NZ753806B2 - An engineered two-part cellular device for discovery and characterisation of t-cell receptor interaction with cognate antigen - Google Patents

Abstract

The present invention relates to a two-part system, wherein a first part is an engineered antigen-presenting cell system (eAPCS), and a second part is an engineered TCR-presenting cell system (eTPCS) forming a combined eAPC:eTPC analytical system. The eAPCS is an engineered antigen-presenting cell (eAPC) lacking endogenous surface expression of at least one family of target analyte antigen presenting complexes (aAPX) and/or target analyte antigenic molecules (aAM), and contains a genomic receiver site for integration of one or two ORFs encoding an aAPX and/or an aAM, and a genetic donor vector which is matched to the genomic receiver site and designed to deliver one or two target ORFs encoding a target aAPX and/or target aAM. The eTPCS is an engineered TCR-presenting cell (eTPC) which lacks endogenous surface expression of at least one family of target analyte antigen-presenting complexes (aAPX) and/or target analyte antigenic molecule (aAM), and lacks endogenous expression of TCR chains alpha, beta, delta and gamma and expresses CD3 proteins that are conditionally presented on the surface of the eTPC only when the eTPC expresses a complementary chain of TCR chains; and contains a genomic receiver site for integration of a single ORF encoding at least one analyte TCR chain of alpha, beta, delta or gamma and/or two ORFs encoding a pair of analyte TCR chains, and a genetic donor vector matched to the genomic receiver site and is designed to deliver a single ORF encoding at least one analyte TCR chain of alpha, beta, delta or gamma and/or two ORFs encoding a pair of analyte TCR chains.

Description

The present invention relates to a two-part system, wherein a first part is an engineered antigenpresenting cell system (eAPCS), and a second part is an engineered TCR-presenting cell system (eTPCS) forming a combined eAPC:eTPC ical system. The eAPCS is an ered antigenpresenting cell (eAPC) lacking endogenous surface expression of at least one family of target analyte antigen presenting complexes (aAPX) and/or target e antigenic molecules (aAM), and contains a genomic receiver site for integration of one or two ORFs encoding an aAPX and/or an aAM, and a genetic donor vector which is matched to the genomic receiver site and designed to deliver one or two target ORFs encoding a target aAPX and/or target aAM. The eTPCS is an engineered TCR-presenting cell (eTPC) which lacks endogenous surface expression of at least one family of target analyte antigen-presenting complexes (aAPX) and/or target analyte antigenic molecule (aAM), and lacks endogenous expression of TCR chains alpha, beta, delta and gamma and expresses CD3 proteins that are conditionally presented on the surface of the eTPC only when the eTPC expresses a complementary chain of TCR chains; and ns a genomic er site for integration of a single ORF ng at least one analyte TCR chain of alpha, beta, delta or gamma and/or two ORFs encoding a pair of analyte TCR chains, and a genetic donor vector d to the genomic er site and is designed to deliver a single ORF encoding at least one analyte TCR chain of alpha, beta, delta or gamma and/or two ORFs encoding a pair of analyte TCR chains. 753806 B2 An engineered two-part ar device for discovery and terisation of T- cell receptor interaction with cognate antigen Field of the invention The present invention s to the construction and use of an engineered cellular de- vice comprising two parts, wherein each part comprises a distinct ered cell sys— tem that are interacted with one another in operation of the . The first part of the two-part cellular device is an antigen—presenting cell (APC) system that is engineered by genome editing to render the APC null for cell surface presentation of human leuko- cyte n (HLA) molecules, HLA-like molecules and ct forms of n-pre- senting molecules and nic molecules. In addition, the ered APC system (eAPCS) contains genomic integration sites for insertion of antigen—presenting mole- cule encoding open reading frames (ORFs), and optionally insertion of genetically en- coded analyte antigens. Genetic donor vectors designed to target the genomic integra- tion sites of the APC as to rapidly deliver analyte antigen molecule- and/or antigen-pre- senting complex encoding ORFs completes the eAPCS. The second part of the two- part cellular device is a T-cell Receptor (TCR)-presenting cell system that is engi- neered by genome editing to render the cell null for surface expression of TCR chains.

This engineered TOR—presenting cell system (eTPCS) s competent to express TCR heterodimeric complexes on the cell e in a CD3 complex context upon intro- duction of TCR-encoding ORFs, and is also competent to respond to TCR ligation in a detectable manner. Genetic donor vectors designed to target the genomic integration sites of the TOR-presenting cell as to rapidly deliver analyte TCR ORFs completes the eTPCS. This engineered two-part cellular device is designed for standardised analysis of TOR/antigen interactions in the native functional context of cell-cell communication.

Introduction to the invention Immune surveillance by T lymphocytes (T—cells) is a central function in the adaptive im— munity of all jawed vertebrates. Immune surveillance by T—cells is ed through a rich functional diversity across T-cell subtypes, which serve to eliminate pathogen-in- fected and neoplastic cells and orchestrate adaptive immune responses to ng pathogens, commensal rganisms, commensal non-self factors such as molecu- lar components of foodstuffs, and even maintain immune tolerance of self. In order to respond to various foreign and self factors, T-cells must be able to specifically detect molecular constituents of these foreign and self factors. Thus T-ceIIs must be able to detect a large cross—section of the self and non—self les that an individual en- counters, with sufficient specificity to mount ent responses against pathogenic or- ganisms and diseased self, while avoiding the mounting of such responses against health self. The highly complex nature of this task becomes clear when considering the practically unlimited diversity of both foreign and self molecules, and that pathogenic organisms are under evolutionary pressure to evade detection by T-cells.

The T-ce/I Receptor (TOR) T—cells are primarily defined by the expression of a T-cell receptor (TCR). The TCR is the component of the T-cell that is responsible for interacting with and sensing the tar- gets of T-cell ve immunity. In general terms, the TCR is comprised of a heterodi- meric protein complex presented on the cell surface. Each of the two TCR chains are composed of two extracellular domains, being the variable (V)-region and the constant (C)—region, both of the immunoglobulin amily (lgSF) , forming antiparallel ts. These are anchored in the cell membrane by a type-l transmembrane do- main, which adjoins a short cytoplasmic tail. The quality of the T-cells to adapt and de- tect e molecular constituents arises from variation in the TCR chains that is gen- erated during T-cell genesis. This variation is generated by somatic recombination in a similar manner to antibody genesis in B-cells.

TCR chain diversity The T cell pool consists of several functionally and phenotypically heterogeneous sub- populations. However, T cells may be broadly classified as (18 or v6 according to the somatically rearranged TCR form they s at their e. There exist two TCR chain pair forms; TCR alpha (TRA) and TCR beta (TRB) pairs; and TRC gamma (TRG) and TCR delta (TRD) pairs. T-cells expressing TRAzTRB pairs are ed to as 08 T- cells, while s expressing TRG:TRD pairs are often referred to as v6 T—cells.

TCRs of both 08 and v6 forms are responsible for recognition of diverse ligands, or ‘an- tigens’, and each T-cell generates dB or v6 receptor chains de novo during T-cell matu- ration. These de novo TCR chain pairs achieve diversity of recognition through - tion of receptor ce diversity in a process called somatic V(D)J recombination af- ter which each T-cell expresses copies of a single distinctly rearranged TCR. At the TRA and TRG loci, a number of discrete variable (V) and functional (J) gene segments are available for recombination and juxtaposed to a constant (C) gene segments, thus ed to as VJ recombination. Recombination at the TRB and TRD loci additionally includes a diversity (D) gene segment, and is referred to as VDJ recombination.

Each recombined TCR possess ial for unique ligand specificity, determined by the structure of the ligand-binding site formed by the q and B chains in the case of GB T-cells or v and 6 chains in the case of v6 T-cells. The structural diversity of TCRs is largely confined to three short hairpin loops on each chain, called complementarity—de— termining regions (CDR). Three CDRs are buted from each chain of the receptor chain pair, and collectively these six CDR loops sit at the membrane—distal end of the TCR ellular domain to form the antigen-binding site.

Sequence diversity in each TCR chain is achieved in two modes. First, the random se- lection of gene segments for recombination provides basal sequence ity. For ex- ample, TRB recombination occurs between 47 unique V, 2 unique D and 13 unique J germline gene segments. In l, the V gene segment contributes both the CDR1 and CDR2 loops, and are thus germline encoded. The second mode to generate se- quence diversity occurs within the ariable CDR3 loops, which are generated by random deletion of template tides and addition of non-template nucleotides, at the junctions between recombining V, (D) and J gene segments.

TCR:CDS Complex Mature dB and v6 TCR chain pairs are presented at the cell e in a complex with a number of accessory CD3 subunits, denoted s, v, 6 and C. These subunits associate with 08 or yo TCRs as three dimers (av, 56, (C). This TCR:CD3 complex forms the unit for initiation of cellular signalling responses upon engagement of a GB or v6 TCR with cognate antigen. The CD3 accessories associated as a TCR:CD3 complex contribute signalling motifs called immunoreceptor tyrosine-based activation motifs ).

CD38, CD3v and CD36 each contribute a single ITAM while the CD3( homodimer con- tains 3 ITAMs. The three CD3 dimers (av, 86, (C) that assemble with the TCR thus con- tribute 10 ITAMs. Upon TCR ligation with cognate antigen, phosphorylation of the tan- dem tyrosine es creates paired docking sites for proteins that contain Src homol- ogy 2 (8H2) s, such as the critical C-chain-associated protein of 70 kDa (ZAP- 70). Recruitment of such proteins initiate the formation of TCR:CD3 signalling com- plexes that are ultimately responsible for T-cell activation and differentiation. (7,8 T-cel/s dB T-cells are lly more abundant in humans than their yo T-cell counterparts. A majority of GB T-cells interact with peptide antigens that are presented by HLA com- plexes on the cell surface. These peptide-H LA (pH LA)—recognising T—cells were the first to be described and are by far the best characterised. More rare forms of GB T-cells have also been described. Mucosal-associated invariant T (MAIT) cells appear to have a relatively limited or and [3 chain ity, and recognise bacterial metabolites rather than protein fragments. The invariant l killer s (iNK T-cells) and germline— d l-reactive T-cells (GEM T-cells) are restricted to recognition of glycoli- pids that are cross-presented by non—H LA molecules. iNK T—cells are largely consid— ered to interact with CD1d-presented glycolipids, whereas GEM s interact with CD1b-presented glycolipids. Further forms of T-cells are thought to interact with glycoli- pids in the context of CD1a and CD1c, however, such cells are yet to be terised in significant detail.

Conventional (7,8 T-cel’ls The key feature of most 08 T—cells is the recognition of peptide antigens in the context of HLA molecules. These are often referred to as ‘conventional’ 08 T-cells. Within an individual, self—HLA les present peptides from self and foreign proteins to T- cells, providing the ial basis for adaptive immunity against malignancies and for- eign pathogens, adaptive tolerance towards sal organisms, foodstuffs and self.

The HLA locus that s HLA proteins is the most gene-dense and polymorphic re- gion of the human genome, and there are in excess of 12,000 alleles bed in hu- mans. The high degree of polymorphism in the HLA locus ensures a diversity of pep- tide antigen presentation between individuals, which is important for immunity at the population level.

HLA class I and II There are two forms of classical HLA complexes: HLA class I (HLAI) and HLA class II (HLAII). There are three classical HLAI genes: HLA-A, HLA-B, HLA-C. These genes encode a membrane-spanning d-chain, which associates with an invariant BZ-micro— globulin (82M) chain. The HLAI d-chain is composed of three domains with an immuno- globulin fold: 0:1, 02 and (13. The 03 domain is membrane-proximal and largely invari— ant, while the 01 and 02 domains together form the rphic membrane-distal anti- gen-binding cleft. There are six classical HLAII genes: HLA-DPA 1, HLA-DPB1, HLA- DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. These genes encode paired DP, DO and DR heterodimeric HLA complexes comprising a n and a B-chain. Each chain has two major structural domains with an immunoglobulin fold, where the (12 and [52 domain comprise membrane-proximal and largely invariant modules similar to that of HLAI 03 domain. The HLAll 02 and [32 domains er form the membrane-distal an- tigen-binding cleft and are regions of high polymorphism.

The antigen-binding cleft of HLAI and HLA" comprises two anti-parallel d—helices on a platform of eight anti-parallel B-sheets. In this cleft the peptide antigen is bound and presented in an extended conformation. The peptide-contacting es in HLAI and HLA" are the location of most of the sequence polymorphism, which constitutes the molecular basis of the diverse peptide repertoires presented by different HLA alleles.

The peptide makes extensive contacts with the antigen-binding cleft and as a result each HLA allele imposes distinct sequence aints and preferences on the pre- sented peptides. A given peptide will thus only bind a limited number of HLAs, and re- ciprocally each allele only accommodates a particular fraction of the peptide collection from a given protein. The set of HLAI and HLA" alleles that is present in each individ- ual is called the HLA haplotype. The polymorphism of HLAI and HLA" genes and the co-dominant expression of inherited s drives very large diversity of HLA haplotype across the human population, which when coupled to the enormous sequence diversity of GB TCRs, ts high obstacles to standardisation of analysis of these HLA-anti- gen-TCR interactions. (1,8 TCR engagement of HLAI and HLA/I dB TCRs recognize peptides as part of a mixed pHLA binding interface formed by resi- dues of both the HLA and the peptide n (altered self). HLAI complexes are pre- sented on the surface of nearly all nucleated cells and are lly considered to pre- sent peptides derived from endogenous proteins. T-cells can thus interrogate the en- dogenous ar proteome of an HLAI-presenting cell by sampling pHLAl xes of an interacting cell. Engagement of HLAI requires the sion of the TCR co—re- ceptor CD8 by the interacting , thus HLAI sampling is restricted to CD8+ 08 T- cells. In contrast, the surface presentation of HLA" complexes is largely restricted to professional APC, and are generally considered to present es derived from pro- teins exogenous to the presenting cell. An interacting T—cell can therefore interrogate the proteome of the extracellular microenvironment in which the presenting cell resides.

The engagement of HLA" requires the expression of the TCR co-receptor CD4 by the interacting T-cell, thus HLAll sampling is restricted to CD4+ dB T-cells.

Thymic selection of GB TCRs The role of GB TCRs as described above is the detection of pHLA complexes, such that the TCR-presenting T—cell can raise ses germane to the role of that T—cell in es- tablishing immunity. It should be considered that the GB TCR repertoire generated within an dual must account for the immense and unforeseen diversity of all for— eign antigens likely to be encountered in the context of a specific haplotype and prior to their actual occurrence. This outcome is achieved on a background where extremely e and numerous dB TCRs are generated in a quasi-randomised manner with the potential to recognise unspecified pHLA complexes while only being specifically in- structed to avoid strong interactions with self pHLA. This is carefully orchestrated dur- ing T-cell maturation in a process call thymic selection.

During the first step of T-cell tion in the thymus, s bearing (18 TCRs that are incapable of cting with self-pH LA complexes with sufficient affinity, are de— prived of a survival signal and eliminated. This step called positive selection assures that the ing T-cells carry a TCR repertoire that is at least potentially capable of recognizing foreign or altered peptides presented in the right HLA context. Subse— quently, dB TCR that strongly interact with self-pHLA and thus have the potential to drive autoimmunity are actively removed through a s of ve selection. This combination of positive and negative selection results in only T-cells bearing 08 TCRs with low affinity for self-pHLA populating the periphery. This establishes an 06 T-cell oire that is self-restricted but not self-reactive. This highly dualised nature of T-cell genesis against HLA haplotype underscores the challenges in standardised anal- ysis dB TCR-antigen-HLA interactions. Moreover, it forms the basis of both graft rejec- tion and graft versus host disease and the general principle that 08 TCRs identified in one individual may have completely different effect in a second individual, which has clear implications for TCR-based and T-cell based therapeutic and diagnostic strate- gies emerging in clinical practice.

Unconventional (7,8 T-cel/s The non-HLA—restricted, or ‘unconventional’, forms of GB s have very different mo- lecular n targets. These unconventional dB T-cells do not engage classical HLA xes, but rather engage ved HLA-like proteins such as the CD1 family or MR1. The CD1 family comprises four forms involved in antigen cross—presentation (CD1a,b,c and d). These cell surface complexes have an o-chain resembling HLAI, which forms heterodimers with BZ-M. A small hydrophobic pocket presented at the membrane distal surface of the o-chain forms a binding site for pathogen-derived lipid- based antigens. Innate like NK s (iNK T-cells) form the best-understood example of lipid/CD1 family recognition with GEM T-cells enting another prominent exam- ple. ‘Type I’ iNK T-cells are known to interact strongly with the lipid d-GalCer in the con- text of CD1d. These iNK s display very limited TCR diversity with a fixed TCR or— chain (Vd10/Jd18) and a limited number of B-chains (with restricted VB usage) and they have been likened to innate pathogen—associated molecular patterns (PAMPS) recogni- tion receptors such as Toll-like and ke receptors. In contrast, ‘type II’ NK T-cells present a more diverse TCR oire, and appear to have a more diverse mode of CD1d-lipid complex engagement. GEM T-cells recognise mycobacteria-derived glycoli- pids presented by CD1b, however, the molecular s of antigen presentation by CD1a, b and c as well as their T-cell recognition are only beginning to be understood.

MAIT cells largely s an invariant TCR d-chain (TRAV1-2 ligated to TRAJ33, TRAJZO, or TRAJ12), which is capable of pairing with an array of TCR B-chains. In- stead of peptides or lipids MAIT TCRs can bind pathogen-derived folate- and riboflavin- based metabolites presented by the HLAl-like le, MR1. The limited but signifi- cant diversity in the TCRs observed on MAIT TCRs appear to enable the recognition of diverse but related lites in the context of the conserved MR1.

It is not well-understood how non-classical HLA-restricted dB T-cell TCRs are selected in the thymus during maturation. However, it appears likely that the fundamental pro- cess of negative and positive selection outlined above still applies and some evidence suggests that this occurs in specialized niches within the thymus. yo T-cel/s In contrast to the detailed mechanistic understanding of GB TCR genesis and pHLA en- gagement, relatively little is known about the antigen targets and context of their v6 T- cell counterparts. This is in part due to their vely low abundance in the ating T-cell compartment. However, it is broadly considered that v6 T-cells are not strictly HLA restricted and appear to recognize surface antigen more freely not unlike antibod- ies. Additionally, more recently it has become appreciated that v6 T—cells can te the resident T-cell compartment of epithelial tissues, the main interaction site of the im- mune system with foreign n. In addition, various isms for v6 T—cell tumour immunuosurveillance and llance of other forms of dysregulated-self are begin- ning to emerge in the literature. The specific antigen targets of both innate-like and adaptive v6 T-cells remain poorly defined but the tissue distribution and fast recognition of PAMPs suggests a fundamental role for v6 T-cells both early in responses to foreign ns as well as early in life when the adaptive immune system is still maturing.

The diverse functions of v6 T—cells appear to be based on different Vv V6 gene seg— ment usage and can be broadly understood in two main categories in which v6 T-cells with largely invariant TCRs mediate innate-like recognition of PAMPs very early during infection. Beyond PAMPs these type of v6 T-cells are furthermore believed to recog- nize self—molecules, including oantigens that could provide very early signatures of cellular stress, infection and potentially neoplastic development. Recognition of PAMPs and such led danger associated molecular patterns ) as well as the large numbers of tissue-restricted innate-like v6 T-cells strongly suggests that these cells are suited to respond rapidly to antigenic challenge without the need for prior acti- vation, homing and clonal expansion.

A second form of v6 T-cells are considered to be more ve in nature, with a highly diverse v6 TCR oire and the ability to peripherally circulate and access lymphoid s directly. Such antigen—specific v6 T-cells to common human pathogens such as CMV have been described and they appear to form a memory response. r, it has also been observed that yo T-cells show only relatively limited clonal proliferation after activation and little data is ble on the extent of TCR diversity and specific re- sponses of v6 T-cells in eral circulation, or in tissues. Furthermore, while it is generally considered that v6 TCRs do not interact with pH LA complexes, and thus do not engage with peptide antigens in this context only few n targets of v6 s have been characterised and the underlying molecular framework is only poorly under- stood.

The low frequency of peripheral v6 T—cells and the difficulty to study tissue-resident T- cells in humans has limited our knowledge of how this important and diverse type of T- cells participate in adaptive immune responses. This emerging area of research would require more reliable logies with which to capture and characterise rare v6 T- cells, isolate their TCR pairs, and to identify their cognate antigens.

Antigens and Antigen-presenting cells In the context of T-cells and TCRs, antigens may be defined as any molecule that may be engaged by a TCR and ing in a signal being transduced within the T-cell. The most well characterised T-cell antigens are peptides presented in an HLAl and HLAll complex, and which are engaged by conventional 08 T-cells. However, in recent years it has become apparent that non-conventional dB T-cells and v6 T-cells are able to en- gage a wide range of ecules as antigens, including , lipopeptides, glyco- peptides, glycolipds and a range of metabolites and catabolites. In addition, it has emerged that yo T—cells may be able to engage fully folded proteins directly in an anti- body-like fashion. Therefore, the view of T-cell antigens being largely restricted to HLA- presented peptides has expanded over the past two decades to include almost any bio- molecule. With this concept in mind, it is relevant to define what may be considered an antigen-presenting cell (APC).

As defined in the above sections, HLAI and HLAII have a disparate expression profiles across cell types. It is widely accepted that nearly all nucleated cells present HLAI com- plexes on the cell e, and are thus competent to present peptide antigens for T- cell sampling. In contrast, HLAII has a restricted expression profile, and at least in steady state conditions is only expressed on the surface of cells that have a specialist role in antigen presentation, including dendritic cells (DC), macrophage and B-cells.

These specialist cell types are often referred to as professional APC. For the purposes of this document, the term APC is used to describe any nucleated cell that is e of presenting an antigen for sampling by 08 or v6 T-cells. Such antigens are not restricted to those presented as ‘cargo’ in specific antigen-presenting complexes such as HLA and HLA-like molecules, but may also include any urface presented moiety that is able to engage a dB or v6 TCR-bearing cell.

Therapeutic use of TCRs Adoptive transfer of y T—cells was first trialled in a clinical setting in the early 1990s, starting with ex vivo expanded s polarised s viral antigens to confer viral immunity in immunocompromised patients. Similar approaches using primary T— cells ed ex vivo against specific cancer antigens were soon after trialled in treat- ment of malignancies. One limitation in these early approaches that ues to be a nge today is a lack of understanding of the nature and diversity of T-cells clash- ing with the need to finely-optimize their composition in the eutic product. At pre- sent, the use of ex vivo expanded primary T-cells has largely been abandoned by the pharmaceutical industry with the exception of a handful of initiatives using primary T— cells with specificity for viral ns.

In recent years the y to reliably introduce genetic material into primary human cells has seen a variety of experimental genetically modi?ed T-cell therapeutics arise. Such therapeutic cell products aim to s the power of T-cell responses and redirect T- cell specificity towards a disease-associated antigen target, for example, an antigen uniquely expressed by malignant cells. These have largely relied on the er of a chimeric antigen receptor (CAR) into recipient T-cells, rather than actual TCR chain pairs. A CAR ents a targeting moiety (most often a single-chain antibody t targeting a e expressed protein of ant cells) grafted to signal receptor ele- ments such as the C-chain of the CD3 complex, to produce a synthetic chimeric recep— tor that mimics CD3-TCR function. These so-called CAR T-cell (CAR-T) ts have met mixed success in al trials to date and despite their potential are not easy to translate beyond tumours with inherent unique molecular targets such as B-cell malig- nancies. Alternatively, the transfer of full-length TCR chain pair ORFs into T-cells is of emerging interest. Such TCR—engineered T-cell therapeutics are at present limited by challenging manufacturing processes, and like the CAR-T products, a dearth of vali- dated antigen targets and targeting constructs. To date this has been focused on the use of GB TCRs for recognition of peptide antigens presented by HLAI on malignant cells and a fundamental challenge of this approach is the need for antigens that are ic to malignant cells.

It has been considered that since the TCR-pH LA interaction is of relatively low-affinity, native TCRs are likely to be suboptimal for TCR-engineered T-cell therapies. Several approaches have been d to affinity-mature TCRs in vitro, in much the same manner as single-chain antibody affinity maturation. These TCR affinity maturation ap- proaches lly also e a single-chain formats, wherein the V-region of one chain is fused to V-region of another chain to make a single polypeptide construct.

Such single polypeptides may then be used in phage- or yeast- display systems adapted from antibody engineering ows, and passed through rounds of selection based on target binding. Two inherent limitations exist in such a single-chain TCR ap- proach in terms of yielding functional TCR chain pairs. Firstly, the selection is based on binding affinity to the target. However, it has been well documented that TCR affinity does not always correlate to the strength or competency of TCR signalling output. Sec- ondly, the ion of single—chain constructs based on affinity does not always trans- late to equivalent affinities once they are reconstituted as full—length receptors.

In a therapeutic context, there exists an onal and crucial limitation in affinity-ma- tured TCR pairs. That is, considering their sequences have been altered, the resulting constructs by definition have no longer been subject to thymic selection, wherein TCRs that react strongly to self-antigens are deleted from the repertoire. Therefore, these modified TCRs carry an inherent risk of being auto-reactive, which is very difficult to rule out in vitro using current methods. For the same reason, any selected or engi- neered TCR for eutic application needs to be individualised. If TCRs are artifi- cially engineered or native TCRs applied across individuals, cross-reactivities have to be ruled out on the basis of the HLA haplotype and presented peptide repertoire of each specific dual in order to avoid potentially catastrophic autoimmunity. This is due to the fact that thymic selection is conducted on a background of all available HLA molecules specific only to that given individual. The likelihood of such cross-reactivity is unclear. r, the ability of our TCR repertoire to recognize pHLA complexes of other individuals of the same species as n is a fundamental property of adaptive immunity and underpins graft rejection and graft versus host disease. Recent clinical trials using a matured TCR chain pair against the cancer-specific ma associated antigen (MAGE) highlighted the potential problem of bypassing thymic selection. When autologous T-cells harbouring the matured TCRs were infused back to two cancer pa- tients, these patients rapidly developed a fatal heart disease. Subsequent studies de- termined that the MAGE-specific d TCRs were cross—reactive with an HLAl-pre- sented peptide from the heart protein titin. This ly suggests that cross—reactivity is a distinct possibility in therapeutic use of TCRs.

A distinct avenue of utilising TCRs for therapeutic purposes is in their use as affinity re- agents in much the same manner as antibody therapeutic substances. Single-chain TCR molecules have been trialled for ry of conjugated drug substances to spe- cific HLA-antigen expressing cell populations. Such an ch is generally consid— ered safer than CAR-T or TCR engineered T-cell therapeutics, as administration of the drug substance may simply be withdrawn. However, the potential for cross-reactivity and off target effects that are ult to predict s a potential tion in this set- ting.

TCR repertoire detection in clinical diagnostics In a related aspect, there is an emerging interest in using the ion of the abun- dance of specific TCR sequences for clinical diagnostic es. With the rise of deep-sequencing methods in particular, it is possible to e the full TCR diversity within an individual globally and for matched 08 pairs in specific contexts. This poten- tially represents a means to diagnose specific conditions and disease states simply by detecting the abundance of expanded T-cell clones, as proxy readout for established immune response against a disease-associated antigen in the patient. However, such global approaches are currently limited to very strong immune responses with estab- lished clinical time-points and suffer from the underlying difficulty in identifying the spe- cific antigen target of any particular TCR identified via sequencing.

Therapeutic and diagnostic use of T—cell antigens The fundamental th of sing adaptive immune responses translates into a central technical challenge in that the exquisite specificity of the TCR-antigen interac- tion requires detailed knowledge of the antigens specifically associated with each path- ogen, cancer cell or autoimmune disease. Furthermore, each antigen may be pre- sented by a specific antigen presenting complex, or allele thereof, such that antigen discovery has be performed for each relevant HLA gene and allele. For several infec- tious diseases like HIV, influenza and CMV that are ated with strong ve im- mune responses and generally display conserved epitope response hierarchies, the most important epitopes have been mapped in context of some common HLA. Simi- larly, the fields of cancer, allergy and autoimmunity have seen sed and system- atic efforts to map the associated T—cell ns. However, these are challenging pro- cedures and the efforts to systematically describe T-cell ns associated with differ- ent clinical contexts are hindered by the e of efficient, robust, fast and scalable protocols.

Specifically, cancer cells represent a challenging and important aspect as most of the peptides presented on the surface of malignant cells are self antigens or very similar to self antigens. Therefore, thymic selection will have deleted TCRs that could strongly ize these es, while at the same time the tumour has evolved to evade im- mune recognition. This means that potent immune ses against ished tu— mours are relatively rare and targets difficult to t or discover. However, these re- sponses do exist and, antly, are generally associated with better outcome. The target of such responses, tumour-associated-antigens (TAA), will in most cases have distinguishing characteristics from self and be derived from proteins that are overex— pressed during cancer development, otherwise absent from the cell type at this stage of development or specifically altered through genetic mutation or post-translational modifications such as phosphorylation.

When available, the dge of such epitopes makes it possible to interrogate the associated T-cell response for fundamental discovery, diagnostic purposes and for ex- ample as a test of e cy. Importantly, they also provide highly specific tar— gets for T-cell zation in allergy and autoimmunity and, crucially, point towards val- uable s for specific immunotherapy and against malignant cells. Malignancies represent a particularly valuable target as the promise of cellular immunotherapies and the progress in the T-cell manipulations are slowed by a lack of validated target TAAs that go beyond the few cases where specific markers for the type of cancer happen to be available.

In the light of the ial of cellular therapy and lack of validated targets the identifica- tion of promising TCR antigens remains one of the most pressing bottlenecks of TCR- based immunotherapy, particularly in the effort to treat cancer.

Technological aspects of TCR and T-ce/I n analyses Overall, the development of TCR-based therapies is still in its early stages, and suc- cess has been limited. Diagnostic approaches, while of immense potential, have sel- dom been deployed into controlled clinical studies that aim to assess patient disease states or response to therapy. Underdeveloped techniques for the reliable capture of native TCR chain pairs, and the systematic analysis of TCR—antigen ctions at high-throughput and in a functional context of cell-cell communication, has been the main hurdle to the development of TCR-based therapies and diagnostics.

Deep sequencing approaches have led to an improved understanding of T-cell receptor ity in heath and disease. r, these approaches have generally focused on short stretches spanning the CDR3 regions, mainly of the TCR B-chain. Most studies have ignored the contribution of the TCR q-chain, and few have sought to e paired dB chains as well as the antigen specificity of TCRs determined to be of interest.

Recent workflows using single cell encapsulation and genetic barcoding has enabled the pairing of native TCR dB or v6 chain pairs and analysis of full-length ces, however, such workflows remain mental.

Isolated TCR chain pairs may be analysed in terms of antigen specificity in either bio- physical or functional modes. Biophysical analysis requires the recombinant production of both the TCR as well as the analyte antigen in soluble form. In the case of HLA-re- stricted TCRs this would thus require the generation of all individual TCRs as well as the cognate pHLA xes. This is cally highly challenging, slow and very low- throughput. Furthermore, such analysis would only provide interaction affinities, which are not well-correlated with functional teristics in predictable ways.

Until recently, the detailed onal analysis of isolated TCR sequences in a cellular context has been limited to laborious protocols of ection of analyte TCR chain pairs into primary s or immortal T—cell lines, and detection of cellular ses by traditional flow cytometric analysis of cell activation, or detection of secreted factors from the transfected cells upon antigen challenge. In a recent publication by Guo et al, rapid cloning, expression, and functional terization of paired TCR chains from single-cells was reported (Molecular Therapy — Methods and clinical development (2016) 3:15054). In this study, analyte human 08 TCR pairs were expressed in a re- porter cell line that lacked dB TCR expression, and which contained a green fluores- cent protein (GFP) reporter system linked to the Nur77 promoter that is activated upon TCR stimulation. This system remains cient due to the lack of standardised TCR integration into the reporter cell line genome, and does not provide a systematic man- ner for cell-bound antigen challenge by an APC element.

Similar to ows for identification of TCRs against known T-cell antigens, the de novo discovery of novel T-cell antigens in health and disease remains highly challeng- ing. Most approaches remain biophysical in nature, and aim to produce candidate anti- gens that may be tested in immunisation protocols, or h identifying cognate TCRs as addressed above. Little or no standardisation exists in the field of T-cell anti- gen discovery, and the field is largely restricted to academic study.

With the accumulating interest in TCRs and their e antigens in both therapeutic and diagnostic use, and the emergence of means to capture significant numbers of na- tive TCR dB and v6 chain pairs, there remains a lack of reliable high—throughput and standardised technologies for the systematic analysis of TCR-antigen interactions. Im- portantly, there is a lack of rdised systems for functional analysis of TCR chain pairs in the native context of cell-cell communication wherein both the TCR and antigen are presented by a viable cell. Moreover, there is a lack of systems that may e TCR candidate selection, and/or affinity maturation of TCR chain pairs, in the relevant context of ell communication.

As described, there is currently a lack of standardised technologies for the high- throughput generation of cells with TCR pairs expressed in a native ar context. It is highly desirable to possess a system in which full-length TCR pairs may be ed as single copies into the genome of a TCR-presenting cell such that said TCRs are presented in a native CD3 cell-surface complex for analysis and selection. A CD3 complex-presented TCR pair assures that affinity analyses are reflective of the actual na- tive TCR composition, which is not the case for single-chain TCR and other non-native TCR-display technology. Moreover, the presentation of TCR pairs in a CD3 complex on a viable cell is an absolute requirement for functional analysis of TCR pairs. Functional analysis, meaning analysis of TCR signalling output, is of critical importance in TCR selection and engineering workflows where signal output is the ter that is gener- ally of greatest importance for use in the context of cellular therapeutics, and is not well correlated with the affinity of a TCR with cognate antigen/HLA as is determined within other display platforms.

Detailed description of the invention The t disclosure relates to the construction and use of an engineered two-part ar device for discovery and characterisation of TCRs and T-cell ns. The two parts of the overall ar device are comprised of distinct engineered cell types that are contacted with one another in operation of the device. The device is used for standardised functional is of responsiveness of analyte TCRs towards analyte anti- gens. The readout of such responsiveness is used for the identification and selection of TCRs, T-cell ns, and ed functional characterisation of the TCR/antigen interactions.

The device presents analyte antigens through a first cell tion, the engineered antigen presenting cell (eAPC) that is prepared using the eAPC system (eAPCS). The device presents analyte TCRs through a second cell population, the en- gineered TCR- ting cell (eTPC) that is ed using the eTPC system (eTPCS).

The two-part device of the present disclosure may contain features that enable a high degree of standardisation, reproducibility and critically restricts the complexity of ana- lyte TCR and T-cell antigens collections. Primarily, this standardisation is achieved through defined and highly controlled integration of analyte antigen-presenting complexes and antigens to the genome of eAPC, and TCR to the genome of eTPC. This permits rapid cycle times to generate analyte cell populations, and greatly reduces costs of t random-integration and viral vector platforms. Moreover, the present disclosure may permit the generation of cell-based arrays of analyte entities in both a eAPC- and eTPC- centric manner, meaning that large libraries of vectors can be integrated into either cell population, and assayed as libraries of cells expressing single analyte entities per cell. Single-copy genomic er sites being engineered into the genome of eAPC and eTPC achieve this, such that when provided by an ORF (for HLA, antigen or TCR chain) within the matched donor vectors, only a single copy of an ORF can be integrated from the vector pool. This means that if the vector pool ses ORFs of mixed identify, each integrated cell will only integrate a single ty; essentially creating cell-based arrays akin to other display rms like bacteriophage. This high level of standardisation and reduction of xity is in st to the current methods using primary or immortal cell cultures and extended wth and endogenous singling responses to achieve s, or in partial systemisation of APC- or T- cell- centric inputs. Importantly, the two-part engineered cell device that represents the present disclosure may achieve standardisation of relevant functional responses in the context of cell-cell communication. This may reduce time and costs of discovery and clinical testing of new TCR and T-cell antigen candidates by sing the predictability of their effects in vivo.

The present disclosure relates to the provision and operation of a two-part device, wherein a first part is an engineered antigen-presenting cell system (eAPCS), and a second part is an engineered TCR-presenting cell system (eTPCS). Overall, this device represents a multicellular analytical system comprised of two distinct types of engineered human cells for functional analyses of TCR recognition of T-cell ns. The primary outputs of this device are cells that express analyte antigen or analyte TCR, which subsequently may be used to determine the terminal outputs of the device as ic T-cell antigens or TCR sequences, respectively (Figure 1).

In some embodiments, the present invention provides a two-part system forming a combined eAPC:eTPC analytical system, n a first part is an engineered antigenpresenting cell system (eAPCS), and a second part is an engineered TCR-presenting cell system ), wherein said eAPCS comprises: a first component which is an engineered antigen-presenting cell (eAPC), designated component 1A, n component 1A lacks endogenous surface expression of at least one family of target analyte antigen presenting complexes (aAPX) and/or target analyte antigenic molecules (aAM), and contains a second ent designated component 1B, a tic genomic receiver site for integration of one or two ORFs encoding an aAPX and/or an aAM, and a third component which is a genetic donor vector, designated component 1C, for ry and ation into component 1B of at least one target ORF encoding at least one target aAPX and/or at least one target aAM, wherein component 1C is matched to component 1B, and wherein component 1C is designed to deliver one or two target ORFs encoding a target aAPX and/or a target aAM, and wherein said eTPCS comprises: a first component which is an engineered esenting cell (eTPC), designated component 2A, wherein component 2A lacks endogenous surface expression of at least one family of target analyte antigen-presenting complexes (aAPX) and/or target analyte antigenic le (aAM), lacks endogenous expression of TCR chains alpha, beta, delta and gamma, and expresses CD3 proteins which are conditionally presented on the e of the eTPC only when the eTPC expresses a complementary pair of TCR chains, and contains a second component designated 2B, a genomic receiver site for integration of a single ORF encoding at least one analyte TCR chain of alpha, beta, delta or gamma, and/or two ORFs encoding a pair of e TCR chains, and a third component which is a genetic donor vector, designated component 2C, for delivery of ORF ng analyte TCR chains, wherein component 2C, is matched to component 2B, and n the component 2C is ed to deliver: a. a single ORF encoding at least one analyte TCR chain of alpha, beta, delta and/or gamma; and/or b. two ORFs encoding a pair of analyte TCR chains.

The two-part device is operated in two phases comprising i. Phase 1, the preparation of analyte antigen and analyte TCR bearing cell populations and their assembly into a combined system contacting two analyte cell populations 16B followed by page 17 ii. Phase 2, the readout of responses intrinsic to either of the contacted cell populations of the combined system to obtain the outputs of the , wherein the eAPCS and the eTPCS provide the means to prepare analyte eAPC and eTPC populations, tively, to be assembled into the combined eAPC:eTPC sys- tem (figure 1, steps i and ii).

The eAPCS is defined as a system to prepare various forms of analyte eAPC popula— tions that are provided to the ed eAPC:eTPC system, wherein the analyte eAPC populations are defined as one of the following i. an eAPC-p ii. an eAPC—a iii. an eAPC-pa iv. Libraries of thereof wherein the selection of the input analyte eAPC tion to the combined eAPC:eTPC system is determined by the nature of the antigens that are the subject of the ion of the device. That is, the required eAPC population combined into the eAPC:eTPC system is defined by the required y output from the device, andlor the required terminal output from the device (figure 1, step i).

The eTPCS is defined as a system to prepare e eTPC populations that are pro- vided to the combined eAPC:eTPC , wherein the analyte eTPC populations are defined as one of the ing i. an eTPC-t ii. Libraries of eTPC-t wherein the selection of input analyte eTPC populations to the combined eAPC:eTPC system is determined by the nature TCRs that are the subject of the operation of the device. That is, the required eTPC tion combined into the eAPC-eTPC system is d by the required primary output from the device, and/or the required terminal output from the device (figure 1, step ii).

Primary outputs from the device are selected cell populations, which have or have not responded to the analyte presented by the reciprocal cell provided in the eAPC:eTPC system. That is, such a primary output may be represented as a single cell, or a pool of cells, that have been selected on the presence or absence of a reported response within the combined TPC system (Figure 1 step v). A response within an ana- lyte eAPC is only provoked by engagement of a cognate TCR presented by a contact— ing eTPC. A response within an analyte eTPC is only provoked by engagement of a cognate antigen presented by a contacting eTPC (figure 1 step iv).

A selection of analyte eAPC and/or analyte eTPC from the combined eAPC:eTPC sys- tem may be made on the basis of a response in the contacting cell. That is, an analyte eAPC may be selected on that basis of a reported response, or lack thereof, in the con- tacting analyte eTPC. sely, an analyte eTPC may be selected on that basis of a reported se, or lack thereof, in the contacting analyte eAPC.

Primary eAPC outputs from the device are selected cells, wherein selection is made based on the presence or absence of a reported signal response, and these cells may comprise one or more of i. an eAPC-p ii. an eAPC-a iii. an eAPC-pa wherein the selected cells may comprise a single cell, a pool of cells of the same iden- tity, a pool of cells of different identities (Figure 1 step v).

Primary eTPC outputs from the device are selected cells, wherein selection is made based on the presence or absence of a reported signal response, and these cells com- prise eTPC-t, wherein selected cells may comprise a single cell, a pool of cells of the same identity, a pool of cells of different identities (Figure 1 step v).

When the two—part ar device is operated, the ion of analyte cell populations to prepare and combine into an eAPC:eTPC system is ined by the nature of an- alyte that each of the eAPC and eTPC tions present.

An eAPC-p is defined as expressing an antigen-presenting complex (aAPX) on the cell surface. An aAPX may have a cargo molecule (CM) loaded as n cargo. An aAPX loaded with antigen cargo is defined as an M complex.

An eAPC-a is defined as expressing an analyte antigen molecule (aAM). Such an aAM may be presented on the cell surface, and/or sed or processed intracellularly such that it represents a cargo aAM that may be loaded into an aAPX.

An eAPC-pa is defined as expressing both an aAPX and an aAM. The aAM may be ex- pressed or processed intracellularly such that it represents a cargo aAM that may be loaded into an aAPX. eAPC-pa thus expresses an aAPX at the cell e, and may also express aAM loaded as cargo into said aAPX, which is defined as an aAPX:aAM complex. It is possible for an eAPC-pa does not load aAM as cargo into an aAPX to form the aAPX:aAM complex. Moreover, due to the nature of biological systems, it is unavoidable that aAPX:CM complexes are also present in the aAPC-pa.

An eTPC-t is defined as possessing an analyte pair of TCR chains that are expressed as TCR surface proteins in complex with CD3 (TCRsp), wherein CD3 represents the e x in which a TCR heterodimeric pair is presented and is defined as pos- g a, v, 6 and C ts that associate with pair of complementary TCR chains as three dimers (av, 86, (C). The sion of CD3 and a pair of complementary TCR chains is required for presentation at the cell surface, thus, the absence of expression of either TCR or CD3 will prevent the reciprocal from presenting on the cell surface. In the preparation of an eTPC-t, the expression of an 08 or v6 TCR pair and/or CD3 is used as a positive selection. ore, regardless of the nature of the TCR o, B, y or 6 chain combinations used to prepare the eTPC-t, only cells with a pair of complemen- tary TCR chains presented on the e of the cell in complex with CD3 are desig- nated eTPC-t.

The above primary outputs all represent cell populations that are derived from a selec- tion made upon their reported response in the combined eAPC:eTPC system. Each an- alyte eAPC presents a set of potential analyte antigens, intrinsic cargo molecules and/or analyte antigen presenting complexes, whereas the eTPC presents analyte TCRsp. It is from the selected cells that the ed analyte molecules may be ob- tained as terminal device outputs (Figure 1, step vi).

An eAPC-p is defined as presenting an aAPX on the cell surface, and may t aAPX:CM complex at the cell surface. Thus an eAPC-p that is selected from the com- bined eAPC:eTPC system based on a reported response, may be used to ine aAPX, CM and/or a aAPC:CM terminal device outputs, wherein these output identities have been selected on the ability of the determined e antigen to form a complex with, and/or report a signal response stimulated by, an analyte TCRsp presented by the analyte eTPC—t contacted with the selected eAPC-p in the combined TPC sys- An eAPC-a is defined as ting an aAM on the cell surface or intracellularly. Thus an eAPC-a that is selected from the combined eAPC:eTPC system based on a re- ported response, may be used to determine an aAM terminal device output, wherein this output identity is selected on the ability of the determined analyte to form a complex with, and/or report a signal response stimulated by, an analyte TCRsp presented by the analyte eTPC-t contacted with the selected eAPC-a in the combined eAPC:eTPC system.

An eAPC-pa is defined as presenting an aAPX, aAM, aAPX, aAPX:aM, or an aAPX:CM at the cell surface. Thus, an eAPC-pa that is selected from the combined eAPC:eTPC system based on a reported response, may be used to ine aAM, aAPX, aAPX:aM and/or aAPX:CM terminal device outputs, wherein this output ty is ed on the ability of the determined e to form a complex with, and/or report a signal response stimulated by, an analyte TCRsp presented by the analyte eTPC-t contacted with the selected eAPC-pa in the combined TPC system.

As addressed above, ion of analyte eAPC may be made on basis of a reported re- sponse in a contacting eTPC. Therefore, any of eAPC-p, eAPC-a and eAPC-pa may be used to indirectly obtain the TCRsp output, if the TCRsp s) to the eTPCS are known prior to preparation of the analyte .

In the present context, an eTPC-t is defined as presenting TCRsp. Thus an eTPC-t that is selected from the combined eAPC:eTPC system based on a reported response, may be used to determine TCRsp terminal device outputs, wherein this output identity is selected on the ability of the determined analyte TCRsp to form a complex with, and/or report a signal response stimulated by, an analyte antigen presented by the analyte eAPC contacted with the selected eTPC in the combined eAPC:eTPC system.

As addressed above, selection of analyte eTPC may be made on basis of a reported response in a contacting eAPC. Therefore, an eTPC may be used to indirectly obtain any of the e antigen outputs, aAM, aAPX, aAPX:aM and/or aAPX:CM, if those analyte antigen input(s) to the eAPCS are known prior to preparation of the analyte eAPC.

Description of the eAPCS As ned above, the present disclosure s to the provision of a two-part cellular device, wherein each part is an engineered cellular system. The first engineered cell system is an engineered multicomponent eAPCS that may be used to e analyte eAPC for combination into a two-part eACP:eTPC system within the device.

The minimal form of eAPCS is a multicomponent system wherein a first component is an eAPC, designated component 1A, containing a second component as a genomic receiver site component B, and a third component as a genetic donor vector, desig- nated component 1C (Figure 2).

An eAPC represents the base component of the eAPCS, to which all other components of the system relate. Therefore, the eAPC contains certain es, that are native or engineered, that make the eAPC suitable for use in both the eAPCS and the combined two-part .

In the present context the eAPC, component 1A i. Lacks endogenous surface expression of at least one family of aAPX and/or aAM and ii. Contains at least one genomic receiver site, designated component 1B n i) may be obtained by selection of a naturally occurring cell population lacking said expression of aAPX and/or aAM, or may be engineered to lack such expression, and ii) which is synthetic and which may be introduced by means of directed or undi- rected genome ation.

The selection of an eAPC cell ate that lacks desired aAPX and/or aAM expres- sion from naturally occurring cell populations can be achieved by methods well known in the art. This may be directly achieved by staining of target cells with affinity reagents specifically for the aAPX and/or aAM that are desired to be lacking from the eAPC, and selection of cells lacking target aAPX and/or aAM expression. ering of cells to lack aAPX and/or aAM expression may be achieved by untar— geted and targeted means. Untargeted mutagenesis of the cell can be achieved by providing a al, radiological or other mutagen to the cell, and then selecting cells lacking target aAPX and/or aAM sion. ed mutation of the genomic loci can be achieved via different means, including but not limited to site directed nesis i. zinc-finger ses ii. CRISPR/Casg mediated targeting iii. Synthetic transcription activator-like effector nucleases (TALEN) wherein said site-directed nucleases induce site-specific DNA-repair error mutagenesis (also known as non-homologous end-joining) at target loci, after which mutated cells are obtained by selecting cells lacking target aAPX and/or aAM expression.

The component 1A, eAPC, may ally include additional T-cell mulation re— ceptors, wherein such features permit robust or varying forms of communication of the analyte eAPC to the analyte eTPC, wherein the le communication is relevant to identification or characterisation of specific e TCRsp and/or analyte ns. In the present context, different forms of CD28 ligation on the eTPC can be promoted by inclusion of one or more of CD80, CD86 and/or further B7 family proteins.

The component 1A ,eAPC, may optionally onally include introduced cell surface adhesion molecule components, or ablation of endogenous cell surface adhesion mole- cules, to promote the eAPC engagement with analyte eTPC and formation of the im- gical synapse, or to avoid tight binding and formation of deleterious cell cluster- ing within the combined eAPC:eTPC system, respectively. Such adhesion molecules that may be introduced as additional ORFs to component 1A, or genetically ablated from 1A, can be selected from the integrin family of on proteins.

An eAPC may optionally possesses the ability to process and load antigen as cargo into aAPX by native processing and loading machinery. An eAPC that possesses the ability to process and load antigen as cargo into aAPX by native processing and loading machinery, will also s and load cargo molecules (CM) that are sic to the eAPC or the culture system in which it is contain, wherein aAPX that is loaded with a CM is designated as an aAPX:CM complex.

The second component of the minimal multicomponent eAPCS is a genetic donor vec- tor, component 1C, which is used for integration of at least one ORF encoding at least one aAPX and/or aAM (Figure 2).

Component 1C is a genetic donor vector that is coupled with the genomic receiver site of Component 18 contained within the genome of the eAPC, Component 1A. Com- ponent 1C is designed for the integration of one or more ORFs encoding an aAPX and/or an aAM, encoded in the genetic donor vector, into the genomic receiver site, 1B, wherein integration results in the expression of aAPX and/or an aAM by the target eAPC.

In the present context, a paired genetic donor vector and genomic receiver site is de- scribed as an ation couple.

In an expanded form or the multicomponent eAPCS, the component 1A eAPC may fur- ther n a second genomic receiver site, designated component 1D, which is cou- pled to a second genomic donor vector, ated ent 1E, that is also added to the system (Figure 3).

A multicomponent eAPCS may further comprise one or more additional integration cou- ples.

A multicomponent eAPCS, comprising an eAPC and either one or two integration cou- ples, is used for preparation of the derivative eAPC forms i. eAPC-p ii. eAPC-a iii. eAPC-pa wherein each genetic donor vector may contain one or more ORFs encoding one or more aAPX and/or an aAM, to integrate said ORFs into the coupled genomic receiver sites, such that i) expresses at least one aAPX, ii) expresses at least one aAM and iii) expresses at least one aAPX and at least one aAM (Figure 4).

The genetic donor vector and genomic receiver sites e as an integration couple tem of the eAPCS. A genetic donor vector must first be combined with target ORFs, such that base donor vector now encodes those target ORFs. The assembled primed donor vector is then introduced to the target eAPC to exchange target ORF(S) to the genomic receiver site, thus integrating the target ORFs to the coupled er site of the target cell (Figure 5).

A multicomponent eAPCS that comprises genetic donor vectors component 10 and/or 1E is combined with at least one ORF ng at least one aAPX and/or aAM to ob- tain component 10’ and/or 1E’, wherein the combination is defined as the ligation of genetic material into the correct coding frame(s), and in the correct orientation(s), of the genetic donor vector.

The combination of one or more ORFs into genetic donor vectors 1O and/or 1E may be performed multiple times with a library of unique ORFs as i. single te reactions to obtain a discrete library of 10’ and/or 1E’ vectors encoding multiple ORFs ii. a singe reaction to obtain a pooled library of 10’ and/or 1E’ vectors encoding multiple ORFS wherein the discrete a discrete y may be combined with component 1A multiple times as to obtain a discrete library of eAPCs with unique ORFs encoding unique aAPX and/or aAM, or a pooled library may be combined with component 1A as a single event as to obtain a pooled library of eAPCs each with unique ORFs encoding unique aAPX and/or aAM.

The efficient integration of a predictable copy number of one or more ORFs into the ge- nomic receiver site is highly advantageous for operation of a standardised eAPC, where analyte eAPC tions may be rapidly prepared and characterised. Thus, the c receiver ) and coupled donor vector(s) are critical to the function of the eAPC. Furthermore, it is strongly desirable to have an eAPC wherein component 18 and 1D, are insulated from one another, such that the donor vector component 10 can- not integrate at component 18, and vice versa. In addition, it is also desirable that the component 1B and/or component 1D are amenable to a method of preparation of an eAPC wherein, the introduction of an analyte antigen is rapid, repeatable, with a high likelihood of correct ation and delivery of the desired number of copies of aAPX and/or aAM.

The c receiver site may be selected from the following i. A synthetic construct designed for recombinase mediated te exchange (RMCE) ii. A synthetic construct ed for site directed homologous recombination iii. A native genomic site for site directed homologous recombination wherein i) is preferred. The RMCE method may employ selected heterospecific sites that are specific for individual recombinase enzymes, such that each component 18 and 1D s insulated specificity.

In the present context the genomic receiver site, component 18 and/or component 1D comprises of at least one of the following genetic ts i. Heterospecific recombinase sites ii. gous arms iii. Eukaryotic promoter iv. Eukaryotic conditional regulatory element v. Eukaryotic terminator vi. Selection marker vii. Splice acceptor site viii. Splice donor site ix. Non-protein coding gene x. tor xi. Mobile genetic element xii. Meganuclease recognition site xiii. Internal ribosome entry site (IRES) xiv. Viral self-cleaving peptide element xv. A kozak consensus sequence.

The red genomic receiver site would comprise of two ent arrangements us- ing the ing selected elements from the previously stated list of element. The first arrangement is for receiving a single ORF encoding one or more aAPX and/or one or more aAM chains and/or a selection mark of integration, via RMCE integration wherein the arrangement is ’ -[A] [B] [C] [D] [E] [F]- 3’ wherein A) is element iii) a constitutive or inducible Eukaryotic promoter B) is element i) heterospecific recombinase site 1 C) is element xv) a Kozak consensus sequence D) is t vi) a FACS and/or MACS compatible encoded protein marker E) is element i) heterospecific recombinase site 2 F) is element v) otic terminator.

The second arrangement is for receiving two ORF encoding one or more aAPX and/or one or more aAM and/or a selection marker of integration, via RMCE integration wherein the arrangement is ’ -[A] [B] [C] [D] [E] [F] [G] [H] [|]- 3’ wherein A) is element iii) a constitutive or inducible Eukaryotic promoter B) is t i) specific recombinase site 1 C) is element xv) a Kozak consensus sequence D) is element vi) a FACS and/or MACS compatible encoded protein marker 1 E) is t v) a Eukaryotic bidirectional riptional terminator F) is element vi) a FACS and/or MACS compatible encoded protein marker 2 G) is element xv) a Kozak consensus sequence H) is element i) heterospecific recombinase site 2 I) is element iii) a tutive or inducible Eukaryotic promoter furthermore, in this second arrangement the elements F, G, and I are encoded in the antisense direction.

Component 1C and/or 1E comprises of at least one of the following genetic elements i. Heterospecific recombinase sites ii. Homologous arms iii. Eukaryotic promoter iv. Eukaryotic conditional regulatory element v. Eukaryotic terminator vi. Selection marker vii. Splice acceptor site viii. Splice donor site ix. Non-protein coding gene x. Insulator xi. Mobile genetic element xii. Meganuclease recognition site xiii. Internal ribosome entry site (IRES) xiv. Viral leaving e element xv. A kozak consensus sequence xvi. Selection marker of integration xvii. An antibiotic resistance cassette xviii. A bacterial origin of replication xix. A yeast origin of replication xx. A cloning site In a preferred embodiment of the genetic donor vector, component 10 and/or compo— nent 1E, would comprise of two different le arrangements using the following se- lected elements from the previously stated list of ts.

The first arrangement is for delivery of a single ORF ng one or more aAPX and/or one or more aAM and/or a selection mark of integration, via RMCE integration wherein the arrangement is ’ - [A] [B] [C] [D] [E] - 3’ wherein A) is element i) specific recombinase site 1 B) is element xv) a Kozak consensus sequence C) is element xx) a cloning site of a single ORF ng one or more aAPX and/or one or more aAM and/or element xvi) a selection marker of integration D) is element i) heterospecific recombinase site 2 E) is element xvii) An otic resistance cassette and element xviii) a bacterial origin of replication, in no specific orientation furthermore, the elements viii and/or xiv may be used to link multiple aAPX and/or one or more aAM and/or element xvi together.

The second arrangement is for delivery of two ORF encoding one or more aAPX and/or aAM and/or a selection mark of integration, via RMCE integration wherein the e- ment is ’ - [A] [B] [C] [D] [E] [F]- 3’ wherein A) is element i) heterospecific recombinase site 1 B) is element xv) a Kozak consensus ce C) is element xx) a cloning site for introduction of two or more ORF, with eukaryotic terminators, encoding one or more aAPX and/or one or more aAM and/or t xvi) a selection marker of integration D) is element xv) a Kozak consensus sequence (antisense direction) E) is element i) heterospecific recombinase site 2 F) is element xvii) An antibiotic resistance cassette and element xviii) a bacterial origin of replication, in no specific orientation furthermore, the elements viii and/or xiv may be used to link multiple aAPX and/or aAM and/or element xvi er within each ORF.

Preparing analyte eAPC populations in the eAPCS The above described eAPCS system may be used in le ways to prepare ct forms of analyte eAPC, or libraries thereof, that serve to present analyte aAPX, aAM, aAPXIaAM and aAPX:CM to the eTPC within the combined eAPC:eTPC system in op- eration of the two-part device.

An eAPCS comprising a single integration couple may be used to prepare an eAPC—p from component 1A in one step, by providing ent 10’ combined with an ORF for an aAPX, such that this aAPX is integrated to site 18, to create 18’. The resulting cell line expresses the provided aAPX, and it is presented at the cell surface (Figure An eAPCS comprising two integration couples may be used to prepare an eAPC-p from ent 1A in one step, by providing component 10’ combined with an ORF for an aAPX, such that this aAPX is integrated to site 18, to create 18’. The resulting cell line expresses the provided aAPX, and it is presented at the cell surface. The sec- ond integration couple 1D/1 E remains unmodified and may be used for downstream in- tegration steps (Figure 7).

An eAPCS comprising a single integration couple may be used to prepare an eAPC-a from component 1A in one step, by providing ent 10’ combined with an ORF for an aAM, such that this aAM is integrated to site 18, to create 18’. The resulting cell line expresses the provided aAM, and is presented either at the cell surface or retained intracellularly (Figure 8).

An eAPCS comprising two integration couples may be used to prepare an eAPC-a from component 1A in one step, by providing ent 10’ combined with an ORF for an aAM, such that this aAM is integrated to site 18, to create 18’. The resulting cell line expresses the provided aAM, and is presented either at the cell surface or retained intracellularly. The second integration couple 1D/1 E remains unmodified and may be used for downstream integration steps (Figure 9).

An eAPCS comprising a single integration couple may be used to prepare an eAPC-pa from ent 1A in one step, by ing component 10’ combined with two ORFs, one encoding and aAPX and the other an aAM, such that both aAPX and aAM are inte- grated to site 18, to create 13’. The resulting cell line expresses the provided aAPX and aAM, and may present an aAPXzaAM at the cell surface (Figure 10).

An eAPCS comprising two integration couples may be used to prepare an eAPC-pa from component 1A in one step, by providing ent 10’ combined with two ORFs, one encoding and aAPX and the other an aAM, such that both aAPX and aAM are inte- grated to site 18, to create 18’. The resulting cell line expresses the provided aAPX and aAM, and may present an aAPX:aAM at the cell e. The second integration couple 1D/1 E remains unmodified and may be used for downstream integration steps (Figure 11).

An eAPCS comprising two integration couples may be used to e an eAPC-pa from component 1A in one step, by providing component 10’ and 1E’ each combined with one ORF encoding either an aAPX or an aAM, such that both aAPX and aAM are integrated to site 18 or 1D, to create 1B’ and 1D’. The resulting cell line expresses the provided aAPX and aAM, and may present an aAPX:aAM at the cell surface (Figure An eAPCS sing two integration couples may be used to prepare an eAPC-pa from component 1A in two steps, by first providing component 10’ combined with an ORF encoding an aAPX such that this aAPX is integrated to site 1B, to create 1B’. The resulting cell line expresses the provided aAPX, and it is presented at the cell surface.

The second integration couple 1D/1 E remains unmodified. In the second step 1E’ is provided wherein the donor vector is combined with an ORF encoding an aAM such that this aAM is integrated to site 1E, to create 1E’. The resulting cell line expresses the provided aAM, and this may be processed and loaded as cargo in the aAPX to form an aAPX:aAM complex on the cell surface (Figure 13).

An eAPCS comprising two integration couples may be used to e an eAPC-pa from component 1A in two steps, by first ing ent 10’ combined with an ORF encoding an aAM such that this aAM is integrated to site 18, to create 18’. The resulting cell line expresses the provided aAM (eAPC—a intermediate). The second integration couple 1D/1 E remains unmodified. In the second step 1E’ is provided wherein the donor vector is combined with an ORF encoding an aAPX such that this aAPX is integrated to site 1E, to create 1E’. The resulting cell line expresses the provided aAPX, which is presented on the cell surface. The aAM integrated in the first step may be processed and loaded as cargo in the aAPX to form an aAPX:aAM x on the cell surface e 14).

In the abovementioned es of preparing analyte eAPC-p, eAPC-a and eAPC-pa populations from eAPC, the eAPCS system is used to provide known aAPX and aAM ates in a defined manner to prepare discrete populations of analyte eAPC ex- pressing d aAPX and/or aAM. Such a process may be repeated many times to build ies of eAPC-p, eAPC-a and eAPC-pa to provide to the combined eAPC:eTPC system in operation of the device. An alternative approach is to take pooled libraries of candidate aAPX and/or aAM ORFs combined with genetic donor vectors, and integrate these in a single reaction to obtain pooled libraries of analyte eAPC-p, eAPC-a or eAPC-pa that express multiple aAPX, aAM and/or aAPX:aAM.

This process of converting a pool of vectors to a pool of eAPC—p, -a, and/or —pa will be ed to as shotgun ation. This is particularly useful when analysing large li- braries of candidate aAM against a fixed aAPX, or vice versa.

An eAPCS comprising two integration couples may be used to prepare an eAPC-pa from component 1A in two steps, by first providing component 10’ combined with an ORF encoding an aAPX such that this aAPX is integrated to site 18, to create 18’. The resulting cell line expresses the provided aAPX on the cell surface p in- ite). The second integration couple 1D/1E remains unmodified. In the second step a library of multiple 1E’ is provided wherein the library of donor vectors comprises a pool of vectors each combined with a single ORF encoding an aAM such that each aAM is integrated to site 1E, to create 1E’, within single cells. The resulting pool of cells contains a collection of cells, n each cell has integrated a single random aAM ORF from the original pool of s. The aAM integrated in the second step may be processed and loaded as cargo in the aAPX integrated in the first step to form an AM complex on the cell surface (Figure 15).

An eAPCS comprising two integration couples may be used to prepare an eAPC-pa from ent 1A in two steps, by first providing component 10’ combined with an ORF encoding an aAM such that this aAM is ated to site 18, to create 18’. The resulting cell line expresses the provided aAM (eAPC-a intermediate). The second ation couple 1D/1 E remains unmodified. In the second step a library of multiple 1E’ is provided wherein the library of donor vectors comprises a pool of vectors each com- bined with a single ORF encoding an aAPX such that each aAPX is integrated to site 1E, to create 1E’, within single cells. The resulting pool of cells contains a collection of cells, wherein each cell has integrated a single random aAPX ORF from the original pool of vectors. The aAM integrated in the first step may be processed and loaded as cargo in the aAPX integrated in the second step to form an aAPX:aAM complex on the cell surface (Figure 16).

An eAPCS comprising two integration couples may be used to prepare an eAPC-pa from component 1A in one steps, by providing component 10’ and 1E’ each combined with a library of ORFs ng either a library of aAPX or a library of aAM, such that both aAPX and aAM are integrated to site 18 or 1D, to create 18’ and 1D’. The result- ing pool of cells contains a collection of cells wherein each cell has integrated a single random aAPX ORF and a single random aAM ORF from the original pool of vectors.

Within each cell in the pooled library, an integrated aAM may be processed and loaded as cargo in the aAPX integrated into the same cell to form an aAPX:aAM complex on the cell surface. Such a pooled library would contain all le combinations of aAPX:aAM from the set of aAPX and aAM provided (Figure 17).

In the above-mentioned shotgun integration methods for providing pooled libraries of eAPC-pa, the ness of the system relies on a single copy of the genomic receiver site. This is to ensurejust a single analyte may be introduced into each cell via the inte- gration couple. This single-copy genomic receiver site is an optional aspect of an eAPCS, as multiple copies of the same genomic receiver25 site may be beneficial in providing integration steps where multiple ‘alleles’ from a library of provided vectors may be ed in the prepared eAPC.

In the present context, an aAPX may be selected from one of the following i. One or more members of HLA class I ii. One or more members of HLA class M iii. One or more non-HLA antigen—presenting complex An aAPX may be selected from one of the following i. a polypeptide or x of polypeptides provided as analyte antigen ii. a e derived from a polypeptide provided as analyte antigen iii. a peptide provided as analyte antigen iv. a metabolite ed as analyte antigen v. a polypeptide or complex of ptides translated from the analyte antigenic molecule ORF(s) vi. a peptide derived from a ptide translated from the analyte antigenic mole- cule ORF(s) vii. a peptide derived from altering the component A proteome viii. a polypeptide derived from ng the component A proteome ix. a metabolite derived from altering the component A metabolome Description of the eTPCS As mentioned above, the present disclosure relates to the ion of a two-part cellular device, n each part is an ered cellular . The first engineered cell system is the eAPCS as described above. The second engineered cell system is an engineered multicomponent eTPCS that is used to e analyte eTPC for combina- tion into a two-part eACP:eTPC system within the device.

The minimal form of eTPCS is a omponent system n a first component is an eTPC, designated component 2A, and a second component is a genetic donor vector, designated component 2C (Figure 18).

An eTPC ents the base component of the eTPCS, to which all other components of the system relate. Therefore, the eTPC contains certain features, that are native or engineered, that make the eTPC suitable for use in both the eTPCS and the combined two-part device.

The eTPC, component 2A, i. Lacks endogenous expression of TCR chains alpha, beta, delta and gamma, ii. Expresses CD3 proteins which are conditionally presented on the surface of the cell only when the cell expresses a complementary pair of TCR chains and iii. ns a further component designated 2B, a genomic receiver site for integration of a single ORF ng at least one analyte TCR chain of alpha, beta, delta or gamma, and/or two ORFs encoding a pair of analyte TCR chains wherein i may by obtained by selection of a naturally occurring cell population lacking said expression, or may be engineered to lack such expression; ii may by obtained by selection of a naturally occurring cell population comprising said expression, or may be engineered to comprise such expression; iii may be or introduced to the genome by means of genetic engineering.

The selection of an eTPC cell candidate that lacks TCR chains alpha, beta, delta and gamma from naturally occurring cell populations can be achieved by methods well known in the art. Staining of target cells with affinity reagents cally for TCR chains alpha, beta, delta and gamma, and selection of cells TCR chains alpha, beta, delta and gamma may directly achieve this.

Engineering of eTPC to lack TCR chains alpha, beta, delta and gamma sion may be achieved by untargeted and targeted means. eted mutagenesis of the cell can be achieved by providing a chemical, radiological or other mutagen to the cell, and then selecting cells lacking target aAPX and/or aAM expression. Targeted muta— tion of the genomic loci can be achieved via different means, including but not limited to site directed mutagenesis via i. zinc-finger nucleases ii. CRISPR/CasQ mediated ing (correct and acronym) iii. Synthetic transcription activator-like effector nucleases (TALEN) wherein said site-directed ses induce site-specific DNA-repair error nesis at target loci, after which mutated cells are obtained by selecting cells lacking TCR al- pha, beta, delta and gamma expression.

Options for Integration of CD3 and the components B and/or D are well known to those skilled in the art but may include homology directed recombination (HDR) and/or ran- dom ation methods, n HDR may be promoted by targeted on of the genomic loci at which HDR is to occur, and can be achieved via different means, in— cluding but not limited to site directed mutagenesis via i. zinc-finger nucleases ii. CRISPR/Cas9 mediated targeting iii. Synthetic transcription activator-like effector nucleases (TALEN) wherein said site-directed nucleases induce site-specific DNA-repair by HDR at target loci. After such events, a proportion of cells will have incorporated HDR vector, an can be selected and/or determined via any combination of the ing, iv. Non-destructive phonotypical sion analysis v. Destructive phonotypical expression analysis vi. Genetic analysis Wherein iv and vi are the red methods for selection and determination of suc- cessful genomic integration events.

Alternatively, viral vectors could be used to deliver the required components in a site- directed or undirected manner.

Considering that the eTPC component 2A is designed to be used in conjunction with the above described eAPC component 1A, within a combined eAPC:eTPC system, for the purpose of analyses of TCR and T—cell antigen interactions (Figure 1), in the pre— ferred aspect the eTPC contains features that minimise the eTPC presenting factors that would interfere in such analyses.

The eTPC component 2A optionally lacks endogenous surface expression of at least one family of aAPX and/or aAM, wherein the lack of surface sion is selected as to minimise interference in matched analyses of target e ns.

The family of aAPX may be any of the following i. HLA class I ii. HLA class M iii. non-HLA antigen-presenting complex.

An aAM is selected from i. a polypeptide or complex of polypeptides translated from the analyte antigenic molecule ORF(s) ii. a peptide d from a polypeptide translated from the analyte antigenic mole- cule ORF(s) iii. a peptide derived from altering the ent A proteome iv. a ptide derived from altering the ent A proteome v. a metabolite d from altering the component A metabolome The component 2A eTPC may optionally additionally include T-cell co-receptors, wherein such features permit robust or varying forms of communication of the analyte eTPC to the analyte eAPC, wherein the tuneable communication is relevant to identifi- cation or characterisation of ic e TCRsp and/or analyte antigens.

The eTPC component 2A may optionally express CD4 and/or CD8, wherein expression of CD4 or CD8 restrict eTPC to engaging aAPX of type HLAll and HLAl, respectively.

In the present context, the eTPC component 2A may optionally expresses CD28 and/or CD45, wherein CD28 and CD45 contribute to signal sensitivity h positive feed forward effects on signalling, whereas they may also contribute to signal specificity through negatie feed back effects on signalling, as it relates to signalling though an ex- pressed analyte TCRsp.

The component 2A eTPC may optionally additionally include introduced cell surface adhesion molecule components, or ablation of endogenous cell surface adhesion mole- cules, to e the eTPC ment with analyte eAPC and formation of the im— munological synapse, or to avoid tight binding and formation of deleterious cell cluster- ing within the combined TPC system, respectively.

Such on molecules that may be introduced as additional ORFs to component 2A, or genetically ablated from 2A, can be selected from the integrin family of adhesion proteins.

The second component of the minimal multicomponent is a genetic donor vector, com- ponent 2C, which is used for integration of at least one ORF encoding at least one an- alyte TCR chain (Figure 18).

Component 2C is a genetic donor vector that is coupled with the c receiver site of the, ZB, contained within the genome of the eTPC, Component 2A. Component 2C is designed for the ation of one or more ORFs encoding an e TCR chain, encoded in the genetic donor vector, into the genomic receiver site, 2B, wherein inte- gration results in the expression of e TCR chains by the target eTPC.

A paired genetic donor vector and genomic receiver site is described as an integration couple.

An eTPC is designed to respond to stimulation by cognate antigen, as presented by the eAPC within the combined TCP system. It is thus desirable to have a standard- ised reporter readout for signalling response from stimulation of the expressed TCRsp.

In the t context, the eTPC component 2A, further contains a component desig- nated 2F, a synthetic genomic TCR-stimulation response element selected from i. A single component synthetic construct containing at least one native promoter and/or at least one synthetic er and at least one reporter ii. A multi-component synthetic construct designed with at least one native pro- moter and/or at least one tic promoter and at least one reporter wherein tion ofi and/or ii is dependent on at least one signal transduction path— way ed from a synthetic pathway, a native y or a combination thereof.

A eTPC is designed to assay ment of the presented TCRsp with a analyte anti- gen. The readout of engagement may be achieved through induction of a synthetic re— porter element that responds to TCRsp engagement. Therefore, the eTPC, may further contain a component ated 2F, a synthetic genomic TOR-stimulation response element selected from i. A single component synthetic construct containing at least one native er and/or at least one synthetic promoter and at least one reporter ii. A multi-component synthetic construct designed with at least one native pro- moter and/or at least one synthetic promoter and at least one reporter wherein activation ofi and/or ii is dependent on at least one signal transduction path- way selected from a synthetic y, a native pathway or a combination thereof.

Synthetic TCR—stimulation response elements (component 2F), with synthetic as op- posed to native promoters, are less likely to mimic a natural TCR ation response and thus represent a more distant approximation to natural T-cell stimulation. However, synthetic promoters provide greater flexibility for tuning and adjustment of the er response for the robust identification of eTPC-t presenting TCRsp that productively en- gage analyte antigen.

Single component synthetic constructs provide limited space for amplification or modu- lation of the signal er se, but are more straightforward to implement and predict the outcome. Multi-component constructs have the age of having a virtu- ally unlimited space to construct complex response networks, however, this comes with the cost of being more difficult to implement and predict. A network also provides in- stances to initiate feedback loops, repression and activation of secondary response re- porters.

Synthetic promoters can be linked to artificial synthetic ling components, which can be either downstream of the initial natural TCR signalling pathway or substitute it.

In such an instance, the CD3 complex can be coupled in-part or wholly to an artificial synthetic signalling pathway and se network. Such artificial pathways provide the advantage of being very modular and adaptable, for greater fine tuning or in- creased variety of responses.

The preferred embodiment utilises a multi-component synthetic construct with synthetic promoters and at least a partial tic signalling pathway. This provides the most ro- bust means to ensure d resting state signal response but with high coupled re— porter signal when ligation of analyte antigen to the TCRsp occurs.

Regardless of the exact architecture of coupling the TCRsp stimulation to a component 2F the desired end outcome is a detectable reporter. In the preferred embodiment, a reporter that is amenable to fluorescent detection (i.e. FACS) and/or al selection methods such as Magnetic Activated Cell Soting (MACS) is d. atively, the reporter could be a secreted or intracellular molecule for detection by ometric, fluorescent or luminescent assays, preferably in a non-destructive , wherein the populations can subsequently be selected based on the presence or absence of the re- porter molecule. In on, the response is not limited to a single reporter type but could be a combination of one or more different reporters.

In one context, a multi—component synthetic construct with synthetic promoters and a partial synthetic ling pathway (Driver—Activator/Repressor + Amplifier—Reporter) would comprise of: Driver—Activator/Repressor: a) a tic promoter b) a Kozak sequence 0) a transcription factor, d) a first terminator Amplifier-Reporter e) a second synthetic promoter f) a Kozak sequence g) a reporter h) a second terminator wherein; a) Represents a synthetic promoter (Driver) that is designed to be coupled to the initial natural signalling pathway generated by TCRsp ligation to the e anti- gen. A minimum driver comprises of one or more transcription factor binding site and a core promoter, wherein the core promoter comprises, at a minimum, of a TATA Box, Initiator element (lnr) and transcriptional start site; b) Represents a Kozak sequence for ent translational initiation of the transcription factor; c) Represents an ORF encod- ing a synthetic or natural transcription factor (Activator, or Repressor), which can bind to the DNA ce of the second synthetic promoter; d) Represents a transcriptional ator for efficient transcription and polyadenylation of the transcription factor mRNA transcript, and optional one or more untranslated 3’ genetic element(s). These genetic elements may include, but are not d to, transcript izing or destabiliz- ing elements, and unique identifier ces; e) Represents a second synthetic pro- moter (Amplifier), which encodes one or more recognition sites for binding of the c) transcription factor and a core promoter, wherein the core er comprisess, at a minimum, of a TATA Box, lnr and transcriptional start site; f) Represents a Kozak se- quence for efficient translational initiation of the reporter; g) Represents an ORF encod- ing a reporter protein; h) Represents a transcriptional terminator for efficient transcrip- tion and polyadenylation of the reporter mRNA ript, and optional one or more un- translated 3’ genetic t(s). These genetic ts may e, but are not lim- ited to, transcript stabilizing or destabilizing elements, and unique fier sequences.

It is important to recognise that the Driver-Activator/Repressor coupled to Amplifier—Re- porter paradigm can be extended to include additional Driver—Activator/Repressor and Amplifier-Reporter pairs and/or additional Amplifier-Reporter units. Furthermore, Acti- vators can be replaced with Repressors to shutdown Amplifier-Reporter units and/or Driver—Activator/Repressor units, for ve selection methods and/or negative-feed- back loops.

In a second context, a single-component synthetic construct with synthetic promoters (Driver—Reporter) would comprise of: a) a synthetic promoter b) a Kozak sequence c) a reporter d) a terminator wherein; a) Represents a synthetic promoter (Driver) that is designed to be coupled to the initial natural signalling pathway generated by TCRsp ligation to the e anti- gen. A minimum driver comprises of one or more transcription factor binding site and a core er, wherein the core promoter comprises, at a m, of a TATA Box, Inr and transcriptional start site; b) Represents a Kozak sequence for ent translational initiation of the reporter; c) Represents an ORF encoding a reporter protein; d) Repre- sents a transcriptional terminator for efficient ription and polyadenylation of the reporter mRNA transcript, and optional one or more untranslated 3’ genetic element(s).

These genetic ts may include, but are not limited to, transcript stabilizing or de— stabilizing ts, and unique identifier sequences.

In an expanded form or the multicomponent eTPCS, the component 2A eTPC may fur- ther contain a second genomic receiver site, designated component 2D, which is cou- pled to a second genomic donor , designated component 2E, that is also added to the system (Figure 19).

An eTPCS component 2A may further comprise one or more additional ation couples.

An eTPCS, comprising an eTPC and either one or two integration couples, is used for preparation of the eTPC-t used for assembly of the combined eAPC:eTPC system.

An eTPC-t may be prepared by integration of two complementary TCR chains to form a TCRsp, or may alternatively may be prepared by two steps by providing each comple- mentary chain sequentially (Figure 20).

The genetic donor vector and genomic receiver sites operate as an integration couple subsystem of the eTPCS. A genetic donor vector must first be combined with target ORFs, such that base donor vector now encodes those target ORFs. The assembled primed donor vector is then introduced to the target eTPC to exchange target ORF(s) to the c receiver site, thus integrating the target ORFs to coupled receiver site of the target cell (Figure 21).

An eTPCS that ses genetic donor vectors component 20 and/or 2E is combined with at least one ORF encoding at least one analyte TCR chain to obtain component 20’ and/or 2E’, wherein the combination is defined as the ligation of genetic material into the correct coding frame(s), and in the correct orientation(s), of the genetic donor vector.

The combination of one or more ORFs into c donor vectors 20 and/or 2E may be performed multiple times with a library of unique ORFs as i. single te reactions to obtain a te library of ZC’ and/or 2E’ vectors encoding multiple ORFs ii. a singe reaction to obtain a pooled library of 20’ and/or 2E’ vectors encoding multiple ORFs wherein a discrete y may be combined with ent 2A multiple times as to ob- tain a discrete library of eTPCs with unique ORFs encoding unique TCR chains, or a pooled library may be combined with component 2A one or more times as to obtain a pooled library of eTPC-t wherein each eTPC-t ates a ised ORFencoding analyte TCR chains derived from the original vector pool, such that each eTPC-t eX- presses a single random unique TCRsp The efficient integration of a table copy number of one or more ORFs into the ge- nomic receiver site is highly advantageous for ion of a standardised eTPC, where analyte eTPC populations may be rapidly prepared and terised. Thus, the genomic receiver site(s) and coupled donor vector(s) are critical to the function of the eTPC. Furthermore, it is strongly desirable to have an eTPC wherein component ZB and 2D, are insulated from one another, such that the donor vector component ZC can- not integrate at component ZB, and vice versa. In addition, it is also desirable that the component 28 and/or component 2D are amenable to a method of preparation of an eTPC—t wherein, the introduction of a single pair of complementary TCR chains is rapid, repeatable, with a high likelihood of correct integration and delivery of only a single pair.

The genomic receiver site may be selected from the following i. A synthetic construct designed for recombinase ed cassette exchange (RMCE) ii. A synthetic construct ed for site directed homologous recombination wherein i) is the preferred form a genomic receiver site for RCME. The RMCE method may employ selected heterospecific sites that are specific for individual recombinase enzymes, such that each component ZB and 2D possess insulated specificity.

The genomic receiver site, component ZB and/or component 2D comprises of at least one of the following genetic elements i. Heterospecific recombinase sites ii. Homologous arms iii. Eukaryotic promoter iv. Eukaryotic conditional regulatory element v. Eukaryotic ator vi. Selection marker vii. Splice acceptor site viii. Splice donor site ix. Non-protein coding gene x. Insulator xi. Mobile genetic element xii. Meganuclease recognition site xiii. Internal ribosome entry site (IRES) Viral self-cleaving peptide element xiv. A kozak consensus sequence.

A preferred genomic receiver site would comprise of two different ements using the following selected elements from the previously stated list of element.

The first arrangement is for receiving a single ORF ng one or more TCR chains and/or a selection mark of integration, via RMCE ation wherein the arrangement ’ -[A] [B] [C] [D] [E] [F]- 3’ wherein A) is element iii) a constitutive or inducible Eukaryotic promoter B) is element i) heterospecific recombinase site 1 C) is element xv) a Kozak consensus sequence D) is element vi) a FACS and/or MACS compatible encoded protein marker E) is element i) heterospecific recombinase site 2 F) is element v) otic terminator The second arrangement is for receiving a two ORF encoding one or more TCR chains and/or a selection mark of integration, via RMCE ation n the arrangement 5’ -[A] [B] [C] [D] [E] [F] [G] [H] [|]- 3’ wherein A) is element iii) a tutive or inducible Eukaryotic promoter B) is element i) heterospecific recombinase site 1 C) is element xv) a Kozak consensus sequence D) is element vi) a FACS and/or MACS compatible encoded protein marker 1 E) is element v) a Eukaryotic bidirectional transcriptional terminator F) is element vi) a FACS and/or MACS ible encoded protein marker 2 G) is element xv) a Kozak consensus sequence H) is element i) heterospecific recombinase site 2 l) is element iii) a constitutive or inducible otic er furthermore, in this second arrangement the elements F, G, and l are d in the antisense direction Component 2C and/or 2E ses of at least one of the following genetic elements i. Heterospecific recombinase sites ii. Homologous arms iii. Eukaryotic promoter iv. otic conditional regulatory element v. Eukaryotic ator vi. ion marker vii. Splice acceptor site viii. Splice donor site ix. Non-protein coding gene x. Insulator xi. Mobile genetic element xii. Meganuclease recognition site xiii. Internal ribosome entry site (IRES) xiv. Viral self-cleaving peptide element xv. A kozak consensus sequence xvi. Selection marker of integration xvii. An antibiotic resistance cassette xviii. A bacterial origin of replication xix. A yeast origin of replication xx. A cloning site A preferred genetic donor vector, component C andior component E, would comprise of two different possible arrangements using the following selected elements from the previously stated list of elements.

The first arrangement is for receiving a single ORF ng one or more TCR chains and/or a selection mark of integration, via RMCE integration wherein the arrangement ’ - [A] [B] [C] [D] [E] - 3’ wherein A) is element i) heterospecific recombinase site 1 B) is element xv) a Kozak consensus sequence C) is element xx) a cloning site of a single ORF encoding one or more TCR chains and/or element xvi) a selection marker of ation D) is element i) heterospecific recombinase site 2 E) is element xvii) An antibiotic resistance cassette and element xviii) a bacterial origin of replication, in no specific orientation furthermore, the elements viii and/or xiv may be used to link multiple TCR chains and/or element xvi together.

’ - [A] [B] [C] [D] [E] [F]- 3’ A) is element i) heterospecific inase site 1 B) is element xv) a Kozak consensus sequence C) is element xx) a cloning site for introduction of two or more ORF, with eukary- otic terminators, encoding one or more TCR chains and/or element xvi) a selec- tion marker of integration D) is element xv) a Kozak consensus sequence (antisense direction) E) is t i) heterospecific recombinase site 2 F) is element xvii) An antibiotic resistance cassette and element xviii) a bacterial origin of replication, in no specific ation furthermore, the elements viii and/or xiv may be used to link multiple TCR chains and/or element xvi together within each ORF. ing e eTPC populations in the eTPCS The above described eTPCS system may be used in multiple ways to prepare distinct forms of analyte eTPC populations, or ies thereof, that serve to present analyte TCRsp to the eAPC within the combined eAPC:eTPC system in operation of the two- part device.

The eTPC-t populations that are created need to derive analyte TCR chains from cer- tain sources with which to analyse candidate antigens.

The sources of analyte TCR chain encoding sequences can be derived from i. Paired cloning of TCR chain ORF ce(s) from primary T-cells ii. Unpaired cloning of TCR chain ORF sequence(s) from y T-cells iii. Synthetic TCR chain ORF sequence(s) n i is able for discovery of native TCRsp that are not likely to be generally cross reactive against self aAPX and/or the aAPX cargo due to thymic selection; ii may be used to identify candidate TCR affinity reagents; may be used in affinity maturation of TCR affinity reagents.

An eTPCS comprising a single integration couple may be used to prepare an eTPC-t from component 2A in one step, by providing component 20’ ed with an ORF for complementary pair of TCR chains, such that this analyte TCR pair is ated to site ZB, to create 2B’. The resulting cell line expresses the provided TCR pair, and it is presented at the cell surface as a TCRsp (Figure 21).

An eTPCS comprising two integration couples may be used to e an eTPC-t from component 2A in one step, by providing component 20’ combined with an ORF for complementary pair of TCR chains, such that this analyte TCR pair is integrated to site 28, to create 28’. The resulting cell line expresses the provided TCR pair, and it is pre- sented at the cell surface as a TCRsp. The second integration couple 1D/1E remains unmodified and may be used for downstream integration steps (Figure 22).

An eTPCS comprising two integration couples may be used to prepare an eTPC-t from component 2A in one step, by providing component 20’ and 2E’ each ed with one ORF encoding one chain of a complementary TCR chain pair, such that both ana- lyte TCR chains are integrated to site 28 or 2D, to create 28’ and 2D’. The resulting cell line expresses the provided TCR pair, and it is ted at the cell surface as a TCRsp. (Figure 23).

An eTPCS comprising two integration s may be used to prepare an eTPC-x from component 2A in one step, by providing component 20’ combined with an ORF for a single analyte TCR chain, such that this analyte TCR chain is integrated to site 28, to create 28’. The resulting cell line expresses the provided TCR chain, but lacks a com- tary chain and thus does not express the any surface TCR. The second integra- tion couple 1D/1 E remains unmodified and may be used for downstream integration steps (Figure 24).

An eTPCS comprising two integration couples may be used to prepare an eTPC-t from component 2A in two steps, by first providing component 20’ combined with an ORF for a single e TCR chain, such that this analyte TCR chain is integrated to site ZB, to create 28’. The resulting cell line expresses the provided TCR chain, but lacks a complementary chain and thus does not express the any surface TCR (eTPC-x inter- mediate). The second integration couple 1D/1 E s unmodified. In the second step 2E’ is provided, wherein the vector is combined with an ORF for a second single ana- lyte TCR chain, mentary to the previously integrated TCR chain, such that this second complementary analyte TCR chain is integrated to site 2D, to create 2D’. The resulting cell line expresses the provided complementary analyte TCR chain pair and it is presented at the cell surface as a TCRsp. (Figure 25).

In the abovementioned examples of preparing analyte eTPC-x and/or eTPC-t popula- tions from eTPC, the eTPCS system is used to provide known analyte TCR chains in a d manner to prepare discrete populations of analyte eTPC expressing defined TCRsp. Such a process may be repeated many times to build ies of eTCP-x and/or eTCP-t to provide to the combined eAPC:eTPC system in operation of the de- vice. An alternative approach is to take pooled libraries of analyte eTPC-t, wherein each eTPC-t integrates a single random pair of TCR ORF from the original vector pool, and thus each eTPC-t expresses a single random TCRsp, but the collectively the pool encodes multiple species of TCRsp represented in the original vector pool. The same shotgun principle can be applied to create pools of eTPC-x. This is particularly useful when analysing large libraries of candidate TCRsp against analyte antigens.

An eTPCS comprising one integration couple may be used to prepare an eTPC-t pool from component 2A in one step, by providing component 20’ ed with a library of multiple ORF encoding a pool analyte of analyte TCR pairs, such that a pair is integrated to site B, to create B’, within each cell and wherein each eTPC—t integrates a single random pair of TCR ORF from the original vector pool, and thus each eTPC-t ex- presses a single random TCRsp, but the tively the pool s le species of TCRsp represented in the al vector pool. The resulting pool of cells contains a collection of cells that collectively express multiple analyte TCRsp (Figure 26).

An eTPCS comprising two integration couples may be used to prepare an eTPC-t pool from component 2A in one step, by providing component 20’ combined with a library of multiple ORF encoding a pool of analyte TCR pairs, such that a pair is integrated to site B, to create B’, within each cell and n each eTPC integrates a single random pair of TCR ORF from the original vector pool, and thus each eTPC-t expresses a single random TCRsp, but the collectively the pool s multiple species of TCRsp repre— sented in the original vector pool. The resulting pool of cells contains a collection of cells that collectively express multiple analyte TCRsp. The second integration couple 1D/1E s fied (Figure 27).

An eTPCS comprising two integration couples may be used to prepare an eTPC-t pool from component 2A in one step, by providing component 20’ ed with a library of multiple ORF encoding a pool of single analyte TCR chains such that each eTPC inte- grates a single randomised TCR chain ORF encoded in component 20’ to site 2B, to create ZB’. Simultaneously, providing component 2E’ combined with a library of multi- ple ORF encoding a pool of single analyte TCR chains complementary to first library provided in 2C’, such that each eTPC—t integrates a single ised complementary TCR chain ORF encoded in component 2E’ into site 2D, to create 2D’. Each resulting cell in the eTPC-t pool has a single pair of randomised complementary TCR chain ORF, such that each cell in the pool expresses a randomised TCRsp. Such a pooled library would contain all possible combinations of provided complementary TCR chains from the sets proceed in components 20’ and 2E’ (Figure 28).

In the present t, an eTPCS comprising two integration s may be used to prepare an eTPC-t pool from a previously obtained e—TPC-x in one step, wherein the site ZB has been converted to ZB’ and contains the single analyte TCR chain. An eTPC—t is prepared by providing component 2E’ combined with a library of le ORF encoding a pool of single analyte TCR chains complementary to the already inte- grated chain, such that each such that each eTPC-t integrates a single randomised TCR chain ORF of the provided 2E’ library in to site 2D, to create 2D’. Each resulting cell in the eTPC—t pool has the analyte TCR chain provided by the ng eTPC-x, and a ion of each single complementary analyte TCR chain, such that each cell in the pool expresses a random pair of ORF as an TCRsp. Such an approach is used when analysing the effect of g a single chain against a fixed chain in a complementary TCR chain pair (Figure 29).

An eTPC-x may be prepared from an eTPC-t by providing either one of further integra- tion vectors, components 2Y or 22, which encode markers of integration or no ORF.

Combination of component 2Y or 22 to an eTPC-t would ge either of the sites to obtain a single TCR chain expressing eTPC-x.

A preferred c integration vector, component 2Y and component 22, for - sion of eTPC-t to eTPC-x would se the same integration vector requirements as component 20 and 2E above, though not encoding any TCR chain ORF, and prefera- bly encoding a marker of integration.

Contacting analyte eAPC and eTPC The present invention relates to the provision of an engineered two—part cellular device.

The two parts of the device, eAPCS and eTPCS, are used to assemble analyte eAPC and analyte eTPC, respectively. These analyte cell populations are then assembled into a ed TPC system from which the device outputs may be obtained.

The eAPC:eTPC system is comprised of a selection of one or more of analyte eAPC populations, and one or more eTPC populations (Figure 1) that are ed as de— scribed above (Figures 4 to 29). The eAPC:eTPC system is provided in a format that permits physical contact between the analyte eAPC and analyte eTPC populations, wherein such contact is permissive of complex formation between one ore more ana- lyte antigen and TCRsp of one or more analyte eTPC—t, wherein the analyte antigen is any of the following i. aAPX and/or ii. aAM and/or iii. aAPXzaAM and/or iv. CM and/or 3O v. aAPX:CM presented by the analyte eAPC, with an analyte TCRsp presented by the analyte eTPC, such that complex formation may lead to stabilisation of such a complex and wherein the ion of signalling within the e eAPC and/or the analyte eTPC may be reported and measured.

A contact between an e eAPC and analyte eTPC is performed in a permissive cell culture system, wherein said system ses cell culture media that is permis- sive to function of both eAPC and eTPC cells.

An analyte eTPC obtained from the ed TPC system is used for charac- terisation of a signal response of the analyte eTPC, expressing e TCRsp, to an analyte antigen presented by the analyte eAPC, wherein such a signal response may be either binary or graduated, and may be measured as intrinsic to the eTPC (Figure ) or intrinsic to the eAPC (Figure 31).

The method for selecting one or more analyte eTPC from an input analyte eTPC or a library of analyte eTPC, from the combined eAPC:eTPC system, to obtain one or more analyte eTPC wherein the expressed TCRsp binds to one or more analyte n pre- sented by the analyte eAPC comprises i. Combining one or more analyte eTPC with one or more analyte eAPC resulting in a contact n an analyte TCRsp with an analyte antigen and at least one ii. Measuring a formation, if any, of a x between one or more analyte TCRsp with one or more analyte antigen and/or iii. Measuring a signal response by the analyte eTPC, if any, induced by the for- mation of a complex between one or more analyte TCRsp with one or more an- alyte antigen and/or iv. Measuring a signal response by the analyte eAPC, if any, induced by the for- mation of a complex between one or more analyte TCRsp with one or more an- alyte antigen and v. Selecting one or more analyte eTPC based on step b, c and/or d wherein the selection is made by a positive and/or negative measurement n i, iii and v comprise the preferred arrangement.

An analyte eAPC ed from the combined eAPC:eTPC system is used for - terisation of a signal response of the analyte eAPC, expressing e antigen, to an TCRsp presented by the analyte eTPC, wherein such a signal response may be either binary or graduated, and may be measured as intrinsic to the eTPC (Figure 30) or in- trinsic to the eAPC (Figure 31).

The method for selecting one or more analyte eAPC from an input analyte eAPC or a library of analyte eAPC, from the combined eAPC:eTPC system, to obtain one or more e eAPC wherein the expressed analyte n binds to one or more analyte TCRsp presented by the analyte eTPC ses i. Combining one or more analyte eAPC with one or more analyte eTPC, resulting in a contact between an analyte antigen ted by the analyte eAPC with analyte TCRsp of one or more analyte eTPC and ii. Measuring a formation, if any, of a complex between one or more analyte anti— gen with one or more analyte TCRsp and/or iii. Measuring a signal response in the one or more analyte eTPC, if any, induced by the formation of a complex between the analyte TCRsp with the analyte anti- gen and/or iv. ing a signal response, if any, by the analyte eAPC induced by the for- mation of a complex n one or more analyte TCRsp with one or more an- alyte antigen and v. Selecting one or more analyte eAPC from step b, c and/or d wherein the selec- tion is made by a positive and/or negative measurement wherein i, iii and v comprise the preferred arrangement. ion of eTPC or eAPC may be from an eAPC:eTPC system comprising binary cul- ture system wherein a single prepared analyte eAPC tion and single ed analyte eTPC population are provided, and the responding eAPC or eTPC are selected on the basis of a binary or graduated response within the eTPC (Figure 30) and/or the eAPC (Figure 31).

An eAPC:eTPC system may comprise an input of prepared single analyte eAPC and a prepared pooled library of eTPC (Figure 32).

An eAPC:eTPC system may comprise an input of ed single analyte eTPC and a prepared pooled library of eAPC (Figure 33).

Within the combined eATP:eTPC system, measuring a signal response in the one or more analyte eTPC or one or more eAPC, if any, which may be induced by the for- mation of a complex between the analyte TCRsp with the analyte antigen is critical to selection of primary device outputs, wherein the primary device outputs are single cells or pools of cells of one or more of the following i. eAPC-p ii. eAPC-a iii. eAPC-pa iv. eTPC-t wherein the selection of cells may be made on the presence or absence of a ed signal response in either, or both, of the contacted analyte eAPC or analyte eTPC cells.

In the present t, a method for selecting analyte eTPC and/or analyte eAPC from the combined eAPC:eTPC system on the basis of a reported signal response within the eTPC comprises i. Determining a native signalling response and/or ii. Determining a synthetic signalling se, if the eTPC contains component 2F, and if the eAPC contains an equivalent tic reporter element.

An induced native or synthetic signal response that is intrinsic to eAPC and/or eTPC is measured by detecting an increase or decrease in one or more of the following i. a secreted biomolecule ii. a secreted chemical iii. an intracellular biomolecule iv. an intracellular chemical v. a surface expressed biomolecule vi. a cytotoxic action of the analyte eTPC upon the analyte eAPC vii. a paracrine action of the analyte eTPC upon the analyte eAPC such that a sig- nal response is induced in the analyte eAPC and is determined by detecting an increase or decrease any of a to e viii. a proliferation of the analyte eTPC ix. an immunological synapse between the analyte eTPC and the analyte eAPC wherein said detected signal responses are compared to the non-induced signal re- sponse state intrinsic to analyte eAPC and/or analyte eTPC prior to assemble of the combined eAPC:eTPC system and/or a parallel assembled combined system wherein e eAPC and/or analyte eTPC may present control analyte n and/or e TCR species that are known not to the induce a signal response within the ed eAPC:eTPC system in use. ing primary device outputs from the eAPC:e TPC system The present ion relates to the provision of an ered two—part cellular device.

The two parts of the device, eAPCS and eTPCS, are used to assemble analyte eAPC and analyte eTPC, respectively. These analyte cell populations are then assembled into a combined eAPC:eTPC system from which the device outputs may be obtained.

The eAPC:eTPC system is comprised of a selection of one or more of analyte eAPC populations, and one or more eTPC populations e 1) that are prepared as de- scribed above (Figures 4 to 29). The eAPC:eTPC system is provided in a format that permits physical contact between the analyte eAPC and analyte eTPC populations, wherein such contact is permissive of complex formation between one ore more ana— lyte antigen and TCRsp of one or more e eTPC-t, wherein the e antigen is any of the following i. aAPX and/or ii. aAM and/or iii. aAPX:aAM and/or iv. CM and/or v. aAPX:CM presented by the analyte eAPC, with an analyte TCRsp presented by the analyte eTPC, such that complex formation may lead to stabilisation of such a complex and wherein the induction of signalling within the e eAPC and/or the analyte eTPC may be reported and measured.

The modes of d signal response ing are described above, and it is these reported responses that are required to be measured in obtaining the y output of the two-part device.

Primary outputs from the device are selected cell populations, which have or have not responded to the analyte presented by the reciprocal cell provided in the eAPC:eTPC system. That is, such a primary output may be ented as a single cell, or a pool of cells, that have been selected on the ce or absence of a reported response within the combined eAPC:eTPC system (Figure 1 step v). A response within an ana- lyte eAPC is only ed by engagement of a cognate TCR presented by a contacting eTPC. A response within an analyte eTPC is only provoked by engagement of a cognate antigen presented by a contacting eAPC (figure 1 step iv).

A selection of analyte eAPC and/or analyte eTPC from the ed eAPC:eTPC sys- tem may be made on the basis of a response in the contacting cell. That is, an analyte eAPC may be selected on that basis of a reported response, or lack thereof, in the con- tacting analyte eTPC. Conversely, an analyte eTPC may be selected on that basis of a ed response, or lack thereof, in the contacting analyte eAPC.

In the t context, primary eAPC outputs from the device are selected cells, wherein selection is made based on the presence or absence of a reported signal re— sponse, and these cells may comprise one or more of i. an eAPC-p ii. an eAPC-a iii. an eAPC—pa wherein the selected cells may comprise a single cell, a pool of cells of the same iden- tity, a pool of cells of ent identities (Figure 1 step v).

Primary eTPC outputs from the device are selected cells, wherein selection is made based on the presence or absence of a reported signal response, and these cells com- prise eTPC-t, wherein selected cells may comprise a single cell, a pool of cells of the same identity, a pool of cells of different identities (Figure 1 step v).

The reported s in the analyte eAPC and/or analyte eTPC in a combined TPC system is used to select analyte cell populations to provide the primary outputs.

A primary output of eAPC and/or eTPC types may be achieved in a an instance wherein the combined eAPC:eTPC system is of binary culture nature (e.g. Figure 30 and Figure 31) by selecting the desired analyte eAPC and/0r analyte eTPC population from the binary system.

A y output of eAPC and/or eTPC types may be achieved from an instance wherein the combined eAPC:eTPC system is of fixed eAPC and pooled library analyte eTPC nature (e.g. Figure 32), or from an instance wherein the combined eAPC:eTPC system is of fixed eTPC and pooled library analyte eAPC nature (e.g. Figure 33) by se- lecting the desired analyte eAPC and/or analyte eTPC population from the combined culture .

There are several ct modes in which the primary outputs may be obtained, n each mode entails a step of cell sorting. Cell sorting may be achieved h scence-activated cell sorting (FACS) and/or magnetic-activated cell sorting (MACS) and/or distinct affinity-activated cell sorting methods.

Primary output eAPC and/or eTPC cells may be ed by single cell sorting to ob- tain a single cell and/0r cell sorting to a pool to obtain a pool of cells.

Primary output eAPC and/or eTPC cells may be obtained by single cell sorting to ob— tain a single cell, and subsequent outgrowth of the single cells to obtain monoclonal pool of ed eAPC or eTPC cells.

Primary output eAPC and/or eTPC cells may be obtained by cell sorting to a pool to ob- tain a pool of cells, and subsequent outgrowth of the pool of cells to obtain a pool of se- lected eAPC and/or eTPC cells.

Obtaining terminal device outputs from the eAPC:eTPC system Subsequent to the above-described methods of obtaining primary outputs, wherein pri- mary outputs are selected analyte eAPC and/or eTPC cells that are selected on the ba- sis of a measured signal response, the terminal s of the two part cellular device may be obtained via further processing of the selected eAPCa and/or eTPC primary outputs.

Terminal outputs from the two-part cellular device are the identities of i. aAPX and/or ii. aAM and/or iii. aAPX:aAM and/or iv. CM and/or v. aAPX:CM an/or vi. TCRsp presented by the analyte eAPC (i to v) or analyte eTPC (vi), and obtained as primary outputs from the two-part device by their selection from the combined eAPC:eTPC sys- Within the two-part cellular device, it is often the case that e molecules that are presented by the analyte eAPC and analyte eTPC are genetically encoded. ore, to identify the analyte les presented by the analyte eAPC or eTPC, genetic se- quencing of the prepared analyte molecules from the eAPC or eTPC may be per- formed. eAPC may be sed such that genetic sequence is obtained for component 18’ and/or component 1D’ of the sorted and/or expanded , eAPC-a or eAPC-pa cells to determine the identity of i. aAPX and/or ii. aAM and/or iii. aAPX:aAM wherein the ed identities represent al outputs from the two-part cellular de- vice. eAPC may be processed such that genetic sequence is obtained for the genome of the sorted and/or expanded eAPC-p, eAPC-a or eAPC-pa cells to determine the identity of i. aAPX and/or ii. aAM and/or iii. aAPX:aAM iv. CM and/or v. aAPX:CM n the obtained identities ent terminal outputs from the two-part cellular de- vice. eTPC may be processed such that genetic ce is obtained for component 28’ and/or component 2D’ of the sorted and/or expanded eTPC-t cells to determine the identity of TCRsp, wherein the obtained identity of TCRsp represents a terminal output from the two-part cellular device. eTPC may be processed such that genetic sequence is obtained for the genome of the sorted and/or expanded eTPC—t cells to determine the identity of TCRsp, wherein the obtained identify of TCRsp ents a terminal output from the two-part cellular de- vice.

Genetic sequencing can be achieved by a range of modes, and from arrange genetic material sources, with and without specific processing.

In the present context, the sequencing step may be preceded by i. Extracting of genomic DNA and/or ii. Extracting of components 18’ and/or 1D’ andlor ZB’ and/or 2D’ RNA transcript and/or iii. Amplifying by a PCR and/or a RT-PCR of the DNA and/or RNA ript of component 18’ and/or 1 D’ and/or ZB’ and/or 2D’.

The sequencing step may be destructive to the eAPC or eTPC, or pool thereof, ob- tained as primary s from the two-part device.

If it is desirable to obtain primary s from the two-part device wherein the se- quencing step has been destructive to the primary output eAPC or eTPC, the sequence information ed as terminal output of the two-part device may be used to prepare equivalent output eAPC and eTPC as analyte eAPC or analyte eTPC through use of the eAPCS and eTPCS, tively.

In the above-described scenarios of genetically encoded analyte molecules, the termi- nal outputs of the rt device may be ed by obtaining sequence information from component 18’ and/or 1D’ and/or ZB’ and/or 2D’, and/or from the cell genome.

However, in some embodiments the antigen information will not be cally en- coded. Post—transnationally modified antigens, antigens provided to the combined eAPC:eTPC system h non-genetic means, antigens that are emergent from an induced or modified state of the analyte eAPC proteome or metabolite, and CM intrin- sic to the eAPC:eTPC system, may not reasonably be identified through genetic means.

In the important case of aAM that may be provided to the TPC system by non- genetic means, there are two ct modes through which an eAPC-p or eAPC-pa may present a provided aAM as an aAPX:aAM complex. In the first scenario the aAM is provided in a form that may directly bind to the aAPX and forms an aAPX:aAM com- plex at the cells surface (Figure 34). An example of such an aAM would be a peptide antigen for an HLA complex. In the second scenario, the aAM is provided is in a form that may be taken up by the analyte eAPC and processed such that it is loaded as cargo in the aAPX and forms an aAPX:aAM complex at the cells surface (Figure 35).

In the present context, a method to select and identify an aAM cargo or a CM cargo, wherein the cargo is a metabolite and/or a peptide, that is loaded in an aAPX of an eAPC selected and obtained by as a a primary output of the two-part device, com- pnses i. isolating an aAPX:aAM or an aAPX:CM or the cargo aM or the cargo CM and ii. identifying the loaded cargo wherein the identified loaded cargo ent terminal outputs of the two-part device.

There are generally two modes through which a cargo molecule may be identified from a selected eAPC. First, a forced release of the cargo from the aAPX:aAM or aAPX:CM results in isolation of the aAM or CM that is available for uent identification (Fig- ure 36). An example of this would be acid-washing of the eAPC to liberate peptide aAM from HLA complexes. Secondly, the capture of the aAPX:aAM or aAPX:CM, for example, by liberation of the complex and immunoaffinity isolation methods, results in isolation of the aAPX:aAM or aAPX:CM compelxes, such that aAM or CM can be iden- tified (Figure 37). 1O Methods for identifying isolated aAM and/or CM directly, or from the isolated aAPX:aAM or an aAPX:CM complexes, can se i. Mass-spectrometry analysis ii. Peptide cing analysis wherein the ontain aAM and/or CM identities are terminal outputs from the two-part cel- lular device.

Legends to ?gures The invention is illustrated in the following non-limiting figures.

Figure 1 - Operation of the rt device comprised of eAPC and eTPC sys- The two-part engineered cellular device is comprised of two omponent cell sys- tems, the eAPCS and the eTPCS, which are contacted as a combined eAPC:eTPC system. Operation of the overall device comprises two phases, the preparation phase, and the analytical phase. In one aspect of Phase 1, the eAPCS system is used to pre- pare cells expressing analyte n-presenting complex (aAPX), and/or analyte anti- genic molecule (aAM) at the cell surface (step i). An eAPC presenting aAPX alone is termed . An eAPC presenting aAM alone is termed eAPC-a. An eAPC pre- senting an aAM presented as cargo in an aAPX is termed an eAPC-pa. These ana— lytes are collectively referred to as the analyte antigen. In a te aspect of Phase 1, the eTPCS system is used to prepare cells expressing analyte TCR chain pairs (TCRsp) at the cell surface (step ii). An eTPC ting a TCRsp at the cell surface is termed an eTPC-t. Phase 2 of the overall system is the contacting of the analyte— bearing cells prepared in Phase 1, to form the ed eAPC:eTPC system (step iii). Contacted analyte eAPC present analyte antigens to the analyte eTPC. Within the combined TPC system, the responsiveness of the analyte TCR chain pair to- wards the provided e antigen is determined by readout of a contact-dependent analyte eAPC and/or eTPC response, as denoted by * and the shaded box to repre- sent an altered signal state of these reporting analyte cells (step iv). As an outcome of the eAPC:eTPC system specific eAPC and/or eTPC-t can be ed based on their response and/or their ability to drive a response in the other contacting analyte cell. It is thus selected single cells, or populations of cells, of the type, , eAPC-a, eAPC- pa and/or eTPC-t that are the primary outputs of the device operation (step v). By obtaining the analyte cells from step v, the presented analyte aAPX, aAM, aAPX:aAM, 1O CM, aAPX:CM and/or TCRsp may be identified from these cells as the al out- put of the device operation (step vi).

Figure 2 - Description of the components of a single integration couple eAPCS.

An example of an eAPCS comprising three components. The first component 1A is the eAPC line itself with all required engineered features of that cell. The eAPC 1A con- tains one further component 13, which is a genomic integration site for ation of aAPX and/or aAM. One additional component, 1C represents a genetic donor vector for site-directed integration of ORFs into sites 13, wherein the arrow indicates coupled specificity. The paired integration site / donor vector couple may be formatted to inte- grate a single ORF or a pair of ORFs to introduce aAPX and/or aAM expression.

Figure 3 - Description of the components of a dual integration couple eAPCS.

An example of an eAPCS comprising five components. The first component 1A is the eAPC line itself with all required engineered es of that cell. The eAPC 1A con- tains two further components, 1B and 1D, which are genomic integration sites for inte- gration of aAPX and/or aAM. Two additional components, 1C and 1E, represent ge- netic donor vectors for irected integration of ORFs into sites 1B and 1D, respec- tively, wherein arrows indicate paired icity. Each paired integration site / donor vector couple may be formatted to integrate a single ORF or a pair of ORFs to intro— duce aAPX and/or aAM expression.

Figure 4 - Compilation of different analyte antigen presenting eAPC The eAPCS system begins with the eAPC and uses a donor vector(s) to create cells expressing analyte antigen-presenting x , and/or analyte antigenic mole- cule (aAM) at the cell surface. An eAPC presenting aAPX alone is termed eAPC-p, and may be created by introduction of aAPX encoding ORF(s) to the eAPC (step i). An eAPC expressing aAM alone is termed eAPC-a, wherein aAM may be expressed at the cell surface and ble for TCR engagement, or require processing and loading as cargo into an aAPX as the AM complex. An eAPC 1A may be created by in- troduction of aAM encoding ORF(s) to the eAPC (step ii). An eAPC presenting an aAM as cargo in an aAPX is termed an eAPC-pa. An eAPC-pa be produced either; in- troduction of aAM and aAPX encoding ORFs to an eAPC aneously (step iii); in— troduction of aAM encoding ORF(s) to an eAPC-p (step iv); introduction aAPX encod- ing ORF(s) to an eAPC-a (step v). 1O Figure 5 - Operation of the genetic donor vector and genomic receiver site inte- gration couple A genetic donor vector and genomic er site form an integration couple, wherein one or more ORFs encoded within the genetic donor vector can integrated specifically to its coupled c receiver site. Step 1 in operation of the integration couple is to introduce one or more target ORFs to the donor vector. The initial donor vector is de- noted X, and is modified to a primed donor vector X’, by introduction of target .

Step 2 entails combination of the primed donor vector, X’, with a cell harbouring a ge- nomic receiver site, Y. Introduction of the ORF encoded by the primed donor vector into the receiver site results in the creation of a cell harbouring an integrated site, Y’.

Figure 6 - Example of compilation of an eAPC-p in one step with one integration couple eAPC 1A contains genomic receiver site 1B. Primed genetic donor vector 1C’ is cou- pled to 1B and encodes an aAPX. When the 1A eAPC is ed with the 1C’ donor vector. The resulting cell has the ORF of 1C’ exchanged to the 1B genomic receiver site to create site 13’ and introduce aAPX expression. This results in expression of the aAPX on the cell surface and creation of an eAPC-p.

Figure 7 - Example of ation of an eAPC-p in one step with one integration couple and one unused integration site eAPC 1A contains genomic receiver sites 13 and 1D. Primed genetic donor vector 1C’ is coupled to 1B and encodes an aAPX. When the 1A eAPC is combined with the 1C’ donor vector. The resulting cell has the ORF of 1C’ exchanged to the 1B genomic re— ceiver site to create site 1B’ and introduce aAPX expression. This s in expression of the aAPX on the cell surface and on of an eAPC-p. c receiver site 1D remains unused.

Figure 8 - Example of compilation of an eAPC-a in one step with one integration couple eAPC 1A contains genomic receiver site 1B. Primed genetic donor vector 1C’ is cou— pled to 1B and encodes an aAM. When the 1A eAPC is combined with the 1C’ donor vector. The resulting cell has the ORF of 1C’ exchanged to the 1B genomic receiver site to create site 1B’ and introduce aAM expression. This results in one of two forms of , expressing aAM at the cell surface or ellularly. 1O Figure 9 - Example of compilation of an eAPC-a in one step with one integration couple and one unused integration site eAPC 1A contains genomic receiver sites 13 and 1D. Primed c donor vector 1C’ is coupled to 1B and encodes an aAM. When the 1A eAPC is combined with the 1C’ donor . The resulting cell has the ORF of 1C’ exchanged to the 1B genomic re- ceiver site to create site 1B’ and introduce aAM expression. This results in one of two forms of eAPC-a, sing aAM at the cell surface or intracellularly. Genomic re- ceiver site 1D s unused.

Figure 10 - Example of compilation of an eAPC-pa in one step with one integra- tion couple eAPC 1A contains genomic receiver site 1B. Genetic donor vector 1C’ is coupled to 1B. Donor vector 1C’ encodes an aAPX as well as an aAM.

The 1A eAPC is combined with donor vectors 1C’. The resulting cell has the ORFs 1C’ exchanged to the 1B genomic receiver site to create site 1B’ and deliver an ORF for an aAPX and an aAM. This results in expression of the aAPX on the cell surface, aAM in- tracellularly, and thus loading of the aAM as cargo in the aAPX in formation of the aAPX:aAM complex at the cell surface.

Figure 11 - Example of compilation of an eAPC-pa in one step with one integra- tion couple and one unused integration site eAPC 1A contains distinct genomic receiver sites 1B and 1D. Genetic donor vector 1C’ is d to 1B. Donor vector 1C’ encodes an aAPX as well as an aAM. The 1A eAPC is combined with donor vectors 1C’. The resulting cell has the ORFs 1C’ ex- changed to the 1B genomic er site to create site 1B’ and deliver an ORF for an aAPX and an aAM. Genomic receiver site 1D remains unused. This results in expres- sion of the aAPX on the cell e, aAM intracellularly, and thus loading of the aAM as cargo in the aAPX in formation of the aAPX:aAM complex at the cell surface. This creates an eAPC-pa cell line. c receiver site 1D remains unused.

Figure 12 - Example of compilation of an eAPC-pa in one step with two a- tion couples eAPC 1A contains distinct genomic receiver sites 13 and 1D. Distinct genetic donor vectors 1C’ and 1E’ are independently coupled to 1B and 1D, respectively. Donor vec- tor 1C’ encodes an aAPX and donor vector 1E’ encodes an aAM. The 1A eAPC is combined with donor vectors 1C’ and 1E’ simultaneously. The resulting cell has the 1O ORF 1C’ exchanged to the 1B genomic receiver site to create site 13’ and deliver an ORF for an aAPX. Simultaneously, the ORF of 1E’ exchanged to the 1D genomic re- ceiver site to create site 1D’ and deliver an ORF for an aAM. This s in expression of the aAPX on the cell surface, aAM intracellularly, and thus loading of the aAM as cargo in the aAPX in formation of the AM complex at the cell surface. This cre- ates an eAPC-pa cell line.

Figure 13 - Example of compilation of an eAPC-pa in two steps with two integra- tion couples via eAPC-p eAPC 1A contains distinct genomic receiver sites 13 and 1D. Distinct genetic donor vectors 1C’ and 1E’ are independently coupled to 1B and 1D, respectively. Donor vec- tor 1C’ encodes an aAPX and donor vector 1E’ s an aAM. In STEP1 the 1A eAPC is combined with the 1C’ donor vector. The resulting cell has insert 1C’ ex- changed to the 1B genomic receiver site to create site 1B’ and deliver an ORF for an aAPX. This results in expression of the aAPX on the cell surface and creation of an eAPC-p. Genomic receiver site 1D remains unused. In STEP2 the eAPC-p created in STEP1 is combine with the 1E’ donor vector. The resulting cell has insert 1E’ ex- changed to the 1D genomic receiver site to create site 1D’ and r an ORF for an aAM. This results in expression of the aAM on the cell e as cargo of the ex- pressed aAPX, and creation of an eAPC-pa.

Figure 14 - Example of compilation of an eAPC-pa in two steps with two integra- tion couples via eAPC-a eAPC 1A contains distinct c receiver sites 13 and 1D. Distinct genetic donor s 1C’ and 1E’ are independently coupled to 1B and 1D, respectively. Donor vec- tor 1C’ encodes an aAM and donor vector 1E’ encodes an aAPX. In STEP1 the 1A eAPC is combined with the 1C’ donor vector. The resulting cell has insert 1C’ ex- changed to the 1B c receiver site to create site 1B’ and deliver an ORF for an aAM. This results in expression of the aAM on the cell surface and creation of an . Genomic receiver site 1D remains unused. In STEP2 the eAPC-a created in STEP1 is combine with the 1E’ donor vector. The resulting cell has insert 1E’ ex- changed to the 1D genomic receiver site to create site 1D’ and deliver an ORF for an aAPX. This results in expression of the aAPX on the cell surface with the aAM as cargo and creation of an eAPC-pa. 1O Figure 15 - Shotgun compilation of an eAPC-pa pool from an eAPC-p The eAPC-p contains the exchanged c receiver site 1B’ expressing an aAPX and the distinct genomic receiver site 1D. The pool of genetic donor vectors 1E’ i-iii are coupled to 1D. Donor vectors 1E’ i-iii each encode a single aAM gene. The eAPC-p is combined with donor vectors 1E’ i, 1E’ ii, 1E’ iii simultaneously. The resulting cell pool has either of inserts 1E’ i-iii exchanged to the 1D genomic receiver site in multiple in- dependent instances to create sites 1D’ i-iii each delivering a single ORF for an aAM gene. The resulting eAPC-pa cell pool comprises a mixed population of three distinct cell cohorts each expressing a discrete combination of 1B’ presenting as aAPX:aAM either of the aAM genes contained in the l vector library.

Figure 16 - Shotgun compilation of an eAPC-pa pool from an eAPC-a eAPC-a contains the exchanged genomic er site 1B’ expressing an aAM and the distinct genomic receiver site 1D. The pool of genetic donor vectors 1E’ H" are coupled to 1D. Donor vectors 1E’ i-iii each encode a single aAPX gene. The eAPC-a is com- bined with donor vectors 1E’ i, 1E’ ii, 1E’ iii simultaneously. The resulting cell pool has either of inserts 1E’ i-iii exchanged to the 1D genomic receiver site in multiple inde- pendent ces to create sites 1D’ i-iii each delivering a single ORF for an aAPX gene. The resulting eAPC-pa cell pool comprises a mixed population of three distinct cell cohorts each expressing a te combination of the aAM encoded in 13’ and ei- ther of the aAPX genes ned in the l vector library.

Figure 17 - Shotgun compilation of pooled eAPC-pa libraries from eAPC contain- ing atorial paring of aAM and aAPX genes eAPC 1A contains distinct genomic receiver sites 13 and 1D. Distinct c donor s 1C’ and 1E’ are coupled to 1B and 1D, respectively. Donor vectors 1C’ i and 1C’ ii each encode a single aAM gene, and donor vectors 1E’ i and 1E’ ii each encode a single aAPX gene. The eAPC 1A is combined with donor vectors 1C’ i, 1C’ ii, 1E’ i and 1E’ ii simultaneously. The resulting cell pool has insert 1C’ i or 1C’ ii exchanged to the 1B genomic receiver site multiple independent instances to create sites 1B’ i and 1B’ ii, each delivering a single ORF for an aAM. The resulting cell pool r has in— sert 1E i or 1E ii exchanged to the 1D genomic receiver site multiple independent in- stances to create sites 1E’ i and 1E’ ii, each delivering a single ORF for an APX gene.

The resulting eAPC-pa cell pool comprises a mixed population of four distinct cell co- horts each expressing a discrete randomised aAPX:aAM pair at the e comprised of one of each gene contained in the initial vector library.

Figure 18 - Description of the components of a single integration couple eTPCS An example of an eAPCS comprising four components. The first component 2A is the eTPC line itself with all required engineered features of that cell. The eTPC 2A con— tains a second ent, 23, which is a genomic integration site for integration of a pair of complementary analyte TCR chain ORFs. A third component included in the eTPC, 2A, is a synthetic reporter construct that is induced upon TCR ligation, 2F. One additional independent component, 2C, represents a genetic donor vectors for site-di- rected ation of ORFs into site 23, where arrow indicates coupled specificity.

Figure 19 - ption of the components of a dual integration couple eTPCS An example of an eAPCS comprising six components. The first ent 2A is the eTPC line itself with all required engineered features of that cell. The eTPC 2A con- tains three further components, two of which are ZB and 2D, which are genomic inte- gration sites for integration of an analyte TCR chain pair. A third component included in the eTPC, 2A, is a tic reporter construct that is induced upon TCR ligation, 2F.

Two additional independent components, 2C and 2E, represent genetic donor vectors for site—directed integration of ORFs into sites 2B and 2D, respectively, where arrows indicate coupled icity. Each paired integration site / donor vector couple may be formatted to integrate a single ORF or a pair of ORFs to introduce analyte TCR chain pair expression by different means.

Figure 20 - Compilation of different analyte TCRsp presenting eTPC The eTPCS begins with the eTPC and uses a donor vector(s) to create cells s— ing e TCRsp, or single analyte TCR chains. An eTPC presenting TCRsp is termed eTPC-t, and may be created by introduction of two complimentary TCR chain encoding ORFs to the eTPC (step i). An eTPC sing a single analyte TCR chain alone is termed an eTPC-x, wherein a, and may be created by introduction of a single TCR chain encoding ORF(s) to the eTPC (step ii). A eTPC-t may alternatively be cre- ated from an eTPCx, n a second complimentary TCR chain encoding ORF is introduced to an existing eTPC-x (step iii).

Figure 21 - ation of a eTPC system with a one-step and one-vector format eTPC 2A contains ct c receiver site. The genetic donor vectors 2C is cou- pled to 2D. Donor vector 2C encodes a TCR chain pair. The eTPC 2A further contains a TCR signal response element 2F. The eTPC 2A is combined with donor vector 2C. 1O The resulting cell has insert 2C exchanged to the 2B genomic receiver site to create site 2C’ and r the two ORFs for a TCR chain pair. This cell is capable of present- ing a TCRsp at the surface, and is thus designated a eTPC-t.

Figure 22 - Compilation of a eTPC-t in one step with one vector and an unused receiver site eTPC 2A contains distinct c receiver sites 2B and 2D. The genetic donor vec- tors 2C’ is d to 2D. Donor vector 2C’ encodes a TCR chain pair. The eTPC 2A further ns a TCR signal response element 2F. The eTPC 2A is combined with donor vector 2C’. The resulting cell has insert 2C’ exchanged to the 2B c re— ceiver site to create site 2B’ and deliver the two ORFs for a TCR chain pair. Genomic receiver site 2D remains unused. This cell is capable of presenting a TCRsp at the sur- face, and thus designated a eTPC-t.

Figure 23 - Compilation of a eTPC-t in one step with two vectors and two a- tion couples eTPC 2A contains distinct genomic receiver sites 23 and 2D. The eTPC 2A further contains a TCR signal response element 2F. Distinct genetic donor vectors 2C’ and 2E’ are independently coupled to 2B and 2D, respectively. Donor vector 2C’ encodes a single TCR chain, and donor vector 2E’ encodes a second reciprocal TCR chain. The eTPC 2A is combined with donor vectors 2C’ and 2E’. The resulting cell has insert 2C exchanged to the 2B genomic receiver site to create site 2B’ and deliver an ORF for a first TCR chain. In addition, the resulting cell line has insert 2E’ exchanged to the 2D genomic receiver site to create site 2D’ and deliver an ORF for a second TCR chain.

This cell is capable of presenting a TCRsp at the surface, and is thus designated a eTPC-t.

Figure 24 - Compilation of a eTPC-x in one step with one vector and an unused receiver site eTPC 2A contains distinct genomic receiver sites ZB and 2D. The genetic donor vec- tors 2C’ is coupled to 2D. Donor vector 2C’ encodes a single TCR chain. The eTPC 2A further contains a TCR signal response element 2F. The eTPC 2A is combined with donor vector 20’. The resulting cell has insert 2C’ exchanged to the ZB genomic receiver site to create site 2B’ and deliver a single TCR chain ORF. Genomic receiver site 2D remains . This cell expresses only a single TCR chain and is thus des— ignated a eTPC-x.

Figure 25 - Compilation of a eTPC-t in two steps with two vectors eTPC 2A contains distinct genomic receiver sites ZB and 2D. The eTPC 2A further contains a TCR signal response element 2F. Distinct genetic donor vectors 2C’ and 2E’ are independently coupled to ZB and 2D, respectively. Donor vector 2C’ encodes a single TCR chain, and donor vector 2E’ encodes a second reciprocal TCR chain. In STEP 1 a eTPC 2A is ed with donor vector 2C’. The resulting cell has insert 2C’ exchanged to the ZB c receiver site to create site 2B’ and deliver an ORF for a first TCR chain. This cell expresses only a single TCR chain and is thus designated a eTPC-x. Genomic receiver site 2D s unused. In STEP 2, the eTPC-x is com— bined with donor vector 2E’. The resulting cell has insert 2E’ ged to the 2D ge- nomic receiver site to create site 2D’ and deliver an ORF for a second complementary TCR chain. This cell is capable of presenting a TCRsp at the surface, and is thus des- ignated a eTPC-t.

Figure 26 - Shotgun compilation of an eTPC-t pool from an eTPC with paired ana- lyte TCR chains in single replicate vector to express discrete TCR chain pairs from a selected TCR chain pair library eTPC 2A contains a genomic er site 23. The genetic donor vectors 2C’ is cou- pled to 2B. Donor vector 2C’ i, 2C’ ii and 20’ iii encode a distinct TCR chain pair and constitutes a mixed vector library of discrete TCR chain pairs. The eTPC 2A further contains a TCR signal response element 2F. The eTPC 2A is combined with donor vectors 2C’ i, 2C’ ii and 2C’ iii simultaneously. The resulting cell pool has insert 2C ex- d to the 2B genomic er site multiple independent instances to create site 23’ i, 2B’ ii and 23’ iii ring two ORFs for each discrete TCR chain pair con- tained in the initial vector library. This eTPC-t cell pool comprises a mixed population of three distinct cell clones each expressing a distinct TCR chain pairs, denoted TCRsp i, ii and iii, forming an eTPC-t pooled library.

Figure 27 - Shotgun compilation of an eTPC-t pool from an eTPC with paired analyte TCR chains in single replicate vector to express discrete TCR chain pairs from a selected TCR chain pair library and an unused receiver site eTPC 2A contains distinct genomic receiver sites 2B and 2D. The genetic donor vec— tors 2C’ is coupled to 2B. Donor vector 2C’ i, 2C’ ii and 2C’ iii encode a ct TCR chain pair and constitutes a mixed vector library of discrete TCR chain pairs. The eTPC 2A further contains a TCR signal response element 2F. The eTPC 2A is com- 1O bined with donor vectors 20’ i, 2C’ ii and 2C’ iii simultaneously. The resulting cell pool has insert 2C exchanged to the 2B genomic receiver site multiple ndent in- s to create site 23’ i, 23’ ii and ZB’ iii delivering two ORFs for each discrete TCR chain pair ned in the initial vector library. This eTPC-t cell pool comprises a mixed population of three distinct cell clones each expressing a distinct TCR chain pairs, denoted TCRsp i, ii and iii, forming an eTPC-t pooled library. Genomic receiver site 2D remains unused.

Figure 28 - Shotgun compilation of an eTPC-t pool from an eTPC with two vec- tors to express random combinations of paired TCR chain pairs from a selected single TCR chain library eTPC 2A contains distinct genomic receiver sites 23 and 2D. Distinct genetic donor s 2C’ and 2E’ are independently coupled to 23 and 2D, tively. Donor vec- tors 2C’ i and 2C’ ii each encode a single TCR chain, and donor vectors 2E’ i and 2E’ ii each encode a reciprocal single TCR chain. The eTPC 2A further contains a TCR signal response element 2F. The eTPC 2A is combined with donor vectors 2C’ i, 2C’ ii, 2E’ i and 2E’ ii simultaneously. The resulting cell pool has insert 2C’ i or 2C’ ii ex- changed to the 2B genomic receiver site multiple independent ces to create sites 23’ i and 23’ ii, each delivering a single ORF for a TCR chain. The resulting cell pool further has insert 2E i or 2E ii exchanged to the 2D genomic er site multiple inde- pendent instances to create sites 2E’ i and 2E’ ii, each delivering a single ORF for a TCR chain reciprocal to those at sites 2C’i and 2C’ii. The resulting eTPC-t cell pool comprises a mixed population of four distinct cell cohorts each expressing a discrete randomised TCRsp at the surface comprised of one of each reciprocal TCR chain con- tained in the initial vector library.

Figure 29 - Shotgun compilation of an eTPC-t pool from an eTPC-x with unpaired analyte TCR chains to express random combinations of paired TCR chain pairs from a ed single TCR chain library eTPC-x contains the exchanged genomic receiver site ZB’ expressing a single TCR chain and the distinct genomic er site 2D. Distinct genetic donor vectors 2E’ i and 2E’ ii are coupled to 2D, respectively. Donor vectors 2E’ i and 2E’ ii each encode a single TCR chain. The eTPC-x further contains a TCR signal response element 2F. The eTPC-x is combined with donor vectors 2E’ i and 2E’ ii simultaneously. The resulting cell pool has insert 2E’ i or 2E’ ii exchanged to the 2D genomic receiver site multiple independent ces to create sites 2E i and 2E’ii, each delivering a single 1O ORF for a TCR chain. The ing eTPC-t cell pool comprises a mixed population of 2 distinct cell cohorts expressing a discrete TCRsp at the surface comprised of the TCR chain expressed from 23’ paired with one of each TCR chain contained in the in- itial vector library.

Figure 30 - Operation of a combined eAPC:eTPC system showing possible eTPC- t-output states The analyte eAPC contains sites 1C’ and 1E’ integrated with one ORF each to encode one aAPX and one aAM, with the aAM loaded as cargo in aAPX at the cell surface.

The analyte eTPC contains sites 2C’ and 2E’ each integrated with one ORF encoding a ocal TCRsp at the surface. The eTPC-t further contains a TCR signal re- sponse element 2F. When eTPC-t and eAPC-pa tions are contacted, four eTPC-t response states can be ed, one negative and three positive. The nega- tive state is the resting state of the eTPC-t, with no signal th at the 2F t, denoting failure of the eAPC aAPX:aAM complex to stimulate the eTPC-t presented chain pair. Three positive states show increasing signal strength from the 2F. States 2F’+, 2F’++ and 2F’+++ denote low, medium and high signal strength, respectively.

The gene product of ZF denoted as hexagons accumulates to report signal th of each cell state, as denoted by darker shading of the cells. This indicates a graded re- sponse of analyte TCRsp expressed by eTPC-t population towards e aAPX:aAM presented by the eAPC-pa.

Figure 31 - Operation of a combined eAPC:eTPC system showing possible eAPC- pa output states The analyte eAPC-pa contains sites 1C’ and 1E’ integrated with one ORF each to en- code one aAPX and one aAM, with the aAM loaded as cargo in aAPX at the cell sur- face. The analyte eTPC contains sites 2C’ and 2E’ each integrated with one ORF en- coding a reciprocal TCRsp at the surface. The eTPC-t further contains a TCR signal response element 2F. When analyte eTPC and eAPC-pa populations are contacted, four eAPC response states can be achieved, one negative and three positive. The neg- ative state is the resting state of the analyte eAPC, denoting failure of the TCRsp chain pair to stimulate the aAPX:aAM complex ted by the analyte eAPC. Three posi— tive states show increasing signal strength from the ted aAPX:aAM. The re- ported signal strength of each cell state, is denoted by *, ** and **, and also denoted by darker shading of the cells. This indicates a graded response of e aAPX:aAM to- 1O wards the analyte TCRsp chain pair.

Figure 32 - Combined operation of the two-part analyte APC:eTPC system to fy TCR chain pairs reactive with analyte aAPX:aAM from a library of eTPC-t expressing discrete e TCR chain pairs The eTPC-t pool contains cells harboring sites 2C’ i, ii or ii, wherein each integrated with two ORFs ng a reciprocal TCR chain pair, and thus each cell cohort in the population expresses a discrete TCRsp at the surface. The eTPC-t further contains a TCR signal response element 2F. The analyte eAPC contain sites 1C’ and 1E’ inte- grated With a distinct set of ORF to encode one aAPX and one aAM, with the aAM loaded as cargo in aAPX at the cell surface. In the present example, only the TRC chain pair expressed from 2C’ i is specific for the aAPX:aAM presented by the ana- lyte APC, such that when eTPC-t pool and analyte APC population are contacted, only the cell cohort of the eTPC-t that bears 2C’ i reports TCRsp engagement through state 2F’.

Figure 33 - Combined operation of the two-part eAPC:eTPC system to identify aAM reactive with analyte eTPC from a library of analyte eAPC expressing dis- crete analyte aAPX:aAM complexes.

The analyte eAPC contain sites 1C’ and 1E’ integrated with a distinct set of ORF each to encode one aAPX and one aAM, with the aAM loaded as cargo in aAPX at the cell e. The analyte eTPC contain the exchanged genomic receiver site 2C’ express— ing a TCRsp at the surface. It further contains a TCR signal se t 2F. In the present e, only the complex aAPX:aAM i is specific for the TCR presented by the analyte eTPC, such that when analyte eAPC pool and analyte eTPC population are contacted, only the cell cohort expressing aAM i express a distinct signal *.

Figure 34 - Generation of a eAPC-p + aAM from a eAPC-p and the addition of a soluble, presentable antigen aAM eAPC-p contains the exchanged genomic receiver site 1B’ expressing an aAPX. A sol- uble, directly table antigen aAM is combined with the eAPC-p. This results in the formation of the aAPX:aAM complex on the cell surface and the generation of a eAPC-p + aAM.

Figure 35 - Generation of an eAPC-p + aAM from an eAPC-p and soluble aAM eAPC-p contains the exchanged genomic receiver site 1B’ expressing an aAPX.A sol- 1O uble antigen aAM is combined with the eAPC-p, this results in expression of the aAPX on the cell surface, the ce of aAM intracellularly, and thus g of the aAM as cargo in the aAPX in formation of the aAPX:aAM complex on the cell surface and the generation of a eAPC-p + aAM.

Figure 36 - Identification of the aAM presented by an eAPC-p + aAM through forced release of the aAM eAPC-p + aAM contains the exchanged genomic receiver site 1B’ sing an aAPX as well as internalized aAM that is presented on the surface as aAPX:aAM com- plex.The aAM is ed from the aAPX:aAM surface complex through incubation and the released aAM available for identification.

Figure 37 - fication of the aAM presented by an eAPC-p + aAM through cap- ture of the aAPX:aAM complex eAPC-p + aAM contains the exchanged c receiver site 1B’ expressing an aAPX as well as internalized aAM that is presented on the surface as aAPX:aAM com- plex.The aAPX:aAM surface x is captured for identification of loaded aAM.

Figure 38 - Selection of cells with targeted mutagenesis of the HLA-A, HLA-B and HLA-C loci in HEK239 cell line a) GFP fluorescence signal in two independent cell tions 48 hours after transfec- tion with plasmids encoding Cas9—P2A—GFP and gRNAs ing the HLA-A, HLA—B and HLA-C loci (grey histogram) compared to HEK293 control cells (dashed lined his- togram). Cells that had a GFP signal within the GFP subset gate were sorted as a poly- clonal population. b) Cell surface HLA-ABC signal observed on the two sorted polyclo- nal populations when labelled with a PE-Cy5 anti-HLA-ABC conjugated antibody (grey histogram). Single cells that showed a low PE-Cy5 anti-HLA—ABC signal and were dis- played within the sort gate were sorted to establish monoclones. Non—labelled HEK293 cells d line histogram) and PE-Cy5 LA-ABC labelled HEK293 cells (full black lined histogram) served as controls.

Figure 39 - Phenotypic analysis of HLA-ABC""" monoclones: Monoclone popula— tions were stained with the PE-Cy5 anti-HLA—ABC conjugated dy, and were ana- lysed by flow cytometry (grey ram). Non—labelled HEK293 cells (dashed lined his- togram) and PE-Cy5 anti-HLA-ABC labelled HEK293 cells (full black lined histogram) 1O served as controls. All three one lines showed a scent signal matching to non-labelled controls trating that each line lacked HLA-ABC surface expres- sion.

Figure 40 - Genetic characterization of a selection of monoclones lacking surface HLA-ABC expression demonstrating a genomic deletion in the targeted HLA al- leles. PCR amplicons were generated with s that spanned the gRNA genomic target sites of a specific HLA alleles and their size determined by electrophoresis. The expected size of the wild type HLA-A amplicon is 1067 bp, HLA-B amplicon is 717 bp and HLA-C amplicon is 1221 bp.

Figure 41 - Selection of cells with targeted genomic integration of synthetic Com- ponent B with or without synthetic Component D a) GFP fluorescence signal 48 hours after transfection with ds encoding CasQ- P2A—GFP, gRNAs targeting the AAVSt locus and component B c elements flanked by AAVS1 left and right gy arms (grey histogram). HEK293 cells server as a GFP negative control (dashed line histogram). Cells that had a GFP signal within the GFP+ gate were sorted as a polyclonal population. b) GFP fluorescence signal 48 hours after transfection with plasmids encoding Cas9-P2A—GFP, gRNAs targeting the AAVS1 locus and component B and D, both flanked by AAVS1 left and right homology arms (grey histogram). HEK293 cells server as a GFP negative l (dashed line histogram). Cells that had a GFP signal within the GFP+ gate were sorted as a polyclo- nal population c) Maintained BFP but no detectable RFP signal observed in the D1 sorted polyclonal population. Single cells that showed high BFP signal in quadrant 03 were sorted to establish eAPC containing synthetic component B monoclones. d) Main- tained BFP and RFP signal observed in the D2 sorted polyclonal population. Single cells that showed high BFP and RFP signals in quadrant 02 were sorted to establish eAPC monoclones containing synthetic component B and synthetic component D.

Figure 42 - Phenotypic analysis of eAPC monoclones a and b) Monoclone populations that display maintained BFP expression suggest the integration of tic component B. c) Monoclone populations that y main- tained BFP and RFP expression suggest the integration of both synthetic component B and synthetic component D.

Figure 43 - Genetic characterization of a selection of monoclones for integration 1O of Component B or Component B and D in the AAVS1 locus. a) PCR amplicons were generated with primers that prime within component B and/or D and size determined by electrophoresis. The expected size of a positive on is 380bp indicating stable integration of component B and/or D. b) PCR amplicons were generated with s that prime on AAVS1 genomic sequence distal to region en- coded by the homologous arms and the SV40 pA terminator encoded by component B and/or D and size determined by electrophoresis. The expected size of a positive am- plicon is 660 bp indicating integration of component B and/or D occurred in the AAVS1 site.

Figure 44 - Selection of cells with targeted genomic integration of component C’ into component B a) GFP scence signal 48 hours after transfection with plasmids encoding CasQ- P2A—GFP, gRNAs targeting the AAVS1 locus and component OWN/"24:02 (left panel) or component C’HLA'EV'OT02 (right panel). Cells that had a GFP signal within the GFP+ gate were sorted as a polyclonal population AOL-303 or 5. b) e HLA cell surface expression observed on the two sorted polyclonal popula- tions when labelled with a PE—Cy5 anti-HLA-ABC conjugated antibody (grey histo- gram). Single cells that showed a high PE-Cy5 anti-HLA—ABC signal and were displayed within the right sort gate were sorted to establish monoclones. Signal detected from PE-Cy5 anti-HLA-ABC labelled AOL-128, the HLA-ABC"""and HLA-DR,DP,DQ""" eAPC cell line (dashed line ram) served as controls.

Figure 45 - Phenotypic analysis of eAPC-p monoclones sing analyte HLA class I n on the cell surface Monoclone populations were stained with the PE-Cy5 anti-HLA-ABC conjugate anti- body, and were ed by flow cytometry (grey ram). AOL-128, the HLA- ABCnull and ,DP,DQ""" eAPC cell line (dashed line histogram) served as con- trols. AOL-321 and AOL-331 monoclone cell lines showed a stronger fluorescent signal compared to the HLA-ABC""" and HLA-DR,DP,DQ""" eAPC cell line control, demon- strating that each line expresses their analyte aAPX, HLA-A*24:02 or HLA-B*—07:02 ORF, respectively, and therefore are eAPC-p cell lines.

Figure 46 - Genetic characterization of a selection of monoclones demonstrating that their genomes integrated component C’, and that the integration occurred in the AAVS1 genomic receiver site, ting component B’ 1O a) PCR amplicons m the presence of HLA insert, a band of 810 bp indicated cor- rect CMV promoter amplicon and 380 bp is the on ted from SV40pA ter- minator. b) PCR amplicons were generated with two set of primers that primed on AAVS1 genomic sequence distal to region encoded by the homologous arms and a pri- mer that is unique to the SV40 pA terminator linked to the analyte HLA ORF. The ex- pected size of a positive on 1 kb and 1.1 kb indicate generation of component B’.

Figure 47 - Selection of cells with targeted genomic integration of component C’ into component B a) GFP fluorescence signal 48 hours after transfection with plasmids encoding Cas9— P2A—GFP, gRNAs targeting the AAVS1 locus and component C’HLADRN‘"=01"HLA"3R31*01101 (left panel) or component C’ HLA'DPA1*01103/HLA'DPB1*04101 (right . Cells that had a GFP signal within the GFP+ gate were sorted as a polyclonal population. b) Analyte HLA cell surface expression observed on the two sorted polyclonal - tions when labelled with an Alexa 647 anti-HLA—DR,DP,DQ conjugated antibody (grey histogram). Single cells that showed a high Alexa 647 anti-HLA—ABC signal and were displayed within the right sort gate were sorted to establish monoclones. Signal de- tected from Alexa 647 anti-HLA—ABC labelled AOL-128 (HLA—ABC""" and HLA- DR,DP,DQ""" eAPC cell line) (dashed line histogram) and ARH wild type cell line (full black lined histogram) served as controls Figure 48 - ypic analysis of eAPC-p monoclones expressing analyte HLA class II n on the cell surface Monoclone populations were stained with a Alexa 647 anti-HLA-DR,DP,DQ conju- gated antibody, and analysed by flow cytometry (grey histogram). AOL-128 (HLA- ABC"""and HLA-DR,DP,DQ""" eAPC cell line) (dashed line histogram) and ARH wild type cell line (full black lined histogram) served as controls. 1 and AOL-350 monoclone cell lines showed a stronger fluorescent signal compared to the HLA- ABC""" and HLA-DR,DP,DQ""" eAPC cell line control, demonstrating that each line ex- pressed their analyte aAPX, HLA-DRA*01 :01/HLA-DRBl*01 :01 or HLA- DPA1*01:03/HLA-DPB1*04:01, respectively, and therefore are eAPC—p cell lines.

Figure 49 - eAPC-p monoclones generated by RMCE integration of analyte HLA class I protein a) eAPC—p monoclone populations AOL—421 and AOL—422 lost BFP fluorescence (grey histogram). Their parent eAPC cell line AOL—385 (full black line histogram) and the BFP negative ARH wild type cell line (dash line histogram) served as a control b) eAPC-p monoclone populations AOL-421 and AOL-422 gained HLA-A*02:01 ex- on when stained with the PE-Cy5 anti-H LA-ABC conjugate antibody (grey histo- gram). Their parent 5 HLA-ABC"""and ,DP,DQ""" eAPC cell line (dash line histogram) and ARH wild type cell line (full black line histogram) served as nega- tive and positive PE-Cy5 anti-HLA-ABC labeling control, respectively.

These results strongly indicated a successful RMCE occurred between the BFP ORF and HLA-A*02:01 ORF in both 1 and AOL-422 cell lines.

Figure 50 - Genetic characterization of a selection of monoclones confirmed HLA-A*02:01 ation by RMCE An amplicon of 630 bp indicated presence of HLA-A2 in monclones AOL-421 and 422 but not in the control line, AOL-128.

Figure 51 - Phenotypic analysis of eAPC-pa monoclones sing analyte HLA class I protein on the cell surface and aAM a) eAPC-p Monoclone populations were stained with the PE-Cy5 anti-HLA—ABC conju- gated antibody, and were analysed by flow cytometry (grey ram). AOL—128, the HLA-ABC""" and HLA-DR,DP,DQ""" eAPC cell line (dashed line histogram) served as control. 1 and AOL-331 monoclone cell lines showed stronger fluorescent sig— nal ed to ls demonstrating that each line expressed their analyte aAPX, HLA-A*02:01 or HLA-B*35:01 ORF, respectively, and therefore were eAPC—p cell lines. b) eAPC-pa Monoclone populations were assessed for GFP scence by flow cy- tometry (grey histogram). AOL—128, the HLA—ABC"""and HLA—DR,DP,DQ""" eAPC cell line (dashed line histogram) served as control. 1 and AOL-395 monoclone cell lines showed a stronger fluorescent signal compared to controls demonstrating that each line expresses analyte aAM selection marker and therefore inferred aAM expres- sion, in a cell line which also expressing —A*02:01 or HLA-B*35:01 ORF, re— spectively. Therefore ACL-391 and AOL-395 were eAPC-pa lines.

Figure 52 - Genetic characterisation of monoclones containing components 1D and/or 1 B Two tables are presented summarising the genetic characterisation of eAPC generated to contain Component 1B, or Component 1B and 1D, tively. 1O Figure 53 - An eACP-p constructed in one step n Component 1C’ d a single HLAI ORF.

An eAPC-p was created h RMCE by electroporation of the cell line 2 with the plasmid that encodes expression of the Tyr—recombinase, Flp (v4.1.8), together with one Component 1C’ plasmid encoding an aAPX, selected from either HLA- A*02:01 (V4.H.5 or HLA—A*24:02 6). At 10 days post electroporation, individual cells positive for HLAI surface expression and diminished fluorescent protein signal, RFP, encoded by Component 1B selection marker, were sorted. Resulting monoclonal eAPC—p lines were analysed by flow cytometry in parallel with the parental eAPC line, and two examples are presented a) Individual outgrown monoclone lines (AOL-900 and AOL-963) were analysed by flow cytometry for loss of RFP, presence of BFP and gain of HLA—ABC . Left-hand plots display BFP vs RFP, the parental cell has both BFP and RFP (QZ, top plot, 99.2%), whereas ACL-900 (Q3, middle plot, 99.7%) and AOL-963 (Q3, bottom plot, 99.9%) both lack RFP signal, indicating integration couple between Component 1B/1C’ has occured. Right-hand plots display BFP vs HLA-ABC (aAPX), wherein both AOL-900 (02, top plot, 99.2%) and AOL-963 (02, bottom plot, 99.2%) show strong signal for HLA-ABC (aAPX), further reinforcing that 1B/1C’ integra- tion. Critically, both ACL-900 and AOL—963 have strong BFP signal, indicating that ent 1D remains open and isolated from the Component 1B/1 C’ integration couple. b) To further characterize 0 and AOL-963, and a third eAPC-p not pre- sented in a) ACL-907, genomic DNA was extracted and PCR ted using primers that target adjacent and internal of ent 18’ (Table 5, 8.8.3, 15.H.2), thereby se- lectively amplifying only successful integration couple events. Comparison is made to an unmodified parental line, ACL-3 n the Component 1B is lacking. on products specific for Component 18’ were produced for all three eAPC-p monoclones whereas no product was ed in the ACL-3 reaction, confirming the specific inte- n couple event between Component 1B and Component 10’ had occurred.

Figure 54 — An eAPC-pa constructed from eAPC-p in one step, wherein Compo- nent 1D’ encodes a single analyte antigen molecule (aAM) ORF.

Multiple eAPC—pa were constructed from a parental eAPC-p 05) in parallel, wherein the c receiver site, Component 1D, is ed for integration by a primed genetic donor vector, Component 1E’, comprising of a single ORF that en- codes an aAM. The eAPC-p (AOL-900, example 8) was independently combined with a vector encoding expression of the RMCE recombinase enzyme (Flp, v4.1.8) and each ent 1E’ of either V9.E.6, V9.E.7, or V9.E.8 by electroporation. At 10 days post 1O electroporation, individual eAPC-pa were ed and single cell sorted (monoclones) based on diminished signal of the selection marker of integration BFP, encoded by Component 1D. Resulting monoclonal eAPC-pa lines were analysed by flow cytometry in parallel with the parental eAPC line, and three examples are presented. In addition, resulting monoclones were also genetically characterized to confirm the integration couple event. a) Monoclones for eAPC-pa, AOL-1219, AOL—1227 and AOL—1233, were analysed and selected by flow cytometry for loss of BFP signal and retention of the HLA-ABC signal. Plots of BFP vs SSC are yed with a BFP- gate. An increase in the number of BFP- events compared to parental eAPC-p is observed, indicating that an integration couple between Component 1D/1E’ has occurred. Single cells from the BFP- gate were selected, sorted and outgrown. b) Selected ones of AOL-1219, AOL-1227, AOL-1233 were analysed by flow cytometry to confirm loss of BFP and re- tention of HLA—ABC signals. Plots of BFP vs HLA-ABC are presented, n all three monoclones can be observed having lost the BFP signal in comparison to parental eAPC-p (right most plot), indicating a successful integration couple event. c) To demonstrate that the monoclones contained the correct fragment size for aAM ORF, a polymerase chain reaction was conducted, utlising primers targeting the aAM ORF and representative e gel is presented. Results from two monoclones representing each aAM ORF are shown. Lane 1: 2_log DNA marker, Lanes 2—3: pp28 ORF (ex- pected size 0.8kb), Lane 4: 2_log DNA , Lanes 5-6: pp52 ORF (expected size 1.5kb), Lane 7: 2_log DNA marker, Lanes 8-9: pp65 ORF ted size 1.9kb), Lane : 2_log DNA marker. All monoclones analysed had the expected amplicon size for the respective aAM, further indicating the integration couple had occurred.

Figure 55 — Shotgun integration of multiple antigens into eAPC-p to create a pooled a y in a single step A pooled library of a were generated from a pool of primed Component 1E s (Component 1E’) collectively encoding le aAM ORF (HCMVpp28, 52 and 65) by integration in a single step into the parental eAPC—p, wherein each individual cell integrates a single random analyte antigen ORF derived from the original pool of vectors, at ent 1D’, such that each generated eAPC- pa expresses a single random aAM, but tively the pooled library of eAPC-pa rep- resents all of aAM ORF encoded in the original pooled library of vectors. The library of eAPC-pa was generated by electroporation by combing the eAPC-p (AOL-905, aAPX: 02:O1) with a pooled vector library comprised of individual vectors encoding an ORF for one of HCMVpp28, HCMVpp52 or HCMVpp65 (V9.E.6, V9.E.7, and V9.E.8), and being mixed at a lar ratio of 1:1:1. Resulting eAPC-pa populations were an- alysed and selected by flow cytometry, in parallel with the parental eAPC-p line. a) At days post electroporation putative eAPC-pa cells (Transfectants) were analysed and ed by flow cytometry, compared in parallel with the parental line (AOL-905). Plots display BFP vs 880, gated for BFP- populations, wherein an increase in BFP- cells are observed in the BFP- gate compared to the parental line. Bulk cells were sorted form the transfectants based on BFP- gate, denoted AOL-1050. b) After outgrowth, ACL- 1050 cells were analysed by flow cytometry for loss of BFP. Plots displayed are BFP vs 880, wherein AOL-1050 has been enriched to 96.4% BFP- compared to parental line ~4% BFP-. Subsequently, single cells were sorted from the BFP- pollution of ACL- 1050. c) To demonstrate that the polyclone AOL-1050 was comprised of a mixture of HCMVpp28, HCMVpp52 and HCMVpp65 encoding cells, 12 ones were se- lected at random, wn and were used for genetic characterisation. Cells were characterised by PCR utilising primers targeted to the aAM ORF (Component 1D’), to amplify and detect integrated aAM. All 12 monoclones ed by PCR have detecta- ble amplicons and are of the expected size for one of pp28 (0.8kb), pp52 (1 .5kb) or pp65 (1 .9kb). In addition, all 3 aAMs were represented across the 12 monoclones. In comparison, amplicons from three discrete monoclones, wherein in the aAM was known, were amplified in parallel as controls; all three controls produced the correct sized amplicons of pp28 (0.8kb), pp52 (1 .5kb) and pp65 (1 .9kb). Thus, it is confirmed that the pool is comprised of eAPC-pa wherein each cell has a single randomly se— lected aAM form the original pool of three s.

Figure 56 - Demonstration of eTPC-t generation from parental eTPC A model TCR alpha/beta pair (JGQ-TCR), which has a known specificity for a HCMV antigen presented in 02:01 was selected for integration to an eTPC parental line. The JGQ-TCR-alpha ORF was cloned in a ent 2E’ context, and JGQ-TCR- beta in a 20’ context. An eTPC-t was created through RMCE by transfection of Com- ponent 20’ and 2E’ plasmids and a construct encoding flp recombinase into the eTPC line AOL-488, which harbours two genomic integration sites, ZB and 2D, encoding re- porters BFP and RFP, tively. 10 days after transfection, individual cells dimin— ished for the BFP and RFP signals, encoded by Components 2B and 2D ion markers, were sorted as single cells. Resulting monoclonal eTPC-t 1 were ana- lysed in parallel with the parental eTPC, and a single example presented. a) and b) Parental eTPC cell line AOL-488 and an example monoclonal was analysed by flow cy- tometry for BFP and RFP signals. The plot displays live single cells as BFP versus RFP, showing the eTPC cell line is ve for selection markers present in Compo- nent 28 and 2D (a), and resulting monoclone has lost these markers as expected for integration couple events between 2B/ZC and 2D/2E (b). Percentage values represent the percentage of double positive cells in a) and double negative cells in b). c) to f) eTPC AOL—488 and monoclone eTPC-t AOL-851 were stained with antibodies for CD3 and TCR alpha/beta (TCRab) and HLA multimer t ic for the JGQ-TCR (Dex HLA-A*02:O1—NLVP) and ed by flow cytometry and gated for live single cells.

The parental eTPC line showed no positive staining for CD3 or TCR on the cell surface (0), and was also negative for staining with HLA multimer reagent (d). In contrast, the resulting one showed positive staining for both CD3 and TCR on the cell sur— face (e) and showed positive staining with the multimer reagent specific for the ex- pressed R. Percentage values represent the percentage of Rab double positive cells in c) and e), and CD3/HLA—multimer double positive cells in d) and f). g) Genomic DNA was prepared from monoclonal eTPC—t AOL-851 and subjected to PCR with primers specific for the JGQ-TCR-alpha chain encoded by Component 2D’, or the JGQ-TCR-beta chain encoded by ent ZB’. PCR products were resolved by agarose gel and observed as expected band size. h) Genomic DNA was prepared from monoclonal eTPC-t AOL-851 and subjected to digital drop PCR with primers and probes specific for the JGQ-TCR-alpha chain encoded by Component 2D’, or the JG9- TCR-beta chain encoded by Component 28’. A reference on primer/probe pair for an intron of the TCR alpha constant (TRAC) was included. The table ts ratios of reference to TCR alpha and TCR beta. A ratio of close to 0.33 indicates that a single copy of each TCR alpha and beta chain is present in the eTPC-t line AOL-851, which is a triploid line.

Figure 57 - Demonstration of eTPC-x reversion from eTPC-t A parental eTPC-t cell line AOL-851, expressing a TCR alpha and beta chain at site 2D’ and 28’, respectively was reverted to a eTPC-x line by exchanging ent 2D’ with a donor vector encoding GFP (Component 22). Component 22 contained recom- binase heterospecific F14/F15 sites flanking the GFP ORF, and was thus ible with Component 2D’. eTPC-t line 1 was transfected with Component 22 along with a construct encoding flp recombinase. 7 days after transfection, individual cells ve for GFP signals were sorted and grown as ones. Resulting monoclonal eTPC—x lines were analysed by flow cytometry in parallel with the parental eTPC-t, and a single example presented. a) and b) The monolcone eTPC-x (AOL-987) derived from parental eTPC-t ACL-851 was analysed by flow cytometry for GFP expression along with the parental line. Plots display SSC versus GFP parameters of gated live single cells. The parental cell line has no GFP expression (a), while the monoclone AOL-987 has gained GFP as expected (b), indicating exchange of the TCR alpha ORF for a GPF ORF. c) and d) The monolcone eTPC-x AOL-987 derived from parental ACL- 851 along with the parental eTPC-t AOL-851 were stained with antibodies for CD3 and TCRab and analysed by flow cytometry. Plots y CD3 versus TCRab parameters gated on live single cells. The parental cell showed positive staining for both CD3 and TCRab (c), while the derived monoclone showed negative staining for both (d); con- firming loss of TCR alpha ORF in the derived eTPC-x line.

Figure 58 - tration of shotgun integration into eTPC-x to create pool of eTPC-t An eTPC-t pool was d from an eTPC-x parental line expressing a single TCR beta chain in Component 28’. The eTPC-x line expressed GFP as the reporter at avail- able site 2D. A pool of 64 variant TCR alpha chains, including the parental chain, were constructed. The parental TCR chain pair represents the JGQ-TCR with known specific- ity for a HCMV antigen presented in HLA-A*02:O1. The ent 2E pool was trans- fected into the parental eTPC—x AOL-987 along with a construct encoding flp recom— binase. A polyclonal line was selected by sorting for GFP positive cells 10 days after transfection. The resulting AOL-988 polyclonal eTPC—t was subsequently sorted on the basis of negative staining for GFP and positive or negative ng for HLA multimer reagent (DEX HLA-A*02:01-NLVP). red single cells were sequenced to identify the encoded TCR-alpha chains and compared to a parallel analysis of each of the TCR-alpha chain ts paired with the native TCR—beta chain in terms of staining with an HLA multimer reagent specific for the parental TCR chain pair. a) and b) Pa- rental eTPC-x AOL-987 line and resulting polyclone eTPC-t AOL-988 line were ana— lysed by flow cytometry for GFP expression. Plots display SSC versus GFP parameters of live single cells. Parental cell line shows positive signal for GFP, indicating intact Component 2D (a). Derived polyclonal line shows half positive and half negative for GFP (b), indicating that half of the cells in the polyclonal population have potentially ex- changed the GFP ORF at D for TCR alpha ORF to form Component 2D’. 0) and d) Pa- rental eTPC-x AOL-987 line and resulting polyclone eTPC-t ACL-988 line were stained with and CD3 antibody and HLA multimer with specificity for the parental JGQ-TCR (DEX HLA-A*02:01-NLVP), and analysed by flow cytometry. Plots display CD3 versus HLA multimer parameters of live single cells. The parental cell line is negative for both CD3 and HLA multimer ng (c). The left hand panel of d) displays gated GFP-neg- ative events, and the right hand GFP—positive events. Only gative , where the Component 2D is converted to D’, shows CD3 positive staining, of which a subset shows positive staining for HLA multimer. Single cells from the gated HLA multi- mer negative and positive gate were sorted and the integrated ORF at Component 2D’ sequenced to determine identity of TCR alpha ORF. e) All 64 JGQ-TCR-alpha variants were cloned into an expression construct that permitted each to be independently transfected to al eTPC—x (AOL—987). Relative staining units (RSU) against the HLA-A*02:01-NLVP tetramer reagent was determined for each. RSU is ated as the ratio of the mean fluorescence intensity (MFI) of 02:01-NLVP tetramer sig- nal for the CD3 positive tion over the CD3 ve population, and is indicative of the binding strength of each TCR chain pair variant to the HLA multimer reagent.

Each point plotted in Figure e) represents the observed RSU for each 64 variants.

Open circles correlate to the sequenced cells recovered from the gative/H LA multimer-positive gate. Open triangles ate to the sequenced cells recovered from the GFP-negative/H LA multimer-negative gate. 3O Figure 59 - Functional demonstration of Component 2F in eAPC:eTPC system us- ing eAPC-p and exogenous aAM The eTPC-t cell line carrying a ent 2F (AOL-1277), wherein the TCR chains at Component 28’ and 2D’ encode a TCR pair that is specific for HMCV antigenic peptide NLVPMVATV presented in HLA-A*02:01. The Component 2F reporter was RFP. This eTPC-t was ted for 24 hours with various eAPC lines of differing —p characteris- tics in the presence and absence of model e antigens, and the contact cultures analysed by flow cytometry. Flow cytometry ram plots show event counts against RFP signal of viable single T—cells fied by antibody staining for a specific surface marker that was not presented by the eAPC. a) and b) eAPC-p cells sing only HLA-A*02:01 (AOL-209) were pulsed with NLVPMVATV (a) or VYALPLKML (b) pep— tides and subsequently co-cultured with eTPC-t for 24 hrs. c) and d) eAPC-p cells ex- pressing only HLA-A*24:02 (AOL-963) were pulsed with NLVPMVATV (c) or WALPLKML (d) peptides and subsequently co-cultured with eTPC-t for 24 hrs. e) eAPC—p cells expressing only HLA-A*02:O1 (AOL-209) were left without peptide pulsing and subsequently co-cultured with eTPC-t for 24 hrs. f) eAPC parental cells that ex- press no HLA on the cell surface (ACL-128) were pulsed with NLVPMVATV and subse- quently co-cultured with eTPC-t for 24 hrs. RFP signal was significantly sed in the eTPC-t 77 only in the presence of HLA-A*02:01 expressing eAPC-p pulsed with NLVPMVATV, representing the known target of the expressed TCR. Histogram gates and values reflect percentage of events in the RFP positive and RFP negative gates. This indicates the specific response of Component 2F to engagement of eTPC-t expressed TCRsp with cognate HLA/antigen (aAPXzaAM).

Figure 60 - Functional demonstration of Component 2F in eAPC:eTPC system us- ing eAPC-pa with integrated aAM The eTPC-t cell line carrying a Component 2F (AOL-1150), n the TCR chains at Component 2B’ and 2D’ encode a TCR pair that is specific for HMCV antigenic peptide NLVPMVATV presented in HLA-A*02:01. The Component 2F reporter was RFP. This eTPC-t was contacted for 24 hours with various eAPC lines of differing —pa character- istics in the absence of exogenous antigen.a) eAPC-pa line 044) expressing 02:01 and the full-length ORF for HCMV n pp52. pp52 does not n antigenic sequences ised by the JGQ-TCR. b) eAPC-pa line (AOL-1046) ex- pressing HLA—A*02:01 and the full-length ORF for HCMV protein pp65. pp65 contains antigenic sequence recognised by the JGQ-TCR, when ted in HLA-A*02:01. c) eAPC—pa line (AOL-1045) expressing 07:02 and the full-length ORF for HCMV protein pp52. pp52 does not contain antigenic sequences recognised by the JGQ-TCR. d) eAPC—pa line (AOL—1048) expressing HLA—B*07:O2 and the full-length ORF for HCMV protein pp65. pp65 contains antigenic sequence recognised by the JGQ-TCR, when presented in HLA—A*02:O1. After independent co—culture of each eAPC—pa line with the eTPC-t for 48 hrs, RFP expression in the eTPC-t was determined by flow cy- tometry. RFP signal was significantly increased in the eTPC-t ACL-1150 only when contacted with eAPC—pa AOL-1046. This was the only eAPC-pa with both the recog- nised antigenic peptide sequence encoded by the integrated aAM ORF, and the correct HLA restriction. ram gates and values reflect percentage of events in the RFP positive and RFP negative gates. This indicates the specific response of Component 2F to engagement of eTPC-t expressed TCRsp with cognate HLA/antigen (aAPX:aAM).

Materials and methods All cell lines used in this application are either on the ARH or HEK293 background.

They are denoted by AOL followed by a number. A y of the cell lines used in this application is presented as Table 1. ection of cells One day prior to transfection] electroporation, cells were seeded at a density of 1.2—1.4 x106 cells/60mm dish in 90% DMEM + 2mML-glutamine + 10% Hl-FBS (Life Technolo- gies). The following day, cells with 65% ency were transfected with a total amount of 5ug DNA and jetPEl ® (Polyplus transfection reagent, Life Technologies) at a N/P ratio of 6. Stock solutions of DNA and jetPEl ® were diluted in sterile 1M NaCl and 150mM NaCl respectively. The final volume of each solution was equivalent to 50% of the total mix volume. The PEI solution was then added to the diluted DNA and the e was ted at room temperature for 15min. Finally, the DNA/PEI mix- tures were added to the 60-mm , being careful not to t the cell film. The cells were incubated for 48 hours at (37 °C, 5% C02, 95% relative humidity) prior to DNA delivery marker expression analysis. The medium was replaced before transfec- tion.

Fluorescence activated cell sorting (FACS) Single cell sorting or polyclone sorting was achieved through standard cell sorting methodologies using a BDInf/ux instrument. Briefly, ACL cells were harvested with Try- pLETM Express Trypsin (ThermoFisher Scientific) and resuspended in a le vol- ume of DPBS 1X (Life Technologies) prior to cell sorting, in DMEM 1X medium contain- ing 20% Hl-FBS and Anti-Anti 100X (Life logies). Table 2 summarises the anti- bodies and multimers used in this application for FACS.

Table 2: BD Influx filters Progin Fluongchronge CasQ/GFP 4884530/40 HLA—A, B, C 561-670/30 BFP 405-460/50 RFP 561-585/29 TCRab (R63) 640-670/30 CD3 (R78) 640—750LP CD3 (R71) 640-760/30 DEX 02:O1-NLVP 561—585/29 AOL-3 AOL—128 - AOL-191 18’, 1D HLA—A*02:O1 — eAPC--p ACL-209 18’, HLA—A*02:01 — eAPC--p ACL—341 ACL-390 18’, 1D’ HLA—A*02:01 pp65 ORF eAPC--pa ACL—402 ACL-488 ACL—851 1B’, 1D’ R-beta JG9-TCR-alpha eTPC-t ACL-900 1B’, 1D HLA—A*02:01 ACL—905 18’ HLA—A*02:01 ACL-963 18’, 1D HLA—A*24:02 _ eAPC--p 7 18’ 1D JGQ—TCR-beta _ eTPC-x ACL-988 1B’, 1D’ JGQ-TCR-beta JGQ-TCR-alpha eTPC-t (pool) 64x variants ACL—1050 1B’, 1D’ HLA-A*02:01 pp28, pp52, pp65 eAPC--pa ACL—1043 18’, 1D’ 02:01 pp28 ORF eAPC-pa ACL-1044 1B’, 1D’ HLA—A*02:01 eAPC-pa ACL—1045 1B’, 1D’ HLA—B*07:02 a ACL-1046 1B’, 1D’ HLA—A*02:01 eAPC-pa AOL—1048 1B’, 1D’ HLA—B*07:02 pp65 ORF eAPC-pa AOL-1063 23, 20, 2F Selection eTPC with remarker 1 marker 2 sponse ele- ACL-1150 23’, 20’, 2F TCR-alpha eTPC-t, with response ele- AOL-1219 18’, 1D’ HLA—A*02:01 pp28 ORF eAPC-pa AOL—1227 1 B’, 1D’ HLA—A*02:O1 pp52 ORF eAPC-pa 33 1 B’, 1D’ HLA—A*02:01 pp65 ORF eAPC-pa AOL-1277 28’, 2D’, F TCR-alpha ta , with response ele- diated integration of HLA-A*02:01 sequences in eAPC cell line eAPC cells were electroporated with vectors encoding Flp, DNA encoding a marker to track delivery (vector encoding GFP) and a vector containing 02:01. The HLA- A*02:O1 sequence also encoded a linker and 3xMyc— tag at the 3’end. The electro- poration conditions used were 258 V, 12.5 ms, 2 pulses, 1 pulse interval. Ratio be- tween each integrating vector and the Flp-vector was 1:3. Cells electroporated with only GFP-vector and no electroporated cells were used as controls respectively in or- der to set the gates for GFP sort after two days. On the following day (2 days after electroporation), cells were analyzed and sorted based on GFP expression. Cells were sorted using the BD Influx Cell Sorter.

At 3 days after electroporation, a sort based on GFP-expression was performed in or- der to enrich for electroporated cells. 7-8 days after electroporation, the cells were har- vested and surface d for C expression. BFP+ve RFP-ve HLA+ve cells were single cell sorted for monoclonal.

To genotype the cells, 100 ng of DNA was used as template to run a PCR reaction to check if ations had occurred at the expected integration site. A forward primer tar- geting the integration cassettes (Pan_HLA_GT_F1) and a reverse primer (SV40pA_GT_R1) targeting just outside the integration site was used and the PCR product was run on a 1% agarose gel.

Flp-mediated integration of HCMV ORF sequences in eAPC-p cell line eAPC-p cells were electroporated with vectors encoding Flp, DNA encoding a marker to track delivery (vector encoding GFP) and vectors containing HCMV pp28, pp52 or pp65 aAM-ORF. The HCMV-ORF sequences also encoded a linker and 3xMyc- tag at the 3’end. The electroporation conditions used were 258 V, 12.5 ms, 2 pulses, 1 pulse interval.

Ratio between each integrating vector and the Flp-vector was 1:3. Cells electroporated with only GFP-vector and no electroporated cells were used as ls respectively in order to set the gates for GFP sort after two days. On the ing day (2 days after electroporation), cells were analyzed and sorted based on GFP expression. Cells were sorted using the BD Influx Cell Sorter.

Flp-mediated shotgun integration of 3 HCMV ORF sequences in eAPC-p cell line eAPC-p cells were electroporated with vectors encoding Flp, DNA encoding a marker to track delivery (vector encoding GFP) and vectors containing HCMV pp28, pp52 or pp65 aAM-ORF. The RF sequences also encoded a linker and 3xMyc- tag at the 3’end. The electroporation conditions used were 258 V, 12.5 ms, 2 pulses, 1 pulse interval. For the shotgun integration, the vectors containing HCMV-ORFs were pooled in a ratio 1:1 :1 and the mixture was oporated into the eAPC-p cell. The resulting eAPC-pa cells were polyclonal. Individual one cells were sorted and genetically characterized to demonstrate that the polyclone was made up of cells containing all three HCMV-ORFs.

Genetic characterization of the monoclones PCR reactions to assess the ntegration of the HCMV ORFs into Compo- nent D Primers used to assess integration of the HCMV ORF ed to the linker (forward primer 10.D.1) and EF1aplha er (reverse primer 15.H.4). Expected size was 0.8kb for pp28, 1.5kb for pp52, 1.9kb for pp65. PCR products were run on a 1% Aga— rose gel in 1XTAE buffer, using the PowerPac Basic (Bio-Rad), stained with 10,000 di- lution of sybersafe and analyzed with Fusion SL (Vilber Lourmat).

Table 3: PCR reagents for assess integration of the aAM ORF on Cogponeng gyolumegper reagction " 5xPhusion buffer 4 ul DNTPs 0,2 ul Phusion DNA polymerase 0,15 ul .D.1 0,5 ul .H.4 0,5 ul H20 up to 20 ul DNA (100ng) 1 ul (100 ng/ul) DMSO 3% 0.6 ul Table 4: PCR cycle ions Step Temperature Willi > initial ratio‘n 36" sec cycles 98°C 10 sec 60°C 10 sec 72°C 15 sec Final ion 72°C 10 min RMCE n a paired integration couple For RMCE integration, cells were transfected with 0.6 pg of DNA vectors encoding FLP, (V4.|.8), 2 pg of Component C/Y, 2 pg of Component E/Z, 0.4 pg of DNA encod- ing a marker to track DNA delivery. 2 days after transfection cell positive for the DNA delivery marker, either GFP or RFP positive, were sorted by FACS. 4-10 days after transfection, individual cells displaying diminished fluorescent protein signal, encoded by Components D and B selection markers were sorted by FACS. The exception being for generating AOL—987 where individual cells displaying GFP positivity were sorted by FACS.

Transient expression of TCR chain pairs to characterization of their RSU For transient expression, cells were ected with DNA vectors encoding FLP, (V4.|.8), JGQ-TCR-alpha variant (VP.7751.RC1.A1 to VP.7751.RC1.H8), JGQ-TCR— beta WT chain (V3.C.5), and DNA vector vehicle (V1.C.2) . 2 days after transfection, all cells were d with HLA-A*02:01-NLVP tetramer and anti-CD3 antibodies. RSU were calculated as the ratio of the mean fluorescence intensity (MFI) of HLA-A*02:01- NLVP tetramer signal for the CD3 positive population over the CD3 negative - tion, and was indicative of the binding strength of each TCR chain pair variant.

HLA multimer staining Cells were stained with HLA-multimer reagent on ice for 10 mins, then with CD3 and/or TCRab antibodies. Detection of ic cell fluorescent properties by the BD/nflux in- strument are defined in table 6. g of single cells for monoclonal generation, the cells displaying the phenotype in- terest were deposited into 96-well plates, containing 200 ul of growth medium. One to two plates were sorted per . Polyclonal cell sorts were directed into FACS tubes, containing media, using the Two-way sorting setting in the cell sorter T'VI (BD Biosciences).

Single cells sorts for molecular characterization of their JGQ-TCR-alpha variant were sorted to PCR plate pre—loaded with 5 uL of nuclease—free water. Specimens were rozen until subsequent processing.

Genomic DNA extraction for genetic characterization DNA was ted from 5x106 cells using the QlAamp DNA Minikit (Qiagen). DNA was stored in 1XTE (10mM Tris pH8.0 and 0.1mM EDTA).

Table 5: Vectors ‘IP‘WW'; ?amwwe '-’.Flc>l1§lc>l;\l/‘/ GLAA M EcDNA§A_GF5 vrAe pcDNA3.1_RFP vrc2 pMA—SV40pA vace pMA—CS—JGQ-TCRbeta V4H9 pMA—F14—TurboGFP-F15 vrAe pMA—F14-TCR—JG9 -F15 VIAA pMA—FRT—TCR—JG9 -F3 V&FB F14-TCRaF15 gen.64mix V4l8 CMVpro—Flp-sv40pA—V2 VP.7751.RC 64 individual vectors, each encode a different ?A1toH8 member of JG9-TRA CDR3 64 variants set V4H5 pMA_F14_H LA—A*02:01-6xHis_F1 5 V4He pMA_F14_H 4:02-6xH is_F1 5 VAHJ pMA_F14_H LA—B*07:02-6xHis_F15 V4He pMA_F14_H LA—B*35:O1-6xHis_F15 V9E6 FRT_HCMVpp28—3XMYC_F3 vaEJ FRT_HCMVpp52—3XMYC_F3 V9.E.8 FRT_HCMVpp52—3XMYC_F3 V1 .A.8 SpCas9—2A—GFP V2.A.1 H LA—A-sg-sp-opti 1 V2.A.7 HLA—B-sg-sp-3 H LA-C-sg-sp-4 V2 .| . 1 0 H x2-3_sg—sp-opti_1 V2 .J .1 H LA-A-ex2-3_sg-sp-opti_2 V2 .J .6 AAVSI_sg-sp-opti_3 PCR ons to assess the RMCE- integration of the F and TRB-ORF into component 2B or 2D.

Primers used to assess integration of the TCR-alpha, annealed to the TRAC segment (forward primer 1.F.7) and the sv40pA terminator (Reverse primer 15.H.2) that is a pre- existing part of the genomic receiving sites. Expected size 566bp. Primers used to as- sess integration of the TCR-beta, annealed to the TRBC segment (forward primer 1.F.9) and the sv40pA terminator (Reverse primer 15.H.2) that is a pre-existing part of the genomic receiving sites. Expected size 610bp. PCR products were run on a 1% 1O Agarose gel in 1XTAE buffer, using the PowerPac Basic (Bio-Rad), stained with 10,000 on of sybersafe and analyzed with Fusion SL (Vilber Lourmat).

Table 6: PCR reagents for assess ation of ORF encoding TCR- alpha and TCR-beta Reactlon Component v Volugge per og TCR—al ha 5XPhusion buffer 4 ul DNTPs 0,2 ul Phusion DNA polymerase 0,15 ul 1.F.7: TRAC—GT-F1 0,5 ul .H.2: sv40pA-GT-R1 0,5 ul H20 up to 20 ul DNA (100ng) 1 III (100 ng/ul) (ReactlonComponen mper reaction TCRBeta 5XPhusion buffer 4 ul DNTPs 0,2 ul Phusion DNA polymerase 0,15 ul 1.F.9: TRBC2-GT-F1 0,5 ul .H.2: sv40pA-GT—R1 0,5 ul H20 up to 20 ul DNA (100 ng) 1 ul (100 ng/ul) Table 7: PCR cycle conditions Step H \ InItIaléDenaturatIon 98°C: 30 sec cycles 98°C 10 sec 60°C 10 sec 72°C 15 sec Final ion 72°C 10 min ddPCR reactions to assess the copy number of TRA-ORF and TRB-ORF in the genome after DNA delivery DNA of selected ACL—851 monoclones was analysed by using specific s and probed targeting the TCR ORF C segment (TRAC) of interest. s and probe used to assess TRA—ORF copy number, annealed to the TRAC segment (forward primer 1.F.7, Reverse primer 1.F.8 and probe . Primers and probe used to assess TRB- ORF copy number, annealed to the TRB-C segment (forward primer 1.F.9, Reverse primer 1.F.10 and probe 1.G.2) In all cases, a reference gene (TRAC) was simultaneously screened to chromosome determine copy numbers, using primers 10.A.9 and 10.A.10 together with the fluores- cent probe 10.B.6 ated with HEX. Integration copy number considered that HEK293 cells are triploid for reference gene (TRAC). Prior to Droplet Digital PCR, DNA was digested with Mfel (NEB) to separate tandem integrations. The reaction setup and cycling conditions were followed according to the protocol for ddPCRT'V' Supermix for Probes (No dUTP) (Bio-Rad), using the QX200T'V' Droplet Reader and Droplet Genera- tor and the C1000 TouchT'V' deep-well Thermal cycler (Bio-Rad). Data was acquired us- ing the QuantaSoftT'VI Software, using cm to detect FAM and Ch2 for HEX.

Table 8: ddPCR conditions mstep, ’iNlWKW ,mlzmw «,mlzwwt \‘A W1-emperatu v Time ulgmw Initial Denaturation 95140 V min 40 cycles 98°C 30 sec 60°C 60 sec Final extension 72°C 10 min n) Cooling 8 C 00 Table 9: ddPCR Primers and probes 1.F.7 TRAC-GT-F1 ATGTGCAAACGCCTTCAAC 1.F.8 TRAC-GT—R1 TTCGGAACCCAATCACTGAC 1.G.1 TRAC-probe-FAM GACCAGCTTGACATCACAGG 1.F.9 TRBC2-GT-F1 AAGTCCAGTTCTACG 1.F.10 TRBC2-GT-R1 CTTGCTGGTAAGACTCGGAG 1.G.2 TRBC2-probe-FAM CAAACCCGTCACCCAGATCGTCA .A.9 TRAC-TCRA-ex1-F1 CTGATCCTCTTGTCCCACAGATA .A.10 TRAC-TCRA-ex1—F1 GACTTGTCACTGGATTTAGAGTCTCT .B.6 TRAC-probe(H EX) ATCCAGAACCCTGACCCTGCCG 21.|.1 HCMVpp65_GT_F2 TCGACGCCCAAAAAGCAC 21.|.2 HCMVpp28_GT_F1 TGCCTCCTTGCCCTTTG 21.|.3 HCMVpp52_GT_F1 CGTCCCTAACACCAAGAAG .H.10 Myc-Tag_GT_R1 AAGGTCCTCCTCAGAGATG .H.9 Linker-Myc_Probe_Fam CTTTTGTTCTCCAGATCCAGATCCACC Sequencing of TCR alpha and beta chains from single T-cells Individual FACS-sorted eTPC-t—cells were subjected to a ep ication pro— cess that entails a V—region specific primer collection for each TRA and TRB, ed by paired nested PCR ons that create TRA and TRB amplicons for sequence analysis. This procedure is described previously (Han et. al. Nat Biotechnol. 2014 32(7): 684-692). The following materials were used in the described procedures: Table 10: Single cell RT-PCR and nested PCR reagents Su "plier Mum- 2x Reaction Mix Thermo Scientific 12574035 5X Phusion HF Buffer Thermo Fisher Scientific F-5498 dNTPs Thermo Fisher Scientific 10297018 Nuclease free water Qiagen 129114 Phusion Hot Start || DNA Polymerase Thermo Fisher Scientific F-5498 SuperScript® ||| One- Step RT—PCR Sys- Thermo ific 35 tem with Platinum® Taq High Fidelity DNA Polymerase Functional demonstration of ent F eTPC—t and eAPC cells were routinely cultured in RPM|+10% heat-inactivated Fetal Calf Serum (complete media) between 0.2 x 10"6 — 1.5 x 10"6 cells/ml, at 37’C, 90% relative humidity and 5% C02. Peptide NLVPMVATV were synthetized by Genescript, and received lyophilized. Peptide y stocks were suspended in 10% DMSO and sorted at -80’C. Working stocks were prepared at the time of administration, at 50 pM in complete media (50x concentrated). The following eAPC-p presenting HLA-A*02:01 1O (AOL-900) or HLA—B*07:02 06) or eAPC-pa with aAPX and exogenous aAM, HLA-A*02:01+HCMVpp52 (ACL-1044) or HLA-A*02:01+HCMVpp65 (ACL-1046) or HLA-B*07:02+HCMVpp52 (AOL-1045) or HLA-B*07:02+HCMVpp65 (AOL-1048), or parent eAPC (ACL-128) were used. Two different eTPC-t cell lines were used; the first eTPC-t, ACL—1277, nent A) was engineered with two unique genomic receiver sites, utilizing native CD3 expression, and harboring a genomic two—component, syn- thetic se element (Component F, RFP reporter) (See Example 14). The second eTPC-t, ACL-1150, (Component A) was engineered with two unique genomic receiver sites, ing native CD3 expression, and harboring a c one-component, syn- thetic response element (Component F, RFP reporter) (See Example 15). Both eTPC-t were loaded with the TCR chain ORF at Component 2B’ and 2E’ encoding a TCR pair that is specific for HLA—peptide complex (H LA), HLA—A*02:01—NLVPMVATV.

Antigen pulsing procedure Actively growing cultures of eAPC cells .0 x 10"6 cells/ml) were suspended, sam- ple taken and counted to determine cell concentration. Subsequently, 1 million cells were harvested, washed once with Dulbecco’s phosphate buffered saline (DPBS, Gibco) followed by suspension in complete media with 1 pM of peptide or no peptide at a cell concentration between 1 to 2 x 10"6 cells/ml. Cells were incubated for 2 h in rd culturing conditions, in a 24—well culture plate. After 2 h the cells were har— vested, pelleted by centrifugation (400 rcf, 3 min), followed by 3 x 10 ml washes with DPBS. Cells were subsequently suspended at 0.2 x 10"6 cells/ml in complete media. e TPC-t harvesting ly g cultures of eTPC-t cells (0.4-1.0 x 10"6 cells/ml) were suspended, sample taken and counted to determine cell concentration. Cells were harvested, washed once with DPBS and then suspended at a concentration of 0.4x10"6 (for en— us assays) or 0.6 x 10"6 cells/ml (or exogenous assays) in complete media.

Contacting eTPC-t and eAPC in an eTPC.'eAPC system with exogenous nic mol- ecu/es To each well of a 96-well bottom plate, 50 pl of complete media, 50 pl of eAPC , followed by 50 pl of eTPC-t were added. This equated to approximately 10,000 eAPC and 30,000 eTPC-t for a ratio of 1:3, at a total cell concentration of approximately 0.27 x 10"6 cells/ml. The cell mixture was then incubated for approximately 24 hours at standard culturing conditions.

Contacting eTPC-t and eAPC in an eTPC.'eAPC system with endogenous antigenic molecules To each well of a 96-well round-bottom plate, 50 pl of complete media, 50 pl of eAPC , followed by 50 pl of eTPC-t were added. This equated to approximately 10,000 eAPC and 20,000 eTPC-t for a ratio of 1:2, at a total cell concentration of approximately 0.2 x "6 cells/ml. The cell mixture was then ted for approximately 48 hours at stand- ard culturing conditions.

Staining and analysis After 24 or 48 hours incubation, the cells were harvested, and transplanted into 0.75 ml V-bottom Micronic tubes, washed once with 500 pl DPBS and subsequently stained with Dead Cell Marker (DCM-APC-H?) as follows; to each well 25 pl of staining solution was added, cells suspended by mixing and then incubated for 15-20 min. The staining solution comprised of 0.5 pl DCM-APC-H7 per 100 pl staining solution. After incuba- tion, cells were washed twice with 500 pl DPBS+2%FCS (Wash Buffer). Cells were then stained for surface markers unique to the eTPC-t; to each well 30 pl of staining solution was added, cells suspended by mixing and then incubated for 30-45 min. The staining solution comprised of 2.5 pl anti-myc-AF647 per 100 pl ng solution (clone 9E10, Santa Cruz Biotech). After tion, cells were washed twice with 500 pl Wash buffer, suspended in 200 pl of Wash buffer and then ed by FACS on a LSRFor— tessa (BD Biosciences).

Examples Example 1: on of an APX gene family by targeted mutagenesis Herein describes how targeted mutagenesis of a family of antigen-presenting complex (APX) encoding genes was achieved to produce the first trait of an engineered antigenpresenting cell (eAPC). The said trait is the lack of surface expression of at least one member of the APX .

In this e, the targeted APX sed the three members of the major HLA class | family, HLA-A, HLA-B and HLA—C in the HEK293 cell line. HEK293 cells were derived from human embryonic kidney cells that showed endogenous surface expres- sion of HLA-ABC. Cytogenetic analysis demonstrated that the cell line has a near trip- loid ype, therefore the HEK293 cells encoded three alleles of each HLA-A, HLA- B and HLA-C gene.

Targeted mutagenesis of the HLA—A, HLA—B and HLA-C genes was performed using an engineered CRISPR/Cas9 system, in which, Cas9 nuclease activity was targeted to the HLA-A, HLA—B and HLA-C loci by synthetic guide RNAs (gRNAs). 4 to 5 unique gRNAs were designed to target conserved nucleotide sequences for each HLA gene locus and the targeted sites were biased towards the start of the gene coding sequence as this was more likely to generate a null allele. The gRNAs efficiency to induce a mutation at their targeted loci was determined and the most efficient gRNAs were selected to gen- erate the HLA—A, HLA—B and HLA-C null (HLA-ABC""") HEK293 cell line.

Plasmid that encoded the optimal gRNAs targeting the HLA—A, HLA—B and HLA-C loci, together with a plasmid that encoded Cas9-P2A—GFP were transfected into HEK293 cells as described in the methods. Cells positive for Cas9-P2A-GFP plasmid uptake were FAC sorted based on GFP fluorescence, 2 days after transfection (figure 38a).

The GFP sorted cells were further expanded for more than 5 days to allow sufficient time for gene editing events to occur, and in the case of a detrimental mutation, to lose of expression of the residual nous HLAI protein. After this growth period, the cells were stained with a A—ABC dy, resulting in the identification of cells with reduced expressed HLA—ABC on their surface (figure 38b). The absence of pan— HLA-ABC antibody staining implied that each HLA-A, HLA-B and HLA-C allele was mu- tated. dual HLA-ABC negative cells were sorted and expanded to represent a col- lection of monoclones.

HLA-ABC""" monoclones were confirmed by lack of surface expression of HLA-ABC. It was demonstrated that a subset of monoclones lacked surface expression of HLA- ABC, of which three example monoclones, AOL-414, AOL-415 and AOL-416 are de— picted in figure 39. Further c characterization of the monoclones that lacked HLAI surface expression was performed by determining that the cell lines possessed an underlying genetic mutation in all s of the HLA—A, HLA-B and HLA—C genes (figure 40). Genetic characterization was performed by PCR with primers that spanned the gRNA genomic target sites, for detection of amplicon size s and/or were used as a template for sequencing. Figure 40 shows a selection of HLA-ABC""" mono- clones that contained genetic deletion in the alleles of HLA-A, HLA—B and HLA—C genes detected by a shorter PCR on compared to the amplicon size of the founding cell line (e.g. ACL-414).

In conclusion, the genetically modified HEK293 cell lines, including, ACL-414, AOL-415 and AOL-416, were demonstrated to lack surface expression of the C and therefore possessed the first trait of an engineered antigen-presenting cell (eAPC).

Example 2: Generation of an eAPC containing Component 1B Herein describes how Component 1B was stably ated into the HLA-ABC""" mon- oclone line ACL-414 to produce the second trait of an eAPC. The said second trait con- tained at least one genomic receiver site for integration of at least one ORF, wherein the genomic receiver site was a synthetic construct ed for recombinase medi— ated cassette exchange (RMCE).

In this example, the genomic integration site, component 18, comprised of selected ge- netic elements. Two unique specific recombinase sites, FRT and F3, which flanked an ORF that encoded the selection marker, blue fluorescent protein (BFP). En- coded 5’ of the FRT site, was an EF1a promoter and 3’ of the F3 site was a SV40 poly- adenylation signal terminator. The benefit of positioning the non-coding cis—regulatory elements on the outside of the heterospecific recombinase sites was so they are not re- quired in the matched genetic donor vector, component 10. Therefore, after ar delivery of the genetic donor vector, no transient expression of the d ORF would be observed. This made the selection of successful RMCE more reliable as the cellular sion of the ORF from the genetic donor vector would mostly likely occur only af— ter correct integration into component 18 as it now contained the riate gu- lator elements (see example 6).

To promote the stable genomic integration of component 18 into the genomic safe har- bour locus, AAVSi, a plasmid was ucted, wherein; the DNA elements of compo- nent 18 were d with AAVS1 left and right homology arms. Each arm comprised of >500 bp of sequence homologous to the AAV81 genomic locus.

Stable integration of ent 13 was achieved h the process of gy di- rected recombination (HDR) at the genomic safe harbour locus, AAVS1. The ACL-414 cell line was transfected with plasmid that encoded the optimal gRNAs ing the AAV81 locus, plasmid that encoded Cas9—P2A—GFP and the plasmid that encoded component 18 genetic elements flanked by AAVS1 left and right homology arms. Cells positive for Cas9—P2A—GFP plasmid uptake were FAC sorted based on GFP fluores- cence, 2 days after transfection (figure 41 a). The GFP sorted cells were further ex- panded for greater than 7 days allowing sufficient time for HDR to occur and to lose ent expression of the selection marker, BFP. After this growth period, the cells were analysed on a FACS machine and individual BFP positive cells were sorted and expanded to represent a collection of monoclones (figure 41c).

Individual monoclone lines were selected as an eAPC on the basis of their maintained BFP expression and for a single integration of component 18 into the desired AAVS1 genomic location. Cell lines AOL-469 and ACL-470 represented monoclones with main- tained BFP expression (figure 42a and b). Genetic characterization was performed on DNA extracted from monoclones AOL-469 and 0 and trated that their genomes integrated component 18, and that component 18 has been ated into the AAVS1 site (figure 43). Confirmation of genomic integration was determined by the detection of a PCR amplicon of the expected size that utilized primers specific for the Component 1B (figure 43a). Confirmation that component 18 ated into the AAVS1 site was determined by the detection of a PCR amplicon of the expected size that utilized primers designed against the AAVS1 genomic sequence distal to the re- gion encoded by the homologous arms and a primer that is unique to the SV40 pA ter- minator encoded by component 18 (Figure 43b). The copy-number of component 18 was determined by digital drop PCR, in which the number of ent 18 and refer— ence gene DNA molecules were measured and the ratio calculated (table 1). Mono- clones AOL-469 and AOL-470 contained a ratio of 1 component 18 molecule to 3 refer- ence gene molecules. When factored in that the ng HEK293 cell line has a near triploid karyotype, this demonstrated a single integration of component 18 in AOL-469 and ACL-470 cell lines.

In conclusion, the genetically modified AOL-469 and AOL-470 cell lines, were HLA— ABC""" and contained a single copy of a synthetic genomic receiver site designed for RMCE and therefore demonstrated the creation of a eAPC with a single synthetic inte- gration receiver site.

Example 3: Generation of an eAPC containing Component 1B and Component 1D Herein describes how Component 1B and Component 1D were stably integrated into the HLA-ABC""" monoclone line ACL-414 to produce the second trait of an eAPC. The 1O said second trait contains two c receiver sites for integration of at least one ORF, wherein the genomic receiver site was a synthetic construct designed for recom- binase mediated cassette exchange (RMCE).

This example uses the same methods and components as bed in example 2 but with the on of a second genomic receiver site, Component 1D. Component 1D genetic elements comprised of two unique heterospecific inase sites, F14 and F15, which were different to component 18. These sites flanked the ORF that encoded the selection marker, the red fluorescent protein (RFP). Encoded 5’ of the F14 site was an EF1a promoter and 3’ of the F15 site was a SV40 polyadenylation signal terminator.

As in e 2, component 1D c elements were flanked with AAVS1 left and right homology arms, each comprised of >500 bp of sequence gous to the AAVS1 genomic locus.

Component 1B and component 1D were integrated into the AAVS1 as described in ex- ample 2 but with the addition of the plasmid that encoded component 1D elements, to the ection mix. Cells positive for Cas9—P2A-GFP plasmid uptake were FAC sorted based on GFP fluorescence, 2 days after transfection (figure 41b). The GFP sorted cells were further expanded for grater than 7 days, after which, the cells were analysed on a FACS machine and dual BFP and RFP positive cells were sorted and ex— panded to represents a collection of monoclones (figure 41d).

Individual one lines were selected as an eAPC on the basis of their maintained BFP and RFP expression and for a single integration of component 18 and a single in- tegration of component 1D into different AAVS1 alleles. Cell line 2 was a repre- sentative monoclone with maintained BFP and RFP expression (figure 42c). As de- scribed in example 2, genetic characterization was performed on DNA extracted from monoclone AOL-472 and demonstrated that their genomes integrated ent 18 and component 1D, and that both components integrated into the AAVS1 site (figure 43). The copy-number of both component 18 and D was determined by digital drop PCR, in which the number of component 18, D and reference gene DNA molecules was measured and the ratio calculated. The monoclone 2 contained a ratio of 2 component 18 and D molecules to 3 reference gene molecules (Table 2). When fac— tored in that the founding HEK293 cell line has a near triploid karyotype, this demon- strated a single integration of ent 18 and a single integration of component 1D into the AOL-472 cell line.

In conclusion, the genetically modified AOL-472 cell line, was C""" and con- tained a single copies of the synthetic genomic receiver site component 18 and compo- nent 1D, designed for RMCE and therefore demonstrated the creation of an eAPC with two unique synthetic integration receiver sites.

Example 4: An eAPC-p constructed in one step with one integration couple wherein component 1C’ encoded a single HLAI ORF Herein describes how an eAPC-p was constructed in one step with one ation cou- ple, wherein, the genomic receiver site, component 18, is a native genomic site and the genetic donor vector, component 10’, comprised a single ORF that encoded one analyte antigen-presenting complex (aAPX).

In this example, the eAPC was a genetically modified ARH-77 cell line, designated AOL-128, wherein, two families of APX, major HLA class | family and HLA class II, were mutated. The founding cell line, , is a B lymphoblast derived from a plasma cell leukemia that showed strong HLA-A,B,C and HLA-DR,DP,DQ cell surface expression. Cytogenetic analysis demonstrated that the founding ARH-77 cell line has a near diploid karyotype, but also displayed a chromosome 6p21 deletion, the region encoding the HLA locus. DNA sequencing of the ARH-77 locus confirmed that ARH—77 encoded only a single allele of HLA-A, HLA-B and HLA-C and HLA-DRA, HLA-DRB, HLA-DQA, B, HLA-DPA and HLA-DPB gene families.

The HLA—ABC""" and HLA-DR,DP,DQ""" cell line AOL—128, was generated by /cas9 ed mutagenesis with gRNA ing the HLA-A, HLA-B and HLA- C and HLA-DRA, HLA-DRB, HLA-DQA, HLA-DQB, HLA-DPA and B gene fam- ilies using the method bed in Example 1. Surface labeling with a pan-anti-HLA- ABC or pan-anti-HLA—DR,DP,DQ med that AOL-128 lacked surface expression of both APX families, figure 44b and 45 and figure 47b, respectively.

In this example, the c receiver site, component 18, was the native AAVS1 ge— nomic site, and the targeted integration was achieved through HDR. The genetic donor vector, component 10, was matched to component 18, by component 10 encoding the AAVS1 left and right homology arms, each sed of >500 bp of sequence - gous to the AAVS1 genomic locus. Between the AAVS1 left and right homology arms, the plasmid encoded a CMV promoter and a SV40 ator. The aAPX of interest was cloned between the promoter and the terminator, generating component 1C’. In this example, component 10’ comprised a single ORF that encoded one aAPX, the HLA-A*24:02 or HLA-B*-07:02, denoted component 1C’HLA'N24102 and component 1C’HLA-B*—07:02 respectively.

The process to construct an eAPC-p was via HDR induced integration of component ’ into component 18 to produce component 1B’. The cell line 8 was electro- d with plasmids that encoded the optimal gRNAs targeting the AAVS1 loci, Cas9-P2A—GFP and ent 10’. Cells positive for 2A—GFP plasmid uptake were FAC sorted based on GFP fluorescence, 2 days after electroporation (figure 44a). The GFP sorted cells were further expanded for grater than 7 days allowing suffi- cient time for HDR to occur and lose transient expression of the aAPX. After this growth period, the cells were stained with a pan-HLA—ABC antibody, resulting in the identification of cells that gained expression of an analyte HLA on their surface (figure 44b). The presence of A-ABC antibody staining implied that the analyte HLA ORF encoded in component 10’ had integrated into the genome. Individual HLA-ABC positive stained cells were sorted and expanded to represent a collection of eAPC-p ones.

Individual monoclone lines were selected as an eAPC—p on the basis of their main- tained analyte HLA e expression and the integration of the e ORF into the genomic er site, creating component 18’. Cell lines AOL-321 and AOL—331 were representative monoclones with maintained analyte HLA surface expression of HLA- A*24:02 or HLA—B*-O7:02 respectively (figure 45). Genetic characterization was per— formed on DNA extracted from selected ones AOL-321, AOL-327, AOL-331 and AOL-332 and demonstrated that their genomes integrated component 10’, and that the integration occurred in the AAVS1 genomic receiver site, generating component 18’ (figure 46). Confirmation of genomic integration was determined by the detection of a PCR on of the expected size using primers specific to the Component 10’ (fig- ure 46a). Presence of component 18’ was med by the detection of a PCR am- plicon of the expected size using primers designed against the AAVS1 genomic se— quence distal to region encoded by the homologous arms and a primer unique to the SV40 pA terminator linked to the analyte HLA ORF (Figure 46b).

In conclusion, the generation of the genetically modified AOL—321 and AOL—331 cell lines, which contained a copy of the aAPX HLA-A*24:02 or HLA-B*—07:02 ORF, respec- tively, within the genomic receiver site, component 18’, resulted in the said e aAPX to be the only major HLA class | member expressed on the cell surface. There- fore, this demonstrated the creation of two defined eAPC-p cell lines using the multi- component system. e 5: An eAPC-p constructed in one step with one integration couple, wherein component 1C’ encoded a paired HLAII ORF Herein describes how an eAPC-p was ucted in one step with one integration cou- ple, wherein, the genomic er site, component 18, was a native c site and the genetic donor vector, component 10’ comprised a single ORF that encoded two aAPX chains.

This example used eAPC, AOL-128, and component 18, both of which are defined in e 4. However component 10’ comprised a single ORF that encoded an HLA— DRA*01 :01 allele linked to an B1*01:01 allele by a viral self-cleaving peptide element, or HLA-DPA1*01 :03 allele linked to an HLA—DPB1*04:01 allele by a viral self- cleaving e element, denoted component 1C’HLADRNW=01mL/H3RBV01:01 and compo- nent 1C’ HLA'DPA1*01iO3/HLA'DPB1*04301 respectively. The viral leaving peptide element encoded a peptide sequence, that when transcribed resulted in self—cleavage of the synthesized peptide and produced two polypeptides defining each HLA chain.

Within example 4, described the process to construct an eAPC-p with the exception that identification of cells that gained expression of an e HLA on their surface were assed by cell surface labelling with a ti-HLA-DR,DP,DQ antibody (figure 47). The presence of pan-anti-HLA-DR,DP,DQ antibody staining implied that the ana- lyte HLA ORF encoded in component 10’ had integrated into the genome. Individual HLA-DR,DP,DQ positive stained cells were sorted and expanded to represent a collec- tion of eAPC-p monoclones.

Individual monoclone lines were ed as an eAPC—p on the basis of their main- tained analyte HLA e expression and the ation of the analyte ORF into the genomic receiver site, creating component 18’ as described in example 4. Cell lines AOL-341 and AOL-350 were the representative monoclones with maintained analyte HLA surface expression of HLA—DRA*01 :01/HLA-DRB1*O1 :01 or HLA— DPA1*01:03/HLA-DPB1*04:01 (figure 48).

In conclusion, the generation of the genetically modified 1 and AOL-350 cell lines, which contained a copy of the aAPX HLA-DRA*01:011HLA-DRB1*O1:01 or HLA- DPA1*01:03/HLA-DPB1*04:01 ORF, respectively, within the genomic receiver site, component 18’, resulted in the said analyte aAPX to be the only major HLA class II member expressed on the cell surface. Therefore, this demonstrated the creation of two defined eAPC-p cell lines using the multicomponent system.

Example 6: An eAPC-p constructed in one step with one integration couple wherein component 1B was a synthetic construct Herein describes how an eAPC-p was ucted in one step with one integration cou- ple, wherein, the genomic receiver site, ent 18, was a synthetic construct de- signed for RMCE genomic site and the genetic donor vector, component 10’ comprised a single ORF that encoded one aAPX.

In this example, the genomic integration site, component 18, comprised of selected ge- netic elements. Two unique heterospecific recombinase sites, FRT and F3, which flanked the ORF that encoded the selection marker, blue scent protein (BFP). En- coded 5’ of the FRT site, was an EF1a promoter and 3’ of the F3 site was a SV40 poly- adenylation signal terminator. The genetic elements of component 18, were integrated in the cell line AOL-128 by electroporation with the same plasmids as described in ex- ample 2. Individual monoclone lines were selected on the basis of their ined BFP expression and were genetically charaterised to contain a single integration of component 18 into the d AAVS1 genomic on as bed in example 2 e 49a). The resulting eAPC cell line, AOL-385, was HLA-ABC""" and HLA- DR,DP,DQ""" and contained a single copy of a synthetic genomic receiver site, compo- nent 18, designed for RMCE The genetic donor vector, component 10 was matched to component 18, as compo— nent 1C encoded the same heterospecific recombinase sites, FRT and F3. The aAPX ORF of interest, additionally encoded a kozak sequence just before the start codon, was cloned between the two heterospecific inase sites, and generated compo- nent 1C’. In this example, ent 10’ comprised a single ORF that encoded one aAPX, the HLA-A*02:01, designated component 1C’FRT1HLA'A*°21011F3.

An eAPC-p was d through RMCE by electroporation of the cell line AOL-385 with plasmid that encoded the Tyr-recombinase, Flp, together with component 1C’FRTIHLA‘ N021011F3. 4-10 days after electroporation, individual cells positive for HLAI e ex- pression and negative/reduced for the fluorescent protein marker, BFP, encoded by component 18 selection marker, were sorted. Individual outgrown monoclone lines were ed on the basis of their maintained HLAI allele expression and loss of BFP florescence, which indicated that the expected RMCE occurred. To identify such mono- clones, both phenotypic and genetic tests were performed. Firstly, all monoclone cell lines were ed for cell surface HLA-ABC expression and lack of BFP florescence (figure 49). Genomic DNA was extracted from such cell lines, e.g. AOL-421 and ACL- 422, and the integration of ent 10’ into component 18 that generated compo- nent 1B’ was confirmed by the detection of a PCR product specific to component 18’ (figure 50).

In conclusion, the generation of the genetically modified AOL—421 and 2 cell lines, which contained a copy of the aAPX HLA-A*02:01 ORF, tively, within the synthetic genomic receiver site, component 18’, resulted in the said analyte aAPX to be the only major HLA class | member expressed on the cell surface. Therefore, this trated the creation of two d eAPC-p cell lines using the multicomponent Example 7: An eAPC-pa constructed in two steps with two integration couples Herein describes how an eAPC—pa was constructed in two steps. Step 1, wherein the genomic receiver site, component 18, was the native genomic site and the genetic do- nor vector, component 10’ comprised a single ORF that encoded one aAPX. Step 2 the genomic receiver site, component 1D, was a second native genomic site and the genetic donor vector, component 1E’ comprised a single ORF that encoded one ana- lyte n molecule (aAM).

In this example, step 1 was performed, wherein, the eAPC was AOL—128, the genomic receiver site, component 18, was the mutated HLA—A allele genomic site, ated HLA—Am‘", and the targeted integration was ed through HDR. The genetic donor vector, component 10 was matched to component 18, by the component 10 encoding the HLA—Anu" left and right homology arms, each comprised of >500 bp of sequence homologous to the HLA—Anu" genomic locus. Between the HLA—Anu" left and right homol- ogy arms, the plasmid d a CMV er and SV40 terminator. The aAPX of interest was cloned between the promoter and terminator, generating component 10’.

In this example, component 10’ comprised a single ORF that encoded one aAPX, the HLA-A*02:01 or HLA—B*—35:01, denoted component 1C’HLAW02:01 component 1C’HLA'B*' "" respectively.

The integration of component 10’ into component 1B, and selection of monoclone eAPC-p cell lines was as described in example 4, with the exception that a gRNA tar- geting the HLA-A""" genomic locus was used to promote HDR integration of component ’ into component 18. Monoclone eAPC-p AOL-191 and AOL-286 expressed HLA- A*02:01 or —35:01 on the cell surface, respectively (figure 51a).

In this example, step 2 was performed, n, the genomic receiver site, component 1D, was the native AAVS1 genomic site, and the ed integration was achieved through HDR. The genetic donor vector, component 1E was matched to component 1D, by the component 1E that encoded the AAVS1 left and right homology arms, each sed of >500 bp of sequence homologous to the AAVS1 c locus. Be- tween the AAVS1 left and right gy arms, the plasmid encoded a CMV promoter and SV40 ator. The aAM of interest was cloned between the promoter and ter- minator, generating Component 1E’. In this example, ent 1E’ comprised a sin- gle ORF that encoded the selection marker, GFP, linked to the aAM ORF, encoding hCMV—pp65, denoted component 12A1PP63. The viral self—cleaving peptide element encoded a peptide ce, that when transcribed resulted in self—cleavage of the synthesized peptide and produced two polypeptides, GFP and the intracellular hCMV- pp65 protein.

The integration of ent 1E’ into component 1D, was as described in example 4.

Individual monoclone lines, AOL-391 and AOL-395, were selected as an eAPC-pa on the basis of their maintained selection marker GFP expression (figure 51b).

In conclusion, the genetically modified AOL-391 and AOL-395 cell lines, which con— tained a copy of the aAPX HLA-A*02:01 or HLA-B*—35:01 ORF, tively, within the genomic receiver site, component 18’, and aAM ORF pp65 within the c receiver site component 1D’ were generated. These genetic modifications resulted in the said aAPX to be the only major HLA class | member expressed on the cell surface of a cell that also sed the said aAM. ore, this demonstrated the creation of two de- fined eAPC-pa cell lines using the multicomponent system.

Example 8: An eACP-p constructed in one step wherein ent 1C’ encoded a single HLAI ORF.

Herein describes the conversion of an eAPC to an eAPC-p in one step, via a single in- tegration couple event, to integrate a single HLAI ORF encoding analyte antigen-pre- senting complex (aAPX), and wherein the eAPC contains two tic genomic re— ceiver sites Component 1B and ent 1D designed for RMCE based genomic integration. The created eAPC—p has one genomic receiver site occupied by the HLAI ORF (Component 1B’), while the remaining Component 1D is available for an addi- tional integration couple event.

This example used the eAPC generated in example 3 (AOL-402) containing Compo- nents 1B and 1D, wherein Component 1B ses two unique heterospecific re- combinase sites, F14 and F15, which flank the ORF that encodes the selection marker, red fluorescent protein (RFP). Encoded 5’ of the F14 site is an EF1a er and 3’ of the F15 site is a SV40 polyadenylation signal terminator. Component 1D comprises of two unique heterospecific recombinase sites, FRT and F3, flanking the ORF that en- codes the selection marker, blue fluorescent protein (BFP). Encoded 5’ of the FRT site, is an EF1a promoter and 3’ of the F15 site is a SV40 polyadenylation signal ator.

This example utilizes a Component 1C genetic donor vector, comprising of heterospe- cific recombinase sites, F14 and F15 and thus is matched to Component 13. Two in- dependent Component 1C’ were generated from Component 1C, wherein one vector (V4.H.5) comprises of a Kozak sequence, start codon and aAPX ORF encoding HLA- A*02:01 between the F14/F15 sites, and wherein the second vector (V4.H.6) comprises a Kozak ce, start codon and aAPX ORF encoding HLA-A*24:02 between the F14/F15 sites.

The eAPC (AOL-402) was independently combined with vector encoding expression of the RMCE recombinase enzyme (Flp, v4.1.8) and each ent 1C’ of either V4.H.5 or V4.H.6 by electroporation. Cells were cultured for 4-10 days, whereupon cells were selected and sorted based on loss of the selection marker of ation, RFP, and gain of HLAI on the surface of the cell. Subsequently, individual wn one lines were characterized, confirmed and selected on the basis of the gain of HLAI surface expression and the loss of the RFP fluorescence, which indicated that the expected conversion of Component 13 to 13’ had occurred. Selected eAPC—p mono— clones AOL-900 (V4.H.5, HLA-A*02:01) and AOL-963 (V4.H.6, HLA—A*24:02) are neg- 1O ative for RFP compared to the parental AOL-402 cell line and maintain HLAI surface expression (Figure 53a). Furthermore, both monoclones retain expression of the BFP selection marker of integration, indicating that Component 1D remains uncoupled and isolated from Component 1B integration couple events. To further characterize the eAPC-p monoclones, genomic DNA was extracted from the cells, and confirmation of the integration couple between Component 1C’ and Component 1B, generating Component 1B’, was ted by detection of a PCR product specific to Compo- nent 1B’ (Figure 53b). The primers were designed to target a region nt to the c receiver site (primer ID 8.8.3), and a region within the integration couple event (primer ID 15.H.2). ication occurred only in cases of specific integration, while no product was generated from the l (AOL-3) or from rget recombination.

In summary, this example demonstrates two specific examples of conversion of an eAPC to an eAPC-p, using the multicomponent system, n two different aAPX are individually delivered (Component 1C’) and integrated into a single genomic receiver site (Component 1B) by RMCE genomic integration , uently creating a d library comprising two discrete eAPC-p. Furthermore, it was demonstrated that second genomic receiver site (Component 1D) was insulated and unaffected by the Component 1BlComponent 1C’ integration couple.

Example 9: An a constructed from eAPC-p in one step, wherein Compo- nent 1D’ encodes a single analyte antigen molecule (aAM) ORF.

The present example describes how multiple eAPC-pa are constructed from a parental eAPC—p (described in example 8) in parallel, wherein the genomic receiver site, Component 1D, is targeted for integration by a primed genetic donor vector, Component 1E’, comprising of a single ORF that encodes an aAM.

In the present example, the parental eAPC-p line used was AOL-900, which expresses a single aAPX (HLA-A*02:01 ) that is integrated at ent 1B’ (described in exam- ple 8). The eAPC-p Component 1D remains open and comprises of two unique heter- ospecific recombinase sites, FRT and F3, which flank the ORF that encodes the selec- tion , blue fluorescent protein (BFP). Encoded 5’ of the FRT site, is an EF1a pro- moter, and 3’ 0f the F15 site is a SV40 polyadenylation signal terminator. The genetic donor vector, ent 1E was used in this example and comprises of two heteros- pecific recombinase sites, F14 and F15, thus being matched to Component 1D. In this example, the Component 1E was further primed with one aAM ORF of interest se- 1O lected from HCMVpp28 (V9.E.6), HCMVpp52 (V9.E.7), or HCMVpp65 8), which also each encode a C-terminal e-serine rich linker and c-myc tag. Furthermore, each Component 1E’ further comprises of Kozak sequence and start codon immedi- ately 5’ of the aAM ORF. Thus, a small discrete library of Component 1E’ was created, comprising of three vectors.

The eAPC-p (AOL-900) was independently combined with a vector encoding expres- sion of the RMCE recombinase enzyme (Flp, V4.1.8) and each Component 1E’ of ei- ther V9.E.6, V9.E.7, or V9.E.8 by electroporation. Cells were incubated for 4-10 days to allow for the integration couple to occur, whereupon, dual eAPC-pa were selected and single cell sorted (monoclones) based on diminished signal of the selection marker of integration BFP, encoded by Component 1D (Figure 54a). Subsequently, the indi- vidual outgrown monoclone eAPC-pa, 19 (pp28), AOL-1227 (pp52) and ACL- 1233 (pp65), were characterized, confirmed and selected on the basis of the loss of BFP expression and maintained surface expression of HLAI (aAPX at Component 1B’) (Figure 54b), which indicated that the expected conversion of Component 1D to 1D’ had occurred. Furthermore, the maintained surface expression of the aAPX indi- cated that Component 1B’ was unaffected and isolated from the integration couple event between ent 1D and Component 1E’. To further characterize the se- lected eAPC-pa monoclones, genomic DNA was extracted, and mation of the in- tegration couple n Component 1E’ and Component 1D, generating Compo- nent 1D’ was conducted by ion of a polymerase chain reaction (PCR) amplicon product specific to Component 1D’ (10.D.1, 15.H.4). In Figure 540 two monoclones representing each of the three a are shown wherein amplicon products of the expected size for aAM ORF pp28 (0.8kb), pp52 (1 .5kb) and pp65 (1 .9kb) are observed, r confirming that the expected integration event has occurred.

In summary, this example demonstrates three specific examples of conversion of an eAPC—p to an eAPC-pa, using the multicomponent system, n three different aAM are individually delivered (Component 1E’) and integrated into a single genomic re- ceiver site (Component 1D) by RMCE genomic integration method, subsequently cre- ating a small library of three discrete eAPC-pa carrying three different aAM ORF. Fur- thermore, it was demonstrated that the prior loaded second genomic receiver site (Component 1B’) was insulated and unaffected by the Component 1DIComponent 1E’ integration couple. 1O Example 10: Shotgun integration of multiple analyte antigen molecule ORF into eAPC-p to create a pooled eAPC-pa library in a single step Herein describes how a pool of primed Component 1E vectors (Component 1E’) col- lectively ng multiple aAM ORF (HCMVpp28, HCMVpp52 and HCMVpp65) were integrated in a single step into the parental eAPC-p (described in example 8) to create a pooled eAPC-pa library, wherein each individual cell ates a single random ana- lyte antigen ORF derived from the original pool of vectors, at Component 1D’, such that each eAPC-pa expresses a single random aAM, but collectively the pooled library of eAPC-pa represents all of aAM ORF encoded in the original pooled library of vec- tors. This method of creating a pool of a each expressing a single random ORF from a pool of s is referred to as shotgun integration.

In this e, the parental eAPC-p line used was 5 expressing an aAPX (HLA-A*02:01) on the cell surface (the construction of the cell line is described in ex— ample 8), Component 1D and ent 1E’ were as described in example 9. In this example, the individual Component 1E’ vectors of example 9, V9.E.6, V9.E.7, and , sing of aAM ORFs ng HCMVpp28, HCMVpp52 and HCMVpp65, respectively, were mixed together in a 1:1 :1 molar ratio to create a vector pool. The eAPC-p (AOL-905) was combined with the vector pool and a vector encoding expres- sion of the RMCE recombinase enzyme (Flp, V4.1.8) by electroporation. Cells were in- cubated for 4-10 days, whereupon, cells were bulk sorted on the basis of having dimin- ished signal for the selection marker of integration, BFP, encoded by Component 1D (Figure 55a) generating the pooled cell population AOL-1050 (Figure 55b).

To confirm that the eAPC-pa pool AOL-1050 was comprised of a mixture of eAPC-pa each encoding one of HCMVpp28, HCMVpp52 or HCMVpp65 at Component 1D’, indi- vidual cells were single cell sorted from the polyclonal tion and 12 were selected at random for genetic characterisation. Amplification of the Component 1D’ was con- ducted using primers that span each aAM (10.D.1 and 15.H.4). In Figure 550, the am- s generated for the 12 cells are presented, with ls, wherein for all 12 cells a single amplicon product consistent with the expected size for one of the aAM ORF, pp28 (0.8kb), pp52 (1.5kb) and pp65 (1 .9kb) is observed. Furthermore, each aAM ORF is identified at least once indicating that the eAPC-pa pool is comprised of a mixture eAPC-pa wherein each eAPC-pa in the pool has ated a single random aAM ORF from the original pool of three vectors. 1O In conclusion, this example trates the use of the multicomponent system for conversion of an eAPC-p into a pooled library of eAPC-pa in a single step, by combin- ing the eAPC-p with a pooled library of three vectors encoding three different analyte antigen molecules (Component 1E’) and ing a RMCE based shotgun integration ch. Furthermore, this example demonstrates that each a within the gen- erated pool of eAPC-pa has integrated a single random aAM ORF from the original vector pool by an integration couple event between Component 1D and Component 1E’, and that all three aAM ORF are represented within the generated pooled eAPC-pa library.

Example 11: Demonstration of eTPC-t generation in one step The present example describes the generation of eTPC-t in a standardised manner, wherein the parental eTPC contains distinct synthetic genomic receiver sites Compo- nents ZB and 2D. All eTPC parental lines described in this example and all further ex- amples were ted with the same ques as were the eAPC lines presented in above examples. The genetic donor vectors Components 2C’ and 2E’ comprised a single chain of a TCR pair (JG9—TCR) known to engage with the antigenic peptide NLVPMVATV (NLVP) derived from Human Cytomegalovirus polypeptide 65 (HCMV pp65) when presented in HLA-A*02 alleles. Components 0’ and E’ are designed for 3O RMCE, and derived from parental Components 2C and 2E.

This example uses a parental eTPC cell line AOL-488, which is TCR null, HLA null, CD4 null and CD8 null, and r containing Component 2B and 2D. Component 2B comprises two unique heterospecific recombinase sites, FRT and F3 that flank a Kozak sequence and ORF encoding the selection marker, blue fluorescent protein (BFP). d 5’ of the FRT site, is an EF1a promoter and 3’ of the F3 site is a SV4O polyadenylation signal terminator. Component 2D ses two unique heterospe- cific recombinase sites, F14 and F15, which were different to Component ZB. These sites flank a Kozak sequence and ORF that s the selection marker, the red fluo- rescent protein, (RFP). Encoded 5’ of the F14 site is an EF1a promoter, and 3’ of the F15 site is a SV40 polyadenylation signal terminator.

This example uses genetic donor vectors, Component 2C’ and Component 2E’, each comprising of two heterospecific recombinase sites, FRT/F3 (2C’) and F141F15 (2E’), thus being matched to Component 2B and 2D, tively. Component 2C’ further 1O comprises, n the FRTIF3 sites, of a Kozak sequence, start codon and TCR ORF encoding JG9—TCR-beta chain. Component 2E’ further ses, between the F14/F15 sites, of a Kozak sequence, start codon and TCR ORF encoding JG9-TCR— alpha chain.

An eTPC-t was created through RMCE by electroporation AOL-488 (eTPC). Four to ten days after electroporation, individual cells displaying diminished fluorescent protein sig- nal, BFP and RFP, encoded by Components 2D and 2B selection markers, were sorted by FACS. Individual monoclones were out grown and then phenotypically as- sessed. The resulting monoclone, AOL-851, was BFP and RFP negative (Figure 56 a and b). AOL-851 also showed TCR and CD3 surface expression while the parental cell line did not (Figure 56 c and e). Furthermore, the introduced JGQ-TCR showed specific staining with the HLA-A*02:01- NLVP tetramer, ting that it is a functional TCRsp on the surface of the eTPC-t (Figure 56 d to f). 1 was confirmed by PCR to con- tain the TCRsp encoded by Component 2B’ and ent 2D’ integrated into the genome (Figure 56 g and h).

In y, an eTPC was converted to an eTPC-t, by use of an RMCE based integra- tion method to integrate TCR ORF delivered in Component 2C’ and 2E’, such that Components ZB and 2D were ted into Component ZB’ and 2D’, and where by this eTPC-t expressed a functional TCRsp on the e of the cell. Furthermore, this example demonstrates operation of a simple eTPC:A system, where a binary composi- tion of an eTPC-t and analyte antigen were combined and the eTPC-t selected based on a complex formation between the soluble analyte antigen (HLA multimer: HLA- A*02:01-NLVPMVATV).

Example 12: Demonstration of conversion of eTPC-t to an eTPC-x The present example describes sion of an eTPC-t to an eTPC-x, wherein the eTPC—X has component 2B’ encoding a TCR chain ORF and Component 2D is availa- ble for integration of complementary TCR chain ORF. Conversion of Component 2D’ of the eTPC-t to Component 2D of the eTPC-x is achieved by use of a genetic donor vec- tor (Component ZZ) matched to Component 2D’.

In this example, the parental eTPC-t cell line ACL-851 ted in example 11 was used. Component 22 is a plasmid vector comprised of two heterospecific recombinase sites, F14/F15 matched to Component 2D’, a Kozak sequence, start codon and an ORF encoding a green fluorescent protein (GFP) as a selection marker of integration.

The eTPC-t was combined with Component 22 and a vector encoding RMCE recom- binase enzyme by electroporation, whereupon the cells were subsequently selected for loss of CD3 presentation and gain of the GFP selection marker of integration. The monolcone AOL-987 was phenotypically characterised by FACS, and it was observed that the AOL-987 has gained GFP and lost CD3 and TCRab (Figure 57b, d), indicating successful exchange of R-alpha with the GFP ORF and conversion of Compo- nent 2D’ to Component 2D, and thus generation of an eTPC—x. In comparison the pa- rental eTPC-t, AOL-851, is lacking GFP expression and has CD3 and TCRab surface expression (Figure 57 a, c).

In summary, this example trates conversion of an eTPC-t to an , with removal of the JGQ-TCR-alpha TCR ORF at Component 2D’ in exchange for the GFP selection marker of integration thereby creating Component 2D, for further ation coupling events of alternative mentary TCR chain ORF. This conversion was conducted using the RMCE method for genomic integration. e 13: Demonstration of shotgun integration into eTPC-x to create pool of eTPC-t The present e describes how a pool of vectors encoding 64 single JGQ-TCR—al- pha variants (as Component 2E’) were integrated as a single step into a parental eTPC—X cell line (described in example 12) to create a pooled eTPC—t library n each dual cell ated a single random TCR ORF encoding alpha chain from the original pool of vectors, such that each eTPC-t expresses a single random pair of TCR ORF as TCRsp. Combined together the individual eTPC-t comprises a pooled a library of eTPC-t wherein the pool of cells potentially represents all possible combina- tions of the 64 TCR-alpha paired with constant original TCR beta chain. Such a method is now referred to as ‘shotgun’ integration. The 64 JG9-TCR—alpha ts have been created by modifying the CDR3 sequence which falls at thejunction of the V and J fragments.

In this example, the parental eTPC-x cell line AOL-987 (see example 12), with non-sur- face expressed JGQ-TCR-beta (Component ZB’) and CD3 complex was used. Com- ponent 2D encodes GFP, a selection marker, as described in example 12. In this ex- ample, the 64 JGQ-TCR—alpha variant fragments were cloned into the ent 2E donor vector, creating Component 2E’, d by F14/ F15 sites, matching to Compo- nent 2D. The 64 vectors were subsequently combined into a single pool of s.

An eTPC-t pool was created via RMCE based c integration, wherein the eTPC- x (AOL-987) and 64 Components 2E’ and RMCE recombinase vector were ed by electroporation. Polyclones were selected on the basis of the GFP expression. The ing polyclone, AOL-988, comprised of both GFP positive and GFP negative cell populations, unlike the al line which comprised of only GFP positive cells e 58 a and b). However, only GFP negative population showed tently strong CD3 sion, indicating successful conversion of Component 2D into Component 2D’ and therefore the eTPC-x has been converted into a pool of eTPC-t (Figure 58 c and d). Furthermore, AOL-988 GFP negative populations showed two distinct intensities when stained with the JG9—TCR specific multimer reagent (DEX HLA-A*02:01-NLVP), suggesting that this tion comprises of cells that express TCR variants with varying binding efficiency.

In parallel, all 64 JG9—TCR-alpha variants were cloned into an expression construct that permitted transient transfection to a parental eTPC-x (AOL-987) and relative stain- ing units (RSU) against the HLA-A*02:01-NLVP multimer t to a reference for each TCR pair presented in the above-described pooled eTPC-t expressing variant JGQ-TCR were determined. RSU were calculated as the ratio of the mean fluorescence intensity (MFI) of HLA multimer signal for the CD3 ve population over the CD3 negative population, and was indicative of the binding strength of each TCR chain pair variant. After the independent transfection of the parental AOL-987 line with each JG9- TCR-alpha variant, the cells were stained with antibodies against CD3 and with the HLA-multimer reagent and analysed by flow cytometry. Each point plotted in Figure 58e represent the observed RSU for each 64 variants.

Individual cells from the pool of ACL—988 were single cell sorted, from the HLA-multi- mer positive population and the HLA—multimer negative population. The Component 2D’ encoding the variant JG9—TCR-alpha ORF for each single cell were amplified and sequenced and compared to the results of the ent expressions RSU units de- d above (Figure 58e). Indeed, individual AOL-988 cells that were HLA-multimer positive encoded JGQ—TCR-alpha variants that predominantly showed high RSU results in the individually tested variants (Figure 58e, open circles). er, individual ACL- 988 cells that were pHLA-multimer negative encoded JGQ-TCR-alpha variants that pre- dominantly showed low RSU results (Figure 58e open triangles).

In conclusion, this example demonstrates use of the component system for con- version of an eTPC-x and a pooled library of vectors (component 2E’) into a pooled li- brary of eTPC—t containing multiple different TCRsp. This was achieved in a single step using shotgun integration. Moreover, this example demonstrated that the pool of eTPC- twere combined with an analyte antigen (as a soluble affinity reagent) into an eTPC:A , wherein it was demonstrated that single cells of the pool could be selected on the basis of complex formation with the analyte antigen ultimer) and wherein the subsequent TCR chain ORF encoded in Component 2D’ were extracted and DNA sequences obtained.

Example 14: Functional demonstration of component 2F in combined eAPC:eTPC system with exogenous aAM Herein describes an eTPC cell line (AOL-1063, Component 2A) engineered with two unique genomic receiver sites (Components 23, 2D), engineered to be HLA Null, utiliz- ing native CD3 expression, and harbouring an engineered genomic two-component, synthetic response element (Component 2F). In this example, the eTPC cell line was converted to an eTPC—t cell line (AOL—1277) as described previously in example 11, n the TCR chain ORF at ent 28’ and 2E’ encode a TCR pair that is spe- cific for HLA-peptide complex (H LA), HLA-A*02:01-NLVPMVATV. This eTPC—t was combined with described eAPC-p in the presence of ous aAM in the form of soluble peptide to assemble a eAPC:eTPC systems. The readout of these combined systems was an RFP signal within the eTPC-t, which was the reporter from component The response ts d as Component 2F comprised of a Driver-Activator component and an Amplifier-Report ent, wherein both units utilized synthetic promoters. The Driver is a tic promoter that is responsive to the native TCR sig- nalling ys, ng three sets of tandem transcription factor binding sites for NFAT-AP1-NFkB (3xNF-AP-NB). Upon transcriptional activation, the Driver induces ex- pression of the tor protein, a synthetic designed transcription factor derived by fu- sion of the Herpes VP16 activation domain, the GAL4 DNA binding domain and two nu- clear localization signals at the N- and C-terminals (NV1GG4N), to which the cognate DNA recognition sequence is present 6 times in tandem in the Amplifier promoter. Both the Driver and Amplifier promoters utilized the core promoter sequence (B recognition element (BRE), TATA Box, Initiator (INR) and transcriptional start site) from HCMV IE1 promoter, immediately 3’ of the respective transcription factor binding sites. The Ampli- fier upon transcriptional activation drives expression of the reporter, red fluorescent protein (RFP).

The eTPC-t cell line was then challenged against eAPC-p presenting HLA—A*02:01 (AOL-209) or HLA-A*24:02 (AOL-963) or was HLA-null parental eAPC (ACL- 128). Wherein analyte eAPC-pa were prepared by g eAPC-p with analyte antigen of either e NLVPMVATV or VYALPLKML, or no peptide. Subsequently, an eTPC- t and analyte eAPC-pa were compiled into an eAPC:eTPC system consisting of 30,000 eTPC-t co-cultured with 10,000 eAPC-pa for 24h. After 24h the cells were harvested, , stained with s specific for the eTPC-t and analyte eAPC-pa in order to guish the populations, and analysed by flow cytometry. Strong activation of the eTPC-t, Component 2F (was only observed in eTPC-t challenged with analyte eAPC- pa presenting the known cognate antigen pHLA complex, i.e. the eAPC-pa with HLA- A*02:01 and NLVPMVATV (Figure 59a). In contrast only resting state RFP expression was observed in eAPC:eTPC compilations comprised of non—specific analyte eAPC—pa (Figure 59b, 0, d) or control parental eAPC lacking HLA (Figure 59f) or eAPC-p lack- ing exogenous aAM peptide (Figure 59e).

In conclusion, an eTPC—t cell line ning a functional ent 2F was engi- neered, and subsequently used to create an eTPC-t. Upon interaction of the eTPC-t with analyte eAPC-pa presenting its cognate target T—cell antigen, provided as exoge— nous soluble aAM, a response was measurable as an increase in RFP expression.

Conversely, when contacted with analyte eAPC or eAPC-p or eAPC—pa not presenting a e T-cell antigen and HLA, or no HLA, no measurable increase in RFP expres- sion above ound was exhibited by the eTPC-t. Furthermore, this example trates an eAPC:eTPC system wherein analyte eAPC-pa and analyte eTPC-t are compiled in discrete binary compositions and the eTPC-t response is used to iden- tify both the e eAPC-pa and eTPC-t wherein a co—operative complex between the TCRsp and analyte antigen occurs.

Example 15: Functional demonstration of ent 2F in combined eAPC:eTPC system with aAM ORF integrated to eAPC-pa state The t e describes the use of an eTPC-t (AOL-1150), (Component 2A) en- gineered with two unique genomic receiver sites, loaded with the TCR chain ORF at Component 28’ and 2E’ encoding a TCR pair that is specific for ptide x 1O (HLA), HLA-A*02:01-NLVPMVATV, utilizing native CD3 expression, and harbouring a genomic one-component, synthetic response element (Component 2F), ed into an eAPC:eTPC system with eAPC-pa that were generated by integration with aAM en- coding ORF integrated at site 1D’. In this example, four different eAPC-pa variants were assembled with two different HLA alleles, and two different aAM ORF derived from the HCMV genome; ting four distinct eAPC-pa lines.

The response elements defined as Component 2F comprised of a Driver-Reporter component. The Driver is a synthetic promoter that is responsive to the native TCR sig- nalling pathways, encoding six sets of tandem ription factor binding sites for NFAT-AP1 AP) and utilizes the core promoter sequence (B recognition element (BRE), TATA Box, Initiator (INR) and transcriptional start site) from HCMV IE1 pro- moter, immediately 3’ of the respective transcription factor binding sites. Upon tran- scriptional activation, the Driver induces expression of the reporter, red fluorescent pro- tein (RFP).

The first eAPC-pa line (AOL-1046) expresses HLA-A*02:O1 and the full-length ORF for HCMV n pp65, wherein pp65 contains the antigenic peptide recognised by the eTPC-t TCRsp, when presented in HLA-A*02:01. The second eAPC-pa line (ACL- 1044) expresses HLA—A*02:01 and the full-length ORF for HCMV protein pp52. The third eAPC-pa line ACL-1045 expresses HLA-B*07:02 and the full-length ORF for HCMV n pp52. The fourth eAPC—pa line (AOL-1048) expresses HLA-B*07:02 and the full-length ORF for HCMV protein pp65. The second, third and fourth eAPC-pa ex- press aAPX:aAM complexes not recognised by the eTPC-t TCRsp.

The eTPC-t cell line AOL-1150 was compiled into ndent eAPC:eTPC system with each of the four eAPC-pa as described above. After 48h the cells were harvested, washed, stained with s specific for the eTPC-t and e eAPC-pa in order to distinguish the populations, and analysed by flow cytometry. Strong activation of the eTPC-t, Component 2F, was only observed in eTPC-t challenged with analyte eAPC- pa presenting the known cognate antigen pHLA complex, i.e. the eAPC-pa with HLA— A*02:01 and pp65 e 60b). In contrast only resting state RFP expression was ob- served in eAPC:eTPC compilations comprised of non—specific analyte a (Figure 60a, c, d).

In conclusion, an eTPC-t cell line containing a functional component 2F was engi- neered, and subsequently used to create an eTPC-t. Upon interaction of the eTPC-t with analyte eAPC-pa presenting its cognate target T-cell antigen, provided as endoge- nous aAM ORF by integration, a response was measurable as an increase in RFP ex- pression. Conversely, when ted with analyte eAPC-pa not presenting a cognate T-ceII n and HLA, no measurable increase in RFP expression above background was exhibited by the eTPC-t. Furthermore, this example trates an eAPC:eTPC system wherein analyte eAPC-pa and analyte eTPC-t are compiled in discrete binary compositions and the eTPC-t response is used to fy both the analyte eAPC-pa and eTPC-t wherein a co-operative complex n the TCRsp and analyte antigen OCCUI'S.

SEQUENCE LISTING <110> Genovie AB <120> An engineered two-part cellular device for discovery and characterisation of T- cell or interaction with cognate antigen <130> ANO15 <160> 104 <170> BiSSAP 1.3 <210> 1 <223> Analyte Antigenic Peptide <210> 2 <223> Analyte Antigenic Peptide <210> 3 <223> HCMV Antigen <210> 4 <223> HCMV Antigen <210> 5 <223> pcDNA3.1_GFP vector V1 .A.4 <210> 6 <223> pcDNA3.1_RFP vector V1 .A.6 <210> 7 <223> pMA—SV40pA vector V1 .C.2 <210> 8 <223> pMA—CS—JGQ—TCRbeta vector V3.C.5 <210> 9 <223> pMA—F14-GFP-F1 5 vector V4.H9 <210> 10 <223> pMA—F14-TCR-JG9—alpha-F15 vector V7.A.3 <210> 1 1 <223> pMA—FRT-TCR-JGQ—beta-F3 vector V7.A.4 <210> 12 <223> F14—TCRaF15 CDR3degen.64mix vector V8.F.8 <210> 13 <223> CMVpro-FIp—sv40pA—V2 vector V4.|.8 <210> 14 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_A1 <210> 15 <223> CDR3 ce of a JG9—TRA 64 variant VP.7751.RC1_A2 <210> 16 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_A3 <210> 17 <223> CDR3 sequence of a A 64 variant VP.7751.RC1_A4 <210> 18 <223> CDR3 sequence of a JG9-TRA 64 variant VP.7751.RC1_A5 <210> 19 <223> CDR3 sequence of a JG9—TRA 64 t VP.7751.RC1_A6 <210> 20 <223> CDR3 sequence of a JG9-TRA 64 t VP.7751.RC1_A7 <210> 21 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_A8 <210> 22 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_B1 <210> 23 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_BZ <210> 24 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_B3 <210> 25 <223> CDR3 sequence of a A 64 variant VP.7751.RC1_B4 <210> 26 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_B5 <210> 27 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_B6 <210> 28 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_B7 <210> 29 <223> CDR3 sequence of a A 64 variant VP.7751.RC1_B8 <210> 30 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_C1 <210> 31 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_C2 <210> 32 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_C3 <210> 33 <223> CDR3 sequence of a JG9—TRA 64 variant 1.RC1_C4 <210> 34 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_C5 <210> 35 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_C6 <210> 36 <223> CDR3 sequence of a JG9-TRA 64 variant 1.RC1_C7 <210> 37 <223> CDR3 ce of a JG9-TRA 64 variant VP.7751.RC1_B1 <210> 38 <223> CDR3 sequence of a JG9-TRA 64 variant VP.7751.RC1_D2 <210> 39 <223> CDR3 ce of a JG9—TRA 64 variant VP.7751.RC1_D3 <210> 40 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_D4 <210> 41 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_D5 <210> 42 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_D6 <210> 43 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_D7 <210> 44 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_D8 <210> 45 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_E1 <210> 46 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_E2 <210> 47 <223> CDR3 sequence of a JG9—TRA 64 t VP.7751.RC1_E3 <210> 48 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_E4 <210> 49 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_E5 <210> 50 <223> CDR3 sequence of a JG9—TRA 64 t VP.7751.RC1_E6 <210> 51 <223> CDR3 ce of a JG9—TRA 64 variant 1.RC1_E7 <210> 52 <223> CDR3 sequence of a A 64 variant VP.7751.RC1_E8 <210> 53 <223> CDR3 sequence of a JG9—TRA 64 t VP.7751.RC1_F1 <210> 54 <223> CDR3 sequence of a JG9-TRA 64 variant VP.7751.RC1_F2 <210> 55 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_F3 <210> 56 <223> CDR3 sequence of a JG9-TRA 64 variant VP.7751.RC1_F4 <210> 57 <223> CDR3 ce of a JG9—TRA 64 variant 1.RC1_F5 <210> 58 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_F6 <210> 59 <223> CDR3 sequence of a A 64 t VP.7751.RC1_F7 <210> 60 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_F8 <210> 61 <223> CDR3 sequence of a A 64 variant VP.7751.RC1_G1 <210> 62 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_G2 <210> 63 <223> CDR3 sequence of a JG9—TRA 64 t VP.7751.RC1_GB <210> 64 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_G4 <210> 65 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_G5 <210> 66 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_G6 <210> 67 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_G7 <210> 68 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_G8 <210> 69 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_H1 <210> 70 <223> CDR3 sequence of a JG9—TRA 64 t VP.7751.RC1_H2 <210> 71 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_H3 <210> 72 <223> CDR3 sequence of a JG9-TRA 64 variant VP.7751.RC1_H4 <210> 73 <223> CDR3 sequence of a JG9-TRA 64 variant VP.7751.RC1_H5 <210> 74 <223> CDR3 sequence of a JG9-TRA 64 variant VP.7751.RC1_H6 <210> 75 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_H7 <210> 76 <223> CDR3 sequence of a JG9—TRA 64 variant VP.7751.RC1_H8 <210> 77 <223> pMA_F14_HLA-A*02:01-6XHis_F15 vector V4.H.5 <210> 78 <223> pMA_F14_HLA-A*24:02-6XHis_F15 vector V4.H.6 <210> 79 <223> pMA_F14_HLA-B*07:02—6XHis_F15 vector V4.H.7 <210> 80 <223> pMA_F14_HLA-B*35:01-6XHis_F15 vector V4.H.8 <210> 81 <223> FRT_HCMVpp28-3XMYC_F3 vector V9.E.6 <210> 82 <223> FRT_HCMVpp52-3XMYC_F3 vector V9.E.7 <210> 83 <223> FRT_HCMVpp52-3XMYC_F3 vector V9.E.8 <210> 84 <223> SpCas9—2A—GFP Vector V1 .A.8 <210> 85 <223> HLA—A-sg-sp—opti1 vector V2.A.1 <210> 86 <223> HLA—B-sg-sp—3 vector V2.A.7 <210> 87 <223> sg-sp—4 vector V2.B.3 <210> 88 <223> HLA—A-ex2-3_sg-sp—opti_1 vector V2.|.10 <210> 89 <223> HLA—A—ex2-3_sg-sp—opti_2 vector V2.J.1 <210> 90 <223> AAVSI_sg-sp—opti_3 vector V2.J.6 <210> 91 <223> TRAC-GT-F1 ddPCR primer/probe <210> 92 <223> TRAC-GT-R1 ddPCR primer/probe 1.F.8 <210> 93 <223> TRAC-probe-FAM ddPCR primer/probe <210> 94 <223> TRBC2-GT—F1 ddPCR primer/probe 1.F.9 <210> 95 <223> TRBC2-GT—R1 ddPCR /probe 1.F.10 <210> 96 <223> TRBC2-probe-FAM ddPCR primer/probe 1.G.2 <210> 97 <223> TRAC-TCRA-eX1-F1 ddPCR primer/probe 10.A.9 <210> 98 <223> TRAC-TCRA—eX1-F1 ddPCR primer/probe 10.A.10 <210> 99 <223> TRAC-probe(HEX) ddPCR primer/probe 10.B.6 <210> 100 <223> HCMVpp65_GT_F2ddPCR primer/probe 21 .|.1 <210> 101 <223> HCMVpp28_GT_F1 ddPCR primer/probe 21 .|.2 <210> 102 <223> HCMVpp52_GT_F1 ddPCR primer/probe 21 .|.3 <210> 103 <223> g_GT_R1 ddPCR /probe 20.H.10 <210> 104 <223> Linker-Myc_Probe_Fam ddPCR /probe 20.H.9 List of abbreviations aAPX Analyte antigen-presenting complex aAM Analyte antigenic molecule aCT Analyte TCR APC Antigen-presenting cell APX Antigen-presenting complex BFP Blue fluorescent protein CAR-T CAR T-cell CM Cargo molecules CRISPR Clustered Regularly Interspaced Short Palindromic Repeats gRNA Cas9 guide RNA CAR ic antigen receptor CDR Complementarity-determining regions C-region Constant region CMV Cytomegalovirus DAMPS Danger associated molecular patterns DC Dendritic cells DNA Deoxyribonucleic acid D-region Diversity region eAPC ered antigen-presenting cell eAPC-p ered antigen-presenting cell that present an analyte antigen- presenting complex eAPC-pa Engineered antigen-presenting cell that presents an analyte anti- gen—presenting complex and analyte antigenic molecule eAPC-a Engineered antigen-presenting cell expressing an analyte antigenic molecule eAPCS Engineered antigen-presenting cell system eTPC Engineered TCR-presenting cell eTPCS Engineered TCR-presenting cell system eTPC-t Engineered TCR-presenting cell that present full-length TCR pairs FACS Fluorescence-activated cell sorting GEM T-cells Germ line-encoded mycolyl-reactive s GFP Green fluorescent protein HLAI HLA class I HLAll HLA class II HDR Homology directed recombination HLA Human leukocyte antigen lgSF lmmunoglobulin amily IRES Internal ribosome entry site iNK T-cells ant natural killer T—cells J-region Joining region MACS Magnetic-activated cell sorting MAGE Melanoma associated antigen MAIT Mucosal-associated ant T NCBP Non-cell based particles ORF Open g frame PAMPS Pathogen-associated molecular patterns PCR Polymerase chain reaction RMCE Recombinase mediated cassette exchange RFP Red fluorescent protein DNA Ribonucleic acid SH2 Src homology 2 T-cells T lymphocytes TCR T-cell or TRA TCR alpha TRB TCR beta TRD TCR delta TCRsp TCR surface ns in complex with CD3 TALEN Transcription activator-like effector ses TRG TRC gamma TAA —associated-antigens V-region Variable region [52M BZ-microglobulin ZAP-70 C—chain-associated protein of 70 kDa Definitions A pair of complementary TCR chains: two TCR chains wherein the translated pro- teins are capable of forming a TCRsp on the surface of a TCR presenting cell Affinity: Kinetic or equilibrium parameter of an interaction between two or more mole- cules or proteins Affinity reagent: Any reagent designed with specific affinity for an analyte. Often used in the context of affinity for tigen complex Allele: Variant form of a given gene AM: Analyte antigenic molecule. Generally, a protein but could also be a metabolite that is expressed by a cell from their genomic DNA and/or a specific introduced c sequence. The AM is expressed in the cell and a fragment can then be presented on the cell surface by an APX as cargo or on its own. Either as cargo or not, the AM can then be the target of T—cell receptor bearing cells or related affinity reagents.

Amplicon: a piece of DNA or RNA that is the source and/or product of artificial amplifi- cation using various methods including PCR.

Analyte: an entity that is of interest to be identified and/or measured and/or queried in the combined system Antibody: Affinity molecule that is expressed by specialized cells of the immune sys- tem called B-cells and that contains of two chains. B—cells s a very large and very diverse repertoire of antibodies that do generally not bind self proteins but can bind and neutralize ens or toxins that would threaten the host. Natural or artifi- cially engineered antibodies are often used as ty reagents.

Antigen: any le that may be engaged by a TCR and results in a signal being transduced within the T-cell, often presented by an n-presenting complex Analyte antigen: collectively the eAPC:eTPC system enting any entity present- ing an antigen for analytical determination APC: Antigen—presenting cell. A cell baring on the surface of the cell an AM, APX, APX APX: Antigen-presenting complex. A protein that is expressed and presented on the cell surface by nucleated cells from genes/ORF ng genomic DNA and/or a spe- cific introduced genetic sequence. The APX presents a cargo, being either a peptide or other metabolite molecules.

C-Region: Constant region. One of the gene segments that is used to assemble the T- cell receptor. The c-region is a distinct segment that rather than driving diversity of the TCR, s its general function in the immune .

Cargo-loading machinery: Cellular set of proteins that generate and load cargo molecules on APX from proteins or other presented molecules found in the cell.

CDR: complementarity-determining regions. Short sequences on the antigen-facing end of TCRs and antibodies that perform most of the target binding function. Each anti- body and TCR contains six CDRs and they are generally the most variable part of the molecules allowing detection of a large number of diverse target molecules.

CM: Cargo molecules. peptide or metabolite that is presented by an antigen-present- ing complex for example a HLA | or HLA II. The CM can be expressed by the cell intrinsically from the genomic DNA, introduced into the culture medium or expressed from a specifically introduced genetic sequence. umber: The whole number ence of a defined sequence encoded within the genome of a cell Cytogenetic: The study of tance in relation to the structure and function of chro- mosomes, i.e. ine the karyotype of a cell Cytotoxic/Cytotoxicity: Process in which a T-cells es factors that directly and specifically damage a target cell.

D-region: Diversity region. One of the gene segments that is used to assemble the T- cell receptor. Each individual has a large number of ent variations of these regions making it le for each individual to arm T-cells with a very large variety of different DNA: Desoxyribonucleic acid. Chemical name of the molecule that forms genetic ma- terial encoding genes and proteins. eAPC system: eAPCS, the system by which eAPC-pa, eAPC-p and eAPC-a cells, or libraries thereof, are prepared for combination in the eAPC:eTPC system. eTPC system: eTPCS, the system by which eTPC-t cells, or ies f, are pre- pared for combination in the eAPC:eTPC system eAPC:eTPC system: the system by which e antigen presented by eAPC and an- alyte TCR presented by eTPC are combined nous: Substance that originated from within a cell Engineered Cell: A cell whereby the genome has been engineered through genetic modification modified.

Eukaryotic conditional tory element: A DNA sequence that can influence the activity of a promoter, which may be induced or repressed under defined conditions Eukaryotic Promoter: A DNA sequence that encodes a RNA polymerase biniding site and response elements The sequence of the er region ls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining where and when your gene of interest will be expressed.

Eukaryotic terminator/Signal ator: A DNA sequence that are recognized by protein factors that are ated with the RNA polymerase II and which trigger the ation process of transcription. It also encodes the poly-A signal FACSfFlow Cytometry: Fluorescence-activated cell sorting. Analytical technique by which individual cells can be analyzed for the expression of specific cell surface and in- tracellular markers. A variation of that technique, cell sorting, allows cells that carry a defined set of markers to be retrieved for further analysis.

Family of APX: A set of several similar genes that encode functionally related pro— teins, which constitute an antigen pressing complex Fluorescent (protein) marker: Molecule that has specific extinction and emission characteristics and can be detected by copy, FACS and related techniques.

Genetic Donor vector: A genetic based vector for delivery of genetic material to the c er site Genomic Receiver Site: A site within the genome for targeted integration of donor ge- netic material d within a Genetic Donor Vector.

Heterospecific recombinase sites: A DNA sequence that is recognized by a recom- binase enzyme to promote the crossover of two DNA molecules HLA I: Human yte n class I. A gene that is expressed in humans in all nucleated cells and exported to the cell surface where it presents as cargo short frag- ments, peptides, of internal proteins to T-cell receptors. As such it presents fragments of potential ongoing infections along with sic proteins. The HLA I can additionally present as cargo peptides that are added to the culture , generated from pro— teins expressed form introduced genetic elements or generated from proteins that are taken up by the cell. HLA class I genes are polymorphic meaning that different individu- als are likely to have variation in the same gene leading to a variation in presentation.

Related to HLA class II.

HLA ll: Human Leukocyte Antigen Class II. A gene that is expressed in humans in speci?c cells that are coordinating and helping the adaptive immune response for ex- ample dendritic cells. Related to HLA class I. HLA class N ns are exported to the cell surface where they present as cargo short fragments, peptides, of external proteins to T-cell receptors. As such it presents fragments of potential ongoing infections along with intrinsic proteins. The HLA II can onally present as cargo peptides that are added to the culture medium, generated from proteins expressed form uced ge— netic elements or generated from proteins that are taken up by the cell. HLA class II genes are polymorphic meaning that different individuals are likely to have variation in the same gene leading to a variation in presentation.

Homologous arms: A stretch of DNA that has near identical sequence identity to a complement homologous arm and ore e the exchange of two DNA mole- cules by the cellular process, homology directed repair.

Immune surveillance: Process in which the immune system detects and becomes ac- tivated by infections, malignancies or other potentially enic alterations.

Insulator: A DNA sequence that prevents a gene from being influenced by the activa- tion or repression of nearby genes. Insulators also prevent the spread of heterochro- matin from a silenced gene to an actively transcribed gene.

Integration: The physical ligation of a DNA sequence into a chromosome of a cell ation couple: A paired genetic donor vector and genomic receiver site Internal ribosome entry site (IRES): A DNA sequence that once transcribed encodes a RNA element that allows the initiation of translation in a cap-independent manner J-region: Joining region. One of the gene segments that is used to assemble the T- cell receptor. Each dual has a large number of ent variations of these regions making it le for each individual to arm T-cells with a very large variety of different Karyotype: The chromosome composition of a cell Kozak Sequence: Short sequence required for the ent initiation of translation Major HLA class I: a Family of APX that comprise of the genes HLA-A, HLA-B and HLA-C Matched: When two components encode genetic elements that direct and restrict the interaction between the complemented components Meganuclease ition site: A DNA sequence that is recognized by a endodeox- yribonuclease, commonly referred to as a meganuclease Metabolite: A molecule created or altered through metabolic pathways of the cell Mobile genetic element: A DNA ce that can permit the integration of DNA with the activity of transposases enzymes Monoclone cell line: A defined group of cells ed from a single ancestral cell by repeated cellular replication Native: a entity that is naturally occuring to the cell ding gene: A non protein coding DNA ce that is transcribed into func- tional ding RNA molecules ORF: Open g frame. Stretch of genetic material that encodes a ation frame for synthesis of a protein (polypeptide) by the ribosome Paracrine: Signalling through soluble factors that directly act on neighboring cells.

PCR: Polymerase chain reaction in which a specific target DNA molecule is exponen- tially amplified Peptide: short string of amino acids between 6 - 30 amino acids in length Phenotypic analysis: Analysis of the observable characteristics of a cell.

Polymorphic: Present in different forms in individuals of the same species through the presence of different alleles of the same gene.

Polypeptide: Protein consisting of a stretch of peptides, forming a three-dimensional structure.

Primary Outputs: eAPC cells and eTPC cells from which the terminal outputs can be derived and/or determined from Primer: Short DNA sequence that allows ic recognition of a target DNA se- quence for example during a PCR.

Promoter: Regulatory DNA element for the controlled initiation of gene expression Selectable : A DNA sequence that confers a trait suitable for cial selection methods Shotgun Integration: The process whereby a library of vectors is introduced to a pop- ulation of cells, whereby only a single copy of any given vector insert may be integrated to the genome of each single cell. Used to refer to pooled vector integration to a given cell population via an integration couple Slice acceptor site: A DNA ce at the 3' end of the intron AM, APX CM or affin- ity reagent for interaction with cells with TCRsp on the surface, or TCRsp based rea— gents Slice donor site: A DNA sequence at the 5' end of the intron Synthetic: an entity that is cially generated and introduced to a cell T-cell: T lymphocyte. White blood cell that expresses a T-cell receptor on its surface.

Selected by the immune system to not react with the own body but have the potential to recognize infections and malignancies as well as reject grafts from most members of the same species.

TCR: T-cell Receptor. Affinity molecule sed by a subgroup of lymphocytes called T—lymphocytes. In humans the TCR recognizes cargo presented by APX CM or APX AM, including fragments from virus or bacterial ions or cancerous cells.

Therefore, the TCR recognition is an al part of the adaptive immune system. The TCR consists of two chains that are paired on the cell surface. The TCR expressed on the e of each cells is assembled at random from a large pool of varied genes (the v,d,j and 0 regions) and thus each individual has a pool of T-cells expressing a very large and diverse repertoire of ent TCRs.

TCRsp: A pair of complementary TCR chains that express as surface proteins in com- plex with CD3 or a pair of complementary TCR chains expressed as proteins in the form of a soluble reagent, an immobilised reagent or present by NCBP.

Terminal Outputs: analyte n and TCR sequences, in the form of AM, APX, APX:CM, APX:AM, or TCRsp TRA: TCR alpha encoding locus. One of the four different locus encoding genes that can form a VDJ recombined TCR chain. Translated TCR alpha chain proteins typically pair with translated TCR beta chain ns to form alpha/beta TCRsp.

TRB: TCR beta encoding locus. One of the four different locus encoding genes that can form a VDJ recombined TCR chain. Translated TCR beta chain proteins typically pair with TCR alpha chain proteins to form alpha/beta TCRsp.

TRD: TCR delta encoding locus. One of the four different locus ng genes that can form a VDJ recombined TCR chain. Translated TCR delta chain proteins typically pair with translated TCR gamma chain proteins to form gamma/delta TCRsp.

TRG: TCR gamma encoding locus. One of the four different locus encoding genes that can form a VDJ recombined TCR chain. Translated TCR gamma chain proteins typi- cally pair with translate TCR delta chain proteins to form delta TCRsp.

V-region: Variable region. One of the gene segments that is used to assemble the T- cell receptor. Each individual has a large number of different variations of these regions making it possible for each individual to arm T-cells with a very large variety of different Items 1. A two-part device, wherein a first part is an engineered antigen-presenting cell system (eAPCS), and a second part is an engineered TCR-presenting cell system (eTPCS). 2. A two-part device according to item 1 wherein eAPCS provides the one or more of analyte eAPC selected from a. eAPC—p and/0r b. eAPC-a, and/or 0. eAPC-pa, and/or d. one or more libraries of a and/or b and/or 0. 3. A two—part device according to item 2, wherein an eAPC-p, eAPC-a or eAPC- pa ses an analyte antigen selected from a. an aAPX or b. an aAM or c. an aAPX:aAM or d. an aAPX:CM or e. a combination thereof. 4. A two-part device according to item 1 or 2 n eTPCS es the one or more analyte eTPC selected from a. eTPC-tand/or b. one or more libraries thereof.

A two-part device according to item 4, wherein an e pair of TCR chains are expressed as TCR surface proteins in complex with CD3 (TCRsp) by an e eTPC.

A two-part device according to any of the preceding items wherein the one or more analyte eAPC, is combined with the one or more analyte eTPC.

A two—part device according to item 6, wherein the combination results in a con- tact between an analyte TCRsp and an analyte antigen as defined in item 3.

A two-part device according to item 7 wherein the contact can result in the for- mation of a complex n the analyte TCRsp and the analyte antigen.

A two-part device ing to item 8 n a formation of a complex, if any, can induce a signal response in the analyte eTPC and/or the analyte eAPC.

. A two-part device according to item 9, wherein the response is used to select an e eTPC or a pool of analyte eTPC with or without a signal response and/or analyte eAPC or a pool of analyte eAPC with or without a signal re- sponse. 11. An analyte eTPC obtained from the two-part device according to any of the pre- ceding items for use in terisation of a signal response of the analyte eTPC, expressing analyte TCRsp, to an analyte antigen. 12. A method for ing one or more analyte eTPC from an input analyte eTPC or a library of analyte eTPC, to obtain one or more analyte eTPC wherein the expressed TCRsp binds to one or more analyte antigen as defined in item 3, wherein the method comprises a. Combining one or more analyte eTPC with one or more analyte eAPC resulting in a contact between an analyte TCRsp with an e antigen and at least one of b. Measuring a formation, if any, of a complex between one or more ana— lyte TCRsp with one or more analyte antigen and/or c. Measuring a signal response by the analyte eTPC, if any, induced by the formation of a complex between one or more e TCRsp with one or more analyte n and/or d. Measuring a signal response by the analyte eAPC, if any, induced by the formation of a complex between one or more analyte TCRsp with one or more e antigen and e. Selecting one or more analyte eTPC based on step b, c and/or d wherein the selection is made by a positive and/or negative measure- ment. 13. A method according item 12 wherein the selection step e is performed by single cell sorting and/or cell sorting to a pool. 14. A method according to item 13 wherein the sorting is followed by ion of the sorted single cell.

. A method according to item 13 wherein the sorting is followed by expansion of the sorted pool of cells. 16. A method according to any of items 13 to 15 further comprising a step of se- quencing component ZB’ and/or ent 2D’ of the sorted and/or ed cell(s). 17. A method according to item 16 wherein the sequencing step is preceded by the following a. Extracting of genomic DNA and/or b. Extracting of component ZB’ and/or component 2D’ RNA transcript and/or c. ying by a PCR and/or a RT-PCR of the DNA and/or RNA tran- script of component ZB’ and/or component 2D’. 18. A method according to item 16 or 17 wherein the sequencing step is destruc— tive to the cell and wherein the sequencing information obtained is used for pre- paring the analyte eTPC selected in step e of item 12. 19. A method according to any of items 12, 13, 14, 15, 18 wherein the selected an- alyte eTPC is subjected to characterisation of the signal se wherein the method further comprises a. Determining a native signalling response and/or b. Determining a synthetic signalling response, if the eTPC contains com- ponent 2F.

. A method according to item 19 wherein the induced signal response is deter- mined by detecting an increase or decrease in one or more of the following a. a secreted biomolecule a secreted chemical an intracellular ecule an intracellular chemical a surface expressed biomolecule a cytotoxic action of the analyte eTPC upon the analyte eAPC a paracrine action of the analyte eTPC upon the analyte eAPC such that a signal response is induced in the analyte eAPC and is determined by detecting an increase or decrease any of a to e a proliferation of the analyte eTPC an immunological synapse between the analyte eTPC and the analyte eAPC compared to the non-induced signal response state. 21. An analyte eAPC, obtained from the two-part device as d in items 1 to 10 to fy the analyte n that induces a signal response of one or more an- alyte eTPC sing an analyte TCRsp to the expressed analyte antigen. 22. A method for selecting one or more analyte eAPC from an input analyte eAPC or a library of analyte eAPC, to obtain one or more analyte eAPC that s a signal response of one or more e eTPC expressing an analyte TCRsp to the expressed analyte antigen as d in item 3, wherein the method comprises a. Combining one or more analyte eAPC with one or more analyte eTPC, resulting in a contact between an analyte antigen presented by the analyte eAPC with analyte TCRsp of one or more analyte eTPC and b. Measuring a formation, if any, of a complex between one or more e antigen with one or more analyte TCRsp and/or 0. Measuring a signal response in the one or more analyte eTPC, if any, induced by the formation of a complex between the analyte TCRsp with the analyte antigen and/or d. ing a signal response, if any, by the analyte eAPC induced by the formation of a x n one or more analyte TCRsp with one or more e antigen and e. Selecting one or more analyte eAPC from step b, c and/or d n the selection is made by a positive and/or negative measurement. 23. A method according item 22 wherein the selection step e is performed by single cell sorting and/or cell sorting to a pool. 24. A method according to item 23 wherein the sorting is followed by expansion of the sorted single cell. 25. A method according to item 24 wherein the sorting is followed by expansion of the sorted pool of cells. 26. A method according to any of items 23 to 25 further comprising a step of se- quencing component 18’ and/or component 1D’ of the sorted and/or expanded ce||(s). 27. A method according to item 26 wherein the sequencing step is preceded by the following a. Extracting of genomic DNA and/or b. Extracting of component 18’ and/or ent 1D’ RNA transcript and/or C. Amplifying by a PCR and/or a RT-PCR of the DNA and/or RNA tran- script of component 13’ and/or component 1D’. 28. A method according to item 26 or 27 wherein the sequencing step is destruc- tive to the cell and wherein the sequencing information obtained is used for pre- paring the analyte eAPC selected in step e of item 22. 29. A method ing to any of items 22, 23, 24, 25, 28 wherein the selected an- alyte eAPC is select based on the signal response of an analyte eTPC wherein the method further comprises a. ining a native signalling se and/or b. Determining a synthetic signalling response.

. A method according to item 29 wherein the induced signal response is deter- mined by detecting an increase or decrease in one or more of the following a. a secreted biomolecule a secreted chemical an intracellular ecule . an intracellular al a surface expressed biomolecule a cytotoxic action of the analyte eTPC upon the analyte eAPC a paracrine action of the analyte eTPC upon the analyte eAPC such that a signal response is induced in the analyte eAPC and is determined by detecting an se or decrease any of a to e . a proliferation of the analyte eTPC an immunological synapse between the analyte eTPC and the e eAPC compared to the non-induced signal response state. 31. A method to select and fy an aAM cargo or a CM cargo, wherein the cargo is a metabolite and/or a e, that is loaded in an aAPX of an analyte eAPC selected and obtained by methods defined in items 22 to 30 n the method comprises a. isolating an aAPX:aAM or an aAPX:CM or the cargo aM or the cargo CM and b. identifying the loaded cargo. 32. A method according to item 31 wherein step b comprises subjecting the isolated aAPX:aAM or an aAPX:CM to one or more a. Mass-spectroscopy analysis b. Peptide sequencing analysis. 33. A pair of TCR chain sequences or library of pairs of TCR chain ces se- lected by the method as d in items 12 to 20 for use in at least one of the following a. diagnostics b. medicine c. cosmetics d. research and development. 34. An antigenic molecule and/or ORF encoding said antigenic molecule, or librar- ies thereof selected by the method as defined in items 22 to 32 for use in at least one of the following a. diagnostics b. medicine c. cosmetics d. research and development.

. A antigen-presenting complex loaded with an antigenic molecule as cargo and/or ORF(s) encoding said complex, or libraries thereof selected by the method as defined in items 22 to 32 for use in at least one of the following a. diagnostics b. medicine c. cosmetics d. research and development. 36. An eAPC, or library of eAPC selected by the method as defined in items 22 to for use in at least one of the following a. diagnostics b. medicine 0. cosmetics d. research and development. 37. An eTPC, or library of eTPC selected by the method as defined in items 12 to for use in at least one of the following a. diagnostics b. medicine c. cosmetics d. research and development. 38. A device according to any of items 1-10 for use in at least one of the ing a. diagnostics b. medicine c. cosmetics d. ch and development.

Claims (13)

Claims

1. A two-part system forming a combined eAPC:eTPC analytical system, wherein a first part is an engineered antigen-presenting cell system (eAPCS), and a second part is an engineered TCR-presenting cell system ), 5 wherein said eAPCS comprises: a first ent which is an engineered antigen-presenting cell (eAPC), ated component 1A, wherein ent 1A lacks endogenous surface expression of at least one family of target e antigen ting complexes (aAPX) and/or target analyte antigenic molecules (aAM), and contains a second component designated component 1B, a synthetic 10 genomic receiver site for integration of one or two ORFs ng an aAPX and/or an aAM, a third component which is a genetic donor vector, designated component 1C, for delivery and integration into component 1B of at least one target ORF encoding at least one target aAPX and/or at least one target aAM, wherein component 1C is matched to component 1B, 15 and wherein component 1C is designed to deliver one or two target ORFs encoding a target aAPX and/or a target aAM, and wherein said eTPCS ses: a first component which is an engineered TCR-presenting cell (eTPC), designated ent 2A, wherein component 2A lacks endogenous surface expression of at least one 20 family of target analyte antigen-presenting complexes (aAPX) and/or target analyte antigenic molecule (aAM), lacks endogenous sion of TCR chains alpha, beta, delta and gamma, and expresses CD3 proteins which are conditionally presented on the surface of the eTPC only when the eTPC expresses a complementary pair of TCR chains, and ns a second component designated 2B, a genomic receiver site for integration of a single ORF encoding 25 at least one analyte TCR chain of alpha, beta, delta or gamma, and/or two ORFs encoding a pair of analyte TCR chains, and a third component which is a genetic donor vector, designated component 2C, for delivery of ORF encoding analyte TCR chains, wherein component 2C, is matched to component 2B, and wherein the component 2C is designed to deliver: 30 a. a single ORF encoding at least one analyte TCR chain of alpha, beta, delta and/or gamma; and/or b. two ORFs encoding a pair of analyte TCR chains.

2. The two-part system according to claim 1, wherein the one or two target ORFs of component 1C further encode a selection marker of integration, such that the target aAPX and/or the 5 target aAM can be sed by component 1A.

3. The two-part system ing to claim 1 or claim 2, wherein a and/or b encodes a ion marker of ation, such that the analyte TCR chains can be expressed as TCR surface protein in complex with the CD3 (TCRsp) on component A.

4. The two-part system according to any one of claims 1 to 3, wherein eAPCS can provide the 10 one or more of analyte eAPC selected from: a. an eAPC presenting an aAPX (eAPC-p); b. an eAPC presenting an analyte antigenic molecule (eAPC-a); c. an eAPC presenting an aAPX and an analyte antigenic molecule (eAPC-pa); and/or 15 d. one or more libraries of a, b and/or c.

5. The rt system according to claim 4, wherein the eAPC-p, eAPC-a or eAPC-pa can express an analyte antigen selected from: a. an aAPX; b. an aAM; 20 c. an aAPX:aAM; d. an aAPX:cargo le (aAPX:CM); or e. a combination thereof.

6. The two-part system according to any one of claims 1 to 5, wherein the eTPCS can provide one or more analyte eTPC selected from: 25 a. an eTPC expressing an analyte pair of TCR chains in complex with CDR3 t); and/or b. one or more libraries thereof.

7. The two-part system according to claim 6, wherein an analyte pair of TCR chains can be expressed as TCR surface proteins in complex with CD3 (analyte TCRsp) by an analyte 5 eTPC.

8. The two-part system according to claim 7, wherein the eTPC contains a component 2F, representing a synthetic TCR signal response element engineered to the genome of the eTPC, and which can be used to report the formation of complex between analyte TCRsp and a e antigen presented by eAPC –p, -a or –pa, and which results in a signal response in 10 the eTPC.

9. The rt system according to any one of claims 1 to 8, n one or more analyte eAPC can be combined with one or more analyte eTPC.

10. The two-part system according to claim 9, wherein the combination s in a contact between an analyte TCRsp and an e antigen as defined in claim 5, and wherein the 15 contact may or may not result in a signal response.

11. The two-part system according to claim 10, wherein the contact results in the formation of a complex between the analyte TCRsp and the analyte antigen.

12. The two-part system according to claim 11, wherein the formation of the complex, if any, can induce a signal response in the analyte eTPC and/or the analyte eAPC. 20

13. The two-part system according to any one of claims 9 to 12, n the signal response can be used to select an analyte eTPC or a pool of analyte eTPC with or without a signal response and/or an e eAPC or a pool of analyte eAPC with or without a signal response.