we varied the target density of immobilized antigen, since functional avidity depends on high target density and high valency 3 ( Figure 11 ).

[0202] If functional avidity is operable, it would be well appreciated that the effect would gradually decrease with decreasing target density when probed with multivalent antigen binding units such as HV display (Crothers and Metzger, 1972, Immunochemistry 9(3):341-357). However, we did not observe any difference in binding strength for either anti-phOx scFv or anti-NIP scFv when decreasing antigen density, regardless of whether unmodified pIX variants or M1L variants were used as display scaffold. Thus, the M1L modification does not seem to change the beneficial properties the original pIX design already has in this respect 1 .

[0203] In summary, the results clearly point to the finding that the "leaky" free pIX phenotype observed with the original pIX display system 1 , is coupled to the different translation initiation of the two independent polypeptides encoded by the single scFv-pIX fusion expression cassette Figure 3 , probably by the use of the scFv start codon and the pIX start codon. Moreover, this effect can be abolished or attenuated by changing the native N-terminal pIX methionine to an alternative amino acid. All alternative amino acids show good results. However, for some applications, an alternative amino acid is preferably leucine, isoleucine, or glycine, as these three residues provide the best compromise in improving antibody display efficiency (improved functional display) while preserving the original high infectivity virion production.

[0204] Example 2: Improved antibody discovery by using M1L modified pIX display

[0205] In Example 1, we identified that the native N-terminal methionine of the pIX capsid in filamentous phage display can be beneficially exchanged with an alternative amino acid, and in particular with leucine, to obtain improved heterologous fusion protein display. In these experiments, we used a monoclonal evaluation that allowed a predictable performance. However, one of the main applications of phage display is as a combinatorial engineering and discovery tool 15,18 In phage display for antibody discovery, the library consists of a collection of artificially diversified antibodies or endogenously diversified antibodies fused to the phage coat, and these antibodies differ in their biophysical, biochemical, and target binding properties. Such libraries are then used to identify those variants with the target properties by a cyclic process called panning, in which each clone in the library competes against each other to enrich the favorable variants 19 In the case of the original pIX phagemid display system, this has been extensively tested in panning, and the effect was found to be very good 1、4、20至22In a first attempt to test whether the apparently improved phenotype of the M1L modification also translated into further improvements in putative panning performance, we performed a single-cycle target enrichment assay in which the anti-NIP scFv was displayed on unmodified pIX or on the M1L variants (Table 1). HV versions of the two scFv variants were prepared by Delta phage rescue from amber non-suppressive E. coli strains SS320 or TOP10F'. The phage were mixed in a tentative 1:1 blend and panned against immobilized target (NIP-BSA), followed by sequencing of random single clones before and after panning to reveal whether one version was preferentially enriched at the expense of the other clone.

[0206] Table 1: Assessment I of preferential target-specific enrichment between unmodified pIX and pIX-M1L

[0207] Phage displaying NIP-specific scFvs on pIXwt or pIX-M1L were rescued from two amber non-suppressor E. coli strains, 10F' or SS320, using Delta phage (providing HV display), titrated and mixed in approximately 50 / 50 blends, followed by a round of selection on immobilized NIP-BSA. Both the input blend and the eluate were used to infect E. coli, and 20 to 24 random single clones from each group were sequenced. Results are shown as the pIX identity of all clones that generated resolved sequencing results.

[0208] Sequence analysis showed only some minor changes in the initial spike ratios, but after panning, we could see that the M1L variant

[0209] We then extended this spiking approach by adding anti-NIP scFv at a 1:10 7 Horizontal spiking into a large background of unrelated scFvs simulates a medium-sized diverse antibody library to represent a more realistic library background. We prepared matched pools in which antibodies were displayed on unmodified pIX or M1L and in HV display format, and then performed three rounds of continuous rounds of panning to determine how to efficiently recover specific scFvs ( Figure 12 ).

[0210] Ten to twenty-four random monoclonals from each round of selection (R1 to R3) were sequenced to reveal clonal identity and the results showed a clear preference for the M1L version of the HV display format as only here was an efficient enrichment of anti-NIP scFv's obtained. Thus, both tagging experiments clearly showed an improved and highly beneficial effect of the M1L modification of pIX coupled to the ability to recover the desired antibodies by panning, also at low abundance in the initial library.

[0211] Encouraged by these promising results, we then reformatted the previously reported fully human scFv antibody library of diversified pIX display 1 . This library has an estimated diversity of about 3 x 10 8 unique antibody clones and thus can serve as a suitable source to discover potential new antibody specificities for further drug development. The original antibody library was kept and based on transformation frequency sequence analysis, the new library was estimated to have roughly the same diversity as before reformatting. To address the putative difference in functional scFv display between the original and the reformatted library, we rescued both libraries from E. coli SS320 in the standard LV (using M13K07) display format and the HV (using Delta phage) display format. We then tested each library for the normalized amount of binding to the conformation-specific superantigen protein L (pL) in serial dilutions in a phage capture ELISA (based on infectious titer) Figure 13 ).

[0212] In fact, the results showed that both libraries were highly reactive in the HV display format and the M1L variant was clearly superior. Moreover, in line with the previous monoclonal analysis Figure 10 ), both libraries showed about similar and significantly lower reactivity in the LV format.

[0213] We then proceeded with a comprehensive antibody discovery campaign against two clinically validated unrelated pHLA targets equipped with tumor associated antigen (TAA) peptides overexpressed in human cancer. Each library was used in the HV format by R1 and R2, while we split both display formats in R3 into the standard LV format or the HV format. Panning was essentially performed according to the previously described protocol 21 , followed by a polyclonal pHLA target specific phage capture ELISA Figure 14 ) comparing the unselected primary library (R0) and the final R3 output sample. Two different pHLA complexes were used as mutual negative controls for putative pHLA cross-reactivity or non-specific HLA binding.

[0214] In line with previous results using the determined model scFv clones, we here also observed an enrichment of the modified pIX-M1L library that was predominantly restricted to the two targets. Notably, there was a clear difference in the apparent target-specific signal between the two targets, which could indicate a stronger enrichment of more specific clones and / or a higher affinity to the pHLA TAA target 1 compared to TAA target 2. To elucidate this aspect, we therefore picked 96 random single clones from each individual panning experiment that focused on TAA target 1, expanded them (using Delta phage rescue to maximize target binding sensitivity) and tested them for specific target binding in a pHLA phage capture ELISA Figure 15 ).

[0215] Here, the results clearly show an improved hit rate from M1L variants panned in LV and HV format as well as a significantly better target binding and specificity compared to the unmodified pIX version.

[0216] In summary, our results clearly explain why full-length scFv-pIX and pIX lacking fusion were observed under HV display conditions where free pIX should not be expressed. The inclusion of the native N-terminal methionine of pIX in the original design appears to have produced two replacement ORFs encoded by the same phagemid expression cassette, and the amino acid substitution at the M1 position is a single determinant that regulates this feature. By changing the substitution of the amino acid at position 1 in pIX, this effect can be reduced or eliminated. It seems that substitutions that allow for multiple substitutions with different effects can be stratified into two key parameters that are important for the use of phage display as an engineering tool and a discovery tool: phage production and fusion protein functionality. From a theoretical point of view, one would like to combine the highest possible phage yield with the highest possible fusion protein functionality. This would ensure the ability to cover the greatest possible functional diversity in any fusion protein library, which should maximize the ability to recover and identify the desired new fusion proteins generated by library selection. In this regard, the exchange of methionine with leucine (M1L) appears to translate into an optimal blend of these two different but connected features. This effect was particularly evident in the limited diversity spike-in model selection, as the M1L modification clearly demonstrated the best efficiency in specific antibody recovery, and this was particularly observed using the HV display format. When the M1L modification was further tested side-by-side with its unmodified counterpart in two fully mature, diverse human antibody discovery campaigns using pHLA target baits, we also observed a significant improvement in the apparent selection efficacy of the M1L variant, which indeed translated into an enhanced ability to identify an expanded number of target-specific clones with equally excellent target reactivity. Here, this effect was also seen in the classic LV version, supporting a generally beneficial effect of the pIX display system independent of the helper phage system employed.

[0217] Although we currently do not fully understand the precise mechanism behind the dual ORF phenomenon, which can be explained by ribosomal skipping, wobble or independent translation initiation, this will translate into the same conclusions regarding the design for practical applications to obtain improved functionality, i.e. changing the M1 position preferably to M1L, M1I or M1G. Moreover, our results point towards a finding that using M1L can best obtain the optimal compromise of phage production and fusion protein functionality if the largest and most flexible diversity space is to be covered in general. In cases where e.g. large fusion protein diversity is less important, e.g. in case of a smaller library is sufficient (in which case high phage production is not needed), other M1x substitutions can be considered to maximize fusion protein functionality. In such a case, our results suggest that M1E or M1D can represent a choice that gives very high target reactivity (M1F, M1W or M1Y, or M1N or M1Q would be other choices; M1P, M1R, M1K or M1H would be another choice). Or, in case high phage production has higher importance (but fusion protein functionality is of lower priority, e.g. in case of not necessarily high affinity clones are needed), then other M1x substitutions can be considered to maximize phage production. The results here suggest that in such a case M1C or M1S or M1T or M1A can represent a suitable choice.

[0218] We have also shown that the improvements seen with the modified pIX system described herein are not limited to antibody phage display libraries. For example, similar results and improvements have been observed with both T cell receptor phage display libraries and peptide MHC (pMHC) phage display libraries.

[0219] References

[0220] 1 L. S. et al. Multivalent pIX phage display selects for distinct and improved antibody properties. Sci. Rep. 6, 39066, doi:10.1038 / srep39066 (2016).

[0221] 2 Nilssen, N. R. et al. DeltaPhage - a novel helper phage for high-valence pIX phagemid display. Nucleic acids research 40, e120, doi:10.1093 / nar / gks341 (2012).

[0222] 3 O'Connell, D., Becerril, B., Roy-Burman, A., Daws, M. & Marks, J. D. Phage versus Phagemid Libraries for Generation of Human Monoclonal Antibodies. Journal of molecular biology 321, 49-56. (2002).

[0223] 4 G. Roos, N., Bogen, B. & Sandlie, I. Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage. PLoS ONE 6, e17433 (2011).

[0224] 5 Graille, M. et al. Complex between Peptostreptococcus magnus protein Land a human antibody reveals structural convergence in the interaction modes of Fab binding proteins. Structure 9, 679-687. (2001).

[0225] 6 Hust, M. et al. Single chain Fab (scFab) fragment. BMC biotechnology 7, 14 (2007).

[0226] 7 Endemann, H. & Model, P. Lcoation of Filamentous Phage Minor Coat Proteins in Phage and in Infected Cells. Journal of molecular biology 250, 496-506 (1995).

[0227] 8 Blumer, K.J., Ivey, M.R. & Steege, D.A. Translational control of phage f1 gene expression by differential activities of the gene V, VII, IX and VIII initiation sites. Journal of molecular biology 197, 439-451 (1987).

[0228] 9 Cruz-Teran, C.A., Tiruthani, K., Mischler, A. & Rao, B.M. Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae. ACS Synthetic Biology 6, 2096-2107, doi:10.1021 / acssynbio.7b00144 (2017).

[0229] 10 Goldman, E., Korus, M. & Mandecki, W. Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display. FASEB J 14, 603-611 (2000).

[0230] 11 Passaretti, P., Sun, Y., Dafforn, T.R. & Oppenheimer, P.G. Determination and characterisation of the surface charge properties of the bacteriophage M13 to assist bio-nanoengineering. RSC Adv 10, 25385-25392, doi:10.1039 / d0ra04086j (2020).

[0231] 12 Lamboy, J. A. et al. Phage wrapping with cationic polymers eliminates nonspecific binding between M13 phage and high pi target proteins. Journal of the American Chemical Society 131, 16454-16460, doi:10.1021 / ja9050873 (2009).

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[0234] 15 Ponsel, D., Neugebauer, J., Ladetzki-Baehs, K. & Tissot, K. High affinity, developability and functional size: the holy grail of combinatorial antibody library generation. Molecules 16, 3675-3700 (2011).

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[0237] 18 Rakonjac, J., Bennett, N. J., Spagnuolo, J., Gagic, D. & Russel, M. Filamentous Bacteriophage: Biology, Phage Display and Nanotechnology Applications. Curr Issues Mol Biol 13, 51-76 (2011).

[0238] 19 Ledsgaard, L. et al. Advances in antibody phage display technology. Drug discovery today 27, 2151-2169, doi:10.1016 / j.drudis.2022.05.002 (2022).

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[0241] 22 Frick, R. et al. A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation. Science Immunology 6, eabg4925, doi:10.1126 / sciimmunol.abg4925 (2021).

Claims

1. A vector construct comprising an open reading frame comprising a nucleic acid sequence encoding a modified pIX filamentous bacteriophage coat protein, wherein, the methionine (M) residue at position 1 of the modified pIX filamentous phage coat protein is replaced by an alternative amino acid residue.

2. The vector construct of claim 1, wherein, the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T, A or V.

3. The vector construct of claim 1 or claim 2, wherein, the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T or A.

4. The vector construct of any one of claims 1 to 3, wherein, the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T or A.

5. The vector construct of any one of claims 1 to 4, wherein, the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T or A.

6. The vector construct of any one of claims 1 to 5, wherein, the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T or A.

7. The vector construct of any one of claims 1 to 6, wherein, the alternative amino acid residue is selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T or A.

8. The vector construct of any one of claims 1 to 7, wherein, the modified pIX filamentous phage coat protein corresponds to a pIX coat protein from M13 phage, fd phage or fl phage or a variant thereof, with the proviso that the methionine (M) residue at position 1 is replaced by an alternative amino acid residue.

9. The vector construct of any one of claims 1 to 8, wherein, the modified pIX filamentous phage coat protein comprises SEQ ID NO: 1 (MSVLVYSFASFVLGWCLRSGITYFTRLMETSS) or a sequence having at least 70% identity to SEQ ID NO: 1, with the proviso that the methionine (M) residue at position 1 is replaced by an alternative amino acid residue.

10. The vector construct of claim 9, wherein, the open reading frame further comprises a sequence of a protein of interest, the sequence encoding the protein of interest being fused to the sequence encoding the modified pIX filamentous phage coat protein.

11. The vector construct of any one of claims 1 to 10, wherein, the protein of interest is an antibody, a T cell receptor or an MHC molecule. the vector is a phagemid or a phage vector.

12. A nucleic acid molecule encoding a modified pIX filamentous phage coat protein as defined in any one of claims 1 to 8, or a fusion protein as defined in claim 9 or claim 10.

14. A library of bacteriophage particles, wherein, 13. A phage particle comprising a vector or nucleic acid molecule as defined in any one of claims 1 to 12 and expressing a modified pIX filamentous phage coat protein or a modified pIX fusion protein on the surface. the phage particle is as defined in claim 13 and wherein a plurality of different proteins of interest are expressed on the surface of the phage particle.

15. A phage display system comprising a vector construct or nucleic acid molecule as defined in any one of claims 1 to 12.

17. The phage display system according to claim 16, wherein:

16. The phage display system according to claim 15, further comprising a helper phage and / or an E. coli host strain. the helper phage encodes a pIX filamentous phage coat protein and wherein the pIX filamentous phage coat protein encoded by the helper phage is capable of complementing the modified pIX filamentous phage coat protein encoded by the vector construct or nucleic acid molecule as defined in any one of claims 1 to 12.

18. The phage display system of claim 16, wherein, The helper phage encodes a pIX filamentous phage coat protein, and wherein the pIX filamentous phage coat protein encoded by the helper phage is unable to complement the modified pIX filamentous phage coat protein encoded by the vector construct or nucleic acid molecule as defined in any one of claims 1 to 12.

19. The phage display system according to any one of claims 16 to 18, wherein, The helper phage encodes a pIX filamentous phage coat protein, and wherein the pIX filamentous phage coat protein encoded by the helper phage is expressed under the control of one or more conditional mutations, for example under the control of one or more suppressible mutations.

20. The phage display system of claim 19, wherein, The suppressible mutation is an amber stop codon, preferably selected from the group consisting of an amber stop codon, an ochre stop codon and an opal stop codon.

21. The phage display system according to any one of claims 16 to 20, wherein, The helper phage is a Delta phage.

22. The phage display system according to any one of claims 18 to 21, further comprising a suitable non-suppressible E. coli host strain that does not allow expression of the pIX filamentous phage coat protein encoded by the helper phage.

23. The phage display system of claim 22, wherein, The non-suppressible E. coli host strain is an amber non-suppressible strain, preferably SS320 or TOP-10F’.

24. The phage display system according to any one of claims 15 to 17, wherein, The E. coli host strain is a suppressible strain, preferably an amber suppressible strain, more preferably XL-1 Blue, TGI or ER2738.

25. A method for producing phage particles, the method comprising using a vector construct or nucleic acid molecule as defined in any one of claims 1 to 12 or a phage display system according to any one of claims 15 to 24.

26. A method of phage display, the method comprising: a. providing an E. coli host strain comprising a vector construct of claim 9 or claim 10, wherein expression of the vector construct results in production of a protein-of- interest-modified pIX fusion protein; b. providing a helper phage; c. infecting the E. coli host strain with the helper phage under conditions such that the host strain produces phage particles displaying the protein-of-interest- modified pIX fusion protein.

27. The method of claim 26, wherein, The method is a method for high value phage display, wherein the pIX filamentous phage coat protein encoded by the helper phage is unable to complement the modified pIX filamentous phage coat protein encoded by the vector construct of claim 9 or claim 10.

28. The phage display method of claim 26 or claim 27, wherein, The method is a method for high value phage display, comprising: a. providing a non-suppressible E. coli host strain comprising a vector construct of claim 9 or claim 10, wherein expression of the vector construct results in production of a protein-of-interest-modified pIX fusion protein; b. providing a helper phage, wherein expression of the pIX filamentous phage coat protein of the helper phage is under the control of one or more suppressible mutations; c. infecting the non-suppressor E. coli host strain with the helper phage under conditions such that the pIX filamentous phage coat protein encoded by the helper phage is not expressed, such that the non-suppressor host strain produces phage particles displaying multiple copies of the target protein-modified pIX fusion protein.

29. The method of claim 26, wherein, The method is a method for low cost phage display, wherein the pIX filamentous phage coat protein encoded by the helper phage is capable of complementing the modified pIX filamentous phage coat protein encoded by the vector construct of claim 9 or claim 10.

30. The phage display method of claim 26 or claim 29, wherein, The method is a method for low cost phage display, comprising: a. providing a suppressor E. coli host strain comprising the vector construct of claim 9 or claim 10, wherein expression of the vector construct results in production of a target protein-modified pIX fusion protein; b. providing a helper phage, wherein expression of the pIX filamentous phage coat protein of the helper phage is controlled by one or more suppressor mutations; c. infecting the suppressor E. coli host strain with the helper phage under conditions such that the pIX filamentous phage coat protein encoded by the helper phage is expressed, such that the suppressor host strain produces phage particles displaying a single copy or a low copy of the target protein-modified pIX fusion protein.

31. The method of any one of claims 25-30, wherein, using a library of the vector constructs encoding a plurality of target proteins.

32. The method of any one of claims 25 to 31, for selecting a protein that binds to a desired target molecule.

33. The method of claim 32, further comprising the step of manufacturing or producing the protein or a component, fragment, variant, or derivative of the protein, and optionally formulating the manufactured or produced protein with at least one pharmaceutically acceptable carrier or excipient.

34. A kit comprising the vector construct of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the phage particle of claim 13 or claim 14, or the phage display system of any one of claims 15 to 24.

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