Immune cells with enhanced 53BP1 and engineered 53BP1 for use in methods of treating cancer

Abstract

The present invention provides polypeptides, polynucleotides, constructs and / or vectors comprising activating mutations in 53BP1. In particular, 53BP1 mutation at amino acid position 334 that makes the resulting protein resistant to phosphorylation and inactivation by GSK3β is provided. Also provided are lymphocytes comprising the mutant 53BP1 or a 53BP1 resistant to GSK3β phosphorylation and inactivation and methods of making and using the same.

Description

[0001] PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0002] IMMUNE CELLS WITH ENHANCED 53BP1 AND ENGINEERED 53BP1 FOR USE IN METHODS OF TREATING CANCER

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] 5 This application claims priority to U.S. Provisional Application No. 63 / 716,545 filed on November 5, 2024, and U.S. Provisional Application No. 63 / 800,984 filed on May 6, 2025, the contents of which are incorporated by reference in their entireties.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under grant number R01CA247947 and DP5OD031863 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0007] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0008] 15 The contents of the electronic sequence listing (16985200191. xml; Size: 41,363 bytes; and Date of Creation: November 3, 2025) is herein incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] Immunotherapy, such as immune checkpoint inhibitors and cell-based therapies such as

[0011] 20 chimeric antigen receptor (CAR) T cells, have greatly improved survival rates for a subset of patients across many tumor types, particularly hematological tumors. However, response is limited to less than half of patients in most cancers, especially in solid tumors. CAR T cells are widely used and commercially available, however, treatment response is reduced in solid tumors. In head and neck squamous cell carcinoma (HNSCC), the overall immune checkpoint blockade (ICB) response rate is only 11-18% at 6 months. Despite the success of immunotherapy in a subset of patients, a better understanding of how to overcome immunotherapeutic resistance is needed to improve current immunotherapy treatments. Accordingly, there is a remaining need in the art for novel therapies to enhance immunotherapy treatments against solid tumors.

[0012] 30 SUMMARY PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0013] This disclosure describes a 53BP1 mutation which inhibits GSK3p-mediated phosphorylation and inactivation of 53BP1. Thus, this mutation leads to increased 53BP1 activity including increased DNA repair through non-homologous end joining. One aspect of the present invention provides an engineered human 53BP1 polypeptide comprising at least one substitution

[0014] 5 mutation at a position corresponding to an amino acid residue at position 222, 294, 334, 379, 380, 552, 809, 862, 1056, 1609 or 1678 of the 53BP1 sequence. The substitution mutation may be any one amino acid other than serine, threonine or tyrosine. The substitution mutation may block phosphorylation at the mutated position. In some embodiments, the 53BP1 polypeptide comprises one of SEQ ID NO: 1-3 or sequences having at least 90% identity to at least one of SEQ ID NO: 1-3. In some embodiments, the 53BP1 mutant comprises SEQ ID NO: 4-6 or 24 or sequences having at least 90% identity to at least one of SEQ ID NO: 4-6 or 24. Polynucleotides, constructs and vectors encoding the provided 53BP1 polypeptides are also provided.

[0015] The present disclosure also provides a cell and, in some embodiments, a lymphocyte comprising a polynucleotide encoding a 53BP1 polypeptide. The polynucleotide encoding the

[0016] 15 53BP1 polypeptide may be operably linked to a heterologous promoter. The 53BP1 polypeptide may comprise at least one substitution mutation at a GSK30 phosphorylation site, which makes the 53BP1 resistant to GSK30 inactivation. In some embodiments, the GSK3 phosphorylation site is T334, S222, S294, S379, S380, S809, S862, S552, T1056, T1609, or S1678 of the 53BP1 polypeptide. In some embodiments, the lymphocyte further comprises a second heterologous

[0017] 20 promoter operably linked to a second polynucleotide encoding a chimeric antigen receptor.

[0018] The present disclosure also provides a method of treating cancer in a subject. In some embodiments, the method comprises administering a therapeutically effective amount of a lymphocyte described herein and a pharmaceutically acceptable excipient, carrier and / or diluent to the subject to treat the cancer.

[0019] The present disclosure also provides a method for increasing resistance to DNA damage or enhancing DNA repair. In some embodiments, the method comprises introducing an engineered polypeptide described herein, a polynucleotide described herein, a construct described herein or a vector described herein into a cell.

[0020] The present disclosure also provides a composition of guide RNAs (gRNAs) capable of

[0021] 30 introducing at least one substitution mutation into a 53BP1 polypeptide that has at least 95% identity to SEQ ID NO: 1. In some embodiments, the at least one mutation is selected from the PCT / US25 / 54216 05 November 2025 (05.11.2025) group consisting of T334, S552, T1056, T1609, S1678 and combinations thereof. In some embodiments, the gRNA comprises a target region comprising at least one of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22.

[0022] 5

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present technology can be better understood by reference to the following drawings. The drawings are merely exemplary to illustrate certain features that may be used singularly or in combination with other features and the present technology should not be limited to the embodiments shown.

[0025] FIG. 1 Inhibition of the GSK3P-53BP1 Axis Improves T Cell Response in Head and Neck Squamous Cell Carcinoma. As described herein, activation of 53BP1 will enhance T cell function by promoting increased DNA repair (non-homologous end joining (NHEJ)) instead of signaling apoptosis mechanisms (cell death). This can aid in the use of immune checkpoint

[0026] 15 blockade therapies (ICB) and allow for adoptive cell transfer (ACT) with tumor infiltrating lymphocytes (TIL) or gene modified T cells expressing modified T cell receptors or chimeric antigen receptors (CARs).

[0027] FIG. 2. GSK3P phosphorylates 53BP1 at T334 to inhibit NHEJ. A) The sequence alignment of 53BP1. Letters in shading and lower case mark the putative phosphorylatable

[0028] 20 threonine that match the GSK30 consensus sequence. Human sequence is SEQ ID NO: 25, mouse and rat sequence is SEQ ID NO: 26. B) Direct phosphorylation of 53BP1 by GSK30. In vitro kinase assay of GSK3P co-incubation with WT 53BP1, or T334A 53BP1 that was expressed in U2OS cells and purified by IP, and isotope-labelled phosphate with or without ATP. C) Schematic of I-SceINHEJ reporter assay. Assessment of NHEJ repair of DSB as measured by %GFP positive 53BP1-KO U2OS cells reconstituted with WT or T334A mutant 53BP1. n=3. D) Representative images of 53BP1 and H2AX foci in WT or GSK30-KO MEF cells reconstituted with WT or T334A 53BP1. E) Percentage of 53BP1 foci per MEF cell. n=50. F) Percentage ofH2AX foci per MEF cell. n=50 **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0029] FIG. 3. Knock-in of 53BP1 T336A mutation in a mouse model. (A, B). a homozygous

[0030] 30 T336A knock-in mouse TA) in a C57BL / 6 background (InGenious targeting laboratory, NY) using CRISPR / Cas9 genome editing to introduce an ACA to GCT nucleotide substitution for PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0031] T336. Fig. 3 A human sequence is SEQ ID NO: 27, mouse is SEQ ID NO: 28; Fig. 3B WT sequence is SEQ ID NO: 29, KI sequence is SEQ ID NO: 30.

[0032] FIG. 4. Mouse model to study tongue tumor response to IR. A) Image of mouse holder used for irradiation. B) A lead plate is placed to protect mouse brains from x-ray treatment. C)

[0033] 5 Image of oral cavities of four mice for irradiation treatment.

[0034] FIG. 5. Ablation of the GSK3P-53BP1 axis enhances response to HNSCC. (A) M0C2 tumor cells were injected into the tongues of WT or 53BP1 TA mutant mice and monitored tumor growth after 16 days (B). In addition, we included an irradiation regimen of 3Gy on days 5, 6, and 7, as radiation therapy is commonly used to treat solid tumors, including HNSCC. (C). Tumors were harvested at day 16 and sectioned transversely followed by immunocytochemistry analysis. Tumor volume was plotted based on measurements from the largest tumor section per mouse. *P<0.05, ***P<0.001.

[0035] FIG. 6. 53BP1 T336A mutant mice have T-cell dependent delayed tumor formation. (A) T cell depletion experiments within the M0C2 tumor model. We find that depletion of any

[0036] 15 type of T cell resulted in similar tumor volumes between wildtype or 53BP1 TA mice, suggesting that T cells are responsible for the increased response in this model. (B) We found that depletion of any subset of T cells (CD3+, CD4+, or CD8+) resulted in the loss of tumor suppression in the 53BP1 TA mutant mice.

[0037] FIG. 7. 53BP1 T336A mutant T cells have increased tumor infiltration in vivo. Tumors

[0038] 20 were harvested at day 17 and analyzed using immunohistochemistry (IHC) for (A) and flow cytometry (B) for CD3+ T cells. For flow cytometry, CD3+ T cells were identified by the staining pattern DAPI-, CD45+, CD19-, CD3+.

[0039] FIG. 8. 53BP1 T336A mutant T cells have less DNA damage in vitro and in vivo. Comet assays (Trevigen) on sorted tumor-infiltrating T cells to quantify double-stranded breaks in vitro (A) and in vivo (B).

[0040] FIG. 9. 53BP1 T336A mutant T Cells have enhanced survival after DNA damage. TA mutant T cells in vitro, were subjected WT or TA mutant CD8+ T cells to DNA damage (0-2 Gy IR) followed by analysis using Cell Titer Gio (A) and Incucyte (B). CD4+ T cells were also analyzed for Cell Titer Gio.

[0041] 30 FIG. 10. 53BP1 T336A mutation enhances immune checkpoint blockade in HNSCC. (A) Tumors were injected into the mouse tongue on Day 0, and immune checkpoint blockade PCT / US25 / 54216 05 November 2025 (05.11.2025) therapy began on day 3 after tumor injection. aCTLA4 was administered at I OOpg / mL every three days, while aPDl was administered at I OOpg / mL every two days. ICB greatly enhanced the tumor response in both WT and TA mutant mice, with near complete control of tumors in TA mutant mice (B-D). Survival data is shown in Fig. 19.

[0042] 5 FIG. 11. Chronic stimulation models T cell exhaustion (TEX). (A) Schematic of exhaustion timeline. All analyses occurred on Day 8; (B) Gene array (Taqman) analysis of exhaustion and memory markers; (C) Flow cytometry analysis of surface inhibitory markers. Mean fluorescent Intensity (MFI) was used to calculate relative changes; (D) Metabolic flux analysis (Seahorse) of acute or chronically stimulated T cells. *P<0.05, **P<0.01.

[0043] FIG. 12. DNA damage in TEX. (A) yH2AX during in vitro exhaustion by Western blotting with densitometry analysis; (B) Comet analysis of tail moment in acute vs. chronically stimulated T cells on Day 8; (C) Flow cytometry analysis of yH2AX or (D) exhaustion markers in acute or chronically stimulated T cells with / without treatment with 200pM hydroxyurea (HU). *P<0.05, **P<0.01.

[0044] 15 Figure 13. Reduced TEX in tumors from TA mice. (A) UMAP plots of GBM single cell RNA sequencing data from WT or TA mice, highlighting CD3+ T cell cluster; (B) Expression levels of TEX and memory markers in tumors from WT or TA mice.

[0045] Fig. 14. Enhanced CD8+ T cell survival after IR treatment. Survival of activated murine CD8+ T cells following 2Gy IR was measured at the indicated time points by (A) growth

[0046] 20 rate assessed using Incucyte, and (B) viability assay using Cell Titer Gio (Promega). *P<0.05, **P<0,01.

[0047] Fig. 15. Retroviral transduction of OT-1 T cells does not interfere with tumor control. OT1 T cells were retrovirally transduced and cocultured with MC38.SIINFEKL target cells at a ratio of 2: 1. Remaining cells were determined every 2 hours for 72 hours.

[0048] Fig. 16. TA mutant mice are highly responsive to ICB. M0C2 tumors were treated with combination ICB. For each 100 pL dose (suspended in PBS), 100 pg of CTLA-4 (9D9), 250 pg of PD-1 (RMP114), 250 pg of Rat IgG, and 100 pg of mIgG2b were used. ICB and corresponding IgG controls were injected every other day starting on Day 3, until Day 15. Tumor growth was monitored every 3 days by Luciferase expression quantified using in vivo imaging system (IVIS)

[0049] 30 spectrum (Bioluminescence and Fluorescence) (A) and quantitated (B). #p<0.05, **p<0.01, ***p<0.001. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0050] Fig. 17. In vitro characterization of T334A T cells shows increased cytotoxicity and survival after irradiation. Mouse in vitro killing assay. CD8+ T cells were isolated from spleens of OT-1 x WT or OT-1 x 53bpl T336A mutant mice and activated for three days prior to coculture. T cells were plated at a ratio of 4:1 with B16-0VA or M0C2-0VA cells on a flat, glass

[0051] 5 bottom imaging plate (60,000 T cells to 15,000 tumor cells). Plate was imaged every 4 hours for two days.

[0052] Fig. 18. Adapting 53BP1 T334A mutation into human models for adoptive cell therapy. Human in vitro persistent CAR killing assay. CD8+ T cells were isolated from a healthy donor, activated, and transduced with virus encoding GFP-labeled CD 19 targeting 28z CAR. On day 7, CAR T cells were sorted on GFP expression and left to rest one day. Cells were then electroporated with either Scrambled RNA or crRNA or gRNA including a donor strand designed to introduce the T334A mutation into genomic 53BP1. Mutation was verified using amplicon sequencing (Azenta). On day 12, T cells were plated at a ratio of 4:1 with NALM6 cells on a flat, glass bottom imaging plate (60,000 T cells to 15,000 tumor cells). Plate was imaged every 4 hours

[0053] 15 for two days. After 48 hours, wells were resuspended and half of the volume was transferred onto fresh tumor cells plated at the same density as the original co-culture. Replating was continued until all T cells were unable to control tumor cell outgrowth.

[0054] Fig. 19. Mouse Survival after MOC2 Inoculation. A Tumors were injected into the mouse tongue on Day 0, and immune checkpoint blockade therapy began on day 3 after tumor

[0055] 20 injection. aCTLA4 was administered at lOOug / mL every three days, while aPDl was administered at lOOug / mL every two days. IgG antibody control were administered to control groups. Mouse weight was measured daily. B. Survival ofWT or TA mice with or without ICB treatment. Weight loss of 20% was used as a cutoff for survival. ** p<0.0012, **** p<0.0001.

[0056] Fig. 20. 53BP1 Phosphorylation Sites Identified By Phospho-TMT Mass Spectrometry. For proteomics analysis, in vitro acute and chronic stimulation was performed on CD8+ T cells isolated from 3 unique healthy donors, as in Figure 11 A. A. Following acute or chronic stimulation, samples were collected on day eight for tandem-mass-tag (TMT) and phospho-TMT mass spectrometry analysis through the IDeA National Resource for Proteomics, located in-house at UAMS. B. Heat map of selected DNA damage response proteins identified in the TMT data as

[0057] 30 differentially regulated during chronic stimulation. C. 53BP1 phosphorylation sites proteins identified in the phosphoTMT data as differentially regulated during chronic stimulation. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0058] DETAILED DESCRIPTION

[0059] While chimeric antigen receptor (CAR) T cells are widely used and commercially available, treatment response is reduced in solid tumors relative to hematologic malignancies. The inventors have shown that T cells accumulate DNA damage during activation and chronic

[0060] 5 stimulation, contributing to T-cell dysfunction. The inventors have also shown the 53BP1 T334A (human) / T336A (mouse) -hereafter 53BP1 TA - mutation to inhibit GSK3b-mediated phosphorylation and inactivation of 53BP1 reduces the DNA damage and maintains T cell function. Thus, this mutation leads to increased 53BP1 activity including increased DNA repair through non-homologous end joining. To improve CAR T cell resistance to DNA damage, the inventors have generated a mutant mouse expressing the 53BP1 T336A mutant protein. The inventors have shown that this mutation increases T-cell efficacy against head and neck squamous cell carcinoma (HNSCC) in a murine tongue tumor model. Additionally, the inventors have shown that combination of this mutation with irradiation or immune checkpoint blockade (ICB) further increases their efficacy in the murine HNSCC model. In addition, other 53BP1 activating

[0061] 15 mutations can be included to further enhance DNA repair. The inventors have identified other 53BP1 phosphorylation sites, including S222, S294, S379, S380, S809, S862, S552, T1056, T1609, and SI 678, that also may be targeted to maintain T cell function. The invention can enhance immunotherapy treatments against solid tumors by engineering CAR T cells to express this novel mutation with or without combinatorial treatment. Overall, the invention improves CAR

[0062] 20 T cell function, increase T cell infiltration in solid tumors, reduces T-cell dysfunction, and promotes increased tumor cell death.

[0063] An engineered human 53BP1 polypeptide may comprise a substitution mutation at a position corresponding to an amino acid residue selected from the group consisting of 222, 294, 334, 379, 380, 552, 809, 862, 1056, 1609, 1678 and combinations thereof. The wild-type 53BP1 polypeptide may comprise any one of SEQ ID NO: 1-3 or sequences with at least 90% identity to SEQ ID NO: 1-3. SEQ ID NO: 1 is the human 53BP1 wildtype, isoform 1 protein sequence, SEQ ID NO: 2 is the human 53BP1 wildtype, isoform 2 protein sequence, SEQ ID NO: 3 is the human 53BP1 wildtype, isoform 3 protein sequence. SEQ ID NO: 4 is the human 53BP1 T334A, isoform 1 protein sequence, SEQ ID NO: 5 is the human 53BP1 T334A, isoform 2 protein sequence, SEQ

[0064] 30 ID NO: 6 is the human 53BP1 T334A, isoform 3 protein sequence. The 53BP1 polypeptides include other isoforms or 53BP1 from non-human sources and may have at least 90%, 92%, 94%, PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0065] 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1-6 outside of the single substitution mutation at the positions listed above. The substitution mutation may change the amino acid at the position to an amino acid other than serine, tyrosine or threonine. In some cases, the substitution mutation may be an alanine substitution. The T334A mutation may correspond to a threonine to

[0066] 5 alanine substitution at position 334 of the human 53BP1 sequence.

[0067] The amino acid at the substitution mutation may not be phosphorylated by GSK30. Wildtype 53BP1 may be subject to phosphorylation by GSK30 at multiple sites. The substitution mutations may prevent GSK30-mediated phosphorylation at these sites by replacing phosphorylatable amino acids with non-phosphorylatable amino acids such as alanine. The engineered polypeptide may have reduced inhibition of DNA repair as compared to wild-type 53BP1. In some cases, the engineered 53BP1 may demonstrate enhanced non-homologous end joining DNA repair pathway.

[0068] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms

[0069] 15 refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an

[0070] 20 isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain. The term “amino acid” refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers, unless otherwise indicated, if their structures allow such stereoisomeric forms.

[0071] 30 As used herein, the term “peptide” refers an oligomer to short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, PCT / US25 / 54216 05 November 2025 (05.11.2025) polypeptides, etc.), peptides are of about 50 amino acids or less in length. A peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and / or modified amino acids. A peptide may be a subsequence of naturally occurring protein or a non-natural (artificial) sequence.

[0072] 5 Regarding the polypeptides disclosed herein, the phrases “% sequence identity,” “percent identity,” or “% identity” refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment

[0073] 15 Search Tool (BLAST® alignment tool), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST® alignment tool software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0074] Polypeptide or polynucleotide sequence identity may be measured over the length of an

[0075] 20 entire defined polypeptide or polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide or polynucleotide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0076] Polynucleotides may encode the engineered 53BP1 polypeptides described herein. Such polynucleotides may comprise DNA or RNA sequences that encode 53BP1 polypeptides having substitution mutations at GSK3P phosphorylation sites. The polynucleotides may be codon-

[0077] 30 optimized for expression in specific cell types, such as human T cells or other lymphocytes. The polynucleotides may be synthesized using standard molecular biology techniques or may be PCT / US25 / 54216 05 November 2025 (05.11.2025) derived from naturally occurring 53BP1 sequences that have been modified to introduce the desired substitution mutations.

[0078] The term "construct" or "polynucleotide construct" is a polynucleotide which allows the encoded sequence to be replicated and / or expressed in the target cell. A construct may contain an

[0079] 5 exogenous promoter, operably linked to any one of the polynucleotides described herein. As used herein, a polynucleotide is “operably connected” or “operably linked” when it is placed into a functional relationship with a second polynucleotide sequence. As used herein, the terms “heterologous promoter,” “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5’ or 3’ side of a polynucleotides described herein, or within the coding region of said polynucleotides. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3’ direction) coding sequence. The typical 5’ promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels

[0080] 15 detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The heterologous promoter may be selected from various promoter types depending on the intended application and target cell type. In some cases, the promoter may be a constitutive promoter such as a

[0081] 20 cytomegalovirus (CMV) promoter. The promoter may alternatively be an inducible promoter that allows for controlled expression of the 53BP1 polypeptide

[0082] In some embodiments, the construct is an expression construct, a vector or a viral vector. A vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence, typically DNA into another cell, where it can be replicated and / or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Expression constructs comprise a heterologous promoter and the nucleic acid sequence encoding protein of interest (e.g., 53BP1) which is capable of expression in the cell in which it is introduced. The expression constructs include vectors which are capable of directing the expression of exogenous genes to which they are operatively linked.

[0083] 30 Such vectors are referred to herein as "recombinant constructs," "expression constructs," "recombinant expression vectors" (or simply, "expression vectors" or "vectors") and may be used PCT / US25 / 54216 05 November 2025 (05.11.2025) interchangeably. Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA

[0084] 5 loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the mutant a-gal protein. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Viral vectors are incorporated into viral particles that are then used to transport the viral polynucleotide encoding the protein of interest into the target cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., lentiviral vectors). Moreover, certain vectors are capable of directing the expression of exogenous genes to which they are operatively linked. In general, vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification "vector" include

[0085] 15 expression vectors, such as viral vectors (e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (AAV)), which serve equivalent functions.

[0086] The vectors are heterogeneous exogenous constructs containing sequences from two or more different sources. Suitable vectors include, but are not limited to, plasmids, expression

[0087] 20 vectors, lentiviruses (lentiviral vectors), adeno-associated viral vectors (rAAV), among others and includes constructs that are able to express the protein of interest in lymphocytes. A preferred vector is a lentiviral vector or adeno-associated vector. Suitable methods of making viral particles are known in the art to be able to transform cells in order to express the protein of interest in lymphocytes described herein.

[0088] Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-preferred, tissue-specific promoters and cell- type specific. The heterologous promoter may be a plant, animal, bacterial, fungal, or synthetic promoter. Suitable promoters are known and described in the art.

[0089] 30 In mammalian cells, typical promoters include, without limitation, promoters for Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 PCT / US25 / 54216 05 November 2025 (05.11.2025) virus, as well as the translational elongation factor EF-la promoter or ubiquitin promoter. Other promoters include the T3, T7 and SP6 promoter sequences, which are often used for in vitro transcription of RNA.

[0090] Within the vector may be an expression cassette. An expression cassette is a distinct

[0091] 5 component of vector DNA consisting of a gene and regulatory sequence to be expressed by a transfected cell. In some embodiments an expression vector is introduced into a cell for purposes of expressing the protein encoded by the expression vector. The expression vector can be introduced into the cell by any means known in the art. These means may include transfection or transduction. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process whereby foreign DNA is introduced into another cell via a viral vector. These are common tools to introduce a foreign gene into host cells. Among others, additional means include transformation, and conjugation.

[0092] The polypeptides described herein are “engineered,” meaning that they have been altered by the hand of man. Specifically, the engineered 53BP1 polypeptide of the present invention has

[0093] 15 been altered to comprise a mutation, generally here a substitution mutation. The term “mutation” as used herein indicates any modification of a nucleic acid and / or polypeptide which results in an altered nucleic acid or polypeptide (i.e., relative to the wild-type nucleic acid or polypeptide sequence). Mutations include, for example, point mutations, substitutions, deletions, or insertions of single or multiple residues in a polynucleotide (or the encoded polypeptide), which includes

[0094] 20 alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, an insertion, or a deletion of part or all of a gene. In certain embodiments, a portion of a genetically modified organism's genome may be replaced with one or more heterologous (exogenous) polynucleotides. In some embodiments, the mutations are the results of artificial selection pressure. In still other embodiments, the mutations in the organism genome are the result of genetic engineering, through use of CRISPR / Cas based genetic engineering or introduction of an expression cassette.

[0095] In some embodiments, the mutation comprises a mutation at the 334 position of the protein

[0096] 30 sequence in a human isoform of 53BP1, which corresponds to the 336 position in mice. In some embodiments, T334 of the human 53BP1 gene is mutated such that the mutant form of the protein PCT / US25 / 54216 05 November 2025 (05.11.2025) is non phosphorylated by GSK30. Thus the substitution mutation at this position may be any amino acid that is not a target for phosphorylation, in particular by GSK3b. Any amino acid other than a serine, threonine or tyrosine may be used. In some embodiments, the mutation is T334A. In some embodiments, 53BP1 may comprise more than one mutation. In some embodiments, the

[0097] 5 53BP1 may comprise a mutation at T334 as well as other activating mutations. An activating mutation is also known as a gain of function mutation. In some embodiments, the 53BP1 protein including the mutation may comprise SEQ ID NO: 4-6, which all include the T334A mutation. Also included are 53BP1 proteins having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 4-6 and including the T334A mutation. Also included are 53BP1 proteins having a mutation at position 334 which makes the resulting 53BP1 protein resistant to phosphorylation by GSK3[3 and these proteins may have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or over 99% sequence identity to any one of SEQ ID NO: 1-3.

[0098] The inventors also identified other 53BP1 phosphorylation sites by comparing activated

[0099] 15 T cells to exhausted T cells. These sites include, position S222, S294, S379, S380, S809, S862, S552, T1056, T1609, and S1678 with positions relative to SEQ ID NO: 1. Generally, these positions may be mutated such that the site may no longer be phosphorylated or may be differentially phosphorylated. In some embodiments, these positions are mutated to S552A, T1056A, T1609A and S1678A. Without wishing to be bound by any one theory, these 53BP1

[0100] 20 phosphorylation sites may be differentially regulated during chronic stimulation and may enhance nonhomologous end joining repair in a cell or may enhance 53BP1 activity relative to wild-type 53BP1. Mutations at these sites may prevent them from being phosphorylated by a kinase. In some embodiments an engineered human 53BP1 polypeptide may comprise a combination of substitution mutations at positions corresponding to an amino acid residue selected from 334, 222, 294, 379, 380, 552, 809, 862, 1056, 1609, 1678. In some embodiments, the combination of mutations may be based on site function, for example a combination of mitotic sites; based on location, or based on the reside, for example all serine, or threonine sites.

[0101] Mutations described herein may be the result of genetic engineering, through use of CRISPR / Cas based genetic engineering. CRISPR gene editing works by delivering a Cas nuclease

[0102] 30 enzyme complexed with a synthetic guide RNA (gRNA) into a cell. A CRISPR gRNA is a guide molecule that directs the Cas enzyme to a specific DNA sequence, while a homology directed PCT / US25 / 54216 05 November 2025 (05.11.2025) repair (HDR) template is a piece of DNA designed to be used by the cell's natural repair process to introduce precise edits like mutations, insertions, deletions, or substitutions. Together, the gRNA / Cas complex creates a double-strand break at a targeted location, and the HDR template provides a new sequence for the cell to use to repair the break, resulting in a precise genome

[0103] 5 modification. The present disclosure provides gRNAs and HDR templates to introduce phosphorylation mutations into the 53BP1 protein.

[0104] In some embodiments, the crRNA sequence of SEQ ID NO: 7 or the gRNA of SEQ ID NO: 23 may be used to introduce the T334A mutation into cells and SEQ ID NOs: 8, 21 or 22 may be used as a homology donor template sequence for homology-directed repair. In some embodiments, either SEQ ID NO: 13 or SEQ ID NO: 14 are used as gRNAs along with the HDR template of SEQ ID NO: 12 to introduce the S552A mutation into 53BP1.

[0105] In some embodiments, either SEQ ID NO: 16 or SEQ ID NO: 17 are used as gRNAs along with the HDR template of SEQ ID NO: 15 to introduce the T1056A mutation into 53BP1.

[0106] In some embodiments, either SEQ ID NO: 19 or SEQ ID NO: 20 are used as gRNAs along

[0107] 15 with the HDR template of SEQ ID NO: 18 to introduce the T1609A mutation into 53BP1.

[0108] In some embodiments, either SEQ ID NO: 22 or SEQ ID NO: 23 are used as gRNAs along with the HDR template of SEQ ID NO: 21 to introduce the A1678A mutation into 53BP1.

[0109] The engineered 53BP1 polypeptides of the present invention comprise one or more mutations relative to the corresponding wild-type polypeptide (i.e., the wild-type version of the

[0110] 20 same 53BP1 polypeptide). The term “wild-type” is used to describe the non-mutated version of a polypeptide that is most typically found in nature. A wild-type 53BP1 polypeptide may comprise any isoform of 53BP1. The wild-type 53BP1 protein may comprise SEQ ID NO: 1-3, which corresponds to wide-type isoforms 1-3. Wild-type isoforms 1-3 with a T334A mutation correspond to SEQ ID NO: 4-6 respectively. In some embodiments, an engineered 53BP1 polypeptide may comprise SEQ ID NO: 1 with at least one mutation at a position selected from the group consisting of S222, S294, T334, S379, S380, S552, S809, S862, T1056, T1609, S1678 and combinations thereof. In some embodiments, an engineered 53BP1 polypeptide may comprise SEQ ID NO: 1 with at least one mutation at a position selected from the group consisting of T334A, S552A, T1056A, T1609A, S1678A and combinations thereof SEQ ID NO: 24 of the

[0111] 30 present disclosure comprises the human 53BP1 peptide (SEQ ID NO: 1) with mutations (X) at position 222, 294, 334, 379, 380, 552, 809, 862, 1056, 1609, and 1678. In some embodiments, the PCT / US25 / 54216 05 November 2025 (05.11.2025) engineered 53BP1 polypeptide may comprise SEQ ID NO: 24, or a sequence with at least 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO: 24, wherein at least one of positions 222, 294, 334, 379, 380, 552, 809, 862, 1056, 1609, and 1678 is mutated relative to the amino acid at the position in the wild-type sequence. Also included are cells, suitably lymphocytes, that are

[0112] 5 engineered to overexpress a mutant or wild-type isoform of 53BP1. Over expression of wild-type 53BP1 by a cell may be sufficient to overcome inactivation by GSK30.

[0113] A single construct or vector may further comprise a second heterologous promoter operably linked to a second polynucleotide encoding a chimeric antigen receptor (CAR). Such dual-promoter constructs may allow for simultaneous expression of both the engineered 53BP1 polypeptide and a CAR in the same cell. Alternatively, a cell may have both the 53BP1 construct or vector and a CAR containing construct or vector introduced into the cell. The second heterologous promoter may be the same as or different from the first promoter. The chimeric antigen receptor encoded by the second polynucleotide may target various tumor-associated antigens. In some cases, the CAR may target antigens such as EGFR, CD 19, HER2, PSMA, GD2,

[0114] 15 or mesothelin. For example, the engineered human 53BP1 polypeptide comprising a substitution mutation may be combined with a CAR specific for GD2 or EGFR. The CAR may comprise an extracellular antigen-binding domain, a transmembrane domain, and one or more intracellular signaling domains.

[0115] The term "chimeric antigen receptor" or “chimeric receptor” or "CAR" or "CARs" as used

[0116] 20 herein refers to a polypeptide having a pre-defined binding specificity to a desired target and operably connected to (e.g., as a fusion or as separate chains linked by one or more disulfide bonds, etc.) the intracellular part of a T-cell activation domain. More particularly, CAR are engineered receptors, which, when expressed graft an antigen specificity onto a cytotoxic cell, for example T cells, NK cells or macrophages. For example, CAR proteins are engineered to give T cells the new ability to target a specific protein. The CARs of the present invention may comprise an extracellular domain with at least one antigen specific targeting region, a transmembrane domain (TM), and an intracellular domain (ID) including one or more co-stimulatory domains (CSD) in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic domain are not naturally found

[0117] 30 together on a single receptor protein. Further, the chimeric receptor is different from the TCR expressed in the native T cell lymphocyte. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0118] An extracellular domain is external to the cell or organelle and functions to recognize and respond to a ligand. A transmembrane domain spans the membrane of a cell, and an intracellular domain is situated inside a cell. Intracellular co-stimulatory domains provide secondary signals to the cell. They can recruit signaling molecules, cytoskeletal mobilization or induce cell

[0119] 5 proliferation, differentiation or survival. In the present disclosure a CAR may include an antigen specific extracellular domain, a transmembrane domain and one or more intracellular domains with one or more co-stimulatory domains.

[0120] The CAR of the present disclosure may comprise a transmembrane and hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be positioned between the antigen recognition moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0121] The transmembrane domain may be derived either from a natural or from a synthetic

[0122] 15 source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon , CD45, CD4, CD5, CD8 (e.g. , CD8 alpha, CD8 beta), CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, 0X40, CD2,

[0123] 20 CD27, LFA-1 (CD11 a, CD18) , ICOS (CD278) , 4-1 BB (CD 137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1 ) , CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11 d, ITGAE, CD103, ITGAL, CD11 a, LFA- 1, ITGAM, CD11 b, ITGAX, CD11 c, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, DNAM 1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM 1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, and PAG / Cbp. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a

[0124] 30 synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the PCT / US25 / 54216 05 November 2025 (05.11.2025) endoplasmic domain of the CAR. In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain or can be different transmembrane domains.

[0125] The CAR of the present disclosure may comprise at least one intracellular signaling

[0126] 5 domain, region or co-stimulatory molecule. The intracellular signaling domain may be a costimulatory domain. A costimulatory domain is required for an efficient antigen response in immune cells. In particular embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3i^ (TCRzeta, GenBank aceno. BAG36664.1). T-cell glycoprotein CD3 zeta (CD3^ chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene. The CAR of the present invention may also optionally comprise additional co-stimulatory domains, including CD28, 4-1BB, OX-40, ICOS or other members of the TNF receptor superfamily or immunoglobulin (Ig) superfamily. Members of the TNF superfamily form trimeric structures, and their monomers are composed of beta-strands that orient themselves into a two-sheet structure. The TNF superfamily ligands include lymphotoxin alpha, tumor necrosis factor, lymphotoxin beta, 0X40 ligand, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, CD137 ligand, TNF-related apoptosis-inducing ligand, receptor activator of nuclear factor kappa-B ligand, TNF-related weak inducer of apoptosis, a proliferation-inducing ligand, B-cell activating factor, LIGHT, vascular endothelial growth factor, TNF superfamily member 18 and ectodysplasin A. These ligands then bind to receptors in the TNF superfamily. Ig superfamily members are characterized based on shared structural features with immunoglobulins (aka antibodies), including an immunoglobulin domain with a characteristic Ig- fold. The Ig domain is reported to be one of the most populous family of proteins in the human genome with over 700 members identified and known in the art. These co-stimulatory domains may be used in isolation or in any combination.

[0127] 25 While the CAR of the present disclosure is exemplified with the above mentioned co- stimulatory molecules, other co-stimulatory domains, including CD27, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA- 1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2 can be used alone or in combination with other co-stimulatory molecules. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0128] The CAR of the present disclosure may comprise a signal sequence or signaling domain. A signal sequence plays a determinant role in protein distribution and can allow the CAR to be glycosylated and anchored in the cell membrane.

[0129] The antigen binding domain of a CAR may bind to a single target, or multiple targets. CAR

[0130] 5 T cells of the present invention may include, without limitation, traditional second, third or fourth generation CAR T cells including a 41BB / CD28 receptor.

[0131] This disclosure includes CAR T cells engineered to overexpress 53BP1 or express a mutant version of the DNA repair protein 53BP1. This mutation abolishes inhibitory phosphorylation by the kinase GSK3[3, allowing increased rates of DNA repair. This addition will enhance T cell cytotoxicity in solid tumors and in combinatorial therapies by increasing DNA repair, preventing T cell dysfunction. Thus, these modifications should improve solid tumor response to CAR T cell therapy. An example of a CAR T cell of the present invention is shown in Figure 18, which includes a CD19-28z CAR T cell which has been modified with a crRNA (SEQ ID NO: 7) or gRNA (SEQ ID NO: 9) to introduce the T334A mutation into genomic 53BP1. In some

[0132] 15 embodiments, a CAR T cell of the present disclosure may be modified with a gRNA to introduce a mutation into genomic 53BP1 at a least one position, wherein the position is selected from S222, S294, T334, S379, S380, S552, S809, S862, T1056, T1609, S1678 and combinations thereof. For example, the gRNA of SEQ ID NO: 13 or SEQ ID NO: 14 may be used to introduced the S552A mutation; the gRNA of SEQ ID NO: 16 or SEQ ID NO: 17 may be used to introduce the T1056A

[0133] 20 mutation; the gRNA of SEQ ID NO: 19 or SEQ ID NO: 20 may be used to introduce the T1609A mutation; or the gRNA of SEQ ID NO: 22 or SEQ ID NO: 23 may be used to introduce the S1678A mutation in 53BP1 in a CAR T cell. These 53BP1 mutated CAR T cells lead to persistent in vitro killing of a human tumor cell line.

[0134] Methods of making engineered cells:

[0135] The present disclosure provides methods of generating an engineered lymphocyte described herein. In some embodiments, the method comprises introducing a construct or vector described herein into an ex-vivo lymphocyte. In some embodiments, the construct is introduced via transduction into the ex-vivo lymphocyte. The construct may comprise a heterologous promoter operably linked to a polynucleotide encoding a 53BP1 polypeptide, wherein the 53BP1

[0136] 30 polypeptide comprises at least one substitution mutation at a GSK30 phosphorylation site. The vector may comprise the polynucleotide encoding the engineered 53BP1 polypeptide or the PCT / US25 / 54216 05 November 2025 (05.11.2025) construct described herein. In some embodiments the lymphocyte is genetically engineered using CRISPR / Cas based methods as described more fully below.

[0137] The ex-vivo lymphocyte may be isolated from a subject using standard cell separation techniques. In some cases, lymphocytes may be obtained from peripheral blood, lymph nodes,

[0138] 5 spleen, or other lymphoid tissues. The lymphocytes may be purified using density gradient centrifugation, magnetic-activated cell sorting (MACS), or fluorescence-activated cell sorting (FACS) to obtain specific lymphocyte populations. The cells may be activated or otherwise manipulated ex vivo to enhance the cells or the ability to act as effectors once re introduced to the subject. The lymphocytes may be autologous to the subject.

[0139] The construct or vector may be introduced via transduction into the ex-vivo lymphocyte. Transduction may involve the use of viral vectors such as lentiviral vectors, retroviral vectors, or adeno-associated virus (AAV) vectors. Lentiviral transduction may be performed by incubating the activated lymphocytes with lentiviral particles containing the construct encoding the engineered 53BP1 polypeptide.

[0140] 15 Alternatively, the construct may be introduced via CRISPR / Cas gene editing of the ex-vivo lymphocyte. CRISPR / Cas gene editing may involve the delivery of guide RNAs (gRNAs), Cas proteins, and homology-directed repair templates to the lymphocytes. The gRNAs may target specific sites in the endogenous 53BP1 gene, while the Cas proteins may create double-strand breaks at these sites. The homology-directed repair templates may provide the desired mutant

[0141] 20 sequences for incorporation into the endogenous 53BP1 locus. The CRISPR / Cas components may be delivered to the ex-vivo lymphocytes using various methods, including electroporation, nucleofection, or lipofection.

[0142] Following genetic modification, the engineered lymphocytes may be expanded in culture to generate sufficient numbers for therapeutic applications. The expansion process may involve culturing the cells in media supplemented with cytokines such as interleukin-2 (IL-2), interleukin- 7 (IL-7), or interleukin- 15 (IL-15) to promote cell proliferation and survival. The expansion period may range from several days to several weeks, depending on the desired cell numbers and the specific application.

[0143] In some cases, the method may involve co-transduction or co-editing to introduce both the

[0144] 30 engineered 53BP1 construct and additional genetic modifications such as chimeric antigen receptors (CARs). The dual modification approach may require careful optimization of the PCT / US25 / 54216 05 November 2025 (05.11.2025) delivery conditions to ensure efficient introduction of both genetic elements while maintaining cell viability and function.

[0145] The engineered lymphocytes generated through these methods may demonstrate enhanced resistance to DNA damage and reduced susceptibility to T cell exhaustion compared to unmodified

[0146] 5 lymphocytes. These properties may make the engineered lymphocytes particularly suitable for adoptive cell therapy applications, especially in challenging tumor microenvironments where DNA damage and T cell exhaustion may limit the effectiveness of conventional cell therapies. Compositions for genetic engineering

[0147] Another aspect of the present disclosure provides a composition comprising a gRNA or set of gRNAs capable of introducing at least one mutation into a 53BP1 polypeptide with at least 95% identity to SEQ ID NO: 1. The mutation may be selected from the group consisting of T334, S552, T1056, T1609, SI 678 and combinations thereof. Such compositions may enable precise gene editing of endogenous 53BP1 sequences to introduce substitution mutations that prevent GSK30 phosphorylation at specific sites. In some embodiments, the 53BP1 polypeptide comprises at least

[0148] 15 one mutation at a position corresponding to SEQ ID NO: 1 selected from the group consisting of T334, S552, T1056, T1609, S1678 and combinations thereof. In some embodiments, the composition comprises a gRNA comprising at least one of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and combinations thereof. The HDR or homology directed repair

[0149] 20 sequences SEQ ID NO:s: 8, 10, 11, 12, 15, 18 and 21 may also be included and introduced into cells with the gRNA and Cas protein.

[0150] A composition described herein may further comprising a Cas protein or a polynucleotide encoding a Cas protein. The Cas protein may be Cas9, Cas 12, Casl3, or other CRISPR-associated nucleases capable of creating double-strand breaks at the target sites specified by the gRNAs. In some cases, the Cas protein may be provided as a purified protein that forms a ribonucleoprotein complex with the gRNA. Alternatively, the Cas protein may be encoded by a polynucleotide that may be co-delivered with the gRNA to target cells. Generally, any Cas nuclease capable of introducing a mutation into 53BP1 may be used in the present disclosure. Cas nucleases include, for example Cas9 or a variant of Cas9 such as a high-fidelity Cas9 variant or another Cas protein

[0151] 30 with similar function such as Cas 12a. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0152] The engineered lymphocytes may be generated using homology-directed repair templates with specific sequences for precise gene editing. These templates may comprise DNA sequences that correspond to the desired mutant 53BP1 sequences, flanked by homology arms that match the genomic sequences surrounding the target sites. The homology-directed repair templates may be

[0153] 5 designed to introduce specific nucleotide changes that result in the desired amino acid substitution. The homology directed repair templates are provided as SEQ ID NO:s: 8, 10, 11, 12, 15, 18 and 21 for several of the mutations described herein.

[0154] The compositions may include additional components to enhance gene editing efficiency or cell viability. Such components may include small molecule enhancers of homology-directed repair, cell permeabilization agents, or protective compounds that reduce cellular stress during the editing process. The compositions may be formulated for various delivery methods, including electroporation, lipofection, or viral transduction.

[0155] Methods of treatment

[0156] The inventors have tested the function of 53BP1 mutant T cells in the head and neck

[0157] 15 squamous cell carcinoma (HNSCC) murine tongue model and found that these T cells have increased infdtration based on IHC and flow cytometry staining. The inventors have also shown that these T cells in vitro have increased resistance to irradiation.

[0158] The present disclosure provides methods of treating cancer in a subject. In some embodiments, the method comprises administering a therapeutically effective amount of a

[0159] 20 lymphocyte, or engineered lymphocyte described herein and a pharmaceutically acceptable excipient, carrier and / or diluent. The therapeutically effective amount is the amount required to treat the cancer in the subject. In some embodiments, the method further comprises administering an additional cancer therapy, which may include but is not limited to an immune checkpoint inhibitor, radiation or another cancer therapeutic. The additional cancer therapy may be administered before, at the same time as or after the administration of the lymphocytes or engineered lymphocytes described herein. The cancer may be a solid tumor and may be selected from the group consisting of head and neck squamous cell carcinoma, breast cancer, lung cancer, pancreatic cancer, and glioblastoma. Solid tumors may present particular challenges for immunotherapy due to the immunosuppressive tumor microenvironment and physical barriers that

[0160] 30 may impede effective T cell infiltration and function. The engineered lymphocytes expressing PCT / US25 / 54216 05 November 2025 (05.11.2025) mutant 53BP1 may demonstrate enhanced survival and function within solid tumor environments compared to conventional lymphocytes.

[0161] A “subject in need thereof’ as utilized herein may refer to a subject in need of treatment for a disease or disorder associated with a suspected tumor or cancer such as a head and neck

[0162] 5 squamous cell carcinoma. A subject in need thereof may include a subject having a cancer that is characterized by gross abnormality visible by X-ray, computerized tomography (CT), or magnetic resonance imaging (MRI), but which has not been diagnosed as a central nervous system (CNS) tumor by histology or immunofluorescence. In some embodiments, the tumor comprises a solid tumor.

[0163] The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. As used herein, “subject” or "patient" refers to both mammals and non-mammals. The term “subject” does not denote a particular age or sex. In one embodiment, the subject is a human.

[0164] As used herein, "treatment,” “therapy” and / or “therapy regimen” refer to the clinical

[0165] 15 intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition. As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the

[0166] 20 probability of developing a disease, disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition. The term "effective amount" or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0167] As used herein, “cancer” is a term for diseases in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body. Without limitation, cancer includes, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, Gastric cancer, head & neck cancers, Hodgkin’s lymphoma, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, non-hodgkin’s lymphoma, ovarian cancer, pancreatic cancer, prostate

[0168] 30 cancer, rectal cancer, renal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine PCT / US25 / 54216 05 November 2025 (05.11.2025) cancer and any other cancer or solid tumor. In some embodiments, the cancer is head and neck squamous cell carcinoma.

[0169] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation or composition to a subject comprising the one or more

[0170] 5 polypeptides, constructs or lymphocytes described herein. Such methods are well known to those skilled in the art and include, but are not limited to, transdermal administration, administration by inhalation, nasal administration, and parenteral administration, including injectable such as intramuscular administration, intertumoral administration, intradermal administration, and subcutaneous administration.

[0171] Treating cancer in a subject includes reducing, repressing, delaying or preventing cancer growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. Treating cancer in a subject also includes the reduction of the number of tumor cells within the subject. The term "treatment" can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting

[0172] 15 the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; and (d) reducing or ameliorating at least one symptom of cancer. In some embodiments, the optimum effective amount can be readily determined by one skilled in the art using routine experimentation.

[0173] As used herein, the term “carrier” refers to a pharmaceutically acceptable solid or liquid

[0174] 20 fdler, diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and / or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories, may be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990).

[0175] Some examples of the materials which can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository

[0176] 30 waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and PCT / US25 / 54216 05 November 2025 (05.11.2025) polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and

[0177] 5 lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions, according to the desires of the formulator.

[0178] Examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0179] In another embodiment, the present formulation may also comprise other suitable agents

[0180] 15 such as a stabilizing delivery vehicle, carrier, support or complex-forming species. The coordinate administration methods and combinatorial formulations of the instant invention may optionally incorporate effective carriers, processing agents, or delivery vehicles, to provide improved formulations for delivery of the polypeptides, constructs or lymphocytes described herein.

[0181] The composition may additionally include a biologically acceptable buffer to maintain a

[0182] 20 pH close to neutral (7.0-7.3). Such buffers preferably used are typically phosphates, carboxylates, and bicarbonates. More preferred buffering agents are sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate. The buffer may comprise about 0.0001-5% (w / v) of the vaccine formulation, more preferably about 0.001-1% (w / v). Other excipients, if desired, may be included as part of the final vaccine formulation.

[0183] The present disclosure also provides a method for increasing resistance to DNA damage. In some embodiments, the method comprises introducing the engineered polypeptide, or construct described herein to a cell. DNA damage is any change to the chemical structure of DNA that alters its coding properties or interferes with cell function. In some embodiments, the DNA damage is

[0184] 30 double stranded DNA breaks. In some embodiments, the DNA damage is caused by chemotherapy, radiation therapy, immunotherapy, or other cancer treatment. The inventors have found T cells PCT / US25 / 54216 05 November 2025 (05.11.2025) accumulate DNA damage during activation and chronic stimulation, contributing to T-cell dysfunction. Without wishing to be bound by any theory, the 53BP1 mutation described herein abolishes inhibitory phosphorylation by the kinase GSK30, allowing increased rates of DNA repair. Thus, the 53BP1 polypeptides comprising the substitution mutations may work well in

[0185] 5 combination with other cancer therapeutics whose use may be limited in combination with CAR T cells due to antagonism between the two treatments which may be alleviated by inclusion of the 53BP1 mutant polypeptides.

[0186] The cells provided herein may be administered in combination with immune checkpoint inhibitors. Immune checkpoint inhibitors which may be used according to the invention are any that disrupt the inhibitory interaction of cytotoxic T cells and tumor cells. These include but are not limited to anti-PD-1 antibody, anti-PD-Ll antibody, anti-CTLA4 antibody, anti- LAG- 3 antibody, and / or anti-TIM-3 antibody. Commercially available and approved checkpoint inhibitors in the U.S. include Atezolizumab (PD-L1), ipimilumab (CTLA-4), pembrolizumab (PD-1), nivolumab (PD-1), avelumab (PD-L1), durvalumab (PD-L1), cemiplimab (PD-1), and

[0187] 15 tislelizumab PD-1). The inhibitor need not be an antibody, but can be a small molecule or other polymer. Structures and potencies of several small molecule inhibitors of PD-L1 have been published. If the inhibitor is an antibody it can be a polyclonal, monoclonal, fragment, single chain, or other antibody variant construct. Inhibitors may target any immune checkpoint known in the art, including but not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA,

[0188] 20 HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and the B-7 family of ligands. Combinations of inhibitors for a single target immune checkpoint or different inhibitors for different immune checkpoints may be used. Additionally, colony stimulating factor 1 receptor (CSF-1R) blockade may be used in combination or as an alternative to immune checkpoint inhibitor(s), to ensure generation of potent and sustained immunity that effectively eliminates distant metastases and recurrent tumors.

[0189] Checkpoint inhibitors that comprise anti-PDl antibodies or anti-PDLl- antibodies or fragments thereof are known to those skilled in the art, and include, but are not limited to, cemiplimab, nivolumab, pembrolizumab, MEDI0680 (AMP-514), spartalizumab, camrelizumab, sintilimab, toripalimab, dostarlimab, and AMP-224. Checkpoint inhibitors that

[0190] 30 comprise anti-PD-Ll antibodies known to those skilled in the art include, but are not limited to, atezolizumab, avelumab, durvalumab, and KN035. The antibody may comprise a monoclonal PCT / US25 / 54216 05 November 2025 (05.11.2025) antibody (mAb), chimeric antibody, antibody fragment, single chain, or other antibody variant construct, as known to those skilled in the art. PD-1 inhibitors may include, but are not limited to, for example, PD-1 and PD-L1 antibodies or fragments thereof, including, nivolumab, an anti- PD-1 antibody. Fragments of PD-1 or PD-L1 antibodies include those fragments of the antibodies

[0191] 5 that retain their function in binding PD-1 or PD-L1 as known in the art.

[0192] The method may include combination with radiation therapy, specifically fractionated ionizing radiation at doses of 3 Gy x 3 daily regimen. The engineered lymphocytes expressing mutant 53BP1 may demonstrate increased resistance to radiation-induced DNA damage compared to wild-type lymphocytes, allowing for more effective combination therapy approaches. The method may include combination with chemotherapy agents such as cisplatin

[0193] The term "combination therapy" is used in its broadest sense and means that a subject is administered at least two agents. More particularly, the term "in combination" with respect to therapy administration refers to the concomitant administration of two (or more) active agents for the treatment of a disease state. As used herein, the active agents may be combined and

[0194] 15 administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms.

[0195] 20 Further, the presently disclosed compositions can be administered alone or in combination with adjuvants that enhance stability of the agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase activity, provide adjuvant therapy, and the like, including other active ingredients. In some embodiments, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0196] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents

[0197] 30 relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0198] The agents, whether administered alone or in combination, may be administered multiple times, and if administered as a combination, may be administered simultaneously or not, and on the same schedule or not. By way of example, a therapeutic composition may be administered multiple times per day, once per day, multiple times per week, once per week,

[0199] 5 multiple times per month, once per month, or as often as a doctor prescribes.

[0200] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms "synergy," "synergistic," "synergistically" and derivations thereof, such as in a "synergistic effect" or a "synergistic combination" or a "synergistic composition" refer to circumstances under which the biological activity of a combination of an agent and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually. In some embodiments, combination therapy may comprise a mutation 53BP1 protein in combination with one or more other cancer therapy which may include one or more of a CAR T cell, immune checkpoint inhibitor, adoptive cell therapy, other immunotherapy, chemotherapy, radiation therapy, and or surgery.

[0201] 15 Methods for increasing resistance to DNA damage or enhancing DNA repair in a cell may comprise introducing an engineered polypeptide, polynucleotide, construct, or vector into the cell. The engineered polypeptide may comprise 53BP1 with substitution mutations at GSK30 phosphorylation sites as described herein. The polynucleotide may encode such engineered 53BP1 polypeptides. The DNA damage may be double stranded DNA breaks. Double stranded DNA

[0202] 20 breaks may represent the most severe form of DNA damage and may be particularly relevant in the context of cancer therapy and T cell exhaustion. The engineered 53BP1 may enhance non- homologous end joining repair of double stranded DNA breaks. The DNA damage may be caused by chemotherapy, radiation therapy, or immunotherapy. Chemotherapy agents may induce various forms of DNA damage including cross-links, alkylation, and strand breaks. Radiation therapy may cause double strand breaks and other DNA lesions through direct ionization and indirect free radical formation. Immunotherapy may indirectly cause DNA damage through activation of immune effector mechanisms and inflammatory responses. The engineered lymphocytes may demonstrate enhanced survival and function under all of these DNA damage-inducing conditions.

[0203] Additional definitions

[0204] 30 The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are PCT / US25 / 54216 05 November 2025 (05.11.2025) capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in

[0205] 5 the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps.

[0206] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0207] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean

[0208] 15 “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0209] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or

[0210] 20 minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0211] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0212] 30 Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated PCT / US25 / 54216 05 November 2025 (05.11.2025) herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible

[0213] 5 combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0214] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g, “a system having at least one of A, B and C” would include

[0215] 15 but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B”

[0216] 20 will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0217] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0218] 30 Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become PCT / US25 / 54216 05 November 2025 (05.11.2025) apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0219] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0220] EXAMPLES

[0221] Example 1- Inhibition of GSK3p-mediated 53BP1 T334 phosphorylation in T Cells Enhances Infiltration and Cytotoxicity Against Head and Neck Squamous Cell Carcinoma

[0222] Immunotherapy has greatly improved survival rates for a subset of patients across many tumor types, particularly hematological tumors. However, response is limited to less than half of patients in most cancers, especially in solid tumors. In head and neck squamous cell carcinoma (HNSCC), the overall immune checkpoint blockade (ICB) response rate is only 11-18% at 6 months1. Despite the success of ICB in a subset of patients, a better understanding of how to overcome resistance to ICB is needed to improve current immunotherapy treatments. Recent studies have highlighted the association of DNA repair protein expression with lymphocyte proliferation, activation, and response to PD1 checkpoint blockade. In this study, we explore the enhancement of ICB and adoptive T cell therapy via modulation of the DNA repair protein 53BP1. 53BP1 is a critical, rate-limiting factor for non-homologous end joining (NHEJ) repair of DNA double- stranded breaks. Moreover, 53BP1 binds and regulates the function of tumor suppressor p53, determining cell fate following genotoxic stress. Our previous work demonstrated that Glycogen Synthase Kinase-30 (GSK30) binds to and phosphorylates 53BP1 at amino acid T334 (murine T336) to suppresses NHEJ-mediated repair of DSBs. Our in vitro assessment of this site suggests that modulation of this phosphorylation site can enhance DNA repair efficacy. We then explored the therapeutic potential of modulating 53BP1 activity using a novel murine model harboring a threonine to alanine mutation at 53BP1 T336, eliminating GSK3P phosphorylation. We utilize this model in conjunction with orthotopic murine HNSCC tumors and combination PCT / US25 / 54216 05 November 2025 (05.11.2025) therapy with radiation and immune checkpoint blockade. Overall, this study highlights the potential of 53BP1 modulation to enhance T cell therapies.

[0223] Inhibition of the GSK3f)-53BP 1 Axis Improves T Cell Response in Head and Neck Squamous Cell Carcinoma. We hypothesize that activation of 53BP1 will enhance T cell function

[0224] 5 by promoting increased DNA repair instead of signaling apoptosis mechanisms. In this study we inhibit the phosphorylation of 53BP1 by the kinase GSK30, which is known to inhibit the function of 53BP1 in DNA repair. These changes in cellular signaling ultimately allow T cells to respond more effectively in a murine model head and neck squamous cell carcinoma, with co-treatment of radiation therapy or immune checkpoint blockade.

[0225] GSK3P inhibits 53BP1 function in NHEJ-mediated DNA damage repair

[0226] GSK3fi phosphorylates 53BP1 at T334 to inhibit NHEJ. We first identified a novel GSK3P phosphorylation site in 53BP1 which modulates its activity. Fig 2A) The sequence alignment of 53BP1. Letters in blue mark the putative phosphorylatable threonine that match the GSK30 consensus sequence. Fig 2B) Direct phosphorylation of 53BP1 by GSK3|3. In vitro kinase assay

[0227] 15 of GSK30 co-incubation with WT 53BP1, or T334A 53BPlthat was expressed in U2OS cells and purified by IP, and isotope-labelled phosphate with or without ATP. C) Schematic of LScel NHEJ reporter assay. Assessment of NHEJ repair of DSB as measured by %GFP positive 53BP1-KO U2OS cells reconstituted with WT or T334A mutant 53BP1. n=3. Fig 2D) Representative images of 53BP1 and H2AX foci in WT or GSK3|3-KO MEF cells reconstituted with WT or T334A

[0228] 20 53BP1. Fig 2E) Percentage of 53BP1 foci per MEF cell. n=50. Fig 2F) Percentage of H2AX foci per MEF cell. n=50 **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0229] Generation of 53BP1 T336A Mutant Mouse Model

[0230] Knock-in of53BPl T336A mutation. To investigate the function of the GSK3P-53BP1 axis in vivo, we developed a homozygous T336A knock-in mouse TA) in a C57BL / 6 background (InGenious targeting laboratory, NY) using CRISPR / Cas9 genome editing to introduce an ACA to GCT nucleotide substitution for T336 (Fig 3A). Pups expressing the mutant were identified by sequencing and further bred with C57BL / 6J to develop the colony. Mice were validated by sequencing genotype of the targeted site (Fig 3B). These animals are bom at Mendelian ratios and develop normally.

[0231] 30 Mouse Model of Irradiation Therapy PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0232] Mouse model to study tongue tumor response to IR. An X-rad 200 x-ray irradiation cabinet is used to treat mice. Shelf distance from source is 40cm. Fig 4A) Image of mouse holder used for irradiation. Fig. 4B) A lead plate is placed to protect mouse brains from x-ray treatment. Fig. 4C) Image of oral cavities of four mice for irradiation treatment.

[0233] 5 Ablation of the GSK3P-53BP1 axis enhances response to HNSCC

[0234] Experimental Design: To test the efficacy of 53BP1 TA mutation in vivo, we used the orthotopic HNSCC mouse model in mouse tongue (Fig. 5 A). M0C2 is a weakly immunogenic murine head and neck squamous cell carcinoma cell line that typically has poor responses to immune checkpoint blockade. For this experiment, we injected M0C2 tumor cells into the tongues of WT or 53BP1 TA mutant mice and monitored tumor growth after 16 days (Fig. 5B). In addition, we included an irradiation regimen of 3Gy on days 5, 6, and 7, as radiation therapy is commonly used to treat solid tumors, including HNSCC.

[0235] Results: We found that systematic ablation of the GSK3f!-53BPl axis in mice resulted in significant suppression of tumor progression and improvement of the tumor radiation treatment

[0236] 15 response in the orthotopic HNSCC model (Fig. 5C). Tumors were harvested at day 16 and sectioned transversely followed by immunocytochemistry analysis. Tumor volume was plotted based on measurements from the largest tumor section per mouse. *P<0.05, ***P<0.001.

[0237] 53BP1 T336A mutant mice have T-cell dependent delayed tumor formation

[0238] Rationale and Experimental Design: To test whether these results were due to an enhanced

[0239] 20 T cell response, we performed T cell depletion experiments within the M0C2 tumor model. For this experiment, we depleted either CD4+ T cells, CD8+ T cells, or all (CD3+) T cells (Fig. 6A). We find that depletion of any type of T cell resulted in similar tumor volumes between wildtype or 53BP1 TA mice, suggesting that T cells are responsible for the increased response in this model.

[0240] Results: We found that depletion of any subset of T cells (CD3+, CD4+, or CD8+) resulted in the loss of tumor suppression in the 53BP1 TA mutant mice (Fig. 6B). These results support our hypothesis that the observed tumor suppression in 53BP1 TA mice is dependent on T-cell activity. 53BP1 T336A mutant T cells have increased tumor infiltration in vivo

[0241] Rationale and Experimental Design. Because 53BP1 TA cells exhibited increased tumor response in vivo, we next investigated the infiltration capability of wildtype or 53BP1 TA cells

[0242] 30 using the orthotopic M0C2 model. To assess tumor infiltration, tumors were harvested at day 17 and analyzed using immunohistochemistry (IHC) for (Fig. 7A) and flow cytometry (B) for CD3+ PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0243] T cells. For flow cytometry, CD3+ T cells were identified by the staining pattern DAPI-, CD45+, CD19-, CD3+.

[0244] Results: Quantification of CD3+ cells (Aperio ImageScope) shows an increase in tumor infiltrating T cells in 53BP1 TA mice versus control. To further assess the status of the tumor

[0245] 5 infiltrating cells, we used flow cytometry to quantify infiltration of CD3+ cells. In agreeance with our IHC results, tumor infiltration analysis by flow cytometry also shows an increase in infiltrating CD3+ cells in 53BP1 TA mice versus control (Fig. 7B).

[0246] 53BP1 T336A mutant T cells have less DNA damage in vitro and in vivo

[0247] Rationale and Experimental Design: Because 53BP1 TA cells in vitro exhibit less DNA damage, we quantified the amount of DNA damage in TILs collected from the M0C2 tumor samples. For this, we sorted tumor-infiltrating T cells and performed comet assays (Trevigen) to quantify double-stranded breaks in vitro (Fig. 8 A) and in vivo (Fig. 8B). For in vitro analysis, CD4+ or CD8+ WT or TA T cells were subjected to culturing conditions mimicking T cell exhaustion via chronic co-stimulation, similar to conditions experienced in the tumor

[0248] 15 microenvironment.

[0249] Results: We find that 53BP1 TA T cells sustain less DNA breaks by comet assay in vitro and in vivo, suggesting 53BP1 TA T cells can better recover from DNA damage sources in the tumor microenvironment.

[0250] 53BP1 T336A mutant T Cells have enhanced survival after DNA damage

[0251] 20 Experimental Design: To test the DNA repair capabilities of 53BP1 TA mutant T cells in vitro, we subjected WT or TA mutant CD8+ T cells to DNA damage (0-2 Gy IR) followed by analysis using Cell Titer Gio (Fig. 9A) and Incucyte (Fig. 9B). CD4+ T cells were also analyzed for Cell Titer Gio.

[0252] Results: Cell Titer Gio results show that 53BP1 TA T cells have increased viability after 48 hours at all doses of irradiation. Incucyte data also shows increased survival and proliferation at various low doses of irradiation. These results support that the 53BP1 T336A mutation improve the DNA damage response specifically in mouse T cells.

[0253] 53BP1 T336A mutation enhances immune checkpoint blockade in HNSCC

[0254] Experimental Design and Rationale: We found that 53BP1 TA T cells showed decreased

[0255] 30 DNA damage after exhaustion. Immune checkpoint blockade (ICB) therapy is used clinically to reverse T cell exhaustion and therefore improve T cell function. We investigated the potential of PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0256] ICB therapy in our 53BP1 TA mutant model. Tumors were injected into the mouse tongue on Day 0, and immune checkpoint blockade therapy began on day 3 after tumor injection. aCTLA4 was administered at lOOug / mL every three days, while aPDl was administered at lOOug / mL every two days (Fig. 10A). IgG antibody control were administered to control groups. All tumors were

[0257] 5 harvested on day 16 after tumor injection. Tumor volume was measured by ICC and luminescence intensity at endpoint.

[0258] Results: ICB greatly enhanced the tumor response in both WT and TA mutant mice, with near complete control of tumors in TA mutant mice (Fig. 10B-D).

[0259] In this study we investigated the potential of ablation of the GSK30-53BP1 axis to improve the function of T cells in vitro and in vivo. For this, we used a mouse model with a threonine to alanine mutation to inhibit 53BP1 phosphorylation by GSK3P at that site. Our preliminary studies show that blockage of this phosphorylation mark increases the DNA damage response through non-homologous end joining. We find that mice harboring this 53BP1 mutation have greatly improved tumor response. Using T cell depletion, we discover that this response is dependent on

[0260] 15 the actions of 53BP 1 mutant T cells. We find that these T cells are better able to resist DNA damage from irradiation, exhibit improved infiltration in the M0C2 head and neck squamous cell carcinoma model, and show increased response to immune checkpoint blockade.

[0261] To accompany these experiments, we will perform in vivo analysis on the tumor-infiltrating lymphocytes (TILs) for DNA damage and phenotypic markers via flow cytometry, comet assay,

[0262] 20 and scRNAseq. We also are currently crossing these 53BP1 TA mutant mice with the OT-1 mouse strain. The OT-1 mouse model is engineered for specific targeting of all T cells against the OVA antigen. Using this model, we will isolate OVA-specific 53BP1 WT or TA T cells and test their ability to kill OVA-expressing tumor cells in vitro and in vivo. This model will help us understand the importance of the T cells in isolation from the rest of the immune system. We also plan to generate adoptive cell therapies expressing this 53BP1 mutation along with chimeric antigen receptors (CARs) against multiple tumor cell targets to assess the efficacy of this mutation in human T cells and amongst other tumor models. This will include both in vitro and in vivo experiments using humanized mice. This project highlights the potential of modulating the DNA damage response to increase the efficacy of immunotherapies in solid tumors.

[0263] 30 Overall, these experiments will give further insight into the mechanism by which 53BP1 activation is allowing increased infiltration of T cells into these tumors. These experiments PCT / US25 / 54216 05 November 2025 (05.11.2025) presented here will help us achieve our goal to increase efficacy of adoptive T cell therapies by manipulating the GSK30-53BP1 axis.

[0264] Disruption of the GSK30-53BP1 axis in T cells leads to increased T cell function in solid tumors

[0265] 5 Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cause of cancer death globally 1. Around 50% of patients with locally advanced HNSCC develop recurrence and / or distant metastasis (R / M) within 2 years of standard treatment. R / M tumors are generally incurable, with a median survival of only 10-12 months. Advances in understanding of the immune anti-tumor response has redefined clinical care for HNSCC with the use of immune checkpoint blockade (ICB)2. Nevertheless, the overall ICB response rate in HNSCC is only 11- 18% at 6 months3. Adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) or gene-modified T cells expressing novel T cell receptors or chimeric antigen receptors is another promising immune anti-tumor strategy despite limited success in solid tumors when compared with hematological malignancies4. Research has shown that T cell exhaustion (TEX),

[0266] 15 characterized by limited cytotoxicity and proliferative potential, is a significant challenge that must be overcome to improve the clinical efficacy of immunotherapies5, including ICB and ACT. This proposal aims to better understand the molecular pathways modulated during TEX and to investigate innovative approaches to promote T cell function in solid tumors.

[0267] Preliminary studies modeling TEX with chronic stimulation revealed that exhausted T

[0268] 20 cells accumulate unrepaired DNA double-strand breaks (DSBs), as shown by increased yH2AX and fragmented chromosomes. 53BP1 is a key DNA damage response protein that controls both the non- homologous end-joining (NHEJ), a critical pathway for the repair of DNA DSBs, and cell survival following DSBs. We found that glycogen synthase kinase 30 (GSK30) directly phosphorylates human 53BP1 at threonine 334 (T334), which inhibits its function in NHEJ- mediated DSB repair. However, in vivo systemic ablation of the GSK30-53BP1 axis in homozygous 53BP1 T336A (equivalent to T334A in human) mutant knock-in mice significantly suppressed tumor progression in syngeneic orthotopic HNSCC (M0C2) and enhanced the tumor response to ionizing radiation (IR) treatment. Immunohistochemistry (IHC) revealed increased tumor infiltrating T lymphocytes (TILs) in tumors from T336A mutant mice, indicating an

[0269] 30 elevated host immune response, while CD4+ or CD8+ depletion eliminated the suppression of tumor progression in mutant mice, indicating a direct involvement of the host immune system. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0270] Importantly, ICB combination therapy (antibodies to PD1 and CTLA-4) was significantly more effective in mutant than wild type (WT) mice bearing M0C2 tumors. Furthermore, 53BP1 T336A mutant T cells were resistant to DNA damage, TEX, and exhibited decreased expression of genes associated with TEX in vivo as shown by single cell RNA sequencing analysis in

[0271] 5 tumors grown in WT or mutant 53BP1 mice.

[0272] Modeling TEX in human T cells. To stimulate TEX in vitro, isolated CD8+ T cells from three individual healthy donors were activated either once (acute stimulation), or once every two days until Day 8 (chronic stimulation) using the CD3 / 28 activator cocktail (StemCell Technologies), as previously described49 (Fig. 11A). Gene array analysis confirmed the upregulation of genes associated with inhibitory receptors (Ctla4, Lag3, Pdcdl, Tigit) and exhaustion- associated transcription factors (Nrd4al, Tox2), with simultaneous downregulation of memory-associated transcription factors (Klrgl, TCF7) (Fig. 11B). Flow cytometry analysis confirmed the increased expression of the surface inhibitory markers, CTLA-4, TIM3, LAG3, and PD1 (Fig. 11C). Lastly, Seahorse analysis confirms the metabolic phenotype of chronically

[0273] 15 stimulated T cells, which are known to utilize higher rates of glycolysis than acutely stimulated T cells (Fig. HD). These data confirm that in vitro chronic stimulation recapitulates the exhausted T cell phenotype.

[0274] Accumulation of DNA damage during TEX. To investigate the DNA damage response during TEX, we performed a series of DNA damage phenotyping experiments on acute or

[0275] 20 chronically stimulated T cells using the in vitro T cell exhaustion model detailed above. Western blot analysis and intracellular staining via flow cytometry both showed increased staining of yH2AX in exhausted T cells (Fig. 12A, C). Additionally, we see increased chromosomal breaks using alkaline comet assay (Fig 12B). To simulate DNA damage in primary T cells during activation, we used hydroxyurea (HU), which inhibits the formation of deoxyribonucleic acids by ribonucleotide reductase and thus eliminates circulating nucleotide pools, leading to the accumulation of DNA damage (Fig. 12C). HU treatment showed increased gH2AX, as expected, and increased expression of exhaustion markers, even in the absence of chronic stimulation (Fig. 12D) Overall, these data suggest that nucleoside deficiencies cause replication stress, leading to DNA damage that precedes TEX.

[0276] 30 GSK3P phosphorylates and inhibits 53BP1 function in NHEJ-mediated DNA damage repair. GSK30, a serine / threonine kinase, plays a key role in many cellular processes, including PCT / US25 / 54216 05 November 2025 (05.11.2025) insulin signaling, cell differentiation and apoptosis. We have demonstrated that GSK3 inhibition protects hippocampal neurons from ionizing radiation (IR)-induced cytotoxicity through enhancement of the NHEJ38,39. Importantly, in vitro (Fig. 2B) and in vivo kinase assay after immunoprecipitation of 53BP1 with an antibody specific for the T334 phosphorylation site,

[0277] 5 generated by us (data not shown), confirmed that GSK3J3 directly phosphorylates 53BP1 on T334 in humans (Fig. 2A-B) and inhibits 53BP1 activity, leading to the downregulation of NHEJ as measured by chromosomal NHEJ repair of DSBs (Fig. 2C), nuclear foci formation of 53BP1, and yH2AX abundance (Fig. 2D-F). In contrast to other key stress kinases (e.g., ATM / ATR / DNA-PK) that phosphorylate 53BP1 to enhance NHEJ activity, phosphorylation of 53BP1 by GSK30 inhibits its function, thereby representing a previously undescribed regulatory mechanism of NHEJ.

[0278] 53bp 1 T336A mouse generation. To further investigate the function of the GSK30-53BP1 axis in vivo, we developed a homozygous T336A knock-in mouse (named as TA mouse throughout the rest of the proposal) in a C57BL / 6 background (InGenious targeting laboratory, NY) using CRISPR / Cas9 genome editing to introduce an ACA to GCT nucleotide substitution for

[0279] 15 T336, which is the consensus site in the mouse 53BP1 sequence corresponding to the human T334 (Fig. 3A). Pups expressing the mutant were identified by sequencing and further bred with C57BL / 6J to develop the colony. Mice were validated by sequencing genotype of the targeted site (Fig. 3 A-B) These animals are bom at Mendelian ratios and develop normally. Interestingly, we found that the systemic ablation of the GSK30-53BP1 axis in mice resulted in significant

[0280] 20 suppression of tumor progression and improvement of the tumor radiation treatment response in the orthotopic HNSCC (M0C2) model (Fig. 4,5). These in vivo data indicate that the primary underlying mechanism is in the host and its TME.

[0281] Enhanced control of HNSCC tumors in TA mice is dependent on the immune system. Both GSK30 and 53BP1 have been reported to have intimate connection with immune function in the context of tumor, infection, and inflammation processes50-53. To determine if the immune system is responsible for the tumor growth reduction (Fig. 5C), we used monoclonal antibodies to systemically deplete all T cells (CD3+), helper T cells (CD4+) or cytotoxic T cells (CD8+) T cells prior to injecting M0C2 tumor cells into the tongue of 53BP1 mutant mice. T cell depletion was verified prior to tumor inoculation and at endpoint using FACS analysis of peripheral blood.

[0282] 30 Tumor size was determined using H&E staining on Day 16 after tumor injection (Fig. 6B). Surprisingly, depletion of CD4+ or CD8+ T cells completely reverted the suppression of M0C2 PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0283] HNSCC tumor growth, suggesting an improved immune control in the 53BP1 TA mice. Consistent with this finding, tumors from TA mice had an increase in CD3+ tumor infiltrating lymphocytes (TILs), quantified using IHC (Fig. 6, 7). These data support the hypothesis that disruption of the GSK3|3-53BP1 axis in T cells leads to increased T cell function in solid tumors.

[0284] 5 Mutant 53BP1 T cells resist exhaustion in vitro and in vivo. As shown in Figure 12, significant accumulation of DNA damage is associated with TEX under chronic stimulation. Given that the phosphodeficient mutant 53BP1 results in increased DNA repair (Fig. 2), we hypothesized that TILs in TA mutant mice are resistant to exhaustion in the TME and therefore able to better control tumor progression. To test this, we used a glioblastoma (GBM) model where tumor cells reside in the brain tissue. Single cell RNA sequencing (scRNA seq) was performed on tumors grown in WT or 53BP1 mutant mice. T cell clusters were identified in both tumors by expression of CD45, CD3, and CD4 / 8 (Fig. 13A). The T cell clusters were compared for differential expression of the genes associated with TEX and memory function (Fig. 13B). Consistent with our hypothesis, 53BP1 mutant T cells had reduced expression of the inhibitory

[0285] 15 receptor PD-1 and increased expression of the functional memory makers, Jun and Trafl. For rigor, we also performed FACS analysis on TILs from subcutaneous flank HNSCC tumors. In agreement with the IHC analysis, mutant mice had an increase in T cell numbers using FACS analysis (data not shown). These data suggests that disruption of the GSK3[3-53BP1 axis in T cells protects T cells from exhaustion in solid tumors.

[0286] 20 Our data demonstrate the importance of the GSK3P-53BP1 axis in TEX and the immune response to cancer, strongly supporting our central hypothesis that blocking the GSK3b-53BPl axis in T cells will enhance DNA repair, promote survival, limit TEX, and increase immune effector function in HNSCC. Thus, our goal is to directly test this hypothesis through a series of mechanistic and clinically relevant animal models. This study will be the first to investigate the GSK3P-53BP1 axis in the immune response to cancer and will provide a new understanding of TEX. The premise to our studies is that the accumulation of DNA damage precedes TEX and is a key hurdle in immunotherapy. Enhancing DNA repair to prevent TEX is a novel approach with the potential to improve the efficacy of immunotherapy for HNSCC and solid tumors.

[0287] Experimental systems: The model systems and endpoints used to carry out the proposed

[0288] 30 studies are listed below and have been validated in our published38,39,56,57 and extensive preliminary studies. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0289] In vitro cell models: (a) Primary human T cells: CD4+ and CD8+ T cells isolated from peripheral blood mononuclear cells (PBMCs) (Fig. 11-12); (b) Primary mouse CD4+ and CD8+ T cells isolated from mouse spleen.

[0290] In vivo animal models: We will use WT C57BL / 6 mice, 53BP1 TA knock-in (KI) mice,

[0291] 5 and 53BP1 knockout (KO) mice (Jackson Laboratory, strain #006495)58. These mice will be used for: (a) collection of T cells for in vitro DNA damage response; (b) in vivo T cell survival in response to DNA damage and TEX; (c) testing of syngenetic M0C1 and M0C2 orthotopic tumor (Fig. 3-7) response to ICB treatment, and to ACT therapy, either alone, or combined with radiation (3Gy x 3), or with the GSK3 |3i (10-30mg / kg body weight lithium, 50-150mg / kg Elraglusib)59,60; (d) generation of adoptive T cells for immunocompetent ACT modeling. We will use NSG mice for human SCC25 and FaDu orthotopic tongue and oral buccal mucosal tumors growth and response to CAR T cell therapy studies.

[0292] Syngenetic models. M0C1 and M0C2 are murine oral cancer (MOC) models extensively used in HNSCC research, particularly in the field of immunotherapy61. Derived from tumors in

[0293] 15 C57BL / 6 mice, these models express the HPV-16 E6 and E7 oncogenes, making them relevant for studies on HPV-associated cancers. M0C1, which forms well-differentiated squamous cell carcinomas and has a high mutation burden, is considered more immunogenic and is used to investigate immune responses, vaccine efficacy, and the dynamics of tumor growth and pathology. On the other hand, M0C2 produces poorly differentiated tumors and is less immunogenic, making

[0294] 20 it suitable for studying aggressive cancer behaviors and therapeutic resistance. The compatibility of these models with syngeneic hosts allows for detailed exploration of tumor-immune system interactions, enhancing our understanding of immune evasion and the potential effectiveness of new immunotherapeutic agents to treat HNSCC. For clinical relevance, tumor cells will be injected into the tongue tumor as in Figure 3-7 or alternatively orthotopically under oral buccal mucosa.

[0295] Human cell xenograft models. We will use the FaDu and SCC25 human tumor cell lines that are extensively used in mouse xenograft experiments modeling HNSCC62,63. FaDu, derived from a pharyngeal carcinoma, has aggressive growth characteristics, and is commonly used to model more invasive aspects of HNSCC. This cell line, along with SCC25, which originates from a tongue squamous cell carcinoma, often expresses high levels of EGFR (Epidermal Growth Factor

[0296] 30 Receptor). EGFR is an existing target for CAR T cell therapies due to its prominent role in promoting tumor growth and survival. These models provide crucial insights into the biology of PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0297] HNSCC and are instrumental in the development and testing of targeted therapies. These cell lines will be injected in the tongue and / or oral buccal mucosa, the clinically relevant tissue.

[0298] Endpoints: (a) DNA damage response (DRR) Signaling endpoints: they will be monitored by (i) GSK30 kinase activity assessed by immunoblot and / or IHC using a specific antibody to

[0299] 5 phospho-Glycogen Synthase (p-GS), a classical direct target of GSK30 (Cell Signaling, #9323); (ii) physical binding of 53BP1 to its downstream functional partner, RIF1 for NHEJ, or p53 for apoptosis will be determined biochemically using reciprocal co-immunoprecipitation (co-IP) assays57; (iii) recruitment of 53BP1 and RIF1 to DSBs will be assessed by IHC staining to demonstrate colocalization with gH2AX nuclear foci, an in situ marker (Fig. 2, 12); (iv) DNA damage-induced 53BP1 and RIF1 binding to chromatin will be measured by immunoblotting of chromatin fractions after irradiation (IR) and Cisplatin; (v) resection of DSB ends in irradiated cells will be measured by BrdU labeling (ThermoFisher) followed by immunofluorescence analysis for foci detection.64

[0300] DNA double-strand break (DSB) repair endpoints will include: (i) unrepaired DNA DSBs,

[0301] 15 quantified by levels of gH2AX nuclear foci, Western blotting, or flow cytometric assay with a gH2AX specific antibody (Fig. 2, 12); (ii) fragmented chromosomes, measured by alkaline / neutral comet assays (BioTechne / R&D, 4250-050-K) (Fig. 12); (iii) NHEJ DSB repair activity, assessed using chromosomal (Fig. 2d) and episomal NHEJ-specific plasmid reporters65.

[0302] T cell survival endpoints will include: (i) growth rate (Fig. 14A); (ii) survival fraction (Fig.

[0303] 20 14B); (iii) apoptosis assessed via FACS analysis of the apoptotic markers, Annexin V / PI, using standard techniques56.

[0304] TEX endpoints', (i) In vitro TEX will be assessed in primary CD8+ T cells isolated from mouse splenocytes and human PBMC. (Fig. 11, 12); (ii) For in vivo TEX in mice, we will utilize the lymphocytic choriomeningitis virus (LCMV) Clone 13 strain, which is known to induce chronic infection and TEX66.The mice will be infected intravenously (i.v.) with 1x106 plaqueforming units (PFU) of LCMV Clone 13 propagated using baby hamster kidney 21 cells (BHK21 [C13] (ATCC® CCL10). Following administration of the virus, viral load and immune response will be periodically monitored in the blood to quantify viral titers by RT-PCR. Simultaneously, the level of TEX will be assessed by flow cytometry, measuring the expression of TEX markers

[0305] 30 such as PD-1, LAG-3, and Tim-3 in CD8+ T cells. Furthermore, the functional impairment of T PCT / US25 / 54216 05 November 2025 (05.11.2025) cells, indicative of exhaustion, will be evaluated on day 3 post infection using FACS analysis for intracellular cytokine staining and proliferation tests.

[0306] Determine how GSK3P inhibits 53BP1 in NHEJ-dependent DSB repair, T cell survival, and TEX.

[0307] 5 Recent evidence suggests that GSK3P plays a role in lymphocyte fitness during DNA DSBs40,41. 53BP1 integrates NHEJ DNA repair and p53-dependent cell fate via distinct mechanisms26. Our preliminary studies revealed a novel functional connection between GSK30 and DNA damage repair through direct phosphorylation at T334 of human 53BP1 and inhibition of NHEJ activity in neurons38,39 and cancer cells (Fig. 2). Interestingly, the TA mutation in mouse CD4 / CD8 T cells resulted in increased survival following IR- induced DNA damage (Fig. 14). Importantly, suppression of orthotopic tongue M0C2 tumor progression and enhancement of tumor response to IR in TA mutant mice (Fig. 3,4) were associated with increased T cell infiltration in the tumor and decreased TEX (Fig. 6-7, 13).

[0308] Determine whether GSK3B phosphorylation of 53BP1 regulates physical and functional

[0309] 15 interactions with its RIF I to control NHEJ DSB repair activity, thereby affecting T cell resistance to To confirm that the GSK3P 53BP1 axis indeed inhibits NHEJ

[0310] DSB repair in CD4+ and CD8+ T cells, we will use a panel of genetically modified CD4+ and CD8+ T cells from selected transgenic mouse models: (i) WT C57BL / 6; (ii) 53BP1 TA KI; (iii) 53BP1 knockout (KO) mice; (iv) T cells from (iii) with retroviral re- expression (Fig. 15) of well

[0311] 20 characterized 53BP128A mutant which is functional deficient in NHEJ DSB repair55. This panel of T cells will be activated in culture and treated with low dose (0-3 Gy) of IR or 1-4 pg / mL Cisplatin to mimic clinical HNSCC treatment. To determine the effect of T336 phosphorylation on 53BP1 interaction with RIFI, we will measure (11 53BP1 binding with RIFI before and after IR, or Cisplatin. The kinetics of 53BP1 and RIFI binding will be determined at 1, 2, 4, 8, and 24 hours after DNA damage by reciprocal co IP using RIFI and 53BP1 antibodies: (2) recruitment of 53BP1 to DSBs and chromatin binding as detailed under NHEJ signaling endpoints.' (3) DNA damage repair endpoints,' (4) T cell survival endpoints. WT CD4+ and CD8+ T cells will serve as positive controls, while T cells that are deficient in NHEJ DSB repair due to 53BP1 KO or expressing 53BP128A mutant55 will serve as negative controls for 53BP1 function in NHEJ.

[0312] 30 Similar expression of 53BP1 and GSK3P will be verified for comparison of the results here. PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0313] We will confirm the role of the human T334 phosphorylation in the regulation NHEJ DSB repair in human CD4+ and CD8+ T cells. We will knockout endogenous 53BP1 with a CRISPR / Cas9-based approach, followed by lentiviral re-expression of the T334A mutant in human T cells, and examination of the same endpoints as described above in mouse T cells.

[0314] 5 To confirm that inhibition of 53BP1 NHEJ function is specifically dependent on GSK30 kinase activity, we will lit pharmacologically inhibit the kinase activity of GSK30 by pretreating the panel of CD4 / CD8 cells mentioned above with the GSK30 inhibitors (GSK30i), Elraglusib (0.5-5 pM) and Lithium (10 pg / ml) or with vehicle; ( ii) genetically inhibit GSK3[3 using CRISPR / Cas9- mediated knockout approaches. We will examine the same endpoints described above.

[0315] Determine if GSK3ft phosphorylation regulates 53BP1 physical and functional interaction with p53 to control and directlv decreases T cell survival. Both GSK30 and 53BP1 play a role in T cell death54. 53BP1 binds p53 to promote apoptosis by regulating p53-mediated transcriptional activation67,68. To investigate this, we will use the panel of 53BP1 -modified mouse T cells described above to measure; (1) the effect of equivalent mouse T336

[0316] 15 phosphorylation (pT336) on binding of 53BP1 with p53 before and after 0-3 Gy IR, or 1- 4 pg / mL

[0317] Cisplatin. The kinetics of 53BP1 and p53 binding will be determined at 1, 2, 4, 8, and 24 hours after DNA damage by reciprocal co-IP using p53 and 53BP1 antibodies: (2) the effect of pT336 on 53BP1 -mediated p53 transactivation of pro-apoptotic proteins, including PUMA and BAX69,70 measured by immunoblotting with their respective specific antibodies: (3) the effect of

[0318] 20 pT336 on DNA damage-induced and p53-dependent apoptosis, measured as described under T cell survival endpoints. CD4+ and CD8+ T cells from WT mice will be the positive controls and

[0319] WT / TA T cells with retroviral CRISPR / Cas9-mediated p53 KO will be negative controls.

[0320] Determine whether the GSK33-specific phosphorylation of 53BP1 is necessary and sufficient to control TEX in response to genotoxic stress using in vitro and in vivo human and murine models. For in vitro TEX study, we will utilize the panel of mouse and human T cells to determine the effect of phosphorylation of 53BP1 on TEX in response to accumulation of persistent DNA damage with and without IR, or Cisplatin as described in Figure 2-3 and in TEX endpoints. Positive and negative controls for these experiments are as described above. Inhibition of GSK30 kinase via genetic KO or GSK30i in the panel of 53BP1 manipulated T cells will serve

[0321] 30 as a confirmation of GSK30-dependent effect; £ 2) For in vivo TEX, we will infect the TA mice with the lymphocytic choriomeningitis virus (LCMV) Clone 13 strain as detailed under TEX PCT / US25 / 54216 05 November 2025 (05.11.2025) endpoints. WT C57BL / 6 and 53BP1 KO mice will serve as positive and negative controls, respectively. GSK3pi or vehicle treatment of infected mice will serve as confirmation of GSK30 kinase activity dependence of in vivo TEX.

[0322] Expected outcomes. Based on our Preliminary Data and previously characterized

[0323] 5 regulatory mechanisms of GSK3J3 and 53BP1 in DNA damage repair and cell fate / death, we propose that the molecular mechanisms of GSK3P-53BP1 axis in control of TEX in response to DNA damage include inhibition of NHEJ DSB repair and subsequent decrease of T cell survival through interaction with RIF1, and / or directly promoting of apoptosis through interaction with D53. 11) If GSK3P functions primarily to regulate 53BP1 in NHEJ pathway, then we would expect the 53BP1 phosphorylation mutant to show significantly increased binding to RIF1 and a modest increase in cell survival following low dose IR / Cisplatin, no increase in p53 binding. On the other hand, if GSK30 primarily regulates 53BP1 in apoptosis, then we would expect the phosphorylation mutant to show significantly decreased binding to p53, a robust increase in T cell survival regardless of IR / Cisplatin, and moderate or no increase in RIF1 binding. In both cases,

[0324] 15 phosphorylation mutant-expressing T cells will display increased survival of T cells and decreased TEXi (2) The two working models on the effects of GSK30 phosphorylation of 53BP1, namely direct inhibition of 53BP1-RIF1 interaction and NHEJ DSB repair and / or direct promotion of 53BPl-p53 interaction and cell apoptosis, are NOT mutually exclusive. 53BP1 NHEJ DNA repair and p53 -regulatory roles are distinct and separable26. Identifying a 53BP1 mutation that separates

[0325] 20 its binding to RIF1 from binding to p53 would help to elucidate the possibility ; (3) Understanding the molecular signaling processes for GSK30-53BP1 regulation of TEX through interaction with its downstream effectors RIF1 and / or p53 provides mechanistic support and may help determine how to target the GSK30-53BP1 axis to improve HNSCC response to immune checkpoint blockade therapy, and take advantage of TA mutant T cells resistant DNA damage-induced cell death and TEX, thereby promoting ATC therapy against HNSCC.

[0326] Determine the effect of blocking the GSK3P-53BP1 axis on tumor infiltrating lymphocyte (TIL) function and ICB response

[0327] Our Preliminary Data indicate that the disruption of the GSK3P-53BP1 axis enhances T cell tumor infiltration into solid tumors (Fig. 6,7), reduces TEX (Fig. 13), and ultimately leads to

[0328] 30 better control of tumor progression. These data suggest that the inhibition of this phosphorylation event has the potential to enhance immunotherapy response. In a pilot experiment, we determined PCT / US25 / 54216 05 November 2025 (05.11.2025) that M0C2 tongue in mice expressing the 53BP1 mutant (TA) are indeed more responsive to ICB (Fig. 10, 16). Furthermore, TA mutation rendered cells resistant to DNA damage insult, opening the door to combining ICB with standard of care such as radiotherapy, which can drastically sensitize the tumor to immune mediated control73,74.

[0329] 5 -53 B P l axis on HNSCC tumor environment and immune infiltration. To further validate our findings, we will use in vivo preclinical models of HNSCC to determine how genetic disruption of the GSK30-53BP1 axis alters the TIL phenotype.

[0330] TIL analysis. To determine the impact of blocking the host GSK30-53BP1 axis in TILs, tumor cell lines (M0C1 or M0C2), will be injected into the tongue, (5xl04 / mouse) as in Figure 3, 4. Tumors will be harvested on Day 10 to avoid highly necrotic tumor tissue. Tumors will be mechanically and enzymatically dissociated into single cell suspensions suitable for FACS analysis. We will perform multi-color flow cytometry on each harvested tumor. Full spectrum flow cytometry (Cytek Northern Lights) will be used to determine the percentage of helper T cells,

[0331] 15 regulatory T cells and cytotoxic T cells. For all cell subsets, we will determine the percentage of total cells (including live and dead tumor cells) and percentage of immune cells (CD45+). Additionally, expression of T cell exhaustion markers will be assessed, including CTLA-4, PD1, TIM3, and LAG3 (see Fig. 11). The groups for this experiment will be as follows: WT-M0C1, WT-M0C2, TA-MOC1, TA-MOC2 (N=6 per condition; 3F / 3M). Flow cytometry will be

[0332] 20 performed by the UAMS Flow Core and analyzed by Dr. Koss (MPI), who has >10 years flow cytometry experience. These experiments will provide genetic evidence establishing that GSK30- specific phosphorylation- deficient 53BP1 mutant alters the TIL phenotype.

[0333] TIL DNA damage analysis. We will assess the accumulation of DNA damage in mutant T cells within solid tumors. TILs will be enriched using magnetic-activated cell sorting positive selection for the T cell marker CD3. Following enrichment, T cells will then be labeled for fluorescence-activated cell sorting (FACS) of cytotoxic T-cells (CD45+, CD3+, CD8+) and helper T-cells (CD45+, CD3+, CD4+). Sorting will be performed with a FacsAria IIIu in the UAMS Flow Cytometry Core Facility. Immune cells will be used as in the DNA DSB repair endpoints detailed previously. These subsets of immune cells will be variable depending on the level of tumor

[0334] 30 inflammation. As shown in Figure 6, 7, tumors from mutant mice will have significantly more TILs. We anticipate that ~5% of tumor cells will be immune cells, while the majority will be PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0335] HNSCC cells. The groups for this experiment will be as described in the TIL analysis section above. These experiments will provide genetic evidence establishing that GSK3p-specific phosphorylation-deficient 53BP1 mutant alters TIL accumulation of DNA damage.

[0336] V(D)J scRNA sequencing. Single-cell RNA sequencing (scRNA-seq) coupled with V(D)J

[0337] 5 sequencing will be used to analyze TCR repertoires and transcriptomes of individual TILs75. Single-cell suspensions will be prepared from M0C1 and M0C2 tumors (Day 10) from either WT or TA mice. Viable cells will be enriched using a dead cell removal kit (Miltenyi). Viable cells will then be divided into nanoliter-scale Gel Beads-in-emulsion (GEMs) using the Chromium Controller (lOx Genomics), where cell lysis and barcoded reverse transcription (RT) of RNA will occur, capturing both mRNA and V(D)I-enriched cDNA. Following GEM-RT, the cDNA will be amplified, and libraries will be constructed for both 5' gene expression and V(D)J sequencing according to the manufacturer's protocol (lOx Genomics). The quality and quantity of the libraries will be validated using a Bioanalyzer (Agilent Technologies) and Qubit fluorometric quantitation (Thermo Fisher Scientific). The libraries will then be sequenced on an Illumina platform, aiming

[0338] 15 for a depth that ensures comprehensive coverage of both transcriptomes and TCR sequences. The resulting reads will be processed using the Cell Ranger software suite (lOx Genomics), which will provide demultiplexing, barcode processing, and gene counting. The V(D)J sequences will be specifically analyzed to reconstruct full-length TCR sequences, allowing for clonotype identification. Data integration and downstream analyses, including clustering and differential

[0339] 20 expression analysis, will be conducted using Seurat and custom scripts to elucidate the functional states and clonal expansion within the T-cell population. This approach will provide a high- resolution view of the T-cell heterogeneity. Dr. William Lu (Col) has 10+ years of experience using scRNAseq and his laboratory will perform sample preparation and data analysis. These experiments will provide genetic evidence establishing that GSK3p-specific phosphorylationdeficient 53BP1 mutant alters the TME, with a focus on TILs.

[0340] Determine the impact of genetic and pharmacological blockade of the GSK3B-53BP1 axis on

[0341] ICB alone and in combination with IR using preclinical mouse HNSCC models.

[0342] We will leverage our TA mutant mouse to directly test the impact of mutant 53BP1 on ICB response. Our approach will also include a clinically relevant combinatorial approach using IR and

[0343] 30 ICB74. We hypothesize that 53BP1 mutant mice will be highly responsive to this approach due to resistance to IR-induced T cell death (Fig. 14). Furthermore, we will use FDA approved GSK30i PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0344] Elraglusib and Lithium (in combination with ICB as a pharmacological approach to inhibiting 53BP1 phosphorylation.

[0345] ICB treatment. To assess the ability of the GSK30-53BP1 blockade to potentiate ICB therapy, we will leverage the M0C1 and M0C2 tumor models detailed above. To validate that the

[0346] 5 effects are not limited to M0C1 (Fig. 16), we will include M0C2 orthotopic models (tongue and oral buccal mucosa). On Day 3 after tumor injection, WT and TA mutant mice will be treated with IgG control antibodies or ICB combination as in Figure 16 and 10. Tumor volume will be calculated at endpoint (Day 16) as in Figure 6. These animal experiments have been approved by the UAMS IACUC, (See Vertebrate Animal Section). These experiments will provide evidence establishing that GSK30-specific phosphorylation-deficient 53BP1 mutant alters the ICB response.

[0347] Combination radiation and ICB treatment. T cells expressing TA mutant are resistant to radiation-induced damage (Fig. 14), providing an opportunity to explore the use of standard of care radiation therapy in combination with ICB while limiting any off-target effects. To mimic

[0348] 15 clinic radiotherapy regimens, these experiments will be carried out in two different ways: 1) Neoadjuvant (IR will precede ICB); and 2) Concurrent (IR and ICB will be administered on the same day). For all experiments, a fractionated IR (3 x 3 Gy) daily regimen will start on Day 3 post tumor inoculation using the X-RAD 320 as in Figure 3, 4. When ICB is delayed, injections will begin on Day 6 after tumor inoculation. Both M0C1 and M0C2 tumor cell lines will be used.

[0349] 20 Tumor size will be calculated at endpoint as described in Figure 5 and the analysis plan. These experiments will provide evidence establishing that GSK3P-specific phosphorylation-deficient 53BP1 mutant alters the ICB response when paired with IR.

[0350] Combination GSK3 inhibition and ICB treatment. We appreciate that the TA mutation has limited translatability when it comes to combination ICB approaches and serves primarily as a model system to explore the relevance of enhanced T cell function in preclinical models. To provide a translational approach, we will use ICB in combination with GSK30i such as Lithium, or 9-ING-41 / Elraglusib, a newly developed GSK30 specific inhibitor. Both have been used in clinical and preclinic settings. GSK30i have been used as monotherapy in other cancers but have not been explored as adjuvant or neoadjuvant therapy to ICB in HNSCC. Experiments will be

[0351] 30 performed in a similar fashion as the ICB experiments detailed above where tumor bearing mice (Day 3 after injection) will be treated with combination ICB and GSK3pi IgG control antibodies PCT / US25 / 54216 05 November 2025 (05.11.2025) or vehicle groups will be used as controls. Elraglusib (50-150 mg / kg body weight) or Lithium (10- 30 mg / kg body weight) will be administered every day until Day 1659,76. These experiments will provide evidence that pharmacological targeting of the GSK30-53BP1 axis using GSK30i can greatly enhance HNSCC tumor response to ICB treatment.

[0352] 5 Expected outcomes. Based on our preliminary findings, we expect to further delineate the influence of the GSK30-53BP1 axis on the functionality of TILs and the response to ICB with or without combination with IR. By inhibiting this axis, we anticipate several crucial findings, including an enhancement in the survival and proliferation of TILs within the TME, leading to increased T cell infiltration and reduced exhaustion, thereby fostering a more robust immune response. Additionally, we expect that immune cells with a TA mutation, including CD4+ and CD8+ T cells, will exhibit increased resistance to the off-target effects of IR. Both treatments are commonly used in combination with ICB in clinical settings. This increased resistance is anticipated to enhance the efficacy of the combined IR and ICB therapy. We also anticipate a significant potentiation of ICB by GSK30i, preventing tumor progression in HNSCC models.

[0353] 15 Comprehensive immune profiling (e.g., B cells, natural killer cells, tumor-associated macrophages, and fibroblasts) using single-cell RNA sequencing, will provide a detailed map of the immune landscape changes within the TME influenced by TA mutation. These insights will enhance our understanding of how the GSK30-53BP1 axis modulates immune dynamics and how its targeting could improve the efficacy of cancer immunotherapies, potentially leading to novel

[0354] 20 treatment approaches for HNSCC and other solid tumors.

[0355] Test the use of 53BP1 mutant T cells in adoptive cell transfer (ACT) therapies for HNSCC

[0356] ACT therapies for HNSCC are limited by the immunosuppressive TME and by physical barriers that impede effective T cell infiltration and function. Our Preliminary Data showed that disrupting the GSK30-53BP1 axis through the TA mutant enhanced DNA repair, and reduced TEX.

[0357] Demonstrate the use of TA mutant T cells in ACT therapy (immune competent). To determine if the TA mutant can enhance ACT in an immune competent setting, we will perform a series of in vitro and in vivo tumor killing experiments. These experiments are designed to directly test this hypothesis in an immune competent pre-clinical model of HNSCC.

[0358] In vitro tumor-; :. TA mutant mice will be crossed to the OT-1 transgenic line

[0359] 30 (Jackson Labs) to generate tumor specific T cells bearing the endogenous TA mutation. Purified and activated OT-1 CD8+ T cells will be co-cultured with M0C1 or M0C2 target cells expressing PCT / US25 / 54216 05 November 2025 (05.11.2025) the class I OT1 antigen SIINFEKL and nuclear red fluorescent protein (RFP; IncuCyte® NucLight) for live cell imaging. Target cells will be plated for 16 hr. prior to culturing with T cells. The co-cultures will be conducted at target: effector (T:E) ratios of 8:1 down to 0.5:1 in 96-well live cell imaging plates (Millipore). Plates will then be automatically imaged every 2 hours with

[0360] 5 an ImageXpress confocal HT.ai microscope (Molecular Devices) in the RFP channel. At each timepoint, nine unique sites will be imaged, and the total number of RFP+ (target) cells quantified. Plates will be imaged for up to 72 hr., and the values will be normalized to target cell counts at t = 0. OT-1 T cells will be rechallenged with new tumor cells every 72 hours until T cells fail to control tumor growth.

[0361] _ In vivo tumor killing. For in vivo tumor killing experiments, we will leverage the allelic variants of the CD45 protein tyrosine phosphatase receptor (CD45.1 and CD45.2) to distinguish host vs. transferred T cells. Purified and activated OT1 T cells (CD45.2) will be adoptively transferred (4 million / mouse) into mice (CD45.1) bearing either M0C1. SIINFEKL or M0C2. SIINFEKL tongue tumors (Day 3) (N=20 per condition, 10F / 10M). Tumor volume will be

[0362] 15 calculated at endpoint (Day 16) as in Figure 6, 7. Additionally, the percent of CD8+ / CD45.2+ vs. CD8+ / CD45.1+ tumor infiltrating lymphocytes will be determined from tumors at endpoint by flow cytometry.

[0363] Demonstrate the use of 53BP1 mutant T cells in CAR T cell therapy (Immune compromised). CAR T cell therapy is currently limited to the treatment of hematological

[0364] 20 malignancies and has failed to be effective in solid tumors. Here, we will directly test the ability of the TA mutant to enhance the function and persistence of CAR T cells in vitro and in vivo using human orthotopic models of HNSCC.

[0365] Human T cells with CRISPR 53BP1 KO and reconstitution of 53BP1 TA. To genetically modify 53BP1 in human T cells, we will use isolated CD8+ T cells, which will be activated and transduced with lentiviral particles simultaneously using methods established in the Koss laboratory. Two viral vectors will be used in a co- transduction approach. One vector contains the EGFR targeted CAR with a RFP reporter, and one contains the mutant 53BP1 fused with GFP as in Figure 2. At 24 hours post-activation, nucleofection (Lonza 4D Nucleofector System) will be used to introduce recombinant CAS9 bound to the 53BP1 guide RNA. For selection purposes, we

[0366] 30 will simultaneously target Thyl, a surface protein routinely used to select for successful transfer of CAS9 complexes into T cells. On Day 7 post-activation, T cells will be selected for RFP+ PCT / US25 / 54216 05 November 2025 (05.11.2025)

[0367] (CAR), GFP+ (53BP1 mutant), and Thyl- (KO control). This approach will allow us to replace endogenous 53BP1 with the TA mutant 53BP1. CAR T cells will be expanded until Day 10 and used in the in vitro and in vivo experiments detailed below.

[0368] CAR-T cell in vitro killing. To determine cytotoxic activity of CAR T cells, we will

[0369] 5 perform in vitro tumor-specific killing experiments as detailed herein. Exogenous expression of EGFR targeted CAR constructs (CD3z, 28z, or BBz) will provide the specificity required for T cell-mediated killing of EGFR+ tumor cells (FaDu and SCC25). CAR T cells will be purified prior to co-culture. Target tumor cells will be engineered to express RFP (IncuCyte® NucLight™ Red) for live cell imaging. To assess the ability of mutant CAR T cells to resist DNA damage while performing cytotoxic activities, we will perform killing assays in conjunction with exogenous DNA damage assays. Co-cultures will be set up as described previously, but with or without addition of IR (1 or 3 Gy) prior to plating in co-culture. Co-cultures will then be performed and analyzed as described herein.

[0370] _ In vivo CAR T tumor killing. To validate the therapeutic potential of TA mutant CAR T

[0371] 15 cells in vivo, we will perform a series of adoptive transfer experiments into tumor-bearing mice. CD8+ T cells will be isolated, activated, transduced, and sorted as described previously. CD8+ CAR T cells (CD3z, 28z, or BBz) will be adoptively transferred (4 million / mouse) into NOD / SCID / IL2Rgc-KO (NSG) mice bearing subcutaneous EGFR+ tumors (FaDu and SCC25). T cells will be intravenously injected 3 days after tumor injections when tumors are established.

[0372] 20 Tumor volume will be calculated on Day 16 as in Figure 6, 7.

[0373] Expected outcomes. Based on our preliminary findings, we expect 53BP1 mutant T cells to perform better in both syngeneic (immunocompetent) and human xenograft (immune- compromised) models of ACT. Transferred T cells expressing mutant 53BP1 should better control solid tumor growth. The prevention of T cell exhaustion in the use of ACT for solid tumors is the approach. The use of adoptive TA T cells in combination with bridging radiotherapy in vivo is also envisaged80.

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[0467] Example 2: Mutant 53BP1 and CAR T Cells

[0468] Mutant 53BP1 and CAR T Cells

[0469] This disclosure includes CAR T cells engineered to express a mutant version of the DNA repair protein 53BP1. This mutation abolishes inhibitory phosphorylation by the kinase GSK3b, allowing increased rates of DNA repair. Thus, this mutation leads to increased 53BP1 activity including increased DNA repair through non-homologous end joining. The inventors have shown that T cells accumulate DNA damage during activation and chronic stimulation, contributing to T- cell dysfunction. To improve CAR T cell resistance to DNA damage, the inventors have generated

[0470] 30 a mutant mouse expressing the 53BP1 T336A mutant. The inventors have shown that this mutation increases T-cell efficacy against head and neck squamous cell carcinoma (HNSCC) in a murine PCT / US25 / 54216 05 November 2025 (05.11.2025) tongue tumor model. Additionally, the inventors have shown that combination of this mutation with irradiation or immune checkpoint blockade further increases their efficacy in the murine HNSCC model. In addition, the inventors will include other 53BP1 activating mutations expected to enhance DNA repair. The inventors hope to enhance immunotherapy treatments against solid tumors by engineering CAR T cells to express this novel mutation with or without combinatorial treatment. Overall, the inventors expect to improve CAR T cell function, increase T cell infiltration in solid tumors, reduce T-cell dysfunction, and promote increased tumor cell death.

[0471] Figure 18 demonstrates that CAR T cells transduced with a gRNA (or crRNA for combination with a tracrRNA) designed to introduce the T334A mutation into genomic 53BP1 results in persistent CAR T cell killing of a human leukemia tumor cell line. CD8+ T cells were isolated from a healthy human donor, activated, and transduced with virus encoding GFP-labeled CD 19 targeting 28z CAR. On day 7, CAR T cells were sorted based on GFP expression and left to rest one day. Cells were then electroporated with either control RNA (scrambled) or crRNA designed to introduce the T334A mutation into genomic 53BP1. The sequence of the crRNA used in Figure 18 to introduce the CAR T cells is SEQ ID NO: 7, and the donor template for homology- directed repair is SEQ ID NO: 8. On day 12, T cells were plated at a ratio of 4:1 with NALM6 cells on a flat, glass bottom imaging plate (60,000 T cells to 15,000 tumor cells). Plate was imaged every 4 hours for two days. After 48 hours, wells were resuspended and half of the volume was transferred onto fresh tumor cells plated at the same density as the original co-culture. Replating was continued until all T cells were unable to control tumor cell outgrowth.

Claims

CLAIMSWhat is claimed:

1. An engineered human 53BP1 polypeptide comprising a substitution mutation at a position corresponding to an amino acid residue selected from the group consisting of 334, 222, 294, 379, 380, 552, 809, 862, 1056, 1609, 1678 and combinations thereof, wherein the wild-type 53BP1 polypeptide comprises any one of SEQ ID NO: 1-3 or sequences with at least 90% identity to SEQ ID NO: 1-3.

2. The engineered polypeptide of claim 1, wherein the substitution mutation changes the amino acid at the position to an amino acid other than serine, tyrosine or threonine.

3. The engineered polypeptide of any one of claims 1 or 2, wherein the substitution mutation is selected from the group consisting of T334A, S552A, T1056A, T1609A, S1678A and combinations thereof.

4. The engineered polypeptide of any one of the preceding claims, wherein the amino acid at the substitution mutation is not phosphorylated by GSK30.

5. The engineered polypeptide of any one of the preceding claims, wherein the engineered polypeptide has reduced inhibition of DNA repair as compared to wild-type 53BP1.

6. The engineered polypeptide of any one of the preceding claims, wherein the engineered polypeptide comprises any one of SEQ ID NOs: 4-6 or 24 or sequences with at least 90% identity to SEQ ID NOs: 4-6 or 24.

7. A polynucleotide encoding the engineered polypeptide of any one of the preceding claims.

8. A construct comprising a heterologous promoter operably linked to a polynucleotide encoding a 53BP1 polypeptide, wherein the 53BP1 polypeptide comprises at least one substitution mutation at a GSK3P phosphorylation site.

9. The construct of claim 8, wherein the substitution mutation is at a position corresponding to an amino acid residue selected from the group consisting of 334, 222, 294, 379, 380, 552, 809, 862, 1056, 1609, 1678 and combinations thereof, wherein the wild-type 53BP1 polypeptide comprises any one of SEQ ID NO: 1-3 or sequences with at least 90% identity to SEQ ID NO: 1- 310. The construct of claim 8 or 9, wherein the polynucleotide encoding a 53BP1 polypeptide comprises any one of SEQ ID NOs: 4-6, or 24 or sequences with at least 90% identity to SEQ ID NOs: 4-6 or 24.

11. The construct of any one of claims 8-10, further comprising a second heterologous promoter operably linked to a second polynucleotide encoding a chimeric antigen receptor.

12. A vector comprising the polynucleotide of claim 7 or the construct of any one of claims 8-11.

13. The vector of claim 12, wherein the vector comprises a lentiviral, retroviral or AAV vector.

14. A cell comprising the engineered polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the construct of any one of claims 8-11, or the vector of any one of claims 12-13.

15. The cell of claim 14, wherein the cell is a lymphocyte.

16. The cell of claim 15, wherein the lymphocyte is a T cell.

17. A lymphocyte comprising a heterologous promoter operably linked to a polynucleotide encoding a 53BP1 polypeptide, optionally wherein the 53BP1 polypeptide comprises at least one substitution mutation at a GSK3P phosphorylation site.

18. A lymphocyte genetically engineered to express a 53BP1 polypeptide comprising at least one substitution mutation at a GSK3f} phosphorylation site, wherein the lymphocyte is optionally engineered using CRISPR / Cas gene editing.

19. The lymphocyte of claim 17 or 18, wherein the GSK3 phosphorylation site and the position of the substitution mutation is selected from the group consisting of T334, S222, S294, S379, S380, S809, S862, S552, T1056, T1609, or S1678 of the 53BP1 polypeptide.

20. The lymphocyte of any one of claims 17-19, wherein the polynucleotide encoding a 53BP1 polypeptide comprises SEQ ID NOs: 1-6 or 24, or a sequence with at least 90% identity to SEQ ID NOs: 1-6 or 24.

21. The lymphocyte of any one of claims 17-20, further comprising a second heterologous promoter operably linked to a second polynucleotide encoding a chimeric antigen receptor (CAR) and optionally the CAR targets an antigen selected from the group consisting of EGFR, CD 19, HER2, PSMA, GD2, and mesothelin.

22. The lymphocyte of any one of claims 17-21, wherein the lymphocyte is selected from the group consisting of a CD8+ T lymphocyte and a CD4+ T lymphocyte.

23. The lymphocyte of any one of claims 17-22, wherein the lymphocyte has increased DNA repair as compared to a control lymphocyte comprising a wild-type 53BP1 polypeptide.

24. The lymphocyte of claim 23, wherein the DNA repair is non-homologous end joining (NHEJ) repair.

25. A method of generating an engineered lymphocyte comprising introducing the construct of any one of claims 8-1 lor the vector of any one of claims 12-13 into an ex-vivo lymphocyte.

26. The method of claim 25, wherein the construct is introduced via transduction into the ex- vivo lymphocyte or via CRISPR / Cas gene editing of the ex-vivo lymphocyte.

27. A method of treating cancer in a subject, the method comprising administering the lymphocytes of any one of claims 17-24 and a pharmaceutically acceptable excipient, carrier and / or diluent to the subject to treat the cancer.

28. The method of claim 27, wherein the cancer is a solid tumor and is optionally selected from the group consisting of head and neck squamous cell carcinoma, breast cancer, lung cancer, pancreatic cancer, and glioblastoma.

29. The method of claim 27 or 28, additionally comprising administering an immune checkpoint inhibitor, radiation, or other cancer therapy to the subject.

30. The method of any one of claims 27-29, wherein the cancer is head and neck squamous cell carcinoma (HNSCC).

31. A method for increasing resistance to DNA damage or enhancing DNA repair in a cell, the method comprising introducing the engineered polypeptide of any one of claims 1-6, the polynucleotide of claim 7, the construct of any one of claims 8-11 or the vector of any one of claims 12 or 13 into the cell.

32. The method of claim 31, wherein the DNA damage is double stranded DNA breaks.

33. The method of claim 31 or 32, wherein the DNA damage is caused by chemotherapy, radiation therapy, or immunotherapy.

34. A composition comprising at least one gRNA or polynucleotide encoding a gRNA, wherein the gRNA is capable of introducing at least one mutation into a 53BP1 polypeptide that has at least 95% identity to SEQ ID NO: 1, wherein the mutation is selected from the group consisting of T334, S552, T1056, T1609, S1678 and combinations thereof.

35. The composition of claim 34, wherein the gRNA comprises a target region comprising at least one of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:

22.

36. The composition of claim 34 or 35, further comprising a Cas protein or a polynucleotide encoding a Cas protein.

Images