SYNTHETIC PATHWAY ACTIVATORS

Abstract

Provided herein are novel synthetic pathway activators comprising multimerization regions, transmembrane domains, and intracellular signaling domains.

Description

SYNTHETIC PATHWAY ACTIVATORS CROSS-REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 489842, filed March 13, 2023, U.S. Provisional Application No. 63 / 495865, filed April 13, 2023; and U.S. Provisional Application No. 63 / 613713, filed December 21, 2023, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a Sequence Listing which is incorporated herein by reference in its entirety. The XML copy, created on February 27, 2024, is named ANB-219WO_SL and is 252,803 bytes in size. FUNDAMENTALS

[0003] Cancer is a disease characterized by the uncontrolled growth of cells. Many approaches to treating cancer have been attempted, including drugs and radiation therapy. Recent cancer treatments have sought to use the body's own immune cells to attack cancer cells. One promising approach uses T cells taken from a patient and genetically modified to produce chimeric antigen receptors, or CARs, receptor proteins that give T cells a new ability to target a specific protein. The receptors are chimeric because they combine antigen-binding and T-cell activation functions in a single receptor.

[0004] CAR-T cell immunotherapy is promising because the modified T cells have the potential to recognize cancer cells in order to attack and destroy them more effectively.

[0005] After T cells are manipulated with CARs, the resulting CAR-T cells are introduced into patients to attack tumor cells. CAR-T cells can be derived from T cells in the patient's own blood (autologous) or derived from T cells from another healthy donor (allogeneic). After CAR-T cells are infused into a patient, they Petition 870250081011, dated 09 / 09 / 2025, page 26 / 230 2 / 166 come into contact with their target antigen on a cell. CAR-T cells bind to the antigen and are activated. After antigen engagement, CAR-T cells can proliferate exponentially, initiate the production of antitumor cytokines, and achieve tumor cell death.

[0006] However, some concerns and limitations remain regarding CAR-T cell-based immunotherapy. Clinically effective adoptive T-cell therapy for the treatment of solid tumors will utilize robust T-cell expansion, persistence, and potency. Therefore, additional therapies that enhance T-cell expansion, persistence, and potency remain desirable. SUMMARY

[0007] In one aspect, synthetic pathway activator (SPA) peptides comprising a chimeric polypeptide comprising are provided here: i. optionally, an extracellular domain; ii. a lipid anchor or a transmembrane domain; iii. an intracellular signaling domain; and iv. a multimerization region.

[0008] In some embodiments, multimerization of the chimeric polypeptide through the multimerization region results in constitutive activity of the intracellular signaling domain.

[0009] In some embodiments, the multimerization region comprises at least one unpaired cysteine ​​residue, a leucine zipper, a BCR domain, and a VASP domain.

[0010] In some embodiments, the multimerization region comprises at least one unpaired cysteine ​​residue.

[0011] In some embodiments, the multimerization region comprises at least one unpaired cysteine ​​residue and a leucine zipper.

[0012] In some forms, the multimerization region is intracellular when expressed by a cell.

[0013] In some modalities, the region of multimerization is Petition 870250081011, dated 09 / 09 / 2025, page 27 / 230 3 / 166 extracellular when expressed by a cell.

[0014] In some modalities, the intracellular signaling domain induces phosphorylation of STAT1, STAT3, or STAT5.

[0015] In some embodiments, the intracellular signaling domain comprises a peptide motif of the type I cytokine receptor superfamily box1 (IWPNVDP (SEQ ID NO: 106)) or box2 (VSVVEIEANDKKP (SEQ ID NO: 107)).

[0016] In some embodiments, the intracellular signaling domain comprises a tyrosine phosphorylation motif comprising YXXQ or YXPQ.

[0017] In some forms, the intracellular signaling domain comprises a polypeptide sequence of an interleukin receptor.

[0018] In some forms, the interleukin receptor comprises an intracellular signaling domain gp130.

[0019] In some embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising amino acids 642 to 918 of gp130 (SEQ ID NO: 59).

[0020] In some embodiments, the intracellular signaling domain comprises a polypeptide sequence that includes the sequence presented as SEQ ID NO: 60.

[0021] In some forms, the interleukin receptor comprises a truncated intracellular signaling domain gp130.

[0022] In some embodiments, the truncated intracellular signaling domain gp130 comprises a deletion of amino acids 771 to 811 of gp130 (SEQ ID NO: 59).

[0023] In some embodiments, the truncated gp130 intracellular signaling domain comprises the truncated gp130 intracellular domain of a sequence selected from the group presented in SEQ ID NOs: 10-16 and 71-77.

[0024] In some embodiments, the intracellular signaling domain gp130 additionally comprises a Y759F mutation of gp130 (SEQ ID NO: Petition 870250081011, dated 09 / 09 / 2025, page 28 / 230 4 / 166 59).

[0025] In some forms, the intracellular signaling domain also includes a prenylation motif at the C-terminus.

[0026] In some embodiments, the lipid anchor or a transmembrane domain comprises a gp130 transmembrane domain, a CD8-alpha transmembrane domain, a prenylation motif, or a myristoylation domain derived from src, fyn, or lck.

[0027] In some embodiments, the transmembrane domain comprises a gp130 transmembrane domain.

[0028] In some embodiments, the transmembrane domain comprises a polypeptide sequence comprising amino acids 620 to 641 of gp130 (SEQ ID NO: 59)

[0029] In some embodiments, the transmembrane domain comprises a polypeptide sequence comprising the sequence presented as SEQ ID NO: 61.

[0030] In some embodiments, SPA peptides also comprise a CD8-alpha hinge domain.

[0031] In some embodiments, the extracellular domain comprises one or more CD34 epitopes, a CD34 ectodomain, a BCR ectodomain, a thrombopoietin receptor (TpoR) ectodomain, or an erythropoietin receptor (EpoR) ectodomain.

[0032] In some embodiments, the thrombopoietin receptor (TpoR) ectodomain or the erythropoietin receptor (EpoR) ectodomain comprises an unpaired cysteine.

[0033] In some modalities, the extracellular domain transmits constitutive activity to the intracellular signaling domain.

[0034] In some embodiments, comprising, from the N-terminus to the C-terminus, an extracellular domain comprising a CD34 epitope, a multimerization region comprising an unpaired cysteine ​​residue, a gp130 transmembrane domain, and an intracellular signaling domain. Petition 870250081011, dated 09 / 09 / 2025, page 29 / 230 5 / 166 gpi30.

[0035] In some embodiments, the SPA peptide comprises a sequence selected from the sequences presented in SEQ ID NOs: 1-58 or 63-104.

[0036] In some forms, the SPA sequence comprises a sequence presented in SEQ ID NO: 20.

[0037] In another aspect, multimers of the SPA peptides disclosed here are provided here.

[0038] In another aspect, nucleic acids encoding the SPA peptides disclosed here are provided.

[0039] In another aspect, vectors comprising the nucleic acids disclosed in this document are provided here.

[0040] In another aspect, systems comprising the following are provided here: i. a first chimeric polypeptide comprising an initiation receptor; ii. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and iii. the SPA peptides disclosed herein.

[0041] In some modalities, CAR induces the expression of the SPA peptide.

[0042] In some modalities, the SPA peptide is constitutively expressed.

[0043] In another aspect, cells or cell populations comprising the SPA peptides disclosed here, the multimers disclosed here, the nucleic acids disclosed here, the vectors disclosed here or the systems disclosed here are provided here.

[0044] In some embodiments, the cell is an immune cell, optionally where the immune cell is a primary human immune cell. Petition 870250081011, dated 09 / 09 / 2025, page 30 / 230 6 / 166

[0045] In another aspect, pharmaceutical compositions comprising the cell or cell population disclosed herein and a pharmaceutically acceptable excipient are provided herein.

[0046] In another aspect, pharmaceutical compositions comprising the nucleic acids disclosed herein or the vectors disclosed herein and a pharmaceutically acceptable excipient are provided herein.

[0047] In another aspect, methods for editing a cell are provided here, comprising: i. to provide a ribonucleoprotein (RNP)-nucleic acid complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the nucleic acid comprises the nucleic acids disclosed herein, and wherein the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the immune cell genome; ii. to introduce the RNP-nucleic acid complex into the immune cell in a non-viral manner, wherein the guide RNA specifically hybridizes with a target region of the primary immune cell genome, and wherein the nuclease domain cleaves the target region to create the insertion site in the immune cell genome; and iii. to edit the immune cell by inserting the nucleic acids disclosed herein into the insertion site in the immune cell genome.

[0048] In another aspect, methods of treating a disease in a subject are provided herein, comprising administering the cells disclosed herein or the pharmaceutical compositions disclosed herein to the subject.

[0049] In one aspect, methods are provided here for inhibiting a target cell in a subject, comprising administering the immune cell disclosed herein to the subject, wherein the immune cell inhibits the target cell.

[0050] In another aspect, methods for modulating the activity of an immune cell are provided here, comprising: Petition 870250081011, dated 09 / 09 / 2025, page 31 / 230 7 / 166 i. obtain a cell that includes 1. SPA peptides disclosed here; 2. the systems disclosed here; 3. the nucleic acids disclosed here; and / or 4. the vectors disclosed here; and ii. placing the immune cell in contact with a target cell, where the activator of the synthetic pathway modulates the activity of the immune cell. [005 1] In another aspect, methods are provided here for modulating the activity of an immune cell comprising: i. obtain a cell that includes 1. SPA peptides disclosed here; 2. the systems disclosed here; 3. the nucleic acids disclosed here; and / or 4. the vectors disclosed herein; and ii. placing the immune cell in contact with a target cell expressing an initiation receptor antigen and a CAR antigen, wherein the binding of the initiation receptor to the initiation receptor antigen on the target cell induces activation of the initiation receptor and expression of the chimeric antigen receptor, wherein the binding of the chimeric antigen receptor to the CAR antigen on the target cell modulates the activity of the immune cell, and wherein the synthetic pathway activator also modulates the activity of the immune cell.

[0052] In some respects, methods of treating a disease in a subject in need thereof are provided herein, comprising: determining or having determined the expression of CD11c in a cell comprising the synthetic pathway activator peptide (SPA) disclosed herein or the nucleic acid disclosed herein; and administering or having administered the cell to the subject.

[0053] In some respects, methods are provided here for determining the expression of an SPA in a cell, comprising expressing one or more SPA peptides disclosed here in the cell and determining the expression of CD11c in the cell. Petition 870250081011, dated 09 / 09 / 2025, page 32 / 230 8 / 166

[0054] In some embodiments, CD11c expression in the cell comprises either a CD11c mRNA expression level or a CD11c protein expression level.

[0055] In some embodiments, the cell is an immune cell, a primary human immune cell, a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell progenitor. BRIEF DESCRIPTION OF THE VARIOUS VIEWS OF THE DRAWINGS

[0056] These and other features, aspects and advantages of the present disclosure will be better understood in relation to the following description and attached drawings, in which:

[0057] Figure 1 provides a diagram of the various transgenic ICT cassettes expressing Logic Gate 1-5 ICs, shRNA and SPAs.

[0058] Figure 2A shows exemplary synthetic pathway activators and that synthetic pathway activators increase the potency and memory phenotype of the T-stem. Figure 2B shows additional exemplary synthetic pathway activators.

[0059] Figure 3 shows that all ICT cells constitutively expressed the PrimeR construct.

[0060] Figure 4 shows that ICT cells induced CAR expression when co-cultured with cell lines expressing the primeR antigen.

[0061] Figure 5 shows that ICT cells expressing SPA exhibit approximately two logs higher pSTAT3 expression when compared to PrimeR- cells without SPA (EGFRt).

[0062] Figure 6A shows cytotoxicity against parental K562 cells that express neither the CAR antigen nor the primeR antigen; Figure 6B shows cytotoxicity against K562 cells that express only the CAR antigen; Figure 6C shows cytotoxicity against K562 cells that express only the primeR antigen. Figure 6D shows cytotoxicity against cells Petition 870250081011, dated 09 / 09 / 2025, page 33 / 230 9 / 166 K562 cells express both the primeR antigen and the CAR antigen.

[0063] Figure 7 shows the production of IFN-γ from ICTs expressing Logic Gates 1-5 only in supernatants taken from co-cultures where the target cells expressed both the primeR antigen and the CAR antigen.

[0064] Figure 8A shows that ICTs expressing LG 1-5 ICs demonstrated in vitro cytotoxicity against the endogenous antigen cell line +CAR / +primeR. Figure 8B shows the secretion of IFNγ, TNFα, GMCSF, and IL-2 by ICT cells after co-culture with endogenous antigen cells +CAR / +primeR.

[0065] Figure 9 shows that co-culture with HUVEC-primeR antigen cells induced CAR protein expression in ICT cells and specific death of CAR+ antigen cells.

[0066] Figure 10A shows the tumor volume after tumor implantation in mice treated with ICTs expressing Logic Gates 1-5, RNP, or PBS generated from donor 1. Figure 10B shows the total T cells and ICT expansion on day 12 after inoculation, followed by contraction on day 21. Figure 10C shows the total T cells expressing the initiation receptor on days 12 and 21. Figure 10D shows the tumor volume after tumor implantation in mice treated with ICTs expressing Logic Gates 1-5, RNP, or PBS generated from donor 2. Figure 10E shows the total T cells and ICT expansion on day 12 after inoculation followed by contraction on day 21. Figure 10F shows the total T cells expressing the initiation receptor on days 12 and 21.

[0067] Figure 11A shows the inhibition of tumor growth (TGI) on the flank of single CAR antigen-positive. Figure 11B shows the inhibition of tumor growth (TGI) on the flank of double CAR antigen-positive primeR / car antigen-positive.

[0068] Figure 12A shows the level of pSTAT3 signaling induced by SPA expression indicated in a T cell after serum deprivation. A Petition 870250081011, dated 09 / 09 / 2025, page 34 / 230 10 / 166 Figure 12B shows the level of pSTATI signaling induced by SPA expression indicated in a T cell after serum deprivation.

[0069] Figure 13 provides a pSTAT1 vs pSTAT3 heat map for the indicated SPAs.

[0070] Figure 14 shows the levels of granzyme B (top panel) and IL10 (bottom panel) induced by the indicated SPA.

[0071] Figure 15 shows the clearance of tumor cells (top panel) and the expansion of T cells (bottom panel) of tumor cells expressing logic gate antigens in a repetitive stimulation assay.

[0072] Figure 16 shows the percentage of ICT cells that express the indicated SPA and that expressed the indicated cellular markers after a repetitive stimulation assay.

[0073] Figure 17 shows the tumor volume in vivo after treatment with ICTs expressing the indicated SPA.

[0074] Figure 18A shows CD11c RNA and cell surface expression and SPA expression in CD4+ and CD8+ cells on Day 7 in tumor and spleen cells. Cells with increased CD11c RNA and cell surface expression correlated with cells expressing SPA. Figure 18B shows CD11c (ITGAX) expression in spleen or tumor cells from mice treated with cells expressing an ICT of SPA (two donors) compared to cells not expressing an ICT of SPA (one donor) collected on Day 0 and Day 7 after treatment. DETAILED DESCRIPTION Definitions

[0075] The terms used in the claims and descriptive report are defined as set out below, except where otherwise specified.

[0076] As used herein, the term “gene” refers to the basic unit of heredity, which consists of a segment of DNA arranged along a chromosome that codes for a specific protein or protein segment. A gene typically includes a promoter, a 5' untranslated region, or Petition 870250081011, dated 09 / 09 / 2025, page 35 / 230 11 / 166 plus coding sequences (exons), optionally introns, and a 3' untranslated region. The gene may also include a terminator, enhancers, and / or silencers.

[0077] As used herein, the term locus refers to a specific, fixed physical location on a chromosome where a gene or genetic marker is located.

[0078] The term “safe harbor locus” refers to a locus where genes or genetic elements can be incorporated without disrupting the expression or regulation of adjacent genes. These safe harbor sites are also called safe harbor sites (SHS). As used herein, a safe harbor locus refers to an “integration site” or “knockin site” where a sequence encoding a transgene, as defined herein, can be inserted. In some embodiments, insertion occurs with the substitution of a sequence that is located at the integration site. In some embodiments, insertion occurs without the substitution of a sequence at the integration site. Examples of additional integration sites are provided in Table D.

[0079] As used herein, the term “insert” refers to a nucleotide sequence that is integrated (inserted) into a target locus or safe harbor site. Insertion may be used to refer to genes or genetic elements that are incorporated into the target locus or safe harbor site using, for example, homology-directed repair (HDR), CRISPR / Cas9 genome editing, or other methods to insert nucleotide sequences into a genomic region known to those with common skill in the technique.

[0080] The term insertion refers to a manipulation of a nucleotide sequence to introduce a non-native sequence. This is done, for example, through the use of restriction enzymes and ligases, whereby the DNA sequence of interest, usually encoding the gene of interest, can be incorporated into another nucleic acid molecule by digesting both molecules with appropriate restriction enzymes to create compatible overlaps and then using a ligase to join the molecules. A Petition 870250081011, dated 09 / 09 / 2025, page 36 / 230 12 / 166 An individual versed in the technique is very familiar with such manipulations, and examples can be found in Sambrook et al. (Sambrook, Fritsch, & Maniatis, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory, 1989), which is incorporated here by reference in its entirety, including any drawings, figures, and tables.

[0081] The CRISPR / Cas system refers to a widespread class of bacterial systems for defense against foreign nucleic acid. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include subtypes I, II, and III. Wild-type II CRISPR / Cas systems use an RNA-mediated nuclease, Cas9, in complex with guide RNA and activator to recognize and cleave foreign nucleic acid. Guide RNAs having the activity of both a guide RNA and an activator RNA are also known in the art. In some cases, these dual-activity guide RNAs are called small guide RNAs (sgRNAs).

[0082] Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to, bacteria from the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae, Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes and Thermotogae. An exemplary Cas9 protein is the Cas9 protein of Streptococcus pyogenes. Additional Cas9 proteins and homologs thereof are described in, for example, Chylinski, et al., RNA Biol. May 1, 2013; 10(5): 726-737; Nat. Rev. Microbiol. June 2011; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci US A. September 24, 2013; 110(39):15644-9; Sampson et al., Nature. May 9, 2013; 497(7448):254-7; and Jinek, et al., Science. August 17, 2012;337(6096):816-21. The Cas9 nuclease domain can be optimized for efficient activity or enhanced stability in the host cell.

[0083] As used herein, the term Cas9 refers to an RNA-mediated nuclease (e.g., of bacterial or archaeological origin, or derived therefrom). Examples of RNA-mediated nucleases include those Petition 870250081011, dated 09 / 09 / 2025, p. 37 / 230 13 / 166 previous Cas9 proteins and their homologs, and include, but are not limited to, CPF1 (See, for example, Zetsche et al., Cell, Volume 163, Issue 3, pages 759-771, October 22, 2015). Similarly, as used herein, the term Cas9 ribonucleoprotein complex and similar refers to a complex between the Cas9 protein and a crRNA (e.g., guide RNA or small guide RNA), the Cas9 protein and a trans-activating crRNA (tracrRNA), the Cas9 protein and a small guide RNA, or a combination thereof (e.g., a complex containing the Cas9 protein, a tracrRNA, and a crRNA guide RNA).

[0084] As used herein, the term “immune cell” includes all cell types that can give rise to immune cells, including hematopoietic cells such as hematopoietic stem cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs). In some embodiments, the immune cell is a B cell, a macrophage, a natural killer (NK) cell, an induced pluripotent stem cell (iPSC), a human pluripotent stem cell (HSPC), a T cell or a T cell progenitor cell, or a dendritic cell. In some embodiments, the cell is an innate immune cell.

[0085] As used herein, the term “primary” in the context of a primary cell or primary stem cell refers to a cell that has not been transformed or immortalized. These primary cells may be cultured, subcultured, or passed a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, primary cells are adapted to in vitro culture conditions. In some cases, primary cells are isolated from an organism, system, organ, or tissue, optionally sorted and used, for example, directly, without culture or subculture. In some cases, primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contact with (e.g., culture in the presence of) CD3, CD28, IL-2, IFN-γ agonists, or a combination of them. Petition 870250081011, dated 09 / 09 / 2025, page 38 / 230 14 / 166 same.

[0086] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to cells that have completed maturation in the thymus and identify certain foreign antigens in the body. The terms also refer to the major types of leukocytes that play various roles in the immune system, including activation and deactivation of other immune cells. A T cell can be any T cell, such as a cultured T cell, for example, a primary T cell, or a T cell derived from a cultured T cell lineage, for example, a Jurkat, SupT1, etc., or a T cell obtained from a mammal. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof.A T cell can be a CD3+ cell. T cells can be CD4+, CD8+, or both CD4+ and CD8+. A T cell can be any type of T cell: double-positive CD4+ / CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), PBMCs, peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, γδ T cells, etc. It can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells).The term AT cell can also refer to a genetically modified T cell, such as a T cell that has been modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also be differentiated from stem cells or progenitor cells.

[0087] CD4+ T cells refer to a subset of T cells that Petition 870250081011, dated 09 / 09 / 2025, page 39 / 230 15 / 166 express CD4 on their surface and are associated with a cellular immune response. CD4+ T cells are characterized by a post-stimulation secretion profile that may include the secretion of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, and IL-10. “CD4” is a 55 kDa glycoprotein originally defined as a differentiation antigen in T lymphocytes, but has also been found in other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin superfamily and has been implicated as an associative recognition element in restricted MHC (major histocompatibility complex) class II immune responses. In T lymphocytes, the CD4 antigen defines a helper / inducer subset.

[0088] “CD8+ T cells” refers to a subset of T cells that express CD8 on their surface, are restricted to MHC class I, and function as cytotoxic T cells. The “CD8” molecule is a differentiation antigen present on thymocytes, as well as on cytotoxic and suppressor T lymphocytes. The CD8 antigen is a member of the immunoglobulin superfamily and is an associative recognition element in the major class I restriction interactions of the major histocompatibility complex.

[0089] As used herein, the term “hematopoietic stem cell” refers to a type of stem cell that can give rise to a blood cell. Hematopoietic stem cells can give rise to cells of the myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are found predominantly in the bone marrow, although they can be isolated from peripheral blood or a fraction thereof. Several cell surface markers can be used to identify, classify, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are identified as c-kit+ and lin-. In some cases, human hematopoietic stem cells are identified as CD34+, CD59+, Thy1 / CD90+, CD38lo / -, C-kit / CD117+, lin-. In some cases, human hematopoietic stem cells are identified as CD34-, CD59+, Thy1 / CD90+, CD38lo / -, C-kit / CD117+, lin-. In some cases, hematopoietic stem cells Petition 870250081011, dated 09 / 09 / 2025, page 40 / 230 16 / 166 human hematopoietic stem cells are identified as CD133+, CD59+, Thy1 / CD90+, CD38lo / -, Ckit / CD117+, lin-. In some cases, mouse hematopoietic stem cells are identified as CD34lo / -, SCA-1+, Thy1+ / lo, CD38+, C-kit+, lin-. In some cases, hematopoietic stem cells are CD150+CD48CD244-.

[0090] As used herein, the term “hematopoietic cell” refers to a cell derived from a hematopoietic stem cell. The hematopoietic cell may be obtained or provided by isolation from an organism, system, organ, or tissue (e.g., blood or a fraction thereof). Alternatively, a hematopoietic stem cell may be isolated and the hematopoietic cell obtained or provided by differentiation from the stem cell. Hematopoietic cells include cells with limited potential to differentiate into other cell types. These hematopoietic cells include, but are not limited to, multipotent progenitor cells, restricted lineage progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or erythroid megakaryocyte progenitor cells. Hematopoietic cells include cells of the lymphoid and myeloid lineages, such as lymphocytes, erythrocytes, granulocytes, monocytes, and thrombocytes.

[0091] As used herein, the term “construct” refers to a complex of molecules, including macromolecules or polynucleotides.

[0092] As used herein, the term “integration” refers to the process of stable insertion of one or more nucleotides of a construct into the cell’s genome, i.e., covalent linkage to a nucleic acid sequence in the cell’s chromosomal DNA. It may also refer to nucleotide deletions at an integration site. When there is a deletion at the insertion site, “integration” may further include the replacement of the deleted endogenous sequence or nucleotide by one or more inserted nucleotides.

[0093] As used herein, the term “exogenous” refers to a molecule or activity that has been introduced into a host cell and is not native to that cell. The molecule may be introduced, for example, by the introduction of the acid Petition 870250081011, dated 09 / 09 / 2025, page 41 / 230 17 / 166 coding nucleic acid in the host genetic material, either by integration into a host chromosome or as non-chromosomal genetic material, such as a plasmid. Therefore, the term, when used in connection with the expression of a coding nucleic acid, refers to the introduction of the coding nucleic acid into a cell in an expressible form. The term endogenous refers to a molecule or activity that is present in a host cell under natural, unedited conditions. Similarly, the term, when used in connection with the expression of coding nucleic acid, refers to the expression of coding nucleic acid that is contained within the cell and not introduced exogenously.

[0094] The term “heterologous” refers to a nucleic acid or polypeptide sequence or domain that is not native to a flanking sequence, for example, where the heterologous sequence is not found in nature coupled to nucleic acid or polypeptide sequences that occur at one or both ends.

[0095] The term “homologous” refers to a nucleic acid or polypeptide sequence or domain that is native to a flanking sequence, for example, where the homologous sequence is found in nature coupled to nucleic acid or polypeptide sequences that occur at one or both ends.

[0096] As used herein, a “polynucleotide donor construct” refers to a nucleotide sequence (e.g., DNA sequence) that is genetically inserted into a polynucleotide and is exogenous to that polynucleotide. The polynucleotide donor construct is transcribed into RNA and optionally translated into a polypeptide. The polynucleotide donor construct may include prokaryotic sequences, eukaryotic mRNA cDNA, genomic DNA sequences from eukaryotic DNA (e.g., mammalian), and synthetic DNA sequences. For example, the polynucleotide donor construct may be a miRNA, shRNA, natural polypeptide (i.e., a naturally occurring polypeptide) or fragment thereof, or a polypeptide. Petition 870250081011, dated 09 / 09 / 2025, page 42 / 230 18 / 166 variant (for example, a natural polypeptide with less than 100% sequence identity to the natural polypeptide) or fragments thereof.

[0097] As used herein, the term “complementary” or “complementarity” refers to the specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), and G and C. The guide RNAs described herein may comprise sequences, for example, DNA targeting sequences that are perfectly complementary or substantially complementary (e.g., having 1-4 mismatches) to a genomic sequence in a cell.

[0098] As used herein, the term transgene refers to a polynucleotide that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. It is optionally translated as a polypeptide. As used, transgene can refer to a polynucleotide that codes for a polypeptide.

[0099] The terms “protein”, “polypeptide” and “peptide” are used interchangeably here.

[0100] As used herein, the term “operationally linked” or “operationally bound” refers to the linking of a nucleic acid sequence to a single nucleic acid fragment such that one function is affected by the other. For example, if a promoter is able to affect the expression of a coding sequence or functional RNA (that is, the coding sequence or functional RNA is under transcriptional control of the promoter), the promoter is operatively linked to it. Coding sequences can be operatively linked to control sequences in both sense and antisense orientations.

[0101] As used herein, the term “states of cellular development” refers, for example, to states in which the cell is inactive, actively expressing, differentiating, senescent, etc. The state of cellular development may also refer to a cell in a state Petition 870250081011, dated 09 / 09 / 2025, page 43 / 230 19 / 166 precursor (e.g., a T cell precursor).

[0102] As used, the term encoding refers to a nucleic acid sequence that codes for a protein or polypeptide of interest. The nucleic acid sequence can be a DNA or RNA molecule. In preferred embodiments, the molecule is a DNA molecule. In other preferred embodiments, the molecule is an RNA molecule. When present as an RNA molecule, it will comprise sequences that direct the host cell ribosomes to initiate translation (e.g., a start codon, ATG) and direct the ribosomes to terminate translation (e.g., a stop codon). Between the start codon and the stop codon there is an open reading frame (ORF). These terms are well known to those skilled in the art.

[0103] As used in this document, the term “subject” refers to a mammalian subject. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs, and sheep. In certain embodiments, the subject is a human being. In some embodiments, the subject has a disease or condition that can be treated with a modified cell provided herein or a population thereof. In some embodiments, the disease or condition is cancer.

[0104] As used herein, the term “promoter” refers to a nucleotide sequence (e.g., DNA sequence) capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and distal upstream elements, the latter often being called enhancers. A promoter may be derived from natural genes in their entirety or may be composed of different elements from different promoters found in nature and / or may comprise synthetic DNA segments. A promoter, as contemplated herein, may be endogenous to the cell of interest or exogenous to the cell of interest. It is appreciated by those skilled in the art that different promoters may induce the expression of a gene in different tissues or types. Petition 870250081011, dated 09 / 09 / 2025, page 44 / 230 20 / 166 of cells, at different stages of development or in response to different environmental conditions. As is known in the art, a promoter can be selected according to the strength of the promoter and / or the conditions under which the promoter is active, for example, constitutive promoter, strong promoter, weak promoter, inducible / repressible promoter, tissue-specific or developmentally regulated promoters, cell cycle-dependent promoters and the like.

[0105] A promoter can be an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, an HNFIa promoter, etc.). The promoter can be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some embodiments, the promoter can be a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.). See, for example, US Publication No. 20180127786, the disclosure of which is incorporated herein by reference in its entirety.

[0106] Gene editing, as contemplated herein, may involve a knockout or knockout of a gene (or nucleotide sequence). As used herein, the term “knockin” refers to the addition of a DNA sequence, or fragment thereof, to a genome. Such DNA sequences to be inserted may include an entire gene or genes, may include regulatory sequences associated with a gene, or any portion or fragment thereof. For example, a polynucleotide donor construct encoding a protein may be inserted into the genome of a cell carrying a mutant gene. In some embodiments, a knockin strategy involves replacing an existing sequence with the supplied sequence, for example, replacing a mutant allele with a wild-type copy. Conversely, the term “knockout” refers to the deletion of a gene or the expression of a gene. For example, a gene may be deleted by the deletion or addition of a sequence. Petition 870250081011, dated 09 / 09 / 2025, p. 45 / 230 21 / 166 nucleotide that leads to frame interruption. As another example, a gene can be eliminated by replacing a part of it with an irrelevant (e.g., non-coding) sequence.

[0107] As used herein, the term “non-homologous end joining” or NHEJ refers to a cellular process in which cut or notched ends of a DNA strand are directly joined without the need for a homologous template nucleic acid. NHEJ may lead to the addition, deletion, substitution, or a combination thereof, of one or more nucleotides at the repair site.

[0108] As used in this document, the term “homology-directed repair” or HDR refers to a cellular process in which the cut or nicked ends of a DNA strand are repaired by polymerization of a homologous template nucleic acid. Thus, the original sequence is replaced by the template sequence. The homologous template nucleic acid may be provided by homologous sequences elsewhere in the genome (sister chromatids, homologous chromosomes, or repeated regions on the same chromosome or on different chromosomes). Alternatively, an exogenous template nucleic acid may be introduced to achieve a specific HDR-induced sequence alteration at the target site. In this way, specific mutations can be introduced at the cleavage site.

[0109] As used herein, a single-stranded DNA template or a double-stranded DNA template refers to a DNA oligonucleotide that can be used by a cell as a template for HDR. Generally, the single-stranded DNA template or a double-stranded DNA template has at least one homology region with a target site. In some cases, the single-stranded DNA template or the double-stranded DNA template has two homologous regions flanking a region containing a heterologous sequence to be inserted into a target cleavage site.

[0110] The terms “vector” and “plasmid” are used interchangeably and, as used herein, refer to polynucleotide carriers useful for Petition 870250081011, dated 09 / 09 / 2025, page 46 / 230 22 / 166 introduce genetic material into a cell. Vectors can be linear or circular. Vectors can integrate into the target genome of a host cell or replicate independently in a host cell. Vectors may comprise, for example, an origin of replication, a multicloning site, and / or a selectable marker. An expression vector typically comprises an expression cassette. Vectors and plasmids include, but are not limited to, integration vectors, prokaryotic plasmids, eukaryotic plasmids, synthetic plant chromosomes, episomes, cosmids, and artificial chromosomes.

[0111] As used herein, the term “introduction” in the context of the introduction of a nucleic acid or a complex comprising a nucleic acid, for example, an RNP-DNA template complex, refers to the translocation of the nucleic acid sequence or the RNP-DNA template complex from outside a cell into the cell. In some cases, introduction refers to the translocation of the nucleic acid or complex from outside the cell into the cell nucleus. Various methods of such translocation are contemplated, including, but not limited to, electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, translocation via cell-penetrating peptides, liposome-mediated translocation, and the like.

[0112] As used herein, the term “expression cassette” is a recombinantly generated or chemically synthesized polynucleotide construct comprising regulatory sequences operatively linked to a selected polynucleotide to facilitate the expression of the selected polynucleotide in a host cell. For example, the regulatory sequences may facilitate the transcription of the selected polynucleotide in a host cell, or the transcription and translation of the selected polynucleotide in a host cell. An expression cassette may, for example, be integrated into the genome of a host cell or be present in an expression vector. Petition 870250081011, dated 09 / 09 / 2025, page 47 / 230 23 / 166

[0113] As used herein, the expression “subject in need of the same” refers to a subject who exhibits and / or is diagnosed with one or more symptoms or signs of a disease or disorder as described herein.

[0114] A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include antihormonal or endocrine therapeutic agents that act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth.

[0115] The term composition refers to a mixture containing, for example, an artificial cell or protein contemplated in this document. In some embodiments, the composition may contain additional components such as adjuvants, stabilizers, excipients and the like. The term “composition” or “pharmaceutical composition” refers to a preparation which, in such form, enables the biological activity of an active ingredient contained therein to be effective in the treatment of a subject, and which does not contain additional components that are unacceptably toxic to the subject in the quantities provided in the pharmaceutical composition.

[0116] The term “in situ” refers to processes that occur in a living cell that grows separately from a living organism, for example, growing in tissue culture.

[0117] The term “in vivo” refers to processes that occur in a living organism.

[0118] As used herein, the term “ex vivo” generally includes experiments or measurements performed on or in living tissue, preferably in an artificial environment outside the organism, preferably with minimal differences from natural conditions.

[0119] The term “mammal” as used in this document includes both human and non-human beings and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines and porcines. Petition 870250081011, dated 09 / 09 / 2025, page 48 / 230 24 / 166

[0120] The term percentage of identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum match, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to qualified individuals) or by visual inspection. Depending on the application, the percentage of identity may exist in a region of the sequence being compared, for example, in a functional domain, or alternatively, over the entire length of the two sequences being compared.

[0121] For sequence comparison, typically one sequence acts as a reference sequence, with which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and sequence algorithm program parameters are assigned. The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequence(s) with respect to the reference sequence, based on the assigned program parameters.

[0122] The ideal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., below).

[0123] An example of an algorithm that is suitable for determining the Petition 870250081011, dated 09 / 09 / 2025, p. 49 / 230 The BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990), is a 25 / 166 percentage of sequence identity and similarity assessment. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov / ).

[0124] The term “sufficient quantity” means a quantity sufficient to produce a desired effect, for example, a quantity sufficient to modulate protein aggregation in a cell.

[0125] The term “therapeutically effective quantity” is a quantity that is effective in improving a symptom of a disease.

[0126] The term “improvement” refers to any therapeutically beneficial outcome in the treatment of a disease state, for example, a cancer disease state, worsening in severity or progression, remission or cure thereof.

[0127] As used herein, the term “effective amount” refers to the amount of a compound (e.g., compositions described in the present invention, cells described in the present invention) sufficient to produce beneficial or desired results. An effective amount may be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration.

[0128] As used herein, the term “treatment” includes any effect, for example, diminishing, reducing, modulating, improving or eliminating, that results in the improvement of the condition, disease, disorder and the like, or in the improvement of a symptom thereof.

[0129] The terms modulated and modulation refer to reducing or inhibiting or, alternatively, activating or increasing a recited variable.

[0130] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more in a recited variable.

[0131] The terms “reduce” and “inhibit” refer to a decrease of 10%, Petition 870250081011, dated 09 / 09 / 2025, page 50 / 230 26 / 166%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times or more in a recited variable.

[0132] With respect to the binding of an antibody to a target molecule, the terms “bind,” “specific binding,” “binds specifically to,” “specific for,” “binds selectively,” and “selective for” a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen means binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). For example, an antibody that binds selectively or specifically to an antigen is a chemical moiety that binds to the antigen with high affinity and does not bind significantly to other unrelated antigens. Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the recognized epitope on the target molecule.In this case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.

[0133] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used in this document, affinity refers to intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (Kd). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including but not limited to surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry. Petition 870250081011, dated 09 / 09 / 2025, page 51 / 230 27 / 166 (e.g., FORTEBIO®).

[0134] The term hypervariable region or HVR, as used in this document, refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops (hypervariable loops). Generally, four-chain native antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues of the hypervariable loops and / or form the complementarity-determining regions (CDRs), the latter being of greater sequence variability and / or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also called “complementarity-determining regions” (CDRs), and these terms are used interchangeably here in reference to portions of the variable region that form the antigen-binding regions.This specific region was described by Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987), where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to a CDR of an antibody or its variants is intended to be within the scope of the term as defined and used in this document. The exact number of residues comprising a specific CDR will vary depending on the sequence and size of the CDR. Skilled individuals can routinely determine which residues comprise a particular CDR given the amino acid sequence of the antibody's variable region.

[0135] The amino acid sequence limits of a CDR can be determined by a specialist in the technique using any of several known numbering schemes, including those described by Kabat et al., supra (Kabat numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., Petition 870250081011, dated 09 / 09 / 2025, p. 52 / 230 28 / 166 273:927-948 (numbering scheme “Chothia”); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (numbering scheme “Contato”); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (numbering scheme “IMGT”); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 (numbering scheme “AHo”); each of which is incorporated by reference in its entirety.

[0136] Table A provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia numbering schemes. For CDR-H1, the residue numbering is provided using the Kabat and Chothia numbering schemes.

[0137] CDRs can be assigned, for example, using antibody numbering software, such as Abnum, available at bioinf.org.uk / abs / abnum / , and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety.

[0138] The “EU numbering system” is generally used when referring to a residue in a constant region of an antibody heavy chain (e.g., as reported in Kabat et al., supra). Unless otherwise indicated, the EU numbering scheme is used to refer to residues in the constant regions of the antibody heavy chain described herein.

[0139] As used in this document, the term single-chain refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In particular, in such an embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule. As described in more detail herein, a scFv has a variable light chain (VL) domain connected from its C-terminus to the N-terminus of a variable heavy chain (VH) domain by a polypeptide chain. Alternatively, the scFv is composed of a polypeptide chain where the C-terminus of the VH is connected to the N-terminus of the VL by a polypeptide chain.

[0140] The “Fab fragment” (also known as the antigen-binding fragment) contains the constant domain (CL) of the light chain and the first Petition 870250081011, dated 09 / 09 / 2025, page 53 / 230 29 / 166 constant domain (CH1) of the heavy chain, along with the variable domains VL and VH in the light and heavy chains, respectively. The variable domains comprise the complementarity-determining loops (CDRs, also called hypervariable regions) that are involved in antigen binding. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

[0141] “F(ab')2” fragments contain two Fab' fragments joined, near the hinge region, by disulfide bonds. F(ab')2 fragments can be generated, for example, by recombinant or synthetic methods or by pepsin digestion of an intact antibody. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0142] The “Fv” fragments comprise a non-covalently linked dimer of a variable heavy chain domain and a variable light chain domain.

[0143] The “single-chain Fv” or “scFv” comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide additionally comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For an analysis of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, volume 113, Rosenburg and Moore eds., Springer-Verlag, New York, pages 269 to 315 (1994). HER2 antibody scFv fragments are described in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0144] The term single-domain antibody or sdAb refers to a molecule in which one variable domain of an antibody binds specifically to an antigen without the presence of the other variable domain. Single-domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference herein. Petition 870250081011, dated 09 / 09 / 2025, page 54 / 230 30 / 166 totality. Single-domain antibodies are also known as sdAbs or nanobodies. Sdabs are quite stable and easy to express as a fusion partner with the Fc chain of an antibody (Harmsen MM, De Haard HJ (20“7). Properties, production, and applications of camelid single-domain antibody fragments. Appl. Microbiol Biotechnol. 77(1): 13-22).

[0145] It should be noted that, as used in this descriptive report and the attached claims, the singular forms “a”, “an”, “the” and “the” include plural referents, except where the context clearly indicates otherwise. Synthetic pathway activators

[0146] In several respects, the systems disclosed here employ one or more “synthetic pathway activator” (SPA) peptides. Immune cells expressing CARs may be limited by the need for in vivo expansion after infusion. To achieve robust expansion, T cells use three signals: antigen stimulation, co-stimulation, and cytokine-induced stimulation. Activation of CARs is sufficient to induce the first two signals, but cannot recapitulate cytokine signaling. Furthermore, the tumor microenvironment is often immunosuppressive and devoid of pro-inflammatory cytokines. SPA peptides can therefore be used to stimulate robust in vivo expansion and enhance desirable properties (such as increased survival, persistence, and potency) of T cells, for example, by expressing initiation receptors and / or CARs as described here.

[0147] In one aspect, synthetic pathway activator (SPA) peptides comprising a chimeric polypeptide comprising: a lipid anchor or a transmembrane domain; an intracellular signaling domain; and a multimerization region are provided herein. In another aspect, synthetic pathway activator (SPA) peptides comprising a chimeric polypeptide comprising: an extracellular domain, a lipid anchor or a transmembrane domain; an intracellular signaling domain; and a multimerization region are provided herein. In some embodiments, an SPA peptide Petition 870250081011, dated 09 / 09 / 2025, page 55 / 230 31 / 166 additionally comprises a CD8-alpha hinge domain. In some embodiments, the SPA peptide is constitutively expressed in a cell. In some embodiments, the expression of the SPA peptide in a cell is induced, for example, by the activation of T cells. For example, an inducible SPA can be expressed after the engagement of a CAR T cell with the CAR cognate ligand in a target cell. In some embodiments, the expression of the SPA peptide in a cell is induced by the CAR initiation and / or signaling receptor. SPA Structure

[0148] In several embodiments, an SPA peptide mimics the activation of interleukin signaling. Interleukin receptors are cytokine receptors that signal via Signal Transducer and Activator of Transcription (STAT) transcription factors (e.g., STAT1, STAT3, and STAT5). Interleukin receptors typically function by dimerization in response to ligand binding. Once dimerized, the receptors can bind to Janus-associated kinases (JAKs) to induce JAK cross-phosphorylation and downstream “JAK / STAT” signaling. Consequently, induced receptor agonism or ligand-independent dimerization of the receptors can be used so that activators of the synthetic pathway induce constitutive receptor activity and thus constitutive cytokine signaling.

[0149] In several embodiments, SPA peptides comprise interleukin receptors or functional fragments thereof. In some embodiments, SPAs comprise or are derived from intracellular signaling domains of the interleukin receptor or functional fragments thereof. In some embodiments, SPA peptides comprise or are derived from interleukin-6 (IL6ST) signal transducer polypeptides or functional fragments thereof. The interleukin-6 (IL6ST) signal transducer is also known as glycoprotein 130 (gp130). An SPA may comprise multimerized SPA peptides, for example, two SPA peptides that are dimerized, for example, homodimerized. Multimerization encompasses dimerization, trimerization, tetramerization, or higher-order combinations of Petition 870250081011, dated 09 / 09 / 2025, page 56 / 230 32 / 166 SPA peptides that interact with each other. Intracellular signaling domain

[0150] In some embodiments, the intracellular signaling domain induces phosphorylation of STAT1, STAT3, and / or STAT5. In some embodiments, a functional SPA induces phosphorylation of STAT1, STAT3, and / or STAT5. In some embodiments, the intracellular signaling domain comprises a peptide motif of the type I cytokine receptor superfamily box1 (IWPNVDP (SEQ ID NO: 106)) or box2 (VSVVEIEANDKKP (SEQ ID NO: 107)). In some embodiments, the intracellular signaling domain comprises a tyrosine phosphorylation motif comprising YXXQ or YXPQ. In some embodiments, the intracellular signaling domain comprises one or more minimal STAT-binding motifs of a STAT1, STAT3, and / or STAT5 protein. In some forms, the intracellular signaling domain comprises one or more minimal STAT-binding motifs of a STAT1 and STAT3 protein.In some embodiments, the intracellular signaling domain comprises one or more minimal STAT-binding motifs of a STAT1 and STAT5 protein. In some embodiments, the intracellular signaling domain comprises one or more minimal STAT-binding motifs of a STAT3 and STAT5 protein.

[0151] In some forms, the intracellular signaling domain comprises a polypeptide sequence of an interleukin receptor.

[0152] In some embodiments, the interleukin receptor comprises an intracellular signaling domain gp130. In some embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising amino acids 642 to 918 of gp130 (SEQ ID NO: 59).

[0153] In some embodiments, the interleukin receptor comprises a truncated gp130 intracellular signaling domain. In some embodiments, SPA peptides comprise truncated gp130 intracellular domains. In some embodiments, the truncated gp130 intracellular signaling domain comprises the truncated gp130 intracellular domain of a sequence selected from the group presented in SEQ ID NOs: 10-16 and 71-77. Petition 870250081011, dated 09 / 09 / 2025, page 57 / 230 33 / 166 In some embodiments, the truncated gp130 intracellular signaling domain comprises a sequence with at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the gp130 intracellular signaling domain as provided in the sequence presented in SEQ ID NO: 60.

[0154] In some embodiments, the truncated intracellular gp130 domain comprises a GP130Δ771-811 fragment. In some embodiments, the truncated intracellular gp130 domain comprises a GP130Δ707755 fragment. In some embodiments, the truncated intracellular gp130 domain comprises a GP130Δ818-901 fragment. In some embodiments, the truncated intracellular gp130 domain comprises one or more selected truncations from the group consisting of GP130Δ707-755, GP130Δ771-811, and GP130Δ818-901. In some embodiments, the truncated intracellular gp130 signaling domain comprises a deletion of amino acids 771 to 811 of gp130 (SEQ ID NO: 59). In some embodiments, the truncated intracellular signaling domain gp130 comprises a deletion of amino acids 707 to 755 of gp130 (SEQ ID NO: 59). In some embodiments, the truncated intracellular signaling domain gp130 comprises a deletion of amino acids 818 to 901 of gp130 (SEQ ID NO: 59).In some embodiments, the truncated gp130 intracellular signaling domain comprises one or more (e.g., one, two, or three) amino acid deletions selected from the group consisting of amino acids 707 to 755, 771 to 811, and 818-901 of gp130 (SEQ ID NO: 59). In other embodiments, the gp130 intracellular domain comprises a Y759F mutation (resulting in a SOCS-proof mutant). In some embodiments, the gp130 intracellular signaling domain additionally comprises a Y759F mutation of gp130 (SEQ ID NO: 59). In other embodiments, the gp130 intracellular domain comprises a Y759F mutation and a Δ771-811 truncation (gp130Y759FΔ771-811) of gp130 (SEQ ID NO: 59). In other forms, the intracellular gp130 domain comprises a Y759F mutation and a truncation. Petition 870250081011, dated 09 / 09 / 2025, page 58 / 230 34 / 166 Δ707-755 (gp130Y759FΔ707-755) of gp130 (SEQ ID NO: 59). In other embodiments, the intracellular gp130 domain comprises a Y759F mutation and a Δ818-901 truncation (gp130Y759FΔ818-901) of gp130 (SEQ ID NO: 59).

[0155] In some embodiments, the intracellular signaling domain gp130 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence presented in SEQ ID NO: 60. In some embodiments, the intracellular signaling domain gp130 comprises the amino acid sequence presented in SEQ ID NO: 60.

[0156] In some embodiments, an SPA peptide may comprise a ligand agonist (e.g., a cytokine, e.g., an interleukin) that enables constitutive activation of the SPA. In some embodiments, the cytokine receptor and a soluble agonist are expressed simultaneously. In some embodiments, the cytokine receptor and a membrane-bound agonist are expressed simultaneously. Lipid anchors and transmembrane domains

[0157] In several embodiments, SPA peptides are anchored to the cell membrane via a lipid anchor. In other embodiments, SPA peptides comprise a transmembrane domain. In several embodiments, an SPA peptide comprising a lipid anchor or a transmembrane domain comprises a gp130 transmembrane domain, a CD8-alpha transmembrane domain, a prenylation motif, or a myristoylation domain derived from src, fyn, or lck.

[0158] In some embodiments, an SPA peptide comprises an src-derived myristoylation domain, a fyn-derived myristoylation domain, or an lck-derived myristoylation domain. In other embodiments, an SPA peptide comprises a prenylation motif. In some embodiments, an SPA peptide comprises an extracellular domain, a Petition 870250081011, dated 09 / 09 / 2025, page 59 / 230 35 / 166 transmembrane domain and an intracellular signaling domain. In some embodiments, an SPA peptide comprises a transmembrane domain of an interleukin receptor.

[0159] In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence presented in SEQ ID NO: 61. In some embodiments, the transmembrane domain comprises the amino acid sequence presented in SEQ ID NO: 61. Multimerization Domains

[0160] In several embodiments, one or more structural changes can be made to confer constitutive activity to a SPA or a functional fragment thereof. In some embodiments, structures or mutations can be added to induce SPA multimerization (e.g., dimerization, trimerization, tetramerization, or higher-order multimers). In some embodiments, one or more amino acids can be mutated to a cysteine ​​to allow the formation of one or more disulfide bonds, for example, between two receptor monomers. In some embodiments, one or more amino acids can be inserted into a wild-type receptor polypeptide to promote dimerization, for example, through the formation of one or more disulfide bonds. In some embodiments, the multimerization domain comprises one or more unpaired cysteine ​​residues.The one or more mutated amino acids with an unpaired cysteine ​​may be in the extracellular domain, the transmembrane domain, a hinge domain, an intracellular signaling domain, or any ligand used to link such domains. In some embodiments, the multimerization domain comprises one or more VASP domains. In some embodiments, the multimerization domain comprises one or more VASP tetramerization domains. In some embodiments, the multimerization domain comprises one or more. Petition 870250081011, dated 09 / 09 / 2025, page 60 / 230 36 / 166 leucine zippers. In some forms, the multimerization domain is intracellular or extracellular.

[0161] In some embodiments, an exogenous polypeptide is operatively linked to a cytokine receptor or a functional fragment thereof to cause its multimerization or dimerization. In some embodiments, a leucine zipper polypeptide is operatively linked to a cytokine receptor or a functional fragment thereof. In some embodiments, the leucine zipper polypeptide is a c-Jun leucine zipper. In some embodiments, an exogenous scaffold is operatively linked to a cytokine receptor or a functional fragment thereof. In some embodiments, the exogenous scaffold is a CD34 ectodomain (e.g., SEQ ID NO: 242), erythropoietin receptor (EpoR) ectodomain, or thrombopoietin receptor (TpoR) ectodomain.

[0162] In some embodiments, multimerization of the chimeric polypeptide through the multimerization region results in the constitutive activity of the intracellular signaling domain. In some embodiments, the SPA peptide comprises one or more multimerization regions. In some embodiments, the SPA peptide comprises two or more multimerization regions. In some embodiments, the multimerization region comprises at least one of one or more unpaired cysteine ​​residues, a leucine zipper, a BCR domain, and a VASP domain. An exemplary BCR domain is provided in SEQ ID NO: 239. An exemplary VASP domain is provided in SEQ ID NO: 240. In some embodiments, the multimerization region comprises at least one or more unpaired cysteine ​​residues. In some embodiments, the multimerization region comprises at least one or more unpaired cysteine ​​residues and a leucine zipper.In some embodiments, the SPA peptide comprises one or more VASP domain polypeptides to promote multimerization (e.g., SEQ ID NO: 240). In some embodiments, the VASP domain is the tetramerization domain of VASP. In some embodiments, the BCR domain. Petition 870250081011, dated 09 / 09 / 2025, page 61 / 230 37 / 166 comprises a spiral tetramerization region. In such embodiments, the BCR ectodomain can result in multimerization through non-covalent interactions.

[0163] In some embodiments, the multimerization region is intracellular when expressed by a cell. In some embodiments, the multimerization region is extracellular when expressed by a cell. For example, the SPA peptide may comprise, in an N-terminus to C-terminus direction, i) one or more multimerization domains - an extracellular domain - a transmembrane domain - an intracellular signaling domain, ii) an extracellular domain - one or more multimerization domains - a transmembrane domain - an intracellular signaling domain, iii) one or more multimerization domains - a transmembrane domain - an intracellular signaling domain, iv) one or more multimerization domains - a lipid anchor - an intracellular signaling domain, v) an extracellular domain - a transmembrane domain - one or more multimerization domains - an intracellular signaling domain,vi) a lipid anchor - one or more multimerization domains - an intracellular signaling domain, vii) an extracellular domain - a transmembrane domain - an intracellular signaling domain - one or more multimerization domains, or viii) a lipid anchor - an intracellular signaling domain - one or more multimerization domains, or any combination thereof. Extracellular domain

[0164] An SPA peptide disclosed herein may also comprise an extracellular domain. In some embodiments, the extracellular domain transmits constitutive activity to the intracellular signaling domain. In some embodiments, the extracellular domain comprises a CD34 ectodomain (e.g., a CD34 extracellular domain, SEQ ID NO: 238). In some embodiments, the extracellular domain comprises a CD34 epitope (e.g., a QBEND10 epitope, SEQ ID NO: 238). In some embodiments, the extracellular domain comprises a receptor extracellular domain. Petition 870250081011, dated 09 / 09 / 2025, page 62 / 230 38 / 166 type I cytokine (e.g., a thrombopoietin receptor (TpoR) ectodomain or an erythropoietin receptor (EpoR) ectodomain). In some embodiments, the extracellular domain of the type I cytokine receptor (e.g., a thrombopoietin receptor (TpoR) ectodomain or an erythropoietin receptor (EpoR) ectodomain) further comprises a transmembrane domain of the type I cytokine receptor (e.g., a transmembrane domain of the thrombopoietin receptor (TpoR) or a transmembrane domain of the erythropoietin receptor (EpoR)). In some embodiments, the extracellular domain comprises one or more CD34 epitopes (e.g., a QBEND10 epitope, SEQ ID NO: 238), a CD34 ectodomain (SEQ ID NO: 242), a BCR ectodomain (SEQ ID NO: 239), a thrombopoietin receptor (TpoR) domain (SEQ ID NO: 243), or an erythropoietin receptor (EpoR) ectodomain (SEQ ID NO: 241).In several embodiments, the thrombopoietin receptor (TpoR) ectodomain or erythropoietin receptor (EpoR) ectodomain further comprises one or more unpaired cysteines. In some embodiments, the BCR ectodomain comprises a spiral tetramerization region. In such embodiments, the BCR ectodomain may result in multimerization through non-covalent interactions. Examples of SPAs

[0165] In some embodiments, the SPA peptide comprises a leucine-gp130 zipper (referred to herein interchangeably as “L-gp130” or “gp130”) or an L-gp130 intracellular signaling domain. L-gp130 comprises a homodimer, with each monomer comprising (a) an extracellular domain comprising an inserted cysteine ​​residue that forms a disulfide bond with another monomer and a c-Jun leucine zipper; and (b) an IL6ST (GP130) transmembrane domain and an intracellular signaling domain. The cysteine ​​residue and the leucine zipper in each polypeptide can induce the formation of stable homodimers that mimic the constitutive activation of IL-6R. Further details on the construction of L-gp130 are described in StuhlmannLaeisz et al. Mol Biol Cell. July 2006;17(7):2986-95 and in document no. Petition 870250081011, dated 09 / 09 / 2025, page 63 / 230 39 / 166 WO2020200325, which are incorporated herein by reference in their entirety. Diagrams of L-gp130 and other exemplary SPAs described herein are provided in Figure 2B.

[0166] In some embodiments, the SPA peptide comprises, from the N-terminus to the C-terminus, an extracellular domain comprising the CD34 epitope or the CD34 extracellular domain, a multimerization region comprising one or more unpaired cysteine ​​residues, a GP130 (IL6ST) transmembrane domain, and a GP130 (IL6ST) intracellular signaling domain. In some embodiments, the SPA peptide further comprises an N-terminus leader sequence. In some embodiments, the leader sequence is a CD8a signal sequence, a GP130 (IL6ST) signal sequence, a CD34 signal sequence, or an erythropoietin receptor signal sequence. (EpoR). In some modalities, the leader sequence comprises MALPVTALLLPLALLLHAARP (SEQ ID NO: 108), MLVRRGARAGPRMPRGWTALCLLSLLPSGFM (SEQ ID NO: 109), MDHLGASLWPQVGSLCLLLAGAAW (SEQ ID MLTLQTWLVQALFIFLTTESTG (SEQ ID NO: 111). NO: 110) or

[0167] In some embodiments, the SPA peptide comprises a sequence selected from the group presented in SEQ ID NOs: 1-58 or 63-104. SPAs with an N-terminus leader sequence are provided in SEQ ID NOs: 1-58. SPAs without an N-terminus leader sequence are provided in SEQ ID NOs: 63-104. In some embodiments, the SPA peptide comprises a sequence as presented in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 104. In some embodiments, the SPA peptide comprises a sequence selected from the group shown in SEQ ID Nos: 1-58 or 63-104. In some embodiments, the SPA peptide comprises Petition 870250081011, dated 09 / 09 / 2025, p. 64 / 230 40 / 166 a sequence with approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with a sequence as presented in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 104. In some embodiments, the SPA sequence comprises the sequence presented in SEQ ID NO: 20. In some embodiments, the SPA sequence comprises the sequence presented in SEQ ID NO: 81. In some embodiments, the SPA peptide comprises a sequence with approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with a sequence as presented in SEQ ID NOs: 20 or 81. Logic Gate Systems

[0168] As used herein, a logic gate, circuit, circuit receptor, system, or system receptor refers to a two-part protein expression system comprising an initiation receptor and a chimeric antigen receptor. The system may be encoded in at least one nucleic acid inserted into a cell, where the initiation receptor is expressed in the cell. The intracellular domain of the initiation receptor is cleaved from the transmembrane domain after binding of the initiation receptor to its target antigen. The intracellular domain is then able to translocate to a cell nucleus where it induces the expression of the chimeric antigen receptor.

[0169] In one aspect, systems comprising an initiation receptor that binds to a target antigen and a chimeric antigen receptor that binds to a target antigen are provided here, wherein the transcription factor of the intracellular domain of the initiation receptor is capable of inducing the expression of CAR and / or SPA. Such systems are alternatively referred to as gates. Petition 870250081011, dated 09 / 09 / 2025, page 65 / 230 41 / 166 logic or circuits. In some respects, the system is encoded by nucleic acid transgenes inserted into an immune cell. The system may be encoded in a single nucleic acid insertion or fragment comprising both transgenes, or it may be encoded in two nucleic acids that encode the system's transgenes individually. The initiation receptor and the CAR of the system may be placed in any order on the single nucleic acid. For example, the initiation receptor may be at the 5' end and the CAR may be at the 3' end, or the CAR may be at the 5' end and the initiation receptor may be at the 3' end.

[0170] A first constitutive promoter can be operationally linked to the nucleotide sequence encoding the initiation receptor and / or SPA. An inducible promoter can also be operationally linked to the nucleotide sequence encoding the CAR. In some embodiments, when the system is encoded in a single recombinant nucleic acid insert or fragment comprising both transgenes, the nucleic acid may comprise, in a 5' to 3' direction, the constitutive promoter; the nucleotide sequence encoding the initiation receptor; the inducible promoter; and the nucleotide sequence encoding the chimeric antigen receptor. Alternatively, the nucleic acid may comprise, in a 5' to 3' direction, the inducible promoter; the nucleotide sequence encoding the chimeric antigen receptor; the constitutive promoter; and the nucleotide sequence encoding the initiation receptor.In some embodiments, the inducible promoter comprises one or more HNF^ enhancing elements (for example, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more HNF^ enhancing elements). In some embodiments, the constitutive promoter comprises an EFW promoter. Initiation Recipients

[0171] Preparation receptors comprising an extracellular antigen-binding domain that specifically binds to target antigens and one or more intracellular domains of or derived from a Petition 870250081011, dated 09 / 09 / 2025, p. 66 / 230 42 / 166 transcriptional regulator and / or a DNA-binding domain.

[0172] In certain aspects of the present disclosure, the initiation receptor is a synthetic receptor based on the Notch protein. The binding of a natural Notch receptor to a cognate ligand, such as those of the Delta protein family, causes intramembrane proteolysis that cleaves an intracellular fragment of the Notch protein. This intracellular fragment is a transcriptional regulator that only functions when cleaved from Notch. Cleavage can occur by sequential proteolysis by the metalloproteinase ADAM and the gamma-secretase complex. This intracellular fragment enters the nucleus of a cell and activates cell-to-cell signaling genes. In contrast to a natural Notch protein, a synthetic Notch initiation receptor replaces the natural intracellular Notch fragment with one that causes a gene encoding a protein of choice, such as a CAR, to be transcribed after the release of the intracellular fragment from the initiation receptor.

[0173] Notch receptors possess a modular domain organization. The ectodomains of Notch receptors consist of a series of N-terminal epidermal growth factor (EGF)-like repeats that are responsible for ligand binding. In synthetic Notch receptors or initiation receptors, the Notch ligand-binding domain is replaced by a ligand-binding domain that binds to a selected target ligand or antigen. The EGF repeats are followed by three LIN-12 / Notch repeat (LNR) modules, which are unique to Notch receptors and are widely reported to participate in preventing premature receptor activation. The heterodimerization (HD) domain of Notch1 is cleaved by furin cleavage, such that its N-terminal portion terminates the extracellular subunit, and its C-terminal portion constitutes the beginning of the transmembrane subunit.Following the extracellular region, the receptor has a transmembrane segment and an intracellular domain (ICD), which includes a transcriptional regulator.

[0174] Multiple forms of initiation receptors can be used in Petition 870250081011, dated 09 / 09 / 2025, page 67 / 230 43 / 166 methods, cells, and nucleic acids as described herein. One type of initiation receptor contemplated for use in the methods and cells of this document comprises a heterologous extracellular ligand-binding domain, a binding polypeptide possessing substantial sequence identity with a Notch receptor including the NRR, a TMD, and an ICD. Fn Notch receptors comprise a heterologous extracellular ligand-binding domain, a binding polypeptide with substantial sequence identity with a Robo receptor (such as a mammalian Robo1, Robo2, Robo3, or Robo4), followed by 1, 2, or 3 fibronectin (“Fn”) repeats, a TMD, and an ICD. Mini Notch receptors comprise a heterologous extracellular ligand-binding domain, a binding polypeptide with substantial sequence identity with a Notch receptor (without the NRR), a TMD, and an ICD.Minimal ligand Notch receptors comprise a heterologous extracellular ligand-binding domain, a ligand polypeptide with no substantial sequence identity with a Notch receptor (e.g., a synthetic (GGS)n polypeptide sequence), a TMD, and an ICD. Hinge Notch receptors comprise a heterologous extracellular ligand-binding domain, a hinge sequence comprising an oligomerization domain (i.e., a domain that promotes higher-order dimerization, trimerization, or multimerization with a synthetic receptor and / or an existing host receptor), a TMD, and an ICD. All these receptor classes are synthetic, recombinant, and do not occur in nature.In some embodiments, the unnaturally occurring receptors disclosed herein bind to a ligand displayed on the target cell surface, which triggers proteolytic cleavage of the receptors and the release of a transcriptional regulator that modulates a customized transcriptional program in the cell. In some embodiments, the initiation receptor does not include a LIN-12-Notch repeat (LNR) and / or a heterodimerization (HD) domain of a Notch receptor. Extracellular Domain of the Initiation Receptor

[0175] In some forms, the extracellular domain includes the portion Petition 870250081011, dated 09 / 09 / 2025, page 68 / 230 44 / 166 of ligand binding to a receptor. In some embodiments, the extracellular domain includes an antigen-binding chemical portion that binds to one or more target antigens. In some embodiments, the antigen-binding chemical portion includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding chemical portion is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody or a minibody, an F(ab')2 fragment, a Fab fragment, a single-chain variable fragment (scFv) and a single-domain antibody (sdAb) or a functional fragment thereof. In some embodiments, the antigen-binding chemical portion comprises an scFv.The chemical portion binding to the antigen may include naturally occurring amino acid sequences or may be manipulated, designed, or modified to provide desired and / or improved properties, for example, increased binding affinity.

[0176] In several embodiments, an initiation receptor comprises means for binding a target protein, optionally binding a human target protein. In some embodiments, the medium binds a target protein. In some embodiments, the medium binds a human target protein. In some embodiments, the medium is an antibody or antigen-binding fragment or equivalent thereof (e.g., a full-length antibody or an F(ab')2 fragment, a Fab fragment, a single-chain variable fragment (scFv), and a single-domain antibody (sdAb) or a functional fragment thereof). Transmembrane Domain

[0177] In some modalities, the initiation receiver also comprises a hinge domain. In some modalities, the hinge domain is a CD8a hinge.

[0178] As described above, the initiation receptor comprises a transmembrane domain (TMD) comprising one or more ligand-inducible proteolytic cleavage sites. Petition 870250081011, dated 09 / 09 / 2025, page 69 / 230 45 / 166

[0179] In some embodiments, the TMD comprises a Notch1 transmembrane domain.

[0180] Generally, the appropriate TMD for the chimeric receptors disclosed here can be any transmembrane domain of a Type 1 transmembrane receptor including at least one gammasecretase cleavage site. A detailed description of the structure and function of the gammasecretase complex, as well as its substrate proteins, including amyloid precursor protein (APP) and Notch, can, for example, be found in a recent review by Zhang et al, Frontiers Cell Neurosci (2014). Suitable non-limiting TMDs of Type 1 transmembrane receptors include those of CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HLA-A, and IFNAR2, where the TMD includes at least one gamma secretase cleavage site.Additional suitable TMDs for the compositions and methods described herein include, but are not limited to, transmembrane domains of Type 1 transmembrane receptors IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBOl, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, YASN, FLT1, CDH5, PKHD1, NECTINl, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd and PTPRM. In some embodiments, the TMD of chimeric polypeptides or Notch disclosure receptors is a TMD derived from a member of the calsintenin family, such as alpha alkalinein and gamma alkalinein. In some embodiments, the TMD of chimeric polypeptides or Notch disclosure receptors is a known TMD for Notch receptors. In some embodiments, the TMD of chimeric polypeptides or Notch disclosure receptors is a TMD derived from a different Notch receptor.For example, in a Mini Notch based on a human Notch1, the TMD Notch1 can be replaced by a TMD Notch2, TMD Notch3, TMD Notch4, or a TMD Notch from a non-human animal, such as Danio. Petition 870250081011, dated 09 / 09 / 2025, p. 70 / 230 46 / 166 rerio, Drosophila melanogaster, Xenopus laevis or Gallus gallus.

[0181] In some embodiments, the initiation receptor comprises a Notch cleavage site, such as S2 or S3. Additional proteolytic cleavage sites suitable for the compositions and methods disclosed herein include, but are not limited to, ADAM10, a metalloproteinase cleavage site for an MMP selected from collagenase-1, 2 and 3 (MMP-1, 8 and 13), gelatinase A and B (MMP-2 and 9), stromelysin 1, 2 and 3 (MMP-3, 10 and 11), matrilysin (MMP-7) and membrane metalloproteinases (MT1-MMP and MT2-MMP). Another example of a suitable protease cleavage site is a plasminogen activator cleavage site, for example, a urokinase plasminogen activator (uPA) cleavage site or a tissue plasminogen activator (tPA) cleavage site. Another example of a suitable protease cleavage site is a prolactin cleavage site. Specific examples of uPA and tPA cleavage sequences include sequences comprising Yal-Gly-Arg.Another example of a protease cleavage site that can be included in a proteolytically cleavable ligand is a protease cleavage site from tobacco mosaic virus (TEV), for example, Glu-Asn-Leu-Tyr-Thr-Gln-Ser (SEQ ID NO: 112), where the protease cleaves between glutamine and serine. Another example of a protease cleavage site that can be included in a proteolytically cleavable ligand is an enterokinase cleavage site, for example, AspAsp-Asp-Asp-Lys (SEQ ID NO: 113), where cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable ligand is a thrombin cleavage site, for example, Leu-Val-Pro-Arg (SEQ ID NO: 114).Additional suitable ligands comprising protease cleavage sites include sequences cleavable by the following proteases: a PreScission™ protease (a fusion protein comprising human rhinovirus protease 3C and glutathione-S-transferase), a thrombin, cathepsin B, Epstein-Barr virus protease, MMP-3 (stromelysin), MMP-7 (matrilysin), MMP-9; thermolysin-like MMP, matrix metalloproteinase 2 (MMP-2), cathepsin L; cathepsin D. Petition 870250081011, dated 09 / 09 / 2025, page 71 / 230 47 / 166 Matrix metalloproteinase 1 (MMP-1), urokinase-type plasminogen activator, membrane matrix metalloprotemase type 1 (MT-MMP), stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin-1, matrix metalloproteinase 13 (collagenase-3), tissue-type plasminogen activator (tPA), human prostate-specific antigen, kallikrein (hK3), neutrophil elastase, and calpain (calcium-activated neutral protease). Proteases that are not native to the host cell in which the receptor is expressed (e.g., TEV) can be used as an additional regulatory mechanism, whereby receptor activation is reduced until the protease is expressed or otherwise supplied. Furthermore, a protease may be associated with tumors or diseases (expressed to a significantly higher degree than in normal tissue) and serve as an independent regulatory mechanism.For example, some matrix metalloproteinases are highly expressed in certain types of cancer.

[0182] In some embodiments, the amino acid substitution(s) within the TMD includes one or more substitutions within a “GV” motif of the TMD. In some embodiments, at least one of these substitutions comprises an alanine substitution. Additional sequences and substitutions are described in WO2021061872, which is incorporated herein by reference in its entirety. Intracellular Domain

[0183] In some embodiments, the initiation receptor comprises one or more intracellular domains of, or derived from, a transcriptional regulator and / or a DNA-binding domain. In some embodiments, the intracellular domain comprises means for modulating the transcription of one or more genes. In some embodiments, the means for modulating the transcription of one or more genes comprise a transcriptional regulator, for example, a transcriptional regulator provided herein or an equivalent thereof. In some embodiments, the initiation receptor comprises one or more intracellular domains of, or derived from, a transcriptional regulator and / or a DNA-binding domain. Petition 870250081011, dated 09 / 09 / 2025, page 72 / 230 48 / 166 In some forms, the intracellular domain comprises an HNF1a / p65 domain or a Gal4 / VP64 domain.

[0184] Transcriptional regulators activate or repress the transcription of cognate promoters. Transcriptional activators typically bind close to transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Transcriptional repressors bind to transcriptional promoters and sterically prevent transcriptional initiation by RNA polymerase. Other transcriptional regulators serve as activators or repressors depending on where they bind and cellular conditions. Thus, as used herein, a transcriptional activation domain refers to the domain of a transcription factor that interacts with transcriptional control elements and / or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerases, etc.) to increase and / or activate the transcription of one or more genes.Non-limiting examples of transcriptional activation domains include: a VP16 activation domain of the herpes simplex virus, VP64 (which is a tetrameric derivative of VP16), HIV TAT, an NFkB p65 activation domain, p53 1 and 2 activation domains, a CREB (cAMP response element-binding protein) activation domain, an E2A activation domain, an NFAT (activated T-cell nuclear factor) activation domain, yeast Gal4, yeast GCN4, yeast HAP1, MLL, RTG3, GLN3, OAF1, PIP2, PDR1, PDR3, PHO4, a LEU3 glucocorticoid receptor transcription activation domain, B-cell POU homeodomain protein Oct2, plant Ap2, or any others known to one skilled in the art. In some embodiments, the transcriptional regulator is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-YP64, Gal4KRAB, and HAP1-VP16. In some embodiments, the transcriptional regulator is Gal4-VP64.A transcriptional activation domain may comprise a wild-type or naturally occurring sequence, or it may be a modified, mutant, or derived version of the original transcriptional activation domain that has the desired ability to enhance and / or activate the transcription of one or more genes. In some embodiments, the transcriptional regulator may include... Petition 870250081011, dated 09 / 09 / 2025, page 73 / 230 49 / 166 is still a nuclear location signal.

[0185] In some embodiments, the initiation receptor comprises one or more intracellular DNA-binding domains (or DB domains). Such DNA-binding domains refer to sequence-specific DNA-binding domains that bind a particular element of the DNA sequence. Thus, as used herein, a sequence-specific DNA-binding domain refers to a portion of the protein domain that has the ability to selectively bind DNA having a specific, predetermined sequence. A sequence-specific DNA-binding domain may comprise a wild-type or naturally occurring sequence, or it may be a modified, mutant, or derived version of the original domain that has the desired ability to bind to a desired sequence. In some embodiments, the sequence-specific DNA-binding domain is manipulated to bind a desired sequence.Non-limiting examples of proteins with sequence-specific DNA-binding domains that can be used in synthetic proteins described herein include HNF1a, Gal4, GCN4, tetracycline reverse receptor, THY1, SYN1, NSE / RU5', AGRP, CALB2, CAMK2A, CCK, CHAT, DLX6A, EMX1, zinc finger proteins or domains thereof, CRISPR / Cas proteins such as Cas9, Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu196 and TALES.

[0186] In those embodiments where a CRISPR / Cas-like protein is used, the CRISPR / Cas-like protein may be a wild-type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild-type or modified CRISPR / Cas protein. The CRISPR / Cas-like protein may be modified to increase the Petition 870250081011, dated 09 / 09 / 2025, page 74 / 230 50 / 166 affinity and / or specificity of nucleic acid binding, alter an enzymatic activity and / or alter another property of the protein. For example, the nuclease domains (i.e., DNase, RNase) of the CRISPR / Cas-like protein can be modified, deleted, or inactivated. Alternatively, the CRISPR / Cas-like protein can be truncated to remove domains that are not essential for the functions of the systems described herein. For example, a CRISPR enzyme that is used as a DNA-binding protein or its domain can be mutated relative to a corresponding wild-type enzyme, so that the mutated CRISPR or its domain lacks the ability to cleave a nucleic acid sequence containing a DNA-binding domain target site. For example, a D10A mutation can be combined with one or more H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially devoid of all DNA cleavage activity. Juxtamembrane Domain

[0187] ECD and TMD, or TMD and ICD, can be linked together with a linking polypeptide, such as a juxtamembrane domain. “SynNotch” or synthetic notches comprise a heterologous extracellular ligand-binding domain, a linking polypeptide with substantial sequence identity to a JMD Notch receptor (including NRR), a TMD, and an ICD. Fn Notch receptors comprise a heterologous extracellular ligand-binding domain, a linking polypeptide with substantial sequence identity to a Robo receptor (such as a mammalian Robo1, Robo2, Robo3, or Robo4), followed by 1, 2, or 3 fibronectin (“Fn”) repeats, a TMD, and an ICD. “Mini Notch” receptors comprise a heterologous extracellular ligand-binding domain, a ligand polypeptide with substantial sequence identity to a JMD Notch receptor but lacking the NRR modules (the LIN-12-Notch repeat (LNR) and heterodimerization domain), a TMD, and an ICD."Minimal ligand notch" receptors comprise a domain that binds to extracellular ligands. Petition 870250081011, dated 09 / 09 / 2025, page 75 / 230 51 / 166 heterologous, a binding polypeptide without substantial sequence identity with a Notch receptor (e.g., without limitation, having a synthetic polypeptide sequence (GGS)n), a TMD, and an ICD. “Hinge Notch” receptors comprise a heterologous extracellular ligand-binding domain, a hinge sequence comprising an oligomerization domain (i.e., a domain that promotes higher-order dimerization, trimerization, or multimerization with a synthetic receptor and / or an existing host receptor), a TMD, and an ICD.

[0188] In some embodiments, the initiation receptor comprises a juxtamembrane domain (JMD) peptide between the extracellular and transmembrane domains. In some embodiments, the initiation receptor comprises a juxtamembrane domain (JMD) peptide between the transmembrane and intracellular domains. In some embodiments, the JMD peptide comprises an LWF motif. The use of LWF motifs in receptor constructs is described in U.S. Patent No. 10,858,443, which is incorporated herein by reference in its entirety. In some embodiments, the JMD peptide has substantial sequence identity with the JMD of Notch1, Notch2, Notch3, and / or Notch4. In some embodiments, the JMD peptide has substantial sequence identity with the JMD of Notch1, Notch2, Notch3, and / or Notch4, but does not include a LIN-12-Notch repeat (LNR) and / or a heterodimerization (HD) domain of a Notch receptor.In some embodiments, the JMD peptide does not possess substantial sequence identity with the JMD of Notch1, Notch2, Notch3, and / or Notch4. In some embodiments, the JMD peptide includes an oligomerization domain that promotes the formation of dimers, trimers, or higher-order clusters of the receptor. Such JMD peptides are described in document WO2021061872, which is incorporated herein by reference in its entirety.

[0189] In the Mini Notch receptor, the binding polypeptide is derived from a Notch JMD sequence after deletion of the NRR and HD domains. The Notch JMD sequence can be the Notch1, Notch2, Notch3, or Notch4 sequence and can be derived from a non-human homolog, such as those Petition 870250081011, dated 09 / 09 / 2025, page 76 / 230 52 / 166 of Drosophila, Gallus, Danio and similar species. Four to 50 amino acid residues of the remaining Notch sequence can be used as a polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are varied to alter the orientation and / or proximity of the ECD and TMD to each other to achieve a desired chimeric polypeptide activity, such as signal transduction when the linker is induced or in the absence of a linker.

[0190] In the minimal ligand Notch receptor, the binding polypeptide does not possess substantial sequence identity with a Notch JMD sequence, including the Notch JMD sequence of Notch1, Notch2, Notch3, or Notch4, or a non-human homolog thereof. Four to 50 amino acid residues may be used as the polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are varied to alter the orientation and / or proximity of the ECD and TMD to each other to achieve a desired chimeric disclosure polypeptide activity. The minimal ligand sequence may be manipulated to include or omit a protease cleavage site and may include or omit a glycosylation site or sites for other types of post-translational modification. In some embodiments, the minimal ligand does not comprise a protease cleavage site or a glycosylation site.

[0191] In some embodiments, the initiation receptor further comprises a hinge. The hinge ligands that can be used in the initiation receptor may include an oligomerization domain (e.g., a hinge domain) containing one or more polypeptide motifs that promote the formation of oligomers of chimeric polypeptides through intermolecular disulfide bonds. In these cases, within the chimeric receptors disclosed herein, the hinge domain generally includes a flexible polypeptide connector region disposed between the ECD and the TMD. Thus, the hinge domain provides flexibility between the ECD and the TMD and also provides sites for intermolecular disulfide bonds between two or more Petition 870250081011, dated 09 / 09 / 2025, page 77 / 230 53 / 166 chimeric polypeptide monomers to form an oligomeric complex. In some embodiments, the hinge domain includes motifs that promote the formation of dimers of the chimeric polypeptides disclosed herein. In some embodiments, the hinge domain includes motifs that promote the formation of trimers of the chimeric polypeptides disclosed herein (e.g., a hinge domain derived from OX40). The hinge polypeptide sequences suitable for the compositions and methods of disclosure may be naturally occurring hinge polypeptide sequences (e.g., those of naturally occurring immunoglobulins) or may be manipulated, manipulated, or modified to provide desired and / or enhanced properties, e.g., by modulating transcription.Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as the IgG1 hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or a functional variant thereof. In some embodiments, the hinge polypeptide sequence contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence contains one or more CPPC motifs (SEQ ID NO: 115).

[0192] Hinge polypeptide sequences can also be derived from a CD8a hinge domain, a CD28 hinge domain, a CD152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an OX40 hinge domain, and their functional variants. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD8a hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an OX40 hinge domain or a functional variant thereof. In some embodiments, the hinge domain Petition 870250081011, dated 09 / 09 / 2025, page 78 / 230 54 / 166 includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof.

[0193] The Fn Notch binding polypeptide is derived from the Robo1 JMD, which contains a fibronectin (Fn) repeat domain, with a short polypeptide sequence between the Fn repeats and the TMD. The Fn Notch binding polypeptide does not contain a Notch negative regulatory region (NRR) or the Notch HD domain. The Fn binding polypeptide may contain 1, 2, 3, 4, or 5 Fn repeats. In some embodiments, the chimeric receptor comprises an Fn binding polypeptide with about 1 to about 5 Fn repeats, about 1 to about 3 Fn repeats, or about 2 to about 3 Fn repeats. The short polypeptide sequence between the Fn repeats and the TMD may have from about 2 to about 30 amino acid residues. In some forms, the short polypeptide sequence can have between about 5 and about 20 amino acids, of any sequence.In some embodiments, the short polypeptide sequence may have between about 5 and about 20 naturally occurring amino acids of any sequence. In some embodiments, the short polypeptide sequence may have between about 5 and about 20 amino acids of any sequence, but not having more than one proline. In some embodiments, the short polypeptide sequence may have between about 5 and about 20 amino acids, and about 50% or more of the amino acids are glycine. In some embodiments, the short polypeptide sequence may have between about 5 and about 20 amino acids, where the amino acids are selected from glycine, serine, threonine, and alanine. In some embodiments, the length and amino acid composition of the Fn-linking polypeptide sequence may be varied to alter the orientation and / or proximity of the ECD and TMD relative to each other to achieve a desired chimeric polypeptide disclosure activity. Transfer Stop Sequence

[0194] In some modes, the initiation receiver also includes a stop-transfer sequence (STS) between the domain Petition 870250081011, dated 09 / 09 / 2025, page 79 / 230 55 / 166 transmembrane and intracellular domains. The STS comprises a charged lipophobic sequence. Without being tied to any theory, the STS serves as a membrane anchor and is believed to prevent the passage of the intracellular domain to the plasma membrane. The use of STS domains in initiation receptors is described in document WO2021061872, which is incorporated herein by reference in its entirety.Non-limiting illustrative STS sequences include STS sequences of APLP1, APLP2, APP, TGBR3, CSF1R, CXCL16, CX3CL1, DAG1, DCC, DNER, DSG2, CDH1, GHR, HLA-A, IFNAR2, IGF1R, IL1R1, ERN2, KCNE1, KCNE2, CHL1, LRP1, LRP2, LRP18, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKTFD1, NECTIN1, KL, IL6R, EFNB1, CD44, CLSTN1, LRP8, PCDHGC3, NRG1, LRP1B, JAG2, EFNB2, DLL1, CLSTN2, EPCAM, ErbB4, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA4, IL1R2, KCNE4, SCN2B, Nradd, PTPRM, Notch1, Notch2, Notch3, and Notch4. In some embodiments, the STS is heterologous to the transmembrane domain. In some embodiments, the STS is homologous to the transmembrane domain. The STS sequences are described in document WO2021061872, which is incorporated herein by reference in its entirety.

[0195] Chimeric Antigen Receptors

[0196] In another aspect, chimeric antigen receptors comprising an extracellular antigen-binding domain that specifically binds to a target antigen or ligand are provided here.

[0197] In some embodiments, the chimeric antigen receptor includes an extracellular portion comprising an antigen-binding domain. The antigen recognition domain of a receptor, such as a CAR, may be linked to one or more intracellular signaling components, such as signaling components that mimic activation via an antigen-receptor complex, such as a TCR complex in the case of a CAR, and / or Petition 870250081011, dated 09 / 09 / 2025, page 80 / 230 56 / 166 signaling through another cell surface receptor. Thus, in some embodiments, the extracellular binding component (e.g., ligand-binding or antigen-binding domain) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that is naturally associated with one of the domains on the receptor is used, for example, CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different cell surface membrane proteins to minimize interactions with other members of the receptor complex.

[0198] In some aspects, the chimeric antigen receptor includes an extracellular portion comprising an antigen-binding domain described herein and an intracellular signaling domain. In some embodiments, an antibody or fragment includes a scFv, a VH, or a single-domain VH antibody, and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a zeta-chain signaling domain of a CD3-zeta chain (CD3). In some embodiments, the chimeric antigen receptor includes a transmembrane domain that links the extracellular domain and the intracellular signaling domain.

[0199] In some embodiments, the transmembrane domain contains a transmembrane portion of CD8a or CD28. The extracellular and transmembrane domains may be linked directly or indirectly. In some embodiments, the extracellular and transmembrane domains are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and the intracellular signaling domain. In some embodiments, the T cell costimulatory molecule is CD28 or 41BB. Chimeric Antigen Receptor Extracellular Domain Petition 870250081011, dated 09 / 09 / 2025, page 81 / 230 57 / 166

[0200] In some embodiments, the extracellular domain includes the ligand-binding portion of a receptor. In some embodiments, the extracellular domain includes an antigen-binding chemical portion that binds to one or more target antigens. In some embodiments, the antigen-binding chemical portion includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding chemical portion is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody or a minibody, an F(ab')2 fragment, a Fab fragment, a single-chain variable fragment (scFv), and a single-domain antibody (sdAb) or a functional fragment thereof. In some embodiments, the antigen-binding chemical portion comprises an scFv.The chemical portion binding to the antigen may include naturally occurring amino acid sequences or may be manipulated, designed, or modified to provide desired and / or improved properties, for example, increased binding affinity.

[0201] In several embodiments, a CAR comprises means for binding a target protein. In some embodiments, the medium binds a target protein. In some embodiments, the medium binds a human target protein. In some embodiments, the medium is an antibody or antigen-binding fragment or equivalent thereof (e.g., a full-length antibody or an F(ab')2 fragment, a Fab fragment, a single-chain variable fragment (scFv), and a single-domain antibody (sdAb) or a functional fragment thereof) means for binding a target protein. CAR Transmembrane Domain

[0202] The transmembrane domain in some embodiments is derived from a natural or synthetic source. When the source is natural, the domain, in some respects, is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprising at least the transmembrane region(s) of) Petition 870250081011, dated 09 / 09 / 2025, page 82 / 230 58 / 166 alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and / or CD154. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain predominantly comprises hydrophobic residues, such as leucine and valine. In some aspects, a trio of phenylalanine, tryptophan, and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is made by ligands, spacers, and / or transmembrane domain(s).

[0203] In some embodiments, the receptor transmembrane domain, for example, the CAR, is a human CD28 transmembrane domain or variant thereof, for example, a 27 amino acid transmembrane domain of a human CD28 (Accessory number: P10747.1).

[0204] In some modes, the CAR comprises a CD8a or CD28 TMD. CAR hinge

[0205] In some embodiments, the CAR also includes a spacer, which may be or include at least a portion of a constant immunoglobulin region or a variant or modified version thereof, such as a hinge region, for example, a CD8a hinge region, an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is from a human IgG, such as IgG4 or IgG1. In some respects, the constant region portion serves as a spacer region between the antigen recognition component, for example, scFv, and the transmembrane domain. The spacer may have a length that provides increased cell responsiveness after antigen binding, compared to the absence of the spacer. In some examples, the spacer is about 12 amino acids long or no more than 12 amino acids long.Exemplary spacers include those that have at least about 10 to 229 amino acids, approximately. Petition 870250081011, dated 09 / 09 / 2025, p. 83 / 230 59 / 166 to 200 amino acids, approximately 10 to 175 amino acids, approximately 10 to 150 amino acids, approximately 10 to 125 amino acids, approximately 10 to 100 amino acids, approximately 10 to 75 amino acids, approximately 10 to 50 amino acids, approximately 10 to 40 amino acids, approximately 10 to 30 amino acids, approximately 10 to 20 amino acids, or approximately 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region is approximately 12 amino acids or less, approximately 119 amino acids or less, or approximately 229 amino acids or less. Exemplary spacers include the CD8a hinge, the IgG4 hinge alone, the IgG4 hinge linked to the CH2 and CH3 domains, or the IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or publication of international patent application number WO2014031687.In some embodiments, the CAR hinge comprises a CD8a CD8α, truncated CD8α, or CD28 hinge domain.

[0206] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a co-stimulatory receptor, and / or a signal through a co-stimulatory receptor alone. In some embodiments, a short oligo- or polypeptide ligand, for example, a ligand between 2 and 10 amino acids in length, such as one containing glycines and serines, for example, a glycine-serine doublet, is present and forms a link between the transmembrane domain and the cytoplasmic signaling domain of the receptor. Intracellular domain of CAR

[0207] In some embodiments, after CAR binding, the cytoplasmic domain or the intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, for example, T cells engineered to express the receptor. In some embodiments, the CAR comprises means to activate at least one of the Petition 870250081011, dated 09 / 09 / 2025, page 84 / 230 60 / 166 normal effector functions or immune cell responses, for example, a T cell manipulated to express the receptor. For example, in some contexts, the receptor induces a T cell function, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal.In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR) and, in some respects, also those of coreceptors that in the natural context act in conjunction with such receptor to initiate signal transduction after antigen receptor engagement and / or any derivative or variant of such molecules and / or any synthetic sequence having the same functional capacity. In some embodiments, the means for at least one of the normal effector functions or responses of the immune cell comprises an intracellular activation domain of CAR, for example, an intracellular activation domain provided herein or an equivalent thereof.In some embodiments, the means for at least one of the effector functions or normal responses of the immune cell comprise an intracellular activation domain of CAR and a co-stimulatory domain of CAR, for example, a co-stimulatory domain provided herein or an equivalent thereof.

[0208] In some respects, the receptor includes a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In Petition 870250081011, dated 09 / 09 / 2025, p. 85 / 230 61 / 166 In some embodiments, the cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, a portion thereof, or a CD3 zeta-derived sequence. In some embodiments, the intracellular activation domain comprises a CD3Z domain.

[0209] In some embodiments, the intracellular signaling domain comprises a human CD3 zeta-stimulating signaling domain or a functional variant thereof, such as a cytoplasmic 112 AA domain of the human CD3 zeta isoform 3 (Accessory No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993.

[0210] The receptor, for example, CAR, may include at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a CD3 chain of TCR that mediates T cell activation and cytotoxicity, for example, CD3 zeta chain. Thus, in some aspects, the extracellular domain is linked to one or more cell signaling modules. In some embodiments, the cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, the receptor, for example, CAR, further includes a portion of one or more additional molecules, such as Fc-gamma receptor, CD8, CD4, CD25, or CD16. For example, in some aspects, CAR includes a chimeric molecule between CD3-zeta or Fc-gamma receptor and CD8, CD4, CD25, or CD16.

[0211] In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 41BB or a functional variant or portion thereof, such as a cytoplasmic domain of 42 amino acids of a human 4-1BB (accession number Q07011.1) or a functional variant or portion thereof.

[0212] In some modalities, the receptor encompasses one or more, for example, two or more co-stimulatory domains and an activation domain, by Petition 870250081011, dated 09 / 09 / 2025, page 86 / 230 62 / 166 example, primary activation domain, in the cytoplasmic portion. Examples of receptors include intracellular components of CD3-zeta, CD28, and 4-1BB. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some respects, the T cell costimulatory molecule is 4-1BB.

[0213] In some embodiments, the receptor includes a signaling domain and / or transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40, DAP10 and ICOS. In some respects, the same receptor includes both activating and co-stimulatory components.

[0214] In certain embodiments, the intracellular signaling domain comprises a transmembrane and CD8a signaling domain linked to an intracellular CD3 domain (e.g., CD3-zeta). In some embodiments, the intracellular signaling domain comprises 4-1BB co-stimulatory domains (CD137, TNFRSF9) linked to an intracellular CD3 zeta domain. In some embodiments, the CAR comprises a 4-1BB co-stimulatory domain.

[0215] In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which can be used to confirm transduction or manipulation of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a nerve growth factor receptor (NGFR), or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operationally linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence or a ribosomal jump sequence, e.g., T2A. See WO2014031687.In some embodiments, the introduction of a construct encoding CAR and EGFRt separated by a T2A ribosome exchange can express two proteins of the same construct, so that EGFRt can be used as a marker to detect cells expressing such a construct. In some embodiments, Petition 870250081011, dated 09 / 09 / 2025, p. 87 / 230 63 / 166 a marker and, optionally, a linker sequence, may be any as disclosed in published patent application no. WO2014031687. For example, the marker may be a truncated EGFR (tEGFR) that is optionally linked to a linker sequence, such as a T2A ribosomal jump sequence.

[0216] In some modalities, the marker is a molecule, for example, a cell surface protein, not found naturally in T cells or not found naturally on the surface of T cells, or a portion thereof.

[0217] In some embodiments, the molecule is a non-self molecule, for example, a non-self protein, that is, one that is not recognized as self by the immune system of the host to which the cells will be adoptively transferred.

[0218] In some embodiments, the marker has no therapeutic function and / or produces no effect other than being used as a marker for genetic engineering, for example, to select successfully manipulated cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, such as a ligand for a cell to be encountered in vivo, such as a co-stimulatory or immune checkpoint molecule to enhance and / or attenuate cell responses after adoptive transfer and encounter with the ligand.

[0219] The CAR may comprise one or more synthetically modified amino acids in place of one or more naturally occurring amino acids. Examples of modified amino acids include, but are not limited to, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylphalanine, 4-carboxyphenylalanine, 3-phenylserine, 3-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetra Petition 870250081011, dated 09 / 09 / 2025, page 88 / 230 64 / 166 hydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbomano)carboxylic acid, α,γ-diaminobutyric acid, α,γ-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0220] For example, in some embodiments, the CAR includes an antibody or fragment thereof, including single-chain antibodies (sdAbs, for example, containing only the VH region), VH and scFvs domains, described herein, a spacer, such as a CD8a hinge, a CD8a transmembrane domain, a 4-1 BB intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, including sdAbs and scFvs described herein, a spacer such as a CD8a hinge, a CD8a transmembrane domain, a 4-1 BB intracellular signaling domain, and a CD3 zeta signaling domain.

[0221] Transgenes expressing the initiation receptor and CAR system can be introduced into cells, such as a T cell, using, for example, a site-specific technique. With site-specific integration of transgenes (e.g., initiation receptor and CAR), the transgenes can be targeted to a safe harbor or TRAC locus. Examples of site-specific techniques for integration into safe harbor loci include, but are not limited to, homology-dependent engineering using nucleases and homology-independent targeted insertion using Cas9.

[0222] Manipulated cells have applications in immuno-oncology. The initiation receptor and CAR, for example, can be selected to target different specific tumor antigens. Examples of cancers that can be effectively targeted using such cells are blood cancers or solid cancers. In some modalities, immune cell therapy can be used to treat solid tumors. Nucleic Acids and Vectors Petition 870250081011, dated 09 / 09 / 2025, p. 89 / 230 65 / 166

[0223] In another aspect, one or more nucleic acids are provided herein, wherein the one or more nucleic acids encode a synthetic pathway activator described herein. In another aspect, one or more nucleic acids are provided herein, wherein the one or more nucleic acids encode a sequence selected from the group consisting of SEQ ID NOS: 1-58 or 63-104.

[0224] In some embodiments, the one or more recombinant nucleic acids further comprise a 5' homology-directed repair arm and / or a 3' homology-directed repair arm complementary to an insertion site on a host cell chromosome. In some embodiments, the one or more nucleic acids comprise the 5' homology-directed repair arm and / or the 3' homology-directed repair arm. In some embodiments, the one or more nucleic acids are incorporated into an expression cassette or an expression vector. In some embodiments, the expression cassette or expression vector further comprises a constitutive promoter upstream of the one or more recombinant nucleic acids.

[0225] In some embodiments, the initiation receptor, CAR, and the synthetic pathway activator are incorporated into a single expression cassette or a single expression vector. In some embodiments, the initiation receptor, CAR, and the synthetic pathway activator are incorporated into two or more expression cassettes or expression vectors. In some embodiments, the expression vector (or expression vectors) is a nonviral vector.

[0226] In some embodiments, the present disclosure includes nucleic acid DNA template inserts comprising one or more transgenes encoding synthetic pathway activators as described herein. In some embodiments, the DNA template insert encodes a synthetic pathway activator. In some embodiments, the nucleic acid DNA template further comprises initiation receptors and / or CARs. In some embodiments, the DNA template insert encodes an initiation receptor transgene. In some embodiments, the DNA template insert encodes a chimeric antigen receptor transgene. In some embodiments, the template insert Petition 870250081011, dated 09 / 09 / 2025, pp. 90 / 230 The 66 / 166 DNA sequence comprises a synthetic pathway activator, an initiation receptor transgene, and a chimeric antigen receptor transgene.

[0227] In some embodiments, one or more nucleic acids are encoded in a single DNA template insert. In some embodiments, one or more nucleic acids are encoded in multiple DNA template inserts. For example, one or more recombinant nucleic acids may be encoded in two, three, or four DNA template inserts.

[0228] The DNA template insert may also comprise a self-cleaving peptide. Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, for example, a porcine teschovirus-1 peptide (P2A), a Thata asigna virus peptide (T2A), an equine rhinitis A virus peptide (E2A), or a foot-and-mouth disease virus peptide (F2A). Self-cleaving 2A peptides allow the expression of multiple gene products from a single construct. (See, for example, Chang et al. Cleavage efficiency 2A peptides for high level monoclonal antibody expression in CHO cells, MAbs 7(2): 403-412 (2015)).

[0229] The DNA template insertion may also comprise a WPRE element. WPRE elements are generally described in Higashimoto, T., et al. Gene Ther 14, 1298-1304 (2007); and Zufferey, R., et al. J Virol. April 1999; 73(4): 2886-92., both incorporated herein by reference.

[0230] The DNA template insert may also comprise a polyA tail of SV40. Cells

[0231] Also provided here are cells or immune cells comprising at least one DNA template inserted non-virally into a target region of the cell genome, wherein the DNA template encodes one or more of the activators of the synthetic pathway as described herein. In some embodiments, the DNA template further encodes an initiation receptor and a CAR system, as described herein. Petition 870250081011, dated 09 / 09 / 2025, page 91 / 230 67 / 166

[0232] A cell comprising a DNA template insert at a target locus or safe harbor site, as described in this disclosure, may be referred to as a manipulated cell. In some embodiments, the cell or immune cell is any cell that can give rise to a pluripotent immune cell. In some embodiments, the immune cell is a primary immune cell. In some embodiments, the immune cell may be an induced pluripotent stem cell (iPSC) or a human pluripotent stem cell (HSPC). In some embodiments, the immune cell comprises primary hematopoietic cells or primary hematopoietic stem cells.In some modalities, this manipulated cell is a stem cell, a human cell, a primary cell, a hematopoietic cell, an adaptive immune cell, an innate immune cell, a natural killer (NK) cell, a T cell, a CD8+ cell, a CD4+ cell, or a T cell progenitor cell. In some modalities, the immune cells are T cells. In some modalities, the T cells are regulatory T cells, effector T cells, or naive T cells. In some modalities, the T cells are CD8+ T cells. In some modalities, the T cells are CD4+CD8+ T cells.

[0233] In some embodiments, the manipulated cell is a stem cell, a human cell, a primary cell, a hematopoietic cell, a hematopoietic stem cell, an adaptive immune cell, an innate immune cell, a T cell, or a T cell progenitor. Non-limiting examples of immune cells contemplated in this disclosure include T cells, B cells, natural killer (NK) cells, NKT / iNKT cells, macrophages, myeloid cells, and dendritic cells. Non-limiting examples of stem cells contemplated in this disclosure include pluripotent stem cells (PSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic stem cells derived from nuclear transfer (ntES; ES of Petition 870250081011, dated 09 / 09 / 2025, page 92 / 230 68 / 166 nuclear transfer), male germline stem cells (GS cells), embryonic germ cells (EG cells), hematopoietic / progenitor stem cells (HSPCs), somatic stem cells (adult stem cells), hemangioblasts, neural stem cells, mesenchymal stem cells, and stem cells from other cells (including osteocytes, chondrocytes, myocytes, cardiac myocytes, neurons, tendon cells, adipocytes, pancreocytes, hepatocytes, nephrocytes, and follicular cells, and so forth). In some embodiments, the manipulated cells are a T cell, NK cells, iPSC, and HSPC. In some embodiments, the manipulated cells used in the present disclosure are human cell lines cultured in vitro (e.g., deliberately immortalized cell lines, cancer cell lines, etc.).

[0234] Also provided in this document are cell populations comprising a plurality of cells or immune cells. In some embodiments, the genome of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the cells comprises the initiation receptor and CAR system as described in this document. Treatment Method for Diseases Related to the Immune System

[0235] In another aspect, the disclosure provides methods for treating an immune system-related condition (e.g., cancer) in an individual comprising administering to the individual an effective amount of a composition comprising a synthetic pathway activator described herein, as a cell comprising a synthetic pathway activator described herein. In some embodiments, the composition further comprises an initiation receptor that specifically binds to a target antigen and a chimeric antigen receptor that specifically binds to a target antigen. In another aspect, the disclosure provides methods for enhancing an immune response in an individual comprising administering to the individual an effective amount of a composition comprising an activator of Petition 870250081011, dated 09 / 09 / 2025, page 93 / 230 69 / 166 synthetic pathway described herein, as a cell comprising a synthetic pathway activator described herein. In some embodiments, the composition further comprises an initiation receptor that specifically binds to a target antigen and a chimeric antigen receptor that specifically binds to a target antigen.

[0236] In some embodiments, the methods provided here are useful for treating an immune system-related condition in an individual. In one embodiment, the individual is a human being.

[0237] In some embodiments, the methods provided herein (as methods of enhancing an immune response) are useful for the treatment of cancer, and as such, an individual receiving the synthetic pathway activator described herein has cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is immune-evasive. In some embodiments, the cancer is immune-responsive. In some embodiments, the cancer is immune-responsive. In specific embodiments, the cancer is renal cancer, renal cell carcinoma, clear cell renal cell carcinoma (ccRcc), colorectal cancer, or lung cancer. In some embodiments, the cancer is mesothelioma.

[0238] In another aspect, the disclosure provides methods of inhibiting (e.g., to exterminate, disable, or prevent the growth or expansion of) a target cell that expresses both the CAR antigen and the initiation receptor antigen. In another aspect, the invention provides methods for exterminating a target cell that expresses both the CAR antigen and the initiation receptor antigen. In some embodiments, the target cell is a cancerous cell.

[0239] In some modalities, treatment results in a decrease in the volume or size of the cancer. In some modalities, treatment is effective in reducing the volume of the cancer compared to the volume of the cancer before antibody administration. In some modalities, treatment results in a decrease in the rate of cancer growth. In some Petition 870250081011, dated 09 / 09 / 2025, page 94 / 230 In 70 / 166 modalities, the treatment is effective in reducing the cancer growth rate compared to the cancer growth rate before antibody administration. In some modalities, the treatment is effective in eliminating the cancer.

[0240] In some embodiments, the CAR antigen and / or the initiation receptor antigen are expressed at a higher level in cancer compared to a non-cancerous cell. The levels of CAR antigen and / or initiation receptor antigen can be assessed by any technique known in the field, including, but not limited to, protein assays or nucleic acid assays, such as FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique and FISH, and combinations thereof. Immune Modulation Method

[0241] Methods of administering a cell comprising a synthetic pathway activator described herein that may result in modulation of an immune response. Modulation may be an increase or decrease in an immune response. In some modalities, modulation is an increase in an immune response.

[0242] In one aspect, administration of a cell comprising a synthetic pathway activator described herein may result in the induction of pro-inflammatory molecules, such as cytokines or chemokines. Generally, the induced pro-inflammatory molecules are present at levels higher than those achieved with isotype control. Such pro-inflammatory molecules, in turn, result in the activation of antitumor immunity, including, but not limited to, T cell activation, T cell proliferation, T cell differentiation, M1-like macrophage activation, and NK cell activation. Thus, administration of a cell comprising a synthetic pathway activator described herein may Petition 870250081011, dated 09 / 09 / 2025, page 95 / 230 71 / 166 induce multiple antitumor immunological mechanisms that lead to tumor destruction. In some modalities, the cell's immunological activity is cytolytic activity.

[0243] In another aspect, methods for increasing an immune response in an individual are provided in this document, comprising administering to the individual an effective amount of a cell comprising a synthetic pathway activator described in this document. In some embodiments, the method of increasing an immune response in a subject comprises administering to the subject a cell comprising a synthetic pathway activator described herein.

[0244] In some embodiments, the cell is present in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0245] In all and any aspects of increasing an immune response as described herein, any increase or decrease or alteration of a characteristic or function(s) is compared to a cell that does not comprise a composition comprising a synthetic pathway activator described herein.

[0246] Enhancing an immune response can mean increasing an immune response or inducing an immune response. For example, enhancing an immune response encompasses both the initiation or start of an immune response, and the enhancement or amplification of an existing or ongoing immune response. In some modalities, treatment induces an immune response. In some modalities, the induced immune response is an adaptive immune response. In some modalities, the induced immune response is an innate immune response. In some modalities, treatment intensifies an immune response. In some modalities, the intensified immune response is an adaptive immune response. In some modalities, the intensified immune response is an innate immune response. In some modalities, treatment Petition 870250081011, dated 09 / 09 / 2025, page 96 / 230 72 / 166 enhances the immune response. In some modalities, the enhanced immune response is an adaptive immune response. In some modalities, the enhanced immune response is an innate immune response. In some modalities, the immune response is initiated or started by administration of a cell comprising a synthetic pathway activator described herein. In some modalities, the immune response is enhanced by administration of the cell comprising a synthetic pathway activator described herein. In some modalities, the immune response is enhanced by administration of the cell comprising a synthetic pathway activator and an initiation receptor and CAR system described herein.

[0247] In another aspect, the present invention provides methods of genetically editing a cell with a synthetic pathway activator described herein, resulting in the modulation of the cell's immune function. The modulation may be an increase in an immune response. In some embodiments, the modulation is an increase in immune function. In some embodiments, the modulation of function leads to the expression of cytokines or interleukins. In some embodiments, the modulation of function leads to the activation of an immune cell.

[0248] In some forms, the cell is a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell progenitor.

[0249] In some embodiments, modulation of cell function comprising a synthetic pathway activator as described herein leads to an increase in the cells’ capabilities to stimulate native and activated T cells, for example, by increasing the secretion of cytokines or chemokines by cells expressing the synthetic pathway activator described herein. In some embodiments, modulation of function enhances or increases the ability of cells to produce cytokines, chemokines, CARs, or co-stimulatory or activating receptors. In some embodiments, modulation enhances the T cell-stimulating function of cells expressing a synthetic pathway activator described herein, including, for example, capabilities Petition 870250081011, dated 09 / 09 / 2025, p. 97 / 230 73 / 166 of the cells to trigger T cell receptor (TCR) signaling, T cell proliferation, or T cell cytokine production.

[0250] In some embodiments, the increased immune response is the secretion of cytokines and chemokines. In some embodiments, the synthetic pathway activator described herein induces increased expression of at least one cytokine or chemokine in a cell compared to an isotype control cell. In some embodiments, at least one cytokine or chemokine is selected from the group consisting of: IL-2 and IFNγ. In some embodiments, the cytokine or chemokine is IL-2. In some embodiments, the cytokine or chemokine is IFNγ. In some modalities, cytokine or chemokine secretion is increased by approximately 1-100 times 1.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 times compared to an untreated cell or a cell treated with an isotype control antibody.In some modalities, the chemokine is IL-2 and secretion is increased between approximately 1-100 times, 1-10 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1-10 times, 10-20 times, 20-30 times, 30-40 times, 40-50 times, 50-60 times, 60-70 times, 70-80 times, 80-90 times, or 90-100 times compared to an untreated cell or a cell treated with an isotype control antibody. In some modalities, the cytokine is IFNγ and secretion is increased between approximately 1-100 times, 1-10 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1-10 times, 10-20 times, 20-30 times, 30-40 times, 40-50 times, 50-60 times, 60-70 times, 70-80 times, 80-90 times, or 90-100 times compared to an untreated cell or a cell treated with an isotype control antibody.

[0251] In some modalities, the enhanced immune response is the recruitment and activation of antitumor immune cells.

[0252] In some modalities, the cell expressing the synthetic pathway activator described here induces a memory immune response in Petition 870250081011, dated 09 / 09 / 2025, p. 98 / 230 74 / 166 comparison with an isotype control cell. In general, a memory immune response is a protective immune response following subsequent exposure to pathogens or antigens that the immune system has previously encountered. Examples of memory immune responses include the immune response after infection or vaccination with an antigen. In general, memory immune responses are mediated by lymphocytes, such as T cells or B cells. In some modalities, the memory immune response is a protective immune response against cancer, including the growth, proliferation, or metastasis of cancer cells. In some modalities, the memory immune response inhibits, prevents, or reduces the growth, proliferation, or metastasis of cancer cells. Cell Editing Methods

[0253] The terms “gene editing” or “genome editing,” as used herein, refer to a type of genetic manipulation in which DNA is inserted, replaced, or removed from the genome using artificially manipulated nucleases or molecular scissors. They are a useful tool for elucidating the function and effect of specific gene or protein sequences or for altering cellular behavior (e.g., for therapeutic purposes).

[0254] Currently available genome editing tools include zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for incorporating genes into safe harbor loci (e.g., the safe harbor locus of the adeno-associated virus 1 (AAVS1) integration site). The DICE (double integrase cassette exchange) system, which utilizes integrase phiC31 and integrase Bxb1, is a tool for target integration. Additionally, clustered regularly interspaced short palindromic repeat / Cas9 (CRISPR / Cas9) techniques can be used for targeted gene insertion.

[0255] Site-specific gene editing approaches may include homology-dependent mechanisms or homology-independent mechanisms. Petition 870250081011, dated 09 / 09 / 2025, page 99 / 230 75 / 166

[0256] All known methods in the art for targeted insertion of genetic sequences are contemplated in the methods described here for inserting constructs into genetic targets or safe harbor loci.

[0257] Methods are provided here for inserting nucleotide sequences longer than about 5 kilobases into the genome of a cell in the absence of a viral vector. In some embodiments, the nucleotide sequence longer than about 5 kilobases can be inserted into the genome of a primary immune cell in the absence of a viral vector.

[0258] The integration of large nucleic acids, for example nucleic acids larger than 5 kilobases, into cells can be limited by low integration efficiency, off-target effects, and / or loss of cell viability. Methods and compositions are described here for achieving the integration of a nucleotide sequence, for example, a nucleotide sequence larger than about 5 kilobases in size, into the genome of a cell. In some methods, integration efficiency is increased, off-target effects are reduced, and / or loss of cell viability is reduced.

[0259] The plasmid can be introduced into an immune cell with a nuclease, such as a CRISPR-associated system (Cas). The nuclease can be introduced in a ribonucleoprotein format with a guide RNA (gRNA) that targets a specific site in the immune cell genome. The nuclease cuts the genomic DNA at this specific site. The specific site can be a portion of the genome that encodes an endogenous immune cell receptor. Therefore, cutting the genome at this site will cause the immune cell to no longer express an endogenous immune cell receptor.

[0260] The plasmid may include repair arms targeted by 5' and 3' homology complementary to sequences at a specific location in the immune cell genome. The complementary sequences are on either side of the nuclease-cut site, which allows the plasmid to be Petition 870250081011, dated 09 / 09 / 2025, pages 100 / 230 76 / 166 is incorporated into a specified insertion site in the immune cell genome. Once the plasmid is incorporated, the cell will express the SPA peptide. In examples where the SPA peptide is also co-expressed with a system comprising an initiation receptor and CAR, the initiation receptor is also expressed by the cell. However, as explained, the transgene cassette design ensures that non-virus-delivered circuit system receptors do not express CAR until the initiation receptor binds to its cognate ligand and releases the cleavable transcription factor.

[0261] Initially, a T cell is activated. The T cell can be obtained from a patient. Thus, the present disclosure provides methods in which immune cells, such as T cells, are harvested from a patient. Then, the plasmid encoding the CAR and the initiation receptor are introduced into a T cell. Advantageously, the plasmids in the present disclosure can be introduced using electroporation. By introducing the plasmid via electroporation, the nuclease can also be introduced. By using electroporation, the methods in the present disclosure avoid the use of viral vectors for transgene introduction, which is a known bottleneck in immune cell engineering. The T cells are then expanded and co-cultured to create a sufficient quantity of engineered immune cells to be used as a therapeutic treatment.

[0262] Methods for editing a cell's genome may include a) providing a complex Cas9 ribonucleoprotein (RNP)-DNA template comprising: (i) the RNP, wherein the RNP comprises a Cas9 nuclease domain and a guide RNA, wherein the guide RNA specifically hybridizes with a target region of the cell's genome and wherein the Cas9 nuclease domain cleaves the target region to create an insertion site in the cell's genome; and (ii) a double-stranded or single-stranded DNA template, wherein the size of the DNA template is greater than about 200 nucleotides, wherein the 5' and 3' ends of the DNA template comprise nucleotide sequences that are homologous to the genomic sequences flanking the insertion site, and wherein the molar ratio Petition 870250081011, dated 09 / 09 / 2025, page 101 / 230 The ratio of 77 / 166 RNP to DNA template in the complex is approximately 3:1 to approximately 100:1; and b) introduction of the RNP-DNA template complex into the cell.

[0263] In some embodiments, the methods described herein provide an efficiency of RNP-DNA template complex delivery of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, 99% or higher. In some cases, efficiency is determined in relation to the number of cells that are viable after the introduction of the RNP-DNA template into the cell. In some cases, efficiency is determined in relation to the total number of cells (viable or non-viable) into which the RNP-DNA template is introduced.

[0264] As another example, delivery efficiency can be determined by quantifying the number of genome-edited cells in a cell population (compared to the total number of cells or the total number of viable cells obtained after the introduction step). Several methods for quantifying genome editing can be used. These methods include, but are not limited to, the use of a specific mismatch nuclease, such as T7 endonuclease I; sequencing of one or more target loci (e.g., by Sanger sequencing of cloned target locus amplification fragments); and high-throughput deep sequencing.

[0265] In some embodiments, the loss of cell viability is reduced compared to the loss of cell viability after the introduction of naked DNA into a cell or the introduction of DNA into a cell using a viral vector. The reduction may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between these percentages. In some embodiments, off-target integration effects are reduced compared to off-target integration after the introduction of naked DNA into a cell or the introduction of DNA into a cell using a viral vector. The reduction may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage between these percentages.

[0266] In some cases, the methods described here provide high Petition 870250081011, dated 09 / 09 / 2025, page 102 / 230 78 / 166 cell viability of cells into which the RNP-DNA template was introduced. In some cases, the viability of the cells into which the RNP-DNA template was introduced is at least approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, 99% or higher. In some cases, the viability of the cells to which the RNP-DNA template was introduced is from about 20% to about 99%, from about 30% to about 90%, from about 35% to about 85% or 90% or more, from about 40% to about 85% or 90% or more, from about 50% to about 85% or 90% or more, from about 50% to about 85% or 90% or more, from about 60% to about 85% or 90% or more, or from about 70% to about 85% or 90% or more.

[0267] In the methods provided herein, the molar ratio between RNP and nucleic acid (e.g., DNA template) can be from about 3:1 to about 100:1. For example, the molar ratio can be from about 3:1 to 10:1, from about 3:1 to about 15:1, 3:1 to about 20:1; 3:1 to about 25:1; from about 3:1 to 50:1, from about 3:1 to 75:1, from about 3:1 to 100:1; from about 5:1 to 10:1, from about 5:1 to about 15:1, 5:1 to about 20:1; 5:1 to about 25:1; from about 5:1 to 50:1, from about 5:1 to 75:1, from about 5:1 to 100:1; from about 8:1 to about 12:1; from about 8:1 to about 15:1, from about 8:1 to about 20:1, from about 8:1 to about 25:1, from about 8:1 to 50:1, from about 8:1 to 75:1, from about 8:1 to 100:1; from about 10:1 to about 15:1, 10:1 to about 20:1, 10:1 to about 25:1; from about 10:1 to 50:1, from about 10:1 to 75:1, or from about 10:1 to 100:1.

[0268] In some embodiments, the DNA template is at a concentration of about 2.5 pM to about 25 pM. For example, the concentration of the DNA template could be approximately 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25 pM, or any concentration between these concentrations.

[0269] In some embodiments, the size or length of the acid Petition 870250081011, dated 09 / 09 / 2025, p. 103 / 230 79 / 166 nucleic (e.g., DNA template) is larger than about 4.5 kb, 5.0 kb, 5.1 kb, 5.2 kb, 5.3 kb, 5.4 kb, 5.5 kb, 5.6 kb, 5.7 kb, 5.8 kb, 5.9 kb, 6.0 kb, 6.1 kb, 6.2 kb, 6.3 kb, 6.4 kb, 6.5 kb, 6.6 kb, 6.7 kb, 6.8 kb, 6.9 kb, 7.0 kb, 7.1 kb, 7.2 kb,7.3 kb, 7.4 kb, 7.5 kb, 7.6 kb, 7.7 kb, 7.8 kb, 7.9 kb, 8.0 kb, 8.1 kb, 8.2 kb, 8.3 kb, 8.4 kb, 8.5 kb, 8.6 kb, 8.7 kb, 8.8 kb, 8.9 kb, 9.0 kb, 9.1 kb, 9.2 kb, 9.3 kb, 9.4 kb, 9.5 kb, 9.6 kb, 9.7 kb, 9.8 kb, 9.9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb or 16 kb or any nucleic acid size (e.g., DNA template) between these sizes. For example, the size of the DNA template can be approximately 4.5 kb to approximately 15 kb, approximately 4.5 kb to approximately 14 kb, approximately 4.5 kb to approximately 10 kb, approximately 5 kb to approximately 15 kb, approximately 5 kb to approximately 14 kb, approximately 5 kb to approximately 10 kb, approximately 5 kb to approximately 9 kb, approximately 5 kb to approximately 8 kb, approximately 5 kb to approximately 7 kb, approximately 5 kb to approximately 6 kb,approximately 6 kb to approximately 15 kb, approximately 6 kb to approximately 14 kb, approximately 6 kb to approximately 10 kb, approximately 6 kb to approximately 9 kb, approximately 6 kb to approximately 8 kb, approximately 6 kb to approximately 7 kb, approximately 7 kb to approximately 15 kb, approximately 7 kb to approximately kb, approximately 7 kb to approximately 10 kb, approximately 7 kb to approximately 9 kb, approximately 7 kb to approximately 8 kb, approximately 8 kb to approximately 15 kb, approximately 8 kb to approximately 14 kb, approximately 8 kb to approximately 10 kb, approximately 8 kb to approximately 9 kb, approximately 9 kb to approximately 15 kb, approximately 9 kb to approximately 14 kb approximately 9 kb to approximately 13 kb, approximately 9 kb to approximately 12 kb, approximately 9 kb to approximately 11 kb, approximately 9 kb to approximately 10 kb, approximately 10 kb to approximately 15 kb, approximately 10 kb to approximately 14 kb, approximately 10 kb to approximately 13 kb, approximately 10 kb to approximately 12 kb or approximately 10 kb to approximately 11 kb.

[0270] In some embodiments, the amount of template DNA is from about 1 pg to about 10 pg. For example, the amount of template DNA may be from about 1 pg to about 2 pg, about 1 pg to about 3 pg, about 1 pg to about 4 pg, about 1 pg to about 5 pg, about 1 pg to about 6 pg, about 1 pg to about 7 pg, about 1 pg to about 8 pg, about 1 pg to about 9 pg, about 1 pg to about 10 pg. In some embodiments, the amount of template DNA is from about 2 pg to about 3 pg, about 2 Petition 870250081011, dated 09 / 09 / 2025, page 104 / 230 80 / 166 pg to about 4 pg, about 2 pg to about 5 pg, about 2 pg to about 6 pg, about 2 pg to about 7 pg, about 2 pg to about 8 pg, about 2 pg to about 9 pg, or about 2 pg to about 10 pg. In some embodiments, the amount of template DNA is about 3 pg to about 4 pg, about 3 pg to about 5 pg, about 3 pg to about 6 pg, about 3 pg to about 7 pg, about 3 pg to about 8 pg, about 3 pg to about 9 pg, or about 3 pg to about 10 pg. In some embodiments, the amount of template DNA is about 4 pg to about 5 pg, about 4 pg to about 6 pg, about 4 pg to about 7 pg, about 4 pg to about 8 pg, about 4 pg to about 9 pg, or about 4 pg to about 10 pg. In some embodiments, the amount of template DNA is about 5 pg to about 6 pg, about 5 pg to about 7 pg, about 5 pg to about 8 pg, about 5 pg to about 9 pg, or about 5 pg to about 10 pg.In some embodiments, the amount of template DNA is about 6 pg to about 7 pg, about 6 pg to about 8 pg, about 6 pg to about 9 pg, or about 6 pg to about 10 pg. In some embodiments, the amount of template DNA is about 7 pg to about 8 pg, about 7 pg to about 9 pg, or about 7 pg to about 10 pg. In some embodiments, the amount of template DNA is about 8 pg to about 9 pg, or about 8 pg to about 10 pg. In some embodiments, the amount of template DNA is about 9 pg to about 10 pg.

[0271] In some cases, the size of the DNA template is large enough and in sufficient quantity to be as lethal as naked DNA. In some embodiments, the DNA template encodes a heterologous protein or a fragment thereof. In some embodiments, the DNA template encodes at least one gene. In some embodiments, the DNA template encodes at least two genes. In some embodiments, the DNA template encodes one, two, three, four, five, six, seven, eight, nine, ten or more genes.

[0272] In some embodiments, the DNA template includes regulatory sequences, for example, a promoter sequence and / or an enhancer sequence to regulate the expression of the heterologous protein or fragment of Petition 870250081011, dated 09 / 09 / 2025, p. 105 / 230 81 / 166 same after insertion into the genome of a cell.

[0273] In some cases, the DNA model is a linear DNA model. In some cases, the DNA model is a single-stranded DNA model. In some cases, the single-stranded DNA model is a pure single-stranded DNA model. As used herein, “pure single-stranded DNA” means single-stranded DNA that substantially lacks the other DNA strand or the opposite strand. “Substantially lacks” means that pure single-stranded DNA lacks at least 100 times more of one strand than the other DNA strand.

[0274] In some cases, the RNP-DNA template complex is formed by incubating the RNP with the DNA template for less than about one minute to about thirty minutes, at a temperature of about 20oC to about 25oC. For example, RNP can be incubated with the DNA template for approximately 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 16 minutes, 22 minutes, 11 minutes, 17 minutes, 23 minutes, 12 minutes, 18 minutes, 24 minutes, 13 minutes, 19 minutes, 25 minutes, 14 minutes, 20 minutes, 26 minutes, 15 minutes, 21 minutes, 27 minutes, 28 minutes, 29 minutes, or 30 minutes, or any period of time between these times, at a temperature of approximately 20°C, 21°C, 22°C. 23°C, 24°C or 25°C.In another example, the RNP can be incubated with the DNA template for less than about one minute to about one minute, for less than about one minute to about 5 minutes, for less than about one minute to about 10 minutes, for about 5 minutes to 10 minutes, for about 5 minutes to 15 minutes, for about 10 to about 15 minutes, for about 10 minutes to about 20 minutes, or for about 10 minutes to about 30 minutes, at a temperature of about 20°C to about 25°C. In some embodiments, the RNP-DNA template complex and the cell are mixed before the introduction of the RNP-DNA template complex into the cell. Petition 870250081011, dated 09 / 09 / 2025, p. 106 / 230 82 / 166

[0275] In some embodiments, the introduction of the RNP-DNA template complex comprises electroporation. Additional or alternative methods, compositions, and devices for electroporation of cells to introduce an RNP-DNA template complex may include those described in the examples herein. Additional or alternative methods, compositions, and devices for electroporation of cells to introduce an RNP-DNA template complex may include those described in WO / 2006 / 001614 or Kim, JA et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternative methods, compositions, and devices for electroporation of cells to introduce an RNP-DNA template complex may include those described in US Patent Publications No. 2006 / 0094095; 2005 / 0064596; or 2006 / 0087522. Additional or alternative methods, compositions, and devices for electroporation of cells to introduce an RNP-DNA template complex may include those described in Li, LH et al. Cancer Res. Treat.1, 341-350 (2002); US Patent Nos.: 6,773,669; 7,186,559; 7,771,984; 7,991,559; 6485961; 7029916; and. US Patent Application Publications Nos. 2014 / 0017213 and 2012 / 0088842 are all incorporated herein by reference. Additional or alternative methods, compositions, and devices for electroporation of cells to introduce a template RNP DNA complex may include those described in Geng, T. et al. J. Release Control 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010), all of which are incorporated herein by reference.

[0276] In some embodiments, the Cas9 protein may be in an active endonuclease form, such that when bound to the target nucleic acid as part of a complex with a guide RNA or part of a complex with a DNA template, a double-strand break is introduced into the target nucleic acid. The double-strand break can be repaired by NHEJ to introduce random mutations, or HDR to introduce specific mutations. Several Cas9 nucleases can be used in the methods described herein. For example, a Cas9 nuclease that requires a motif adjacent to the NGG protospacer. Petition 870250081011, dated 09 / 09 / 2025, p. 107 / 230 83 / 166 (PAM) immediately 3' from the target region of the guide RNA can be used. Such Cas9 nucleases can be targeted to any region of a genome that contains an NGG sequence. As another example, Cas9 proteins with orthogonal PAM motif requirements can be used to target sequences that do not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificities include, but are not limited to, CFP1, those described in Nature Methods 10, 1116-1121 (2013), and those described in Zetsche et al., Cell, Volume 163, Issue 3, p759-771, October 22, 2015, both incorporated herein by reference.

[0277] In some cases, the Cas9 protein is a nickase, so that when bound to the target nucleic acid as part of a complex with a guide RNA, a double-strand break or cut is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally different guide RNA, can be targeted to two proximal sites of a target genomic region and thus introduce a pair of proximal single-strand breaks into the target genomic region. Nickase pairs can provide enhanced specificity because off-target effects are likely to result in single cuts, which are usually repaired without injury by base excision repair mechanisms. Exemplary Cas9 nickases include Cas9 nucleases with a D10A or H840A mutation.

[0278] In some embodiments, the RNP comprises a Cas9 nuclease. In some embodiments, the RNP comprises a Cas9 nickase. In some embodiments, the RNP-DNA template complex comprises at least two structurally different RNP complexes. In some embodiments, the at least two structurally different RNP complexes contain structurally different Cas9 nuclease domains. In some embodiments, the at least two structurally different RNP complexes contain structurally different guide RNAs. In some embodiments, where the at least two structurally different RNP complexes contain structurally different guide RNAs, each of the RNP complexes Petition 870250081011, dated 09 / 09 / 2025, page 108 / 230 84 / 166 structurally different components include a Cas9 nickase and structurally different guide RNAs that hybridize with opposite strands of the target region.

[0279] In some cases, a plurality of RNP-DNA templates comprising structurally different ribonucleoprotein complexes is introduced into the cell. For example, a Cas9 protein can be complexed with a plurality (e.g., 2, 3, 4, 5 or more, e.g., 2-10, 5-100, 20-100) of structurally different guide RNAs to direct the insertion of a DNA template into a plurality of structurally different target genomic regions.

[0280] In the methods and compositions provided herein, the cells include, but are not limited to, eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, and the like. Optionally, the cell is a mammalian cell, for example, a human cell. The cell may be in vitro, ex vivo, or in vivo. The cell may also be a primary cell, a germ cell, a stem cell, or a precursor cell. The precursor cell may be, for example, a pluripotent stem cell or a hematopoietic stem cell. In some embodiments, the cell is a primary hematopoietic cell or a primary hematopoietic stem cell. In some embodiments, the primary hematopoietic cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a regulatory T cell, an effector T cell, or a naive T cell. In some embodiments, the T cell is a CD4+ T cell.In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a CD4+CD8+ T cell. In some embodiments, the T cell is a CD4-CD8- T cell. Populations of any of the cells modified by any of the methods described herein are also provided. In some embodiments, the methods further comprise expanding the population of modified cells.

[0281] In some cases, cells are removed from a subject, modified using any of the methods described here, and administered to Petition 870250081011, dated 09 / 09 / 2025, page 109 / 230 85 / 166 patient. In other cases, any of the constructs described herein is delivered to the patient in vivo. See, for example, US Patent No. 9737604 and Zhang et al. “Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy”, NPG Asia Materials Volume 9, page e441 (2017), both of which are incorporated herein by reference.

[0282] In some embodiments, the RNP-DNA template complex is introduced into about 1 x 10⁵ to about 2 x 10⁶ cells. For example, the RNP-DNA template complex may be introduced into about 1 x 10⁵ to about 5 x 10⁵ cells, about 1 x 10⁵ to about 1 x 10⁶, 1 x 10⁵ to about 1.5 x 10⁶, 1 x 10⁵ to about 2 x 10⁶, about 1 x 10⁶ to about 1.5 x 10⁶ cells, or about 1 x 10⁶ to about 2 x 10⁶.

[0283] In some cases, the methods and compositions described herein may be used for the generation, modification, use, or control of recombinant T cells, such as chimeric antigen receptor T cells (CAR-T cells). Such CAR-T cells may be used to treat or prevent cancer, an infectious disease, or an autoimmune disease in a subject. For example, in some embodiments, one or more gene products are inserted or knocked into a T cell to express a heterologous protein (e.g., a chimeric antigen receptor (CAR) or an initiation receptor). Insertion Sites

[0284] Methods for editing the genome of a T cell specifically include a method for editing the genome of a human T cell that comprises inserting a nucleic acid sequence or construct into a target region in exon 1 of the TCR-α subunit gene (TRAC) in the human T cell. In some embodiments, the target region is in exon 1 of the constant domain of the TRAC gene. In other embodiments, the target region is in exon 1, exon 2, or exon 3, prior to the start of the sequence encoding the transmembrane domain of TCR-α. In some embodiments, the target region is the GS94 genomic safe harbor. Petition 870250081011, dated 09 / 09 / 2025, pages 110 / 230 86 / 166

[0285] Methods for editing the genome of a T cell also include a method of editing the genome of a human T cell that comprises inserting a nucleic acid sequence or construct into a target region in exon 1 of a TCR-β subunit gene (TRBC) in the human T cell. In some embodiments, the target region is in exon 1 of the TRBC1 or TRBC2 gene.

[0286] Methods for editing the genome of a T cell, specifically, include a method of editing the genome of a human T cell that comprises inserting a nucleic acid sequence or construct into a genomic safe harbor (GSH) target region.

[0287] Methods for editing the genome of a T cell also include a method for editing the genome of a human T cell comprising inserting a nucleic acid sequence or construct into a GS94 target region (chr11 locus: 128340000-128350000).

[0288] In some modes, the target region is the GS94 locus.

[0289] Gene editing therapies include, for example, vector integration and site-specific integration. Site-specific integration is a promising alternative to random viral vector integration, as it attenuates the risks of insertional mutagenesis or insertional oncogenesis (Kolb et al. Trends Biotechnol. 2005 23:399-406; Porteus et al. Nat Biotechnol. 2005 23:967-973; Paques et al. Curr Gen Ther. 2007 7:49-66). However, site-specific integration continues to face challenges such as low knockin efficiency, risk of insertional oncogenesis, unstable and / or anomalous expression of adjacent genes or the transgene, low accessibility (e.g., within 20 kB of adjacent genes), etc.These challenges can be addressed, in part, through the identification and use of safe harbor loci or safe harbor sites (SHS), which are locations where genes or genetic elements can be incorporated without disrupting the expression or regulation of adjacent genes.

[0290] The most widely used human safe harbor site is the AAVS1 site on chromosome 19q, which was initially identified as a site for Petition 870250081011, dated 09 / 09 / 2025, p. 111 / 230 87 / 166 recurrent insertion of adeno-associated viruses. Other potential SHS have been identified based on homology, with sites first identified in other species (e.g., the human homolog of the permissive murine locus Rosa26) or among the growing number of human genes that appear non-essential under some circumstances. One possible such SHS is the chemokine receptor gene CCR5, which, when disrupted, confers resistance to human immunodeficiency virus infection. Other potential genomic SHS have been identified in human cells and other cell types based on viral integration site mapping or gene trap analyses, as well as the original murine Rosa26 locus. The three main SHS, AAVS1, CCR5, and Rosa26, are close to many protein-coding genes and regulatory elements. (See Sadelain, M., et al. (2012). Safe harbors for the integration of new DNA in the human genome.)Nature reviews Cancer, 12(1), 51-58, whose relevant disclosures are incorporated here by reference in full).

[0291] AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 is a known SHS site for hosting transgenes (e.g., DNA transgenes) with expected function. It is located at position 19q13.42. It has an open chromatin structure and is transcriptionally competent. The canonical SHS locus for AAVS1 is chr19: 55.625.24155.629.351. See Pellenz et al. “New Human Chromosomal Sites with Safe Harbor Potential for Targeted Transgene Insertion.” Human gene therapy vol. 30,7 (2019): 814-828, relevant disclosures of which are incorporated herein by reference. An exemplary AAVS1 target gRNA and target sequence are provided below: • AAVS1-gRNA sequence: ggggccactagggacaggatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 116) • AAVS1 target sequence: ggggccactagggacaggat (SEQ ID NO: 117) Petition 870250081011, dated 09 / 09 / 2025, pp. 112 / 230 88 / 166

[0292] CCR5, located on chromosome 3 at position 3p21.31, encodes the major coreceptor of HIV-1. Disruption at this site in the CCR5 gene has been beneficial in HIV / AIDS therapy and has stimulated the development of zinc finger nucleases that target its third exon. The canonical SHS locus for CCR5 is chr3: 46414443-46414942. See Pellenz et al. “Novel Human Chromosomal Sites with Potential Safe Harbor for Targeted Transgene Insertion.” Human Gene Therapy vol. 30,7 (2019): 814-828, relevant disclosures of which are incorporated herein by reference.

[0293] The mouse Rosa26 locus is particularly useful for genetic modification because it can be targeted with high efficiency and is expressed in most cell types tested. Irion et al. 2007 (Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nature biotechnology 25.12 (2007): 1477-1482, relevant disclosure of which is incorporated here by reference) identified the human homolog, human ROSA26, on chromosome 3 (position 3p25.3). The canonical SHS locus for human Rosa26 (hRosa26) is chr3: 9415082-9414043. See Pellenz et al. “New Human Chromosomal Sites with Safe Harbor Potential for Targeted Transgene Insertion.” Human gene therapy vol. 30,7 (2019): 814-828, whose relevant disclosures are incorporated herein by reference.

[0294] Additional examples of safe harbor sites are provided in Pellenz et al. “Novel Human Chromosomal Sites with Potential Safe Harbor for Target Transgene Insertion.” Human Gene Therapy vol. 30,7 (2019): 814-828, relevant disclosures of which are incorporated herein by reference. Examples of additional integration sites are provided in Table D.

[0295] In some embodiments, safe harbor loci allow high transgene expression (sufficient to permit transgene functionality or treatment of a disease of interest) and stable transgene expression over several days, weeks, or months. In some embodiments, deletion of the gene at the safe harbor locus confers a benefit to cell function, or the gene at the safe harbor locus has no known function within the Petition 870250081011, dated 09 / 09 / 2025, p. 113 / 230 89 / 166 cell. In some embodiments, the safe harbor locus results in stable transgene expression in vitro with or without CD3 / CD28 stimulation, negligible off-target cleavage as detected by iGuide-Seq or CRISPR-Seq, less off-target cleavage compared to other loci as detected by iGuide-Seq or CRISPR-Seq, negligible transgene-independent cytotoxicity, negligible transgene-independent cytokine expression, negligible transgene-independent chimeric antigen receptor expression, negligible dysregulation or silencing of nearby genes and positioned outside of a cancer-related gene.

[0296] As used, a nearby gene may refer to a gene that is within about 100 kB, about 125 kB, about 150 kB, about 175 kB, about 200 kB, about 225 kB, about 250 kB, about 275 kB, about 300 kB, about 325 kB, about 350 kB, about 375 kB, about 400 kB, about 425 kB, about 450 kB, about 475 kB, about 500 kB, about 525 kB, about 550 kB away from the safe harbor locus (integration site).

[0297] In some embodiments, the present disclosure includes insertions comprising one or more transgenes. The transgene may encode a therapeutic protein, an antibody, a peptide, or any other gene of interest. The integration of the transgene may result, for example, in enhanced therapeutic properties. These enhanced therapeutic properties, as used herein, refer to an enhanced therapeutic property of a cell when compared to a typical immune cell of the same normal cell type. For example, a T cell with enhanced therapeutic properties has an enhanced, improved, and / or increased treatment outcome when compared to a typical, unmodified, and / or natural T cell.The therapeutic properties of immune cells may include, but are not limited to, cell transplantation, transport, localization, viability, self-renewal, persistence, control and regulation of the immune response, survival, and cytotoxicity. The therapeutic properties of immune cells are also manifested by: expression of... Petition 870250081011, dated 09 / 09 / 2025, page 114 / 230 90 / 166 antigen-directed receptor; HLA presentation or lack thereof; tolerance to the intratumoral microenvironment; induction of bystander immune cells and immune regulation; enhanced target specificity with reduction; resistance to treatments such as chemotherapy.

[0298] As used herein, the term insertion size refers to the length of the nucleotide sequence that is being integrated (inserted) into the target site or safe harbor site. In some embodiments, the insertion size comprises at least about 4.5 kilobase pairs (kb) to about 10 kilobase pairs (kb). In some embodiments, the insertion size comprises about 5000 nucleotides or more base pairs. In some embodiments, the insertion size comprises up to 4.5, 4.8, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 kbp (kilobase pairs) or intermediate sizes. In some modes, the insertion size is greater than 4.5, 4.8, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 kbp or sizes in between. In some modes, the insertion size is within the range of 4.5 to 15 kbp or is any number in that range.In some modes, the insertion size is in the range of 4.8 to 8.3 kbp or is any number within that range. In some modes, the insertion size is in the range of 5 to 8.3 kbp or is any number within that range. In some modes, the insertion size is in the range of 5 to 15 kbp or is any number within that range. In some modes, the insertion size is in the range of 4.5 to 20 kbp or is any number within that range. In some modes, the insertion size is 5 to 10 kbp. In some modes, the insertion size is 4.5-10, 5-10, 6-10, 7-10, 8-10, 9-10 kbp. In some modes, the insertion size is 4.5-11, 6-11, 7-11, 8-11, 9-11 or 10-11 kbp. In some modalities, the insertion size is 4.5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12 kpb. In some modalities, the insertion size is 4.5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, or 12-13 kpb.In some modes, the insertion size is 4.5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, or 13-14 kbp. In some modes, the insertion size is 4.5. Petition 870250081011, dated 09 / 09 / 2025, pages 115 / 230 91 / 166 15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 13-15 or 14-15 kbp. In some modes, the insertion size is 4.5-16, 6-16, 7-16, 8-16, 9-16, 10-16, 11-16, 12-16, 13-16, 14-16 or 15-16 kbp. In some modes, the insertion size is 4.5-17, 6-17, 7-17, 8-17, 9-17, 10-17, 11-17, 12-17, 13-17 or 14-17, 15-17 or 16-17 kbp. In some modes, the insertion size is 4.5-18, 6-18, 7-18, 8-18, 9-18, 10-18, 11-18, 12-18, 13-18, 14-18, 15-18, 16-18, or 17-18 kbp. In some modes, the insertion size is 4.5-19, 6-19, 7-19, 8-19, 9-19, 10-19, 11-19, 12-19, 13-19, 14-19, 15-19, 16-19, 17-19, or 18-19 kbp. In some modes, the insertion size is 4.5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 kbp.

[0299] The insertions in this disclosure refer to nucleic acid or polynucleotide molecules inserted into a target locus or safe harbor site. In some embodiments, the nucleotide sequence is a DNA molecule, for example, genomic DNA, or comprises deoxyribonucleotides. In some embodiments, the insertion comprises a smaller fragment of DNA, such as plastid DNA, mitochondrial DNA, or isolated DNA in the form of a plasmid, a fosmid, a cosmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and / or any other subgenome DNA segment. In some embodiments, the insertion is an RNA molecule or comprises ribonucleotides. The nucleotides in the insertion are contemplated as naturally occurring nucleotides, non-naturally occurring nucleotides, and modified nucleotides.Nucleotides can be chemically or biochemically modified, or they can contain unnatural or derived nucleotide bases, as will be readily apparent to those skilled in the art. Such modifications include, for example, markers, methylation, substitution of one or more of the naturally occurring nucleotides by an analog, and internucleotide modifications. Polynucleotides can be in any topological conformation, including single-stranded, double-stranded, partially duplex, triplex, hairpin, circular, and other three-dimensional conformations contemplated in the [reference to relevant documentation]. Petition 870250081011, dated 09 / 09 / 2025, pages 116 / 230 92 / 166 technique.

[0300] Inserts may have coding and / or non-coding regions. The insert may comprise a non-coding sequence (e.g., control elements, for example, a promoter sequence). In some embodiments, the insert encodes transcription factors. In some embodiments, the insert encodes antigen-binding receptors, such as single receptors, T cell receptors (TCRs), initiation receptors, CARs, mAbs, etc. In some embodiments, the insert is a human sequence. In some embodiments, the insert is chimeric. In some embodiments, the insert is a multigene / multimodule therapeutic cassette. A multigene / multimodule therapeutic cassette refers to an insert or cassette having one or more receptors (e.g., synthetic receptors), other exogenous protein coding sequences, non-coding RNAs, transcription regulatory elements, and / or insulator sequences, etc.

[0301] In some embodiments, the nucleic acid sequence is inserted into the cell genome as an immune cell or T cell via nonviral delivery. In nonviral delivery methods, the nucleic acid may be naked DNA or in a plasmid or nonviral vector. Nonviral delivery techniques may be site-specific integration techniques, as described herein, or known to persons of ordinary skill in the art. Examples of site-specific techniques for integration into safe harbor loci include, without limitation, homology-dependent engineering using nucleases and homology-independent targeted insertion using Cas9 or other CRISPR endonucleases.

[0302] In some embodiments, the insert is integrated into a safe harbor site by introducing into the manipulated cell (a) a target nuclease that cleaves a target region in the safe harbor site to create the insertion site; and (b) the nucleic acid sequence (insert) into which the insert is incorporated by, for example, HDR. Examples of nonviral delivery techniques that can be used in the methods of this disclosure are provided. Petition 870250081011, dated 09 / 09 / 2025, pp. 117 / 230 93 / 166 in US Patents Nos. US11033584B2 and US11814624B2, the relevant disclosures of which are incorporated herein by reference in their entirety.

[0303] Examples of additional integration sites are provided in Table D. Table D: sgRNA sequences sgRNA ID sgRNA sequence SEQ ID NO: GRCH38 of coor. sgRNA initiation loci sgRNA target integration site Median (% modified), summarized from 2 donors, 2 primer sets sgRNA _1 GCACCTGAATACC ACGCCTG 118 chr16:8881181 8 APRT APRT 79.28 sgRNA _2 CGCCTGCGATGT AGTCGATG 119 chr16:8881155 1 ​​APRT APRT 78.60 sgRNA _3 CAGGACGGGCGA GATGTCCC 120 chr16:8881164 0 APRT APRT 85.25 sgRNA _4 CTGAATCTTTGGA GTACCTG 121 chr15:4471542 5 B2M B2M 78.51 sgRNA _5 GGCCACGGAGCG AGACATCT 122 chr15:4471155 0 B2M B2M 94.75 sgRNA _6 AAGTCAACTTCAA TGTCGGA 123 chr15:4471551 5 B2M B2M 70.97 sgRNA _7 GCTTGGAGGCCT GATCAGCG 124 chr19:3614111 1 CAPNS1 CAPNS1 89.34 sgRNA _8 CTTATCTCTTCGC AGCGAGG 125 chr19:3614230 TTCCGCAAAATAG AGCCCCA 127 chr3:10574601 9 CBLB CBLB 91.55 sgRNA _11 TGCACAGAACTAT CGTACCA 128 chr3:10575162 2 CBLB CBLB 91.43 sgRNA GCAATAAGACTCT 129 chr3:10585347 CBLB CBLB 76.18 Petition 870250081011, dated 09 / 09 / 2025, pp. 118 / 230 94 / 166 _12 TTAAAGA 0 sgRNA _13 CAAAGAGATTACG AATGCCT 130 chr1:11675465 8 CD2 CD2 89,80 sgRNA _14 CAAGGCACCCCA GGTTTCCA 131 chr1:11675466 3 CD2 CD2 92,70 sgRNA _15 TTACGAATGCCTT GGAAACC 132 chr1:11675466 6 CD2 CD2 92,82 sgRNA _16 CAGAGACGCATCT GACCCTC 133 chr11:1183155 40 CD3E CD3E 90,96 sgRNA _17 CATGCAGTTCTCA CACACTG 134 chr11:1183137 15 CD3E CD3E 87,47 sgRNA _18 GTGTGAGAACTG CATGGAGA 135 chr11:1183137 15 CD3E CD3E 86,65 sgRNA _19 TCTCATTTCAGGA AACCACT 136 chr11:1183497 48 CD3G CD3G 87,24 sgRNA _20 AGTCATACACCTT AACCAAG 137 chr11:1183497 54 CD3G CD3G 87,99 sgRNA _21 TTCAAGGAAACCA GTTGAGG 138 chr11:1183524 58 CD3G CD3G 86,55 sgRNA _22 GAGCCTTGCCTG GAAATCTG 139 chr11:6111817 7 CD5 CD5 84,03 sgRNA _23 AAGCGTCAAAAGT CTGCCAG 140 chr11:6111832 4 CD5 CD5 89,19 sgRNA _24 CGTTCCAACTCGA AGTGCCA 141 chr11:6111812 1 CD5 CD5 83,11 sgRNA _25 GAGCGACTGGGA CACGGTGA 142 chr9:13686624 6 EDF1 EDF1 88,84 sgRNA _26 GCTGCGCAAGAA GGGCCCTA 143 chr9:13686621 1 EDF1 EDF1 91,04 sgRNA _27 TTGTTCTGGCCAG CAGCCCC 144 chr9:13686343 3 EDF1 EDF1 85.98 sgRNA _28 CTTCCAGAGCCAC ATCATCG 145 chr19:4896579 1 FTL FTL 93.10 sgRNA _29 GGGACTCACCAG AGAGAGGT 146 chr19:4896560 1 FTL FTL 88.86, Petition 870250081011, dated 09 / 09 / 2025, pp. 119 / 230 95 / 166 sgRNA _30 CGGTCGAAATAGA AGCCCTA 147 chr19:4896577 0 FTL FTL 93.14 sgRNA _31 AAAAGGATATTGT GCAACTG 148 chr10:8793301 5 PTEN PTEN 92.37 sgRNA _32 TGTGCATATTTAT TACATCG 149 chr10:8793318 3 PTEN PTEN 90.64 sgRNA _33 TTTGTGAAGATCT TGACCAA 150 chr10:8793308 7 PTEN PTEN 85.36 sgRNA _34 TGTCATGCTGAAC CGCATTG 151 chr18:1283097 2 PTPN2 PTPN2 87.94 sgRNA _35 CCACTCTATGAGG ATAGTCA 152 chr18:1285921 9 PTPN2 PTPN2 92.45 sgRNA _36 TTGACATAGAAGA GGCACAA 153 chr18:1283682 8 PTPN2 PTPN2 93.96 sgRNA _37 GAGTACTACACTC AGCAGCA 154 chr12:6952098 PTPN6 PTPN6 89.61 sgRNA _38 TCACGCACAAGAA ACGTCCA 155 chr12:6954872 PTPN6 PTPN6 82.74 sgRNA _39 AGGTCTCGGTGA AACCACCT 156 chr12:6951610 PTPN6 PTPN6 91.27 sgRNA _40 AGCATTATCCAAA GAGTCCG 157 chr1:19869687 3 PTPRC PTPRC 88.88 sgRNA _41 ATATTAATTCTTAC CAGTGG 158 chr1:19869237 0 PTPRC PTPRC 88.95 sgRNA _42 AGCTTTAAATCAA GGTTCAT 159 chr1:19875617 6 PTPRC PTPRC 96.89 sgRNA _43 ATCCCGAGCCCTA AGGTGCA 160 chr11:6743632 5 PTPRCAP PTPRCAP 84.95.08 sgRNA _44 GGCAGCGCGGAG GACAGCGT 161 chr11:6743628 5 PTPRCAP PTPRCAP 97.74 sgRNA _45 CTCAGGGGGCTA CTACCACC 162 chr11:6743617 0 PTPRCAP PTPRCAP 91.50 sgRNA _46 GTCACCGACGAG ACCAGAAG 163 chr5:82277810 RPS23 RPS23 79.40 sgRNA GTCGGTGGACTTC 164 chr5:82277843 RPS23 RPS23 83.07, Petition 870250081011, dated 09 / 09 / 2025, pages 120 / 230 96 / 166 _47 GTACTGCT sgRNA _48 TAA III IIAGGCA AGTGTCG 165 chr5:82277860 RPS23 RPS23 61.94 sgRNA _49 TTAGCTGTTAGAC TTGAATA 166 chr14:5199381 0 RTRAF RTRAF 85.50 sgRNA _50 CGAGAGCCGTCA ACTTGCGT 167 chr14:5198965 2 RTRAF RTRAF 85.64 sgRNA _51 CGGCTTCAACTGC AAAGGTG 168 chr14:5198970 0 RTRAF RTRAF 88.77 sgRNA _52 TATGAAAAAGCAG AGCGACT 169 chr15:4379302 5 SERF2 SERF2 89,61 sgRNA _53 TCTGGCGGGCGA GCTCACGC 170 chr15:4379298 9 SERF2 SERF2 86,73 sgRNA _54 CTCACGCTGGTTA CCGCCTA 171 chr15:4379297 7 SERF2 SERF2 80,57 sgRNA _55 AAAGATTACGAAC TTCCCTG 172 chr12:4620755 9 SLC38A1 SLC38A1 92,24 sgRNA _56 GTTAAAAACAGAC ATGCCTA 173 chr12:4622923 2 SLC38A1 SLC38A1 91,51 sgRNA _57 ATGCCTAAGGAG GTTGTACC 174 chr12:4622924 6 SLC38A1 SLC38A1 79,48 sgRNA _58 CTCCAGGTATCCC ATCGAAA 175 chr18:4786941 8 SMAD2 SMAD2 79,53 sgRNA _59 CACCAAATACGAT AGATCAG 176 chr18:4787053 2 SMAD2 SMAD2 86,61 sgRNA _60 TGGCGGCGTGAA TGGCAAGA 177 chr18:4789672 9 SMAD2 SMAD2 82,91 sgRNA _61 TAGGATGGTAGCA CACAACC 178 chr16:1125547 8 SOCS1 SOCS1 92.25 sgRNA _62 CAGCAGCAGAGC CCCGACGG 179 chr16:1125543 2 SOCS1 SOCS1 83.79 sgRNA _63 CGGCGTGCGAAC GGAATGTG 180 chr16:1125529 6 SOCS1 SOCS1 84.24 sgRNA _64 TATAGACGCTGCC CGACGTC 181 chr15:4003889 5 SRP14 SRP14 95.12 Petition: 870250081011, on September 9, 2025, page. 121 / 230 97 / 166 sgRNA _65 TCCAAAGAAGGGT ACTGTGG 182 chr15:4003836 8 SRP14 SRP14 92,14 sgRNA _66 ACAGTACCCTTCT TTGGAAT 183 chr15:4003835 8 SRP14 SRP14 65,82 sgRNA _67 GCGACGGGCGCA TCTACGTG 184 chr12:1204695 72 SRSF9 SRSF9 83,68 sgRNA _68 CCCGACCTCCATA AGTCCTG 185 chr12:1204657 00 SRSF9 SRSF9 92,56 sgRNA _69 GGGGTCCTCGAA GCGCACGA 186 chr12:1204694 26 SRSF9 SRSF9 89,94 sgRNA _70 TGCTCTGTTTAGA AGATGAC 187 chr5:32591641 SUB1 SUB1 79,36 sgRNA _71 ATATTCIIIICIAG TTAAAG 188 chr5:32591566 SUB1 SUB1 70,93 sgRNA _72 CCTGTAAAGAAAC AAAAGAC 189 chr5:32591614 SUB1 SUB1 93,66 sgRNA _73 TGGAGAAAGACG TAACTTCG 190 chr4:10523431 5 TET2 TET2 83,53 sgRNA _74 TCTGCCCTGAGGT ATGCGAT 191 chr4:10523474 7 TET2 TET2 90.97 sgRNA _75 ATTCCGCTTGGTG AAAACGA 192 chr4:10523565 6 TET2 TET2 89.62 sgRNA _76 CAGGCACAATAGA AACAACG 193 chr3:11429557 1 TIGIT TIGIT 92.65 sgRNA _77 CCATTTGTAATGC TGACTTG 194 chr3:11429570 0 TIGIT TIGIT 60.75 sgRNA _78 CTGGGTCACTTGT GCCGTGG 195 chr3:11429563 4 TIGIT TIGIT 87,99 sgRNA _79 GTCAGGGTTCTG GATATCTG 196 chr14:2254750 8 TRAC TRAC 98.20 sgRNA _80 TGGATTTAGAGTC TCTCAGC 197 chr14:2254754 1 TRAC TRAC 88.15 sgRNA _81 CTGCGGCTGTGG TCCAGCTG 198 chr14:2255066 1 TRAC TRAC 94.77 sgRNA ACAAAACTGTGCT 199 chr14:2254765 TRAC TRAC 87.86, Petition 870250081011, dated 09 / 09 / 2025, pages 122 / 230 98 / 166 _82 AGACATG 8 sgRNA _83 TTCTTCCCCAGCC CAGGTAA 200 chr14:2254777 8 TRAC TRAC 89,85 sgRNA _84 CGTCATGAGCAG ATTAAACC 201 chr14:2255062 5 TRAC TRAC 95,81 sgRNA _85 GAGAGCGCCTGC GACCCGAG 202 chr19:5854498 0 TRIM28 TRIM28 89,44 sgRNA _86 CCAGCGGGTGAA GTACACCA 203 chr19:5854486 9 TRIM28 TRIM28 94,79 sgRNA _87 GGAGCGCIIIIC GCCGCCAG 204 chr19:5854483 9 TRIM28 TRIM28 91,81 sgRNA _88 TGAGGCCTGGAC CTTATGCA 205 chr10:3313419 3 chr10:331 30000- 33140000 desert_1 (GS88) 69,44 sgRNA _89 CCTGGTGGAGTG AACCATGA 206 chr10:3313291 7 chr10:331 30000- 33140000 desert_1 (GS89) 95,25 sgRNA _90 CAAGCACTTAGGT TCCCCTG 207 chr10:3313463 3 chr10:331 30000- 33140000 desert_1 (GS90) 91,13 sgRNA _91 GGTCTCCCTACAA TTCAGCG 208 chr10:7229456 8 chr10:722 90000- 72300000 desert_2 (GS91) 92,02 sgRNA _92 CACAGCGCGTGA CTGCAATG 209 chr10:7229826 8 chr10:722 90000- 72300000 desert_2 (GS92) 90,22 sgRNA _93 TCTGGGGCACCA ATTCTAGG 210 chr10:7229278 6 chr10:722 90000- 72300000 desert_2 (GS93) 86,35 sgRNA _94 GAGCCATGCTTG GCTTACGA 211 chr11:1283425 76 chr11:128 34000012835000 0 desert_3 (GS94) 91.24 sgRNA _95 GTACAAGTACTTA TCTCATG 212 chr11:1283435 92 chr11:128 340000- desert_3 (GS95) 89.02, Petition 870250081011, dated 09 / 09 / 2025, pp. 123 / 230 99 / 166 chr11:1283471 70 chr11:128 34000012835000 0 desert_3 (GS96) 96.47 sgRNA _97 CATATTCCATAGT CTTTGGG214 chr11:6542500 0 chr11:654 25000- 65427000 (NEAT1) desert_4 (GS97) 88.54 sgRNA _98 CTGCCCCTTAGCA ACTTAGG 215 chr11:6542550 7 chr11:654 25000-1. 65427000 (NEAT1) desert_4 (GS98) 92.76 sgRNA _99 TGTTTAAAAATAT GTTGACA 216 chr11:6542626 4 chr11:654 25000- 65427000 (NEAT1) desert_4 (GS99) 90.76 sgRNA _100 CCATTACK ACTCACGC 217 chr15:9283031 5 chr15:928 30000- 92840000 desert_5 (GS100) 87.84 sgRNA _101 GAGGCCGCTGAA TTAACCCG 218 chr15:9283185 0 chr15:928 30000- 92840000 desert_5 (GS101) 85.32 sgRNA _102 ATACACGCACACT TGCAGAA 219 chr15:9283113 1 chr15:928 30000- 92840000 desert_5 (GS102) . 99.92 sgRNA _103 GAGCAGACAGAA ACCCAGGG 220 chr16:1122567 0 chr16:112 20000- 11230000 desert_6 (GS103) 87.92 sgRNA _104 TGAGTCTCCAAAC AGAACAG221 chr16:1122628 4 chr16:112 20000-11230000 desert_6(GS104)88.53 sgRNA _105 TAATATCACTGAC TTCACGG 222 chr16:1122502 9 chr16:112 20000- desert_6 (GS105) 87.65, Petition 870250081011, dated 09 / 09 / 2025, pp. 124 / 230 100 / 166 11230000 sgRNA _106 TACACACAATGTA AGCAGCA 223 chr2:87467461 chr2:8746 0000- 87470000 desert_7 (GS106) 71,79 sgRNA _107 GGGAGCTCAATTC GAAACCA 224 chr2:87468809 chr2:8746 0000- 87470000 desert_7 (GS107) 65,89 sgRNA _108 TTGGACAGGTGA GACAGTCG 225 chr2:87467001 chr2:8746 0000- 87470000 desert_7 (GS108) 72,64 sgRNA _109 AAGCTCACTCAGA TAGTGTG 226 chr3:18651131 6 chr3:1865 10000- 18652000 0 desert_8 (GS109) 76,89 sgRNA _110 CAGGAGAACCAC CTTACACG 227 chr3:18651526 0 chr3:1865 10000- 18652000 0 desert_8 (GS110) 86,31 sgRNA _111 GGACAGACCCTG ATTCACAA 228 chr3:18651965 5 chr3:1865 10000- 18652000 0 desert_8 (GS111) 85,47 sgRNA _112 ACATGGCAGTCTA TGAACAG 229 chr3:59451154 chr3:5945 0000- 59460000 desert_9 (GS112) 87,77 sgRNA _113 CCTATAGAGAGTA CTACTTG 230 chr3:59456416 chr3:5945 0000- 59460000 desert_9 (GS113) 79,33 sgRNA _114 CCAACCGGGTCTT CATTACG 231 chr3:59457029 chr3:5945 0000- 59460000 desert_9 (GS114) 92,21 sgRNA _115 TCAAGCGTAGAGT TCCGAGT 232 chr8:12799300 6 chr8:1279 80000- 12800000 0 desert_10 (GS115) 93.07, Petition 870250081011, dated 09 / 09 / 2025, pp. 125 / 230 101 / 166 sgRNA _116 TCATGCAATTATG GACCCAG 233 chr8:12799466 3 chr8:1279 80000- 12800000 0 desert_10 (GS116) 89.40 sgRNA _117 CGGGAAAGTGAC TGGCCATG 234 chr8:12799676 6 chr8:1279 80000- 12800000 0 desert_10 (GS117) 87.45 sgRNA _118 TGAGATTGAAATC AAATCGG 235 chr9:7974159 chr9:7970 000- 7980000 desert_11 (GS118) 84.84 sgRNA _119 TATGCAATATTCA TCACGCG 236 chr9:7977914 chr9:7970 000- 7980000 desert_11 (GS119) 85.44 sgRNA _120 AATGTGTTAAATC AAATGCA 237 chr9:7976895 chr9:7970 000- 7980000 desert_11 (GS120) 83.48 CRISPR-Cas Edition

[0304] An effective example of gene editing is the CRISPRCas approach (e.g., CRISPR-Cas9). This approach incorporates the use of a guide polynucleotide (e.g., guide ribonucleic acid or gRNA) and a cas endonuclease (e.g., Cas9 endonuclease).

[0305] As used herein, a polypeptide referred to as a Cas endonuclease or with Cas endonuclease activity refers to a CRISPR-related (Cas) polypeptide encoded by a Cas gene, where a Cas polypeptide is a target DNA sequence that can be cleaved when operably linked to one or more guide polynucleotides (see, for example, U.S. Patent No. 8,697,359). Also included in this definition are variants of Cas endonucleases that retain guide polynucleotide-dependent endonuclease activity. The Cas endonuclease used in the donor DNA insertion method detailed herein is an endonuclease that introduces double-strand breaks in the DNA at the target site (e.g., within the target locus or at the port site). Petition 870250081011, dated 09 / 09 / 2025, pp. 126 / 230 102 / 166 insurance).

[0306] As used herein, the term “guide polynucleotide” refers to a polynucleotide sequence capable of complexing with a Cas endonuclease and enabling the Cas endonuclease to recognize and cleave a target DNA site. The guide polynucleotide may be a single molecule or a double molecule. The guide polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). A guide polynucleotide comprising only ribonucleic acid is also referred to as guide RNA. In some embodiments, a polynucleotide donor construct is inserted into a safe harbor locus using a guide RNA (gRNA) in combination with a Cas endonuclease (e.g., Cas9 endonuclease).

[0307] The guide polynucleotide includes a first nucleotide sequence domain (also referred to as a variable targeting domain or VT domain) that is complementary to a nucleotide sequence in the target DNA and a second nucleotide that interacts with a Cas endonuclease polypeptide. It may be a double-stranded guide polynucleotide (also referred to as a double-stranded guide polynucleotide) comprising a sequence domain (referred to as a Cas endonuclease recognition domain or CER domain). The CER domain of this double-stranded guide polynucleotide comprises two separate molecules that hybridize along the complementary region. The two separate molecules may be RNA sequences, DNA sequences, and / or RNA-DNA combination sequences.

[0308] Genome editing using CRISPR-Cas approaches relies on the repair of site-specific DNA double-strand breaks (DSBs) induced by RNA-guided Cas endonuclease (e.g., Cas9 endonuclease). Homology-directed repair (HDR) of these DSBs allows for precise genome editing through the introduction of defined genomic alterations, including base substitutions, sequence insertions, and deletions. Conventional HDR-based CRISPR / Cas9 genome editing involves Petition 870250081011, dated 09 / 09 / 2025, pages 127 / 230 103 / 166 transfection of cells with Cas9, gRNA and donor DNA containing homologous arms corresponding to the genomic locus of interest.

[0309] HITI (homology-independent targeted insertion) uses a homology-independent strategy based on non-homologous end joining (NHEJ) and the method may be more efficient than HDR. Guide RNAs (gRNAs) target the insertion site. For HITI, donor plasmids do not have homology arms and DSB repair does not occur via HDR. The donor polynucleotide construct can be manipulated to include Cas9 cleavage site(s) flanking the gene or sequence to be inserted. This results in Cas9 cleavage in both the donor plasmid and the target genomic sequence. Both the target and donor have blunt ends and the linearized donor DNA plasmid is used via NHEJ, resulting in integration into the DSB genomic site. (See, for example, Suzuki, K., et al. (2016). In vivo genome editing via CRISPR / Cas9 mediated homology-independent targeted integration.)Nature, 540(7631), 144-149, relevant disclosures of which are incorporated herein in their entirety.

[0310] Methods for conducting gene editing using CRISPR-Cas approaches are known to those of common skill in the art. (See, for example, U.S. Applications Nos. U.S.16 / 312,676, U.S.15 / 303,722, and U.S.15 / 628,533, disclosures of which are incorporated herein by reference in their entirety.) In addition, the uses of endonucleases to insert transgenes into safe harbor loci are described, for example, in U.S. Application No. 13 / 036,343, disclosures of which are incorporated herein by reference in their entirety.

[0311] Guide RNAs and / or mRNAs (or DNA) encoding an endonuclease may be chemically linked to one or more chemical moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Non-limiting examples of such chemical moieties include lipid chemical moieties, such as a cholesterol chemical moiety, cholic acid, a thioether, a thiocholesterol, an aliphatic chain (e.g., dodecandiol or undecyl residues), a phospholipid, e.g., dihexadecyl Petition 870250081011, dated 09 / 09 / 2025, pages 128 / 230 104 / 166 rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or polyethylene glycol chain, adamantane acetic acid, a palmityl chemical moiety and an octadecylamine or hexylamino-carbonyltoxicolesterol moiety. See, for example, US Patent Publication No. 20180127786 which is incorporated by reference herein in its entirety. Therapeutic Applications

[0312] For therapeutic applications, manipulated cells, populations thereof, or compositions thereof are administered to a subject, usually a mammal, usually a human, in an effective quantity. Manipulated cells may be administered to a subject by infusion (e.g., continuous infusion over a period of time) or other modes of administration known to those skilled in the art.

[0313] The manipulated cells provided here are used not only in gene therapy, but also in non-pharmaceutical uses, such as, for example, the production of animal models and the production of recombinant cell lines that express a protein of interest.

[0314] The manipulated cells of this disclosure can be any cell, generally a mammalian cell, generally a human cell that has been modified by the integration of a transgene at a safe harbor locus described herein. Exemplary cells are provided in the Recombinant Cells section.

[0315] The cells, compositions, and manipulated methods of this disclosure are useful for therapeutic applications, such as CAR-T cell therapy and TCR T cell therapy. In some embodiments, the insertion of a sequence encoding a transgene within a safe harbor locus maintains TCR expression relative to cases where there is no insertion and allows transgene expression while maintaining TCR function.

[0316] In some modalities, the present disclosure provides methods of treatment for a subject in need of treatment, administering to Petition 870250081011, dated 09 / 09 / 2025, pages 129 / 230 105 / 166 subject to a composition comprising any of the manipulated cells described herein. In some embodiments, administration of the manipulated cell composition results in a desired pharmacological and / or physiological effect. This effect may be partial or complete cure of the disease and / or adverse effects resulting from the disease. In some embodiments, the treatment encompasses any treatment of a disease in a subject (e.g., mammal, e.g., human). In addition, the treatment may stabilize or reduce undesirable clinical symptoms in subjects (e.g., patients). The cells provided herein, populations thereof, or compositions thereof may be administered during or after the occurrence of the disease.

[0317] In certain modalities, the subject has a disease, condition and / or injury that can be treated and / or improved by cell therapy. In some modalities, the subject requiring cell therapy is a subject who has an injury, disease or condition, thus causing the cell therapy (e.g., therapy in which cellular material is administered to the subject). However, it is considered possible to treat, improve and / or reduce the severity of at least one symptom associated with the injury, disease or condition. Administration Method

[0318] An effective amount of the immune cell comprising the SPA peptide can be administered for cancer treatment. The appropriate dosage of the immune cell comprising the SPA peptide can be determined based on the type of cancer being treated, the type of immune cell comprising the SPA peptide, the severity and course of the cancer, the individual's clinical condition, the individual's medical history and response to treatment, and at the discretion of the attending physician. Determining the CD11c Expression

[0319] Methods for treating cancer in a subject in need thereof are also provided herein, comprising: determining or having determined the expression of CD11c in a cell comprising a synthetic pathway activator peptide (SPA) disclosed herein, optionally in Petition 870250081011, dated 09 / 09 / 2025, pages 130 / 230 106 / 166 that the SPA is inserted into a target region of the cell's genome; and administering or having administered to the subject the cell that comprises the SPA.

[0320] CD11c is also known as Integrin Subunit Alpha X, Integrin, Alpha X (Complement Component 3 Receptor Subunit 4) or ITGAX (HGNC: 6152, Gene NCBI: 3687, UniProtKB / Swiss-Prot: P20702).

[0321] In some respects, methods are provided here for determining the expression level of CD11c protein in a sample from a subject, comprising contacting the sample with an anti-CD11c antibody and performing FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, monoplex immunohistochemistry, multiplex immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, surface plasmon resonance, optical spectroscopy, mass spectrometry assay, or any combination thereof. In some respects, methods are provided here for determining the expression level of CD11c mRNA in a sample from a subject, comprising performing qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, Luminex, MSD, or FISH, and combinations thereof.

[0322] In some aspects, methods are provided here for producing a CD11c-positive (CD11c+) cell comprising inserting a nucleic acid encoding any SPA peptide disclosed herein and expressing the SPA peptide in the cell. In other aspects, methods are provided here for detecting an SPA, optionally a functional SPA, in a cell comprising an SPA peptide disclosed herein, optionally wherein the SPA is inserted into the cell genome.

[0323] In some respects, methods are provided here for screening a cell for SPA expression, optionally where the SPA is a functional SPA, comprising expressing one or more SPAs in a cell and detecting CD11c expression in the cell, where detection of CD11c indicates a functional SPA peptide. In such modalities, a functional SPA is one that has functional signaling (e.g., it can stimulate the cell, such as a Petition 870250081011, dated 09 / 09 / 2025, pages 131 / 230 107 / 166 T cell, through phosphorylation of STAT1, STAT3 and / or STAT5).

[0324] In some respects, assays are provided here for detecting cells, such as primary cells and / or immune cells, designed to express an SPA, comprising: determining or having CD11c expression determined in the primary cell, where CD11c expression indicates that the cell expresses the SPA.

[0325] In some respects, methods of treating a patient with a CD11c-expressing T cell are provided herein, comprising: administering a T cell comprising a SPA disclosed herein to the patient.

[0326] In some embodiments, CD11c expression is determined in the T cell, from a biological sample of a patient who received the cell expressing the SPA peptide, or in the tumor of a patient who received the cell expressing the SPA peptide. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a primary immune cell. In some embodiments, the immune cell is a hematopoietic cell, an adaptive immune cell, an innate immune cell, a natural killer (NK) cell, a T cell, a CD8+ cell, a CD4+ cell, or a T cell progenitor cell. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD4+CD8+ T cells.

[0327] In some embodiments, the CD11c expression level comprises the CD11c mRNA expression level. In some embodiments, the CD11c expression level comprises the CD11c protein expression level. In some embodiments, the CD11c expression level is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immuno Petition 870250081011, dated 09 / 09 / 2025, pages 132 / 230 108 / 166 histochemistry, monoplex immunohistochemistry, multiplex immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNAseq, microarray analysis, SAGE, MassARRAY technique, Luminex, MSD and FISH, and combinations thereof. Pharmaceutical compositions

[0328] The recombinant cells provided herein may be administered as part of pharmaceutical compositions. These compositions may comprise, in addition to one or more recombinant cells, an excipient, carrier, buffer, stabilizer, or other pharmaceutically acceptable materials well known to those skilled in the art. Such materials must be non-toxic and must not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal. The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and a professional with ordinary skill in the field is able to select suitable pharmaceutical excipients. Consequently, the pharmaceutical excipients provided below are intended to be illustrative and not limiting.Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), which is incorporated in its entirety by reference.

[0329] Various modes of administration of the additional therapeutic agents are contemplated herein. In some embodiments, the additional therapeutic agent is administered by any suitable mode of administration.

[0330] A composition may be administered alone or in combination with other treatments, simultaneously or sequentially, Petition 870250081011, dated 09 / 09 / 2025, pages 133 / 230 109 / 166 depending on the condition being treated. Kits and Manufacturing Supplies

[0331] This application provides kits comprising any one or more of the SPA peptides or cellular compositions described herein together with instructions for use. The instructions for use may be included in the kits in the form of a package insert, on the packaging label of the kit or its components, or may be in digital format (e.g., on CD-ROM, via an internet link). A kit may include one or more of a genome-targeting nucleic acid, a polynucleotide encoding a genome-targeting nucleic acid, a site-targeting polypeptide, and / or a polynucleotide encoding a site-targeting polypeptide. Additional components within the kits are also contemplated, for example, buffer (such as reconstitution buffer, stabilization buffer, dilution buffer) and / or one or more control vectors.

[0332] In some embodiments, the kits also contain a component selected from any of the secondary antibodies, reagents for immunohistochemical analysis, a pharmaceutically acceptable excipient and an instruction manual, and any combination thereof. In one specific embodiment, the kit comprises a pharmaceutical composition comprising one or more of the antibody compositions described herein, with one or more pharmaceutically acceptable excipients.

[0333] This application also provides articles of manufacture comprising any of the antibody compositions or kits described herein. Examples of an article of manufacture include vials (including sealed vials). EXAMPLES

[0334] Below are examples of specific ways of carrying out this disclosure. The examples are offered for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Efforts have been made to ensure accuracy with respect to Petition 870250081011, dated 09 / 09 / 2025, pp. 134 / 230 110 / 166 numbers are used (e.g., quantities, temperature, etc.), but some experimental errors and deviations must, of course, be taken into account.

[0335] The practice of this disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the knowledge of the art. Such techniques are fully explained in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (Plenum Press) Vols A and B (1992). Example 1: In vitro Synthesis and Characterization of Logic Gate Circuits Comprising Synthetic Pathway Activators

[0336] Materials and Methods

[0337] Expression of ICT construct in T cells

[0338] Integrated circuit T cells (ICTs) were generated via site-directed CRISPR-mediated knockin (KI). T cells were activated for two days using CD3-CD28 microspheres. On day 2, the microspheres were removed, followed by the distribution of the ICT transgene to the GS94 site in the T cell genome. Transgene integration was performed using a CRISPR-based process and an electroporation step that combined activated T cells, CRISPR / Cas9 RNP targeting the GS94 autosomal non-coding integration site, and plasmid DNA constituting a repair template to effect transgenic cassette insertion via cellular DNA repair machinery.

[0339] The GS94 CRISPR / Cas9 RNP used was generated by complexing single guide RNA (sgRNA) with recombinant Streptococcus pyogenes Cas9. Petition 870250081011, dated 09 / 09 / 2025, pages 135 / 230 111 / 166 (SpCas9).sgRNA contained a protospacer sequence directing CRISPR / Cas9 RNP to the GS94 transgene integration site. The plasmid DNA repair template contained the ICT transgenic cassette, flanked by 450 base pair (bp) sequences homologous to the regions flanking the integration site to effect repair-mediated insertion.

[0340] A diagram of five generated ICT transgene cassettes is provided in Figure 1. ICT constructs 1, 2, 3, and 4 comprised a constitutively expressed initiation receptor, an inducible CAR (collectively forming a Logic Gate or “LG”), constitutively expressed shRNAs, and a synthetic pathway activator (SPA). ICT 5 included LNGFR in place of SPA.

[0341] After electroporation, the cells were recovered and expanded in T cell medium for 7 days. When indicated, negative control T cells were generated using a sham electroporation process that edited ribonucleoprotein (RNP) T cells in the absence of a donor plasmid and are referred to as “RNP controls”.

[0342] ICT cells were evaluated for KI transgene and PrimeR and CAR expression using flow-based staining. The constructs contained myc and flag tags in the distal extracellular portion of PrimeR and CAR, respectively, after the signal peptide. ICT cells on day 7 post-activation were stained with myc, flag, and CD3 antibodies for 30 minutes at 4°C. After activation, the cells were washed in FACs buffer and analyzed by flow cytometry. ICTs were analyzed for PrimeR and CAR expression after selection of each sample for live CD3+ cells.

[0343] Induction of CARs by ICT

[0344] ICTs were generated as described above from T cells from 2 donors. On day 11 after activation, ICTs were measured for CAR and PrimeR expression by Flag and Myc staining. % KI was quantified by summing the % of T cells in a sample that were PrimeR+ or CAR+. Before co-culture setup, ICTs were normalized to the same % KI using cell-only addition of matched RNP. Petition 870250081011, dated 09 / 09 / 2025, pp. 136 / 230 112 / 166 donor cells. 1x107 ICTs were co-cultured with 1x107 target cells or medium for 72 hours and stained to calculate the % of CAR+ cells using flag staining. Baseline CAR expression was measured during assay setup.

[0345] Synthetic pathway activators

[0346] Synthetic pathway activators (SPAs) constitutively control STAT signaling without the need for external cytokine input. SPAs can be designed to engage the activity of multiple STAT family transcription factors at varying levels through rational design. Exemplary Class I SPAs primarily increase pSTAT3 activity, and exemplary Class II SPAs primarily increase pSTAT5 activity. Figure 2 shows the structures of exemplary synthetic pathway activators.

[0347] A synthetic pathway activator (SPA) based on gp130 was constructed as shown in SEQ IDs NOS: 20 and 81. SEQ ID NO: 20 includes a leader sequence. The SPA comprises the transmembrane region and the intracellular domain of gp130 linked to an ectodomain derived from the cell adhesion protein CD34. An unpaired cysteine ​​residue was introduced into the receptor ectodomain to allow the formation of a covalent bond and subsequent dimerization of individual synthetic gp130 monomers. The SPA drives the constitutive recruitment and phosphorylation of the transcription factors STAT1 and STAT3. (Figure 5 and data not shown)

[0348] To demonstrate the ability of the SPA module to drive constitutive phosphorylation of STAT3, ICTs expressing the SPA module under unstimulated conditions were fixed, permeabilized, and stained for pSTAT3 and the myc epitope label to distinguish between edited and unedited cells expressing the ICT.

[0349] Cytotoxicity, engineered K562 cells

[0350] ICT cells expressing integrated circuits comprising Logic Gate IC 1, Logic Gate IC 2, Logic Gate IC 3, Logic Gate IC 4 or Logic Gate IC 5 (LG-15 IC) with shRNA and an SPA were Petition 870250081011, dated 09 / 09 / 2025, pages 137 / 230 113 / 166 cells were co-cultured with target antigens K562_EFG, K562_EFG_CAR, K562_EFG_primeR, or K562_EFG_CAR_primeR at different E:T ratios for 72 hours at 37 °C. After incubation, cytotoxicity was measured using a luciferase reporter assay. Data are presented as mean ± standard deviation of 4 donors.

[0351] Cytokine secretion

[0352] To better assess the specificity and function of ICT cells expressing Logic Gates 1-5, supernatants were collected from K562 target cytotoxicity cocultures (Effector:Target ratio of 1:1, 72-hour coculture). After incubation, supernatants were collected at the endpoint and cytokine release levels were measured using a Luminex assay. Data from 4 donors are shown.

[0353] Cytotoxicity in endogenous CAR cells

[0354] ICT cells expressing Logic Gates 1-5 were co-cultured with cells endogenously expressing a CAR target and engineered to express a primeR target at different E:T ratios for 72 hours at 37 °C. After incubation, cytotoxicity was measured using a luciferase reporter assay. Data are presented as mean ± standard deviation of 4 donors. Prior to the luciferase reading described above, supernatants were collected at the endpoint and the cytokines IFN-γ, TNFα, GM-CSF, and IL-2 were measured using a Luminex assay. Data from 4 donors are shown.

[0355] Cytotoxicity of Mixed Co-culture

[0356] ICT cells expressing LG 1-5 ICs were co-cultured with HUVEC cells expressing target primeR+ / target CAR- and luciferase cells expressing target primeR- / target CAR+ (K562-EFG-CAR) at different E:T ratios for 72 hours at 37 °C. After incubation, cytotoxicity was measured using a luciferase reporter assay. Data are from a normal donor. ICT-mediated CAR+ target cell death was assessed relative to an RNP-electroporated negative control using a luciferase reporter assay. Petition 870250081011, dated 09 / 09 / 2025, pages 138 / 230 114 / 166

[0357] Results

[0358] All ICT cells constitutively expressed the PrimeR construct as shown by myc expression (Figure 3). The inducible CAR was not expressed in the basal state in ICT cells, as indicated by the lack of FLAG expression (Figure 3), indicating that the initiation receptor did not induce CAR expression.

[0359] As shown in Figure 4, ICT cells induced CAR expression when co-cultured with cell lines expressing the target antigen primeR. The numbers shown in Figure 4 are (% CAR) / (% KI normalized at baseline)*100. Thus, the logic gate circuit functioned correctly by not expressing CAR in the absence of primeR binding to the target antigen of primeR (Figure 3) and inducing CAR expression after primeR binding to its cognate ligand in a target cell (Figure 4).

[0360] As shown in Figure 5, flow cytometry analysis revealed that ICT cells expressing SPA (LG 1-4 ICs) exhibit approximately one log higher pSTAT3 expression when compared to PrimeR- cells without SPA (EGFRt). T cells edited with an EGFRt module (non-signaling) in place of an SPA do not exhibit increased pSTAT3 staining when compared to PrimeR- cells. Overall, the results indicate that ICTs expressing the SPA module exhibit increased STAT3 phosphorylation.

[0361] ICTs expressing LG 1-5 ICs demonstrated cytotoxicity only against cells expressing both CAR dual target antigen and primeR target antigen compared to unedited control cells (RNPs). Figure 6A shows cytotoxicity against parental K562 cells expressing no target antigen, Figure 6B shows cytotoxicity against K562 cells expressing only CAR target antigen, Figure 6C shows cytotoxicity against K562 cells expressing only primeR target antigen, and Figure 6D shows cytotoxicity against K562 cells expressing both primeR target antigen and CAR target antigen. According Petition 870250081011, dated 09 / 09 / 2025, pages 139 / 230 115 / 166 shown in Figure 6D, the ICTs exhibited cytotoxicity against only cells expressing both the target antigen primeR and the target antigen CAR compared to unedited cells (RNP).

[0362] IFN-γ production from ICTs expressing LG 15 ICs was observed only in supernatants taken from co-cultures where target cells expressed both the target antigen primeR and the target antigen CAR (Figure 7). The cytokine analysis results were consistent with the cytotoxicity data. Together, these data further demonstrate that TIC activity is driven by the co-expression of the target antigen primeR and the target antigen CAR.

[0363] ICTs expressing Logic Gate 1-5 ICs demonstrated in vitro cytotoxicity against the cell line expressing the endogenous CAR target antigen and the manipulated primeR target antigen (Figure 8A). ICTs also secreted cytokines after co-culture with the CAR / primeR target antigen cell line. Figure 8B shows the secretion of IFNγ, TNFα, GM-CSF, and IL-2 by ICT cells after co-culture with +CAR / +primeR target antigen cells. Thus, ICTs expressing Logic Gate 1-5 ICs secreted cytokines and killed ccRCC cell lines expressing endogenous CAR antigen in the presence of the primeR target antigen.

[0364] Co-culture with HUVEC-primeR antigen cells induced CAR protein expression in ICT cells, and specific killing of CAR antigen-positive cells was confirmed (Figure 9). Thus, ICTs expressing Logic Gates 1-5 were able to induce CAR expression through interaction with endothelial cells positive for the target primeR antigen and subsequently specifically engulf and exterminate CAR antigen-positive tumor cells. Therefore, without relying on theory, ICTs can be prepared by binding to endothelial cells expressing the target primeR antigen to express CAR and then exterminate CAR target tumor cells.

[0365] Thus, ICT cells with logic gates were developed and Petition 870250081011, dated 09 / 09 / 2025, pages 140 / 230 116 / 166 activators of signaling pathways that utilize the presence of two antigens to trigger tumor cell death and improve the therapeutic index of CAR T cells, thus increasing tumor specificity. CAR induction was determined by the expression of the target antigen primeR found in the tumor neovasculature of ccRCC. When the initiation receptor (PrimeR) binds to the target antigen of primeR, PrimeR engagement triggers the proteolytic release of a transcription factor that induces the expression of a CAR. The viability of vascular initiation was confirmed using a transwell assay where ICTs were initiated by an endothelial cell line expressing the target antigen primeR and then migrated through the transwell membrane to exterminate RCC cells expressing the CAR antigen.

[0366] When constitutively expressed in ICT cells, SPA resulted in significant enhancements in T cell potency and expansion. Repetitive stimulation assays, in which T cells were challenged with tumor cells every 2 days, show that Class I SPAs result in a 6 log or greater improvement in tumor cell clearance over a 2-week assay period. (data not shown) In several mouse xenograft models (Figures 2, 10A, 10D, 11B and data not shown), ICTs expressing SPA achieve at least 6-fold better tumor growth inhibition. RNAseq and ATACseq analyses indicate alterations in gene expression profiles in T cells expressing Class I SPAs, with maintenance of T stem cell-like phenotypes and restricted accessibility of several marker genes for exhaustion (Figure 2 and data not shown).Importantly, despite significantly higher levels of expansion, SPA-equipped ICTs are not immortalized, showing no signs of cytokine-independent growth (data not shown). Furthermore, SPA-expressing ICT cells contract rapidly after tumor clearance in vivo (Figures 10B, 10C, 10E, 10F).

[0367] Collectively, these results demonstrate that the cells Petition 870250081011, dated 09 / 09 / 2025, pages 141 / 230 117 / 166 primeR / CAR plus SPA ICT can (i) selectively target antigens that generally cannot be safely targeted by conventional CARs; and (ii) overcome multiple suppressive mechanisms in the tumor microenvironment. Example 2: In vivo efficacy of primeR and CAR logic gate T cells expressing a synthetic pathway activator.

[0368] Materials and Methods

[0369] RCC Effectiveness Model

[0370] Human ccRCC cells express endogenous levels of the CAR target antigen and were engineered to express physiological levels of the primeR target antigen. 2 x106 primeR target antigen cells were inoculated into the right dorsal flank of five- to six-week-old female NSG MHC I / II DKO mice. On day 35 after tumor inoculation, the mean tumor volume reached 150 mm3 and tumor-bearing animals were randomized into multiple treatment groups so that the mean tumor volume per group was within 10% of the overall mean. Seven mice / group were intravenously injected with a single dose of 0.15 x106 PrimeR+ ICT cells expressing one of the five LG ICTs described in the RNP or PBS of Example 1 (LG IC 1, LG IC 2, LG IC 3, LG IC 4, or LG IC 5). The study was repeated with ICTs generated from two different normal donors. Tumor volumes and body weight were recorded every two weeks.The tumor volume was calculated using the formula 1 / 2 * L * W², where L is the length of the tumor and W is the width of the tumor.

[0371] Blood pharmacokinetics demonstrated ICT expansion on day 14, followed by complete contraction on day 42 after T cell injection. PrimeR+ ICTs in mouse blood were quantified to track ICT expansion using flow cytometry with bright counting microspheres for T cell quantification / volume. Mean and SEM plotted.

[0372] Double Flank Model

[0373] Human ccRCC 786-O cells were genetically modified to express the CAR target antigen and the primeR target antigen or Petition 870250081011, dated 09 / 09 / 2025, pages 142 / 230 118 / 166 only the CAR target antigen. Two x 10⁶ 786-O-CAR+ and 786-O-CAR antigen+-primeR antigen+ cells were inoculated into the left and right dorsal flanks, respectively, of five- to six-week-old female NSG MHC I / II DKO mice. On day 35 after tumor inoculation, the mean tumor volume reached 150-200 mm³ in each flank, and tumor-bearing animals were randomized into multiple treatment groups so that the mean tumor volume per group was within 10% of the overall mean. Seven mice / group were intravenously injected with a single dose of 0.25 x 10⁶ or 1 x 10⁶ PrimeR+ ICT cells, constitutive CAR T cells, RNP, or PBS control. Tumor volumes and body weight were recorded biweekly. The tumor volume was calculated using the formula %*L*W2, where L is the length of the tumor and W is the width of the tumor. (B) Tumor volumes only on the 786-O CAR flank (left) and (C) Tumor volumes on the 786O-CAR+primeR+ flank (right).The data represent a study with a single donor, with 7 mice per group, with mean and SEM plotted.

[0374] Results

[0375] RCC A498 Effectiveness Model

[0376] ICTs expressing logic gates 1-5 ICs showed tumor elimination in a ccRCC model. Figures 10A and 10D show tumor volume after tumor implantation in mice treated with ICTs expressing logic gates 1-5, RNP, or PBS generated from donor T cells 1 (Figures 10A-C) or donor T cells 2 (Figures 10D-F). Figures 10B and 10E show total T cell and ICT expansion on day 12 post-inoculation, followed by contraction on day 21. Figures 10C and 10F show total T cells expressing the initiation receptor on days 12 and 21. In both replicates, ICT cells demonstrated significant inhibition of tumor growth in mice (P < 0.05).

[0377] Double Flank Model

[0378] ICTs expressing LG 1-5 ICs showed specificity in a double flank model (Figures 11A-B). Greater inhibition was observed. Petition 870250081011, dated 09 / 09 / 2025, pp. 143 / 230 119 / 166 of tumor growth (TGI) in the double flank positive primeR / CAR (Figure 11B) than in the single flank positive CAR (Figure 11A). Thus, the double flank xenograft model shows that controlled logic circuits (CCTs) more selectively exterminate tumors that express both CAR and primeR target antigens, and not tumors that express only CAR. Example 3: Generation and characterization of new synthetic pathway activators

[0379] Methods

[0380] Generation and synthesis

[0381] A library of novel gp130-based STAT1 / 3 synthetic pathway activators (SPAs) was generated through diversification of extracellular and intracellular domains, multimerization modalities, membrane anchoring modalities, and epitope / signaling peptide markers. SPAs were generated to explore optimization and performance enhancement of SPAs, such as enhanced potency, lower immunogenicity, detection capabilities, and smaller gene size. Several forms of the intracellular gp130 domain (ICD) were used, including the full-length gp130 ICD, as well as truncations comprising gp130 ICD deletions Δ707-755, Δ771811, Δ818-901, and combinations thereof. A gp130 Y759F mutation was also included in some ICD constructs. The multimerization modalities used were unpaired cysteines, a leucine zipper, a BCR ectodomain (SEQ ID NO: 239), and a VASP tetramerization domain (SEQ ID NO: 240).The extracellular domains used were the full CD34 ectodomain (SEQ ID NO: 242), a CD34 epitope (SEQ ID NO: 238), a BCR ectodomain (SEQ ID NO: 239), a thrombopoietin receptor domain (SEQ ID NO: 243), and an erythropoietin receptor (EpoR) ectodomain (SEQ ID NO: 241). The membrane anchoring modalities used were prenylation and myristoylation domains derived from src, fyn, or lck. Prenylation modification was also used at the C-terminus of some SPAs. Some SPAs also contained a CD8alpha hinge domain (FACD). In some SPAs, SPA expression... Petition 870250081011, dated 09 / 09 / 2025, pages 144 / 230 120 / 166 was inducible based on T cell activation. Other SPAs were constitutively expressed. The sequences of the new SPAs are provided in SEQ ID NOs: 1-58 and 63-104. The SPAs were screened first by a pSTAT activation screening, then an in vitro functional assessment, and finally an in vivo efficacy assessment.

[0382] The activity of the new pathway activators was assessed by intracellular pSTAT staining after 24 hours of serum fasting. ICTs expressing an SPA construct module were deprived of food for 24 hours, without antigen stimulation or cytokine support, and then fixed, permeabilized, and stained for pSTAT proteins. The MFI values ​​of pSTAT3 and pSTAT1 were measured for two independent donors, and the mean value was plotted on a heat map.

[0383] Cytokine Induction

[0384] A diverse range of ICTs expressing novel STAT1 / STAT3 SPAs were cultured with K562 cells at a 1:1 ratio. After 72 hours of co-culture, the supernatants were isolated and analyzed by Luminex for granzyme B and IL10 production. Representative cytokines exhibiting various levels of expression are shown (grouped according to pSTAT1 levels).

[0385] Repetitive Stimulation and Memory Phenotype

[0386] Logic gate expressing T cells (ICTs) and a diverse range of novel Class I STAT1 / STAT3 SPAs were challenged in a 14-day repetitive stimulation assay with K562 cells expressing primeR and CAR antigens supplemented with IL-2. ICTs and tumor cells were renormalized to a fixed number every two days to maintain a 1:1 E:T ratio. Total tumor cell growth and T cell expansion throughout the experiment were normalized to the EGFRt control and plotted in groups according to pSTAT1 levels. L-gp130 was used as the control SPA. Additional controls included the expression of cJun, EGFRt, mbIL-15, and IL7Ra-IL7 in ICTs. Petition 870250081011, dated 09 / 09 / 2025, pp. 145 / 230 121 / 166

[0387] The memory phenotype was measured by flow cytometry for CD45RA and CD27 expression on day 0 and at the endpoint of the repetitive stimulation assay (day 14) and plotted in groups according to the pSTAT1 profile.

[0388] Results

[0389] Approximately 60% of the new SPAs demonstrated elevated levels of pSTAT1 (Figure 12B) and / or pSTAT3 (Figure 12A) compared to an ICT logic gate expressing an inert truncated EGFR molecule in place of an SPA. The pSTAT1 vs pSTAT3 heat map highlights the combinatorial diversity achieved through modification of the SPA architecture (Figure 13). Thus, without being limited by theory, STAT-inducing cell surface receptors demonstrate remarkable flexibility in their architecture, allowing for the diversification of STAT profiles.

[0390] Diversity in pSTAT1 and pSTAT3 signaling resulted in varied cytokine secretion, including Granzyme B (upper panel of Figure 14) and IL-10 (lower panel of Figure 14). Thus, the pSTAT1 / 3 signaling diversity achieved through the novel SPAs boosted cytokine responses.

[0391] All new STAT1 / STAT3 SPAs demonstrated dramatic levels of tumor clearance in the repetitive stimulation assay. Figure 15 (top panel) shows tumor expansion compared to control ICTs under chronic antigen stimulation. ICTs expressing the new SPAs significantly reduced tumor expansion and resulted in greater tumor clearance compared to control ICTs. Figure 15 (bottom panel) shows T cell expansion. Without being limited by theory, the new Class I SPAs thus demonstrated superior antitumor activity compared to L-gp130 (SEQ ID NO: 62) and also demonstrated a favorable safety profile by rapidly contracting after tumor clearance.

[0392] The new SPAs boosted diverse memory phenotypes and functional phenotypes based on pSTAT profiles (Figure 16). Figure 16 shows Petition 870250081011, dated 09 / 09 / 2025, pages 146 / 230 122 / 166 is the percentage of ICT cells expressing the new SPAs that expressed CD45RA and / or CD27 and the corresponding T cell type (e.g., Tscm, Tcm, Teff) after the repetitive stimulation assay.

[0393] Without wanting to get bogged down in theory, the new SPAs represent a novel, improved and scalable intrinsic T-cell approach to cell fate engineering that results in potent antitumor properties, for example, compared to L-gp130. Example 4: In vivo efficacy of second-primeR and CAR logic gate T cells expressing a synthetic pathway activator.

[0394] Materials and Methods

[0395] In a mesothelioma solid tumor (MSTO) model, mice were grafted with MSTO tumors expressing different primeR and CAR antigens compared to Example 2. 300,000 ICT cells expressing the exemplary primeR and CAR logic gate and novel Class I SPAs (SPAs with SEQ ID NOs: 20, 30, or 16) or the exemplary primeR and CAR logic gate and no additional SPAs were administered intravenously when the tumors reached 100 mm3. T cells expressing the exemplary primeR and CAR logic gate and L-gp130 (SPA001, SEQ ID NO: 62) were used as an additional control. Blood was collected weekly for pharmacokinetic (PK) studies on days 7, 14, 21, and 28 after T-cell injection. Tumor growth inhibition (TGI) was measured 3 times a week for 45 days after T-cell injection.

[0396] Results

[0397] ICT cells expressing the logic gate and the novel Class I SPA variants of SEQ ID NOs: 20, 30, and 16 showed improved antitumor efficacy compared to ICT cells expressing a logic gate and L-gp130 (SPA01, Figure 17). The novel Class I SPA variants also showed improved T cell expansion compared to the base molecule SPA01. Thus, the novel SPAs outperformed L-gp130 in an in vivo experiment evaluating anticancer efficacy. Petition 870250081011, dated 09 / 09 / 2025, pages 147 / 230 123 / 166 Example 5: The expression of SPA in T cells induces the expression of CD11c.

[0398] Materials and Methods

[0399] Female NSG MHC DKO mice aged 5–6 weeks were implanted with 786-O B2M KO tumors expressing the primeR antigen and CAR antigen from Example 2. On day 36 after tumor implantation, mice were intravenously injected with LG 1 IC T cells or non-SPA IC T cells (LG 5 ICT). Tumor and spleen samples were collected from 15 mice treated with LG 1 IC T cells (2 donors) or non-SPA ICT (1 donor) on day 7 after ICT injection. Tumors were lysed in DMEM containing DNase / collagenase / hyaluronidase. Tumor dissociation was performed on gentleMacs (Octo) using a customized program. The spleen was mechanically digested using a syringe plunger handle-end over a 70 µm filter. After digestion, the cell suspension was filtered and subjected to red blood cell lysis and staining with live-dead Zombie NIR dye for 15 minutes in RT in the dark.Following live-dead staining, cells were centrifuged, washed, and stained with a cell surface antibody cocktail containing murine CD45, murine GR-1, human CD3, CAR idiotype (CAR receptor), and Prime idiotype (Prime receptor) in the presence of human and mouse Fc blocks for 30 minutes at room temperature in the dark. Cells were then centrifuged at 400 g for 10 min, washed, and suspended in flow staining buffer (BD Biosciences). Cells were then classified using BD FACS Aria. Receptor-positive cells were further classified and subjected to RNA sequencing and CITE sequencing.

[0400] CITE Labeling Protocol

[0401] The TotalSeq-C Human Universal Cocktail antibody mixture, V1.0 (Biolegend cat. no. 399905) was reconstituted in 26 µl of PBS with 1% BSA. Cells were blocked with Fc Block and 13 µl of the antibody mixture was added to 500,000 cells. After 30 minutes of incubation on ice, the Petition 870250081011, dated 09 / 09 / 2025, pages 148 / 230 124 / 166 cells were washed, resuspended, and transferred for GEM generation and barcoding using the 10x Genomics Chromium Next Gem 5' Single Cell kit (Cat. No. 1000263). The cells were combined with the GEM Master mix, loaded onto a 10X K chip in the Chromium X controller for GEM generation, and then incubated in a thermocycler for reverse transcription. After GEM cleanup and cDNA amplification, gene expression sequencing libraries were generated according to protocols published by 10x Genomics. Using the supernatant fraction from the cDNA amplification cleanup step, CITE-Seq libraries were constructed using the 10x Genomics Dual Index Plate TN Set A (Cat. No. 1000250) with 8 PCR amplification cycles. After the library was prepared, the samples were sequenced on an Illumina NovaSeq 6000.

[0402] Bioinformatics analysis

[0403] Single-cell RNA-seq and CITE-seq data were first analyzed by Cellranger 7.0 to generate genes per cell matrix. Cells were filtered to exclude cells with low UMI, cells with high mitochondrial content, and non-T cells. The filtered matrix was analyzed by scvi 0.20 to generate low-dimensional visualizations for individual cells. The filtered matrix was also analyzed by Seurat 5.0 to define differentially expressed genes, which are genes that are significantly upregulated or downregulated in SPA-positive cells compared to SPA-negative cells (SPA vs. no SPA). Genes that were upregulated in SPA-vs-no SPA in all D0, D7 Tumor, and D7 Spleen samples were intersected to generate a shared list. The same analysis was performed for RNA-seq and CITE-seq to generate two shared lists.The two shared lists were subsequently cross-referenced to name the final list of cell surface protein genes that are most closely associated with SPA expression. CD11c was named by manually examining the final named genes.

[0404] Detection of CD11 c by flow cytometry Petition 870250081011, dated 09 / 09 / 2025, pp. 149 / 230 125 / 166

[0405] Peripheral blood samples were collected in EDTA-coated tubes. Red blood cells were lysed with ammonium chloride, and the remaining cell fraction was stained with a fixable amine-reactive viability dye, and Fc receptors were blocked using anti-CD16 / CD32 monoclonal antibodies. After washing, cell surface antigens were stained using fluorochrome-conjugated monoclonal antibodies against human CD45, murine CD45, murine Gr-1, Flag tag (chimeric antigen receptor), and Myc tag (initiation receptor). Cells were then fixed with Cytofix fixation buffer (BD Biosciences) and permeabilized with Phosflow Perm Buffer III (BD Biosciences), following the manufacturer's recommendations. After permeabilization, cells were stained using fluorochrome-conjugated monoclonal antibodies against CD11c (CD11c is also known as Integrin, alpha X, or ITGAX) and intracellular pSTAT3.The samples were analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0406] Results

[0407] There is a strong correlation between cell surface RNA expression of CD11c and SPA expression in CD4+ and CD8+ cells on D7 in tumor and spleen samples (Figure 18A). Increased CD11c expression was observed in CD8 and CD4 cells expressing SPA compared to CD8 and CD4 cells not expressing SPA. Figure 18B shows CD11c (ITGAX) expression in spleen or tumor cells from mice treated with cells expressing an ICT of SPA (two donors) compared to cells not expressing an ICT of SPA (one donor) collected on Day 0 and Day 7 after treatment. CD11c (i.e., ITGAX) was expressed at higher levels in SPA-containing T cells isolated from tumor and spleen compared to non-SPA-containing T cells (Figure 18B). Thus, quantifying the expression of CD11c (mRNA or protein) can be used as a marker for SPA expression in CD4+ and CD8+ cells.

[0408] Although the revelation was particularly shown and Petition 870250081011, dated 09 / 09 / 2025, pp. 150 / 230 126 / 166 described with reference to a preferred embodiment and several alternative embodiments, it will be understood by those skilled in the relevant art that various alterations in form and details may be made thereto without departing from the spirit and scope of the present revelation.

[0409] All references, issued patents and patent applications cited in the body of this descriptive report are incorporated herein by reference in their entirety, for any purpose. informal sequence listing SEQ ID NO Name Sequence 1 QBEND10Cys-LGP130Y759 F with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QFSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERP EDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDIS HFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQM KMFQEVSAADAFGPGTEGQVERFETVGMEAATDEG MPKSYLPQTVRQGGYMPQ 2 QBEND10Cys-LGP130Y759 ΕΔ771-811 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QFSTVVHSGYRHQQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE Petition 870250081011, dated 09 / 09 / 2025, pages 151 / 230 127 / 166 VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 3 QBEND10Cys-LGP130Δ771811 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 4 QBEND10L-GP130 with leading sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSR IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVM NAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS YLPQTVRQGGYMPQ 5 QBEND10- L- GP130Y759 F with leading sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVM NAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQFST Petition 870250081011, dated 09 / 09 / 2025, pages 152 / 230 128 / 166 VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS YLPQTVRQGGYMPQ 6 QBEND10- L- GP130Y759 ΕΔ771-811 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSR IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVM NAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQFST VVHSGYRHQQQYFKQNCSQHESSPDISHFERSKQVS SVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAA DAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQT VRQGGYMPQ 7 QBEND10L- GP130Δ771- 811 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSR IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVM NAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQQQYFKQNCSQHESSPDISHFERSKQVS SVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAA DAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQT VRQGGYMPQ 8 SPAQBEND10Cys-LGP130 comMALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM Petition 870250081011, dated 09 / 09 / 2025, pp. 153 / 230 129 / 166 leader sequence YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERP EDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDIS HFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQM KMFQEVSAADAFGPGTEGQVERFETVGMEAATDEG MPKSYLPQTVRQGGYMPQ 9 SPA-Lgp130_ICDhKRasCAAX MRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQK VMNNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRH NFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSL DLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENE SSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQ PLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCS QHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHIS QSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVG MEAATDEGMPKSYLPQTVRQGGYMPQMSKDGKKKK KKSKTKCVIM 10 SPAQBEND10Cys-LGP130_ICD _trunc1 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEAND KKP FPETVQYSTVVHSGYR HQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDG GDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSV NEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVR QGGYMPQ 11 SPA- QBEND10- MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLQNSELASTANMLREQVAQL Petition 870250081011, of 09 / 09 / 2025, p. 154 / 230 130 / 166 Cys-LGP130Δ771811(ICD_tru nc2) with leader sequence KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 12 SPAQBEND10Cys-LGP130_ICD _trunc3 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERP EDLQLVDHVDGGDGILPRQQYFKQPKSYLPQTVRQG GYMPQ 13 SPAQBEND10Cys-LGP130_ICD _trunc4 com sequência líder MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEAND KKP FPETVQYSTVVHSGYR HQQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGT EGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYM PQ 14 SPAQBEND10Cys-L- MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKANSQELQLQLQM KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD Petition 870250081011, of 09 / 09 / 2025, p. 155 / 230 131 / 166 GP130_ICD _trunc5 with leader sequence LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQQQYFKQPKSYLPQTVRQGGYMP Q 15 SPAQBEND10Cys-LGP130_ICD _trunc6 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEAND KKP FPETVQYSTVVHSGYR HQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDG GDGILPRQQYFKQPKSYLPQTVRQGGYMPQ 16 SPAQBEND10Cys-LGP130_ICD _trunc7 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVE IEAND KKP FPETVQYSTVVHSGYR HQQQYFKQPKSYLPQTVRQGGYMPQ 17 SPAQBEND10L-GP130 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSR IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVM NAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS Petition 870250081011, dated 09 / 09 / 2025, pages 156 / 230 132 / 166 YLPQTVRQGGYMPQ 18 SPAQBEND10BCR-GP130 with leading sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVS MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELE RCKASIRRLEQEVNQERFRMIYLQTLLAKEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSV QVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILP RQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFV RLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTE GQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMP Q 19 SPAQBEND10cys-GP130 with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIK KHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYS DGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTE GHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQ YSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPE DLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISH FERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMK MFQEVSAADAFGPGTEGQVERFETVGMEAATDEGM PKSYLPQTVRQGGYMPQ 20 SPAQBEND10cysGP130_TM_ ICD with sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTD Petition 870250081011, dated 09 / 09 / 2025, pages 157 / 230 133 / 166 leads HVDGGDGILPRQYFKQNCSQHESSPDISHFERSKQ VSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVS AADAFGPGTEGQVERFETVGMEAATDEGMPKSYLP QTVRQGGYMPQ 21 SPAQBEND10cys-LGP130 sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIK KHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYS DGNFTDVSVVEIEANDKKPPPEDLKSLDLFKKEKINTE GHSSGSSIGSSQSSQSSQSSQSSQSSQMS YSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPE DLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISH FERSKQVSSVNEEDVRLKQQISDHISQSCGSGQMK MFQEVSAADAFGPGTEGQVERFETVGMEAATDEGM PKSYLPQTVRQGGYMPQRIARLEEKVKTLKAQNSEL ASTANMLREQVAQLKQKVMN 22 SPAQBEND10cysGP130_TM_ ICD-L com sequência líder MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGAIVVLPGFLNKLVKFLKVKWKWKVLL VPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTD VSVVEIEANDKKPPPEDLKSLDLFKKEKINTEGHSSGI GGSSCMSSSRPSISSSDENESQNTSSTVQYSTVH SGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD HVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQ VSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVS AADAFGPGTEGQVERFETVGMEAATDEGMPKSYLP QTVRQGGYMPQRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN 23 SPA- QBEND10- cys- MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIK KHRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQ Petition 870250081011, dated 09 / 09 / 2025, pages 158 / 230 134 / 166 GP130_TM_ L-ICD with leader sequence KVMNIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERP EDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDIS HFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQM KMFQEVSAADAFGPGTEGQVERFETVGMEAATDEG MPKSYLPQTVRQGGYMPQ 24 SPAQBEND10GP130_TM_ L-ICD with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHRIAR LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS YLPQTVRQGGYMPQ 25 SPAQBEND10FACDCD8a_TMD_ GP130_ICD with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSF ACDIYIWAPLAGTCGVLLLSLVITLYCNKRDLIKKHIWP NVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFT DVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVH SGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD HVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQ VSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVS AADAFGPGTEGQVERFETVGMEAATDEGMPKSYLP QTVRQGGYMPQ Petition 870250081011, dated 09 / 09 / 2025, pages 159 / 230 135 / 166 26 SPAQBEND10CD8a_hinge TMD_GP130 _ICD with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVST TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRDLIKK HIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSD GNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEG HSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQY STVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPED LQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHF ERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKM FQEVSAADAFGPGTEGQVERFETVGMEAATDEGMP KSYLPQTVRQGGYMPQ 27 SPAQBEND10cysCD8a_TMD_ GP130_ICD with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGIYIWAPLAGTCGVLLLSLVITLYCNKRDLIKKHIW PNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGN FTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTV VHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 28 SPAQBEND10cys- CD8a_TMD_ GP130_ICDL with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGIYIWAPLAGTCGVLLLSLVITLYCNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGN FTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTV VHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE Petition 870250081011, dated 09 / 09 / 2025, pages 160 / 230 136 / 166 VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQRIARLEEKVKTLKAQNSELASTAN MLREQVAQLKQKVMN 29 SPAQBEND10cys-LCD8a_TMD_ GP130_ICD with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNIYIWAPLAGTCGVLLLSLVITLYCNKRDLIKKH IWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS YLPQTVRQGGYMPQ 30 SPA-C7gp130 with leading sequence MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDN NGTATPELPTQGTFSNVSTNVSYQE IIIPSTLGSTSL HPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSV QSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLST TSTS LATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQ GICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADAD AGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLM KKHQSDLKKLGILDFTEQDVASHQSYSQKTPILLTCPT ISILSFFSVALLVILACVLWNKRDLIKKHIWPNVPDPSK SHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEI EANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAF Petition 870250081011, dated 09 / 09 / 2025, pages 161 / 230 137 / 166 GPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQ 31 SPA-CD34long-gp130 with leader sequence MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDN NGTATPELPTQGTFSNVSTNVSYQE IIIPSTLGSTSL HPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSV QSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLST TSTS LATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQ GICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADAD AGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLM KKHQSDLKKLGILDFTEQDVASHQSYSQKTAQGEIEAI VVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPS KSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVE IEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSC MSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAF GPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQ 32 SPA-CD34gp130_ICD with leader sequence MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDN NGTATPELPTQGTFSNVSTNVSYQE IIIPSTLGSTSL HPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSV QSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLST TSTS LATSPTKPYTSSSP ILSDIKAE IKCSGIREVKLTQ GICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLM KKHQSDLKKLGILDFTEQDVASHQSYSQKTAIVVPVC LAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK Petition 870250081011, dated 09 / 09 / 2025, pages 162 / 230 138 / 166 KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSV QVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILP RQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFV RLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTE GQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMP Q 33 SPAQBEND10VASPGP130_ICD with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSP SSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQEL RKRGSPAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKIN TEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERP EDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDIS HFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQM KMFQEVSAADAFGPGTEGQVERFETVGMEAATDEG MPKSYLPQTVRQGGYMPQ 34 SPAQBEND10GP130_ICDVASP with leading sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSA QGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWP NVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFT DVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSG IGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVH SGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD HVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVS AADAFGPGTEGQVERFETVGMEAATDEGMPKSYLP QTVRQGGYMPQPSSSDYSDLQRVKQELLEEVKKEL QKVKEEIIEAFVQELRKRGSP Petition 870250081011, dated 09 / 09 / 2025, pages 163 / 230 139 / 166 35 SPAQBEND10cys-VASPGP130_ICD with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGPSSSDYSDLQRVKQELLEEVKKELQKVKEEIIE AFVQELRKRGSPAQGEIEAIVVPVCLAFLLTTLLGVLF CFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNF NSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDL FKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESS QNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPL LDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQH ESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQS CGSGQMKMFQEVSAADAFGPGTEGQVERFETVGME AATDEGMPKSYLPQTVRQGGYMPQ 36 SPAQBEND10cysGP130_ICD- VASP with leader sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIK KHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYS DGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTE GHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQ YSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPE DLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISH FERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMK MFQEVSAADAFGPGTEGQVERFETVGMEAATDEGM PKSYLPQTVRQGGYMPQPSSSDYSDLQRVKQELLEE VKKELQKVKEEIIEAFVQELRKRGSP 37 SPA-CD34- TpoR-gp130 MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDN NGTATPELPTQGTFSNVSTNVSYQEIIIPSTLGSTSL HPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSV QSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLST TSTS LATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQ GICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADAD AGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLM Petition 870250081011, dated 09 / 09 / 2025, pages 164 / 230 140 / 166 KKHQSDLKKLGILDFTEQDVASHQSYSQKTISLVTALL LVLGLNAVLGLLLLRKQFPAHYRRLRHAIWPNVPDPS KSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVE IEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSC MSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAF GPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQ 38 SPA-TpoRgp130_ICD with leader sequence MALPVTALLLPLALLLHAARPSDPTRVETATETAWISL VTALLLVLGLNAVLGLLLLRKQFPAHYRRLRHAIWPNV PDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDV SVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIG GSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHS GYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDH VDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQV SSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSA ADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQ TVRQGGYMPQ 39 SPA-EpoRgp130_ICD with leader sequence MDHLGASLWPQVGSLCLLLAGAAWAPPPNLPDPKFE SKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVG PGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCS LPTADTSSFVPLELRVTAASGAPRYHRVIHINEVVLLD APVGLVACLADESGHVVLRWLPPPETPMTSHIRYEV DVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSLDLDPLILTL SLILVVILVLLTVLALLSNKRDLIKKHIWPNVPDPSKSHI AQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEAN Petition 870250081011, dated 09 / 09 / 2025, pages 165 / 230 141 / 166 DKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSS SRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVP SVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGIL PRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDF VRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGT EGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYM PQ 40 SPA-EpoRgp130 with leading sequence MDHLGASLWPQVGSLCLLLAGAAWAPPPNLPDPKFE SKAALLAARGPEELLCFTERLEDLVCFWEEAASAGVG PGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCS LPTADTSSFVPLELRVTAASGAPRYHRVIHINEVVLLD APVGLVACLADESGHVVLRWLPPPETPMTSHIRYEV DVSAGNGAGSVQRVEILEGRTECVLSNLRGRTRYTF AVRARMAEPSFGGFWSAWSEPVSLLTPSDLDPAIVV PVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKS HIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIE ANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCM SSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQ VPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGD GILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNE EDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFG PGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQG GYMPQ 41 SPAQBEND10EpoRgp130_ICD with sequence MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSA PPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVC FWE EAASAGVGPGNYSFSYQLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPRY HRVIHINEVVLLDAPVGLVACLADESGHVVLRWLPPP ETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECVL Petição 870250081011, de 09 / 09 / 2025, pág. 166 / 230 142 / 166 líder SNLRGRTRYTFAVRARMAE PSFGG FWSAWS EPVSLL TPSDLDPLILTLSLILVVILVLLTVLALLSNKRDLIKKHIW PNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGN FTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTV VHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 42 SPAQBEND10EpoR-gp130 com sequência líder MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSA PPPNLPDPKFESKAALLAARGPEELLCFTERLEDLVC FWE EAASAGVGPGNYSFSYQLEDEPWKLCRLHQAP TARGAVRFWCSLPTADTSSFVPLELRVTAASGAPRY HRVIHINEVVLLDAPVGLVACLADESGHVVLRWLPPP ETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECVL SNLRGRTRYTFAVRARMAE PSFGG FWSAWS EPVSLL TPSDLDPAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS YLPQTVRQGGYMPQ 43SPAMyr(Src)-Lgp130 MGSSKSKPKDPSQRRRRIARLEEKVKTLKAQNSELA STANMLREQVAQLKQKVMNNKRDLIKKHIWPNVPDP SKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVV Petition 870250081011, dated 09 / 09 / 2025, pp. 167 / 230 143 / 166 EIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSS CMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYR HQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDG GDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSV NEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADA FGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVR QGGYMPQ 44 SPA- Myr(Src)gp130-L MGSSKSKPKDPSQRRRNKRDLIKHIWPNVPDPSKS HIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIE ANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCM SSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQ VPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGD GILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNE EDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFG PGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQG GYMPQRIARLEEKVKTLKAQNSELASTANMLREQVA QLKQKVMN 45 SPAMyr(Fyn)-Lgp130 MGCVQCKDKEATKLTERIARLEEKKVKTLKAQNSELAS TANMLREQVAQLKQKVMNNKRDLIKKKHIWPNVPDPS KSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVE IEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSC MSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAF GPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQ 46 SPA- Myr(Fyn)-MGCVQCKDKEATKLTENKRDLIKKHIWPNVPDPSKSH IAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEA Petition 870250081011, dated 09 / 09 / 2025, pp. 168 / 230 144 / 166 gp130-L NDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMS SSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQV PSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDG ILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEED FVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPG TEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGY MPQRIARLEEKVKTLKAQNSELASTANMLREQVAQLK QKVMN 47 SPA- Myr(Lck)gp130-L MGCGCSSHPEDDWMENNKRDLIKKHIWPNVPDPSK SHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEI EANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSC MSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAF GPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQRIARLEEKVKTLKAQNSELASTANMLREQV AQLKQKVMN 48 SPAMyr(Lck)-Lgp130 MGCGCSSHPEDDWMENRIARLEEKVKTLKAQNSELA STANMLREQVAQLKQKVMNNKRDLIKKHIWPNVPDP SKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVV EIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSS CMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYR HQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDG GDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSV NEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADA FGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 49 SPA- MGSSKSKPKDPSQRRRPSSSDYSDLQRVKQELLEEV Petition 870250081011, dated 09 / 09 / 2025, pages 169 / 230 145 / 166 Myr(Src)VASP-gp130 ICD KKELQKVKEEIIEAFVQELRKRGSPGSNKRDLIKKHIW PNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGN FTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTV VHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 50 SPAMyr(Src)gp130 ICDVASP MGSSKSKPKDPSQRRRNKRDLIKKHIWPNVPDPSKS HIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIE ANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCM SSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQ VPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGD GILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNE EDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFG PGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQG GYMPQGSPSSSDYSDLQRVKQELLEEVKKELQKVKE EIIEAFVQELRKRGSP 51 Myr(Src)-Lgp130 MGSSKSKPKDPSQRRRRIARLEEKVKTLKAQNSELA STANMLREQVAQLKQKVMNNKRDLIKKHIWPNVPDP SKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVV EIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSS CMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYR HQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDG GDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADA FGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVR QGGYMPQ Petition 870250081011, dated 09 / 09 / 2025, pages 170 / 230 146 / 166 52 Myr(Src)gp130-L MGSSKSKPKDPSQRRRNKRDLIKKHIWPNVPDPSKS HIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIE ANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCM SSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQ VPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGD GILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNE EDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFG PGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQG GYMPQRIARLEEKVKTLKAQNSELASTANMLREQVA QLKQKVMN 53 Myr(Fyn)gp130-L MGCVQCKDKEATKLTENKRDLIKKHIWPNVPDPSKSH IAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEA NDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMS SSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQV PSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDG ILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEED FVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPG TEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGY MPQRIARLEEKVKTLKAQNSELASTANMLREQVAQLK QKVMN 54 Myr(Lck)gp130-L MGCGCSSHPEDDWMENNKRDLIKKHIWPNVPDPSK SHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEI EANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSC MSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQRIARLEEKVKTLKAQNSELASTANMLREQV Petition 870250081011, dated 09 / 09 / 2025, pages 171 / 230 147 / 166 AQLKQKVMN 55 Myr(Lck)-Lgp130 MGCGCSSHPEDDWMENRIARLEEKVKTLKAQNSELA STANMLREQVAQLKQKVMNNKRDLIKKHIWPNVPDP SKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVV EIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSS CMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYR HQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDG GDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSV NEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADA FGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVR QGGYMPQ 56 Myr(Src)VASP-gp130 ICD MGSSKSKPKDPSQRRRPSSSDYSDLQRVKQELLEEV KKELQKVKEEIIEAFVQELRKRGSPGSNKRDLIKKHIW PNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGN FTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTV VHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 57 (CAR-SPA) QBEND10Cys-LGP130 induzível com sequência líder MALPVTALLLPLALLLHAARPELPTQGTFSNVSTNVSE LCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRD LIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTV QYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERP EDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDIS Petition 870250081011, dated 09 / 09 / 2025, pages 172 / 230 148 / 166 HFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQM KMFQEVSAADAFGPGTEGQVERFETVGMEAATDEG MPKSYLPQTVRQGGYMPQ 58 myr(Src)- JUN-gp130 MGSSKSKPKDPSQRRRRIARLEEKVKTLKAQNSELA STANMLREQVAQLKQKVMNNKRDLIKKHIWPNVPDP SKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVV EIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSS CMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYR HQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDG GDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSV NEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADA FGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVR QGGYMPQ 59 Gp130 (IL6ST) UniProt P40189 MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVV QLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIP KEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNV YGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRET HLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTV YFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPH NLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDA STWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMK EDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPS HTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKS HLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKS DAA VLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRE SVKKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKG PTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYR TIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTD Petition 870250081011, dated 09 / 09 / 2025, pages 173 / 230 149 / 166 EGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLL GVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPP RHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDL KSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQ 60 Intracellular signaling domain Gp130 (IL6ST) NKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNS KDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFK KEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQN TSSTVQYSTVVHSGYRHQVP SVQVFS RSESTQPLLD SEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHES SPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCG SGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAA TDEGMPKSYLPQTVRQGGYMPQ 61 Gp130 transmembrane domain (IL6ST) AIVVPVCLAFLLTTLLGVLFCF 62 L-gp130 MALPVTALLLPLALLLHAARPDYKDDDDKELCGGRIA RLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN AQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIW PNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGN FTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL Petition 870250081011, dated 09 / 09 / 2025, pages 174 / 230 150 / 166 VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERS KQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQE VSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYL PQTVRQGGYMPQ 63 QBEND10Cys-LGP130Y759 F ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQFSTVVHSGYRHQVPSV QVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILP RQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFV RLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTE GQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMP Q 64 QBEND10Cys-LGP130Y759 ΕΔ771-811 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQFSTVVHSGYRHQQQYF KQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQI SDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVER FETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 65 QBEND10Cys-LGP130Δ771811 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR Petition 870250081011, dated 09 / 09 / 2025, pages 175 / 230 151 / 166 PSISSSDENESSQNTSSTVQYSTVVHSGYRHQQQYF KQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQI SDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVER FETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 66 QBEND10- L-GP130 ELPTQGTFSNVSTNVSRIARLEEKVKTLKAQNSELAS TANMLREQVAQLKQKVMNAQGEIEAIVVPVCLAFLLT TLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHT PPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPE DLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSS DENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRS ESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFK QNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQIS DHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERF ETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 67 QBEND10- L- GP130Y759 F ELPTQGTFSNVSTNVSRIARLEEKVKTLKAQNSELAS TANMLREQVAQLKQKVMNAQGEIEAIVVPVCLAFLLT TLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHT PPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPE DLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSS DENESSQNTSSTVQFSTVVHSGYRHQVPSVQVFSRS ESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFK QNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQIS DHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERF ETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 68 QBEND10- L- GP130Y759 ΕΔ771-811ELPTQGTFSNVSTNVSRIARLEEKVKTLKAQNSELAS TANMLREQVAQLKQKVMNAQGEIEAIVVPVCLAFLLT TLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHT PPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPE DLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSS Petition 870250081011, dated 09 / 09 / 2025, pp. 176 / 230 152 / 166 DENESSQNTSSTVQFSTVVHSGYRHQQQYFKQNCS QHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHIS QSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVG MEAATDEGMPKSYLPQTVRQGGYMPQ 69 QBEND10- L- GP130Δ771- 811 ELPTQGTFSNVSTNVSRIARLEEKVKTLKAQNSELAS TANMLREQVAQLKQKVMNAQGEIEAIVVPVCLAFLLT TLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHT PPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPE DLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSS DENESSQNTSSTVQYSTVVHSGYRHQQQYFKQNCS QHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHIS QSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVG MEAATDEGMPKSYLPQTVRQGGYMPQ 70 SPAQBEND10Cys-LGP130 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSV QVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILP RQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFV RLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTE GQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMP Q 71 SPAQBEND10Cys-LGP130_ICD _trunc1 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNAQGEIEAIVVPPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPETVQYSTVVHSGYRHQVPSVQVFSRSESTQPL Petition 870250081011, dated 09 / 09 / 2025, pp. 177 / 230 153 / 166 LDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQH ESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQS CGSGQMKMFQEVSAADAFGPGTEGQVERFETVGME AATDEGMPKSYLPQTVRQGGYMPQ 72 SPAQBEND10Cys-LGP130Δ771811(ICD_tru nc2) ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQYSTVVHSGYRHQQQYF KQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQI SDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVER FETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 73 SPAQBEND10Cys-LGP130_ICD _trunc3 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSV QVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILP RQQYFKQPKSYLPQTVRQGGYMPQ 74 SPAQBEND10Cys-LGP130_ICD _trunc4 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPETVQYSTVVHSGYRHQQQYFKQNCSQHESSP DISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSG QMKMFQEVSAADAFGPGTEGQVERFETVGMEAATD EGMPKSYLPQTVRQGGYMPQ Petition 870250081011, dated 09 / 09 / 2025, pp. 178 / 230 154 / 166 75 SPAQBEND10Cys-LGP130_ICD _trunc5 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQYSTVVHSGYRHQQQYF KQPKSYLPQTVRQGGYMPQ 76 SPAQBEND10Cys-LGP130_ICD _trunc6 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPETVQYSTVVHSGYRHQVPSVQVFSRSESTQPL LDSEERPEDLQLVDHVDGGDGILPRQQYFKQPKSYL PQTVRQGGYMPQ 77 SPAQBEND10Cys-LGP130_ICD _trunc7 ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPETVQYSTVVHSGYRHQQQYFKQPKSYLPQTVR QGGYMPQ 78 SPA- QBEND10- L-GP130 ELPTQGTFSNVSTNVSRIARLEEKVKTLKAQNSELAS TANMLREQVAQLKQKVMNAQGEIEAIVVPVCLAFLLT TLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHT PPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSS DENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRS ESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFK QNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQIS DHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERF Petition 870250081011, dated 09 / 09 / 2025, pages 179 / 230 155 / 166 ETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 79 SPAQBEND10BCR-GP130 ELPTQGTFSNVSTNVSMVDPVGFAEAWKAQFPDSEP PRMELRSVGDIEQELERCKASIRRLEQEVNQERFRMI YLQTLLAKEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKK HIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSD GNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEG HSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQY STVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPED LQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHF ERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKM FQEVSAADAFGPGTEGQVERFETVGMEAATDEGMP KSYLPQTVRQGGYMPQ 80 SPAQBEND10cys-GP130 ELPTQGTFSNVSTNVSELCGGAQGEIEAIVVPVCLAFL LTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSP HTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPF PEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSIS SSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFS RSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQY FKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQ QISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVE RFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 81 SPAQBEND10cysGP130_TM_ ICD ELPTQGTFSNVSTNVSELCGGAIVVPVCLAFLLTTLLG VLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPR HNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLK SLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSES TQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQN CSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDH ISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFET Petition 870250081011, dated 09 / 09 / 2025, pages 180 / 230 156 / 166 VGMEAATDEGMPKSYLPQTVRQGGYMPQ 82 SPAQBEND10cys-GP130-L ELPTQGTFSNVSTNVSELCGGAQGEIEAIVVPVCLAFL LTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSP HTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPF PEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSIS SSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFS RSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQY FKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQ QISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVE RFETVGMEAATDEGMPKSYLPQTVRQGGYMPQRIA RLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN 83 SPAQBEND10cysGP130_TM_ ICD-L ELPTQGTFSNVSTNVSELCGGAIVVPVCLAFLLTTLLG VLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPR HNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLK SLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDE NESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSES TQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQN CSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDH ISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFET VGMEAATDEGMPKSYLPQTVRQGGYMPQRIARLEEK VKTLKAQNSELASTANMLREQVAQLKQKVMN 84 SPAQBEND10cysGP130_TM_ L-ICD ELPTQGTFSNVSTNVSELCGGAQGEIEAIVVPVCLAFL LTTLLGVLFCFNKRDLIKKHRIARLEEKVKTLKAQNSEL ASTANMLREQVAQLKQKVMNIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKK PFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRP SISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQ VFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPR QQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVR Petition 870250081011, dated 09 / 09 / 2025, pages 181 / 230 157 / 166 LKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEG QVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 85 SPAQBEND10GP130_TM_ L-ICD ELPTQGTFSNVSTNVSELCGGAIVVPVCLAFLLTTLLG VLFCFNKRDLIKKHRIARLEEKVKTLKAQNSELASTAN MLREQVAQLKQKVMNIWPNVPDPSKSHIAQWSPHTP PRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPED LKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQ 86 SPAQBEND10FACDCD8a_TMD_ GP130_ICD ELPTQGTFSNVSTNVSFACDIYIWAPLAGTCGVLLLSL VITLYCNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPR HNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLK SLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDE NESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSES TQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQN CSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDH ISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFET VGMEAATDEGMPKSYLPQTVRQGGYMPQ 87 SPAQBEND10CD8a_hinge TMD_GP130 _ICD ELPTQGTFSNVSTNVS IIIPAPRPPTPAPTIASQPLSL RPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFP EDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISS SDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSR SESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYF KQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQI Petition 870250081011, dated 09 / 09 / 2025, pages 182 / 230 158 / 166 SDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVER FETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 88 SPAQBEND10- cys- CD8a_TMD_ GP130_ICD ELPTQGTFSNVSTNVSELCGGIYIWAPLAGTCGVLLL SLVITLYCNKRDLIKKHIWPNVPDPSKSHIAQWSPHTP PRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPED LKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQ 89 SPAQBEND10cys- CD8a_TMD_ GP130_ICDL ELPTQGTFSNVSTNVSELCGGIYIWAPLAGTCGVLLL SLVITLYCNKRDLIKKHIWPNVPDPSKSHIAQWSPHTP PRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPED LKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQRIARLEE KVKTLKAQNSELASTANMLREQVAQLKQKVMN 90 SPAQBEND10cys-LCD8a_TMD_ GP130_ICD ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNIYIWAPLAGTCGVLLLSLVITLYCNKRDLIKKHIWPNVPDPSKSHIAQWSPH TPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFP EDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISS SDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSR SESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYF KQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQI Petition 870250081011, dated 09 / 09 / 2025, pages 183 / 230 159 / 166 SDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVER FETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 91 SPA-C7gpi30 SLDNNGTATPELPTQGTFSNVSTNVSYQE 1 IIPSTLG STSLHPVSQHGNEATTNITETTVKFTSVITSVYGNT NSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNV SDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIRE VKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEE QADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISS KLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTPI LLTCPTISILSFFSVALLVILACVLWNKRDLIKKHIWPNV PDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDV SVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIG GSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHS GYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDH VDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQV SSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSA ADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQ TVRQGGYMPQ 92 SPA-CD34long-gp130 SLDNNGTATPELPTQGTFSNVSTNVSYQE t HPSTLG STSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNT NSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNV SDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIRE VKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEE QADADAGAQVCSLLLAQSEVRPQCLLVLANRTEISS KLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTA QGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFT DVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSG IGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVH Petition 870250081011, dated 09 / 09 / 2025, pages 184 / 230 160 / 166 SGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD HVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQ VSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVS AADAFGPGTEGQVERFETVGMEAATDEGMPKSYLP QTVRQGGYMPQ 93 SPA-CD34gp130_ICD LDNNGTATPELPTQGTFSNVSTNVSYQE IIIPSTLGS TSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTN SSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVS DLSTTSTLATSPTKPYTSSSPILSDIKAEIKCSGIREV KLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQ ADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSK LQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTAIV VPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSK SHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEI EANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSC MSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAF GPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQ 94 SPAQBEND10- VASPGP130_ICD ELPTQGTFSNVSTNVSPSSDYSDLQRVKQELLEEVK KELQKVKEEIIEAFVQELRKRGSPAQGEIEAIVVPVCL AFLLTTLLGVLFCFNRKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSV QVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILP RQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFV Petition 870250081011, dated 09 / 09 / 2025, pages 185 / 230 161 / 166 RLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTE GQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMP Q 95 SPAQBEND10GP130_ICDVASP ELPTQGTFSNVSTNVSAQGEIEAIVVPVCLAFLLTTLL GVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPP RHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDL KSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQPSSSDY SDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGS P 96 SPAQBEND10cys-VASPGP130_ICD ELPTQGTFSNVSTNVSELCGGPSSSDYSDLQRVKQE LLEEVKKELQKVKEEIIEAFVQELRKRGSPAQGEIEAIV VPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSK SHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEI EANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSC MSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRH QVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGG DGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVN EEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAF GPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQ GGYMPQ 97 SPAQBEND10- cysGP130_ICD- ELPTQGTFSNVSTNVSELCGGAQGEIEAIVVPVCLAFL LTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPF PEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSRPSIS Petition 870250081011, dated 09 / 09 / 2025, pages 186 / 230 162 / 166 VASP SSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFS RSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQY FKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQ QISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVE RFETVGMEAATDEGMPKSYLPQTVRQGGYMPQPSS SDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRK RGSP 98 SPA-CD34- TpoR-gp130 SLDNNGTATPELPTQGTFSNVSTNVSYQE 1 IIPSTLG STSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNT NSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNV SDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIRE VKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEE QADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISS KLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTIS LVTALLLVLGLNAVLGLLLLRKQFPAHYRRLRHAIWPN VPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTD VSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGI GGSSCMSSSRPSISSSDENESSQNTSSTVXYSTVVH SGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD HVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQ VSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVS AADAFGPGTEGQVERFETVGMEAATDEGMPKSYLP QTVRQGGYMPQ 99 SPA-TpoRgp130_ICD SDPTRVETATETAWISLVTALLLVLGLNAVLGLLLLRK QFPAHYRRLRHAIWPNVPDSKSHIAQWSPHTPPRH NFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSRPSISSSDENE SSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQ PLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCS Petition 870250081011, dated 09 / 09 / 2025, pages 187 / 230 163 / 166 QHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHIS QSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVG MEAATDEGMPKSYLPQTVRQGGYMPQ 100 SPA-EpoRgp130_ICD APPPNLPDPKFESKAALLAARGPEELLCFTERLEDLV CFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQA PTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPR YHRVIHINEVVLLDAPVGLVACLADESGHVVLRWLPP PETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECV LSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSL LTPSDLDPLILTLSLILVVILVLLTVLALLSNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDG NFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS YLPQTVRQGGYMPQ 101 SPA-EpoRgp130 APPPNLPDPKFESKAALLAARGPEELLCFTERLEDLV CFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQA PTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPR YHRVIHINEVVLLDAPVGLVACLADESGHVVLRWLPP PETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECV LSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSL LTPSDLDPAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHI WPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGH SSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYST VVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQ Petition 870250081011, dated 09 / 09 / 2025, pages 188 / 230 164 / 166 LVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFER SKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQ EVSAADAFGPGTEGQVERFETVGMEAATDEGMPKS YLPQTVRQGGYMPQ 102 SPA- QBEND10- EpoRgp130_ICD ELPTQGTFSNVSTNVSAPPPNLPDPKFESKAALLAAR GPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSY QLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSF VPLELRVTAASGAPRYHRVIHINEVVLLDAPVGLVACL ADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAG SVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPS FGGFWSAWSEPVSLLTPSDLDPLILTLSLILVVILVLLT VLALLSNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPP RHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDL KSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQ 103 SPAQBEND10EpoR-gp130 ELPTQGTFSNVSTNVSAPPPNLPDPKFESKAALLAAR GPEELLCFTERLEDLVCFWEEAASAGVGPGNYSFSY QLEDEPWKLCRLHQAPTARGAVRFWCSLPTADTSSF VPLELRVTAASGAPRYHRVIHINEVVLLDAPVGLVACL ADESGHVVLRWLPPPETPMTSHIRYEVDVSAGNGAG SVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSLLTPSDLDPAIVVPVCLAFLLTTTLL GVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPP RHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDL KSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD Petition 870250081011, dated 09 / 09 / 2025, pages 189 / 230 165 / 166 ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQ 104 (CAR-SPA) QBEND10Cys-LGP130 induzível ELPTQGTFSNVSTNVSELCGGRIARLEEKVKTLKAQN SELASTANMLREQVAQLKQKVMNAQGEIEAIVVPVCL AFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQ WSPHTPPRHNFNSKDQMYSDGNFTDVSVVEIEANDK KPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSR PSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSV QVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILP RQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFV RLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTE GQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMP Q 105 L-gp130 DYKDDDDKELCGGRIARLEEKVKTLKAQNSELASTAN MLREQVAQLKQKVMNAQGEIEAIVVPVCLAFLLTTLL GVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPP RHNFNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDL KSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSD ENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSE STQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQ NCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISD HISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFE TVGMEAATDEGMPKSYLPQTVRQGGYMPQ 238 QBEND10 (epítopoCD34) ELPTQGTFSNVSTNVS Petition 870250081011, dated 09 / 09 / 2025, pages 190 / 230 166 / 166 239 BCR Domain MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELE RCKASIRRLEQEVNQERFRMIYLQTLLAKE 240 VASP Domain PSSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQE LRKRGSPAQGEIE 241 Cysteine-mutated EpoR Domain APPPNLPDPKFESKAALLAARGPEELLCFTERLEDLV CFWEEAASAGVGPGNYSFSYQLEDEPWKLCRLHQA PTARGAVRFWCSLPTADTSSFVPLELRVTAASGAPR YHRVIHINEVVLLDAPVGLVACLADESGHVVLRWLPP PETPMTSHIRYEVDVSAGNGAGSVQRVEILEGRTECV LSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVSL LTPSDLDP 242 CD34 Ectodomain SLDNNGTATPELPTQGTFSNVSTNVSYQE 1 IIPSTLG STSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNT NSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNV SDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIRE VKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEE QADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISS KLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT 243 Domain TpoR ISLVTALLLVLGLNAVLGLLLL

Claims

Claims 1. Synthetic pathway activator peptide (SPA) characterized in that it comprises a chimeric polypeptide comprising: a. optionally, an extracellular domain; b. a lipid anchor or a transmembrane domain; c. an intracellular signaling domain; and d. a multimerization region.

2. SPA peptide, according to claim 1, wherein the SPA peptide is characterized in that it comprises, from the N-terminus to the C-terminus, an extracellular domain comprising a CD34 epitope, a multimerization region comprising an unpaired cysteine ​​residue, a gp130 transmembrane domain and a gp130 intracellular signaling domain; wherein a. the SPA sequence comprises a sequence presented in SEQ ID NO: 81 or 20; or b. multimerization of the chimeric polypeptide through the multimerization region results in the constitutive activity of the intracellular signaling domain.

3. SPA peptide, according to claim 1 or 2, characterized in that the multimerization region comprises at least one unpaired cysteine ​​residue, a leucine zipper, a BCR domain and a VASP domain.

4. SPA peptide, according to claim 3, characterized in that the multimerization region comprises at least one unpaired cysteine ​​residue.

5. SPA peptide, according to claim 3 or 4, characterized in that the multimerization region comprises at least one unpaired cysteine ​​residue and a leucine zipper.

6. SPA peptide, according to any one of claims 1 - 5, characterized in that the multimerization region is intracellular Petition 870250081011, dated 09 / 09 / 2025, page 192 / 230 2 / 11 when expressed by a cell.

7. SPA peptide, according to any one of claims 1-5, characterized in that the multimerization region is extracellular when expressed by a cell.

8. SPA peptide, according to any one of claims 1-7, characterized in that the intracellular signaling domain induces phosphorylation of at least one of STAT1, STAT3, and STAT5.

9. SPA peptide, according to any one of claims 1-8, characterized in that the intracellular signaling domain comprises a peptide motif of type I cytokine receptor superfamily box1 (IWPNVDP (SEQ ID NO: 106)) or box2 (VSVVEIEANDKKP (SEQ ID NO: 107)).

10. SPA peptide, according to any one of claims 1 and 9, characterized in that the intracellular signaling domain comprises a tyrosine phosphorylation motif comprising YXXQ or YXPQ.

11. SPA peptide, according to any one of claims 110, characterized in that the intracellular signaling domain comprises a polypeptide sequence of an interleukin receptor.

12. SPA peptide, according to any one of claims 1 11, characterized in that the interleukin receptor comprises an intracellular signaling domain gp130.

13. SPA peptide, according to any one of claims 1 12, characterized in that the intracellular signaling domain comprises a polypeptide sequence comprising amino acids 642 to 918 of gp130 (SEQ ID NO: 59).

14. SPA peptide, according to any one of claims 1-13, characterized in that the intracellular signaling domain comprises a polypeptide sequence comprising the sequence presented as SEQ ID NO:

60. Petition 870250081011, dated 09 / 09 / 2025, pp. 193 / 230 3 / 11 15. SPA peptide, according to any one of claims 1 12, characterized in that the interleukin receptor comprises a truncated gp130 intracellular signaling domain.

16. SPA peptide, according to claim 15, characterized in that the truncated intracellular signaling domain gp130 comprises a deletion of amino acids 771 to 811 of gp130 (SEQ ID NO: 59).

17. SPA peptide, according to claim 15, characterized in that the truncated intracellular signaling domain gp130 comprises a deletion of amino acids 707-755, 771-811 or 818-901 of gp130 (SEQ ID NO: 59).

18. SPA peptide, according to claim 15, characterized in that the truncated intracellular gp130 signaling domain comprises the truncated intracellular gp130 domain of a sequence selected from the group presented in SEQ IDs 10-16 and 71-77.

19. SPA peptide, according to any of claims 1 18, characterized in that the intracellular signaling domain gp130 additionally comprises a Y759F mutation of gp130 (SEQ ID NO: 59).

20. SPA peptide, according to any one of claims 1 19, characterized in that the intracellular signaling domain additionally comprises a prenylation motif at the C-terminus.

21. SPA peptide, according to any one of claims 120, characterized in that the lipid anchor or a transmembrane domain comprises a gp130 transmembrane domain, a CD8-alpha transmembrane domain, a prenylation motif or a myristoylation domain derived from src, fyn or lck.

22. SPA peptide, according to claim 21, characterized in that the transmembrane domain comprises a gp130 transmembrane domain.

23. SPA peptide, according to claim 21 or 22, Petition 870250081011, dated 09 / 09 / 2025, pp. 194 / 230 4 / 11, characterized in that the transmembrane domain comprises a polypeptide sequence comprising amino acids 620 to 641 of gp130 (SEQ ID NO: 59).

24. SPA peptide, according to any one of claims 1 23, characterized in that the transmembrane domain comprises a polypeptide sequence comprising the sequence presented as SEQ ID NO:

61.

25. SPA peptide, according to any one of claims 1 24, characterized in that it further comprises a CD8-alpha hinge domain.

26. SPA peptide, according to any of claims 1 25, characterized in that the extracellular domain comprises one or more of a CD34 epitope (e.g., a QBEND10 epitope), a CD34 ectodomain, a BCR ectodomain, a thrombopoietin receptor (TpoR) ectodomain, or an erythropoietin receptor (EpoR) ectodomain.

27. SPA peptide, according to claim 26, characterized in that the thrombopoietin receptor (TpoR) domain or erythropoietin receptor (EpoR) domain comprises an unpaired cysteine.

28. SPA peptide, according to any one of claims 1 27, characterized in that the extracellular domain transmits constitutive activity to the intracellular signaling domain.

29. SPA peptide, according to claim 1, characterized in that it comprises, from the N-terminus to the C-terminus, an extracellular domain comprising a CD34 epitope, a multimerization region comprising an unpaired cysteine ​​residue, a gp130 transmembrane domain and a gp130 intracellular signaling domain.

30. SPA peptide, according to any one of claims 1-29, wherein the SPA peptide is characterized in that it comprises a sequence selected from the sequences presented in SEQ ID Nos: 158 or 63-104. Petition 870250081011, dated 09 / 09 / 2025, pp. 195 / 230 5 / 11 31. SPA peptide, according to claim 30, characterized in that the SPA sequence comprises a sequence presented in SEQ ID NO:

20.

32. SPA peptide, according to claim 30, characterized in that the SPA peptide comprises a sequence presented in SEQ ID NO:

81.

33. Multimer characterized in that it is of the SPA peptide, as defined in any one of claims 1-32.

34. Nucleic acid characterized in that it encodes an SPA peptide, as defined in any one of claims 1-32.

35. Vector characterized in that it comprises the nucleic acid as defined in claim 34.

36. System characterized in that it comprises: a. a first chimeric polypeptide comprising an initiation receptor; b. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR); and c. the SPA peptide, as defined in any one of claims 1-32.

37. System according to claim 36, characterized in that binding to the CAR or initiation receptor induces the expression of the SPA peptide.

38. System according to claim 36, characterized in that the SPA peptide is expressed constitutively or inducibly.

39. A cell or population of cells characterized in that it comprises the SPA peptide, as defined in any one of claims 1-32, the multimer, as defined in claim 33, the nucleic acid, as defined in claim 34, the vector, as defined in claim 35, or the system, as defined in any one of claims 36-38. Petition 870250081011, dated 09 / 09 / 2025, pp. 196 / 230 6 / 11 40. Cell according to claim 39, wherein the cell is characterized in that it is an immune cell, optionally wherein the immune cell is a primary human immune cell.

41. A pharmaceutical composition characterized in that it comprises the cell or population of cells, as defined in any one of claims 39 to 40, and a pharmaceutically acceptable excipient.

42. Pharmaceutical composition characterized in that it comprises the nucleic acid, as defined in claim 34, or the vector, as defined in claim 35, and a pharmaceutically acceptable excipient.

43. A method for editing a cell, characterized in that it comprises inserting nucleic acid, as defined in claim 34, into an insertion site in the cell genome.

44. A method for producing a CD11 c+ cell, characterized in that it comprises inserting the nucleic acid, as defined in claim 34, into an insertion site in the cell genome.

45. Method according to claim 43 or 44, characterized in that the nucleic acid is introduced into the cell in a non-viral manner.

46. ​​Method for editing a cell characterized in that it comprises: a. providing a nuclease domain and a guide RNA, wherein the nucleic acid comprises the nucleic acid according to claim 34, and wherein the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the cell genome; b. introducing the nuclease domain and the nucleic acid into the cell, wherein the guide RNA specifically hybridizes with a target region of the cell genome, and wherein the nuclease domain cleaves the target region to create the insertion site in the cell genome; and c. editing the cell by inserting the nucleic acid into the insertion site in the cell genome.

47. Method according to claim 46, characterized in that the nuclease domain and nucleic acid are introduced into the cell in a non-viral manner.

48. A method for editing an immune cell, characterized in that it comprises: a. providing a ribonucleoprotein (RNP)-nucleic acid complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein the nucleic acid comprises the nucleic acid according to claim 34, and wherein the 5' and 3' ends of the nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the immune cell; b. non-virally introducing the RNP-nucleic acid complex into the immune cell, wherein the guide RNA specifically hybridizes with a target region of the genome of the primary immune cell, and wherein the nuclease domain cleaves the target region to create the insertion site in the genome of the immune cell; and c. editing the immune cell by inserting the nucleic acid according to claim 34 into the insertion site in the genome of the immune cell.

49. A method according to any one of claims 45, 47 or 48, characterized in that the nonviral introduction comprises electroporation.

50. Method, according to any one of claims 46-49, characterized in that the nuclease domain comprises a CRISPR-associated endonuclease (Cas), optionally a Cas9 nuclease.

51. Method, according to any one of claims 46 to 50, characterized in that the target region of the cell genome is a T-cell Receptor Alpha Constant (TRAC) locus or a genomic safe harbor (GSH) locus. Petition 870250081011, dated 09 / 09 / 2025, pp. 198 / 230 8 / 11 52. Method, according to any one of claims 46 to 51, characterized in that the target region is the GS94 locus (chr11 locus: 128340000-128350000).

53. A method according to any one of claims 43 to 52, characterized in that the nucleic acid is either a double-stranded nucleic acid or a single-stranded nucleic acid.

54. A method according to any one of claims 43 to 53, characterized in that the nucleic acid is either a linear nucleic acid or a circular nucleic acid, optionally wherein the circular recombinant nucleic acid is a plasmid.

55. Method, according to any one of claims 48 to 54, characterized in that the cell is an immune cell, optionally a primary human immune cell.

56. A method according to any one of claims 48 to 55, characterized in that the immune cell is an autologous immune cell.

57. Method, according to any one of claims 48 to 55, characterized in that the immune cell is an allogeneic immune cell.

58. A method according to any one of claims 48 to 57, characterized in that the immune cell is a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a progenitor T cell.

59. Method, according to any one of claims 48 to 58, characterized in that the immune cell is a primary T cell.

60. A method according to any one of claims 48 to 59, characterized in that the immune cell is a primary human T cell.

61. Method, according to any one of claims 43 to 60, characterized in that the cell is virus-free. Petition 870250081011, dated 09 / 09 / 2025, pp. 199 / 230 9 / 11 62. Method, according to any one of claims 43 to 61, characterized in that it further comprises determining the expression of CD11c in the cell.

63. A method according to any one of claims 43 to 62, characterized in that it further comprises obtaining an immune cell from a patient and introducing nucleic acid in vitro.

64. A method for treating a disease in a subject, characterized in that it comprises administering the cell or population of cells, as defined in any one of claims 39 or 40, or the pharmaceutical composition, as defined in any one of claims 41 or 42, to the subject.

65. Method according to claim 64, characterized in that the disease is cancer.

66. Method according to claim 65, characterized in that the cancer is either a solid cancer or a liquid cancer.

67. Method according to claim 65 or 66, characterized in that the cancer is renal cancer, clear cell renal cell carcinoma (ccRcc), colorectal cancer or lung cancer.

68. Method for inhibiting a target cell in a subject, characterized in that it comprises administering the cell or population of cells, as defined in any one of claims 39 or 40, or the pharmaceutical composition, as defined in any one of claims 41 or 42, to the subject, wherein the cell inhibits the target cell.

69. Method for modulating the activity of an immune cell characterized in that it comprises: a. obtaining a cell or an immune cell comprising i. SPA peptide, as defined in any of claims 1-32; ii. the system, as defined in any of claims 36-38; Petition 870250081011, dated 09 / 09 / 2025, pp. 200 / 230 10 / 11 iii. the nucleic acid, as defined in claim 34; and / or iv. the vector, as defined in claim 35; and b. placing the cell or immune cell in contact with a target cell, wherein the activator of the synthetic pathway modulates the activity of the cell or immune cell.

70. Method for modulating the activity of a cell or immune cell characterized in that it comprises: a. obtaining a cell or immune cell comprising i. SPA peptide, as defined in any one of claims 1-32; ii. the system, as defined in any one of claims 36-38; iii. the nucleic acid, as defined in claim 34; and / or iv. the vector, as defined in claim 35; and b.to place the cell or immune cell in contact with a target cell that expresses an initiation receptor antigen and a CAR antigen, wherein the binding of the initiation receptor to the initiation receptor antigen on the target cell induces activation of the initiation receptor and expression of the chimeric antigen receptor, wherein the binding of the chimeric antigen receptor to the CAR antigen on the target cell modulates the activity of the cell or immune cell, and wherein the synthetic pathway activator also modulates the activity of the cell or immune cell.

71. A method for treating a disease in a subject in need thereof, characterized in that it comprises: a. determining or having determined the expression of CD11c in a cell comprising the synthetic pathway activator peptide (SPA), as defined in any of claims 1-32, or the nucleic acid, as defined in claim 34; and b. administering or having administered to the subject the cell.

72. Method for determining the expression of an SPA in a cell. Petition 870250081011, dated 09 / 09 / 2025, page 201 / 230 11 / 11, characterized in that it comprises expressing one or more SPA peptides, as defined in any of claims 1-32, in the cell and determining the expression of CD11 c in the cell.

73. Method, according to claim 70 or 72, characterized in that the expression of CD11c in the cell comprises either a CD11c mRNA expression level or a CD11c protein expression level.

74. Method, according to any one of claims 70-73, characterized in that the cell is an immune cell, a primary human immune cell, a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell or a progenitor T cell.

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