Synthetic receptor discovery through pooled t cell display

Abstract

The disclosure describes a pooled T-cell display method for identifying potent synthetic receptors for use in cancer therapy.

Description

PATENT Attorney Docket No.: 081906-1497756-251310PC Client Reference No.: SF-2023-033 SYNTHETIC RECEPTOR DISCOVERY THROUGH POOLED T CELL DISPLAY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Patent Application No. 63 / 574,616, filed April 4, 2024, which is incorporated by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] A conventional approach for testing binding domains for inclusion in chimeric binding proteins, such as chimeric antigen receptors (CARs), is to select several binding domains that are high affinity, incorporate them into receptors, characterize each receptor in vitro, and validate the most promising in vivo. To date, however, binding attributes that promote productive CAR signaling, maintenance of effector function, and persistence are not well understood. Further, these attributes may depend on the specific antigen and contextual factors.

[0003] It has been shown that an important consideration for CARs is binder affinity. Antibody-derived binders are often high affinity. Decreasing affinity is a way to maintain the targeting of tumor cells expressing high levels of antigen while sparing other cells expressing low levels (Caruso et al., 2015; Drent et al., 2017; Liu et al., 2015; Park et al., 2017). Aside from reducing off-tissue effects, affinity modulation can also benefit on-tissue targeting; lowering affinity can normalize microvillar contacts with target cells (Beppler et al., 2023), decrease trogocytosis (Olson et al., 2022), and increase tumor clearance (Ghorashian et al., 2019). A meta-analysis of CAR-T cell clinical trial data proposed that an ideal binder is one that is moderate affinity (Mao et al., 2022).

[0004] Besides binder affinity, other salient characteristics for binding domains include their effect on CAR tonic signaling (Chen et al., 2023; Long et al., 2015), the epitope targeted on the antigen (He et al., 2023; Zhang et al., 2023), and the synaptic distance between effector cell and target cell (James et al., 2008) as well as other signaling aspects.BRIEF SUMMARY OF THE INVENTION

[0005] The present invention provides a method for screening for antigen binding domains for synthetic receptors. The method includes these steps: (a) providing a plurality of T cells comprising T cells that express different synthetic receptors on the cell surface, wherein an individual synthetic receptor comprises an antigen binding domain (ABD) that is different from the ABD expressed by other T cells contained in the plurality of T cells, and wherein the synthetic receptor is encoded by an expression construct introduced into a locus that is monoallelic or can support monoallelic expression; (b) introducing the plurality of T cells into immunodeficient non-human mammalian subjects in vivo, wherein the subjects have engrafted tumors that express the antigen to which the ABD binds; (c) isolating T cells after a sufficient period of time for a T-cell response to be induced following binding of the ABD to the antigen expressed by tumor cells; (d) amplifying nucleic acid extracted from T cells obtained from the bone marrow or other tissue of a subject having an engrafted tumor, and optionally a control subject lacking tumor cells, to obtain amplicons indicating ABD- encoding regions; (e) sequencing amplicons obtained of (d); and (f) determining ABD- encoding regions that are enriched in subjects having engrafted tumors.

[0006] In some embodiments of the claimed method, the locus into which the expression construct introduced is a T-cell receptor alpha (TRAC) locus. In some embodiments, the locus is a T-cell receptor beta (TRBC1 or TRBC2) locus. In some embodiments, the synthetic receptor is a chimeric antigen receptor (CAR). In some embodiments, the synthetic receptor is an HLA-independent T cell (HIT) receptor. In some embodiments, the synthetic receptor is a T cell receptor (TCR)-like receptor. In some embodiments, the ABD comprises a VHH. In some embodiments, the ABD comprises a VH and / or a VL. In some embodiments, the ABD comprises a natural ligand.

[0007] In some embodiments of the claimed method, the T cell is a cytotoxic T cell, helper T cell, memory T cell, regulatory T cell, effector T cell, natural killer T cell, or a T cell derived in vitro from an embryonic or pluripotent stem cell. In some embodiments, the expression construct is introduced into a monoallelic locus or a locus that can support monoallelic expression, such as the T-cell receptor alpha (TRAC) locus or the T-cell receptor beta (TRBC1 or TRBC2) locus, by a gene editing system selected from the group consisting of a CRISPR / Cas gene editing system, a TALEN gene editing system, and a zinc finger nuclease system. In some embodiments, step (d) comprises amplifying genomic DNA. In some embodiments, step (d) comprises amplifying cDNA reverse-transcribed from RNA.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG.1a–b. a, Targeting of a pooled library to the TRAC locus in T cells. EGFRt is a marker for detecting knock-in independent of CAR staining. The homology-directed repair template (HDRT) can be packaged in an adeno-associated virus (AAV) vector. b, Flow cytometry plots indicating knockout following CRISPR / Cas editing and knock-in of the library.

[0009] FIG.2a–d. Library amplification in preparation for sequencing. a, In the HDRT, a region of the left homology arm differs from the corresponding endogenous sequence. The differing region is not retained after HDRT integration. b, This difference enables the use of a forward primer that binds to the endogenous sequence and a reverse primer that binds within the cargo, such that polymerase chain reaction (PCR) amplifies only integrated cargo. In a subsequent PCR, the forward primer is in the a VHH framework region 1 and the reverse primer is in the VHH framework region 4. The resulting amplicon contains the three complementarity-determining regions (CDRs). c, For pooling multiple libraries—such as the same original library subjected to different assays—library-specific indexes are added in a third PCR. d, RNA optionally can be used instead of genomic DNA as input.

[0010] FIG.3. Relative abundance of library members is similar (high correlations) across AAV, genomic DNA, and RNA-derived cDNA. Library composition is maintained and detectable after HDRT knock-in in T cells.

[0011] FIG.4a–b. a, Schematic of editing and in vivo screening for T cell display. b, Rank-ordering of library member enrichment from bone marrow extracted from mice receiving a CAR-T cell library (containing a 1XX stimulatory domain), with NALM6 injection prior to T cell injection. Open circles are mouse replicates, and filled circles are averages.

[0012] FIG.5a–b. CARs were selected for arrayed validation. a, Top hits from the screen were more effective at clearing the NALM6. Instances of antigen-low / negative cells suggest that CARs differ in their effect on antigen escape. b, In general, more CAR-T cells are recovered for high-ranked CARs.

[0013] FIG.6a–b. Validation of individual CARs against NALM6 in vivo at a stress test CAR-T cell dose. a, Mouse survival correlates with the rankings from the screen. The negative control condition (TRAC knockout) is depicted (dark line) in each plot. b, Tumor burden. Each dot is one mouse. Higher-ranked CARs led to lower burden.

[0014] FIG.7a–c. Validation of individual CARs against JEKO1 in vivo at a stress test CAR-T cell dose. a, Mouse survival. The negative control condition (TRAC knockout) is depicted in each plot. b, Tumor burden in the JEKO1 model correlates well with rankings from the screen in NALM6. Each dot is one mouse. c, CAR-T cells in the bone marrow at nine days after T cell injection.

[0015] FIG.8. A library can be subjected to in vitro assays, such as a proliferation assay, in which T cells undergo multiple rounds of co-culture with antigen-positive or antigen-negativecells, and a cytokine assay, in which production of molecules such as TNF and IFN isdetected. Plots indicate correlations between in vitro screens and the in vivo screen. The in vitro screens were at best modest predictors of in vivo enrichment.

[0016] FIG.9a–d. Affinity screening. a, the CAR-T cell library was stained with recombinant antigen protein and flow-sorted into equal-percentage bins. b, Sequencing was used to determine the enrichment of library members in each bin and calculate an affinity score: a metric approximating the relative MFI of individual CARs. This score can be used to rank the whole-cell affinity of library members. This readout of this assay is whole-cell affinity, as opposed to measurement of a binder's KD. An example calculation is shown. c, Validation for individual CARs; d, Correlation between the individual validation and the pooled affinity screen.

[0017] FIG.10. Correlation between affinity score and enrichment from other screens. Affinity was at best a modest predictor of CAR-T cell function. In the left plot: CARs that expand little in vivo (poor function) are weak binders, but CARs that expand greatly in vivo (suggesting good function) are not necessarily strong binders.

[0018] FIG.11. A binder library can be screened on different CAR backbones. Constructs are depicted that have the same binder (library) and a different hinge domain, costimulatory domain, or stimulatory domain.

[0019] FIG.12a–d. A binder identified through T cell display can be used on other types of receptors. a, The HIT uses a binder (that could otherwise be used on a CAR) on a TRAC- targeted TCR constant scaffold. To accommodate a VHH binder and improve receptor expression, the CRISPR / Cas sgRNA and HDRT were modified as compared with the previous report of HIT that used an scFv binder: (i) a disulfide bond was added between theconstant alpha (C ) and constant beta (C ) by mutating a single amino acid in each chain tocysteine, (ii) a different CRISPR / Cas sgRNA cut site in TRAC was identified, downstream ofthe site corresponding to the mutation in C , and (iii) the HDRT homology arms wereadjusted based on the cut site. b, Examples of HIT using two different nanobodies: VHH2 from the Carvykti product (targeting BCMA), and NB06 identified from T cell display. A binder or multiple binders can be placed on the HIT alpha chain, HIT beta chain, or both. For VHH2, dual placement had an additive effect on recombinant antigen protein binding (x- axis), whereas for NB06 it had a synergistic effect. This difference might relate to how HIT engages with a particular antigen; CD72 is a homodimer, and one explanation is that dual binders improve avidity of one HIT receptor binding one antigen homodimer. c, Cytotoxicity in vitro of CAR and HITs containing NB06 at varied effector cell to target cell ratios. HITs containing a binder identified from T cell display are cytotoxic. d, Compared with VHH- CAR, VHH-HIT has greater antigen sensitivity.

[0020] FIG.13a–b. Pooled screening of CAR libraries that have an scFv binder. Binders in the library vary by mutations in the CDRs. HDRTs were targeted to the Trac locus of mouse T cells. a, From the pooled affinity screen, a normalized affinity score is calculated based on the relative abundance of each library member across sorting bins. The score is normalized to the affinity of the original binder. b, An in vitro proliferation screen was conducted by co- culture with a TRP2-expressing target cell line.

[0021] FIG.14. Results from the two types of screens are plotted for 10 example CDR positions. In each plot, the left y-axis is proliferation fold enrichment (lighter dots) and the right y-axis is normalized affinity score (black dots). Library members that are highlighted are those that improved proliferation and span a range of affinities.. DETAILED DESCRIPTION OF THE INVENTION Terminology

[0022] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0023] As used herein, an “antigen binding domain” as used in the present disclosure refers to a polypeptide that selectively binds to a molecule expressed by a cell to be targeted, or that same molecule as a recombinant reagent. Such an antigen binding domain for purposes ofthis disclosure comprises one or more variable regions from an antibody. In some embodiments, the antigen binding domain comprises a VH region in a VHH format. In some embodiments, the antigen binding domain comprises a VH and / or VL region of an antibody. In other embodiments, the binding domain is a natural ligand of the target (e.g., a CD27 domain to target CD70; an APRIL domain to target TACI; or an IL-13 domain to target IL- 13Ra).

[0024] As used herein, the terms “chimeric antigen receptor” and “CAR” refer to a synthetic multi-module receptor that modulates immune cell activation. A CAR typically comprises an extracellular domain that comprises an antigen binding domain, a transmembrane domain and typically one or more intracellular signaling domains.

[0025] The term “heterologous sequence” refers to a sequence not normally found in a given cell in a nature. As such, a heterologous polynucleotide or protein sequence may be: (a) foreign to its host cell (i.e., is exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous) but present at an unnatural quantity in the cell (i.e., at a greater quantity than naturally found in the host cell); or (c) naturally found in the host cell but positioned in a structural conformation, e.g., in a different position relative to adjacent sequences that that observed in nature. A heterologous nucleotide or polypeptide sequence can also mean a sequence that is not found in the native nucleic acid or protein, respectively. For example, relative to a TCR, a CAR comprises an amino acid sequence with some portions that can be derived from sources other than the TCR.

[0026] An “HLA-independent TCR” (also referred to as a HIT receptor or HIT) as used herein is a TCR-like receptor that can recognize an antigen independent of HLA restriction. In an exemplary embodiment, a HIT may bind to an antigen on the cell surface that is not presented by the HLA complex.

[0027] As used herein, the term “antibody” means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an “antibody” as used herein is any form of an antibody of any class or subclass or fragment thereof that exhibits the desired biological activity, e.g., binding a specific target antigen. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and includes antibody fragments such as, but not limited to scFv, VHH, and thelike, as long as they exhibit the desired biological activity. Accordingly, an antigen binding domain can be obtained from any form of antibody.

[0028] As used herein, "complementarity-determining region (CDR)" refers to each of the three hypervariable regions (HVRs) that occur alternating with the four "framework" regions of a variable domain. The CDRs are primary contributors to binding to an epitope of an antigen. The CDRs of are referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.

[0029] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol.196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol.227, 799-817; Al-Lazikani et al., J.Mol.Biol 1997, 273(4)). Definitions of antigen combining sites are also described in the following: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219–221 (2000); and Lefranc,M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268–9272 (1989); Martin, et al, Methods Enzymol., 203, 121–153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141–1721996). Reference to CDRs as determined by Kabat numbering are based, for example, on Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia CDRs are determined as defined by Chothia (see, e.g., Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

[0030] An "antigenic determinant", also referred to herein as an “epitope", refers to a site on an antigen to which an antigen binding domain binds. An antigenic determinant can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3,and more usually, at least 5 or 8–10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, Glenn E. Morris, Ed (1996).

[0031] As used herein, “affinity” with respect to antibody binding to an epitope generally refers to the strength of the interaction of the antibody with the epitope. In the present disclosure, the affinity of antibody for binding to the target polypeptide is represented by theequilibrium dissociation constant (KD).KDis inversely related to the binding affinity.

[0032] As used herein, the term “specifically binds” to a target refers to a binding reaction whereby, for example, an antigen binding domain binds to its cognate antigenic determinant with greater affinity, greater avidity, and / or greater duration than it binds to a different target. In some embodiments, a target-binding protein has at least 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, 1,000-fold, 10,000- fold, or greater affinity for the target compared to an unrelated target when assayed under the same binding affinity assay conditions. For example, an antigen binding domain may bind to an antigenic determinant or set of antigenic determinants on a target cell with a KDthat is at least 100-fold greater than its affinity compared to unrelated antigens expressed by the cell.

[0033] The term “immune cell” as used herein generally refers to lymphoid cells, including T cells, B cells, and natural killer (NK) cells; and myeloid cells, such as neutrophils, eosinophils, basophils, monocytes, macrophages, and dendritic cells.

[0034] The terms “T cell” and “T lymphocyte” as used herein are interchangeable. Examples of T cells include, but are not limited to, cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, effector T cells, natural killer T cells, or T cells derived in vitro from embryonic or pluripotent stem cells. T-cell display

[0035] In one aspect, the disclosure features a pooled T cell display protocol, in which binding domains such as VHHs or scFvs are displayed as a component of synthetic receptors on primary cells, in contrast to existing display methodologies that employ phage, yeast, T cell lines, or other cell lines and are independent of the receptor context. In the methods provided by the present disclosure, receptor transgenes are targeted to a locus that can be monoallelically expressed and is constant in a T cell, e.g., often the TRAC locus, which supports efficient pooled assessment of binder and receptor characteristics. Pooled T celldisplay of the present disclosure provides the ability to assess many candidates in a direct comparison and identifies antigen binding domains that support more efficacious receptor activity in vivo compared to traditional proliferation, cytokine production, and affinity assays employed to select antigen binding domains to generate synthetic receptors.

[0036] In the context of the present disclosure, a T cell employed in T cell display is thus a genetically modified T cell in which an expression construct for a synthetic receptor that comprises an antigen binding domain is integrated into a generally monoallelically expressed locus of a T cell e.g., TRAC, TRBC1, or TRBC2 locus native to the T cell. In some embodiments, the synthetic receptor is a CAR. In some embodiments, a synthetic receptor is a HIT. In some embodiments, the antigen binding domains screened in the pooled T cell display method are ligands of a receptor that is the “antigen” for purposes of this description of pooled T cell display.

[0037] In some embodiments, a synthetic receptor introduced into a T cell may be modified with a mutated TCR beta chain constant region to remove one or more antibody binding epitopes that are commonly recognized by antibodies that bind to wild type TCR beta 1 or TCR beta 2 constant regions. Thus, cells expressing a wild type TCR beta constant chain can be removed from a population of engineered cells generated as described herein. In some embodiments, a HIT receptor can contain a mutated TCR beta chain. Such mutations are described, e.g., in PCT Application No. PCT / US2023 / 068423, which is incorporated by reference for the material relating to mutations in the beta chain constant region. Introducing binding domains into T cell constant region locus

[0038] Any number of binding domains may be incorporated into a synthetic receptor and introduced into a T cell constant region locus for performing a pooled T cell display method of the present disclosure. In some embodiments, the binding domain comprises a VH region, e.g. a VHH. In other embodiments, the binding domain comprises a VH region and a VL region, e.g., an scFv. In some embodiments, the antigen binding domain comprises a ligand of a receptor that is the “antigen” for purposes of this description of pooled T-cell display.

[0039] A plurality of synthetic receptors comprising different antigen binding domains can be introduced into an immune cell, e.g., a T cell, using known technology. In the present disclosure, a pool of receptor constructs comprising antigen binding domains to be evaluated is introduced into a population of T cells in the form of a homology-directed repair template (HDRT). Such a template comprises homology sequences for targeting a synthetic receptorconstruct to a locus of interest in the T cell, e.g., TRAC, TRBC1, or TRBC2, and a transgene encoding the synthetic receptor polypeptide comprising a candidate antigen binding domain such as a VHH, i.e., the cargo for incorporation into the T-cell receptor constant domain locus to replace the endogenous gene. In some embodiments, the template is packaged in a viral vector. In some embodiments, the vector is an adenovirus vector. In many embodiments, the vector is an adeno-associated virus (AAV) vector, such as AAV6, AAV- DJ, AAV5, or any one of those named in WO2023 / 004407.

[0040] Alternatively, in some embodiments, an HDRT can be delivered to a population of T cells by an alternative vehicle, e.g., liposomes, or by electroporation instead of using a viral vector. In some embodiments, a transposase or recombinase system may be employed.

[0041] In an illustrative embodiment, an AAV vector, e.g., an AAV serotype 6 vector comprising an HDRT with a transgene encoding an antigen binding domain, is used to introduce the transgene into the T-cell receptor locus to generate a library of candidate synthetic receptors, e.g., CARs or HITs. In further embodiments, the AAV template additionally comprises a marker gene whose expression facilitates the identification of cells into which the transgene encoding the antigen binding domain is incorporated. The marker thus provides an ability to detect genomic integration of the template independent of direct CAR detection. In some embodiments, the marker is truncated epidermal growth factor receptor (EGFRt). The marker can also be a gene such as for truncated nerve growth factor receptor (NGFRt), LNGFR, RQR8, Thy1.1, Thy1.2, or a fluorescent protein.

[0042] Any targeted genome editing methods using a DNA nuclease can be used to deliver transgenes encoding candidate antigen binding domains. Such methods include CRISPR-Cas systems, meganucleases, transcription activator–like effector-nucleases (TALENs), zinc finger nuclease-based methods, and the like. In some embodiments, a CRISPR-Cas gene editing system is used to introduce the synthetic receptor construct into the T cell to replace the endogenous gene encoded by the target locus, e.g., TRAC, TRBC1, or TRBC2 locus. Synthetic T cell receptor constructs CARs

[0043] Numerous synthetic receptor proteins can be screened using the methods of the claims in the invention. In some embodiments, the synthetic receptor is a CAR construct comprising an antigen binding domain to be assessed in the in vivo display screen, a hinge region (also referred to as a spacer), a transmembrane domain, and a signaling domain, e.g., aCD3 , CD3 , CD3 , or CD3 signaling domain. In some embodiments, the CAR comprisesone or more alternative signaling domains comprising intracellular tyrosine activation motifs (ITAMs) signaling motifs. Additional examples of ITAM-containing primary intracellularsignaling motifs include common FcR , Fc RIIa , FcR , Fc RIb, CD79a, CD79b, DAP10,and DAP12. In some embodiments, the CAR comprises one or more signaling domains comprising mutations to the wild type ITAMs.

[0044] An intracellular signaling domain of a CAR can comprise a primary intracellular signaling domain only, or may comprise additional desired intracellular signaling domain(s) useful in the context of a CAR of the disclosure. For example, the intracellular signaling domain of the CAR can comprise a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell-surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83 and the like. Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8A, CD8B, IL2RB, IL2RG, IL7RA, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD1a, LFA-1, ITGAM, CD1b, ITGAX, CD1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0045] In some embodiments, the synthetic receptor may contain one or more hinge domains that link the antigen binding domain and the transmembrane domain for positioning the antigen binding domain. Such a hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region, e.g., a naturally occurring human immunoglobulin hinge region, or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in a cell receptors include the hinge region derivedfrom the extracellular regions of type I membrane proteins such as CD8 , CD4, CD28, PD1,CD152, and CD7, which may be wild type hinge regions from these molecules or may be altered. In some embodiments, the hinge domain is based on the hinge region of a human immunoglobulin IgG1 or IgG4. In some embodiments, the hinge region includes the IgG1 or IgG4’s CH2 region, which may comprise one or more mutations. In some embodiments, the mutation is L235E, N297Q, or both L235E and N297Q (which is known as the EQ mutation in the IgG4 hinge region). In some embodiments, the hinge domain is derived from CD28.

[0046] Any transmembrane domain suitable for use in a cell receptor construct may be employed for the synthetic receptor. A transmembrane domain incorporated into a cell receptor construct may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. Such transmembrane domains, include, but are not limited to, all or partof the transmembrane domain of TCR , TCR , CD28, CD27, CD3E, CD3Z, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD1a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2RB, IL2RG, IL7RA, ITGAL, VLAL, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1d, ITGAE, CD103, ITGAL, CD1a, LFA-1, ITGAM, CD1b, ITGAX, CD1c, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME, (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In some embodiments, the transmembrane domain is derived from CD28. HIT receptors

[0047] In some embodiments, the synthetic receptor is a HIT. A HIT is introduced by gene editing, e.g., CRISPR / Cas-based systems into a generally monoallelically expressed locus, typically the TRAC locus. The receptor comprises one or more variable domains, e.g., one ormore VHHs, or a VL and VH, linked to the constant regions of TCR alpha and TCR beta thatare expressed using endogenous TCR alpha sequence elements such as TRAC exons. The HIT can assemble with CD3 chains. In some embodiments, the synthetic receptor is a TCR- like receptor other than a HIT.Library analysis and screening

[0048] Once a library is generated, a portion of the library can be assessed for library quality using an amplification reaction. Furthermore, portions of the library can be individually amplified to incorporate barcode sequences such that the individual portions of the library can be used in different assays.

[0049] The libraries are analyzed by sequencing. Either genomic DNA or cDNA transcribed from RNA can be used for sequencing analysis. For amplification, a portion of a homology arm of the HDRT may be designed to differ from the corresponding endogenous sequence. This region is not retained after integration of the construct into the target locus. Thus, one primer that binds to the endogenous sequence and another primer that binds within the region that encodes the synthetic receptor amplifies only integrated constructs. In a subsequent amplification reaction, e.g., PCR, the forward primer can bind to the VHH framework region 1 and the reverse primer can bind to the VHH framework region 4. The resulting amplicon contains the three CDRs. For pooling multiple libraries, e.g., the same original library employed in different assays, library-specific indexes can be added in a third amplification reaction, e.g., PCR. The same principle holds for sequencing VH and VL domains.

[0050] The library of T cells can be introduced into animal models of cancer, e.g., immunodeficient mice into which cancer cells that express the target antigen of interest have been introduced. T cells are later isolated from the bone marrow or other tissue, and genomic DNA or RNA-derived cDNA comprising variable region sequences at the target locus, e.g., TRAC locus, are amplified and sequenced. The relative abundance of the variable region sequences (e.g., VHH, VH, or VL) compared to a control, e.g. pre-injection baseline, can then be assessed. Antigen binding domains can be ranked according to enrichment, and the T cells that express enriched binding domains can be assessed in further assays. For example, activity of the synthetic receptors that are enriched can be assessed to evaluate activity towards cancer cells in the animal model, with measurement of survival and / or tumor burden.

[0051] T cell display can be employed to identify potent synthetic receptors that can be used in CAR-T cells and HIT-T cells to target various types of cancer. Non-limiting examples of cancers include In some embodiments, the cancer is a carcinoma or a sarcoma. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is breast cancer, prostate cancer, testicular cancer, renal cell cancer, bladder cancer,liver cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, colorectal cancer, anal cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, kidney cancer, head and neck cancer, glioblastoma, mesothelioma, melanoma, a chondrosarcoma, or a bone or soft tissue sarcoma. In some embodiments, the cancer is adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal-cell carcinoma, bile duct cancer, bone tumor, brainstem glioma, brain cancer, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, or bronchial adenomas. In some embodiments, the cancer is acute lymphoblastic leukemia, acute myeloid leukemia, Burkitt's lymphoma, central nervous system lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, chronic myeloproliferative disorders, a myelodysplastic syndrome, an adult acute myeloproliferative disorder, multiple myeloma, cutaneous T-cell lymphoma, Hodgkin lymphoma, or non-Hodgkin lymphoma. In some embodiments, the cancer is desmoplastic small round cell tumor, ependymoma, epithelioid hemangioendothelioma (EHE), Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, gastric carcinoid, heart cancer, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, lip and oral cavity cancer, liposarcoma, non-small cell lung cancer, small-cell lung cancer, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, chronic, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma, supratentorial primitive neuroectodermal tumors, pituitary adenoma. plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine sarcoma, Sézary syndrome, non-melanoma skin cancer, melanoma Merkel cell carcinoma, small intestine cancer, squamous cell carcinoma, squamous neck cancer, throat cancer, thymoma, thyroid cancer, transitional cell cancer of therenal pelvis and ureter, trophoblastic tumor, gestational, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, or Wilms tumor. Technical Section

[0052] A pool of VHHs targeting the B cell antigen CD72 that were identified by rounds of yeast display (Nix et al., 2021) were cloned into an AAV HDRT (Fig.1a). This template contains a CAR transgene and an EGFRt marker for detecting knock-in independent of CAR staining. The cargo was targeted using CRISPR / Cas editing to the TRAC locus (Fig.1b), which confers largely monoallelic and homogeneous expression (Eyquem et al., 2017). Following knock-in, expression of the endogenous TCR is replaced by that of the cargo. Screening entails sequencing VHHs (Fig.2a–d), which can employ either genomic DNA or RNA-derived cDNA as input (Fig.3). Use of genomic DNA is illustrated below.

[0053] The library is amplified, e.g., by PCR, in preparation for sequencing. A portion of the left homology arm of the HDRT differs from the corresponding endogenous sequence. This region is not retained after integration. Thus, a forward primer that binds to the endogenous sequence and a reverse primer that binds within the cargo amplifies only integrated cargo. In a subsequent amplification reaction, e.g., PCR, the forward primer binds to the VHH framework region 1 and the reverse primer binds to the VHH framework region 4. The resulting amplicon contains the three (CDRs). For pooling multiple libraries, e.g., the same original library employed in different assays, library-specific indexes can be added in a third amplification reaction, e.g., PCR.

[0054] In the screen, library CAR-T cells were introduced into immunodeficient mice that had been engrafted with NALM6 leukemia cells, which express CD72 (Fig.4a). Enrichment of library members was determined by isolating T cells from the bone marrow, extracting genomic DNA, amplifying VHH sequences integrated at the TRAC locus, performing sequencing, and comparing relative abundances to the pre-injection baseline (Fig.4b). Several binding domains were enriched, with a VHH referred to as NB06 as the most enriched.

[0055] To evaluate the predictive capability of this screen, CARs with various rankings of enrichment were selected for arrayed assessment. In an in vivo experiment using the same parameters as the screen described above, bone marrow analysis indicated that T cells with the top-ranked CARs generally better cleared NALM6 (Fig.5a) and underwent more expansion (Fig.5b). CAR-T cell activity toward NALM6 was also evaluated in a stress testexperiment, in which the CAR-T cell dose is lowered. Higher-ranked CARs conferred better mouse survival (Fig.6a) and lower tumor burden (Fig.6b).

[0056] In a stress test experiment using a JEKO1 lymphoma model, T cells with higher- ranked CARs also generally provided better survival (Fig.7a) and a lower tumor burden (Fig.7b) and underwent more expansion (Fig.7c).

[0057] Library CAR-T cells were evaluated by in vitro assays for proliferation and cytokine production. The in vitro outcomes did not correlate strongly with in vivo outcomes (Fig.8).

[0058] To investigate how binding affinity relates to receptor function, the library was flow-sorted into bins based on recombinant antigen protein staining (Fig.9a). By analysis of the sequencing data, an affinity score was assigned for each CAR that was used to rank-order the whole-cell affinity for antigen (Fig.9b). Scores from the pooled assay correlated well with arrayed measurements (Fig.9c,d). Analysis of the enrichment outcomes across screens indicated that although CARs that did poorly in vivo were low affinity, CARs that did well were not necessarily high affinity (Fig.10). These results thus support the utility of in vivo T cell display, as compared with conventional affinity-based selection, for identifying effective antigen binding domains for chimeric receptors.

[0059] One of skill further understands that although these experiments used a CAR backbone containing the CD28 hinge, transmembrane, and costimulatory domain and a 1XX CD3 zeta signaling domain (Feucht et al., 2019), alternative domains can also be employed (Fig.11).

[0060] T cell display thus accurately identified potent CARs. The results demonstrate that in vivo expansion in response to tumor challenge can be an effective proxy for anti-tumor potency. Performance of T-cell display-identified binding domains in HIT receptors

[0061] We additionally determined whether a binding domain identified from T cell display could be using as the binding domain for another type of chimeric receptor, the HLA- independent TCR (HIT). Modifications were made to the original HIT design that used anscFv binder (Fig 12a), because VHH deviates more from the V and V domains of a TCRthan do the VL and VH domains of an scFv. In one modification, a disulfide bond was added to the HIT constant regions to improve receptor surface expression. As compared with aCAR, which has one chain, the two variable chains of HIT offer several options for binding domain placement. For CD72 targeting, we observed that binding domain placement on both the chain and chain was preferable for antigen binding (Fig.12b), although placement on either chain individually was sufficient for cytotoxicity (Fig.12c). Importantly, it was observed that VHH-HIT exhibited better antigen sensitivity than a CAR in a cytotoxicity assay against a cell line expressing a low level of CD72 (lower than in parental JEKO1 cells) (Fig.12d). In summary, a VHH binder nominated by pooled T cell display on a CAR can be employed in a HIT.

[0062] The above analyses demonstrated that pooled T cell display identified the most effective binders based on CAR-T cell expansion in response to tumor challenge in vivo. VHHs were employed as binding domains. Targeting a library to TRAC provided the benefit of both the physiological regulation conferred by this locus and a uniformity that supported comparisons across library members. Recent studies have used pooled knock-in libraries at TRAC to study the effects of natural and synthetic transcription factors, receptors, and other genes (Blaeschke et al., 2023; Roth et al., 2020) and of CAR intracellular signaling domains (Castellanos-Rueda et al., 2022).

[0063] The illustrative data presented above using T cell display based on CAR and HIT receptors demonstrated in arrayed validation in models of both leukemia and lymphoma that top hits from the screen conferred superior tumor control and mouse survival. Conventional in vitro assays for proliferation, cytokine production, and affinity were less effective at predicting (and in some cases not correlated with) anti-tumor potency. Thus, pooled T cell display provides a superior method over existing approaches for identifying synthetic receptor binding domains. T-cell display-identified scFv-CARs

[0064] T cell display can also be employed for scFvs. The data presented in this section illustrate a library of scFv-CARs targeted to the murine Trac locus that is screened on in vitro proliferation and affinity. In vivo screening as described above can also be performed. Although in this illustrative embodiment the scFv is derived from a TCR-mimic antibody (which targets a peptide-MHC), the methods also apply to other types of binders and antigens.

[0065] Libraries were generated by DNA synthesis and cloning. Library members vary in individual residues of the CDRs of the VL and VH domains of the scFv binder. Mouse T cellswere edited to express the library from the Trac locus. For pooled screening of affinity, cells underwent staining and flow-sorting, genomic was isolated and amplified, and amplicons were sequenced. The results reveal a range of affinities for dextramer (recombinant peptide- MHC reagent), and that different CDR positions tolerate different substitutions (Fig.13a).

[0066] Library CAR-T cells were evaluated in a proliferation assay. The assay was conducted using two week-long rounds of stimulation through co-culture with antigen- expressing target cells. Sequencing analysis indicated correlations in enrichment between target cell lines (relative to pre-assay baseline) (Fig.13b). There were some CDR position- specific trends in proliferation, as well as within-position trends. Among positions that improved proliferation, some retained high affinity similar to that of the original scFv, whereas others had lowered affinity (Fig.14).

[0067] All patents, patent applications, and other publications cited in this application are incorporated by reference in the entirety for all purposes. References Beppler, C., Eichorst, J., Marchuk, K., Cai, E., Castellanos, C.A., Sriram, V., Roybal, K.T., and Krummel, M.F. (2023). Hyperstabilization of T cell microvilli contacts by chimeric antigen receptors. J Cell Biol 222, e202205118. Blaeschke, F., Chen, Y.Y., Apathy, R., Daniel, B., Chen, A.Y., Chen, P.A., Sandor, K., Zhang, W., Li, Z., Mowery, C.T., et al. (2023). Modular pooled discovery of synthetic knockin sequences to program durable cell therapies. Cell 186, 4216–4234. Caruso, H.G., Hurton, L.V., Najjar, A., Rushworth, D., Ang, S., Olivares, S., Mi, T., Switzer, K., Singh, H., Huls, H., et al. (2015). Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity. Cancer Res 75, 3505–3518. 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Claims

WHAT IS CLAIMED IS:

1. A method for screening for antigen binding domains for synthetic receptors, comprising (a) providing a plurality of T cells comprising T cells that express different synthetic receptors on the cell surface; wherein an individual synthetic receptor comprises an antigen binding domain (ABD) that is different from the ABD expressed by other T cells contained in the plurality of T cells; wherein the synthetic receptor is encoded by an expression construct introduced into any locus that is monoallelic or can be monoallelically expressed, such as a T-cell receptor alpha (TRAC) or a T-cell receptor beta (TRBC1 or TRBC2) locus; (b) introducing the plurality of T cells into immunodeficient non-human mammalian subjects in vivo; wherein the subjects can have engrafted tumors that express the antigen to which the ABD binds; (c) isolating T cells after a sufficient period of time for a T-cell response to be induced following binding of the ABD to the antigen expressed by tumor cells; (d) amplifying nucleic acid extracted from T cells obtained from the bone marrow or other tissue of a subject having an engrafted tumor, and optionally a control subject lacking tumor cells, to obtain amplicons indicating ABD-encoding regions; (e) sequencing amplicons obtained of (d); and (f) determining ABD-encoding regions that are enriched in subjects having engrafted tumors.

2. The method of claim 1, wherein the expression construct in introduced into the TRAC locus.

3. The method of claim 1 or 2, wherein the synthetic receptor is a chimeric antigen receptor (CAR).

4. The method of claim 1 or 2, wherein the synthetic receptor is an HLA- independent T cell (HIT) receptor.

5. The method of claim 1 or 2, wherein the synthetic receptor is a TCR- like receptor.

6. The method of any one of claims 1-5, wherein the ABD comprises a VHH.The method of any one of claims 1-5, wherein the ABD comprises a VHand / or a VL.

8. The method of any one of claims 1–5, wherein the ABD comprises a natural ligand.

9. The method of any one of claims 1-7, wherein the T cell is a cytotoxic T cell, helper T cell, memory T cell, regulatory T cell, effector T cell, natural killer T cell, or a T cell derived in vitro from an embryonic or pluripotent stem cell.

10. The method of any one of claims 1-9, wherein the expression construct is introduced into the T-cell receptor alpha (TRAC) locus or T-cell receptor beta (TRBC1 or TRBC2) locus by a gene editing system selected from the group consisting of a CRISPR / Cas gene editing system, a TALEN gene editing system, and a zinc finger nuclease system.

11. The method of any one of the foregoing claims, wherein (d) comprises amplifying genomic DNA.

12. The method of any one of the foregoing claims, wherein (d) comprises amplifying cDNA prepared from RNA.

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