Modified pIX vector constructs

Abstract

The present invention provides a vector construct comprising an open reading frame including a nucleic acid sequence encoding a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with an alternative amino acid residue. Also provided are phage particles or phage display systems comprising the vector construct, as well as methods and kits for phage display.

Description

[Technical Field]

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

[0002] Over the past 30 years, phage display has become a powerful and efficient method for discovering and evolving novel binding proteins. The principle of combinatorial phage display technology is based on the link between genotype and phenotype, achieved by the property that each virion displays on its surface only proteins identical to those encoded by the genome enclosed in its protein coat. Phage particles themselves are highly tolerant to diverse physiochemical conditions, making phage display more versatile than competing combinatorial technologies in many selection regimes. While several competing combinatorial technologies exist, none offer the same level of versatility and ease of use. However, screening for the desired phenotype after phage panning remains challenging, and optimal binders are not always identified.

[0003] Phage display of heterologous polypeptides has been achieved using all five structural proteins present in the filamentous phage coat, with the pIII structural protein becoming the most widely used. pIX display has also been reported and has shown certain advantages over standard pIII display in terms of its effectiveness in identifying target-specific antibody candidates and yielding clones with superior biophysical properties (Hoydahl et al., 2016, Sci. Rep. 6, 39066).

[0004] Therefore, the present inventors have developed an improved pIX phage display system that utilizes an improved pIX expression vector construct, in which a vector is provided that encodes a modified pIX phage coat protein in which the methionine (M) residue at position 1 is replaced with an alternative amino acid residue, rather than the wild-type full-length pIX protein.

[0005] Surprisingly, the inventors have demonstrated that such modified pIX vectors can provide significant improvements in phage display compared to phage display using wild-type pIX proteins, 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), infectious phage particle production (infectious phage titer), or both. Such improvements are highly advantageous for phage display, whether in terms of the ability to successfully select and isolate binders, the quality and / or quantity of selectable binders, or the size of the phage display library that can be constructed. For example, to most effectively utilize phage display as an engineering and discovery tool, it is necessary to achieve both the highest possible functional display capacity and the production of the largest possible number of infectious virions / phage particles. This would allow screening of the largest heterogeneous fusion protein library pool for desired variant properties, increasing the likelihood of identifying proteins with the desired properties. Advantageously, the present invention achieves this combination of properties.

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

[0007] Thus, in one aspect, the present invention provides a vector construct comprising an open reading frame including a nucleic acid sequence encoding a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with an alternative amino acid residue. From another perspective, the present invention provides a vector construct comprising a nucleic acid sequence encoding a pIX phage coat protein in which the methionine (M) residue at position 1 has been replaced with an alternative amino acid residue (modified pIX phage coat protein).

[0008] The vectors of the present invention are expression vectors or expression constructs, i.e., they generally consist of nucleic acid sequences that allow the expression (protein synthesis) of an encoded desired protein component in a suitable host cell.

[0009] As outlined above, the present invention is in the field of phage display on the pIX phage coat protein. Thus, the vectors of the present invention can be phage vectors or phagemid vectors (plasmids), the basic structure and components of which are well known to those skilled in the art and are selected so that expression of the phage protein in a suitable host cell and packaging of phage particles are achieved, and a heterologous or foreign protein (protein of interest, POI) fused to the modified pIX phage coat protein of the present invention is displayed on the surface of the phage particle.

[0010] Thus, when a vector of the present invention is used to transform a suitable host cell, for example a suitable prokaryotic host cell such as a suitable Escherichia coli strain, phage particles are produced that contain the desired POI, which is fused to the modified pIX phage coat protein of the present invention and displayed on the surface of the phage particle, wherein various phage components of the phage genome, as well as vector sequences or other nucleic acid sequences encoding the POI, are contained within the phage particle.

[0011] In the modified pIX phage coat protein of the present invention, the methionine (M) residue at position 1 of a pIX phage coat protein, for example, a wild-type or native pIX phage coat protein, can be substituted or replaced with any alternative amino acid residue, i.e., any amino acid residue other than methionine (M). In other words, the methionine (M) residue at the N-terminus of a pIX phage coat protein, for example, a wild-type or native pIX phage coat protein, can be substituted or replaced with any alternative amino acid residue, i.e., any amino acid residue other than methionine (M). These pIX phage coat proteins used in the present invention or encoded by the constructs of the present invention are referred to herein as modified pIX phage coat proteins of the present invention or non-wild-type pIX phage coat proteins. Thus, the modified pIX phage coat proteins of the present invention do not correspond to the wild-type pIX phage coat protein in terms of amino acid sequence.

[0012] Since it is advantageous in the present invention to have a genetic fusion of the POI and the modified pIX phage coat protein of the present invention within a single ORF, a suitable alternative amino acid to conveniently substitute for M at position 1 of the pIX phage coat protein would be a genetically encoded amino acid, which can be coded for by a nucleic acid sequence and thus easily incorporated into the vectors of the present invention.

[0013] In an embodiment of the present invention, the alternative amino acid residue substituting for methionine (M) is selected from 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).

[0014] In another embodiment of the invention, the alternative amino acid residues are selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, H, C, S, T, or A.

[0015] In another embodiment of the invention, the alternative amino acid residues are selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, or H; or are selected from L, G, I, C, S, T, or A.

[0016] In some preferred embodiments, the alternative amino acid residues are selected from L, G, I, F, W, Y, N, Q, E, D, P, R, K, or H.

[0017] In another preferred embodiment of the invention, said replacement amino acid residue is selected from L, G, or I, with L or G being more preferred, and L being most preferred. While any alternative amino acid residue can be used, the selection may be guided by the desired outcome of the phage display process. For example, in many cases, one would desire both the highest possible phage production and the highest possible fusion protein functionality. This would ensure the ability to cover the greatest functional diversity in any fusion protein library and maximize the ability to recover and identify desired novel fusion proteins resulting from library selection. In this case, the results presented herein indicate that replacing the methionine with leucine (M1L), glycine (M1G), or isoleucine (M1I) appears to provide the optimal balance of these two fundamentally different, yet related, characteristics.

[0018] However, in situations where high diversity of the fusion protein is less important, e.g., where a smaller library is sufficient (high phage production is not necessary), alternatives to M1x may be appropriate to maximize fusion protein functionality. In this case, the results presented herein indicate that M1E or M1D are suitable alternative amino acids that confer extremely high target reactivity, with M1F, M1W, M1Y, M1N, or M1Q being other options. M1P, M1R, M1K, or M1H are further options.

[0019] Alternatively, in situations where high phage production is more important (but functionality of the fusion protein is less of a priority, e.g., when high affinity clones are not necessarily required), other M1x may be appropriate to maximize phage production. In this case, the results presented herein indicate that M1C or M1S or M1T or M1A are suitable replacement amino acids in this scenario. M1V is yet another option.

[0020] In some embodiments, the replacement 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, the replacement amino acid is not R. In some embodiments, it is not K. In some embodiments, it is not D. In some embodiments, it is not S. In some embodiments, it is not A. In some embodiments, it is not V. In some embodiments, it is not T. In some embodiments, it is not C. In some embodiments, it is not H. In some embodiments, it is not P. In some embodiments, it is not E. In some embodiments, it is not Q. In some embodiments, it is not N. In some embodiments, it is not Y. In some embodiments, it is not W. In some embodiments, it is not F. In some embodiments, it is not I. In some embodiments, it is not G. In some embodiments, it is not L.

[0021] In some embodiments, position 2 of the modified pIX phage coat protein of the invention is S. In some embodiments, position 3 of the modified pIX phage coat protein is V. In some embodiments, position 4 of the modified pIX phage coat protein is L. In some embodiments, one or more, two or more, and preferably all of positions 2, 3, and 4 of the modified pIX phage coat protein are S, V, and L, respectively. In some embodiments, position 2 of the modified pIX phage coat protein of the invention is not S. In some embodiments, position 3 of the modified pIX phage coat protein is not V. In some embodiments, position 4 of the modified pIX phage coat protein is not L. In some embodiments, one or more, two or more, and preferably all of positions 2, 3, and 4 of the modified pIX phage coat protein are not S, V, and L, respectively. In some embodiments, position 16 is not C. In some such embodiments, none of the modified sites is M.

[0022] For the avoidance of doubt, a modified pIX phage coat protein of the invention (or a pIX portion / component in a vector construct or nucleic acid molecule of the invention) has (or encodes) an amino acid residue at position 1 of the pIX protein that is not a methionine (M). In other words, the methionine (M) at position 1 of the pIX protein has been substituted or replaced with an alternative amino acid. Simply deleting or removing the methionine (M) at position 1 is not sufficient. Thus, for example, modified pIX proteins or pIX fragments in which the methionine (M) at position 1 has been deleted or removed (e.g., deleted or removed without being replaced with an alternative amino acid residue) are not encompassed by the present invention. Thus, for example, pIX fragments comprising or consisting of positions 2 and beyond, e.g., positions 2 to 32 in the case of a full-length pIX protein, or pIX fragments in which the M at position 1 has been removed are not encompassed by the present invention. In fact, it has been shown that vector constructs lacking the methionine (M) at position 1 of the pIX protein do not exhibit the advantages or improved properties over wild-type pIX that the vector constructs of the present invention possess. It should be noted that the improvements observed when using the vectors and systems of the present invention as described herein typically refer to improvements compared to corresponding vectors, systems, etc. that use wild-type or native pIX phage coat protein.

[0023] The term open reading frame (ORF) as used herein refers to its standard meaning generally recognized in the art. Thus, the term open reading frame (ORF) is used herein to refer to a nucleic acid molecule, typically a span of DNA, between a start codon and a stop codon, or between a translation initiation site and a translation termination site. Such an ORF typically encodes a polypeptide, and herein encodes a polypeptide comprising the modified pIX phage coat protein of the present invention as described herein. A preferred ORF encodes a fusion protein of a POI and a modified pIX phage coat protein of the present invention. Suitable start codons will be well known to those skilled in the art. A typical and exemplary start codon would be ATG, which encodes methionine. The start codon is positioned within the vector (or nucleic acid molecule) of the present invention at an appropriate distance upstream of the sequence encoding the modified pIX phage coat protein so that translation of the modified pIX phage coat protein is initiated under appropriate conditions. If a POI is also encoded by the vector (or nucleic acid molecule) of the invention, the initiation codon is positioned an appropriate distance upstream of the sequence encoding the POI-modified pIX fusion, e.g., adjacent to or directly adjacent to the sequence, so that translation of the POI-modified pIX fusion protein is initiated under appropriate conditions. Suitable stop codons will be well known to those skilled in the art. Typical and exemplary stop codons are TAA, TGA, or TAG. One or more stop codons can be used. In some embodiments, the vector or nucleic acid molecule of the invention comprises a single ORF.

[0024] As used herein, the terms "pIX phage coat protein" or "pIX protein" or "pIX phage protein" or "pIX coat protein" and the like refer to a pIX protein that arises from or is derived from a wild-type or naturally occurring pIX filamentous phage coat protein sequence, or that has a sequence that corresponds to the sequence of such a pIX protein. Preferred filamentous phages from which the pIX protein is derived or corresponds are M13 phage, fd phage, or f1 phage. Any suitable pIX protein can be used, provided it is capable of displaying a POI as a pIX fusion protein on the surface of a phage particle. While wild-type (or native) pIX proteins or wild-type-like pIX proteins, e.g., those containing all the amino acids of wild-type (or native) pIX but incorporating one or more additional amino acids, e.g., in the form of conditional mutations, are used in some embodiments of the invention, e.g., in various helper phages as described herein, pIX proteins encoded by vectors of the invention, or otherwise used in fusion proteins fused to a POI for the purpose of displaying the POI on the phage surface, correspond to pIX proteins in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with an alternative amino acid residue, or in other words, are modified pIX phage coat proteins of the invention as described elsewhere herein.

[0025] Preferably, the modified pIX phage coat protein encoded by the vector of the invention comprises or consists of a full-length pIX phage coat protein in which the methionine (M) residue at position 1 of the full-length pIX filamentous phage coat protein has been replaced with an alternative amino acid residue, for example, as described elsewhere herein. Such a full-length pIX phage coat protein may typically have 32 amino acids.

[0026] Preferably, the modified pIX phage coat protein encoded by the vector of the invention comprises or consists of the following amino acid sequence corresponding to the wild-type pIX protein from VCSM13 helper phage (Genbank AY598820.1): MSVLVYSFASFVLGWCLRSGITYFTRLMETSS (SEQ ID NO: 1) The methionine (M) residue at position 1 of the pIX filamentous phage coat protein (herein SEQ ID NO: 1) has been replaced with an alternative amino acid residue, eg, as described elsewhere herein.

[0027] An exemplary nucleic acid sequence encoding this sequence for incorporation into a vector of the invention is set forth elsewhere herein as SEQ ID NO:2, where again the nucleic acid sequence encoding the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with a nucleic acid sequence encoding an alternative amino acid residue, e.g., as described elsewhere herein.

[0028] Thus, yet another embodiment of the present invention provides a vector construct wherein the encoded modified pIX filamentous phage coat protein corresponds to the pIX coat protein from M13, fd, or f1 phage, or a variant thereof, and the methionine (M) residue at position 1 has been replaced with an alternative amino acid residue, for example, as described elsewhere herein.

[0029] In other embodiments, for example, when a variant of the modified pIX protein is used, e.g., a variant M13, fd, or f1 pIX phage coat protein, the encoded pIX protein comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, or 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% identity, to that of SEQ ID NO: 1, wherein the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced with an alternative amino acid residue. In other words, in modified pIX proteins of the present invention, position 1 of the pIX protein sequence should not be a methionine (M). Preferred alternative amino acid sequences to incorporate at position 1 are described elsewhere herein and can result in improved phage display, e.g., improved production of infectious phage particles (infectious phage titer), and / or improved display of functional proteins, 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 substituting an alternative amino acid for M at position 1 of the pIX protein improves the translation efficiency of the ORF of the vector construct, and in particular positively impacts (i.e., improves) the yield of the POI-modified pIX fusion protein produced.

[0030] Similarly, a nucleic acid molecule encoding a variant of the modified pIX protein of the invention can comprise or consist of, for example, a nucleotide sequence having at least 60%, 65%, 70%, 75%, or 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, to that of SEQ ID NO: 2, in which the encoded methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced with an alternative amino acid residue, e.g., as described elsewhere herein.

[0031] Thus, yet another embodiment of the present invention provides a vector construct of the invention, 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%, etc., identity to SEQ ID NO:1, wherein the methionine (M) residue at position 1 has been replaced with an alternative amino acid residue, e.g., as described elsewhere herein. Exemplary and preferred percent identity values ​​are provided elsewhere herein.

[0032] Other preferred examples of modified pIX sequences, e.g., variant modified pIX sequences, encoded by the vectors or nucleic acid molecules of the invention include pIX sequences, e.g., SEQ ID NO: 1, that contain up to 12, e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, altered amino acids, in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein (e.g., SEQ ID NO: 1) has been replaced with an alternative amino acid residue, e.g., as described elsewhere herein.

[0033] Thus, another embodiment of the present invention provides a vector construct of the present invention, wherein the encoded modified pIX filamentous phage coat protein comprises SEQ ID NO:1 (MSVLVYSFASFVLGWCLRSGITYFTRLMETSS) or a sequence comprising up to 12, e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, altered amino acids in the pIX sequence, e.g., SEQ ID NO:1, wherein the methionine (M) residue at position 1 has been replaced with an alternative amino acid residue, e.g., as described elsewhere herein.

[0034] These modified pIX sequences or variants thereof, e.g., modified or variant pIX sequences of the present invention, should retain or have the functional ability to display a POI as a pIX fusion protein on the surface of a phage particle. Functional C-terminal truncations or functional N-terminal fragments of SEQ ID NO: 1 (or variant sequences thereof) or other pIX sequences can also be used, provided that the methionine (M) residue at position 1 of the pIX filamentous phage 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, no fragments are used. Thus, instead, a full-length pIX protein, e.g., a pIX protein in which all 32 amino acids (or variants thereof, e.g., as described elsewhere herein) are present, can be used, in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein is replaced with an alternative amino acid residue, e.g., as described elsewhere herein.

[0035] As used herein, "sequence identity" refers to a measure of identity at the amino acid level between proteins, and a measure of identity at the nucleotide level between nucleic acids. Protein sequence identity can be determined by aligning the sequences and comparing the amino acid sequences at any position in each sequence. Similarly, nucleic acid sequence identity can be determined by aligning the sequences and comparing the nucleotide sequences at any position in each sequence. When variant molecules are described herein as having a percent identity of, for example, at least 60%, 65%, 70%, etc., such variants should generally be construed as functional variants or functional derivatives, i.e., those that retain or have improved function.

[0036] Methods for determining the percentage identity of two amino acid sequences or two nucleic acid sequences are well known and reported in the art, and any of these methods may be used. For example, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., gaps may be introduced into the sequence of the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). Then, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, these molecules are identical at that position. The percent identity between the two sequences is determined by the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100). In some embodiments, the two sequences are the same length.

[0037] Sequences may be aligned manually and the number of identical amino acids counted. Alternatively, alignment of two sequences to determine percent identity may be achieved using a mathematical algorithm. Such algorithms are incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous (or with a certain percent identity) to the nucleic acid molecules of the invention. BLAST protein searches may be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous (or with a certain percent identity) to the protein molecules of the invention. Gapped BLAST may be used to obtain gapped alignments for comparison purposes. Alternatively, PSI-Blast may be used to perform an iterative search to detect distant relationships between molecules. When using the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of each program may be used. See http: / / www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after aligning sequences using, for example, the BLAST program in the EMBL database (www.ncbi.nlm.gov / cgi-bin / BLAST). Generally, default settings, e.g., for "scoring matrix" and "gap penalty," may be used for alignment. For the present invention, the default settings of BLASTN and PSI BLAST may be advantageous. In calculating percent identity, only exact matches are counted.

[0038] In preferred vectors of the invention, the sequence encoding the modified pIX phage coat protein of the invention is linked, e.g., operably linked, to a sequence encoding a protein of interest (POI). Thus, in a preferred embodiment, the vector construct, or the 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 invention. Thus, preferred vector constructs and nucleic acid molecules of the invention encode a POI-modified pIX fusion protein.

[0039] The terms "fusion protein," "fusion," and the like are used herein to describe the functional association of two or more protein components in the same polypeptide sequence or the same open reading frame (ORF). Because such fusion proteins are encoded by the same nucleic acid sequence, they can also be described as genetic fusions (sometimes called "fusion genes" or "fusion nucleotide sequences"). In such fusion proteins, the two (or more) protein components (or the nucleic acid sequences encoding them) can be directly adjacent to each other, but they can also be linked by a suitable peptide spacer or linker. As is well known in the art, the spacer or linker can be important to enable functional expression of each individual protein component, e.g., to enable them to form the appropriate three-dimensional structure and perform or maintain the desired function.

[0040] Thus, in fusion proteins encoded by vectors or nucleic acid molecules of the present invention, a peptide spacer (or linker) is typically included between the protein of interest (POI) and the modified pIX phage coat protein of the present invention. In other embodiments, such a linker or spacer may be absent or may be included only between some of the components. Thus, in vectors or nucleic acid molecules of the present invention, the sequence encoding the POI can be fused to the sequence encoding the modified pIX phage coat protein of the present invention with or without a spacer or linker sequence between the components. All of these contemplated configurations (i.e., fusion proteins or nucleic acids encoding same, with or without spacer or linker sequences) are still considered direct fusions or direct gene fusions.

[0041] Although this description has focused on the linker or spacer between the modified pIX phage coat protein of the present invention and the POI, linker sequences may be included elsewhere in the vectors of the present invention as needed, for example between other components of a vector as described herein, such as between the VH and VL domains of a POI that is an antibody, or between other POIs that involve or include two or more separate polypeptide components.

[0042] Thus, the term "pIX fusion protein" refers to a pIX protein (pIX phage coat protein, pIX filamentous phage coat protein) fused to a foreign peptide / polypeptide, e.g., a protein of interest (POI). Similarly, the term "modified pIX fusion protein" refers to a modified pIX protein (modified pIX phage coat protein, modified pIX filamentous phage coat protein) of the invention fused to a foreign peptide / polypeptide, e.g., a protein of interest (POI).

[0043] Thus, preferred vectors of the present invention comprise a sequence (nucleic acid sequence) encoding a modified pIX phage coat protein of the present invention (sometimes referred to herein as a POI-modified pIX or POI-modified pIX fusion protein) fused (genetically fused) to a sequence encoding a POI. The POI and modified pIX can be present in any suitable order or spacing within the vector, provided that, upon expression and packaging into a phage particle, a functional fusion protein is formed between the POI and the modified pIX, the modified pIX coat protein component of the fusion protein forms part of the phage coat, and the POI is functionally expressed or displayed on the surface of the phage particle. In this manner, the POI portion of the fusion protein is positioned in-frame with the modified pIX coat protein portion of the fusion protein. This means that the POI and pIX are expressed within the same polypeptide sequence (or as part of the same ORF), or, in other words, as a direct fusion. In vectors of the invention, it is generally preferred that the POI component of the fusion protein be located N-terminal to (ie, at or near the N-terminus of) the modified pIX component of the fusion protein.

[0044] Yet another aspect of the present invention provides a modified pIX filamentous phage coat protein of the present invention. In other words, this aspect of the present invention provides a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with an alternative amino acid residue, for example, as described elsewhere herein. In a preferred embodiment, a fusion protein comprising the aforementioned modified pIX phage coat protein of the present invention is provided, e.g., a fusion protein comprising a POI and the modified pIX phage coat protein of the present invention. In other words, this aspect of the present invention provides a POI fused (preferably N-terminally) to a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with an alternative amino acid residue (e.g., as described elsewhere herein). Nucleic acid molecules encoding such modified pIX filamentous phage coat proteins and fusion proteins are also provided.

[0045] The present invention also extends to nucleic acid sequences or molecules that may be part of, or comprise components of, the vectors of the invention. Accordingly, another aspect of the present invention provides a nucleic acid molecule or sequence comprising an open reading frame that includes a nucleic acid sequence encoding a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with an alternative amino acid residue (e.g., an alternative amino acid residue as described elsewhere herein).

[0046] In addition, the present invention provides a nucleic acid molecule or nucleic acid sequence encoding a modified pIX phage coat protein of the present invention or a fusion protein of the present invention comprising a POI fused to a modified pIX phage coat protein of the present invention.

[0047] In a preferred embodiment of the present invention, one or more ribosome binding sites (RBS) are included in the vector construct. Such components may also be referred to as translation initiation regions (TIRs).

[0048] The RBS sequence is positioned in a vector at a location suitable for its function. The role of the RBS is to recruit ribosomes during protein translation initiation. Therefore, it is advantageous to position the RBS at an appropriate distance upstream of the start codon of the protein to be translated or upstream of the ORF of the protein to be translated. Thus, in the vectors of the present invention, it is advantageous to position the RBS sequence upstream of the sequence encoding the POI-modified pIX fusion protein. For example, in embodiments in which a signal peptide is also part of the ORF, as described elsewhere herein, it is also advantageous to position the RBS sequence upstream of the sequence encoding the signal peptide (or upstream of the start codon of the ORF containing the nucleic acid sequence encoding the signal peptide). In embodiments in which the signal peptide is not present in the ORF, it is advantageous to position the RBS sequence upstream of the sequence encoding the POI-modified pIX fusion protein at an appropriate distance. Appropriate distances are known or can be easily determined by one of skill in the art depending on the RBS selected. Exemplary distances include 7 or 8 nucleotides from the ATG (or other) start codon, although this can be varied.

[0049] The RBS / TIR sequence regulates the translation strength (level of protein expression) of the downstream sequence, and different types of RBS can result in different levels of protein expression, such as weak or strong expression. Weak or strong RBS / TIR sequences are well known in the art and can be easily selected by those skilled in the art depending on the desired protein expression level. As expected, a strong RBS promotes or induces more translation (strong translation) than a weak RBS. Both weak and strong RBS sequences can be used in the vectors of the present invention. In some embodiments, a weak RBS is used.

[0050] In particular, in preferred vectors of the present invention, an RBS is included upstream of (or 5' to or N-terminal to) the start codon of the sequence encoding the POI-modified pIX fusion protein (or, for example, upstream of the start codon of the ORF containing the nucleic acid sequence encoding the POI-modified pIX fusion protein). A preferred RBS for use in the present invention is a Shine-Dalgarno (SD) sequence or an SD-based sequence, which can be included in the vector construct. SD sequences are well known and have been reported in the art, and any of these may be used. For example, the core SD sequence is GAGG (SEQ ID NO: 3), and other consensus sequences are GAGGAG (SEQ ID NO: 4), AGAGGAG (SEQ ID NO: 5), or AGGAGAA (SEQ ID NO: 6), which include, for example, the sequence AGGAG (SEQ ID NO: 7). Therefore, SD sequences containing these core or consensus sequences can be used.

[0051] An exemplary structure of a construct of the invention having a POI-modified pIX fusion protein is shown in Figure 3B. As described elsewhere herein, the POI shown in Figure 3B is an scFv antibody fragment, but this is only one example of a POI or POI library that can be used.

[0052] A preferred vector of the present invention includes a sequence encoding a spacer or linker (typically a peptide spacer or linker) between the sequence encoding the POI and the sequence encoding the modified pIX phage coat protein. Such a sequence is typically a synthetic or artificial (e.g., non-natural or unnatural) linker or spacer sequence, e.g., a sequence that does not encode a functional protein or protein domain. Composite linker sequences can also be used. Such linker or spacer sequences may include tag sequences such as c-Myc or FLAG tags (e.g., DYKDDDDK; SEQ ID NO: 8). Complete or full-length linker or spacer sequences are generally used; for example, such sequences are not generally truncated sequences. The incorporation of such sequences can aid in the folding of the linked proteins, particularly the N-terminal protein (usually referred to herein as the POI). Therefore, the length of the spacer or linker can be adjusted appropriately to enable optimal or sufficient functional folding of both components (i.e., the POI and the modified pIX). The appropriate length can be easily determined by one skilled in the art. However, exemplary lengths include 5 to 15 amino acids (Weiss et al., 2000, Protein Sci., 9:647-654), e.g., 6 to 10 amino acids. A particular linker for use in the present invention is AAAGSKDIR (SEQ ID NO: 12). Alternatively, a linker such as a GS linker could be used, e.g., a linker having a certain number of GS repeats, e.g., a linker having G4S repeats.

[0053] When present in a vector or fusion protein of the invention, such a spacer or linker forms a distinct portion of the vector or fusion protein 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 invention; i.e., the modified pIX phage coat protein of the invention is a distinct or separate component, e.g., there is a junction between the modified pIX phage coat protein and the upstream portion of the vector. Thus, any alternative amino acid residue used to substitute for position 1 of the modified pIX phage coat protein of the invention is part of the pIX component (portion) of the vector, but not part of the spacer or linker sequence (or any other portion of the vector); i.e., the spacer or linker sequence (or POI sequence) is a distinct or separate component. Such distinct portions of a vector construct are often 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 or spacer component (or other portion of the vector) and the modified pIX component.

[0054] As described elsewhere herein, preferred vectors of the invention can encode 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 invention. As described elsewhere herein, the use of modified pIX phage coat proteins of the invention can improve the display of the POI.

[0055] Thus, the POI (and indeed any linker or spacer sequence located between the POI and the modified pIX phage coat protein) is typically foreign or heterologous. By foreign or heterologous protein we mean a protein or peptide not originally contained in the associated phage coat protein, such as the pIX protein, which is fused to the modified pIX phage coat protein of the invention (with or without any linker or spacer amino acids, also foreign or heterologous and not contained or originally contained in the associated phage coat protein), for example fused to the N-terminus of the modified pIX phage coat protein of the invention, e.g. fused to the N-terminal amino acid residue that serves as a substitute for the M residue at position 1 of the pIX phage coat protein.

[0056] Any protein of interest (POI) can be encoded in the vector of the present invention, provided that the protein is suitable for display on phage, particularly as a fusion with the pIX phage coat protein, e.g., a modified pIX phage coat protein of the present invention. Suitable examples are well known and documented in the art. However, preferred examples would be targeting molecules / targeting units or binding partners / binding proteins capable of binding to another entity (target / target entity, e.g., target protein). Some examples of suitable POIs include antibodies or fragments thereof (e.g., Fab, scFv, nanobody), MHC molecules (class I or class II), T cell receptors (TCR), or non-Ig-derived binding proteins such as DARpins, ankyrin family, fibronectin family, knottins, anticalins, etc. (Hosse et al., 2006, Protein Sci 15:14-27), and peptides.

[0057] The appropriate design of the vector of the present invention for displaying a selected type of POI on the surface of a phage can be easily determined by one skilled in the art. For example, if the selected type of POI is in the form of a single polypeptide chain, such as an scFv antibody, a single-chain TCR, or a single-chain MHC molecule, e.g., a single-chain MHC class I or class II, nucleic acid molecules encoding these polypeptides can be simply placed in a vector to produce a fusion protein with the modified pIX phage coat protein of the present invention. Alternatively, if the selected type of POI is in the form of two or more polypeptide chains, such as a Fab antibody fragment, a TCR, or an MHC molecule having two chains, a nucleic acid molecule encoding one of the polypeptides (one of the chains) can be placed in a vector to produce a fusion protein with the modified pIX phage coat protein of the present invention, and the remaining polypeptide chains can be produced separately or independently.

[0058] The vectors of the invention can be used in classical phage display for the purpose of selecting binding partners (e.g., antibodies) for a particular target entity, e.g., a target protein or 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 invention and selected for binding to the target entity by standard, well-known techniques.

[0059] Another preferred component of the vector construct of the present invention is a suitable promoter sequence for controlling the expression of the ORF containing the modified pIX protein of the present invention and the fusion protein containing the modified pIX protein. Suitable promoter sequences are well known to those skilled in the art, and any of these can be used. An exemplary promoter sequence includes the lac promoter, which is inducible, for example, with IPTG. Other promoters include tac, arabB, or psp.

[0060] Optionally, a signal sequence or signal peptide, such as a pelB signal sequence or signal peptide, can be incorporated into an ORF containing a nucleic acid sequence encoding a modified pIX phage coat protein of the present invention. Thus, such a signal sequence or signal peptide may or may not be present in a vector or nucleic acid molecule of the present invention. If present, its appropriate location will be readily determined. Such a signal sequence is generally located upstream (at the N-terminus) of the POI-modified pIX fusion protein of the present invention as part of the same ORF. In some embodiments, a signal sequence or signal peptide is not used or is absent. A signal sequence or signal peptide is sometimes referred to as a leader sequence or leader peptide.

[0061] Other optional features that may be included in the vectors (or nucleic acid molecules) of the present invention will be well known to those skilled in the art. For example, the vectors, such as the phage vectors or phagemid vectors (which may be collectively referred to as phage display vectors or phage display constructs), may optionally further comprise other appropriate components, such as an origin of replication, an inducible or non-inducible promoter / operator for initiating transcription, an enhancer, a termination sequence, an antibiotic resistance gene and marker, a sequence encoding a chaperone protein (e.g., a periplasmic chaperone protein such as FkpA), a signal sequence, a linker, a protease site, a general tag or reporter molecule, restriction enzyme or site-specific recombination sites for cloning and other manipulations (e.g., cloning an appropriate POI into an appropriate position in the vector of the present invention to form a fusion protein with the modified pIX phage coat protein), primer binding sites for amplifying the construct, for example, by PCR, or other desired sequence elements, such as a DNA sequence for enabling discrimination between different libraries, for example, by PCR. The appropriate source and placement within the phage display construct of such additional components so that they perform the desired function is well within the ordinary skill of one of ordinary skill in the art.

[0062] As described elsewhere in this specification, a nucleic acid molecule encoding the modified pIX phage coat protein of the present invention, or a fusion protein of the present invention comprising the aforementioned modified pIX phage coat protein of the present invention, represents a further aspect of the present invention.

[0063] As described elsewhere herein, the vectors of the present invention are primarily used for phage display and may therefore be phagemid or phage vectors. Thus, in a further embodiment of the present invention, the vector construct is a phagemid or phage vector.

[0064] Phage display is a well-known and widely reported technique in the art. In 1985, GP Smith established a method for displaying polypeptides on the surface of filamentous phages, viruses that infect E. coli cells (Smith, GP, 1985, Science 228, 1315-1317). Since then, so-called phage display has evolved into a powerful technique for protein engineering and the selection of peptides and proteins that bind to specific targets (Loset and Sandlie, 2012, Methods 58, 40-46). The filamentous phage M13 is constructed from five distinct structural proteins. Protein VIII (pVIII) is the major coat protein, and the particle is capped at one end with five copies of pIII and pVI and at the other end with five copies of pVII and pIX. The particle infects F-pilus+ E. coli via pIII, and its ssDNA is injected into the bacterial cell. Here, the phage DNA is replicated and transcribed, and new phage particles are assembled and then non-lytically secreted into the growth medium.

[0065] In phage display, a gene encoding a protein of interest (POI) is typically placed between the gene encoding the coat protein (often pIII, but here pIX) and its N-terminal signal sequence to produce a POI-coat protein fusion, although in some embodiments of the invention, no signal sequence is present. A "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 their POI and can be prepared by standard molecular cloning techniques. A library can contain, perhaps as few as 10 10 The method can include more than one member and can be used to select for specific binding agents.

[0066] Thus, the present invention further provides a phage or phage particle comprising a vector or nucleic acid molecule of the present invention and expressing a modified pIX filamentous phage coat protein or a modified pIX fusion protein on its surface. Thus, the phage particle may comprise a phage genome or phagemid, preferably a phagemid. Such a phage or phage particle may be any filamentous phage. Preferred examples include enterobacterial phages, such as M13 phage, fd phage, or f1 phage.

[0067] Another aspect of the present invention provides a library of phage / phage particles, e.g., a library of filamentous phage, produced using and thus comprising the vectors (or nucleic acid molecules) of the invention as described herein. The phage comprise a fusion protein of a POI and a modified pIX phage coat protein as described herein. Thus, the filamentous phage display a POI or library of POIs as fusions with the modified pIX phage coat protein of the invention. As with other phage display libraries, each individual phage particle expresses / displays the same POI, but the presence of multiple particles displaying different POIs allows for the display of multiple (or library-like, or two or more types of) different POIs.

[0068] Thus, in such libraries, a collection of diverse protein fusions, e.g., diverse antibody fusions, with different properties are displayed in the form of a phage display library, e.g., an antibody-phage display library, and selected based on the desired target. In phage display, used for target discovery, e.g., antibody discovery, the library typically consists of a collection of artificially or endogenously diversified target proteins, e.g., antibodies, fused to a phage capsid (here, modified pIX), where these target proteins, e.g., antibodies, differ in their biophysical, biochemical, and target-binding properties. Such libraries are then used to identify variants with the desired properties through a cyclical process called panning, in which clones in the library compete with each other to enrich for the preferred variants.

[0069] Thus, yet another aspect of the present invention provides a library of phage particles, wherein the phage particles comprise a vector (or nucleic acid molecule) of the present invention as described herein, and a plurality of different proteins of interest are expressed on the surface of the phage particles fused to a modified pIX phage coat protein of the present invention.

[0070] For embodiments of the invention involving phage display, reference may be made to general phage display textbooks 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 for relevant techniques and definitions.

[0071] The POI-modified pIX fusion proteins of the present invention can be encoded within a complete phage genome by inserting a sequence encoding the POI-modified pIX fusion protein into the phage genome (phage vector display) or can be encoded on a phagemid (phagemid display). Phagemids are high-copy-number plasmids capable of encoding the POI-modified pIX fusion protein and require superinfection with a helper phage to provide the genetic material necessary for phage production. Thus, in phagemid display, the coat protein used to display the POI (herein, the pIX phage coat protein) typically comes from two sources: a helper phage-encoded pIX protein (e.g., a pIX protein not fused to a POI; unfused pIX protein) and a phagemid-encoded POI-pIX phage coat protein fusion (herein, the POI-modified pIX fusion protein). The new virions produced will have a mixture of the phagemid-derived POI-modified pIX fusion protein and the helper phage-derived pIX coat protein (unfused pIX coat protein). The pIX protein / unfused pIX protein encoded by such a helper phage can be a wild-type (or native) or wild-type-like pIX protein. Similarly, when a phage genome system is used, an unfused, e.g., wild-type or wild-type-like pIX phage protein must also usually be present, although in some embodiments of the present invention, an unfused, e.g., wild-type or wild-type-like pIX coat protein is not present.

[0072] Thus, in the present invention, phage particles can be engineered to display one or more copies of a POI (protein of interest) on a modified pIX coat protein.

[0073] In a phage genome system, such manipulation can be achieved, for example, by modifying the phage genome to contain a sequence encoding a POI-modified pIX fusion protein of the present invention (or an ORF containing such a sequence). If this is the only version / form of the pIX phage coat protein present in the phage genome, multiple copies of the POI will be displayed on the modified pIX coat protein, and there will be no other form of the pIX phage coat protein to compete for surface display, resulting in high-valency (HV) display. On the other hand, if another version / form of the pIX phage coat protein is provided in the system, such that two versions of the pIX phage coat protein are present in the phage genome, further modifying the phage genome to contain, for example, a sequence encoding another pIX protein (a non-fused pIX protein) in addition to the modified pIX phage coat protein of the present invention (or an ORF containing such a sequence), will result in the two forms of pIX competing with each other for surface display, resulting in a mixture of the POI-modified pIX fusion protein and the non-fused pIX protein being displayed on the surface, resulting in low-valency (LV) display.

[0074] In the phagemid system, this can be controlled, for example, by the helper phage used; in a preferred embodiment of the present invention, phage particles are used that display multiple copies of the POI on a modified pIX coat protein. This can be achieved by any suitable method. However, to specifically increase the level of display, thereby allowing multiple copies of the POI on the phage surface (which can also increase binding avidity), modified helper phage, such as a helper phage called DeltaPhage, can be used, which allows high-valency (HV) display on pIX. This is in contrast to the use of conventional helper phage such as M13K07, VCSM13, R408, or similar, which only allow low-valency (LV) display.

[0075] A helper phage called DeltaPhage, reported by Nilssen et al. (2012, Nucleic Acids Research, 40, e120; WO 2011 / 036555), contains at least one (e.g., two) amber mutations inserted near the pIX initiation codon, i.e., near the codon encoding the methionine (M) residue at position 1 of the pIX phage coat protein, thereby conditionally inactivating (conditionally repressing) the pIX encoded by the helper phage. Specifically, these amber mutations are located between positions 2 and 3 of the pIX phage coat protein, i.e., between the S and V residues of the wild-type pIX phage coat protein. However, other positions may be possible as long as the mutations act to conditionally inactivate (conditionally repress) the pIX encoded by the helper phage.

[0076] Next, when this helper phage is superinfected into host cells (e.g., E. coli) transformed with a phagemid encoding a POI-pIX fusion protein, e.g., a POI-modified pIX fusion protein of the present invention, moderate (low) valence display of POI-pIX is observed in host cells that suppress amber mutations (such as amber-suppressing E. coli strains (e.g., supE+ strains)), whereas high valence display of POI-pIX is observed in host cells that do not suppress amber mutations (such as amber-nonsuppressing E. coli strains (e.g., supE- / supE-negative strains)). This is because helper phage-derived pIX (unfused pIX) is produced in amber-suppressing host cell strains (e.g., supE+ E. coli), and as a result, the helper phage-derived pIX (unfused pIX) and the phagemid-derived POI-pIX fusion protein compete for display, resulting in the display of a mixture of both, resulting in moderate (low) valence display. However, production of pIX (unfused pIX) from the helper phage is suppressed or prevented in amber non-suppressing strains (e.g., supE- / supE negative strains), and only the phagemid-encoded POI-pIX fusion, here the POI-modified pIX fusion protein of the present invention, should be present, resulting in high-value display of the fusion protein on the modified pIX.

[0077] Thus, in a preferred embodiment of the present invention, the vector construct is a phagemid vector encoding the POI-modified pIX fusion protein of the present invention, and such a vector construct is used in combination with a helper phage having a conditional mutation, thereby controlling the expression / production of the pIX (non-fused pIX) phage protein encoded by the helper phage, thereby enabling control of the number of POI-modified pIX fusion proteins on the phage surface. In an embodiment in which the conditional mutation is not suppressed, for example, when a non-suppressing strain of E. coli, such as an amber non-suppressing strain, e.g., a supE- / supE-negative strain, is used, the pIX encoded by the helper phage will not be expressed / produced (suppressed), and only the POI-modified pIX will be expressed / produced, and only the POI-modified pIX fusion protein of the present invention will be presented on the surface (high titer, HV, display). In embodiments in which the conditional mutation is suppressed, for example, when a suppressor strain of E. coli, such as an amber suppressor strain, e.g., a supE+ strain, is used, helper phage-encoded pIX is expressed / produced and a mixture of helper phage pIX (unfused pIX) and POI-modified pIX fusion protein is brought to the surface (low valency, LV, display).

[0078] Suitable conditional mutations are well known to those skilled in the art, and helper phage vectors can be easily designed and appropriate host cells selected so that expression of pIX encoded by the helper phage is controlled under the conditional mutation. For example, in a helper phage called DeltaPhage, as described above, conditional mutations in the form of one or more suppressible stop codons (e.g., amber mutation / amber stop codon, or other suppressible stop codons such as ochre or opal mutation / stop codon) are used in conjunction with appropriate host cells that either suppress or do not suppress the conditional mutation.

[0079] Other means for achieving high titer (HV) display using a phagemid (+helper phage) system are also possible. For example, any helper phage that lacks (e.g., has deleted) or does not produce a functional pIX phage coat protein (e.g., due to mutation or truncation) can be used in conjunction with a phagemid of the invention, i.e., a phagemid containing a sequence encoding a POI-modified pIX fusion protein of the invention (or an ORF containing a sequence encoding the POI-modified pIX fusion protein), to achieve high titer display. Again, such a system is designed so that the POI-modified pIX fusion protein of the invention is the only functional pIX coat protein present in the system and, therefore, the only pIX coat protein displayed on the phage surface, resulting in high titer display.

[0080] In some embodiments of the present invention, high-valency display and systems enabling high-valency display are preferred, as the modified pIX vectors of the present invention have been shown to be particularly effective and advantageous when combined with high-valency display. However, the modified pIX vectors of the present invention are equally compatible with low-valency display and systems enabling low-valency display. Methods and systems for achieving low-valency display will be well known to those of skill in the art. For example, the modified pIX vectors of the present invention, e.g., phagemid vectors, can be used with helper phage such as DeltaPhage under repressing conditions, as described above. Alternatively, the modified pIX vectors of the present invention can be used with more conventional helper phage encoding the pIX (unfused pIX) coat protein, such as M13K07 or VCSM13, where expression of the pIX coat protein is not subject to specific control or repression, to achieve low-valency display having a mixture of helper phage (unfused) pIX and the POI-modified pIX fusion protein on the surface. Thus, in some embodiments, low-valency display and systems enabling low-valency display can be used with the modified pIX vectors of the present invention.

[0081] HV display has not traditionally been used to identify high-affinity binders because it is believed that the avidity effect of displaying multiple copies of a POI may impair the accuracy of high-affinity selection. Instead, LV display has been widely used to enable the identification of high-affinity binders. However, it has been discovered that the modified pIX vectors of the present invention can be advantageously used in HV display systems to identify high-affinity binders. The HV display system has the additional advantage of maximizing the functional fraction of phage particles, since more particles carrying a POI fusion protein mean that the functional diversity of the displayed POI is broader, making it easier to find binders. Thus, the HV display system of the present invention, which can simultaneously improve the functional fraction and the functional properties of the displayed candidates, is extremely advantageous, for example, in terms of successfully identifying binders for a target of interest.

[0082] As used herein, the term "high-value (HV) display" refers to a phage display system designed to maximize the number of copies of a specific phage coat fusion protein, herein the number of modified pIX fusion proteins, displayed on the phage surface compared to the number of wild-type (or non-fused) pIX proteins displayed on the surface. Thus, such a system is designed so that all (theoretically all) copies of a specific phage coat protein, herein the pIX phage coat protein, displayed on the phage surface are POI fusion proteins, herein the POI-modified pIX fusion proteins. Thus, in HV display, the system is designed to display five copies of the POI-modified pIX fusion protein on each particle.

[0083] As used herein, the term low valency (LV) display refers to a phage display system designed to display a mixture of coat protein fusion proteins, here modified pIX fusion proteins, and wild-type (or non-fused) pIX proteins on the surface of phage. Thus, such systems are designed so that less than (ideally less than) all copies of a particular phage coat protein, here pIX phage coat protein, displayed on the phage surface, i.e., less than 5 copies, e.g., less than 4 copies, less than 3 copies, less than 2 copies, or less than 1 copy (i.e., a non-maximum or low copy number, e.g., less than 5 copies, less than 4 copies, less than 3 copies, less than 2 copies, or less than 1 copy), are POI fusion proteins, here POI-modified pIX fusion proteins. Such systems are typically configured to provide an average copy number of POI fusion proteins per phage particle of 1 copy or less (although such systems can be configured to achieve a higher average number, if desired). Thus, in a system designed to have an average copy number of POI fusion protein per phage particle of one or less copies, many of the phage particles will not display the POI-modified pIX fusion protein at all.

[0084] The invention described herein is designed for use in prokaryotic systems, not eukaryotic systems, for example. Therefore, suitable host cells are prokaryotic cells, particularly bacterial cells. Suitable bacterial hosts for phage display, which can be used to express the vectors and nucleic acid sequences of the present invention and package and produce phage particles, are well known to those of skill in the art and may be selected accordingly. Preferred bacterial host cells are Gram-negative bacteria, such as Escherichia coli strains. Exemplary E. coli strains include XL-1 blue, TG1, ER2738, AVB100FmkII', MC1061, SS320, TOP10F', and K91K. In some embodiments, non-repressing strains, e.g., amber non-repressing strains, are preferred, examples of which include SS320, TOP10F', AVB100FmkII', MC1061, and K91K. In other embodiments, repressing strains, e.g., XL-1 Blue, TG1, or ER2738, are used.

[0085] The term "phage," often also called bacteriophage, is used herein in its art-recognized form to refer to 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) packaged with phage coat proteins. Secreted filamentous phage particles have a phenotypically filamentous structure. In the present invention, filamentous bacteriophage or filamentous phage are preferably used.

[0086] As used herein, the terms phage, filamentous phage, or filamentous bacteriophage encompass both phage genome-derived virions and phagemid-derived virions. The term "phagemid" is a term of art and refers to a type of cloning vector developed as a hybrid between the filamentous phage Ff and a plasmid to produce a vector that can be propagated as a plasmid and packaged into viral particles as single-stranded DNA. Like plasmids, phagemids can be used to clone DNA fragments and can be introduced into bacterial hosts using a wide range of techniques (e.g., transformation, electroporation). However, infection of the phagemid-containing bacterial host with a 'helper' phage, such as VCSM13 or M13K07, or the aforementioned DeltaPhage, provides the necessary viral components to enable replication of single-stranded DNA and packaging of the phagemid DNA into phage particles.

[0087] The term "helper phage" is a term of the art and refers to a virus that helps a separate, unrelated defective virus, e.g., a phagemid, which is not itself a phage genome or a functional virus, but is merely a plasmid containing one or several elements from a phage genome (here, at least the modified pIX protein of the present invention), by infecting the same host cell already occupied by the defective virus (e.g., phagemid) and assisting the replication of the defective virus by supplying proteins that the defective virus (e.g., phagemid) lacks and needs to complete its life cycle and form virions, e.g., virions containing the phagemid.

[0088] Preferred helper phages for use in the present invention are described elsewhere herein and include M13K07 (Stratagene), Hyperphage (Progen Biotechnik GmbH), R408 (Agilent Technologies), and VCSM13 (Stratagene). In a preferred embodiment, the helper phage may be a helper phage having a conditional (or suppressible) mutation as described herein, for example, DeltaPhage helper phage, or Phaberge, or Ex-phage, as described herein and reported in the art.

[0089] Another aspect provided by the present invention is a phage display system comprising a vector (or nucleic acid molecule) of the invention. A preferred phage display system comprises a vector (or nucleic acid molecule) of the invention, e.g., a phagemid vector of the invention, 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). Another preferred phage display system of the invention comprises a vector (or nucleic acid molecule) of the invention, e.g., a phagemid vector of the invention, and a bacterial host cell, e.g., an E. coli host cell / strain. Suitable host cells / strains are described elsewhere herein and can be included as a component in any of the phage display systems, kits, methods, and uses described herein. Another preferred phage display system comprises a vector (or nucleic acid molecule) of the invention, 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.

[0090] Thus, yet another embodiment of the present invention provides a phage display system of the invention as described elsewhere herein, further comprising a helper phage and / or a bacterial host cell line, for example an E. coli host strain.

[0091] In some embodiments, the pIX phage coat protein encoded by the helper phage can complement or compete with the modified pIX phage coat protein encoded by a vector construct or nucleic acid molecule of the invention. In such embodiments, the pIX phage coat protein encoded by the helper phage can essentially provide an additional copy of the pIX phage coat protein, e.g., an additional copy of a non-fused pIX phage coat protein, e.g., an additional copy of a functional pIX phage coat protein, available for phage coat formation. In some such embodiments, the pIX filamentous 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 suppressor mutations, e.g., suppressor mutations as described elsewhere herein. For example, in a preferred embodiment, the suppressor 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 a system is DeltaPhage, details of which are described elsewhere herein and reported in the art. Suitable and preferred E. coli host strains for use in such embodiments are suppressor strains, preferably amber suppressor strains, more preferably XL-1 Blue, TG1, or ER2738.

[0092] 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 invention. Such inability to complement or compete can occur in any suitable manner. For example, such inability can occur because the pIX phage coat protein encoded by the helper phage is non-functional, e.g., non-functional due to mutation or truncation, or because the pIX phage coat protein encoded by the helper phage is absent, e.g., absent due to deletion.

[0093] Alternatively, such inability may occur because the pIX phage coat protein encoded by the helper phage is not produced or expressed, e.g., because the pIX phage coat protein is produced or expressed under the control of one or more conditional mutations, e.g., one or more suppressor mutations, e.g., suppressor mutations described elsewhere herein, thereby suppressing said production or expression. For example, in a preferred embodiment, the suppressor 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, and the production or expression of the pIX phage coat protein is suppressed by using an appropriate bacterial host strain. A preferred helper phage for use in such a system is DeltaPhage, which is described in detail elsewhere herein. Suitable and preferred E. coli host strains for use in such embodiments are non-suppressing strains that do not tolerate the production or expression of the pIX phage coat protein encoded by the helper phage, preferably amber non-suppressing strains (or ochre non-suppressing strains, or opal non-suppressing strains), more preferably SS320 or TOP-10F'.

[0094] The vectors (or nucleic acid molecules) of the present invention also find utility, i.e., can be used in phage display methods.

[0095] Thus, yet another aspect of the present invention provides a method for producing phage particles, comprising use of a vector construct or nucleic acid molecule of the invention as described herein, or use of a phage display system of the invention. Such phage particles are typically produced by a method comprising the step of introducing a vector construct of the invention, optionally together with a suitable helper phage, into a suitable bacterial host cell as exemplified elsewhere herein.

[0096] Thus, yet another aspect of the present invention is a. Providing a bacterial host cell / strain, e.g., an E. coli host strain, comprising a vector construct of the invention, 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 invention, wherein expression of said vector construct results in the production of a protein of interest-modified pIX fusion protein; b. providing a helper phage; c. infecting the bacterial host cell / strain, e.g., the E. coli host strain, with the helper phage under conditions in which the host strain produces phage particles displaying the protein of interest-modified pIX fusion protein; The present invention provides a method for phage display, comprising:

[0097] In some embodiments, such methods can be used for titered phage display. Thus, in some embodiments, the methods are for titered 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 a vector construct of the invention.

[0098] In particular, the present invention provides a) providing a non-suppressed bacterial host cell / strain, e.g., a non-suppressed E. coli host strain, comprising a vector construct of the invention, 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 invention, and expression of said vector construct results in the production of a protein of interest-modified pIX fusion protein; b) providing a helper phage, wherein expression of said pIX phage coat protein of said helper phage is under the control of one or more suppressor mutations; c) infecting the non-suppressed bacterial host cell / strain, e.g., the E. coli host strain, with the helper phage under conditions in which the pIX phage coat protein encoded by the helper phage is not expressed or produced, so that the non-suppressed host cell / strain produces phage particles displaying multiple copies of the protein-of-interest-modified pIX fusion protein; The present invention provides a method for high titer phage display, comprising:

[0099] As described elsewhere herein, in some embodiments, such high density displays are preferred.

[0100] In other embodiments, the methods of the invention can be used for low-valency phage display, and thus, in such embodiments, the methods are for low-valency 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 a vector construct of the invention.

[0101] In particular, the present invention provides a) providing an inhibiting bacterial host cell / strain, e.g., an inhibiting E. coli host strain, comprising a vector construct of the invention, 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 invention, and expression of said vector construct results in the production of a protein of interest-modified pIX fusion protein; b) providing a helper phage, wherein expression of said pIX phage coat protein of said helper phage is under the control of one or more suppressor mutations; c) infecting the suppressor bacterial host cell / strain, e.g., the E. coli host strain, with the helper phage under conditions in which the pIX phage coat protein encoded by the helper phage is expressed or produced, so that the suppressor host cell / strain produces phage particles displaying one or a small number of copies of the protein-of-interest-modified pIX fusion protein; The present invention provides a method for low-valency phage display, comprising:

[0102] In accordance with the present invention, any method of phage display as described herein, e.g., high valence or low valence phage display, preferably uses a library of vector constructs of the present invention encoding multiple proteins of interest. In another preferred embodiment, the phage display method is used to select proteins that bind to a desired target molecule.

[0103] Once one or more proteins (POIs) have been selected using the methods of the present invention, these proteins, or components, fragments, variants, or derivatives thereof, may be manufactured or produced and, if necessary, formulated with at least one pharmaceutically acceptable carrier or excipient. Such molecules, or components, fragments, variants, or derivatives thereof, once produced, are also encompassed by the present invention. Alternatively, these molecules may take the form of nucleic acids encoding the proteins, which may then be incorporated into a suitable expression vector and / or contained in a suitable host cell. Thus, nucleic acid molecules encoding the proteins, or expression vectors containing the nucleic acid molecules, form further aspects of the present invention.

[0104] Thus, a further aspect of the present invention provides a method for producing or manufacturing a protein (POI), comprising the steps of selecting a protein by a method of the present invention as described herein, producing or generating the protein, or a component, fragment, variant, or derivative thereof, and, optionally, formulating the produced protein with at least one pharmaceutically acceptable carrier or excipient. Alternatively stated, a method of the present invention as described herein, e.g., a method for selecting a protein, may further comprise the steps of producing or generating the protein, or a component, fragment, variant, or derivative thereof, and, optionally, formulating the produced antibody with at least one pharmaceutically acceptable carrier or excipient. The protein variant or derivative may have at least 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to the original polypeptide from which it is derived.

[0105] Another embodiment described herein, e.g., for use in the methods of the invention, is a kit comprising a vector (or nucleic acid molecule) of the invention, or a kit comprising a phage display system of the invention as described above, e.g., a kit comprising a phagemid of the invention and a helper phage, preferably a helper phage as described herein, e.g., a helper phage in which the pIX phage coat protein is expressed under the control of a conditional (or repressible) mutation as described herein, or a kit comprising a vector (or nucleic acid molecule) of the invention, e.g., a phagemid vector of the invention, and a bacterial host cell, e.g., an E. coli host strain as described herein, e.g., a non-repressible E. coli host strain. The kit may also include necessary instructions for use. Kits comprising a phagemid of the invention, a helper phage, and a bacterial host cell as described herein are also provided. Preferred vectors, helper phage, and bacterial host cells of the invention for use in such kits are as described elsewhere herein.

[0106] Preferred vectors (or nucleic acid molecules) for inclusion in such kits of the invention can include the modified pIX phage coat protein vectors of the invention as described herein, and can further include one or more cloning sites (e.g., multiple cloning sites) suitable for cloning in a protein of interest (POI) that is then fused to the modified pIX phage coat protein.

[0107] Thus, a preferred kit can comprise or consist of a collection of reagents for generating phage particles having a fusion protein in which a POI is fused to a modified pIX coat protein of the invention. In addition to the vector of the invention, the kit can include one or more components selected from other phagemids, helper phages, bacterial strains, and instructions. Preferred options for such additional components are described elsewhere herein.

[0108] Yet another aspect of the present invention provides the use of a vector construct, nucleic acid molecule, phage display system, or kit of the present invention for producing phage particles or for use in phage display. Stated alternatively, the present invention provides a method for producing phage particles (or a method of phage display), comprising the use of a vector construct, nucleic acid molecule, phage display system, or kit of the present invention. Such methods for producing phage particles typically include the step of introducing a vector construct or nucleic acid molecule of the present invention, optionally together with a suitable helper phage, into a suitable host cell, such as a bacterial host cell. Examples of bacterial host cells are described elsewhere herein.

[0109] The phage particles of the invention as defined herein may also be used as molecular tools for in vitro applications and assays, said particles may be used in any assay in which it is desired to display a POI on the pIX phage protein.

[0110] Preferred phage particles of the present invention also display a POI, which may be a specific binding partner or targeting unit, such as an antibody as described elsewhere herein, and therefore these phage particles can function as specific binding pair members or targeting reagents, and such phage particles can be used in any assay where said specific binding pair member or targeting unit is required.

[0111] Thus, a further aspect of the present invention provides reagents comprising the phage particles of the invention as defined herein, as well as the use of such phage particles as molecular tools, for example in in vitro assays.

[0112] As used throughout this application, the terms "a" and "an" are used to mean "at least one," "at least a first," "one or more," or "a plurality" of the referenced component or step, unless an upper limit is specifically stated thereafter.

[0113] Furthermore, when the terms "comprise," "comprises," "has," or "having," or other equivalent terms, are used herein, in some more specific embodiments, these terms include "consists of" or "consists essentially of," or other equivalent terms.

[0114] A method comprising certain steps also includes, where appropriate, a method consisting of those steps. In the methods described herein, the steps of the method can be performed in any suitable order.

[0115] The terms "increase" or "improve" or "enhance" (or equivalent terms) used herein include any measurable increase or improvement / modification when compared to an appropriate control. Suitable controls are readily identified by those skilled in the art, and include, for example, the level of a particular parameter determined when using wild-type pIX phage coat protein compared to a modified pIX phage coat protein of the invention. Preferably, the increase or the like will be significant, e.g., statistically significant, when compared to an appropriate control level or control value, e.g., at a probability value of 0.05 or less. Methods for determining the statistical significance of a difference are well known and documented in the art.

[0116] Some of the sequences referred to herein, along with their associated identifiers, are listed in the table below. [ka] All sequences shown in this table are written herein in the 5' to 3' or N-terminal to C-terminal direction, as is customary in the art.

[0117] The invention will now be further described with reference to the following non-limiting examples, with reference to the figures described below. [Brief explanation of the drawings]

[0118] [Figure 1] Figure 1. Polyclonal phage ELISA and single-clone screening for OMV reactivity. (A) Normalized phage samples from R0 and R3 outputs were analyzed for OMV binding by ELISA. Phages of irrelevant specificity (scFv anti-NIP) were included as a control. (B) Random single colonies from R3 were rescued for high-value (HV) display across all libraries. Samples were analyzed for OMV reactivity by ELISA, and a signal-to-background (S / B) ratio of ≥3 was considered positive. The percentage of OMV-positive clones within each library group is shown. Supernatant from empty E. coli SS320 was included as a control. (C) Post-R3 phage libraries were reconstituted for soluble scFv E. coli expression by batch cloning, and random single clones were analyzed for OMV binding by ELISA. (Hoydahl et al., 2016). [Figure 2] Figure 2. SDS-PAGE / Western blot analysis. (Left) Phages from a fully human antibody phagemid library (Hoydahl et al., 2016) and (right) normalized amounts of defined anti-phOx scFv phagemid control clones were separated by 4%-12% SDS-PAGE, followed by anti-pIX Western blot analysis probed with polyclonal rabbit anti-pIX serum. M13K07 helper phage was included as a control (C). Both phagemid samples were packaged for either low-titer (LV, rescued with M13K07) or high-titer (HV, rescued with DeltaPhage) display. pIXwt and scFv-pIX fusions are shown. [Figure 3]Figure 3. (A) Schematic diagram of the genomic region encoding pV, pVII, pIX, and pVIII in the M13 filamentous phage. The pIX ORF has an initiation codon within the pVII ORF and is expressed as a complete protein without post-translational processing. (B) Schematic diagram of the scFv-pIX phagemid expression cassette. The heterologous scFv fusion is linked to the N-terminal end of the complete pIX capsid via an artificial linker / spacer, as published (Loset et al., 2011). The complete phagemid sequence is accessible via GenBank accession code HQ528250. Transcription is controlled by the lac promoter (LacPO) up to the T7 terminator. Following this, translation of the continuous open reading frame (ORF) from mRNA to protein begins at the Shine-Dalgarno (SD) sequence and the initiation codon *, which encodes Met. (C) The amino acid sequences of the start of the scFv (*) and the junction of the linker with native pIX (**) are shown (top), where the start residue methionine (M) of native pIX is shown in bold and underlined (**). This residue was subjected to site-directed mutagenesis (***) as indicated (bottom panel). [Figure 4] Figure 4. Phage production after phagemid rescue was determined by infectious titer (cfuampR / ml) or by total virion content calculated by OD using the formula (((A269nm-A320nm) x 6.083 x 1016) / genome size = virions / ml). [Figure 5]Figure 5. M1x anti-phOx scFv phage was separately produced and titered in E. coli XL1-blue at low titer (A) or in E. coli SS320 at high titer (B). Target binding to phOx-BSA was assessed by phage capture ELISA using phage with similar total virion amounts per sample. The level of target binding (left x-axis) and the corresponding titer (right x-axis) are plotted in the same figure. (C) Anti-phOx scFv phage displayed on pIXwt was produced and titered in E. coli XL1-blue at low titer (LV) or in E. coli SS320 at high titer (HV). Target binding to phOx-BSA was assessed by phage capture ELISA using a low titer (LT) phage concentration of 10 cfuampR / ml and a high titer (HT) phage concentration of 10 cfuampR / ml. (D) M1x anti-phOx scFv-displaying phages were separated by 4%-12% SDS PAGE, followed by anti-pIX Western blot analysis using polyclonal rabbit anti-pIX serum as a probe (top). All phagemids were produced in E. coli SS320 at high viability, whereas wild-type phages were produced in E. coli XL1-blue at low viability. To compare the amount of phage used in the experiments, Western blot analysis was performed using mouse anti-pIII (bottom). [Figure 6] Figure 6. Phage production after phagemid rescue was determined by infectious titer (cfuampR / ml) or total virion content as determined by OD using the formula (((A269nm - A320nm) x 6.083 x 1016) / genome size = virions / ml). Each phage M1x version was produced and titered individually. The phage samples were then grouped based on their biochemical similarity, and the mean ± standard deviation (SD) of the total titer within each group is shown as indicated. [Figure 7]Figure 7. DeltaPhage rescue of M1x anti-NIP scFv phage was performed separately with E. coli XL1-Blue (LV) (A) and E. coli SS320 (HV) (B), and titered. Target binding to NIP-BSA was assessed by phage capture ELISA using serial dilutions of each M1x variant. The variants were then grouped based on their biochemical similarity, and the mean ± standard deviation (SD) of the pooled data for individual binding curves within each group is shown as indicated. Note that in some samples, phage production was poor and only low titers could be tested. [Figure 8] Figure 8. Phage target binding and phage titer were scored from 1 to 10 based on how each value related to the other (A). Because none of the groups reached signal saturation in target binding, specific target binding was defined as the titer that produced a signal twice the baseline value and was scored based on this hierarchical relationship. The M1L_I_G group, which showed the highest score (Q4) for the combination of target binding and phage titer, was divided by each amino acid (B). [Figure 9] Figure 9. Comparison of M1L and M1G. M1L, M1G, and wild-type anti-phOx and wild-type anti-NIP scFv phages were separately produced in E. coli XL1-blue at low liberation or in E. coli SS320 at high liberation. Phages were titered, and target binding to phOx-BSA (A, C) or NIP-BSA (B, D) was assessed by phage capture ELISA with serial dilutions. [Figure 10]Figure 10. Low-valency versus high-valency display. M1L and wild-type anti-phOx and wild-type anti-NIP scFv phages were separately produced in E. coli XL1-blue (LV) or E. coli SS320 (HV). Phages were titered, and target binding to phOx-BSA (A) or NIP-BSA (B) was assessed by phage capture ELISA with serial dilutions. Performance of LV and HV phages was compared to standard low-valency protocols (M1L_standard-LV and wt_standard-LV) using M13K07 rescue and E. coli XL1-blue. [Figure 11] Figure 11. Functional binding versus target concentration. Anti-phOx and anti-NIP scFvs displayed on pIXwt and M1L were produced by hybridization in E. coli SS320. Phages were titered, and target binding to decreasing amounts of phOx-BSA (A) or NIP-BSA (B) was assessed by phage capture ELISA using serial dilutions. [Figure 12] Figure 12. Evaluation of preferential target-specific enrichment between pIXwt and pIX-M1L in spiked panning II. NIP-specific scFvs were prepared in E. coli SS320 (HV) using DeltaPhage helper phage and spiked against target-irrelevant scFvs at a ratio of 1:107. Three rounds of panning with immobilized NIP-BSA were then performed. Then, 40 randomly selected single colonies from each mock library were packaged and tested for target reactivity using antigen-specific phage capture ELISA before the selection round (R0) and after each selection round (R1–R3). Clones were considered positive if they showed a reactivity at least three-fold higher than the background signal. Results are shown as the number of positive clones divided by the total number of clones tested, as shown. [Figure 13]Figure 13. A previously reported fully human scFv antibody phage library displayed on pIXwt (Hoydahl et al., 2016) was reconstituted on pIX-M1L. Both libraries were prepared in E. coli SS320 by standard LV and HV display using M13K07 and DeltaPhage helper phages, respectively. The apparent levels of functionally folded scFv on the phage were then assessed by phage capture ELISA based on binding to the conformation-specific superantigen protein L (pL) contained in serial dilutions of titrated phage. An scFv control phage that does not bind pL was included as a control. [Figure 14] Figure 14. Two versions of the fully human scFv antibody phage library were used to independently select pHLA-specific binders through three consecutive parallel panning rounds (R1–R3) against two unrelated tumor-associated antigen (TAA)-specific pHLA targets using the same protocol. Following panning, equal amounts of polyclonal phage prepared from the R3 output, as well as the unselected library (R0), were tested for target-specific binding by phage capture ELISA. All samples were tested against both targets to serve as both specificity screens and negative controls for each other based on apparent specificity. Results for each sample are shown as the ratio of signal for the specific target to signal for the nonspecific target, which is an indirect measure of target-specific enrichment. An irrelevant scFv control phage was included as a negative control (NC). [Figure 15]Figure 15. Random single clones from the R3 output selected against pHLA TAA target 1 were amplified and rescued with DeltaPhage to generate phage, regardless of the original standard LV or HV format used during selection, to maximize sensitivity in the screen. These phages were assessed separately for binding to matched (TAA target 1) and mismatched targets (TAA targets 2 and 3) by phage capture ELISA. Clones are grouped based on their display capsid (A and B) and the display version used for selection (standard LV or HV). The number of target-specific clones in each version is shown. [Example]

[0119] Improved antibody discovery with modified pIX display Example 1: Identification of modified pIX versions that result in improved antibody display material and method Expression of single clone phage Single clones were packaged in 96-deep-well plates using 400 μl of culture medium for screening experiments or in 50 ml cultures for larger-scale expression. Briefly, clones were inoculated into YT-AG and grown overnight at 37°C. For 96-deep-well expression, 10 μl was transferred to fresh medium in a new plate and grown at 37°C and 600 rpm for 3 hours, after which 10 μl were transferred per well. 9 cfu of DeltaPhage. For larger scale expression, cultures were grown to OD 600nm Reinoculate fresh medium to an OD of 0.05. 600nmAfter culturing at 37°C with vigorous shaking until the RI reached 0.2, cells were superinfected with DeltaPhage or M13K07 at an MOI of 10. Plates and culture flasks were further incubated at 37°C for 30 minutes with gentle agitation, and then incubated for another 30 minutes with vigorous shaking before pelleting the cells and resuspending them in 2x YT-AK. Phage packaging was carried out overnight at 30°C. 100 μL of the clear supernatant from the deep-well packaging was used for screening by ELISA. Meanwhile, phages from larger-scale expression were subjected to PEG precipitation and spot titration. 1 and then used for ELISA and Western blot analysis.

[0120] Single clone phage ELISA using anti-phOx and NIP phages ELISA plates were coated with serial dilutions of phOx-BSA or NIP-BSA in PBS starting at 5 μg / mL, incubated overnight at 4°C, and blocked with 4% nonfat dry milk in PBST for 1 hour at room temperature. Serial dilutions of phage were then added and incubated for 1 hour at room temperature. 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 three times with PBST.

[0121] SDS-PAGE and Western blot 2×10 9 cfu AmpRPhage samples were normalized and heated to 95°C for 5 minutes in Bolt™ LDS sample buffer. Then, electrophoresis was performed on a 4%-12% Tris Plus gel in Bolt MES SDS running buffer (Invitrogen) at 220V for 22 minutes with a broad-range ladder. Proteins were blotted onto Immobilon™-P membranes (Sigma) in Tris-glycine buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) using a semi-dry blotting apparatus and blocked with PBSM. For pIII detection, mouse anti-pIII (MoBiTec, 1:5000) and anti-mouse IgG-HRP (1:10,000) were used. For pIX detection, polyclonal anti-pIX rabbit serum was generated by immunization with a C-terminal pIX peptide (N-CITYFTRLMETSS-C; SEQ ID NO: 9) (AbMART). The anti-pIX serum was used at 1:2000 in combination with anti-rabbit IgG-HRP (1:5000). Western blot detection was performed by reading the chemiluminescent signal.

[0122] Spike Panning Phages displaying NIP-specific scFvs on either pIXwt or pIX-M1L were rescued from either of the two amber-unsuppressed E. coli strains, TOP10F' or SS320, using DeltaPhage, titered, and mixed at approximately 50 / 50 ratios before undergoing one round of selection on immobilized NIP-BSA. Briefly, ELISA wells (NUNC) were coated overnight at 4°C with 5 μg / mL phOx-BSA or NIP-BSA in PBS. Phages were incubated with antigen for 1.5 hours at room temperature with agitation. Wells were washed 10 times with PBST and 5 times with PBS, and bound phage were eluted by incubation with 0.5 ml of 0.5% trypsin for 10 minutes. Both input phage and eluate were used to infect E. coli, and 20–24 random single clones from each group were sequenced (Eurofins Genomics).

[0123] Rebuilding the pIX library Naive human scFv-pIX phage library 2 PCR amplification of the scFv cassette was performed using 10 10 cfu AmpRThe scFv-pIX library phage was directly purified using 0.25 μM each of the forward primer 5'-ATTAAAGAGGAGAAATTAACCATGGCC-3' (SEQ ID NO: 10) and the reverse primer 5'-TTTGGATCCAGCGGCCGC-3' (SEQ ID NO: 11) (Eurofins Genomics), which contain biotinylated primers containing NcoI and NotI RE sites, and 0.05 U / ml Phusion High-Fidelity DNA polymerase. The appropriate bands were extracted from agarose gels, followed by digestion and capture of the biotinylated ends using MyOne Streptavidin T1 magnetic beads (Invitrogen). The scFv cassettes were purified and ligated to the phosphatase-treated vector overnight at 16°C in the presence of polynucleotide kinase, followed by purification using Pellet Paint Coprecipitant (Novagen). 600 ng of library DNA was electroporated into E. coli SS320 (Lucigen) in 350 μl aliquots using an ECM600 electroporator (BTX) essentially as previously described. 3 The transformation mixture was plated onto Bio-Assay dishes (Nunc) and incubated overnight at 30°C. Primary transformants numbered 8.7 × 10 9 The resulting amount was scraped off the plate and rescued (see section on phage rescue and PEG / NaCl precipitation). The library size was limited to the repertoire size of the scFv-pIX library, which had a diversity of 3 x 10 8 It is known to be 2 .

[0124] Phage rescue and PEG / NaCl precipitation Phagemid rescue was performed by transferring scraped material to 2x YT supplemented with 30 μg / ml tetracycline, 100 μg / ml ampicillin, and 0.1 M glucose (2x YT-TAG) at OD 600nmThe cultures were inoculated to an OD of 0.05 and incubated at 37°C with vigorous shaking until an OD of 0.1–0.2 was reached. The cultures were superinfected at an MOI of 20 with the pIX_M1L library, DeltaPhage for R1, R2, and R3, or M13K07 for R3 only, and incubated at 37°C for 60 min with gentle shaking. Then, after 30 min of vigorous shaking, the medium was replaced with 2x YT supplemented with 100 μg / ml ampicillin and 50 μg / 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, resuspended in PBS, and cfu were measured by spot titration. 1

[0125] Protein L ELISA ELISA plates were coated with 5 μg / mL protein L (pL) in PBS, incubated overnight at 4°C, and blocked with 2% nonfat dry milk in PBST for 1 hour at room temperature. Serially diluted phage samples were added and incubated for 2 hours at room temperature. Bound phage particles were detected using an anti-M13 antibody (produced by immunizing chickens with M13 bacteriophage from Norwegian antibodies) conjugated to HRP. The phage samples and antibody were diluted in PBST. The plates were developed with TMB solution and read at 450 nm using a microplate reader. Between each step, the plates were washed three times with PBST.

[0126] Phage selection Selections were performed using both solid- and solution-based panning. Blocked phage samples were incubated for 1 hour with 100 nM biotinylated HLA-A2 loaded with a panel of irrelevant TAAs captured on MyOne Streptavidin T1 beads. Unbound phage were transferred to a new tube and incubated with 100 nM biotinylated HLA-A2:TAA target 1 either pre-captured on beads (solid-based panning for R1) or in solution (solution-based panning for R2 and R3) for 1 hour before capture on beads. The antigen concentration was reduced 10-fold with each round, and washing stringency was increased: 8 washes with PBST + 2 washes with PBS for R1, 13 washes with PBST + 2 washes with PBS for R2, and 18 washes with PBST + 2 washes with PBS for R3. Prior to R2 and R3, phage samples were heat challenged at 65°C for 15 min and then used for panning. Between each wash, the tubes were vortexed briefly. As blocking reagents, 4% (w / v) nonfat dry milk (PBSM) or 2% (w / v) bovine serum albumin (essentially fatty acid-free) was used alternately in the selection rounds. Elution was performed by incubation with 0.5 ml of 0.5% trypsin for 10 min, followed by infection of E. coli with half the eluate. Infected colonies were scraped and rescued (see the Phage Rescue and PEG / NaCl Precipitation sections). A small sample of the infected culture was taken for output measurement.

[0127] Screening of selected outputs Single clones were isolated as described 4 Briefly, clones were inoculated into YT-AG and grown overnight at 37°C and 600 rpm. 10 μl was transferred to a new plate in fresh medium and grown for 3 hours before being plated in 10 wells per well. 9cfu of DeltaPhage were superinfected. Plates were incubated at 37°C for 30 min with gentle agitation, followed by an additional 30 min with vigorous shaking. After that, cells were pelleted and resuspended in 50 μl of 2x YT-AK, and phage packaging was carried out overnight at 30°C. 100 μL of the clarified supernatant was used for screening by ELISA.

[0128] Phage ELISA for single clones obtained by phage selection ELISA plates were coated with 5 μg / mL NeutrAvidin in PBS, incubated overnight at 4°C, and blocked with 5% nonfat dry milk in PBST for 1 hour at room temperature. Biotinylated pHLA variants were captured for 1 hour at room temperature, after which phage was added. Bound phage particles were detected with anti-M13-HRP (Amersham Biosciences, 1:5,000). pHLA, phage samples, and antibodies 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 three times with PBST.

[0129] References 1 Loset, GA, Kristinsson, SG & 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) 2 Hoydahl, LS et al. Multivalent pIX phage display selects for distinct and improved antibody properties. Sci. Rep. 6, 39066, doi:10.1038 / srep39066 (2016). 3 Tonikian, R., Zhang, Y., Boone, C. & Sidhu, SS Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nat Protoc 2, 1368-1386 (2007). 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).

[0130] <Result> We have previously demonstrated that antibody screening using pIX can improve the efficiency of identifying candidate antibodies specific to desired targets and can yield clones with superior biophysical properties compared to those of antibodies discovered by standard pIII display (Figure 1). 1 This beneficial property allows the use of pIX as a scaffold for antibody display and the development of DeltaPhage, which enables high-volume (HV) display of pIX fusion proteins. 2 This was achieved by using a combination of engineered helper phages called

[0131] In this previous study, when comparing the use of pIX display and pIII display as scaffolds for antibody display, a striking discrepancy was evident between the collective output of target-specific signal sampling in target-binding assays after phage library selection and the actual downstream success rate in discovery (Figure 1A vs. Figure 1C). While a higher target signal in the phage selection output would typically be expected to indicate a higher enrichment of specific clones, this correlation proved not to be the case in this case. Furthermore, functional affinity effects (also known as avidity) are thought to impair high-affinity selection in HV phage display, in contrast to low-valency display (LV), which allows for the efficient identification of higher-affinity binders. 3 However, our previous study found this assumption not to be true across two full-scale antibody library selections, with HV pIX display outperforming both LV pIII and HV pIII displays in antibody identification success rates (Figure 1C) and consistently identifying the most potent target binders. 1 There is a strong correlation between phage genome size and virion size. 4 Because pIX is encoded by a much smaller gene than pIII, virions in pIX display have a smaller virion coat than pIII-expressing phage. 4 This would lead to a lower signal in the target binding assay, as shown in Figure 1A. In addition, we have previously found that this effect is further amplified by the higher propensity for the formation of large polyphage virus particles in the pIII system, since target-specific detection is based on virion recognition. 1,4 .

[0132] To better understand whether differences in productive scFv antibody display when using pIX and pIII as display scaffolds might also affect the interpretation of these results, we performed pL (superantigen binding functional antibodies) analysis using pIX and pIII libraries, which represent diverse antibody repertoires. 5 We performed both binding experiments against capsid-specific antibodies (e.g., LV pIII display and HV pIX display) and capsid-specific Western blots to assess the actual display levels. Indeed, the two most successful display routes, LV pIII display and HV pIX display, both elicited seemingly similar antibody levels in these assays. Furthermore, as expected, there was a marked difference in display levels between LV and HV display.

[0133] Further studies focusing on pIX display using Western blot followed by anti-pIX detection indeed showed clear differences in apparent scFv antibody display between the LV and HV formats (Figure 2). However, we made several additional observations in these assays. First, for unknown reasons, pIX lacking the scFv fusion consistently showed two distinct bands of approximately 12.5 kDa and 18 kDa, which are higher than the predicted MW of pIX, approximately 3.6 kDa. However, deviations in SDS-PAGE migration behavior are also well known for other M13 capsids, such as pIII. 6 Second, although the DeltaPhage helper phage system was designed to completely block pIXwt expression in amber-non-suppressing E. coli strains such as TOP10F' and SS320, 2 , and HV samples consistently showed the detection of apparent scFv fusion-defective pIX (Fig. 2, HV samples). This led us to be particularly interested in the latter observation, as it is unlikely to be indicative of nonspecific degradation, which typically yields multiple lower molecular weight intermediates.

[0134] pIXwt displays a complex and only partially characterized translation initiation mechanism that is likely coupled to the upstream pVII capsid due to the inefficiency of the pIX-specific Shine-Dalgarno (SD) site (Fig. 3A). 7,8 Therefore, we reexamined the original scFv-pIX fusion design to explore alternative explanations for the apparent coexistence of both scFv-pIX and free pIX in the HV display sample (Figure 2). Our previous design contained only one SD site, located upstream of the scFv ORF, thus ensuring efficient single translation initiation of the ORF of the complete heterologous fusion protein. However, because we used the complete native pIX ORF, this design included two naturally occurring initiation codons encoding methionine (M): the N-terminal initiation codon of scFv and the native methionine of pIX. We therefore explored the possibility that both products could be translated independently and simultaneously due to previously unreported ribosomal wobbling or potential SD-like sequences inherent in pIX (Figure 3B). To our knowledge, such atypical translation, i.e., skipping or translational wobble, has not been reported previously for pIX in filamentous phage. 9,10 .

[0135] To test the hypothesis of independent translation initiated by the native pIX methionine (hereafter referred to as M1) in our system, we performed M1x targeting experiments limited to alternatives to this M1 (Figure 3C). We investigated, for example, modifications that remove a side chain (M1A and M1G) and modifications that introduce a negatively charged residue consistent with the overall negative charge of the virion coat (M1D and M1E). 11 Several modifications were made to the virion, including the addition of a small hydrophobic side chain (M1L) and the addition of a positively charged R chain (M2L), as this initial study indicated that the positively charged R chain may destabilize the virion in close proximity to the virion coat. 12In the well-characterized phagemid display of anti-phOx scFv reported so far, 4 The various pIX variants were tested. Virions were prepared using phagemid rescue with DeltaPhage in both the amber-suppressed E. coli strain XL1-Blue and the amber-unsuppressed E. coli strain SS320, resulting in LV and HV display, respectively. First, we assessed whether there were any clear differences in virion production between the different M1x versions and unmodified pIX (referred to as wt) (Figure 4).

[0136] Here, infectious particles (cfu) ampR ) and genome number (A 268 ) and the total amount of virions based on 2 In typical phage production, approximately 10%–50% of virions are infectious. 13 On the other hand, in the case of polyphages that produce virions several genome units long, there is a significant discrepancy between the infectious virion titer and the total virion titer. 1 As expected, LV display, which allows expression of the helper phage pIXwt and thus complements the heterologous fusion protein, yielded uniform and nearly identical virion production in both assays (Fig. 4, left panel). However, this was altered in HV display, where all pIX M1x variants showed reduced infectious titers, although to varying degrees (Fig. 4, right panel). The most significant adverse effects were observed for the M1D and M1E variants, which showed approximately 100-fold reductions in infectious titers without a concomitant reduction in total genome numbers. In contrast, the M1A, M1G, and M1L variants showed only small reductions in infectious titers compared to the pIXwt variant, and corresponding reductions in total virion titers.

[0137] We also tested the target binding ability of different phage variants by ELISA (Figure 5). To better visualize the variability in the M1x response, we measured the detection limit (A) of the unmodified pIX display response. 268Based on 8x10 7 A normalized amount of virions, set at 1 / ml (equivalent to 1 / ml, Figure 5C), was used for both LV and HV samples, and the individual total undiluted infectious titers were plotted graphically along with the target binding responses. Consistent with the results in Figure 4, the infectious phage input was similar across all LV samples (Figure 5A), whereas it varied significantly across HV samples (Figure 5B). Furthermore, clear differences in antigen binding were observed for the LV samples, with all M1x variants showing, to varying degrees, better binding than scFvs displayed on unmodified pIX (Figure 5A). Furthermore, two samples, namely, the M1D and M1L variants, clearly stood out for their superior target reactivity, yet at comparable levels. Changing the scFv display format from LV to HV is expected to increase apparent target reactivity due to the increased amount of scFv displayed, as well as the varying degrees of functional avidity effects resulting from multivalent scFv display. 2 Indeed, improved target reactivity was observed for all M1x variants in the HV format compared to both the unmodified pIX and LV formats. This effect was pronounced for both the M1D and M1E variants, but importantly, these samples also showed a large discrepancy between virion titers and infectious titers. Notably, the M1L variant also stood out for its improved target reactivity, without a concomitant severe reduction in infectious titers.

[0138] To understand the actual scFv-pIX display levels, especially for the HV display versions, we further performed pIX-specific Western blot analysis, comparing samples with approximately equal total virion amounts (Figure 5D). This analysis revealed several interesting trends. First, the apparent amount of scFv was comparable across all samples. Second, there was a very clear difference in the amount of free pIX between the different M1x versions, again demonstrating the presence of a significant amount of free pIX lacking fusions in unmodified pIX. This trend also held true, particularly for the M1A variant. The M1G and M1L variants displayed a somewhat intermediate phenotype, with significantly less free pIX than unmodified pIX. In stark contrast, the M1D and M1E variants did not display any free pIX. As mentioned above, it is important to note that these HV samples exhibit strong indications of polyphage formation, with varying degrees of discrepancy between total virion numbers and infectious titers. Regardless of virion length (which varies with polyphage distribution), the stoichiometric ratios of tip capsid pVII / pIX and pIII / pVI are similar to the infectious titer. 14 To account for the actual virion counts, we performed parallel anti-pIII Western blots. This analysis indeed confirmed that the two samples with the highest target reactivity (M1D and M1E) had particularly low infectious virion titers. Therefore, we cannot completely rule out the possibility that even in these samples, trace amounts of free pIX below the detection limit of the assay were present.

[0139] Taken together, these analyses of the anti-phOx scFvs revealed that the M1L variant was the most favorable variant, maintaining overall good virion production under both LV and HV conditions while demonstrating a clear improvement in target reactivity that must be due to the significant reduction in the pIX protein component lacking the scFv. These results also strongly suggest that it is the native N-terminal pIX methionine that allows for the production of this off-target pIX product and that altering this specific residue can largely abolish its effect.

[0140] The primary purpose of antibody phage display is to be used for antibody engineering and discovery, where a collection of diverse antibody fusions with different properties is displayed in the form of an antibody library and selected based on the target. 15 These antibodies inevitably have different inherent display efficiencies, and the human anti-phOx scFv is a relatively well-functioning unit in this regard, since it was obtained through multiple rounds of optimization using phage display technology. 16 Therefore, we broadened the scope of our analysis of the effects of M1x to include hybridoma-derived anti-NIP scFv, which is known to perform poorly in phage display. 2,4 Because initial analysis clearly demonstrated that the effects of substitution of amino acid M1 on pIX off-target product reduction, virion production, and target reactivity differed, we now tested all 20 amino acid variants encoded by the gene. Individual M1x variants were prepared in both LV (XL1-Blue) and HV (SS320) versions, again using DeltaPhage, and the corresponding titers of infectious and total virions were assessed (Figure 6).

[0141] Overall, the results were similar to those for the anti-phOx scFv, with the LV version yielding relatively uniformly high titers (Figure 6, left panel), whereas the HV version yielded considerable variations when examining the number of infectious virions (Figure 6, right panel). To facilitate interpretation, we grouped the data for individual M1x variants into bins that displayed similar trends, consistent with some degree of amino acid biochemistry (e.g., positive vs. negative charge, hydrophobic vs. hydrophilic properties). Several mutations showed clear differences in infectious phage production compared to unmodified pIX, but these differences were primarily observed in the HV format, with the most significant adverse effects observed in the M1E and M1D versions, which resulted in a four-order reduction in infectious titers compared to unmodified (wt) pIX (Figure 6, right panel). The same phage preparations were also used to assess antigen-specific reactivity to NIP-BSA by phage capture ELISA (Figure 7).

[0142] Instead of using a single normalized phage concentration as in the case of anti-phOx scFv, we used serial dilutions for all samples (see Figure 5). Again, data for individual M1x variants were categorized into buckets with similar antigen reactivity profiles. First, as expected, there was a clear difference in overall reactivity between the LV and HV groups (Figure 7A vs. Figure 7B), likely due to an avidity effect associated with HV display. Notably, this effect was primarily limited to M1x variants. Second, the unmodified (wt) pIX version clearly belonged to the lowest target-reactive bucket, regardless of whether it was displayed using LV or HV. Third, the reactivity bucket profiles were somewhat more complex among the various M1x variants, depending on whether they were displayed using LV or HV. However, the unmodified pIX and M1V variants consistently belonged to the lowest target-reactive bucket. As shown, the M1L and M1I variants also consistently belonged to the bucket with the highest target reactivity in both LV and HV formats. As noted above, the effective use of phage display as an engineering and discovery tool requires both maximizing functional display capacity and maximizing the production of infectious virions, thereby enabling the largest heterogeneous fusion protein library pools to be screened for desired variant properties.

[0143] To comprehensively analyze these two key properties based on the HV display data shown in Figures 6 and 7, we separately scored phage production and target binding on a scale of 1 to 10 (10 being the most favorable) and plotted them against each other (Figure 8). Here, the group containing M1L, M1I, and M1G stood out as the best group (Q4), revealing minimal individual variation (Figure 8A). Further stratification of this group based on individual amino acids revealed that the M1L variant offered the best compromise between improved target binding and maintained phage production (Figure 8B). However, the M1G variant also performed quite well, appearing to be slightly superior in target binding. Their similar phenotype was in good agreement with earlier analyses of an unrelated anti-phOx scFv, which showed a potent and comparable reduction in free pIX production (Figure 5D). Further evaluation therefore focused on comparing matched samples to compare the target binding ability of unmodified pIX with the M1L and M1G variants using a phage capture ELISA comparing these two scFvs (Figure 9).

[0144] This focused side-by-side comparison clearly demonstrated that substitution of either M1L or M1G at the M1 position produced very similar effects, although the M1L version likely produced a slightly better effect. For both anti-phOx and anti-NIP scFvs, the M1-modified versions were clearly superior to the unmodified versions. The effect was greater for anti-NIP scFv (Figures 9B and 9D) compared with anti-phOx scFv (Figures 9A and 9C), and for both scFvs, the effect was greater in the HV display format compared with the LV display format.

[0145] To confirm and extend previous analyses, LV and HV rescue was again performed using DeltaPhage for both anti-phOx and anti-NIP scFv constructs, but this time also included phagemid rescue using standard M13K07 helper phage in E. coli XL1-Blue. 17 M13K07 (and other equivalent helper phages, such as VCSM13 and R408) are known to display antibodies with significantly lower efficiency than DeltaPhage, which also leads to a decrease in apparent target sensitivity. 2 To verify whether this was also true under the present conditions, the corresponding phage capture ELISA was performed again (Figure 10).

[0146] As observed in previous analyses, the M1L variant consistently showed the strongest target reactivity with both scFvs in both LV and HV display. Again, the greatest difference was observed with the HV version of the anti-NIP scFv. Furthermore, phage rescued with M13K07 showed clearly inferior target reactivity with both scFvs, regardless of whether the LV or HV version was compared. Interestingly, the difference between unmodified pIX and M1L also appeared to be equalized with M13K07, suggesting that the use of DeltaPhage served as an important helper phage reagent to reveal the distinct profiles underlying this.

[0147] Our previous work showed that there was no clear difference in the extent to which the conventional pIII display system and our proprietary pIX display system were subject to functional affinity effects that reduce the ability to discriminate between high- and low-affinity binders when comparing the LV and HV versions as discovery tools. 1The present M1x modification appears to have improved antibody display levels, especially in the HV version, suggesting that the modified pIX display method may also be subject to functional affinity constraints. To test this hypothesis, we performed a multi-target analysis of the M1x display method, which is based on the fact that functional affinity depends on high target density and high titer. 3 The phage capture ELISA was then repeated with varying target densities of immobilized antigen (FIG. 11).

[0148] It is well recognized that functional affinity, when probed with multivalent antigen-binding units such as HV display, gradually decreases with decreasing target density (Crothers and Metzger, 1972, Immunochemistry 9(3):341-357). However, no difference in binding was observed with decreasing antigen density for either the anti-phOx scFv or the anti-NIP scFv, regardless of whether unmodified pIX or the M1L variant was used as the display scaffold. Thus, the M1L modification may be a useful addition to the advantageous properties already possessed by the original pIX design in this regard. 1 It was thought that this would not impair the

[0149] Taken together, these results suggest that the "leaky" free pIX phenotype observed in the original pIX display system is not a problem. 1This finding clearly suggests that the use of both scFv and pIX initiation codons likely contributes to the separate translation initiation of the two distinct polypeptides encoded by a single scFv-pIX fusion expression cassette (Figure 3). Furthermore, this effect can be eliminated or reduced by substituting alternative amino acids for the native N-terminal pIX methionine. All of these alternative amino acids showed favorable results. However, for certain applications, leucine, isoleucine, or glycine are preferred as the alternative amino acid because these three residues offer the best compromise between maintaining high infectious virion production and improving antibody display efficiency (improving functional display).

[0150] Example 2: Improving antibody discovery using M1L-modified pIX display In Example 1, we demonstrated that replacing the native N-terminal methionine of the pIX capsid with alternative amino acids, particularly leucine, in filamentous phage display advantageously improved the display of heterologous fusion proteins. In these experiments, performance was assessed using predictive single clones. However, one of the major applications of phage display is as a tool for recombinant engineering and discovery. 15,18 In phage display, a library used in antibody discovery consists of a collection of artificially or endogenously diversified antibodies fused to a phage capsid, which differ in their biophysical, biochemical, and target-binding properties. Such libraries are then used to identify variants with desired properties through a cyclical process called panning, in which clones in the library compete with each other to enrich for the preferred variants. 19 The original pIX phagemid display system has been extensively tested in this panning scenario and has been shown to work very well. 1,4,20-22As a first attempt to verify whether the apparent phenotypic improvements of the M1L modifications might lead to further improved performance in panning, we performed a single target enrichment assay in which anti-NIP scFv was displayed on unmodified pIX or on M1L variants (Table 1). The two scFv variants were prepared as hybrid versions from the amber-unsuppressed E. coli strains SS320 or TOP10F' by rescue using DeltaPhage. Phages were initially mixed at a 1:1 ratio and panned against an immobilized target (NIP-BSA). Random single clones were subsequently sequenced before and after panning to determine whether one clone was preferentially enriched relative to the other. [Table 1] Phages displaying NIP-specific scFvs on either pIXwt or pIX-M1L were rescued from either of the two amber-unsuppressed E. coli strains, 10F' or SS320, using DeltaPhage (HV display), titered, and mixed at approximately 50 / 50 ratios before undergoing a single round of selection on immobilized NIP-BSA. Both the input mixture and the eluate were used to infect E. coli, and 20–24 random single clones from each group were sequenced. Results are presented as pIX identity for all clones with discernible sequencing results.

[0151] The sequence analysis revealed only minor variations in the initial spike ratio, but after panning, a very strong preference for the M1L variant was observed, suggesting that panning performance had indeed improved. Next, to mimic a more realistic library situation, we extended this spiking approach by spiking anti-NIP scFv at a 1:10 ratio in a background of abundant irrelevant scFv. 7To mimic a medium-sized diverse antibody library, we generated matched blends in which antibodies were displayed in a hybrid display format on either unmodified pIX or M1L, followed by three consecutive rounds of cyclic panning to examine how efficiently the specific scFvs were recovered (Figure 12).

[0152] Sequencing of 10–24 random single clones from each selection round (R1–R3) revealed their identities and revealed a significant preference for the M1L version in the HV display format, as only under these conditions did anti-NIP scFvs efficiently enrich. Thus, both spiking experiments clearly demonstrated that modifying pIX to M1L has a significant beneficial effect on improving the ability to recover the desired antibody by panning, even when it is present at low abundance in the initial repertoire.

[0153] Motivated by these promising results, we reconstructed our previously reported diverse pIX-displayed fully human scFv antibody library into M1L variants. 1 This library contains approximately 3 × 10 8The resulting library is expected to contain a diversity of 100 unique antibody clones, making it a suitable source for the discovery of promising novel antibody specificities for further drug development. While careful attention was paid to preserving the original antibody repertoire, the new library was estimated to have similar diversity to the original library based on sequence analysis of transformation frequencies. To investigate whether there might be any differences in the display of functional scFvs between the original and reassembled libraries, we rescued both libraries from E. coli SS320 in both standard LV (using M13K07) and standard HV (using DeltaPhage) display formats. Next, normalized amounts (based on infectious titers) of each library were serially diluted and tested for binding to the conformation-specific superantigen protein L (pL) by phage capture ELISA (Figure 13). The results showed that both libraries were highly reactive in the HV display format, with the M1L variant clearly superior. Furthermore, consistent with the previous analysis of single clones (Fig. 10), both libraries showed similar but significantly lower reactivity in the LV format.

[0154] Next, we performed a large-scale antibody discovery project against two clinically validated, unrelated pHLA targets, each featuring a tumor-associated antigen (TAA) peptide overexpressed in human cancers. Each library was used in hybrid variant format in R1 and R2, while in R3, the display format was split into either the standard LV or hybrid variant format. Panning was performed essentially according to previously reported protocols. 21 The procedure was followed by a polyclonal pHLA target-specific phage capture ELISA to compare the unselected initial library (R0) with the output samples from the final round, R3 (Figure 14). The two different pHLA complexes served as reciprocal negative controls for possible pHLA cross-reactivity or nonspecific HLA binding.

[0155] Consistent with previous results using defined model scFv clones, enrichment was again observed for both targets, primarily restricted to the pIX-M1L mutant library. Of note, a clear difference in apparent target-specific signal was observed between the two targets, which may indicate stronger enrichment of more specific clones and / or higher affinity of clones for pHLA TAA target 1 compared to TAA target 2. To clarify this point, we expanded 96 random single clones from each panning experiment focused on TAA target 1 (rescued with DeltaPhage to maximize target-binding sensitivity) and tested them for specific target binding in a pHLA phage-capture ELISA (Figure 15).

[0156] Here, the results obtained clearly showed that the M1L variant resulted in an improved hit rate and apparently better target binding and specificity in pannings in both LV and HV formats compared to the unmodified pIX version.

[0157] Taken together, our results provide a clear explanation for the observation of both full-length scFv-pIX and fusion-defective pIX under HV display conditions that should not result in the expression of free pIX. In the original design, the presence of a natural N-terminal methionine in pIX likely resulted in two separate ORFs encoded on the same phagemid expression cassette, and the amino acid at position M1 was the only determinant controlling this feature. Altering the amino acid at position M1 in pIX could potentially weaken or eliminate this effect. Multiple alternative amino acids appear to be tolerated, each with different effects, and these can be stratified based on two key parameters for using phage display as a tool for engineering and discovery: phage production and fusion protein functionality. From a theoretical perspective, achieving the highest possible phage production and fusion protein functionality is desirable. This should ensure the ability to cover the maximum functional diversity in any fusion protein library and maximize the ability to recover and identify desired novel fusion proteins resulting from library selection. In this case, the substitution of the methionine with leucine (M1L) appears to achieve an optimal balance between these two inherently different but related characteristics. In selections using the limited-diversity spike model, this effect was highly evident, in that the M1L modification clearly demonstrated the best efficiency in recovering specific antibodies, and this effect was particularly observed in the hybrid display format. When the M1L modification was tested side-by-side with the unmodified version in two fully fledged diverse human antibody discovery campaigns using pHLA as the target bait, again, a significant improvement in apparent selection efficiency was observed for the M1L variant, which indeed translated into an improved ability to identify more target-specific clones, which also had superior target reactivity.Here, the effect was also confirmed in the conventional LV format, confirming a comprehensive beneficial effect on the entire pIX display system, independent of the employed helper phage system.

[0158] Although we have not yet fully elucidated the exact mechanism underlying the dual ORF phenomenon, which is likely explained by ribosome skipping, fluctuation, or independent translation initiation, we would likely conclude that substituting M1 with M1L, M1I, or M1G is preferable for designing to improve functionality for practical applications. Furthermore, our results suggest that the best compromise between phage production and fusion protein functionality may be achieved by using M1L when comprehensively covering the broadest and most flexible diversity space. Furthermore, in situations where high fusion protein diversity is less important, such as when a smaller library is sufficient (high phage production is not required), other types of M1x may be considered to maximize fusion protein functionality. In this case, our results indicate that M1E or M1D may be choices that result in very high target reactivity (M1F, M1W, or M1Y, or M1N or M1Q are other options; M1P, M1R, M1K, or M1H are further options). Alternatively, in situations where high phage production is more important (but functionality of the fusion protein is less of a priority, e.g., when high-affinity clones are not necessarily required), other M1x may be considered to maximize phage production. Here, our results indicate that M1C, M1S, M1T, or M1A may be suitable alternative amino acids in this scenario.

[0159] We also demonstrate 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 were observed with both T cell receptor phage display libraries and peptide-MHC (pMHC) phage display libraries.

[0160] References 1 Hoydahl, LS et al. Multivalent pIX phage display selects for distinct and improved antibody properties. Sci. Rep. 6, 39066, doi:10.1038 / srep39066 (2016). 2 Nilssen, NR et al. DeltaPhage-a novel helper phage for high-valence pIX phagemid display. Nucleic acids research 40, e120, doi:10.1093 / nar / gks341 (2012). 3 O'Connell, D., Becerril, B., Roy-Burman, A., Daws, M. & Marks, JD Phage versus Phagemid Libraries for Generation of Human Monoclonal Antibodies. Journal of molecular biology 321, 49-56. (2002). 4 Loset, GA, 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). 5 Graille, M. et al. Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins. Structure 9, 679-687. (2001). 6 Hust, M. et al. Single chain Fab (scFab) fragment. BMC biotechnology 7, 14 (2007). 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). 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). 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). 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). 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). 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). 13 Thomas, W. D. & Smith, G. P. The case for trypsin release of affinity-selected phages. BioTechniques 49, 651-654, doi:10.2144 / 000113489 (2010). 14 Loset, G. A. & Sandlie, I. Next generation phage display by use of pVII and pIX as display scaffolds. Methods 58, 40-46 (2012). 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). 16 Marks, J. D. et al. By-passing immunization: building high affinity human antibodies by chain shuffling. Biotechnology (N Y) 10, 779-783 (1992). 17 Vieira, J. & Messing, J. Production of single-stranded plasmid DNA. Methods in enzymology 153, 3-11 (1987). 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). 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). 20 Huszthy, P. C. et al. B cell receptor ligation induces display of V-region peptides on MHC class II molecules to T cells. PNAS 116, 25850-25859, doi:10.1073 / pnas.1902836116 (2019). 21 Frick, R. et al. Affinity maturation of TCR-like antibodies using phage display guided by structural modeling. Protein Engineering, Design and Selection 35, gzac005, doi:10.1093 / protein / gzac005 (2022). 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 containing a nucleic acid sequence encoding a modified pIX filamentous phage coat protein in which the methionine (M) residue at position 1 of the pIX filamentous phage coat protein has been replaced with an alternative amino acid residue.

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

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

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

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

6. 6. The vector construct of claim 1, wherein the alternative amino acid residue is selected from L, G, or I, more preferably L or G, and most preferably L.

7. 7. The vector construct of claim 1, wherein the modified pIX filamentous phage coat protein corresponds to a pIX coat protein derived from M13 phage, fd phage, or f1 phage, or a variant thereof, in which the methionine (M) residue at position 1 has been replaced with an alternative amino acid residue.

8. 8. The vector construct of claim 1, 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, wherein the methionine (M) residue at position 1 is replaced with an alternative amino acid residue.

9. 9. The vector construct of claim 1, wherein the open reading frame further comprises a sequence encoding a protein of interest fused to the sequence encoding the modified pIX filamentous phage coat protein.

10. 10. The vector construct of claim 9, wherein the target protein is an antibody, a T cell receptor, or an MHC molecule.

11. The vector construct of claim 1 , wherein the vector is a phagemid or phage vector.

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

13. 13. A phage particle comprising the vector or nucleic acid molecule of any one of claims 1 to 12 and expressing a modified pIX filamentous phage coat protein or a modified pIX fusion protein on its surface.

14. A library of phage particles, wherein the phage particles are the phage particles of claim 13 and a plurality of different target proteins are expressed on the surface of the phage particles.

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

16. The phage display system of claim 15, further comprising a helper phage and / or an E. coli host strain.

17. The phage display system of claim 16, wherein the helper phage encodes a pIX filamentous phage coat protein, and 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 of 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 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 of any one of claims 1 to 12.

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

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

21. 21. The phage display system of claim 16, wherein the helper phage is DeltaPhage.

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

23. The phage display system of claim 22, wherein the non-repressing E. coli host strain is an amber non-repressing 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 suppressor strain, preferably an amber suppressor strain, more preferably XL-1 Blue, TG1, or ER2738.

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

26. a. providing an E. coli host strain containing the 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 helper phage; c) infecting the E. coli host strain with the helper phage under conditions in which the host strain produces phage particles displaying the target protein-modified pIX fusion protein.

27. The method described in claim 26, wherein the method is a method for high titer phage display and 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 described in claim 9 or claim 10.

28. A phage display method according to claim 26 or claim 27, wherein the method is a method for high titer phage display, comprising the following a to c: a. Providing a non-repressed E. coli host strain containing the 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 suppressing mutations; c) infecting the non-suppressed E. coli host strain with the helper phage under conditions in which the pIX filamentous phage coat protein encoded by the helper phage is not expressed, thereby allowing the non-suppressed host strain to produce phage particles displaying multiple copies of the protein-of-interest-modified pIX fusion protein.

29. The method described in claim 26, wherein the method is a method for low-valency phage display and 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 described in claim 9 or claim 10.

30. 30. The phage display method according to claim 26 or 29, wherein the method is a method for low-valency phage display, comprising the following steps a) to c) a. Providing an inhibiting 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 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 suppressing mutations; c) infecting the suppressor E. coli host strain with the helper phage under conditions that allow expression of the pIX filamentous phage coat protein encoded by the helper phage, thereby causing the suppressor host strain to produce phage particles that display a single or a small number of copies of the protein-of-interest-modified pIX fusion protein.

31. 31. The method of any one of claims 25 to 30, wherein a library of said vector constructs encoding multiple proteins of interest is used.

32. 32. A method according to any one of claims 25 to 31 for the selection of proteins that bind to a desired target molecule.

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

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

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