EP4791760A1 - Chimeric ap-1 transcription factor polypeptides and methods of use - Google Patents

Abstract

Provided are chimeric AP-1 transcription factor polypeptides. In some aspects, provided are chimeric polypeptides comprising an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor. Such polypeptides may further comprise a C-terminal domain of an AP-1 transcription factor. In other aspects, provided are chimeric polypeptides comprising a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor. Such polypeptides may further comprise an N-terminal domain of an AP-1 transcription factor. In some instances, T cells expressing the chimeric AP-1 transcription factor polypeptides exhibit enhanced T cell function relative to T cells expressing only native wild-type AP-1 transcription factors. Also provided are T cells expressing the chimeric polypeptides and methods of using such cells to treat a condition (e.g., cancer, infection, and/or the like) in a subject in need thereof.

Description

CHIMERIC AP-1 TRANSCRIPTION FACTOR POLYPEPTIDES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/543,984, filed October 13, 2023, which application is incorporated herein by reference in its entirety.

INTRODUCTION

T-cell exhaustion is a broad term used to describe T cell dysfunction resulting from chronic stimulation. Exhausted T cells present with a distinct phenotype including overexpression of inhibitory markers such as PD-1 , LAG-3 and TIM-3 as well as impairment in their ability to release pro-inflammatory cytokines (IFNy and TNFa). Exhaustion commonly occurs in the tumor microenvironment where T cells suffer a loss of their cytotoxic function and become ineffective in their ability to kill cancerous cells.

Adoptive cell therapies, including chimeric antigen receptor (CAR) T cell therapy, engineered T cell receptor (TCR) T cell therapy, and the like, have been successful across multiple hematological cancers but robust responses against solid tumors remain elusive. A major barrier to clearance of solid tumors is intrinsic dysfunction or exhaustion of CAR T cells, where chronic antigen contact and subsequent signaling leads to reduced cytotoxicity. Myriad strategies, including genetic manipulation, receptor optimization and tuning of response to extracellular signals have aimed at preventing the development or decreasing the severity of this dysfunction. Yet, a broadly effective dysfunction-resistance strategy remains to be identified.

SUM ARY

Provided are chimeric AP-1 transcription factor polypeptides. In some aspects, provided are chimeric polypeptides comprising an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor. Such polypeptides may further comprise a C-terminal domain of an AP-1 transcription factor. In other aspects, provided are chimeric polypeptides comprising a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor. Such polypeptides may further comprise an N- terminal domain of an AP-1 transcription factor. In some instances, T cells expressing the chimeric AP-1 transcription factor polypeptides exhibit enhanced T cell function relative to T cells expressing only native wild-type AP-1 transcription factors. Also provided are T cells expressing the chimeric polypeptides and methods of using such cells to treat a condition (e.g., cancer, infection, and/or the like) in a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A-1 B: Schematic illustration of the general structure of an AP-1 transcription factor showing the N-terminal, basic motif-leucine zipper (bZIP) and C-terminal domains (FIG. 1A). Schematic illustration of 500 different AP-1 transcription factors representing combinations of 10 different N-terminal domains, 5 different bZIP domains and 10 different C-terminal domains.

FIG. 2: Schematic illustration of the production of a DNA library encoding chimeric AP-1 transcription factors. Constructs of the library comprise an amplifiable combination of three barcodes each uniquely identifying the particular N-terminal domain, a bZIP domain and a C- terminal domain, respectively, encoded by a particular construct. Barcode amplicons may be sequenced to determine the identity of the N-terminal domain, bZIP domain and C-terminal domain expressed by a particular population of T cells.

FIG. 3: Schematic illustration of a targeted knock-in strategy for producing a library of T cells expressing constructs produced according to the method shown in FIG. 2.

FIG. 4A-4B: Schematic illustration of in vitro repetitive stimulation assays for determining T cell enrichment under conditions of chronic or acute stimulation.

FIG. 5: A volcano plot showing chimeric AP-1 transcription factors enriched by chronic stimulation. The figure shows a summary (n = 3 donors) of the degree and significance of enrichment of chimeric AP1 TF-expressing CAR T cells following 2 weeks of repetitive stimulation. Many chimeric AP1 TFs are enriched above the ’’CAR Only” control (no chimeric AP1 TF) suggesting that these constructs can enhance the function of a clinically used CAR under high antigen load. The labels denote which endogenous gene domains contribute to a particular chimeric AP1 TF (N_bZIP_C). Analysis was performed with DESeq2.

FIG. 6: A volcano plot showing chimeric AP-1 transcription factors enriched by acute stimulation. The figure shows a summary (n = 3 donors) of the degree and significance of enrichment of chimeric AP1 TF-expressing CAR T cells following a single addition of CD19- bearing Nalm6 cells and subsequent culture in the absence of further stimulation (acute stimulation). Many chimeric AP1 TFs are enriched above the ’’CAR Only” control (no chimeric AP1 TF) suggesting that these constructs can augment the proliferative capacity/persistence of a clinically used CAR. The labels denote which endogenous gene domains contribute to a particular chimeric AP1 TF (N_bZIP_C). Analysis was performed with DESeq2.

FIG. 7: Comparison of degree and significance of enrichment across chronic and acute conditions for AP-1 transcription factors.

FIG. 8: A table summarizing the reproducibility of top hits from the chronic stimulation screen and the enrichment of specific 2-domain combinations. Nearly all of the top hits follow one of the two highlighted “N_bZIP” or “bZIP_C” compositions.

FIG. 9: Data showing show donor-level enrichment for chronic and acute stimulation conditions repeated across biological replicates.

FIG. 10A-10F: Development of a scalable, high-fidelity molecular pipeline to iteratively and combinatorially construct new genes from constituent domains, a, Schematic of domain delineation and cloning, b, Alignment of natural AP-1 TF domain amino acid sequences using COBALT c, Top, natural domains classified by AP-1 subfamily. Bottom, natural domains comprehensively rearranged, d, Left, binned histogram to assess balance and representation in the final plasmid library by barcode sequencing. Right, scatter plot to correlate barcode representation in the final plasmid library versus edited T cells from a representative human donor. R² calculated from a y ~ x linear regression, e, Long-read sequencing spanning the full TF and barcode of the final plasmid pool filtered to reads with three barcodes. Left, stacked bar plot of overall barcode linkage fidelity and a breakdown of main error modes. Right, scatter plot correlating barcode linkage fidelity of individual constructs to base-pair (bp) length. R² calculated from a y ~ x linear regression, f) Barcode sequencing of edited T cells from a representative human donor. Left, strip plot of each natural domain’s representation. Right, scatter plot correlating TF representation to bp length. R² calculated from a y ~ x linear regression.

FIG. 11 A-11G: Pooled screening identifies DESynR TFs that outperform natural TFs and outlines domain-use logic, a) Schematic of pooled screening in primary human T cells. The library and controls were subcloned into a vector containing a CD19-28z CAR flanked by homology to the TRAC locus to enable non-viral integration. Barcodes from edited cells were sequenced before (input) and after acute and chronic stimulation with target cells, b) A volcano plot indicating the enrichment or depletion of each TF following chronic stimulation. Analysis subsetted to constructs with at least 10 reads in input samples in all three human donors (n = 12,284). Log₂FC calculations were performed with DESeq2; significance calculated by Wald test and corrected for multiple comparisons using the Benjamini-Hochberg method. Hashed horizontal line indicates p<0.05. c) Log₂FC in individual human donors for selected TFs and controls. Each vertical bar represents one human donor (n = 3). d) Three hits (blue lines) from the pooled screen were individually validated in a competitive chronic stimulation assay against GFP control (black lines). Shapes indicate unique human donors (n = 2). Independent two-tailed Student’s t-test performed after 6 stimulations, e) For 242 significantly enriched DESynR AP-1 TFs, bar plots enumerating utilization of “N”, “bZIP” and “C” domains from left to right. To the left of each bar plot, a heatbar of expression in endogenous T cells of the corresponding domain, binned by quartile. Vertical dashed lines indicate uniform representation. A two-tailed z-test was performed for each domain to assess significant deviations from uniformity, f) Each three-domain DESynR AP-1 TF was re-classified as three separate domain-pair architectures (N+bZIP, bZIP+C,N+C). Heatmap of Log₂FC of domain-pair combinations within each architecture. Heatbars report expression of the corresponding domain in endogenous T cells, binned by quartile, g) Bar plots of Log₂FC of exemplar domain-pairs completed with respective third domain. From left to right, “C” domain for “N+bZIP”, “N” for “C+bZIP” and “bZIP” for “N+C”. Each dot represents one human donor (n = 3). Log₂FC calculations were performed with DESeq2. Significance calculated using Wald test and Corrected for multiple comparisons using the Benjamini-Hochberg method. Kruskal-Wallis test used to assess overall deviation from uniformity. *P< 0.05, **P< 0.01 , ***P< 0.0001 . FIG. 12A-12I: Multi-parameter pooled screening identifies functional DESynR TF archetypes, a-c, A mini pool was functionally screened in vitro and in vivo. Log2FC calculations were performed with DESeq2. Significance was calculated by Wald test and corrected for multiple comparisons using the Benjamini-Hochberg method, a) A strip plot of average Log₂FC scaled to GFP. Unsealed values shown for “CD62L+” where GFP was omitted due to antibody fluorescence overlap with GFP signal (n = 3 human donors for all “Proliferation/Persistence” and “CD62L+” screens, n = 2 human donors for cytokine screens, and n = 1 human donor and 3-4 mice for in vivo screens), b) Bar plots of average Log₂FC from three functional screens with overlaid points representing one human donor/mouse. “FOSL1_BATF_[x]” indicates the “FOSL1_BATF” “N+bZIP” domain pair with a variable C domain, c) Hierarchically clustered heatmap of average LOG2FC. Highlighted TFs subjected to arrayed validation experiments. GFP omitted as not present in “CD62L” screen, d. Scatter plot correlating average Log₂FC in in vitro “Nalm6 Hi” and in vivo “Bone Marrow” screens. R² calculated from a y ~ x linear regression, e-i, Arrayed validation experiments of select TFs and GFP. e) Left, a bar plot of average acute cytotoxicity (E:T = 1 :8 shown) relative to GFP with overlaid points representing average of technical duplicate or triplicate in one human donor (n = 3). Right, a bar plot of degranulation and cytokine production relative to GFP with overlaid points representing average of technical duplicate or triplicate in one human donor (n = 2-5). f) Exemplar flow cytometry plots of degranulation and IFNg secretion from a representative human donor following acute stimulation, g) A line graph of CAR T cell cytotoxicity throughout an arrayed repetitive stimulation assay. Each point represents mean +/- s.e.m. (n = 2-4 human donors). One-way ANOVA performed for each stimulation to test for significant differences in cytotoxicity across constructs, h) Left, a bar plot of average cytotoxicity (E:T = 1 :12 shown) following chronic stimulation relative to GFP with overlaid points representing average of technical duplicate or triplicate in one human donor (n = 2-6). Right, a bar plot of degranulation and cytokine production following chronic stimulation relative to GFP with overlaid points representing average of technical duplicate or triplicate in one human donor (n = 2-8). i) Exemplar flow cytometry plots of degranulation and IFNg secretion from a representative human donor following chronic stimulation. *P< 0.05, **P< 0.01 , ***p< 0.0001 .

FIG. 13A-13J: DESynR TFs program novel T cell states, a-d, Single-cell (sc) RNA- seq following arrayed chronic stimulation (n = 3-5 human donors per construct; xx total cells), a) Uniform manifold approximation and projection (UMAP) of donor-integrated data across all constructs. Ten clusters were identified by the Louvain method and annotated manually, b) UMAPs of scaled expression of selected marker genes, c) UMAPs with red dots indicating cells of selected constructs with all other cells as gray dots, d) A stacked bar plot showing occupancy in each annotated cluster for all constructs, e-j, Pseudobulked (pb) sc-RNA-seq data and bulk ATAC-seq data following arrayed chronic stimulation (n = 3-5 human donors per construct), e) Top, principal-component analysis (PCA) of bulk ATAC-seq data. Bottom, PCA of pb sc-RNA- seq data, f) Hierarchically clustered heatmap of 18,165 differentially accessible peaks (DAPs) from bulk ATAC-seq data. Top color bar indicates normalized z-scores for each DAP. Each module is ordered by accessibility at all peaks (high to low) in GFP as shown by the bottom heatbar. g) Hierarchically clustered heatmap of differentially expressed genes (DEGs) from pb sc-RNA-seq data. Top color bar indicates normalized z-scores for each DEG. Each module is ordered by expression (high to low) in GFP as shown by the bottom heatbar. h) Curated upregulated pathways determined by QIAGEN Ingenuity Pathway Analysis. Heatbar of -log adjusted p-value calculated by Fisher's exact test, i, Heatmap showing chromVAR bias-corrected ATAC-seq deviations of the top 150 most variable TF motifs across all constructs. Clustered using k-means unsupervised analysis. Color bar indicates normalized z-scores for each motif, j) Chromatin accessibility tracks for selected loci along with normalized pb sc-RNA-seq expression. Average of all human donors (n = 3-5).

FIG. 14A-14E: A) Schematic of cloning. B) Long-read sequencing spanning the TF and barcode to assess the fidelity of barcode-linkage. C) Whole plasmid sequencing of individual clones to assess the fidelity of barcode-linkage. D) Flow cytometry of edited cell frequency in three human donors. E) Histograms of library distribution in edited T cells from three human donors by barcode sequencing. Representation inset.

FIG. 15A-15E: A) Composition of pilot library. B) Schematic of pooled screening platform including non-viral integration of TF library, amplification of TF barcodes for NGS on Illumina platforms and the CAR T cell antigen-specific chronic stimulation assay. Coverage of the TF library throughout the experiment is noted. C) Volcano plot of acute stimulation screen. D) Volcano plot of chronic stimulation screen. E) Scatter plot comparing LOG2FC in acute and chronic stimulation screens.

FIG. 16A-16C: A) Volcano plot of acute stimulation screen with comprehensive library. B) LOG2FC individual human donors for selected TFs and controls. Each vertical bar represents one human donor (n = 3). C) Scatter plot comparing LOG2FC in acute and chronic stimulation screens.

FIG. 17A-17C: A) Alignment of natural AP-1 TF domain amino acid sequences using COBALT. B) LOG2FC for select bZIP domains where each cell represents a unique N+C domain combination. Heatbars report expression of the corresponding domain in endogenous T cells, binned by quartile. C) Each three-domain DESynR AP-1 TF was re-classified as three separate domain-pair architectures (N+bZIP, bZIP+C,N+C). Volcano plots of each domain-pair architecture. Log₂FC calculations were performed with DESeq2. Significance calculated using Wald test and Corrected for multiple comparisons using the Benjamini-Hochberg method.

FIG. 18A-18D: A) Schematic of mini pool functional screening. B) Volcano plots of each individual screen. Log₂FC calculations were performed with DESeq2. Significance calculated using Wald test and Corrected for multiple comparisons using the Benjamini-Hochberg method. Horizontal dashed line shows p = 0.05. C) Bar plot of Log₂FC in “Spleen”. Scaled to “GFP” (black). Each overlaid point represents one mouse (n = 3). D) Scatter plot correlating LOG2FC in two representative human donors across all screens. R² calculated from a y ~ x linear regression.

FIG. 19A-19I: A) Proliferation in arrayed chronic stimulation assay. Left, line graph of mean count +/- sem (n = 2-6 human donors). Right, bar plot of average cumulative count following six stimulations (n = 2-6 human donors). B) Exemplar flow cytometry from one representative human donor of degranulation and cytokine secretion following acute stimulation. C) Exemplar flow cytometry from one representative human donor of degranulation and cytokine secretion following chronic stimulation. D) Exemplar flow cytometry from one representative human donor of key surface proteins at baseline (no stimulation). E) Exemplar flow cytometry from one representative human donor of key surface proteins following chronic stimulation. F) Bar plots of average expression of key surface proteins following chronic stimulation. Overlaid points represent average of technical replicates for one human donor (n = 2-6). Left, raw percent positive for indicated markers (see gating top row in “e”). Right, relative to GFP. G) Bar plots of average expression of key surface proteins at baseline. Overlaid points represent average of technical replicates for one human donor (n = 2-3). H) Bar plots of average cytotoxicity relative to GFP in acutely challenged CAR T cells. Overlaid points represent average of technical replicates or triplicates for one human donor (n = 3). E:T’s left to right: 1 :2, 1 :4, 1 :16. I) Cytotoxicity relative to GFP in a terminal challenge following chronic stimulation. Overlaid points represent average of technical replicates or triplicates for one human donor (n = 2-6). E:T’s left to right: 1 :6, 1 :8, 1 :12.

FIG. 20A-20D: A) Schematic of murine xenograft tumor model. B) Kaplan-Meier curve of survival. C) Line graph of bioluminescence imaging of tumor burden. D) Bioluminescence imaging.

FIG. 21A-21 L: A) Overview of sample processing for sc-RNA-seq and bulk ATAC-seq experiments. B) sc-RNA-seq quality control metrics by human donor and construct. C) Uniform manifold approximation and projection (UMAP) colored by donor to show integration of data. D) UMAP colored by construct. E) UMAPs with red dots indicating cells of selected constructs with all other cells as gray dots. F) Detection of mRNA transcripts of TFs. G) Barplot of differentially expressed genes (DEGs) from sc-RNA-seq by human donor and construct. H) Hierarchically clustered heatmap of Jaccard similarity index of constructs and human donors. I) Violin plots of exemplar marker genes by construct and human donor. J) Dot plot percent of cells expressing and scaled average expression for select marker genes by construct. K) Dot plot percent of cells expressing and scaled average expression for select marker genes by manually annotated clusters. L) LOG2FC of occupancy in selected clusters for each construct split by donor.

FIG. 22A-22E: A) Principal component analysis (PCA) of pseudo-bulked RNA-seq data. Top, colored by T cell subset (CD4+ vs. CD8+) and unique shape for each human donor (n = 5). Bottom, colored by construct and unique shape for each human donor (n = 5). B) Scatter plot of correlating LOG2FC of DEGs in CD4+ versus CD8+ for six of eight constructs. R² calculated from a y ~ x linear regression. P-value from Pearson’s correlation. C) Gene mobility analysis. Line graphs connecting points reporting percentage of genes in the bottom 40% of expression in GFP that are in varying percentiles of expression in each construct. D) Permutation test results of the enrichment of the nearest genes of ATAC peaks group that overlap with corresponding DEG group or with random background gene set. E) Rank aggregation plots of TF motifs in ATAC-seq data split by groups determined by hierarchical clustering.

FIG. 23A-23B: A) Chromatin accessibility tracks for selected loci along with normalized pb sc-RNA-seq expression. Average of all human donors (n = 3-5). B) Ingenuity pathway analysis from DEGs for each group determined by hierarchical clustering. Top 10 pathways shown.

DETAILED DESCRIPTION

Before the chimeric polypeptides, compositions and methods of the present disclosure are described in greater detail, it is to be understood that the chimeric polypeptides, compositions and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the chimeric polypeptides, compositions and methods will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the chimeric polypeptides, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the chimeric polypeptides, compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the chimeric polypeptides, compositions and methods.

Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the chimeric polypeptides, compositions and methods belong. Although any chimeric polypeptides, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the chimeric polypeptides, compositions and methods, representative illustrative chimeric polypeptides, compositions and methods are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present chimeric polypeptides, compositions and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the chimeric polypeptides, compositions and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the chimeric polypeptides, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present chimeric polypeptides, compositions and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

CHIMERIC AP-1 TRANSCRIPTION FACTOR POLYPEPTIDES

Aspects of the present disclosure include chimeric AP-1 transcription factor polypeptides. Currently, genetic manipulation of adoptive cell therapies, including gene deletion or knockdown and overexpression, is largely restricted to the endogenous genome. As such, resultant phenotypes predictably exist along the known continuum of canonical T cell differentiation and are commonly described as more “stem-like” or “effector-like”. These cells, however, tend to succumb to similar dysfunctional fates as their unmanipulated counterparts. An approach to this problem, then, is to deliberately engineer cells into a synthetic state where cells do not differentiate into a dysfunctional state in the face of chronic stimulation, but rather persist and remain cytotoxic. T cell states are commonly defined by hallmark patterns of transcription factor (TF) expression. These proteins regulate the expression of genes by binding promoter or enhancer elements and thus work to maintain particular cell states. The chimeric AP-1 transcription factor polypeptides of the present disclosure are based in part on the rationale that installing synthetic transcription factors into T cells would allow unprecedented control over T cell fate and uncover favorable, unevolved T cell phenotypes. The present engineering effort focused on the AP-1 family of transcription factors, which are broadly implicated across T cell differentiation. Prior work of dysfunctional T cells has shown that these factors and the DNA motifs that they bind are highly expressed and accessible, respectively. AP-1 TFs contain a “bZIP” domain that consists of a basic DNA binding domain and a leucine zipper, which enables dimerization (i.e., operate as homo- and/or heterodimers). The bZIP domain is flanked by modestly characterized effector domains, which vary across individual TFs. Moreover, the larger AP-1 family is divided into four subfamilies, JUN, FOS, ATF and MAF, whose constituents share extensive bZIP homology. In sum, an AP-1 TF consists of (from N- to C-terminus) an N-terminal effector domain, bZIP domain and C-terminal effector domain (FIG. 1 A). As proof of concept, the inventors have produced and identified a subset of chimeric AP-1 transcription factors that significantly enhance T cell function relative to T cells expressing native, wild-type AP-1 transcription factors. These chimeric AP-1 TFs represent a novel class of synthetic sequences conferring enhanced cellular function and, with the benefit of the present disclosure, are expected to have broad applications across cellular engineering and cellular therapeutic applications. Details regarding chimeric polypeptides according to embodiments of the present disclosure will now be described.

The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof. As used herein, the term “chimeric” refers to a polypeptide composed of domains of different origins. A chimeric molecule, as a whole, is non-naturally occurring, e.g., synthetic or recombinant, although the domains which comprise the chimeric molecule can be naturally occurring.

In some aspects, provided are chimeric polypeptides comprising an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor. The first AP-1 transcription factor may be a member of the FOS, ATF, JUN or MAF family of AP-1 transcription factors. FOS family members include c-FOS, FOSB, FOSL1 , FRA-1 and FRA-2. ATF family members include ATF-2, ATF-3, ATF-4, ATF-5, ATF-6, ATF-6B, ATF-7, BATF, BATF- 2, BATF-3 and JDP2. JUN family members include c-JUN, JUNB and JUND. MAF family members include c-MAF, MAFA, MAFB, MAFF, MAFG and MAFK. The second AP-1 transcription factor is a different AP-1 transcription factor from the first AP-1 transcription factor and may be a member of the FOS, ATF, JUN or MAF family of AP-1 transcription factors.

In some instances, when the chimeric polypeptide comprises an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor, the first AP-1 transcription factor is a member of the FOS family of AP-1 transcription factors. By way of example, the first AP-1 transcription factor may be FOSL1 .

In certain embodiments, when the chimeric polypeptide comprises an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor, the first AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors. In one non-limiting example, the first AP-1 transcription factor may be BATF3.

According to some embodiments, when the chimeric polypeptide comprises an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor, the first AP-1 transcription factor is a member of the MAF family of AP-1 transcription factors. By way of example, the first AP-1 transcription factor may be MAFF.

In some instances, when the chimeric polypeptide comprises an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor, the second AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors. In some such instances, the second AP-1 transcription factor is BATF, ATF3, BATF2, or JDP2.

In certain embodiments, when the chimeric polypeptide comprises an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor, the chimeric polypeptide further comprises a C-terminal domain of an AP-1 transcription factor. The C-terminal domain may be from an AP-1 transcription factor of the FOS, ATF, JUN or MAF family of AP-1 transcription factors. Non-limiting examples of such C-terminal domains include a FOSL1 C-terminal domain, a FOSL2 C-terminal domain, a BATF C-terminal domain, a BATF2 C-terminal domain, a c-Jun C-terminal domain, a MAFA C-terminal domain, and a MAFF C-terminal domain.

In some instances, when the chimeric polypeptide comprises an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor, the polypeptide comprises an N-terminal domain of an AP-1 transcription factor of the FOS family and a bZIP domain of an AP-1 transcription factor of the ATF family. In one non-limiting example, such a chimeric polypeptide comprises a FOSL1 N-terminal domain and a BATF bZIP domain. Such a chimeric polypeptide may further comprise a C-terminal domain of an AP-1 transcription factor of the FOS, ATF, JUN or MAF family of AP-1 transcription factors, e.g., a FOS, ATF or JUN family member. Accordingly, chimeric polypeptides of the present disclosure that comprise an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor, and wherein the chimeric polypeptides further comprise a C-terminal domain of an AP-1 transcription factor, include but are not limited to chimeric polypeptides comprising: a FOSL1 N-terminal domain, a BATF bZIP domain, and a BATF C-terminal domain; a FOSL1 N- terminal domain, a BATF bZIP domain, and a c-JUN C-terminal domain; and a FOSL1 N-terminal domain, a BATF bZIP domain, and a FOSL1 C-terminal domain.

In some aspects, provided are chimeric polypeptides comprising a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor. The first AP-1 transcription factor may be a member of the FOS, ATF, JUN or MAF family of AP-1 transcription factors. The second AP-1 transcription factor is a different AP-1 transcription factor from the first AP-1 transcription factor and may be a member of the FOS, ATF, JUN or MAF family of AP-1 transcription factors.

In some instances, when the chimeric polypeptide comprises a bZIP domain of a first AP- 1 transcription factor and a C-terminal domain of a second AP-1 transcription factor, the first AP- 1 transcription factor is a member of the ATF family of AP-1 transcription factors. By way of example, the first AP-1 transcription factor may be BATF, BATF2 or ATF3.

According to some embodiments, when the chimeric polypeptide comprises a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor, the second AP-1 transcription factor is a member of the JUN family of AP-1 transcription factors. In one non-limiting example, the second AP-1 transcription factor may be c-JUN. In other embodiments, when the chimeric polypeptide comprises a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor, the second AP-1 transcription factor is a member of the FOS family of AP-1 transcription factors. By way of example, the second AP-1 transcription factor may be FOSL1 .

In certain embodiments, when the chimeric polypeptide comprises a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor, the second AP-1 transcription factor is a member of the MAF family of AP-1 transcription factors. Non-limiting examples of such family members include MAFA and MAFF.

In some instances, when the chimeric polypeptide comprises a bZIP domain of a first AP- 1 transcription factor and a C-terminal domain of a second AP-1 transcription factor, the chimeric polypeptide further comprises an N-terminal domain of an AP-1 transcription factor. The N- terminal domain may be from an AP-1 transcription factor of the FOS, ATF, JUN or MAF family of AP-1 transcription factors. Non-limiting examples of such N-terminal domains include FOSL1 , BATF3, MAF, and MAFF.

According to some embodiments, when the chimeric polypeptide comprises a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor, the polypeptide comprises a bZIP domain of an AP-1 transcription factor of the ATF family and a C-terminal domain of an AP-1 transcription factor of the FOS, ATF or JUN family. Such a chimeric polypeptide may further comprise an N-terminal domain of an AP-1 transcription factor of the FOS, ATF, JUN or MAF family of AP-1 transcription factors, e.g., a FOS family member. Accordingly, chimeric polypeptides of the present disclosure that comprise bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor, and wherein the chimeric polypeptides further comprise an N-terminal domain of an AP-1 transcription factor, include but are not limited to chimeric polypeptides comprising: a FOSL1 N-terminal domain, a BATF bZIP domain, and a BATF C-terminal domain; a FOSL1 N-terminal domain, a BATF bZIP domain, and a c-JUN C-terminal domain; and a FOSL1 N-terminal domain, a BATF bZIP domain, and a FOSL1 C-terminal domain.

In certain embodiments, provided is a chimeric polypeptide comprising: a BATF3 N- terminal domain, a JDP2 bZIP domain, and a BATF2 C-terminal domain. In some instances, provided is a chimeric polypeptide comprising a MAFF N-terminal domain, an ATF3 bZIP domain, and a BATF2 C-terminal domain. According to some embodiments, provided is a chimeric polypeptide comprising a BATF3 N-terminal domain, a BATF2 bZIP domain, and a MAFF C- terminal domain. In certain embodiments, provided is a chimeric polypeptide comprising a FOSL1 N-terminal domain, a BATF bZIP domain, and a MAFA C-terminal domain. In some instances, provided is a chimeric polypeptide comprising a c-Jun N-terminal domain, a cFOS bZIP domain, and a BATF C-terminal domain. According to some embodiments, provided is a chimeric polypeptide comprising a FOSL1 N-terminal domain, a BATF bZIP domain, and a BATF C- terminal domain. In certain embodiments, provided is a chimeric polypeptide comprising a FOSL1 N-terminal domain, a BATF bZIP domain, and a c-JUN C-terminal domain. In some instances, provided is a chimeric polypeptide comprising a FOSL1 N-terminal domain, a BATF bZIP domain, and a FOSL1 C-terminal domain. According to some embodiments, provided is a chimeric polypeptide comprising a FOSL1 N-terminal domain, a BATF bZIP domain, and a FOSL2 C- terminal domain. In certain embodiments, provided is a chimeric polypeptide comprising a FOSL1 N-terminal domain, a cFOS bZIP domain, and a BATF C-terminal domain. In some instances, provided is a chimeric polypeptide comprising a FOSL1 N-terminal domain, a cFOS bZIP domain, and a FOSL1 C-terminal domain. According to some embodiments, provided is a chimeric polypeptide comprising a MAFF N-terminal domain, a cFOS bZIP domain, and a BATF C-terminal domain.

The amino acid sequences of the AP-1 transcription factors referenced herein are known for various mammals. For example, the amino acid sequences of mouse and human AP-1 transcription factors are known and readily available from resources such as UniProt, the National Center for Biotechnology Information (NCBI) (e.g., RefSeq), and the like. In addition, the amino acid sequences of the N-terminal, bZIP and C-terminal domains of various human AP-1 transcription factors are provided in Table 1 below. Table 1 - Amino Acid Sequences of Human AP-1 Transcription Factor Domains

As will be appreciated, the N-terminal, bZIP and/or C-terminal domains of a chimeric polypeptide of the present disclosure may comprise the wild-type (e.g., wild-type human) amino acid sequences of their respective AP-1 transcription factor, or alternatively, one or more of the N-terminal, bZIP and C-terminal domains may comprise a variant amino acid sequence relative to the wild-type domain sequence. When one or more of the N-terminal, bZIP and C-terminal domains comprise a variant amino acid sequence, the amino acid sequence may comprise 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the corresponding wild-type domain. A chimeric polypeptide that comprises one, two or three variant domains will retain AP-1 transcription factor activity. When a population of T cells expresses a chimeric polypeptide comprising one or more variant AP-1 transcription factor domains, in some instances, the T cell population exhibits at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of a desirable property exhibited by a T cell population expressing a chimeric polypeptide comprising the same N-terminal, bZIP and C-terminal domains, but where the N-terminal, bZIP and C-terminal domains comprise the wild-type domain sequences. Nonlimiting examples of desirable properties include enrichment in an in vitro repetitive stimulation assay (see, e.g., Example 2 below), reduced exhaustion as determined in vitro or in vivo as compared to control T cells not expressing the chimeric AP-1 transcription factor polypeptide, enhanced efficacy (e.g., enhanced anti-tumor and/or anti-viral infection efficacy) as determined in vitro or in vivo as compared to control T cells not expressing the chimeric AP-1 transcription factor polypeptide, and/or the like.

In certain embodiments, a variant AP-1 transcription factor domain comprises an N- terminal, bZIP and/or C-terminal domain comprising one or more amino acid substitutions, deletions, or truncations (e.g., a truncation at the N-terminus and/or C-terminus of the domain). Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." More substantial changes are provided in Table 2 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into a polypeptide and the products screened for a desired activity, e.g., retained or improved resistance to cell exhaustion when expressed in therapeutic cells (e.g., CAR T cells, TILs, or the like), retained or improved anti-tumor and/or anti-viral infection efficacy when expressed in therapeutic cells (e.g., CAR T cells, TILs, or the like), and/or the like.

Table 2 - Amino Acid Substitutions

Amino acids may be grouped according to common side-chain properties:

(1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. NUCLEIC ACIDS, EXPRESSION CONSTRUCTS, CELLS AND COMPOSITIONS

Nucleic Acids and Expression Constructs

Aspects of the present disclosure further include nucleic acids and expression constructs. For example, provided are nucleic acids encoding any of the chimeric polypeptides of the present disclosure. Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a chimeric polypeptide of interest provides a description of all the polynucleotides capable of encoding the chimeric polypeptide of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the polypeptides and domains disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the polypeptide of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences of AP-1 transcription factor domains set forth in Table 1 . Non-limiting examples of nucleotide sequences encoding the AP-1 transcription factor domains set forth in Table 1 are provided in Table 3 below.

Table 3 - Nucleotide Sequences Encoding Human AP-1 Transcription Factor Domains

The nucleotide sequences of the nucleic acids of the present disclosure may be codon- optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding a polypeptide to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding a polypeptide may be codon- optimized for optimal production from the host organism selected for expression, e.g., human cells, such as human immune cells (e.g., human T cells).

Also provided are expression constructs comprising any of the nucleic acids of the present disclosure. As used herein, an “expression construct” is a circular or linear polynucleotide (a polymer composed of naturally occurring and/or non-naturally occurring nucleotides) comprising a region that encodes a chimeric polypeptide of the present disclosure, operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the polypeptide is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoter, enhancer, etc., present in the expression construct. In some embodiments, expression of the polypeptide may be controlled by one or more endogenous regulatory elements, e.g., promoter, enhancer, etc., at or near a genomic locus into which the expression construct is inserted or knocked in. In some instances, the one or more endogenous regulatory elements are of the T-cell receptor a constant (TRAC) locus. For example, the nucleic acid encoding the chimeric polypeptide may be knocked into TRAC locus, e.g., integrated inframe at exon 1 of the TRAC locus as described, e.g., by Eyquem et al. (2017) Nature 543:1 13- 117 and Roth et al. (2018) Nature 559(7714) :405-409.

The expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression constructs may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the first subunit, the second subunit, or both. The expression constructs optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

To obtain high levels of expression of a cloned nucleic acid it is common to construct expression constructs which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli 's also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the polypeptide are available using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used. Transducing cells with nucleic acids (e.g., expression constructs) can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector.

In certain embodiments, upon delivery of an expression construct to cells, one or more of the expression constructs are episomal (e.g., extra-chromosomal), where by “episome” or “episomal” is meant a polynucleotide that replicates independently of the cell’s chromosomal DNA. A non-limiting example of an episome that may be employed is a plasmid.

According to some embodiments, upon delivery of an expression construct to cells, the expression construct integrates (e.g., by insertion or knock-in) into the genome of the cell. In certain embodiments, the expression construct is adapted for site-specific integration into the genome. For example, an expression construct may be adapted for site-specific integration into the genome, where the site-specific integration inactivates a gene within the genome of the cell. Functional integration of an expression construct may be achieved through various means, including through the use of integrating vectors, including viral and non-viral vectors. In some instances, a retroviral vector, e.g., a lentiviral vector, may be employed. In some instances, a non-retroviral integrating vector may be employed. An integrating vector may be contacted with the cells in a suitable transduction medium, at a suitable concentration (or multiplicity of infection), and for a suitable time for the vector to infect the target cells, facilitating functional integration of the expression construct. Non-limiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated virus (AAV) vectors, hybrid Ad-AAV vector systems, and the like.

In certain embodiments, an expression construct of the present disclosure comprises a ribosome skipping site to allow for polycistronic expression of the chimeric AP-1 transcription factor polypeptide and one or more additional polypeptides, e.g., a recombinant receptor (e.g., a CAR) as described elsewhere herein. A non-limiting example of a suitable ribosome skipping site which may be incorporated into an expression construct is the P2A ribosome skipping site from porcine teschovirus.

Also provided by the present disclosure are messenger RNAs (mRNAs) encoding any of the polypeptides of the present disclosure. In some instances, the mRNAs are encapsulated within particles, e.g., lipid nanoparticles. Such mRNAs and particles find use, e.g., for expression of a polypeptide in vivo upon delivery of the mRNA to cells of a subject. Strategies for delivering mRNA-based therapeutics are described, e.g., in Kowalski et al. (2019) Mol Then 27(4)710-728; and Hou et al. (2021) Nature Reviews Materials 6:1078-1094.

Cells and Compositions

Aspects of the present disclosure further include cells comprising a nucleic acid of the present disclosure, as well as cells comprising an expression construct of the present disclosure. In certain embodiments, the cells are prokaryotic cells (e.g., bacteria), a yeast cells, insect (e.g, drosophila) cells, amphibian (e.g., frog, e.g., Xenopus) cells, plant cells, etc. According to some embodiments, the cells are mammalian cells. Mammalian cells of interest include human cells, rodent cells, and the like.

In some embodiments, the cells are immune cells. Non-limiting examples of immune cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the cells are T cells. When the immune cells comprise T cells, the T cells may comprise one or any combination of naive T cells (TN), cytotoxic T cells (T_CTL), memory T cells (TMEM), T memory stem cells (TSCM), central memory T cells (T_CM), effector memory T cells (TEM), tissue resident memory T cells (TRM), effector T cells (TEFF), regulatory T cells (TREGS), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha beta T cells (T_ap), gamma delta T cells (T_vo).

Approaches for introducing the nucleic acid or expression construct into cells of interest are known and may include contacting a population of cells with the nucleic acid or expression construct under conditions in which the nucleic acid or expression construct is delivered to cells of the population of cells. The contacting step may comprise contacting the population of cells with the nucleic acid or expression construct, e.g., by combining the cells and the nucleic acid or expression construct in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of the nucleic acid or expression construct into cells of the population of cells.

A variety of suitable approaches and conditions for the delivery of nucleic acids and expression constructs to cells are known. According to some embodiments, delivery is carried out by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, and/or the like. In certain embodiments, the nucleic acid or expression construct is introduced into cells of the population of cells by AAV transduction. The AAV vector may comprise ITRs from AAV2, and a serotype from any one of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10. According to some embodiments, the AAV vector comprises ITRs from AAV2 and a serotype from AAV6. In certain embodiments, the nucleic acid or expression construct is introduced into the cells (e.g., T cells) by lentiviral or retroviral transduction. The lentiviral vector backbone may be derived from HIV-1 , HIV-2, visna- maedi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (Bl V), or simian immunodeficiency virus (SIV). The lentiviral vector may be integration competent or an integrase deficient lentiviral vector (TDLV). In one embodiment, IDLV vectors including an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) are employed. In certain embodiments, a cell of the present disclosure expresses the chimeric polypeptide.

According to some embodiments, a cell of the present disclosure expresses the chimeric polypeptide and also expresses a recombinant receptor on the surface of the cell. Non-limiting examples of such recombinant receptors include a chimeric antigen receptor (CAR), a recombinant T cell receptor (TOR), a synthetic notch receptor (synNotch), a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.

As used herein, with respect to a protein, the term “recombinant” means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein, and cells or organisms that express the protein. With respect to a nucleic acid, the term “recombinant” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant.

In certain embodiments, the recombinant receptor (e.g., a CAR) comprises an extracellular binding domain that binds a tumor antigen expressed on the surface of a cancer cell. Non-limiting examples of such tumor antigens include 5T4, AXL receptor tyrosine kinase (AXL), B7-H3, B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD44, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS- like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1 ), GD2 ganglioside (“GD2”), glycoprotein non-metastatic B (GPNMB), guanylate cyclase 2 C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal- associated membrane protein 1 (LAMP-1 ), Lewis Y, LIV-1 , leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1 ), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), programmed cell death receptor ligand 1 (PD-L1 ), programmed cell death receptor ligand 2 (PD- L2), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1 ), tissue factor (TF), T cell immunoglobulin and mucin protein-1 (TIM-1 ), Tn antigen, trophoblast cell-surface antigen (TROP-2), Wilms’ tumor 1 (WT1 ), and VEGF-A.

As described above, according to some embodiments, the engineered receptor is a CAR. The extracellular binding domain of the CAR may comprise a single chain antibody. The singlechain antibody may be a monoclonal single-chain antibody, a chimeric single-chain antibody, a humanized single-chain antibody, a fully human single-chain antibody, and/or the like. In one non-limiting example, the single chain antibody is a single chain variable fragment (scFv). In some embodiments, the extracellular binding domain of the CAR is a single-chain version (e.g., an scFv version) of an antibody approved by the United States Food and Drug Administration and/or the European Medicines Agency (EMA) for use as a therapeutic antibody. Non-limiting examples of single-chain antibodies which may be employed when the protein of interest is a CAR include single-chain versions (e.g., scFv versions) of Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab,

Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab,

Panitumumab, Patritumab, Pritumumab, Radretumab, Ramucirumab, Rilotumumab,

Robatumumab, Seribantumab, Tarextumab, Teprotumumab, Tovetumab, Vantictumab,

Vesencumab, Votumumab, Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab, Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Cantuzumab, Citatuzumab, Clivatuzumab, Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or an antigenbinding variant thereof.

When the cells are engineered to express a recombinant receptor on the surface thereof, the receptor may include one or more linker sequences between the various domains. A “variable region linking sequence” is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that includes the same light and heavy chain variable regions. A non-limiting example of a variable region linking sequence is a glycine-serine linker, such as a (648)3 linker as described above. In certain embodiments, a linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and/or primary signaling domains. In particular embodiments, the receptor (e.g., CAR) includes one, two, three, four, or five or more linkers. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more amino acids in length.

In some embodiments, when the cells are engineered to express a recombinant receptor on the surface thereof, the antigen binding domain of the receptor (e.g., CAR) is followed by one or more spacer domains that moves the antigen binding domain away from the cell surface (e.g., the surface of a T cell (e.g., a CD8+ or CD4+ T cell) expressing the receptor) to enable proper cell/cell contact, antigen binding and/or activation. The spacer domain (and any other spacer domains, linkers, and/or the like described herein) may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In some embodiments, the spacer domain includes the CH2 and/or CH3 of lgG1 , lgG4, or IgD. Illustrative spacer domains suitable for use in the receptors (e.g., CARs) described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD4, which may be wild-type hinge regions from these molecules or variants thereof. In certain embodiments, the hinge domain includes a CD8a hinge region. According to some embodiments, the hinge is a PD-1 hinge or CD152 hinge. In certain embodiments, the hinge is an lgG4 hinge.

The “transmembrane domain” (Tm domain) is the portion of the receptor (e.g., CAR) that fuses the extracellular binding portion and intracellular signaling domain and anchors the receptor to the plasma membrane of the cell (e.g., T-cell, such as a Treg). The Tm domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the Tm domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell receptor, CD35, CD3^, CD3y, CD36, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1.

In one embodiment, a receptor (e.g., CAR) includes a Tm domain derived from CD28. In certain embodiments, a receptor includes a Tm domain derived from CD28 and a short oligo- or polypeptide linker, e.g., between 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the Tm domain and the intracellular signaling domain of the receptor. A glycine-serine linker may be employed as such a linker, for example.

The “intracellular signaling” domain of a receptor (e.g., a CAR) refers to the part of the receptor that participates in transducing the signal from binding to a target molecule/antigen into the interior of the cell to elicit cell function. Accordingly, the term “intracellular signaling domain” refers to the portion of a protein which transduces the signal and that directs the cell to perform a specialized function. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of a full-length intracellular signaling domain as long as it transduces the signal. The term intracellular signaling domain is meant to include any truncated portion of an intracellular signaling domain sufficient for transducing signal.

Signals generated through the T cell receptor (TCR) alone are insufficient for full activation of the T cell, and a secondary or costimulatory signal is also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex) and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal. As such, a receptor (e.g., CAR) expressed by a genetically modified cell may include an intracellular signaling domain that includes one or more (e.g., 1 , 2, or more) “costimulatory signaling domains” and a “primary signaling domain.”

Primary signaling domains regulate primary activation of the TCR complex either in a stimulatory manner, or in an inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”). Non-limiting examples of ITAM-containing primary signaling domains suitable for use in a receptor of the present disclosure include those derived from FcRy, FcRp, CD3y, CD35, CD3s, CD3^, CD22, CD79a, CD79P, and CD665. In certain embodiments, a receptor includes a CD3^ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.

In some embodiments, when the cells of the present disclosure are engineered to express a recombinant receptor on the surface thereof, the receptor (e.g., CAR) includes one or more costimulatory signaling domains to enhance the efficacy and expansion of immune effector cells (e.g., T cells) expressing the receptor. As used herein, the term “costimulatory signaling domain” or “costimulatory domain” refers to an intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Example costimulatory molecules suitable for use in receptors contemplated in particular embodiments include TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1 BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70. In some embodiments, the receptor (e.g., CAR) includes one or more costimulatory signaling domains selected from the group consisting of 4-1 BB (CD137), CD28, and CD134, and a CD3^ primary signaling domain.

A receptor (e.g., CAR) may include any variety of suitable domains including but not limited to a leader sequence; hinge, spacer and/or linker domain(s); transmembrane domain(s); costimulatory domain(s); signaling domain(s) (e.g., CD3 domain(s)); ribosomal skip element(s); restriction enzyme sequence(s); reporter protein domains; and/or the like.

Also provided by the present disclosure are compositions comprising a population of the therapeutic immune cells of the present disclosure, e.g., any of the therapeutic immune cells described elsewhere herein.

Compositions suitable for adoptive cell therapy may be manufactured by expanding the therapeutic immune cells of the present disclosure. By “expanding” is meant the cells are cultured under conditions in which the cells proliferate. Suitable conditions may vary depending upon, e.g., the type of cells being expanded. Such conditions may include culturing the cell in a suitable container (e.g., a cell culture plate or well thereof, a cassette, tube, bottle or bag suitable for use in an automated therapeutic cell manufacturing system, e.g., a closed automated therapeutic cell manufacturing system such as the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, or the like), in suitable medium (e.g., cell culture medium, such as RPMI, DMEM, IMDM, MEM, DMEM/F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO2, e.g., 3% to 10%, such as 5%.

Methods for activating and expanding cells for therapy (e.g., therapeutic T cells and the like) are known in the art and are described, e.g., in U.S. Patent Nos. 6,905,874; 6,867,041 ; and 6,797,514; and PCT Publication No. WO 2012/079000, the contents of which are hereby incorporated by reference in their entirety. In the example of T cells, such methods may include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC). One example is the Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. In other embodiments, the T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012/129514, the contents of which are hereby incorporated by reference in their entirety.

In certain embodiments, the cells are expanded using an automated system designed for the manufacture of therapeutic cells. Non-limiting examples of such systems include the CliniMACS Prodigy® system by Miltenyi Biotec, the Xuri® cell expansion system by Cytiva, the G-Rex® cell expansion system by Wilson Wolf, the Quantum® cell expansion system from Terumo, the Cocoon® system by Lonza, etc. Detailed guidance and protocols for manufacturing therapeutic cells on such systems are available from the providers of such systems.

Harvested therapeutic immune cell populations may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and/or device. Such populations of cells and pharmaceutical compositions may be prepared and/or used fresh or may be cryopreserved. In some instances, populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g, where the therapeutic cells are present in a suitable diluent and/or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media). Populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like.

In some instances, the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container. Cell therapy doses may be generated through a variety of methods. Aliquoting expanded populations of therapeutic cells into cell therapy doses may be performed by a variety of means. In some instances, a cell therapy dose includes, e.g., at least 10 million, at least 25 million, at least 50 million, at least 75 million, at least 100 million, at least 250 million, at least 500 million, at least 750 million, at least 1 billion, at least 2 billion, at least 3 billion, at least 4 billion, at least 5 billion, at least 6 billion, at least 7 billion, at least 8 billion, at least 9 billion, at least 10 billion, at least 15 billion, at least 20 billion, at least 30 billion, at least 40 billion, at least 50 billion, at least 60 billion, at least 70 billion, at least 80 billion, at least 90 billion, or at least 100 billion therapeutic cells. In certain embodiments, the compositions may include the therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCI₂, KCI, MgSO₄), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3- aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

The compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control. An effective amount can be administered in one or more administrations.

The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and/or the like.

Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, tricresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).

METHODS OF USE

Aspects of the present disclosure further include methods of using the cells of the present disclosure for therapy, e.g., to treat a cell proliferative disorder such as cancer, and/or to treat an infection such as a viral infection. In some instances, the cells are immune effector cells that express a chimeric AP-1 transcription factor polypeptide of the present disclosure. According to some embodiments, the cells further express a recombinant receptor (e.g., a CAR) on their surface as described elsewhere herein.

In certain embodiments, provided are methods of administering an adoptive cell therapy to a subject having cancer. Such methods comprise administering to the subject a composition of the present disclosure (e.g., a composition comprising any of the therapeutic immune cells of the present disclosure) in an amount effective to treat the cancer.

According to some embodiments, the adoptive cell therapy is an adoptive T cell therapy, e.g., a CAR T cell therapy, a TIL therapy, a natural killer T (NKT) cell therapy, a therapy comprising administration of T cells expressing an engineered TCR, or the like. In some instances, the adoptive cell therapy is a natural killer (NK) cell therapy (e.g., a CAR NK cell therapy) or a macrophage therapy.

The therapeutic cells may be autologous/autogeneic (“self”) or non-autologous (“nonself,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells (e.g., T cells) are cells obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human.

As summarized above, in some embodiments, the subject has cancer. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. In some instances, the cancer comprises a solid tumor. According to some embodiments, the solid tumor is a carcinoma, lymphoma, blastoma, or sarcoma. When the solid tumor is a carcinoma, in certain embodiments, the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma.

According to some embodiments, the cancer comprises a hematological malignancy. For example, the subject treated by the methods of the present disclosure may have a hematological malignancy such as a leukemia, a lymphoma, or multiple myeloma.

According to some embodiments, the cancer of the subject is myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), multiple myeloma (MM), Non-Hodgkin’s lymphoma (NHL), non-small cell lung cancer, head and neck squamous cell carcinoma, gastroesophageal junction adenocarcinoma, gastric adenocarcinoma, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, chronic lymphocytic lymphoma (CLL), B cell lymphoma, lung adenocarcinoma, osteosarcoma, ovarian cancer, or leiomyosarcoma.

Particular examples of cancers which the subject may have include renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactinsecreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, KRAS mutated colorectal cancer; colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgendependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer); renal carcinoma; Wilms' tumor; and bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In some embodiments, the cancer is myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinomas.

In certain embodiments, provided are methods of administering an adoptive cell therapy to a subject having an infectious disease. Such methods comprise administering to the subject a composition of the present disclosure (e.g., a composition comprising any of the therapeutic immune cells of the present disclosure) in an amount effective to treat the infectious disease. In some embodiments, the infectious disease is a viral infection. Non-limiting examples of viral infections treatable according to the methods of the present disclosure include Human Immunodeficiency Virus (e.g., in which the cells express a CAR targeting a CD4 binding site on gp-120), Hepatitis C Virus (e.g., in which the cells express a CAR targeting HCV E2 glycoprotein), Hepatitis B Virus (e.g., in which the cells express a CAR targeting S HBV surface protein or HBV surface antigen), and Human Cytomegalovirus infections (e.g., in which the cells express a CAR targeting Glycoprotein B or virally-encoded FcRs). In some instances, the infectious disease is an opportunistic fungal infection. In some embodiments, the opportunistic fungal infection is an invasive aspergillosis (e.g., Aspergillus fumigatus) infection, e.g., in which the cells express a CAR targeting p-glucan.

As demonstrated in the Experimental section below, the chimeric AP-1 transcription factor polypeptides of the present disclosure find use in significantly enhancing T cell function relative to T cells expressing native, wild-type AP-1 transcription factors. Accordingly, provided are methods in which the adoptive cell therapy exhibits: reduced (or prevented) T cell exhaustion as compared to an adoptive cell therapy employing T cells not expressing the chimeric AP-1 transcription factor polypeptide; enhanced efficacy (e.g., enhanced anti-tumor and/or anti-viral infection efficacy) as compared to an adoptive cell therapy employing T cells not expressing the chimeric AP-1 transcription factor polypeptide; and/or the like.

The adoptive cell therapy may be administered via a route of administration independently selected from parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, or epidural injection), oral, topical, or nasal administration.

An “amount effective to treat” a particular condition or a “therapeutically effective amount” may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a pharmaceutical composition of the present disclosure includes from 1x10⁶ to 5x10¹⁰ of the therapeutic immune cells of the present disclosure.

By treatment is meant at least an amelioration of one or more symptoms associated with the condition (e.g., cancer, infectious disease, or the like) of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the condition, or at least the symptoms that characterize the condition. With respect to cancer, in some embodiments, the treatment is effective to slow the growth of a tumor, reduce the size of a tumor, and/or the like. With respect to an infectious disease, in some embodiments, the treatment is effective to slow the growth of the underlying pathogen, reduce the number of cells infected by the pathogen, and/or the like.

For purposes of completeness, non-limiting aspects and embodiments of the present disclosure are further disclosed in the following numbered clauses.

1 . A chimeric polypeptide comprising: an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor.

2. The chimeric polypeptide of clause 1 , wherein the first AP-1 transcription factor is a member of the FOS family of AP-1 transcription factors.

3. The chimeric polypeptide of clause 2, wherein the first AP-1 transcription factor is FOSL1 .

4. The chimeric polypeptide of clause 1 , wherein the first AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors.

5. The chimeric polypeptide of clause 2, wherein the first AP-1 transcription factor is BATF3.

6. The chimeric polypeptide of clause 1 , wherein the first AP-1 transcription factor is a member of the MAF family of AP-1 transcription factors.

7. The chimeric polypeptide of clause 2, wherein the first AP-1 transcription factor is MAFF.

8. The chimeric polypeptide of any one of clauses 1 -7, wherein the second AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors.

9. The chimeric polypeptide of clause 8, wherein the second AP-1 transcription factor is BATF.

10. The chimeric polypeptide of clause 8, wherein the second AP-1 transcription factor is ATF3. 11. The chimeric polypeptide of clause 8, wherein the second AP-1 transcription factor is BATF2.

12. The chimeric polypeptide of any one of clauses 1 -7, wherein the second AP-1 transcription factor is a member of the JDP family of AP-1 transcription factors.

13. The chimeric polypeptide of clause 12, wherein the second AP-1 transcription factor is JDP2.

14. The chimeric polypeptide of any one of clauses 1-13, further comprising a C-terminal domain of an AP-1 transcription factor.

15. The chimeric polypeptide of clause 14, wherein the C-terminal domain is of an AP-1 transcription factor of the FOS, ATF or JUN family of AP-1 transcription factors.

16. The chimeric polypeptide of clause 15, wherein the C-terminal domain is a FOSL1 C- terminal domain.

17. The chimeric polypeptide of clause 15, wherein the C-terminal domain is a FOSL2 C- terminal domain.

18. The chimeric polypeptide of clause 15, wherein the C-terminal domain is a BATF C- terminal domain.

19. The chimeric polypeptide of clause 15, wherein the C-terminal domain is a BATF2 C- terminal domain.

20. The chimeric polypeptide of clause 15, wherein the C-terminal domain is a c-Jun C- terminal domain.

21 . The chimeric polypeptide of clause 14, wherein the C-terminal domain is of a member of the MAF family of AP-1 transcription factors.

22. The chimeric polypeptide of clause 21 , wherein the C-terminal domain is a MAFA C- terminal domain.

23. The chimeric polypeptide of clause 21 , wherein the C-terminal domain is a MAFF C- terminal domain.

24. A chimeric polypeptide comprising: a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor.

25. The chimeric polypeptide of clause 24, wherein the first AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors.

26. The chimeric polypeptide of clause 25, wherein the first AP-1 transcription factor is BATF.

27. The chimeric polypeptide of clause 25, wherein the first AP-1 transcription factor is BATF2.

28. The chimeric polypeptide of clause 25, wherein the first AP-1 transcription factor is ATF3. 29. The chimeric polypeptide of any one of clauses 24-28, wherein the second AP-1 transcription factor is a member of the JUN family of AP-1 transcription factors.

30. The chimeric polypeptide of clause 29, wherein the second AP-1 transcription factor is c- Jun.

31 . The chimeric polypeptide of any one of clauses 24-28, wherein the second AP-1 transcription factor is a member of the FOS family of AP-1 transcription factors.

32. The chimeric polypeptide of clause 31 , wherein the second AP-1 transcription factor is FOSL1 .

33. The chimeric polypeptide of any one of clauses 24-28, wherein the second AP-1 transcription factor is of a member of the MAF family of AP-1 transcription factors.

34. The chimeric polypeptide of clause 33, wherein the second AP-1 transcription factor is MAFA.

35. The chimeric polypeptide of clause 33, wherein the second AP-1 transcription factor is MAFF.

36. The chimeric polypeptide of any one of clauses 24-32, further comprising an N-terminal domain of an AP-1 transcription factor.

37. The chimeric polypeptide of clause 36, wherein the N-terminal domain is of an AP-1 transcription factor of the FOS family of AP-1 transcription factors.

38. The chimeric polypeptide of clause 36, wherein the N-terminal domain is a FOSL1 N- terminal domain.

39. The chimeric polypeptide of clause 36, wherein the N-terminal domain is of an AP-1 transcription factor of the FOS family of AP-1 transcription factors.

40. The chimeric polypeptide of clause 36, wherein the N-terminal domain is of an AP-1 transcription factor of the ATF family of AP-1 transcription factors.

41. The chimeric polypeptide of clause 40, wherein the N-terminal domain is a BATF3 N- terminal domain.

42. The chimeric polypeptide of clause 14, wherein the N-terminal domain is of a member of the MAF family of AP-1 transcription factors.

43a. The chimeric polypeptide of clause 21 , wherein the N-terminal domain is a MAFF N- terminal domain.

43b. The chimeric polypeptide of clause 1 , wherein the chimeric polypeptide comprises: a BATF3 N-terminal domain, a JDP2 bZIP domain, and a BATF2 C-terminal domain; a MAFF N-terminal domain, an ATF3 bZIP domain, and a BATF2 C-terminal domain; a BATF3 N-terminal domain, a BATF2 bZIP domain, and a MAFF C-terminal domain; a FOSL1 N-terminal domain, a BATF bZIP domain, and a MAFA C-terminal domain; a c-Jun N-terminal domain, a cFOS bZIP domain, and a BATF C-terminal domain; a FOSL1 N-terminal domain, a BATF bZIP domain, and a BATF C-terminal domain; a FOSL1 N-terminal domain, a BATF bZIP domain, and a c-JUN C-terminal domain; a FOSL1 N-terminal domain, a BATF bZIP domain, and a FOSL1 C-terminal domain; a FOSL1 N-terminal domain, a BATF bZIP domain, and a FOSL2 C-terminal domain; a FOSL1 N-terminal domain, a cFOS bZIP domain, and a BATF C-terminal domain; a FOSL1 N-terminal domain, a cFOS bZIP domain, and a FOSL1 C-terminal domain; or a MAFF N-terminal domain, a cFOS bZIP domain, and a BATF C-terminal domain;

44. A nucleic acid encoding the chimeric polypeptide of any one of clauses 1-43b.

45. A cell comprising the nucleic acid of clause 44, wherein the cell expresses the chimeric polypeptide.

46. The cell of clause 45, wherein the nucleic acid is operably linked to an endogenous promoter.

47. The cell of clause 46, wherein the endogenous promoter is of the T-cell receptor a constant (TRAC) locus.

48. The cell of clause 45, wherein the nucleic acid is operably linked to an exogenous promoter.

49. The cell of clause 48, wherein the exogenous promoter is a constitutive promoter.

50. The cell of clause 48, wherein the exogenous promoter is an inducible promoter.

51 . The cell of any one of clauses 45-50, wherein the cell is a human cell.

52. The cell of any one of clauses 45-51 , wherein the cell is an immune effector cell.

53. The cell of clause 52, wherein the immune effector cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a macrophage.

54. The cell of any one of clauses 45-53, wherein the cell comprises a nucleic acid encoding a recombinant receptor, wherein the cell expresses the recombinant receptor on its surface.

55. The cell of clause 54, wherein the recombinant receptor is a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), a synthetic notch (synNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.

56. The cell of clause 54, wherein the recombinant receptor is a CAR.

57. The cell of clause 52 or 53, wherein the cell does not comprise a nucleic acid encoding a recombinant receptor.

58. The cell of clause 57, wherein the cell is a tumor infiltrating lymphocyte (TIL).

59. A method of making the cell of one of clauses 45-58, comprising stably introducing into the cell the nucleic acid of clause 44. 60. A composition comprising a population of cells as defined in any one of clauses 45-58.

61 . The composition of clause 60, wherein the composition is formulated for administration to a subject in need thereof.

62. A method comprising administering an effective amount of the composition of clause 61 to a subject in need thereof, wherein the cells express the chimeric polypeptide.

63. The method according to clause 62, wherein the composition comprises a population of immune effector cells as defined in any one of clauses 52-58.

64. The method according to clause 63, wherein the chimeric polypeptide prevents or reduces exhaustion of the immune effector cells upon administration to the subject.

65. The method according to any one of clauses 62-64, wherein the cells express a recombinant receptor as defined in clause 55.

66. The method according to clause 65, wherein the recombinant receptor is a CAR.

67. The method according to clause 65 or 66, wherein the subject has cancer, and wherein the recombinant receptor binds to a tumor antigen present on cancer cells in the subject.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1 - Production of a Chimeric AP-1 Transcription Factor-Encoding T Cell Library

Described in this example is the production of a chimeric AP-1 transcription factorencoding T cell library. The chimeric AP-1 transcription factors expressed by cells of the library comprise an N-terminal domain, a bZIP domain, and a C-terminal domain, where at least two of the domains are from different AP-1 transcription factors. As illustrated in FIG. 1 B, 500 different AP-1 transcription factors were generated representing combinations of 10 different N-terminal domains, 5 different bZIP domains and 10 different C-terminal domains. The N-terminal domains were selected from those of c-JUN, JUNB, JUND, c-FOS, FOSL1 , ATF2, ATF4, BATF, c-MAF and MAFF. The bZIP domains were selected from those of c-JUN, c-FOS, BATF, ATF2 and c- MAF. The C-terminal domains were selected from those of c-JUN, c-FOS, FOSB, FOSL1 , FOSL2, ATF2, ATF4, BATF, c-MAF and MAFF.

DNA constructs encoding the AP-1 transcription factors were generated using a three- round cloning approach as schematically illustrated in FIG. 2. As shown, the starting construct encodes an N-terminal domain and comprises a barcode (“N”) uniquely identifying the N-terminal domain as a c-JUN, JUNB, JUND, c-FOS, FOSL1 , ATF2, ATF4, BATF, c-MAF or MAFF N- terminal domain. Next, a “stuffer” region disposed between the N-terminal domain and the barcode was excised and replaced via ligation with a construct encoding a bZIP domain and comprising a barcode (“BZIP”) uniquely identifying the bZIP domain as a c-JUN, c-FOS, BATF, ATF2 or c-MAF bZIP domain. Next, a “stutter” region disposed between the bZIP domain and the bZIP barcode was excised and replaced via ligation with a construct encoding a C-terminal domain and comprising a barcode (“C”) uniquely identifying the C-terminal domain as a c-JUN, c-FOS, FOSB, FOSL1 , FOSL2, ATF2, ATF4, BATF, c-MAF or MAFF C-terminal domain. The result is a construct encoding an N-terminal domain, a bZIP domain and a C-terminal domain, and comprising an amplifiable combination of three barcodes each uniquely identifying the particular N-terminal domain, a bZIP domain and a C-terminal domain, respectively, encoded by the construct.

T cells expressing Kymriah and a chimeric AP-1 transcription factor were generated using a targeted knock-in approach as schematically illustrated in FIG. 3. A plasmid encoding polycistronic expression of a CD19-targeting CAR (Kymriah) and a barcoded chimeric AP-1 transcription factor, flanked by sequence homologous to the TCR-a constant region (TRAC) locus, was introduced into primary human T cells by electroporation, along with a TRAC-specific Cas9 RNP. Following Cas9 RNP-mediated double-strand break formation at the 5’ end of TRAC Exon 1 , endogenous homology-directed repair machinery enables transgene integration inframe. Thus, the endogenous TCR-a promoter drives expression of the transgene, but endogenous TCR expression is disrupted. The TRAC locus knock-in strategy was generally as described in Eyquem et al. (2017) Nature 543:1 13-1 17 and Roth et al. (2018) Nature 559(7714) :405-409.

Example 2 -Identification of Chimeric AP-1 Transcription Factors Conferring Enhanced T Cell Function

The T cell library generated as described in Example 1 was employed in an in vitro repetitive stimulation assay (schematically illustrated in FIG. 4A-4B) to mimic the chronic antigen environment of solid tumors. In this particular example, CD19-targeting CAR T cells engineered to express a chimeric AP1 transcription factor (TF) were co-cultured with a CD19-expressing human leukemia-derived cell line (Nalm6). Every two to three days for two weeks (6 total stimulations), tumor cells and T cells were counted by flow cytometry and re-set to a baseline CAR to Nalm6 ratio. Genomic DNA was harvested from a subset of the input population (Day 0) and terminal population (Day 14) and barcodes identifying chimeric AP1 TFs were amplified.

By sequencing the barcodes and comparing abundances in the input and terminal populations, chimeric AP1 TFs that endowed CARs with a functional advantage in the face of repetitive stimulation were deduced.

FIG. 5 is a volcano plot generated using DESeq2 showing chimeric AP-1 transcription factors enriched by chronic stimulation. Chimeric AP-1 transcription factors exhibiting particularly robust enrichment include those comprising: a FOSL1 N-terminal domain, BATF bZIP domain and c-JUN C-terminal domain (“FOSL1_BATF_c-JUN”); and a FOSL1 N-terminal domain, BATF bZIP domain and FOSL1 C-terminal domain (“FOSL1_BATF_FOSL1 ”). “Reconstructed BATF” refers to the cells that harbored a chimeric AP-1 TF that had the N-terminal, bZIP and C-terminal domains of BATF, which “recreates” the native gene. “CAR only” refers to Kymriah cells not expressing a chimeric AP-1 TF.

A volcano plot (generated using DESeq2) summarizing the results of the assay mimicking acute stimulation is shown in FIG. 6. Chimeric AP-1 transcription factors exhibiting particularly robust enrichment include those comprising: a FOSL1 N-terminal domain, BATF bZIP domain and c-JUN C-terminal domain (“FOSL1_BATF_c-JUN”); a FOSL1 N-terminal domain, BATF bZIP domain and FOSL1 C-terminal domain (“FOSL1 BATF FOSL1 ”); a FOSL1 N-terminal domain, BATF bZIP domain and BATF C-terminal domain (“FOSL1_BATF_BATF”); and a FOSL1 N- terminal domain, BATF bZIP domain and ATF2 C-terminal domain (“FOSL1_BATF_ATF2”).

FIG. 7 is a comparison of enrichment of AP-1 transcription factors in the chronic and acute stimulation assays. Significant enrichment under both conditions was observed for chimeric AP- 1 transcription factors comprising: a FOSL1 N-terminal domain, BATF bZIP domain and c-JUN C-terminal domain (“FOSL1_BATF_c-JUN”); a FOSL1 N-terminal domain, BATF bZIP domain and FOSL1 C-terminal domain (“FOSL1_BATF_FOSL1 ”); a FOSL1 N-terminal domain, BATF bZIP domain and BATF C-terminal domain (“FOSL1_BATF_BATF”); a FOSL1 N-terminal domain, BATF bZIP domain and ATF2 C-terminal domain (“FOSL1_BATF_ATF2”); a FOSL1 N- terminal domain, BATF bZIP domain and FOSL2 C-terminal domain (“FOSL1_BATF_FOSL2”); and a FOSL1 N-terminal domain, BATF bZIP domain and cMAF C-terminal domain (“FOSL1_BATF_cMAF”).

FIG. 8 summarizes the reproducibility of top hits from the chronic stimulation screen and the enrichment of specific 2-domain combinations. Nearly all of the top hits follow one of the two highlighted “N_bZIP” or “bZIP_C” compositions.

Next, the chronic and acute stimulation assays were repeated across biological replicates, i.e., with edited T cells from different donors. Results are shown in FIG. 9. The panels show donor-level enrichment for the chronic and acute stimulation conditions for T cells expressing CAR-only, c-Jun_cFOS_BATF, FOSL1_BATF_BATF, FOSL1_BATF_c-JUN, FOSL1_BATF_FOSL1 , FOSL1_BATF_FOSL2, FOSL1_cFOS_BATF, FOSL1_cFOS_FOSL1 , and MAFF_cFOS_BATF. Each vertical line represents the enrichment of one donor. The data demonstrates that enrichment of T cells expressing the chimeric transcription factors is robust across biological replicates.

Example 3 - Production of a Comprehensive Domain-Engineered by Synthesis & Recombination (DESynR) AP-1 Transcription Factor Library

Described in this example is the construction of nearly 14,000 non-evolved AP-1 TFs by recombining 72 natural AP-1 TF domains. These TFs, termed DESynR (Domain-Engineered by Synthesis & Recombination) AP-1 TFs were integrated into primary human T cells along with an FDA-approved CD19-targeting Chimeric Antigen Receptor (CAR). Pooled screening in vitro and in vivo across multiple functional axes converged on a collection of hits that offer enhancements beyond those of natural AP-1 TFs. The functional data is complemented by epigenetic and transcriptional profiling, which supports divergence from canonical T cell states.

TFs are organized into families based on a shared functional domain, commonly the DNA- binding domain. Individual members feature a unique composition of trans-activation and repression domains and further interaction domains. To encompass the variable compositions of flanking domains, a TF structure can be simplified to an “N Terminal Effector Domain”, “DNA Binding Domain” and “C Terminal Effector Domain” (FIG. 10A). AP-1 TFs are marked by the basic leucine zipper (bZIP) domain, which includes both the basic DNA-binding motif and a leucine zipper motif that enables dimerization.

A cloning strategy was developed to build and barcode every possible combination of three-domain AP-1 TFs from 24 family members (FIG. 10B). Each domain was separated from an 1 1 -base pair (bp) barcode by an internal Type IIS restriction enzyme-excisable staffer and this cassette flanked by additional external cleavage sites. The 72 total components were synthesized and integrated into a cloning vector and subsequently pooled by TF domain position. In the first round of cloning, internal cleavage of the “N Domain” pool, external cleavage of the “bZIP Domain” pool and subsequent ligation formed 576 barcoded “N + bZIP” combinations. An analogous cloning round formed the final 13,824-member barcoded three-domain DESynR AP- 1 TF library. To enable functional screening in primary human T cells, the library was subcloned into a final vector.

By sequencing the DESynR AP-1 TF-associated barcodes it was confirmed that the plasmid library is nearly comprehensive with 99.8% representation, which was maintained when integrated in primary human T cells (FIG. 10C-1 OE). Moreover, it was determined that bp-length of DESynR AP-1 TFs did not impact integration efficiency (FIG. 10E). The 72 constituent domains were evenly represented in the final library (FIG. 10F). Barcoding enables screening of the DESynR AP-1 TF library using next-generation sequencing, but to assess the fidelity of linkages between barcodes and TFs, amplification was carried out across the whole TF and barcode and sequenced by long-reads. Over 80% of the library was correctly linked, while roughly 13% had barcode-domain mismatches (FIG. 10F). The remaining reads reported cloning failures that resulted in less than three barcodes and thus do not impact barcode-enrichment analysis. Overall, the DESynR AP-1 TF library is comprehensive, well-balanced and retains barcoding fidelity.

Example 4 - DESynR AP-1 TFs outperform native TFs and enhance primary human T cell function

A major barrier to durable responses in CAR T cell-treated patients is the dysfunction caused by repetitive stimulation with tumor antigen. In such settings, CAR T cells are plagued by poor persistence and diminished proliferation and cytotoxicity. To determine whether DESynR AP-1 TFs can support proliferation, a repetitive stimulation screen was performed where a pool of DESynR AP-1 TF-harboring 19-28z CAR T cells were challenged with CD19-expressing Nalm6 leukemia cells six times (FIG. 15B).

To validate the assay and our barcode sequencing pipeline, we first screened a 216- member pilot library (FIG. 14A-14B). High coverage was successfully maintained throughout cloning, editing and the assay. By comparing abundance after a single stimulation (acute) and after six stimulations (chronic) to input, identified were many DESynR AP-1 TFs enriched beyond a “CAR-Only” control and two conserved domain-pair signatures (FIG. 14C-14E). Multiple DESynR AP-1 TFs enriched in both acute and chronic stimulation screens with variable “C” domains featured a FOSL1_BATF “N + bZIP” domain-pair. Conversely, several DESynR AP-1 TFs with variable “N” domains and a conserved cFOSJBATF “bZIP + C” domain-pair were exclusively enriched with chronic stimulation. Following these technical validations, a comprehensive DESynR AP-1 TF screen was then performed.

Along with the DESynR AP-1 TF library, 24 full-length natural AP-1 TFs which contributed domains, several previous overexpression hits (LTBR, IL2RA, TFAP4) and several “CAR Only” variants were integrated (FIG. 1 1 A). Knock-in was detected in three human donors and representation of the library was high, such that this is believed to be the largest pooled knock-in screen conducted (FIG. 14C-14D). Nearly 250 DESynR AP-1 TFs were significantly enriched in the chronic stimulation screen and over 50 out-performed the top natural AP-1 TF JUNB (FIG. 11 B). Enrichment of the top hits was consistent across the three human donors (FIG. 1 1 C). Moreover, three top hits were synthesized en bloc (i.e. not domain-by-domain) and competitive enrichment was validation in chronic stimulation assays versus GFP (FIG. 11 D).

The usage of domains in each position by significantly enriched DESynR AP-1 TFs were analyzed for deviance from equal representation. The FOS subfamily was enriched in the “N” domain position with the three of four members making up the top three: FOSL2, FOSB and FOSL1 (FIG. 11 E, left). On the other hand, the ATF subfamily was enriched at the “bZIP” domain position. All overrepresented “bZIP” domains came from the ATF and FOS subfamilies. An amino acid alignment of the “bZIP” domains suggests that the top two “bZIP” domains, ATF3 and JDP2 are more similar to each other than the rest of the broader AP-1 family (FIG. 17A). Conversely, the depleted “bZIP” domains, largely from the MAF subfamily, cluster together by sequence similarity. More modest trends were seen in the “C” domain position, however BATF3 was featured in almost 15% of constructs.

It was considered that a domain-pair (i.e., “N+bZIP”, “bZIP+C” or “N+C”) could have such an outsized effect that the third component is dispensable. All DESynR AP-1 TFs were reclassified as three separate domain-pairs (FIG. 1 1 F & FIG. 17C). While significantly enriched domain-pairs could be identified for all three architectures, the third domain, regardless of position, was indispensable (FIG. 1 1 G). In fact, trends in the third domain used to complete domain-pairs suggests functional relevance. For example, the FOSL1_BATF “N+bZIP” domainpair was enriched with five of six MAF subfamily “C” domains (FIG. 1 1 G, left). Moreover, all the “bZIP” domains that completed the MAFF_BATF2 “N+C” domain-pair productively came from the ATF and FOS subfamilies, which accords with the preferential usage of these bZIPs in significantly enriched DESynFt AP-1 TFs.

Finally, considered was whether domain components of top DESynFt AP-1 TFs are typically expressed in T cells. The heatbars in “e” and “f” show mRNA expression of corresponding domains in T cells (average across multiple subsets). Many of the top single domains and constituents of domain-pairs are lowly expressed in T cells, suggesting the underutilization of domains that can enhance cellular function.

Example 5 - Multi-parameter functional assays uncover diverse DESynR TF archetypes

Sustained proliferation through chronic antigen exposure is just one desirable function of T cells so we assessed the impacts of DESynR AP-1 TFs on a broader set of T cell functions. A list of 36 top-performing DESynR AP-1 TFs was curated from the comprehensive screen that reflected diversity at the bZIP domain to form a mini pool along with five natural AP-1 TFs (FIG. 12A). In a variety of dysfunction-encoding contexts in vitro and in vivo, the mini pool was screened on proliferation and persistence, effector function and retention of sternness (FIG. 18A).

Confirming the results of the comprehensive screen (FIG. 1 1 B), the majority of DESynR AP-1 TFs out-proliferated natural AP-1 TFs and GFP-control when chronically exposed to Nalm6 leukemia cells. Some variability in top-performers was noted in the absence of exogenous IL-2 supplementation and chronic stimulation with a different leukemic cell line, underscoring the subtle influences of environment on this phenotype. Across different measures of effector function, DESynR TFs consistently out-performed GFP-control, whereas natural counterparts were variably interspersed in each distribution. Enrichment of the mini pool relative to GFP- control was divergent between the two murine organs: most TFs were more abundant in the spleen and less abundant in the bone marrow. Finally, CD62L positivity as a proxy for sternness produced a consensus winner, MAFF_ATF3_BATF2, which was the top hit in the comprehensive screen. Overall, the FOSL1_BATF “N + bZIP” domain-pair was enriched along most of the functional axes.

To understand patterns of differential engagement of these functions, the LOG2FC enrichment across the screens was hierarchically clustered into functional categories and TF groups (FIG. 1 1 C). The TFs and functions each fell into three distinct groups. Group 1 TFs, consisting exclusively of DESynR TFs, were strong proliferators and effectors with a relative decrease in sternness. The more diverse Group 2 TFs were largely poor proliferators with variable effector function and sternness. Finally, Group 3 TFs retained sternness without loss in proliferative capacity. This multi-parameter screening reflects the diversity of functions engaged by T cells and offers a framework for identifying functional profiles desirable in specific contexts.

Interestingly, in vivo abundance in both the spleen and bone marrow clustered with in vitro measures of proliferation. Indeed, L0G2FC in the bone marrow and high-dose Nalm6 screens was strongly correlated (FIG. 12D). As the bone marrow is considered the tumor microenvironment of hematologic malignancies, enrichment there was used as a filter for tumor responsiveness to select TFs for further study. From each of the three TF groups (FIG. 12C), at least one TF was selected that was also enriched in the bone marrow. All of these candidates featured a domain from BATF or the greater ATF subfamily so BATF was chosen as a natural control.

Cytotoxicity is a requisite function for therapeutic T cells, but difficult to measure via pooled screening. Thus, by arrayed screening of selected DESynR AP-1 TFs, we assessed impacts on cytotoxicity at baseline following acute stimulation, throughout chronic stimulation and in a chronically stimulated state.

Following a single challenge across a range of effector to target ratios (E to Ts), DESynR AP-1 TFs killed as well or slightly better than GFP-control and natural BATF (FIG. 12E, left). While degranulation was unchanged, most DESynR AP-1 TFs trended towards lower effector cytokine production (FIG. 12E, right). Expression of several inhibitory receptors (IRs - PD1 , LAG3, CD39) was unchanged and while T cells uniformly adopted a CD45RA+CD62L+ naive- like phenotype, FOSL1_BATF_MAFA consistently trended towards higher per-cell expression of those markers (FIG. 19B).

The challenge of chronic stimulation assay was not enough to effectively parse differences in cytotoxicity between the remaining TFs, so the cells were challenged a final time across a range of E to Ts. Here, two DESynR TFs, FOSL1_BATF_MAFA and cJUN_cFOS_BATF, consistently out-performed GFP-control and BATF (FIG. 12H, left). In parallel, the DESynR TFs showed increased degranulation and effector cytokine secretion following chronic stimulation (FIG. 12H, right). DESynR TFs generally downregulated IRs relative to GFP-control outside of high LAG3 expression by cJUN_cFOS_BATF (FIG. 19F). However, this was juxtaposed by the strongest relative downregulation of CD39. While chronic stimulation skews the differentiation to a CD45RA-CD62L- effector-memory state, FOSL1_BATF_MAFA consistently retained roughly a third of the population in a CD45RA-CD62L+ central-memory state (FIG. 19F).

In a stress-test murine model of CAR T cell function, cJUN_cFOS_BATF and BATF3_BATF2_MAFF-edited cells controlled Nalm6-tumors significantly better than GFP- control, which resulted in remarkable elongation of lifespan (FIG. 20).

Example 6 - DESynR TFs program non-natural transcriptional and epigenetic T cell states

To understand the underpinnings of functional divergence encoded by DESynR AP-1 TFs, single-cell RNA sequencing was first performed. It was desirable to parse whether DESynR AP-1 TFs stochastically impacted a minor subset of edited cells or if remodeling was broad. Unsupervised clustering of nearly 53,000 cells from five human donors across five DESynR AP- 1 TFs, two natural AP-1 TFs and GFP-control following chronic stimulation mainly split cells based on CD4 versus CD8 identity and proliferative versus cytotoxic phenotype (FIG. 13A-13B). A distinct cluster of Treg-like CD4+ cells upregulating FOXP3 and CTLA4 was noted. Two separate CD4+ clusters, an MKI67+ proliferative cluster and PRF1 + cytotoxic cluster, were uniquely marked by FXYD2. FXYD2 encodes the gamma subunit of the sodium-potassium ATPase channel. Though FXYD2 expression is predominantly confined to the kidney, it is upregulated in the earliest T cell-lineage committed thymic progenitors. Moreover, a recent analysis of post-infusion CD19 CAR T cells in children with B-ALL identified FXYD2 as a marker of persisting cells. Mapping of edited TF identity to the UMAP showed that MAFF_ATF3_BATF2 and BATF3_JDP2_BATF2 almost exclusively occupied the FXYD2+ clusters, roughly half of cJUN_cFOS_BATF enriched there and the remaining TFs were nearly absent (FIG. 13C-13D). The three DESynR AP-1 TFs enriched in the FXYD2+ clusters were relatively depleted from the Treg-like cell state.

The transcriptional states being so tightly linked to edited TF identity encouraged pseudobulking (pbulking) the single-cell data for comparison to donor- and construct-matched bulk chromatin profiles. It was confirmed that differences in differentially expressed genes (DEGs) were not attributable to CD4/8+ status (FIG. 22A-22B). Principal component analysis (PCA) of differentially accessible chromatin regions (DARs) largely mirrored the PCA of DEGs (FIG. 13E). A permutation test was performed to determine that changes in chromatin accessibility are associated with changes in gene expression (FIG. 14D). Hierarchical clustering of DEGs and DARs resulted in four linked chromatin accessibility and gene expression modules that were differentially engaged by the TFs (FIG. 13F-13G) and centered around MAFF_ATF3_BATF2, BATF3_JDP2_BATF2 and cJUN_cFOS_BATF. Of interest was to understand whether these gene modules were repressed or absent in GFP-control cells or simply amplified in Group 2-4 TFs. It was found that a sizable fraction of genes relatively increased in expression were lowly expressed by GFP-control, suggesting activation of latent gene programs rather than amplification of existing ones (FIG. 13G). To identify biological processes associated with the gene modules, pathway analysis was then performed (FIG. 13H, FIG. 23B). While genes from Group 1 were associated with common T cell processes such as “PD-1 cancer immunotherapy pathway”, Groups 2-4 were more divergent including “Myelination Signaling Pathway” and “Semaphorin interactions”.

Finally, assessed was which TF binding sites are enriched in open chromatin regions to infer which TFs may be casual in establishing or maintaining these divergent epigenetic states (FIG. 131, FIG. 22E). Accessibility of motifs associated with muscle cells and neuronal processes such as MYOG and ASCL1 , respectively, was enriched in cJUN_cFOS_BATF (FIG. 131).

Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1 . A chimeric polypeptide comprising: an N-terminal domain of a first AP-1 transcription factor and a bZIP domain of a second AP-1 transcription factor.

2. The chimeric polypeptide of claim 1 , wherein the first AP-1 transcription factor is a member of the FOS family of AP-1 transcription factors.

3. The chimeric polypeptide of claim 2, wherein the first AP-1 transcription factor is FOSL1 .

4. The chimeric polypeptide of claim 1 , wherein the first AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors.

5. The chimeric polypeptide of claim 2, wherein the first AP-1 transcription factor is BATF3.

6. The chimeric polypeptide of claim 1 , wherein the first AP-1 transcription factor is a member of the MAF family of AP-1 transcription factors.

7. The chimeric polypeptide of claim 2, wherein the first AP-1 transcription factor is MAFF.

8. The chimeric polypeptide of any one of claims 1 -7, wherein the second AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors.

9. The chimeric polypeptide of claim 8, wherein the second AP-1 transcription factor is BATF.

10. The chimeric polypeptide of claim 8, wherein the second AP-1 transcription factor is ATF3.

11 . The chimeric polypeptide of claim 8, wherein the second AP-1 transcription factor is BATF2.

12. The chimeric polypeptide of claim 8, wherein the second AP-1 transcription factor is JDP2.

13. The chimeric polypeptide of any one of claims 1 -12, further comprising a C-terminal domain of an AP-1 transcription factor.

14. The chimeric polypeptide of claim 13, wherein the C-terminal domain is of an AP-1 transcription factor of the FOS, ATF, JUN or MAF family of AP-1 transcription factors.

15. The chimeric polypeptide of claim 14, wherein the C-terminal domain is a FOSL1 C- terminal domain.

16. The chimeric polypeptide of claim 14, wherein the C-terminal domain is a FOSL2 C- terminal domain.

17. The chimeric polypeptide of claim 14, wherein the C-terminal domain is a BATF C- terminal domain.

18. The chimeric polypeptide of claim 14, wherein the C-terminal domain is a BATF2 C- terminal domain.

19. The chimeric polypeptide of claim 14, wherein the C-terminal domain is a c-Jun C- terminal domain.

20. The chimeric polypeptide of claim 14, wherein the C-terminal domain is a MAFA C- terminal domain.

21 . The chimeric polypeptide of claim 14, wherein the C-terminal domain is a MAFF C- terminal domain.

22. A chimeric polypeptide comprising: a bZIP domain of a first AP-1 transcription factor and a C-terminal domain of a second AP-1 transcription factor.

23. The chimeric polypeptide of claim 22, wherein the first AP-1 transcription factor is a member of the ATF family of AP-1 transcription factors.

24. The chimeric polypeptide of claim 23, wherein the first AP-1 transcription factor is BATF.

25. The chimeric polypeptide of claim 23, wherein the first AP-1 transcription factor is BATF2.

26. The chimeric polypeptide of claim 23, wherein the first AP-1 transcription factor is ATF3.

27. The chimeric polypeptide of any one of claims 22-26, wherein the second AP-1 transcription factor is a member of the JUN family of AP-1 transcription factors.

28. The chimeric polypeptide of claim 27, wherein the second AP-1 transcription factor is c- Jun.

29. The chimeric polypeptide of any one of claims 22-26, wherein the second AP-1 transcription factor is a member of the FOS family of AP-1 transcription factors.

30. The chimeric polypeptide of claim 29, wherein the second AP-1 transcription factor is FOSL1 .

31 . The chimeric polypeptide of any one of claims 22-26, wherein the second AP-1 transcription factor is of a member of the MAF family of AP-1 transcription factors.

32. The chimeric polypeptide of claim 31 , wherein the second AP-1 transcription factor is MAFA.

33. The chimeric polypeptide of claim 31 , wherein the second AP-1 transcription factor is MAFF.

34. The chimeric polypeptide of any one of claims 22-30, further comprising an N-terminal domain of an AP-1 transcription factor.

35. The chimeric polypeptide of claim 34, wherein the N-terminal domain is of an AP-1 transcription factor of the FOS family of AP-1 transcription factors.

36. The chimeric polypeptide of claim 34, wherein the N-terminal domain is a FOSL1 N- terminal domain.

37. The chimeric polypeptide of claim 34, wherein the N-terminal domain is of an AP-1 transcription factor of the ATF family of AP-1 transcription factors.

38. The chimeric polypeptide of claim 37, wherein the N-terminal domain is a BATF3 N- terminal domain.

39. The chimeric polypeptide of claim 13, wherein the N-terminal domain is of a member of the MAF family of AP-1 transcription factors.

40. The chimeric polypeptide of claim 39, wherein the N-terminal domain is a MAFF N- terminal domain.

41 . A nucleic acid encoding the chimeric polypeptide of any one of claims 1-40.

42. A cell comprising the nucleic acid of claim 41 , wherein the cell expresses the chimeric polypeptide.

43. The cell of claim 42, wherein the nucleic acid is operably linked to an endogenous promoter.

44. The cell of claim 43, wherein the endogenous promoter is of the T-cell receptor a constant (TRAC) locus.

45. The cell of claim 42, wherein the nucleic acid is operably linked to an exogenous promoter.

46. The cell of claim 45, wherein the exogenous promoter is a constitutive promoter.

47. The cell of claim 45, wherein the exogenous promoter is an inducible promoter.

48. The cell of any one of claims 42-47, wherein the cell is a human cell.

49. The cell of any one of claims 42-48, wherein the cell is an immune effector cell.

50. The cell of claim 49, wherein the immune effector cell is a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a macrophage.

51 . The cell of any one of claims 42-50, wherein the cell comprises a nucleic acid encoding a recombinant receptor, wherein the cell expresses the recombinant receptor on its surface.

52. The cell of claim 51 , wherein the recombinant receptor is a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), a synthetic notch (synNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, or a generalized extracellular molecule sensor (GEMS) receptor.

53. The cell of claim 51 , wherein the recombinant receptor is a CAR.

54. The cell of claim 49 or 50, wherein the cell does not comprise a nucleic acid encoding a recombinant receptor.

55. The cell of claim 54, wherein the cell is a tumor infiltrating lymphocyte (TIL).

56. A method of making the cell of one of claims 42-55, comprising stably introducing into the cell the nucleic acid of claim 41 .

57. A composition comprising a population of cells as defined in any one of claims 42-55.

58. The composition of claim 57, wherein the composition is formulated for administration to a subject in need thereof.

59. A method comprising administering an effective amount of the composition of claim 58 to a subject in need thereof, wherein the cells express the chimeric polypeptide.

60. The method according to claim 59, wherein the composition comprises a population of immune effector cells as defined in any one of claims 49-55.

61 . The method according to claim 60, wherein the chimeric polypeptide prevents or reduces exhaustion of the immune effector cells upon administration to the subject.

62. The method according to any one of claims 59-61 , wherein the cells express a recombinant receptor as defined in claim 52.

63. The method according to claim 62, wherein the recombinant receptor is a CAR.

64. The method according to claim 62 or 63, wherein the subject has cancer, and wherein the recombinant receptor binds to a tumor antigen present on cancer cells in the subject.