Spatial proximity assay

Abstract

The invention relates to a method to query a molecular interaction between a first and a second partner moiety, wherein the first partner moiety is associated to a first partial effector sequence, and the second partner moiety is associated to a second partial effector sequence. The first and second partial effector sequences constitute a functional split HaloTag system. The first partner is a peptide sequence encoded by a polynucleotide sequence, and is physically associated with the polynucleotide sequence. The method according to the invention comprises the steps of: a) contacting the first and the second partner moiety in the presence of a HaloTag substrate covalently linked to a separation function; b) separating the first partner moiety being attached to the separation function; c) determining a molecular interaction between the first partner moiety and the second partner moiety by detecting the presence of the polynucleotide sequence.

Description

[0001] Spatial Proximity Assay

[0002] This application claims the right of priority of European Patent Application EP23184886.2 filed 11 July 2023, incorporated by reference herein.

[0003] Field

[0004] The present invention relates to methods and systems that enable the interrogation of interactions between molecular entities attached to the respective components of a split HaloTag system.

[0005] Background

[0006] In vitro selection techniques, including phage display, mRNA Display, DNA-encoded chemical libraries and SELEX facilitate the rapid generation of high affinity ligands, such as peptides, antibodies, small molecules or nucleic acids for a broad range of biological targets in a matter of weeks. In an iterative process, diverse libraries of up to 1014variants are selected for their desired properties (i.e., typically binding) to a protein of interest. Despite their overall success, selection techniques largely failed to identify novel binders that selectively target integral membrane proteins, such as ion channels or G-protein coupled receptors (GPCRs), which are of great interest for both the research community and pharmaceutical industry (e.g., for diagnostic or therapeutic applications, antibody-drug conjugates, etc.). This limitation can be attributed to bottlenecks with regards to protein expression, selection bias towards inaccessible or non-native epitopes and enrichment of off-target binders when selections are performed on inhomogeneous antigens (i.e., live cells).

[0007] Similarly, the generation of neutralizing antibodies for small-molecule drugs or diagnostics of metabolites remains a major challenge due to their limited immunogenicity. Thus, the non- immunogenic small-molecule of interest (Hapten) is typically conjugated to an immunogenic carrier protein (e.g., BSA) in order to elicit an immune response and to generate antibodies. However, this strategy often generates binders for only the carrier protein or the protein complex and yields the low affinity antibodies.

[0008] HaloTag is a self-labelling protein tag derived from a bacterial enzyme and able to covalently bind to synthetic ligands comprising a reactive chloroalkane. HaloTag can be split into a cpHaloA protein and a complementing peptide which are active only when brought into close proximity, as described in WO2020212537A1.

[0009] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to investigate large libraries of potential binders against a variety of targets in a simple and efficient protocol that allows for a broad structural freedom for both targets and binders. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0010] Summary of the Invention

[0011] In summary, the invention discloses the application of the split HaloTag labeling system previously published by the inventors, for the generation of binders. This can be applied to look for binders, for example, to integral membrane proteins on live cells, and to challenging antigens in a complex environment (i.e. , haptens, MHC-peptide complexes, selections in lysates).

[0012] To this end, an entity capable of bearing information (i.e., phage, ribosome, mRNA or DNA strand) is modified to co-display cpHaloA - the major protein part of the split HaloTag system - in addition to the potential binder (i.e., peptide, antibody). Halopep is conjugated to the target (e.g., through genetic fusion or linkage to an existing binder) and thereby limits the complementation and activity of split HaloTag to spatial proximity of the protein of interest. Target bound entities are subsequently labeled (e.g., biotinylated) with a modified HaloTag substrate and separated from off-target binders through capture on streptavidin-coated magnetic beads and stringent washing. A variety of Halopep and cpHaloA variants with a broad range of affinities (nM to mM) serve as a homing beacon for challenging targets (initial affinity handle for haptens or transmembrane receptors with low expression levels) and enable minute control over the selection process. Moreover, the position of Halopep on the target receptor enables the generation of binders for various epitopes (epitope mapping).

[0013] In one aspect, the invention relates to a method to query a molecular interaction between a first partner moiety and a second partner moiety, wherein the first partner moiety is associated to a first partial effector sequence, and the second partner moiety is associated to a second partial effector sequence, wherein the first and second partial effector sequences constitute a functional split HaloTag system. The first partner is a peptide sequence, or a nucleic acid, encoded by a polynucleotide sequence, and is physically associated with the polynucleotide sequence.

[0014] The method according to the invention comprises the steps of: a) contacting the first partner moiety and the second partner moiety in the presence of a HaloTag substrate covalently linked to a separation function; b) separating the first partner moiety being attached to the separation function; c) determining a molecular interaction between the first partner moiety and the second partner moiety by detecting the presence of the polynucleotide sequence.

[0015] An alternative of this aspect of the invention relates to a method to generate a binding moiety capable of binding to a target moiety, comprising the steps of:

[0016] A) providing a plurality of candidate entities, wherein each candidate entity comprises a candidate binding moiety that is associated to a first partial effector sequence, and comprises a polynucleotide sequence encoding the candidate entity; the candidate entity further physically associates the candidate binding moiety and the polynucleotide sequence encoding the candidate binding moiety.

[0017] Each member of the plurality of candidate entities is characterized by a different polynucleotide sequence encoding the candidate entity;

[0018] B) contacting the plurality of candidate entities with the target, the target (second partner) moiety being associated or linked to a second partial effector sequence, the first and second partial effector sequences constituting a functional split HaloTag system, in the presence of a HaloTag substrate covalently linked to a separation function;

[0019] C) separating the candidate entities bound to the separation function; and

[0020] D) isolating a plurality of polynucleotide sequences;

[0021] E) optionally, amplifying said plurality of polynucleotide sequences; and

[0022] F) obtaining sequence information about the plurality of polynucleotide sequences.

[0023] Terms and definitions

[0024] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0025] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0027] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0028] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0030] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0031] Sequences

[0032] Sequences similar or homologous (e.g., at least about 90% sequence identity) to the sequences disclosed herein, particularly with regard to the first and second partial effector sequence, are also part of the invention, to the extent that the first and second partial effector sequence retain substantially the same biological activity from the pair of first and second partial effector sequence that they are most closely related to in terms of identity, respectively. In some embodiments, the sequence identity at the amino acid level can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

[0033] In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0034] One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

[0035] Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

[0036] The term having substantially the same activity in the context of the present specification relates to the activity of an effector polypeptide pair, i.e. haloalkane transferase activity. A polypeptide qualified as having substantially the same activity does not necessarily show the same quantity of activity as the reference polypeptide; in the particular case of the present invention, a reduction of (self-) labelling activity with respect to the reference peptide (SEQ ID NO 004 or 005) might indeed be desirable for certain applications. As laid out below, for purposes of distinguishing polypeptides covered by the present inventions from those that are not covered, the inventors propose a threshold of activity of 102s'1M'1in the standard assay as laid out in Example 3, with CA-CPY as the substrate (CA: chloroalkane; CPY: (6-(dimethylamino)-3-(dimethyliminio)-10,10-dimethyl-3,10- dihydroanthracen-9-yl)benzoate; see Wilhelm et al., Biochemistry 2021 , 60, 33, 2560-2575).

[0037] For purposes wherein the above definition of activity is not applicable, 3 standard deviations above background regarding haloalkane transferase activity shall be example taken as the reference threshold for having substantially the same activity. In certain embodiments, at least 5 standard deviations are used as the reference threshold. In certain particular embodiments, at least 10 standard deviations are used as the reference threshold.

[0038] In the context of the present specification, the term amino acid linker or peptide linker refers to a polypeptide of variable length that is used to connect two polypeptides in order to generate a single chain polypeptide. Exemplary embodiments of linkers useful for practicing the invention specified herein are oligopeptide chains consisting of 1 , 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids.

[0039] There is no constraint on the amino acid composition of the linker. In certain embodiments, the linker consists of amino acids selected from the group of G S, A and D. An important characteristic of the conjugate peptide linkers as specified above are low immunogenicity, and a peptide length that allows the domains which are joined by the linker, to interact to form a functional entity as disclosed herein. In particular desirable embodiments of the domain peptide linkers specified above, the sequences are primarily made up of stretches of amino acids such as glycine (G) and serine (S).

[0040] In certain embodiments peptide linker is >15 amino acids in length, particularly 15 to 30 amino acids in length wherein the amino acids are selected from G S, A and D.

[0041] A non-limiting example of an amino acid linker is a monomer or di-, tri- or tetramer of a peptide motif composed of three or four glycine and one serine. Any embodiments relating peptide linkers as disclosed herein, encompass structures in which amino acids with similar characteristics are exchanged, for example, the amino acids V, L, I, P, S, C, or M may replace G, S, or S, and D may be replaced by E.

[0042] General Biochemistry: Peptides, Amino Acid Sequences

[0043] The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

[0044] The term peptide in the context of the present specification relates to a molecule consisting of up to 50 amino acids, in particular s to 30 amino acids, more particularly 8 to 15 amino acids that form a linear chain wherein the amino acids are connected by peptide bonds.

[0045] Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rded. p. 21). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)- amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0046] Binding; Binders, Ligands, Antibodies:

[0047] If not specified more narrowly in the Detailed Description of the Invention, reference to binders and ligands encompasses antibodies, antibody-like molecules and aptamers as defined in the following paragraphs.

[0048] The term specific binding in the context of the present invention refers to a property of ligands that bind to their target with a certain affinity and target specificity. The affinity of such a ligand is indicated by the dissociation constant of the ligand. A specifically reactive ligand has a dissociation constant of < 10'7mol / L (particularly < 10'8mol / L or even < 10'9mol / L) when binding to its target, but a dissociation constant at least three orders of magnitude higher in its interaction with a molecule having a globally similar chemical composition as the target, but a different three-dimensional structure.

[0049] The term aptamer relates to an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers can be created by selecting them from a large random sequence pool. Nucleic acid aptamers can be generated through repeated rounds of in-vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to molecular targets such as small molecules, proteins or nucleic acids through non-covalent interactions. Aptamers offer molecular recognition properties that rival that of antibodies.

[0050] In the context of the present specification, the term antibody refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen-binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region of IgG is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system. Similarly, the term encompasses a so-called nanobody or single domain antibody, an antibody fragment consisting of a single monomeric variable antibody domain.

[0051] The term halogen alkane moiety or HaloTag substrate in the context of the present specification relates to an o-halogen alkyl moiety capable of covalent attachment to HaloTag proteins. In certain embodiments, a HaloTag substrate is a moiety of formula:

[0052] (6-chlorohexyl), wherein R can be any moiety as further defined in the specification. In certain embodiments, R is a detection moiety. In certain particular embodiments, R comprises a fluorescent dye. In certain embodiments, R additionally comprises a linker. The HaloTag substrate can also be an ro -bromo- alkane, and aryl o-halogen-alkyl substrates are known and can take this place (see Shields et al., Thousandfold Cell-Specific Pharmacology of Neurotransmission; BioRxiv Oct. 21 , 2022).

[0053] The term fluorescent dye or fluorophore in the context of the present specification relates to a small molecule capable of fluorescence in the visible or near infrared spectrum. Examples for fluorescent labels or labels presenting a visible color include, without being restricted to, fluorescein, rhodamine and silcon-rhodamine based dyes, allophycocyanine (APC), peridinin chlorophyll (PerCP), phycoerithrin (PE), Alexa Fluors (Life Technologies, Carlsbad, CA, USA), DyLight fluors (Thermo Fisher Scientific, Waltham, MA, USA) ATTO Dyes (ATTO-TEC GmbH, Siegen, Germany), BODIPY Dyes (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene based dyes) and the like. The term fluorescent dye or fluorophore in the context of the present specification also relates to dyes described in W02020115286 and WO2019122269A1 , or US16956596, which are incorporated by reference herein.

[0054] Detailed Description of the Invention

[0055] A first aspect of the invention relates to a method to query a molecular interaction between a first partner moiety and a second partner moiety.

[0056] Therein, the first partner moiety is spatially associated to a first partial effector sequence of a functional split HaloTag system, and the second partner moiety is spatially associated to a second partial effector sequence of a functional split HaloTag system, the first and second partial effector sequences constituting a functional split HaloTag system. “Spatially associated” means that the two partners are physically close. They can be each part of a fusion construct; one that joins the first partner moiety with the first partial effector sequence, and one that joins the second partner moiety with the second partial effector sequence. It may also be that the spatial association is only made to happen indirectly, as laid out below.

[0057] The first partner can be a peptide sequence, or a nucleic acid sequence, encoded by a polynucleotide sequence, and it is physically associated with, in other words linked to, the polynucleotide sequence encoding the first partner.

[0058] In embodiments where the first partner is a peptide, the code that encodes peptide will likely make use of the well-known mechanism of cellular information flow, transcribing and translating the polynucleotide sequence to render the first partner as a cell or virus encodes its constituent protein parts through polynucleotide sequences. This is however not absolutely necessary. The key concept is that the information about the first partner, or binder, is encoded in a retrievable form and physically associated to that first partner in a way that allows recovering information about which binders successfully interact with the second partner -the target- and to read out this information.

[0059] The method according to the invention comprises the following steps: a) In a contacting step, the first partner -i.e. the binder- moiety and the second partner -i.e. the target- moiety contact in the presence of a HaloTag substrate that is covalently linked to a separation function, to yield a contact system that provide conditions that facilitate attachment of the HaloTag substrate to the first partial effector sequence if the second partial effector sequence is present. The HaloTag system and substrates useful in its practice have been described, inter alia, in WO2020212537A1 and US2022275350A1 , incorporated by reference herein. b) In a separation step, the contact system is exposed to conditions facilitating separating the first partner -i.e. the binder- moiety if it has been bound through attachment to the separation function. c) Subsequently, whether a molecular interaction between the first partner -i.e. the binder- moiety and the second partner -i.e. the target- moiety has existed or not, is determined by detecting the presence of the polynucleotide sequence. The polynucleotide sequence can only be detected if the previous steps have led to linking of the first binder to the separation function and subsequent separation.

[0060] The association between the first partner and the polynucleotide sequence is stable under the conditions of step a) and b). The term stable is understood the way that the association is not interrupted under conditions of step a) and b). In certain embodiments, the association is a covalent bond. In certain embodiments, the association is a non-covalent, highly charged interaction. In certain embodiments, the association is a non-covalent, protein-protein interaction. In certain embodiments, the association is a chemical bond (particularly a covalent bond) between the candidate binding moiety and an envelope of the polynucleotide sequence. In certain embodiments, this envelope is a phage. In certain embodiments, this envelope is a cell. In certain embodiments, this envelope is a virus particle. In certain embodiments, the association is a chemical bond (particularly a covalent bond) between the candidate binding moiety and a protein- nucleic acid complex comprising the polynucleotide sequence (particularly wherein the complex comprises a ribosome).

[0061] The terminology “first partner moiety” and “second partner moiety” expresses the generic, abstract nature of the roles of the two entities the interaction of which is to be assessed. This may equally well be expressed in terms of “binder” or “binder candidate” for the first, and in terms of “target” for the second partner moiety. The understanding is that when used as part of a large library of first partner moieties, or binding candidates, not all of the library of first partners will necessarily bind, or at least bind equally well, for which reason the terminology of “first” and “second” is meant to clarify the abstract nature of their roles.

[0062] The split HaloTag system consists of two separate protein parts (partial effector sequences) that together constitute a functional, self-labelling HaloTag protein, as described in WO2020212537A1 and EP23158085.3, both incorporated by reference herein. The first, larger partial effector sequence (also referred to as the “cpHalodelta” or cpHaloA) is active in presence of the second, oligopeptide partial effector sequence, which is sometimes herein referred to as the “Halopep”. This latter second partial effector sequence is located on the target and the cpHaloA part on the part carrying the nucleic acid information about the binder, which in the example is a bacteriophage (also referred to as phage herein). This orientation is important, as the covalent label will be only on the cpHaloA part.

[0063] The Halopep (second partial effector) needs to be linked to the target. cpHaloA is linked to each member / entity of a library of binders (i.e. the first partner) encoded by nucleic acids to which they are physically linked. Thus, in one aspect the invention can be framed as libraries of binders, each binder being connected to a cpHaloA, which libraries can be screened, and individual members thereof selected for binding to a target molecule which is brought in proximity of Halopep.

[0064] The association of cpHaloA (the first effector sequence) to the nucleic acid encoding the first partner moiety should be irreversible (covalent, or at least extremely stable), as non-covalent systems might lead to a) loss of target bound nucleic acid and b) labelling of off-target bound nucleic acid (when e.g. an already labelled cpHaloA would dissociate and then re-associate to another nucleic acid, e.g. phage). Moreover, an irreversible label allows for harsh and efficient washing in the separation step (pulldown on the streptavidin-coated beads).

[0065] Association of the first partner moiety (“binder”) to the first partial effector sequence (cpHaloA) can be achieved by binding it directly or indirectly. The key is the proximity of the Halopep to the target I second partner moiety, such that binding of the binder to the target allows for complementation of cpHaloA. “Direct binding” in this context may mean, for example, the presence of both functionalities, the first partner moiety and the first partial effector sequence, on the same.

[0066] The “contact system” requires both the interaction of the first and the second partner and the presence of a HaloTag substrate. During the separation step (b) (separation on the magnetic beads) the interaction of the first and second partner is no longer necessary. The separation function (for example, biotin) does not have to bind to the first partner (binder) directly, but rather to the partial effector sequence, which is associated to the first partner.

[0067] The invention facilitates running selections for nucleic-acid-encoded binders where binding is linked to selective tagging (directly or indirectly) of the first partner with a separation function that allows its isolation and amplification, or direct sequencing and thus linking information about the physical structure of the binder (first partner) to its ability to interact with the target (second partner). Possible targets include a peptide, a nucleic acid, a cell, a small molecule, in essence anything to which a Halopep can be attached.

[0068] The term “separation function” is used herein to describe a functional part of a molecule that allows separation. Separation may be effected directly, for example by using a biotin moiety as separation function. Equally, separation may be achieved by labelling the first partial effector (cpHaloA) with a substrate bearing an alkyne “click-chemistry handle”. The “clickable” moiety itself does not allow direct separation, but can be turned into a separation function by subsequent specific modification with a readily separable function (e.g. an HA-Tag azide, the HA-Tag which can be used for pulldown on magnetic beads coated with the corresponding anti-HA antibody.

[0069] The term peptide when used in the context of the present specification, refers to any polymer of proteinogenic amino acids, independent of length. A “peptide sequence” is encoded by a polynucleotide, thus can be subject to selection and amplification and mutation. An “oligopeptide” generally refers to a peptide of 50 or less amino acids, whereas a “polypeptide” usually refers to a peptide of more than 50 amino acids. In the Examples, the inventors report on results obtained by genetically encoding the second partial effector (the Halopep) to link it to the target, for example a membrane receptor protein. However, for this interaction it is not essential to have an irreversible association and the inventors are currently exploring alternative strategies, e.g. linkage of the second partial effector (the Halopep) to a small molecule / peptide / protein that binds to the target (second partner moiety; “receptor”) with high affinity (thereby bringing the Halopep in spatial proximity to the target).

[0070] While the examples show direct linking of the second partner (target) moiety to the Halopep (second partial effector), this covalent association as part of the same amino acid chain is only one particular embodiment of this association between second partner (target) and Halopep. An alternative would be the linkage of the Halopep to a regulatory protein, the latter of which is associated to the target itself (for example, a protein complex of the pore-forming unit (target) and a regulatory subunit of an ion channel). Thus, while particular, favourable examples show the second partial effector and the target to be encoded as part of the same polypeptide sequence, the second partial effector (the Halopep) does not have to be irreversibly linked to the target receptor.

[0071] The invention may alternatively be framed as an evolutive method to generate a binding moiety capable of a molecular binding interaction to a target (the second partner) moiety, comprising the following steps:

[0072] A) A plurality of candidate entities is provided, wherein each candidate entity is characterized by the following features: it comprises a candidate binding moiety that is spatially associated to a first partial effector sequence of a functional split HaloTag system, the larger fragment in which the self-labelling activity resides when complemented by the smaller second peptide fragment; it comprises a polynucleotide sequence encoding the candidate entity; it physically associates the candidate binding moiety and the polynucleotide sequence encoding the candidate binding moiety;

[0073] Each member of the plurality of candidate entities is characterized by a different polynucleotide sequence encoding the candidate entity.

[0074] B) In a contacting step, the plurality of candidate entities is brought into contact with the target. The target (second partner) moiety is spatially associated to a second partial effector sequence, the smaller peptide fragment of a functional split HaloTag system. The first and second partial effector sequences together constitute a functional split HaloTag system. This contact is made in the presence of a HaloTag substrate covalently linked to a separation function, and together the partners, the first associated to the first partial effector sequence, and the second partner associated to the second partial effector sequence, together with the HaloTag substrate, constitute a contact system. C) In a separating step, the contact system is exposed to conditions facilitating separating the candidate entities as a function of having bound to the separation function, yielding isolated candidates.

[0075] D) Then, a plurality of polynucleotide sequences are isolated from the isolated candidates, yielding isolated polynucleotide sequences; in a sequence isolation step.

[0076] E) Optionally, the polynucleotides are amplified. Modern sequencing methods may similarly allow reading out the sequence information directly in the subsequent step:

[0077] F) Obtaining sequence information about the plurality of polynucleotide sequences is obtained in the isolation step.

[0078] The association between the candidate binding moiety and the polynucleotide sequence is stable under the conditions of step B) and C). The term stable is understood the way that the association is not interrupted under conditions of step B) and C). In certain embodiments, the association is a covalent bond. In certain embodiments, the association is a non-covalent, highly charged interaction. In certain embodiments, the association is a non-covalent, protein-protein interaction. In certain embodiments, the association is a chemical bond (particularly a covalent bond) between the candidate binding moiety and an envelope of the polynucleotide sequence. In certain embodiments, this envelope is a phage. In certain embodiments, this envelope is a cell. In certain embodiments, this envelope is a virus particle. In certain embodiments, the association is a chemical bond (particularly a covalent bond) between the candidate binding moiety and a protein- nucleic acid complex comprising the polynucleotide sequence (particularly wherein the complex comprises a ribosome).

[0079] In phage display, the candidate binding moiety (e.g. a nanobody) and the polynucleotide sequence are not covalently linked, but the genetic information is located inside the phage, while the candidate binding moiety is linked to a phage coat protein (quasi covalently as a fusion protein). With ribosome display it is also the case that the candidate binding moiety is not covalently bound to the mRNA (polynucleotide sequence) but remains indirectly bound to the ribsosome via electrostatic and non-covalent interaction (i.e. a non-covalent complex of mRNA, protein and ribosome (Hanes and Pliickthun, PNAS, Vol. 94, No. 10, 1997). In other methods, such as mRNA display, there is a covalent link between the mRNA (polynucleotide sequence) and the protein (candidate binding moiety), e.g. mediated via puromycin.

[0080] The skilled person understands that the “first partner moiety” and the “candidate binding moiety” are functionally indistinct circumscriptions of the same functional feature of the method to query an interaction, and the evolutive method to generate a binding peptide, respectively. The only difference is that the method to query an interaction looks at the process from the point of view of a single entity, whereas the evolutive method to generate a binding peptide takes a view of a vast multiplicity of query methods at once, with the aim of selecting multiple successful binding events. Optionally, the selected binders may be then used in a subsequent step to generate further diversity around a more narrowly defined range of substructures, to repeat the evolutive method described herein to obtain even better binders.

[0081] The first partner moiety

[0082] The first partner moiety is the part of the molecule providing variable selectivity to the binding process. In other words, the first partner moiety is the part the interaction of which is being interrogated by the method, and the increase of binding of which is the objective of the method presented herein.

[0083] In certain embodiments, the candidate binding moiety or the first partner moiety is a peptide or polypeptide sequence. There is no conceptual distinction here between (oligo-) peptide and polypeptide. The inventors envision that the method according to the invention may be applied to binding studies and optimization of a broad range of types of peptide-based molecules, including shorter peptides that may act on cellular receptors, but also to antibody-like molecules and antibodies, or other larger structures that specifically bind to a target, as may be in the center of any particular technical issue.

[0084] Similarly, the method may lend itself to optimization of binding of nucleic acids, such as may be RNA single- or double strands, or complex mixtures of secondary structure, as well as DNA molecules. Such so-called aptamers have been shown to engage targets with similar specificity of binding.

[0085] In certain embodiments, the candidate binding moiety or the first partner moiety is a nucleic acid sequence.

[0086] In particular embodiments, subsequent to the sequence isolation step, the isolated polynucleotide sequences are amplified under conditions favouring the introduction of mutations into amplification products obtained by amplifying the isolated polynucleotide sequences, yielding amplified polynucleotide sequences, and a plurality of candidate entities are reconstituted from the amplified sequences and the contacting, separating and sequence isolation steps are repeated.

[0087] Such generation of a secondary library (amplification of the output of the primary library followed by further diversification) is a viable strategy to further improve binders identified from a first selection campaign as performed according to the aspects and embodiments of the invention as disclosed herein.

[0088] In certain embodiments, such generation of a secondary library is performed once binders from the primary library have been identified and characterized.

[0089] In some embodiments, the spatial association between the first partner, or candidate binding moiety, and the first partial effector sequence of the HaloTag system may be simply generated by encoding the two moieties or components as part of the same polypeptide sequence. The SpyTag / SpyCatcher system

[0090] In other embodiments, the spatial association between the first partner, or candidate binding moiety, and the first partial effector (cpHaloA) sequence of the HaloTag system may however also be generated by providing the encoded first partner or candidate peptide with a peptide tag that allows selective covalent association of the first partial effector sequence. The inventors used the ’’SpyTag / SpyCatcher” system (Zakeri et al. (2012). Proc. Nat. Acad. Sci, USA 109 (12): E690-7). Other similarly useful systems include the ones disclosed by Keeble et al. (Angewandte Chemie Int. Ed. (2017) 56(62) 16521-16525); Keeble et al. (Proc. Nat. Sci USA (2019) 116 (52) 26523- 26533); Li et al. J. Mol. Biol. (2014) 426(2) 309-317). All generations of the SpyTag / SpyCatcher technology are cross compatible.

[0091] Keeble et al. (Proc. Nat. Sci USA 2019) give a good overview of the sequences and their crosscompatibilities. The inventors tested SpyCatcher003 (Keeble et al. Proc. Nat. Sci USA (2019) in combination with SpyTag001 , SpyTag002 and SpyTag003. Initial experiments indicated that all combinations give comparable results with regards to output of model selections. The inventors have found that the highest rate of success is attained in their hands with the following combination:

[0092] Truncated “SpyCatcher003” sequence (SEQ ID NO 378); SpyTag001 (SEQ ID NO 379).

[0093] In embodiments that utilize this approach, the first partner / candidate peptide sequence is present on the same polypeptide as the SpyTag sequence, and the first partial HaloTag effector sequence is expressed on the same polypeptide sequence as the SpyCatcher partner that binds to the SpyTag upon contact.

[0094] Thus, the two components, by separation, reduce the size of the polynucleotide encoding the first partner or candidate moiety. The reduced size is clearly an advantage, in particular with regards to the construction of libraries (the smaller the encoded sequence, the easier it is to generate large and diverse libraries). Where embodiments of the method related to the use of phage display technology, the fraction of phages displaying a fusion protein is also strongly correlated to the size of the displayed polypeptide.

[0095] Another advantage is the high modularity of this approach. The inventors were able to use novel, improved cpHaloA variants without re-cloning the whole library, thereby saving a significant amount of work.

[0096] In principle, other strategies to facilitate the covalent linkage of cpHaloA to the phage lend themselves to practicing the invention as disclosed herein, including:

[0097] SpyLigase (derived from SpyTag / SpyCatcher; see Fierer et al., (Proc. Nat. Sci USA (2014) 111 (13) E1176-E1181);

[0098] SnoopTag / SnoopCatcher and SnoopLigase (Veggiani et al., (Proc. Nat. Sci USA (2016) 113 (5) 1202-1207); Keeble, Wood and Howarth Bioconjugate Chem. 2023, 34, 6, 1019— 1036. - Transglutaminases (Hitomi et al., Amino Acids volume 36, pages 619-624 (2009)).

[0099] Other available technologies include cysteine bearing peptides; subtiligase, Native Chemical Ligation / Split-lnteins; Sortase-mediated approaches; Lipoic Acid Ligase, LAP-Tag.

[0100] The SpyTag / SpyCatcher system has proven a very elegant and straightforward design, as it can be fully genetically encoded, has virtually no unspecific interactions, exhibits high kinetics and even more importantly high conjugation rates (>90 %) even at low concentrations, is mild (as water is the only side product) and it requires no chemicals that might be harmful to the phages or other proteins involved in the protocol.

[0101] One advantage accrued for embodiments wherein phage display and the use of the SpyTag / SpyCatcher technology is combined, is that the phage assembly in bacteria processes through pathways that are not suitable for the functional expression of certain proteins. That results in the disadvantage that either the yield of phage particles may drop significantly and / or the protein may misfold on the phage surface. This is circumvented by producing the cpHaloA-SpyCatcher separately (as disclosed herein).

[0102] Thus, in certain embodiments, the first partner or candidate binding moiety is covalently linked or encoded as part of the same peptide chain together with a peptide tag sequence capable of forming a covalent bond to an acceptor polypeptide as part of the same polypeptide sequence, and the first partial effector sequence of a functional split HaloTag system is encoded as part of one polypeptide sequence distinct from the polypeptide of the first partner or candidate binding moiety together with the acceptor polypeptide.

[0103] In particular embodiments, the peptide tag sequence is AHIVMVDAYKPTK (SEQ ID NO 379), and the acceptor polypeptide (truncated spy catcher) is SEQ ID NO 378.

[0104] Phage Display

[0105] In certain embodiments, the first partner or candidate binding moiety is associated to the polynucleotide sequence encoding the first partner or the candidate binding moiety, as part of a phage.

[0106] Most commonly employed in phage display are non-lytic phages (most prominently “M13”, but also “f1” and “fd”). Non-lytic systems facilitate easy downstream purification of phage particles.

[0107] For M13, different capsid proteins may be used for the display (e.g., the major coat protein pVIII which is abundant in ~2700 copies, or the other capsid proteins pill, pVI, pVII and pIX, all of which are abundant in 3-5 copies per phage). By far the most commonly used protein in phage display is pill (p3), which was employed by the inventors in the examples disclosed herein.

[0108] Other phages used for phage display include T4, T7 or lambda phages (all lytic). Any of these systems should in principle work in practicing the invention, howeverthe skilled artisan understands that the virus proteins that are engineered to display the protein of interest (e.g., an antibody or peptide) are different for each species.

[0109] In certain particular embodiments, the nucleotide sequences for the a) first partner moiety (i.e., an antibody antigen binding fragment, or peptide) b) SpyTag c) pill phage protein are encoded in one gene to guarantee that the first partner or candidate binding moiety, for example the antibody, is covalently displayed on the phage and that the genetic information encoding for this antibody is passed on to progeny phage after infection of the bacterial cells. Several other functional elements may be encoded in the associated phagemid (phagemids being plasmids that contain genetic material of phages) as known to the skilled artisan.

[0110] In particular embodiments, the first partner is encoded as the same polypeptide sequence as a phage coat protein.

[0111] In certain embodiments, the first partner or candidate binding moiety is cleaved from the polynucleotide sequence subsequent to the separation step. In particular embodiments, a protease recognition site is present between the first partner or candidate binding moiety and a point of physical association to the polynucleotide sequence. In more particular embodiments, the protease recognition site is a TEV protease site.

[0112] The skilled artisan understands that proteolytic cleavage by TEV (or any other protease) is only possible on a protein level (in other words, cleavage from the polypeptide sequence which in turn is encoded by a polynucleotide sequence). Such cleavage however leads to disassociation of the polynucleotide-encoded information about the first partner moiety I binder, facilitating separation and collection and further processing of the information contained in the polynucleotide.

[0113] In embodiments using pill of M13, the protease site is located N-terminally of pill (as this protein is not only required for display of an antibody, but also for infection of bacterial cells) and C-terminally of the SpyTag nucleotide sequence (as this is the position at which for example the biotin (as separation function) is attached and for an elution from any solid support, e.g., beads to which the system is bound), this part has to be cleaved off). Another advantageous side effect of the introduction of the TEV site as this location is that the infectivity of phage particles is dramatically reduced when large proteins are displayed on pill and that by cleavage, the full infectivity of wild type phages is restored at this stage.

[0114] Alternative ways to couple the genetic information encoding the first partner or candidate binding moiety, and its peptide manifestation, are known to the skilled artisan. They include yeast display systems, bacterial display, ribosome display and mRNA display. The separation function

[0115] Common affinity systems employable in the context of the separation step of the present invention include, but are not limited to the following:

[0116] His-Tag / nickel-NTA

[0117] StrepTag (peptide)ZStrepTactin

[0118] - ArgTag

[0119] - GST-Tag

[0120] - M BP-Tag

[0121] - FLAG-Tag

[0122] - HA-Tag

[0123] Calmodulin-binding peptide

[0124] Cellulose-binding domain

[0125] - SUMO

[0126] - Antibodies / Protein A or Protein G

[0127] Cysteine / Disulfide-based approach

[0128] Coiled Coil Interactions

[0129] Lectins (e.g., concanavalin A)

[0130] - Alkaline Phosphatase

[0131] - Albumin

[0132] Subtilisin based approaches (Profinity eXact Purification and Tag Cleavage; Bio-Rad Laboratories GmbH, Feldkirchen, Germany)

[0133] Spy&Go (Anuar et al. (2019) Nat. Communications 10, Art. No 1734)

[0134] Mahmoodi et al. (2019; Current affinity approaches for purification of recombinant proteins, Cogent Biology, 5:1 , DOI: 10.1080 / 23312025.2019.1665406) discuss these methods in detail.

[0135] In particular embodiments, the separation function is biotin.

[0136] Biotin-tagged HaloTag proteins bound to the candidate entity or first partner as defined herein may be separated from unbound I nonreactive partners by capturing the biotin-bound specimen on streptavidin-coated magnetic beads, or on a streptavidin-coated surface, and washing off unbound supernatant. Other technologies with similar scope are known to the skilled artisan and include avidin, neutravidin as well as the various Strep-Tactin analogues (IBA Lifesciences GmbH, Gottingen, Germany).

[0137] The skilled artisan is aware of a number of alternatives of the biotin-streptavidin system that lend themselves to substitute this role, as exemplified by the list given on the previous page. Virtually any affinity purification method may be used for this purpose. The biotin on the substrate itself might be either coupled to any other avidin / streptavidin-modified affinity reagent, or substituted by an azide / alkyne to then perform “click-chemistry” to attach an affinity handle of interest. In particular embodiments, the HaloTag substrate comprises a fluorescent dye, particularly a rhodamine-type dye.

[0138] The dye is important as it significantly increases the affinity of the ligand for the protein but, indeed, in principle, only a separation function such as a biotin moiety is essential for the principle. See Wilhelm et al., Biochemistry 2021 , 60, 33, 2560-2575, where this aspect is described.

[0139] It is further conceivable that the dye is the only separation function, and the system is isolated by optical methods. While phages are too small to be selected in a Flow Cytometer, such optical separation might be possible foryeast display. To that end, one would perform the labelling of yeast cells displaying the binder and cpHaloA and then sort for the populations that have a high fluorescent signal.

[0140] In certain embodiments, the second partner moiety is covalently associated to the second partial effector sequence as part of the same polypeptide sequence. Alternatively, the second partner moiety may only be indirectly associated to the second partial effector sequence. One non-limiting example is the covalent association of second partner and second partial effector to two non- covalently associated components of a membrane protein complex.

[0141] The first partial effector sequence, cpHaloA

[0142] The first and second partial effector sequence together constitute a circularly permutated haloalkane transferase. The first partial effector sequence corresponds to the larger part, termed cpHaloA. Sequences that can be employed as first and second partial effector sequences are disclosed in WO2020212537A1 and US2022275350A1 .

[0143] In certain embodiments, the first partial effector sequence consists of o an N-terminal first effector sequence part characterized by SEQ ID NO 002 (the original N- terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 002, o a C-terminal first effector sequence part characterized by SEQ ID NO 003 (the original C- terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 003, and o an internal cpHalo linker consisting of 10 to 35 amino acids, wherein the internal cpHalo linker connects the C-terminus of the N-terminal first effector sequence part to the N-terminus of the C-terminal first effector sequence part. In certain embodiments, the internal cpHalo linker consists of 12 to 20 amino acids. In certain embodiments, the internal cpHalo linker consists of ~15 amino acids. In certain embodiments, the cpHalo linker may be selected from SEQ ID NO 344 to 366, or 368.

[0144] The second partial effector sequence (Halopep)

[0145] The second partial effector sequence is the peptide that complements cpHaloA to activity. In certain embodiments, the second partial effector sequence consists of a sequence selected from the group consisting of SEQ ID NO 006-343.

[0146] In certain embodiments, the second partial effector sequence consists of a sequence selected from the group consisting of SEQ ID NO: 6-10, 12,13, 15-26, 28, 30, 31 , 34, 40, 46, 48, 50, 75, 85, 98, 103, 112, 154, 156, 160, 167, 173, 175, 182, 189, 195, 212, 214, 233 and 337-342. Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

[0147] Generating activating or inactivating antibodies

[0148] The method can further be employed to generate antibodies which are able to stabilize a particular conformation of a receptor, which is an active or an inactive conformation of the receptor. For that purpose, the active / inactive conformation of the receptor is stabilized in the selection step. This stabilization may be achieved via:

[0149] • Providing the native ligand or a modified activating / inactivating (i.e. , agonistic / antagonistic or inverse agonistic) ligand;

[0150] • Stabilizing the active / inactive conformation of the receptor from the intracellular side using wild-type or chimeric G-proteins, variants of G-proteins (dominant-negative G-proteins (Liang et al., ACS Pharmacol. Transl. Sci. 2018, 1 , 1 , 12-20), 4A insertion (Jang et al., Nature Chemical Biology volume 19, pages687-694 (2023)) (helical extension), mini G- proteins (Nehme et al. (2017) PLoS ONE 12(4): e0175642.), C-terminal peptide fragments of G-proteins (Scheerer et al., Nature 455, 497-502 (2008))) or other signal transducers and G-protein mimetics (such as arrestins or stabilizing nanobodies (e.g., “Nb35”)) either by co-expression or direct receptor fusion and alone or in combination with apyrase, hydrolysis-resistant or modified analogues of GTP and GDP (e.g., GDPpS, GTPyS);

[0151] • Introducing a or several mutation(s) into the receptor amino acid sequence to stabilize the active / inactive conformation of the receptor;

[0152] • Employing G-protein targeted modulators, e.g. pertussis toxin or FR900359;

[0153] • Replacing flexible loops of receptors with more stable proteins (e.g., BRIL orT4 Lysozyme)

[0154] • modulating the cell’s resting potential by electrophysiological means or co-expression (and modulation) of ion channels (e.g., co-expression of potassium channels and selections in the presence of high potassium concentrations).

[0155] All mentioned ways of stabilization may also be combined.

[0156] After the selection of antibodies binding to the active / inactive conformation of the receptor, there may be applied a negative selection pressure using an inactive / active conformation of the receptor. All antibodies also binding to the inactive / active conformation are deselected from further analysis. The method may be employed to generate antibodies that selectively recognise the inactive conformation of a receptor (these are then referred to as "inverse agonists"). This is interesting, for example, for receptors that have a certain basal activity, i.e. are already signalling without a ligand. The selection scheme would then be corresponding (selection on inactive conformation, depletion on active conformation).

[0157] In certain embodiments, the method generates an antibody capable of stabilizing an active / inactive conformation of a receptor, and wherein the target moiety is an active / inactive conformation of a receptor and the candidate binding moiety is an antibody.

[0158] In certain embodiments, the receptor is stabilized in its active / inactive conformation during step B) of the method.

[0159] In certain embodiments, the receptor is stabilized via:

[0160] • Providing the native ligand or a modified activating / inactivating ligand; and / or

[0161] • Stabilizing the active / inactive conformation of the receptor from the intracellular side using wild-type or chimeric G-proteins, variants of G-proteins, 4A insertion, mini G-proteins, C- terminal peptide fragments of G-proteins or other signal transducers and G-protein mimetics either by co-expression or direct receptor fusion and alone or in combination with apyrase, hydrolysis-resistant or modified analogues of GTP and GDP; and / or

[0162] • Introducing a or several mutation(s) into the receptor amino acid sequence to stabilize the active / inactive conformation of the receptor; and / or

[0163] • Employing G-protein targeted modulators; and / or

[0164] • Replacing flexible loops of receptors with more stable proteins; and / or

[0165] • modulating the cell’s resting potential by electrophysiological means or co-expression of ion channels.

[0166] In certain embodiments, additionally steps B) to F) are repeated with the target moiety being an inactive / active conformation of the receptor, and wherein the resulting candidate entities of the repetition are deselected from further analysis.

[0167] The description further encompasses the following items.

[0168] Items

[0169] 1. A method to query a molecular interaction between a first partner moiety and a second partner moiety, wherein the first partner moiety is associated to a first partial effector sequence, and the second partner moiety is associated to a second partial effector sequence, the first and second partial effector sequences constituting a functional split HaloTag system, wherein the first partner: is a peptide sequence or a small molecule encoded by a polynucleotide sequence; is physically associated with the polynucleotide sequence; the method comprising the steps of: a) contacting the first partner moiety and the second partner moiety in the presence of a HaloTag substrate covalently linked to a separation function; b) separating the first partner moiety being attached to the separation function; c) determining a molecular interaction between the first partner moiety and the second partner moiety by detecting the presence of the polynucleotide sequence. A method to generate a binding moiety capable of binding to a target moiety, comprising the steps of:

[0170] A) providing a plurality of candidate entities, wherein each candidate entity comprises a candidate binding moiety that is associated to a first partial effector sequence, comprises a polynucleotide sequence encoding the candidate entity; physically associates the candidate binding moiety and the polynucleotide sequence encoding the candidate binding moiety; wherein each member of the plurality of candidate entities is characterized by a different polynucleotide sequence encoding the candidate entity;

[0171] B) contacting the plurality of candidate entities with the target, the target (second partner) moiety being associated with a second partial effector sequence, the first and second partial effector sequences constituting a functional split HaloTag system, in the presence of a HaloTag substrate covalently linked to a separation function;

[0172] C) separating the candidate entities bound to the separation function; and

[0173] D) isolating a plurality of polynucleotide sequences;

[0174] E) optionally, amplifying the polynucleotide sequences;

[0175] F) obtaining sequence information about the plurality of polynucleotide sequences obtained. The method according to item 1 or 2, wherein the candidate binding moiety or the first partner moiety is a peptide sequence. The method according to item 1 or 2, wherein the candidate binding moiety or the first partner moiety is a nucleic acid sequence. The method according to any one of the preceding items, wherein subsequent to the sequence isolation step, the isolated polynucleotide sequences are amplified under conditions favouring the introduction of mutations into amplification products obtained by amplifying the isolated polynucleotide sequences, a plurality of candidate entities are reconstituted from the amplified sequences and the contacting, separating and sequence isolation steps are repeated. The method according to any one of the preceding items, wherein the first partner or candidate binding moiety is linked together with a peptide tag sequence capable of forming a covalent bond to an acceptor polypeptide as part of the same polypeptide sequence, and the first partial effector sequence is encoded as part of one polypeptide sequence together with the acceptor polypeptide. The method according to item 6, wherein the peptide tag sequence is AHIVMVDAYKPTK (SEQ ID NO 379), and the acceptor polypeptide is SEQ ID NO 378. The method according to any one of the preceding items, wherein the first partner or candidate binding moiety is associated to the polynucleotide sequence, as part of a phage. The method according to item 4, wherein the first partner is encoded as the same polypeptide sequence as a phage coat protein. The method according to any one of the preceding items, wherein the first partner or candidate binding moiety is cleaved from the polynucleotide sequence subsequent to the separation step, particularly wherein a protease recognition site is present between the first partner or candidate binding moiety and a point of physical association to the polynucleotide sequence, more particularly wherein the protease recognition site is a TEV protease site. The method according to any one of the preceding items, wherein the separation function is biotin. The method according to any one of the preceding items, wherein the HaloTag substrate comprises a fluorescent dye, particularly a rhodamine-type dye. The method according to any one of the preceding items, wherein the second partner moiety is covalently associated to the second partial effector sequence as part of the same polypeptide sequence. The method according to any one of the preceding items, wherein the first partial effector sequence consists of o an N-terminal first effector sequence part characterized by SEQ ID NO 002 (the original N-terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 002, o a C-terminal first effector sequence part characterized by SEQ ID NO 003 (the original C-terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 003, and o an internal cpHalo linker consisting of 10 to 35 amino acids, wherein the internal cpHalo linker connects the C-terminus of the N-terminal first effector sequence part to the N-terminus of the C-terminal first effector sequence part. 15. The method according to any one of the preceding items, wherein the second partial effector sequence consists of a sequence selected from the group consisting of SEQ ID NO 006-343.

[0176] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0177] Description of the Figures

[0178] Fig. 1 shows an exemplary embodiment: the first partial effector and first partner moiety are expressed by phage display.

[0179] Fig. 2 shows an exemplary embodiment using indirect association of first binding partner and first partial effector through the SpyTag / SpyCatcher system. The first and second partners are FKBP and FRB, which dimerize in the presence of rapamycin. Biotin-carrying HaloTag substrates are linked to the phages only in presence of rapamycin, as evidenced by fluorescence polarization readout of a fluorescent dye linked to the HaloTag substrate.

[0180] Fig. 3 schematically shows the continuation of the method according to claim 1 with the system shown in Fig. 2.

[0181] Fig. 4 schematically shows a system allowing selections on live cells with the AlfaTag model system. A nanobody (NbAlfa) (second partner) is presented on the surface of HeLa cells as Halopep fusion, and contacted with phages displaying a corresponding high affinity peptide (AlfaTag, ko =26 pM) (first partner).

[0182] Fig. 5 shows the result of an optimization of Halopep sequences and second partial effector sequences using the system shown in Fig. 4. The sequences shown are SEQ ID NO 6, 25, 23, 21 , 20, 19, 341 , 28, 11 , 339, 337, 340, 17, 5, 41 (from left to right).

[0183] Fig. 6 shows the results of the PD-L1 nanobody selection described in Example 1.5

[0184] Fig. 7 shows the results of a monoclonal Phage ELISA of single domain antibodies against

[0185] Cannabinoid Receptor 1 (CNR1) as described in Example 2.

[0186] Fig. 8 show ELISA binding characterization of selected hits from monoclonal phage ELISA experiments using heavy-chain only antibodies as described in Example 2.

[0187] Fig. 9 shows flow cytometer binding characterization of selected hits from monoclonal phage ELISA experiments using heavy-chain only antibodies as described in Example 2. Fig. 10 shows exemplary embodiments of the HaloTag substrate covalently linked to a separation function. The separation function is biotin in these embodiments. The fluorescent dye is optional.

[0188] Fig. 11 shows the characterization of eight selected hits from the monoclonal phage ELISA that were re-formatted into full length heavy-chain only antibodies and their binding was characterized by ELISA on transiently transfected HEK293 cells expressing the metabotropic glutamate receptor 2 (mGluR2) on their surface. The antibodies show specific binding to the active (i.e., agonist bound) over the inactive (i.e., antagonist bound) receptor state with apparent affinities of 1.1 nM, 106 nM, 42 nM, 33 nM, 38 nM, 8.5 nM, 18 nM and 55 nM, respectively (Clone 1-8). No binding was detected to mock-transfected HEK293 cells.

[0189] Fig. 12 A) illustrates the selection of binders against wild-type receptors employing an alternative tagging strategy. In doing so, the Halopepis fused to an existing GLP-1 R peptide ligand (Exendin-4) which is washed in during the selection procedure. B) shows the characterization of five selected hits from the monoclonal phage ELISA that were re-formatted into full length heavy-chain only antibodies and their binding was characterized by ELISA on HEK293 cells stably expressing the glucagon-like peptide 1 receptor (GLP-1 R) on their surface in the presence of 200 nM Exendin- 4 peptide. The antibodies show specific binding compared to an IgG control antibody with apparent affinities of 398 pM, 118 pM, 1.3 nM, 148 pM and 92 nM respectively (Clone 1-5). No binding was detected to mock-transfected HEK293 cells. C) shows the characterization of two selected hits from the monoclonal phage ELISA that were re-formatted into full length heavy-chain only antibodies and their binding was characterized by flow cytometry on HEK293 cells stably expressing the glucagon-like peptide 1 receptor (GLP-1 R) on their surface. The antibodies show specific binding to target-expressing cells only with apparent affinities of 2.8 nM and 2.9 nM respectively (Clone 2 and Clone 5 respectively).

[0190] Fig. 13 A) illustrates the selection of binders against the tetraspanin membrane protein cluster of differentiation (CD20) employing an alternative tagging strategy. In doing so, the Halopepbearing a terminal cysteine is linked to a methyltetrazine moiety via maleimide chemistry. In addition, cells are transfected with a modified receptor con struct featuring a modified lysine residue (A157K-TCO) bearing a trans- cyclooctene (TCO) side-chain using amber codon suppression following standard protocols. Prior to the selection, the Halopepis “clicked” onto the target receptor via IEDDA (inverse electron demand Diels-Alder reaction). B) shows the characterization of a selected hit from the monoclonal phage ELISA that was reformatted into a full length heavy-chain only antibody and its binding was characterized by ELISA on HEK293 cells transiently transfected with CD20. The antibody shows specific binding with apparent affinities of 1.2 nM, whereas no binding was detected to mock-transfected (PD-L1) HEK293 cells. C) shows the functional characterization of a selected hit from the monoclonal phage ELISA that was re-formatted into a full length heavy-chain only antibody and its cell-killing activity was characterized by an ADCC bioluminescence-based reporter assay on HEK293 cells transiently transfected with CD20 or mock-transfected (PD-L1). The selected antibody shows target specific ADCC activity with an EC50 of 14.3 nM.

[0191] Fig. 14 A) illustrates the model selection for the yeast surface display technology. Yeast cells displaying AlfaTag and cpHaloA2 are incubated with HEK293 cells transiently transfected with either Halopep-NbAlfa-mEGFP or NbAlfa-mEGFP and subsequently labeled with CA-SiR. Labeled cells / complexes are analyzed by flow cytometry. B) shows the flow cytometry analysis of the yeast surface display model selection. Significantly higher SiR signal was observed for HEK293 cells displaying Halopep- NbAlfa-mEGFP compared to NbAlfa-mEGFP only, indicating Halopep-dependent labelling of cpHaloA-displaying yeast cells.

[0192] Fig. 1 schematically shows the principle of the invention in a simple embodiment. Cells are genetically manipulated to express an extracellular protein (the second partner in the parlance of the abstract description used herein), for example a receptor, that displays the Halopep peptide (the second partial effector). The cells are contacted with a plurality of phages that express potential binders (first partner) and cpHaloA (the first partial effector). Addition of a biotinylated HaloTag substrate leads to the phages being biotinylated where first and second partner have engaged and thereby facilitated complementation of the cpHaloA to a self-labelling protein. The biotinylated phages can be separated through streptavidin pulldown and eluted and re-amplified or analysed.

[0193] Examples

[0194] Example 1: Validation of the system as exemplified by phage display

[0195] 1.1 Binding of FKBP and FRB induced by rapamycin (Fig. 2; 3)

[0196] Kinetics and extent of conjugation of cpHaloA-SpyCatcher (15 pM) to phages displaying SpyTag- plll (25 nM, Western Blot analysis with pill antibody) were determined. Rapid conjugation of recombinant cpHaloA-SpyCatcher protein to SpyTag-plll phages was observed within 60 min at room temperature and to ~80-85 % completion, (data not shown).

[0197] Labelling kinetics of cpHaloA-SpyCatcher (20 nM) conjugated to phages co-displaying FKBP (2.5*1012cfu), in the presence of FRB-Halopep fusion (20 nM) and with or without the smallmolecule chemical dimerizer rapamaycin (500 nM) (5 nM CA-TMR-Biotin final) were determined by fluorescence polarization on a microplate reader Spark20M (Tecan Group) in black, flat-bottom low-volume, 384-well polystyrene plates (Corning) with a humidity cassette at 25 °C in a final volume of 40 pL. The result of the measurement is shown in Fig. 2 B. A substantial increase in fluorescence polarization, indicative of labelling of cpHaloA with TMR, was only observed in the presence of rapamycin.

[0198] Enrichments of model selections in solution with F KB P / FRB- rapamycin model system were determined; the workflow is shown schematically in Fig. 3. FKBP-displaying phages (1*1010cfu) conjugated to cpHaloA-SpyCatcher (15 pM) with CA-T MR- Biotin (50 nM) were labelled in the presence of FRB-Halopep (1 pM) and + / - rapamycin (5 pM). Subsequent pulldown and proteolytic elution from streptavidin-coated magnetic beads afforded the labelled phages. FKBP-displaying phages were preferentially labelled and biotinylated in the presence of rapamycin (57-fold enrichment over the condition without rapamycin) and a total recovery of up to 27 % of the input phages.

[0199] 1. 2 AlfaTaq mediated optimization of Halopep and cpHaloA (Fig. 4; 5)

[0200] Phages displaying AlfaTag (5*1011) were conjugated to cpHaloA-SpyCatcher (100 nM) and added to HeLa cells (5*105) expressing NbAlfa-Halopep or NbAlfa only at 4 °C for 30 min, labeled with CA-TMR-Biotin (250 nM) at 4 °C for 2 hours, quenched with an excess of HaloTag protein and finally eluted by cell lysis. The lysate was incubated with streptavidin-coated magnetic beads, allowing biotinylated phages to bind. Stringent washing with TBS + 0.5 % Tween-20 and proteolytic elution (TEV) showed an enrichment of ~10-fold for the parental Halopep (ARETFQAFRT; SEQ ID NO 005). Phages that do not display AlfaTag showed no enrichment and gave ~10,000-fold lower output titers compared to AlfaTag displaying phages.

[0201] Selections with a second model system comprised of a GFP enhancer nanobody (VHHGFP) displayed on phage and EGFP presented to the cell surface gave comparable enrichments, indicating the generalizability of this approach.

[0202] Selections of AlfaTag- and nHcFP-displaying phages on cells stably expressing NbAlfa-Halopep as well as EGFP showed preferred enrichments of target bound phages (AlfaTag) over off-target binders (VHHGFP) of ~60-fold, indicating that indeed the complementation of cpHaloA is spatially restricted to the target receptor.

[0203] Selections with various Halopep variants as N-terminal NbAlfa fusions showed improved enrichments of up to ~200-fold without any substantial increase in enrichment of background phages (i.e., phages not display AlfaTag peptide), with the exemption of WREMFRLFRT (SEQ ID NO 20) showing an approx. 20-fold background enrichment (Fig. 5).

[0204] 1.3 Nanobodies for Programmed Death-Ligand 1 (Fig. 6)

[0205] Aim of this example was to perform selections on HEK293 cells transiently transfected with Halopep-PD-L1 ([SKRDAREMFQAFRT] SEQ ID NO 006, N-terminal fusion) as previously described [Increased stringency in a third round by reducing cell number to 6*105), to screen hits with a monoclonal phage ELISA, and to determine binding affinities of nanobodies to recombinant PD-L1 (extracellular domain) with ELISA. In addition, the functionality of binders in a luciferasebased PD-1 / PD-L1 blockade assay (Promega J 1250 / J 1255) was to be established.

[0206] A strong increase in the output titre was observed after the 2ndround of selection, the output after the 3rdround was lower due to the increased stringency of the selection (R1 : 7.5*103, R2: 1.2*109, R3: 3.1*104).

[0207] Monoclonal ELISA experiments of individual clones after the 2ndround of selection indicated strong enrichment of target specific binders (hit rate21 / 2i with aS / N > 3, as determined by ELISA signal on PD-L1 transfected over non-transfected HeLa cells, which do not express PD-L1 endogenously) (see Fig. 6 A).

[0208] Five selected recombinant nanobodies (R3 output) plus control nanobody were added to immunoplates coated with the extracellular domain of PD-L1. An HRP-coupled Hisex antibody directed towards the Hisex-Tag (SEQ ID NO 383) of the recombinant nanobodies was added and subsequently the HRP substrate was added to the plates. The reaction was stopped after 10 mins and the absorption was plotted against the concentration in order to determine the affinity of the nanobodies (which were as high as 305 nM) (see Fig. 6 B).

[0209] PD-L1 expressing CHO-K1 cells and PD-1 expressing Jurkat cells were co-cultured and different amounts of nanobodies plus mAb control were added to the supernatant. Blockade of the PD- L1 / PD-1 interaction leads to expression of luciferase, which in turn can be visualized upon addition of luciferase substrate. Both nanobodies showed concentration-dependent blockade of the PD- L1 / PD-1 signaling axis with approximately double-digit pM EC50S, showcasing that the generated binders are also functional in a cellular environment (see Fig. 6 C).

[0210] 1.4 Model Selection on mEGFP

[0211] The objective of this example was to perform selections on live cells with a Sybody Library on mEGFP as a model target (overall high immunogenicity and various nanobodies for GFP have been reported in the literature), to screen hits with monoclonal phage ELISA, and to determine binding affinities of top ELISA hits with isothermal titration calorimetry (ITC).

[0212] Three rounds of selections were performed on HEK293 cells (2*107) transiently transfected with mEGFP-Halopep (Halopep [SKRDAREMFQAFRT], SEQ ID NO 006, N-terminal fusion) linked to the PDGFRp transmembrane domain) and sybody displaying phages (input 1.5*1011cfu) that were conjugated to cpHaloA-SpyCatcher. Cells were detached with Versene (PBS + EDTA) and blocked (TBS + 2 % BSA) at 4 °C for 30 min and then cells were resuspended in phage suspension. After 30 min at 4 °C with overhead shaking, cells were centrifuged (250xg at 4 °C for 5 min) and the unbound phages were removed carefully without disturbing the pellet. Cells were washed once (TBS + 0.5 % BSA), resuspended in labelling buffer (250 nM CA-TMR-Biotin in TBS + 0.5 % BSA) and incubated with overhead shaking at 4 °C for 2 hours. Unreacted Halo substrate wash quenched through resuspension in quenching buffer (TBS + 0.5 % BSA + 2 pM HaloTag) and removed by subsequent centrifugation. Cells were resuspended in lysis buffer (GPCR extraction buffer + 0.5 % BSA + 0.1 % Tween-20 + 1x HALT Protease and Phosphatase Inhibitor), resuspended by pipetting and incubated with overhead shaking at 4 °C for 20 min. The lysate was cleared through centrifugation at 21 ,000xg at 4 °C for 15 min and the supernatant was transferred to pre-blocked streptavidin-coated magnetic beads. Biotinylated phages were pulled down through overhead shaking at 4 °C for 20 min and then washed six times with TBS-T (TBS + 0.5 % Tween-20) and twice with TBS only. Beads were resuspended in elution buffer (TBS + 2 % BSA + 0.2 mg / mL TEV protease) and incubated with overhead shaking at room temperature for 30 min. Beads were separated from supernatant with magnetic stand and eluate was removed for re-infection for subsequent rounds and titering.

[0213] An increase in the output titre indicated enrichment of target bound phages (R1 : 6.0*105, R2: 1.9*107, R3: 4.4*108).

[0214] Polyclonal phage ELISA experiments on mEGFP-transfected over untransfected HeLa cells showed an increase in signal (indicative of cell binding) from unselected library to R3 output after selection (the latter which is comparable to signal for positive control VHHGFP displaying phages). Moreover, a polyclonal ELISA from the output of two rounds of selection on HEK293 cells transfected with mEGFP only (no Halopep) indicated that no target specific binders were selected and Halopep on the target is essential for enrichment of target-bound binders.

[0215] Monoclonal ELISA experiments of individual clones after the 2ndround of selection indicated strong enrichment of target specific binders (hit rate41 / 45 with aS / N > 3).

[0216] Sequence analysis of 22 ELISA hits compared with the positive control showed that 6 out 8 positions in the nanobody sequence that are reported to form key interactions with the mEGFP protein (through salt bridges, hydrogen bonding or hydrophobic interactions) are conserved among ELISA positive hits.

[0217] Affinities of four selected nanobodies and the reference nanobody VHH GFP to recombinant mEGFP were determined by means of ITC and ranged from 92 nM to 557 nM (140 nM for the literature-reported reference VHHGFP).

[0218] Example 2: Antibodies for Membrane Proteins

[0219] Methods

[0220] Library Cloning:

[0221] A synthetic nanobody mRNA library (doi.org / 10.7554 / eLife.34317) served as a template and was re-cloned into a modified phagemid that harbors a PelB leader sequence, the nanobody-encoding sequence, a Hisex-Tag (SEQ ID NO 383), an amber stop codon (TAG), the SpyTag001 sequence, a TEV cleavage site (ENLYFQG SEQ ID NO 380) and the sequence of pill from filamentous bacteriophage M13. Reverse transcription, PCR amplification, restriction and ligation were performed according to standard procedures. The three individual libraries were separately transformed into electrocompetent TG1 (Agilent, 200123), yielding a final total library size of 3*108colony-forming units (cfu). The library was further analyzed by Next-Generation Sequencing (Illumina NovaSeq 2x150 bp), indicating a large diversity with a total of 1.6*108unique sequences.

[0222] Phage production:

[0223] A representative aliquot (~400 fold representation) of the concave, loop and convex nanobody library was used to inoculate a 500 mL culture in 2xYT medium (Carl Roth, X966.2) supplemented with 2 % glucose (Carl Roth, 6780.1) and 100 pg / mL Ampicillin (Carl Roth, K029.3) at ODeoo=0.1 and grown at 37 °C and 220 rpm. The libraries were rescued with 2 mL Hyperphage (Progen, PRHYPE; MOI (multiplicity of infection) = 11) each at OD6oo=0.4-0.5 and infected at 37 °C without shaking for 45 minutes and subsequently incubated at 37 °C and 220 rpm for 45 minutes. Superinfected bacterial cells were pelleted at 2,000xg and 22 °C for 20 minutes and re-suspended in 500 mL 2xYT + 100 pg / mL Ampicillin + 50 pg / mL Kanamycin (Supelco, PHR1487). Cultures were grown at 25 °C overnight and bacterial cells were pelleted by centrifugation at 15,000xg and 4 °C for 60 minutes. The supernatant was decanted and % volume (i.e., 125 mL) pre-chilled precipitation buffer (20 % PEG-8000 (Carl Roth, 0263.2) + 2.5 M NaCI in H2O) was added. The centrifugation bottles were inverted a couple times and placed on wet ice overnight. The next day, phages were pelleted by centrifugation at 15,000xg and 4 °C for 60 minutes. The supernatant was removed and the phage pellet was re-suspended in 30 mL ice cold TBS (50 mM Tris-HCI (Carl Roth, 9090.2), 150 mM NaCI (Supelco, 1 .06404), 10 mM MgCI2(Merck, 105833), 1 mM CaCI2(Carl Roth, A.119.1), pH 7.5). Bacterial debris was removed by centrifugation at 15,000xg and 4 °C for 60 minutes and the phage-containing supernatant was transferred to a new falcon tube containing 7.5 mL precipitation buffer. The tube was inverted a couple times and placed on wet ice for at least two hours. Phages were pelleted by centrifugation at 15,000xg and 4 °C for 60 minutes. The phage pellet was re-suspended in 2 mL TBS and centrifuged at 21 ,000xg and 4 °C for 30 minutes to remove any residual cell debris. The supernatant was carefully transferred to a new Eppendorf tube, mixed with 1 volume (i.e., 2 mL) TBS (10x) + 90 % glycerol (Merck, 356350) and stored at - 20 °C.

[0224] Phages were titered according to standard procedures. Briefly, a culture of TG1 cells was inoculated in 2xYT + 2 % glucose at ODeoo=0.1 and grown at 37 °C and 220 rpm. In the meantime, an aliquot of the library (15 pL) was mixed with 1 volume (i.e., 15 pL) TEV protease (1 mg / mL) and incubated at room temperature for 30 minutes. Then 20 pL of this mix was diluted in 180 pL 2xYT + 2 % glucose in row A on a 96 well plate. Ten-fold serial dilutions were performed by transferring 20 pL of phage suspension into 180 pL 2xYT + 2 % glucose in the next row (rows B through H). Subsequently, 20 pL of each phage dilution were transferred to a new 96 well plate and 180 pL of mid-log TG1 culture (OD6oo=0.4-0.5) was added. Infection was performed at 37 °C without shaking for 30 minutes and then 10 pL from each well was plated out on a pre-warmed LB / Agar / Ampicillin plate and incubated at 30 °C overnight. The next morning, colonies were counted and the titer of the stock solution was calculated. The titer of each library was ~1 *1013cfu / mL. Selections:

[0225] Selections were performed on transiently transfected HEK293 cells (DSMZ, ACC 305). Briefly, one day prior to transfection cells were seeded in DMEM (Gibco, 10566016) supplemented with 10 % FBS (Gibco, 10500-064) at 1*107or 3.3*106cells (Round 1 and Round 2 / 3, respectively) in a T75 orT25 flask, respectively. On the next day, cells were transfected with the Halopep-POl (protein of interest) constructs in a pcDNA5 / FRT / TO backbone (Invitrogen, V652020) using Lipofectamine 3000 (Invitrogen, L3000001) according to the manufacturer’s protocol. Approximately 16 hours post-transfection, cells were washed with PBS (Gibco, 10010023) once and supplied with fresh medium. One day prior to selection, the phage library (100-200- fold representation of each individual library, approximately 5*1010cfu in total) was incubated with recombinant SpyCatcher003-cpHaloA2 protein (2 pM final) in TBS pH7.0 + 2 % BSA (Sigma-Aldrich, A3294- 100G) at room temperature for 3 hours and subsequently diluted in TBS pH 7.5 + 2 % BSA and placed for overhead shaking at 4 °C overnight.

[0226] Two days post-transfection, cells were washed with PBS once, detached with 2 mL Versene (Gibco, 15040066) at room temperature for 3-5 minutes and subsequently transferred to a 15 mL Falcon tube by washing the cells off the flask with 9 mL PBS. Cells were pelleted in a pre-cooled centrifuge at 250xg and 4 °C for 5 minutes. The supernatant was carefully decanted, cell pellets were carefully re-suspended in 1 mL pre-chilled TBS + 2 % BSA and placed for overhead shaking at 4 °C for 30 minutes in order to block the cells. Cells were pelleted at 250xg and 4 °C for 5 minutes and the supernatant was carefully removed. The cell pellet was re-suspended in 100 pL pre-chilled, pre-conjugated and blocked phage suspension (see above) and placed for overhead shaking at 4 °C for 30 minutes. Cells were pelleted at 250xg and 4 °C for 5 minutes and the unbound phages were carefully removed. Cells were washed once by re-suspension in 500 pL ice cold TBS + 0.5 % BSA, immediately pelleted at 250xg and 4 °C for 5 minutes and the supernatant was carefully removed. The cell pellet was re-suspended in pre-chilled labelling buffer (250 nM CA-TMR-Biotin in TBS + 0.5 % BSA) and placed for overhead shaking at 4 °C for 15 minutes. Excess HaloTag substrate was quenched upon addition of 400 pL ice cold TBS + 0.5 % BSA + 2 pM HaloTag protein. Cells were pelleted at 1 ,000xg and 4 °C for 5 minutes and the supernatant was carefully removed. The cell pellet was re-suspended in 500 pL lysis buffer (RIPA (Thermo Scientific, 89900) + 0.5 % BSA + 0.1 % Tween-20 (Thermo Scientific, 233362500) + 1x Halt Protease Inhibitor Cocktail without EDTA (Thermo Scientific, 78429)), thoroughly mixed by shaking and placed for overhead shaking at 4 °C for 20 minutes. Subsequently, the cell debris was pelleted at 21 ,000xg and 4 °C for 15 minutes. The supernatant was transferred to a new Eppendorf tube and 100 pL blocked Streptavidin-coated magnetic beads (NEB, S1420) and 400 pL bead wash buffer (TBS + 0.5 % BSA + 0.5 % Tween-20) were added. In order to block of the magnetic beads, 25 pL bead slurry was transferred to an Eppendorf tube and separated in a magnetic separation rack (NEB, S1509S), then beads were wash once with 2 volumes (i.e., 50 pL) bead blocking buffer (TBS + 2 % BSA + 0.2 % Tween-20), and again re-suspend in 4 volumes bead blocking buffer. Finally, beads were placed for overhead shaking at 4 °C for at least 30 minutes. Biotinylated phages and blocked magnetic beads were placed for overhead shaking at 4 °C for 15 minutes and subsequently separated in a magnetic rack. The supernatant was removed and beads were re-suspended in 900 pL bead wash buffer. In total, six bead washes were performed and the Eppendorf tube was exchanged after every other wash. Subsequently, a single bead wash in TBS was performed and finally beads were re-suspended in 200 pL phage elution buffer (TBS + 2 % BSA + TEV protease (200 pg / mL final)). The bead slurry was placed for overhead shaking at room temperature for 30 minutes and beads were separated in a magnetic rack. The phage-containing supernatant was transferred to a 24 deep well plate. An aliquot (20 pL) was removed for titering (see above) and the remaining phages were used to infect 5 mL of mid-log TG1 culture (OD6oo=0.4-0.5, grown in 2xYT + 2 % glucose) for 30 minutes at 37 °C without shaking. Then, the deep well plate was centrifuged at 2,000xg and 22 °C for 20 minutes and 5 mL of supernatant was carefully removed. The remaining supernatant was used to re-suspend the bacterial pellet and infected cells were plated out onto a pre-warmed LB / Agar / Amp plate and grown at 30 °C overnight. The next morning, 1 mL 2xYT + 2 % glucose + 100 pg / mL ampicillin + 15 % glycerol was added to the plate, the colonies were scraped, flash frozen in liquid and stored at -80 °C.

[0227] Modifications for Round2 / 3:

[0228] Depletions on un-transfected HEK293 cells were performed prior to round 2 and 3. For that, 5*106cells were seeded per T75 flask two days prior to selection. On the day of selection, cells were detached and blocked as described previously. Then, the cell pellet was re-suspended in 100 pL phage suspension and placed for overhead shaking at 4 °C for 30 minutes. Cells were pelleted at 21 ,000xg and 4 °C for 5 minutes and the phage-containing supernatant was used to re-suspend the transfected cell pellet for selection.

[0229] Monoclonal Phage ELISA:

[0230] After two to three rounds of selection, individual colonies were used to inoculate a 500 pL 2xYT + 2 % glucose + 100 mg / mL Ampicillin culture in a 96 deep well plate at 37 °C and 850 rpm overnight. The next morning, 500 pL 2xYT + 0.1 % glucose + 100 mg / mL Ampicillin were inoculated with 2.5 pL of the overnight pre-culture in a new 96 deep well plate and grown at 37 °C and 850 rpm. Once the ODeoo reached 0.4-0.5, 5 pL Hyperphage was added with a multi-dispenser and cultures were incubated at 37 °C without shaking for 45 minutes, followed by incubation at 37 °C and 850 rpm for 45 minutes. Then 5 pL Kanamycin (50 pg / mL final) was added with a multi-dispenser and the cultures were grown at 25 °C and 850 rpm overnight. The next morning, the 96 deep well plate was centrifuged at 4,500xg and 4 °C for 1 hour and 400 pL of supernatant was transferred to a separate 96 deep well plate, containing 100 pL TBS + 10 % BSA per well (i.e., 2 % BSA final to block the phages for ELISA).

[0231] For the phage ELISA on cells, 2*104HeLa cells per well were seeded in DMEM + 10 % FBS on a 96 well plate one day prior to transfection. The next day, cells were transfected with the native receptor (no Halopep) or mock-transfected (non-target receptor) using Lipofectamine 3000 according to the manufacturer’s protocol. One day post-transfection, cells were supplied with fresh medium and the M13-HRP antibody (BioCat, AS003-50-ADL; 1 / 5,000 dilution) was blocked in TBS + 2 % BSA overnight.

[0232] On the day of the ELISA, cell medium was physically ejected and plates were placed upside down on a paper towel to remove excess supernatant. Subsequently, cells were blocked by addition of 100 pL TBS + 2 % BSA per well at 4 °C for 30 minutes on a rocking stage. The blocking buffer was physically ejected, 50 pL blocked phages per well were added and cells were placed on a rocking stage at 4 °C for 60 minutes. Unbound phages were completely removed with a multichannel pipette and cells were washed carefully with 100 pL TBS + 0.05 % Tween-20 thrice. Then, 50 pL per well blocked antibody was added and cells were placed on a rocking stage at 4 °C for 60 minutes. Unbound antibody was completely removed with a multichannel pipette and cells were washed carefully with 100 pL TBS + 0.05 % Tween-20 thrice. TMB substrate reagent (BD Biosciences, 555214) was prepared freshly by mixing equal volumes of substrate reagent A and substrate reagent B. Then, 100 pL of the TMB substrate was added per well and cells were placed on an orbital shaker at 22 °C and 350 rpm for 10-20 minutes. Once a deep blue color occurred, the reaction was quenched by addition of 100 pL 0.5 M H2SO4 per well. The supernatant was mixed briefly and 150 pL were transferred to a clear 96 deep well plate. The absorbance of the solution was read at 450 nm (with 620 nm as a reference wavelength). Typically, a S / N (signal to noise) of >3 (POI-transfected over mock-transfected) indicates a hit.

[0233] Antibody Production:

[0234] ELISA Hits were analyzed by Sanger Sequencing and re-formatted into chimeric human heavychain only antibodies by fusion of the nanobody-encoding sequence to the constant domains (CH2 + CH3) of human IgG 1 (AA223-449 of UniProt PODOX5) in a pcDNA3.4 backbone. Antibodies were produced in Expi293F cells (Gibco, A14527) in Expi293 Expression medium (Gibco, A1435101) in a humidified incubator at 37 °C, 8 % CO2 and 125 rpm. Cells were grown to a density of 2-3*106viable cells / mL and then split into 25 mL cultures in 125 mL PETG Erlenmeyer flasks (Thermo Scientific, 4115-0125). Subsequently, cells were transfected using the ExpiFectamine 293 Transfection Kit (Gibco, A14525) according to the manufacturer’s protocol. Approximately 24 hours post-transfection, Enhancer 1 and 2 solutions were added to the cultures. Five days posttransfection, cells were harvested by centrifugation at 4,500xg and 4 °C for 30 minutes and the supernatant was further cleared by filtration through a 0.22 pM syringe-filter (TRP, 99722). Then, ! volume (i.e., 15 mL) of Protein A binding buffer (20 mM Na3PO4 (Merck, 106578), pH 7.0) was added and the antibodies were purified via HiTrap Protein A High performance column (Cytiva, GE17-0403-01 ) on an AktaPure FPLC system (Cytiva) and eluted with a low pH gradient (100 mM glycine-HCI (Gerbu, 1023), pH 2.7) into1 / volumes neutralization buffer (1 M Tris-CI, pH 9.0). Subsequently, the buffer was exchanged to TBS pH 7.5 using a HiPrep 26 / 10 desalting column (Cytiva, GE17-5087-01). Antibody Characterization - Live cell ELISA:

[0235] Antibodies were initially characterized by ELISA on live cells. To that end, 2*104HeLa cells or 4*104HEK293 cells per well were seeded in DMEM + 10 % FBS on a 96 well plate one day prior to transfection. The next day, cells were transfected with the native receptor (no Halopep) or mock- transfected (non-target receptor) using Lipofectamine 3000 according to the manufacturer’s protocol. One day post-transfection, cells were supplied with fresh medium and ProteinG-HRP (Invitrogen, 101223; 1 / 5,000 dilution) was blocked in TBS + 2 % BSA overnight.

[0236] On the day of the ELISA, cell medium was physically ejected and plates were placed upside down on a paper towel to remove excess supernatant. Subsequently, cells were blocked by addition of 100 pL TBS + 2 % BSA per well at 4 °C for 30 minutes on a rocking stage. The blocking buffer was physically ejected, 50 pL blocked antibody dilutions in TBS + 2 % BSA per well were added and cells were placed on a rocking stage at 4 °C for 60 minutes. Unbound antibody was completely removed with a multichannel pipette and cells were washed carefully with 100 pL TBS + 0.05 % Tween-20 thrice. Then 50 pL per well blocked ProteinG-HRP was added and cells were placed on a rocking stage at 4 °C for 60 minutes. Unbound ProteinG-HRP was completely removed with a multichannel pipette and cells were washed carefully with 100 pL TBS + 0.05 % Tween-20 thrice. TMB substrate reagent was prepared freshly by mixing equal volumes of substrate reagent A and substrate reagent B. Then, 100 pL of the TMB substrate reagent mix per well was added and cells were placed on an orbital shaker at 22 °C and 350 rpm for 10-20 minutes. Once a deep blue color occurred, the reaction was quenched upon addition of 100 pL 0.5 M H2SO4 per well. The supernatant was mixed briefly and 150 pL were transferred to a clear 96 well plate. The absorbance of the solution was read at 450 nm (with 620 nm as a reference wavelength) and dose-response curves were plotted in GraphPad Prism 9.

[0237] Antibody Characterization - Flow Cytometry:

[0238] Antibodies were further characterized by flow cytometry. To that end, 1*107HEK293 cells were seeded in DMEM + 10 % FBS in a T75 flask one day prior to transfection. The next day, cells were transfected with the native receptor (no Halopep) or mock-transfected (non-target receptor) using Lipofectamine 3000 according to the manufacturer’s protocol. One day post-transfection, cells were supplied with fresh medium.

[0239] On the day of the experiment, cells were detached with 2 mL Versene at room temperature for 3-5 minutes and subsequently transferred to a 15 mL Falcon tube by washing the cells off the flask with 9 mL PBS. Cells were pelleted in a pre-cooled centrifuge at 250xg and 4 °C for 5 minutes. The supernatant was carefully decanted, cell pellets were carefully re-suspended in 3 mL pre-chilled TBS + 2 % FBS and placed for overhead shaking at 4 °C for 30 minutes. Cells were distributed into a V-shaped 96 well plate (12.5 pL per well) and antibody dilutions (in TBS + 2 % FBS) or lgG1 control (Invitrogen, 31154) were added to the cells (2x stocks, 12.5 pL per well). Cells were incubated at 4 °C on a rocking stage for 60 minutes and subsequently pelleted by centrifugation at 250xg and 4 °C for 10 minutes. The supernatant was physically ejected and cells were washed once by resuspension in TBS + 2 % FBS. Cells were pelleted again, supernatant was removed and cells were re-suspended in secondary antibody (anti-human IgG Fc Cross-Adsorbed Secondary Antibody DyLight™ 650, SA5-10137; 25 pL per well of a 1 / 100 dilution in TBS + 2 % FBS). Cells were incubated at 4 °C on a rocking stage for 60 minutes and subsequently pelleted by centrifugation at 250xg and 4 °C for 10 minutes. The supernatant was physically ejected and cells were washed once by resuspension in TBS + 2 % FBS. Cells were pelleted again, supernatant was removed and cells were re-suspended in 100 pL TBS + 2 % FBS and transferred to a U-shaped 96-well plate.

[0240] Cells were analyzed with a BD LSRFortessa X-20 Flow Cytometer (DyLight650: 640 nm excitation, 670 / 30 nm emission). Data were analyzed using FlowJo (BD). Live cells (SSC-A over FSC-A) and single cells (FSC-H over FSC-A) were gated and the mean fluorescence intensities for the APC channel were calculated and plotted against the concentration of the heavy-chain only antibodies.

[0241] Results

[0242] Monoclonal Phage ELISA:

[0243] Fig. 6 A illustrates the results of a monoclonal Phage ELISA of nanobody against PD-L1 (CD274). Individual clones from round 2 output were tested for their ability to bind to HeLa cells transfected with PD-L1 or mock- transfected. A total number of 21 positive clones (out of 21) selectively bind PD-L1 expressed on HeLa cells with a S / N > 3 (dashed line).

[0244] Fig. 7 illustrates the results of a monoclonal Phage ELISA of nanobody against CNR1 . Individual clones from round 2 and round 3 output were tested for their ability to bind to HeLa cells transfected with CNR1 or a mock-receptor (PD-L1). A total number of 79 positive clones (out of 82 tested clones) selectively bind CNR1 expressed on HeLa cells with aS / N > 3 (dashed line).

[0245] Live Cell ELISA of recombinant heavy-chain only antibodies:

[0246] Fig. 8 A shows ELISA binding characterization of two selected hits from the monoclonal phage ELISA that had been re-formatted into full length heavy-chain only antibodies, on transiently transfected HeLa cells expressing the Cannabinoid Receptor 1 (CNR1) on their surface. The two antibodies show apparent affinities of 0.8 nM and 2.2 nM, respectively (Clone 1 &2).

[0247] Fig. 8 B shows ELISA binding characterization of four selected hits from the monoclonal phage ELISA that had been re-formatted into full length heavy-chain only antibodies, on transiently transfected HEK293 cells expressing a23-1 (CACN2D1) - a regulatory subunit of voltage-gated calcium channels - on their surface. The antibodies show apparent affinities of 3.1 nM, 6.1 nM, 2.5 nM and 1 .6 nM, respectively (Clone 1-4). The IgG 1 isotype control shows no binding to transfected cells.

[0248] Fig. 8 C shows ELISA binding characterization of two selected hits from the monoclonal phage ELISA that had been re-formatted into full length heavy-chain only antibodies, on transiently transfected HEK293 cells expressing the Mu-type Opioid Receptor 1 (OPRM1) on their surface. The antibodies show apparent affinities of 0.44 nM and 3.9 nM, respectively (Clone 1&2). No binding was detected to mock-transfected HEK293 cells (transfected with CNR1 as a related Class A GPCR).

[0249] Fig. 8 D shows ELISA binding characterization of three selected hits from the monoclonal phage ELISA were re-formatted into full length heavy-chain only antibodies and their binding was characterized by ELISA on transiently transfected HEK293 cells expressing the Delta-type Opioid Receptor 1 (OPRD1) on their surface. The antibodies show apparent affinities of 1 .4 nM, 0.38 nM and 1.9 nM, respectively (Clone 1-3). No binding was detected to mock-transfected HEK293 cells (transfected with CNR1 as a related Class A GPCR).

[0250] Flow Cytometry:

[0251] Fig. 9 A shows the results of flow cytometry binding characterization of three selected hits from the monoclonal phage ELISA that had been re-formatted into full length heavy-chain only antibodies. The assay was performed on transiently transfected HEK293 cells expressing PD-L1 on their surface. The antibodies show apparent affinities of 34 nM, 87 nM and 16 nM, respectively (Clone 1-3). No binding was detected to mock-transfected HEK293 cells (transfected with CNR1).

[0252] Fig. 9 B shows the results of flow cytometry binding characterization of the two clones previously tested in ELISA in heavy-chain only antibody format, on transiently transfected HEK293 cells expressing CNR1 on their surface. The antibodies show apparent affinities of 87 nM and 77 nM, respectively (Clone 1&2). No binding to mock-transfected HEK293 cells (transfected with CD274) nor binding of the lgG1 isotype control to HEK293 cells (transiently transfected with CNR1) was detected.

[0253] Further methods

[0254] Modifications for conformation-specific nanobodies:

[0255] Selections were performed on HEK293 cells that were transiently transfected with both mGluR2 as well as chimeric G-protein Gqi9 and in the presence of agonist LY 379268 (5 pM) and PAM BINA (5 pM). Depletions in the 2ndand 3rdround of selection were performed on HEK293 cells that were transiently transfected with mGluR2 and in the presence of antagonist LY 341495 (5 pM) and NAM VU 6001966 (5 pM).

[0256] Modifications for an alternative tagging strategy using existing ligands:

[0257] Selections were performed on HEK293 cells stably expressing wild-type GLP-1 R and in the presence of fusion peptide Exendin-4-Halopep(20 pM in round 1 ; 100 nM in round 2).

[0258] Modifications for an alternative tagging strategy using unnatural amino acids and click chemistry:

[0259] Selections were performed on HEK293 cells that were transiently transfected with an amber codon mutant of CD20 as well as the expression machinery for unnatural amino acid incorporation (tRNApv' + PylRS) and incubated with frans-cyclooct-2-en-L-lysine (250 pM final) for 24 hours. Cells were detached as described previously and subsequently incubated with methyltetrazine-Halopep(100 pM final) for 30 minutes at 4 °C to perform the biorthogonal labeling. Subsequently, excess reagent was wash away and selections were performed as described previously.

[0260] ADCC bioluminescence reporter assay:

[0261] The ADCC-mediated cell killing activity was determined on transiently transfected HEK293 cells (“target cells”) using the ADCC Reporter Bioassay Kit from Promega (G7015) according to the manufacturer’s protocol.

[0262] Yeast Surface Display proof of concept:

[0263] Yeast cells (EBY100) that were maintained in YPD medium (2 % peptone, 1 % yeast extract, 2 % glucose) were transfected with AlfaTag-cpHaloD2-Aga2p-HA-Myc-eUnaG2 and selected in SD- CAA medium (0.67 % yeast nitrogen base without amino acids, 0.5 % yeast synthetic drop-out medium supplement, 38 mM Na2HPO4, 62 mM NaH2PO4, 2 % glucose) at 30 °C and 250 rpm. After 48 hours, cells were diluted 1 / 10 in SG-CAA (0.67 % yeast nitrogen base without amino acids, 0.5 % yeast synthetic drop-out medium supplement, 38 mM Na2HPO4, 62 mM NaH2PO4, 2 % galactose) and induced for 24 hours at 30 °C and 250 rpm. In parallel, HEK293 cells were transiently transfected with either Halopep-gsgx10-NBAIfa-PDGFRp_TM-mEGFP or NBAIfa- PDGFRp_TM-mEGFP.

[0264] The next day, yeast cells were counted and aliquoted to 2.5*107yeast cells per condition. Yeast cells were pelleted at 14,000xg at 22 °C for 1 min, re-suspended in TBS + 2 % BSA and blocked for 2 hours at 22 °C with overhead shaking. In parallel, HEK293 cells were detached and blocked as described previously. Subsequently, yeast cells were mixed with blocked mammalian cells at a ratio of 5:1 (200 pL final) and incubated at 4 °C for 20 mins with overhead shaking. Cells were pelleted at 250xg and 4 °C for 5 mins and re-suspended in TBS + 0.2 % BSA. Cells were pelleted again immediately, re-suspended in ice-cold labeling buffer (200 nM CA-SiR in TBS + 0.2 % BSA) and incubated at 4 °C for 15 mins with overhead shaking. Excess reagent was quenched upon addition of ice-cold HaloTag7 protein (10 pM) in TBS + 0.2 % BSA. Cells were pelleted and resuspended in PBS + 0.1 % BSA. Cells were pelleted again, re-suspended in PBS + 0.1 % and analyzed by flow cytometry by gating for mEGFP positive cells / complexes.

[0265] Cited prior art documents:

[0266] WO2020212537A1 (US2022275350A1)

[0267] EP23158085.3

[0268] Zakeri et al. (2012). Proc. Nat. Acad. Sci, USA 109 (12): E690-7);

[0269] Keeble et al. (Angewandte Chemie Int. Ed. (2017) 56(62) 16521-16525)

[0270] Keeble et al. (Proc. Nat. Sci USA (2019) 116 (52) 26523-26533)

[0271] Li et al. J. Mol. Biol. (2014) 426(2) 309-317) Fierer et al., (Proc. Nat. Sci USA (2014) 111 (13) E1176-E1181)

[0272] Veggiani et al., (Proc. Nat. Sci USA (2016) 113 (5) 1202-1207)

[0273] Hitomi et al., Amino Acids volume 36, pages 619-624 (2009))

[0274] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

Claims

Claims1. A method to query a molecular interaction between a first partner moiety and a second partner moiety, wherein the first partner moiety is associated to a first partial effector sequence, and the second partner moiety is associated to a second partial effector sequence, the first and second partial effector sequences constituting a functional split HaloTag system, wherein the first partner: is a peptide sequence or a small molecule encoded by a polynucleotide sequence; is physically associated with the polynucleotide sequence; the method comprising the steps of: a) contacting the first partner moiety and the second partner moiety in the presence of a HaloTag substrate covalently linked to a separation function; b) separating the first partner moiety being attached to the separation function; c) determining a molecular interaction between the first partner moiety and the second partner moiety by detecting the presence of the polynucleotide sequence; wherein the association between the first partner and the polynucleotide sequence is stable under the conditions of step a) and b), and wherein the first partial effector sequence consists of o an N-terminal first effector sequence part characterized by SEQ ID NO 002 (the original N-terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 002, o a C-terminal first effector sequence part characterized by SEQ ID NO 003 (the original C-terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 003, and o an internal cpHalo linker consisting of 10 to 35 amino acids, wherein the internal cpHalo linker connects the C-terminus of the N-terminal first effector sequence part to the N-terminus of the C-terminal first effector sequence part.

2. A method to generate a binding moiety capable of binding to a target moiety, comprising the steps of:A) providing a plurality of candidate entities, wherein each candidate entity comprises a candidate binding moiety that is associated to a first partial effector sequence, comprises a polynucleotide sequence encoding the candidate entity;physically associates the candidate binding moiety and the polynucleotide sequence encoding the candidate binding moiety; wherein each member of the plurality of candidate entities is characterized by a different polynucleotide sequence encoding the candidate entity;B) contacting the plurality of candidate entities with the target, the target (second partner) moiety being associated with a second partial effector sequence, the first and second partial effector sequences constituting a functional split HaloTag system, in the presence of a HaloTag substrate covalently linked to a separation function;C) separating the candidate entities bound to the separation function; andD) isolating a plurality of polynucleotide sequences;E) optionally, amplifying the polynucleotide sequences;F) obtaining sequence information about the plurality of polynucleotide sequences obtained; wherein the association between the candidate binding moiety and the polynucleotide sequence is stable under the conditions of step B) and C), and wherein the first partial effector sequence consists of o an N-terminal first effector sequence part characterized by SEQ ID NO 002 (the original N-terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 002, o a C-terminal first effector sequence part characterized by SEQ ID NO 003 (the original C-terminal cpHaloA protein) or by a sequence at least (>) 90% identical (particularly >93%, 95%, 97% or >98% identical) to SEQ ID NO 003, and o an internal cpHalo linker consisting of 10 to 35 amino acids, wherein the internal cpHalo linker connects the C-terminus of the N-terminal first effector sequence part to the N-terminus of the C-terminal first effector sequence part.

3. The method according to claim 1 or 2, wherein the candidate binding moiety or the first partner moiety is a peptide sequence.

4. The method according to claim 1 or 2, wherein the candidate binding moiety or the first partner moiety is a nucleic acid sequence.

5. The method according to any one of the preceding claims, wherein subsequent to the sequence isolation step, the isolated polynucleotide sequences are amplified under conditions favouring the introduction of mutations into amplification products obtained by amplifying the isolated polynucleotide sequences, a plurality of candidate entities are reconstituted from the amplified sequences and the contacting, separating and sequence isolation steps are repeated.

6. The method according to any one of the preceding claims, wherein the first partner or candidate binding moiety is linked together with a peptide tag sequence capable of forming a covalent bond to an acceptor polypeptide as part of the same polypeptide sequence, andthe first partial effector sequence is encoded as part of one polypeptide sequence together with the acceptor polypeptide.

7. The method according to claim 6, wherein the peptide tag sequence is AHIVMVDAYKPTK (SEQ ID NO 379), and the acceptor polypeptide is SEQ ID NO 378.

8. The method according to any one of the preceding claims, wherein the first partner or candidate binding moiety is associated to the polynucleotide sequence, as part of a phage.

9. The method according to claim 4, wherein the first partner is encoded as the same polypeptide sequence as a phage coat protein.

10. The method according to any one of the preceding claims, wherein the first partner or candidate binding moiety is cleaved from the polynucleotide sequence subsequent to the separation step, particularly wherein a protease recognition site is present between the first partner or candidate binding moiety and a point of physical association to the polynucleotide sequence, more particularly wherein the protease recognition site is a TEV protease site.

11. The method according to any one of the preceding claims, wherein the separation function is biotin.

12. The method according to any one of the preceding claims, wherein the HaloTag substrate comprises a fluorescent dye, particularly a rhodamine-type dye.

13. The method according to any one of the preceding claims, wherein the second partner moiety is covalently associated to the second partial effector sequence as part of the same polypeptide sequence.

14. The method according to any one of the preceding claims, wherein the second partial effector sequence consists of a sequence selected from the group consisting of SEQ ID NO 006-343.

15. The method according to any one of the preceding claims 2 to 14, wherein the method generates an antibody capable of stabilizing an active conformation of a receptor, and wherein the target moiety is an active conformation of a receptor and the candidate binding moiety is an antibody.

16. The method according to claim 15, wherein the receptor is stabilized in its active conformation during step B) of the method.

17. The method according to claims 15 to 16, wherein additionally steps B) to F) are repeated with the target moiety being an inactive conformation of the receptor, and wherein the resulting candidate entities of the repetition are deselected from further analysis.

18. The method according to any one of the preceding claims 2 to 14, wherein the method generates an antibody capable of stabilizing an inactive conformation of a receptor, and wherein the target moiety is an inactive conformation of a receptor and the candidate binding moiety is an antibody.

19. The method according to claim 18, wherein the receptor is stabilized in its inactive conformation during step B) of the method.

20. The method according to claims 18 to 19, wherein additionally steps B) to F) are repeated with the target moiety being an active conformation of the receptor, and wherein the resulting candidate entities of the repetition are deselected from further analysis.

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