TW202506711A - Switch receptors - Google Patents

Abstract

The present disclosure relates to the fields of molecular biology, more specifically cytokine receptors. The present disclosure also relates to methods of medical treatment and prophylaxis, particularly cellular immunotherapy.

Description

Switching receptors

The present disclosure relates to the field of molecular biology, more specifically to interleukin receptors. The present disclosure also relates to methods of medical treatment and prevention, more specifically to cellular immunotherapy.

T cells expressing chimeric antigen receptors (CARs) have demonstrated clinical efficacy in different hematological malignancies. For solid tumor indications, CAR-T cell therapy has not yet provided meaningful clinical benefit over chemotherapy and immunotherapy. This difference may be attributed to multiple factors, including tumor heterogeneity, limited immune cell tumor infiltration, tumor microenvironment (TME), and the presentation or secretion of immunosuppressive factors.

It is well known that immunosuppressive ligands (such as PD-L1, PD-L2, and CTLA-4) interact with their respective receptors on T cells, leading to T cell exhaustion and impaired anti-tumor responses. In addition, the release of immunosuppressive cytokines (e.g., IL10, IL4, TGFβ) may inhibit T cell function and promote tumor immune escape.

Interleukin 23 (IL23) has emerged as a potential factor with pro-oncogenic properties. IL23 is produced by tumor-associated immune cells and can promote tumor growth and metastasis (Li et al., Carcinogenesis 34, No. 3 (March 2013): 658–66.; Zhang et al., Carcinogenesis 35, No. 6 (June 2014): 1330–40.; Langowski et al., Nature 442, No. 7101 (July 2006): 461–65.), angiogenesis, and mediate tumor immune evasion and suppression (Fu et al., European Urology 75, No. 5 (May 2019): 752–63.); Teng et al. , Proceedings of the National Academy of Sciences 107, No. 18 (May 4, 2010): 8328–33.), and showed potential as a diagnostic marker for lung and breast cancer. (Gangemi et al. Journal of Cellular Biochemistry 113, Issue 6 (June 2012): 2122–25.); Zhang et al. Therapeutics and Clinical Risk Management Volume 18 (April 2022): 429–37.).

For example, one study highlighted that IL23 blockade in cancer treatment destabilizes Tregs within tumors and leads to beneficial outcomes (Wight et al., Proceedings of the National Academy of Sciences 119, No. 18 (May 3, 2022): e2200757119.). This is emphasized by the fact that IL23 and IL12 are part of the same receptor superfamily, but their signaling outcomes can be opposite (Sieve et al., European Journal of Immunology 40, No. 8 (August 2010): 2236–47.; Teng et al., Cancer Research 72, No. 16 (August 15, 2012): 3987–96.; Kortylewski et al., Cancer Cell 15, No. 2 (February 2009): 114–23.).

Although the potent anti-tumor function of IL12 has been well documented, approaches to induce IL12 signaling in tumors remain challenging. For example, approaches involving systemic and/or non-targeted downregulation of IL-23 or upregulation of IL12 signaling would severely interfere with the normal function of the immune system and pose serious consequences and risks to the patient's health. Therefore, strategies to modulate the balance between IL23 and IL12 signaling hold promise for improving the outcomes of CAR-T cell therapy in solid tumors. On the other hand, although some groups have attempted to create chimeric receptors (also called switch receptors) to direct signaling to the desired pathway, one of the challenges associated with this strategy is finding mutations that effectively prevent activation by the original ligand without otherwise compromising the receptor's structure or intrinsic ability to activate gene transcription.

The disclosure provided herein provides solutions to problems existing in previous attempts to manipulate immune cells and potentially provides improved methods for therapies involving cell transfer.

The present invention generally relates to chimeric interleukin receptors (also referred to herein as switching receptors or chimeric receptor polypeptides) that are capable of converting interleukin 23 messages into cellular responses similar to interleukin 12. The chimeric interleukin receptors of the present invention comprise at least the first extracellular domain of the interleukin 23 receptor and the additional extracellular domain of the interleukin 12 receptor β2, the intracellular domain of the interleukin 12 receptor β2, and the transmembrane domain of either receptor. After the chimeric interleukin receptor binds to interleukin 23 and interleukin 12 receptor β1, a chimeric receptor complex is formed that causes an intracellular response similar to interleukin 12 in a dose-dependent manner. The present invention also relates to nucleic acid molecules, vectors, cells, pharmaceutical compositions encoding the chimeric interleukin receptors of the present invention, and uses and methods as described below.

In a first aspect, the present disclosure provides a chimeric receptor polypeptide comprising: (i) an extracellular portion comprising an IL23 binding domain; (ii) an intracellular portion comprising an intracellular domain of IL12Rb2 (interleukin 12 receptor β2); and (iii) a transmembrane domain connecting the extracellular portion and the intracellular portion.

In some embodiments, the IL23 binding domain of the chimeric receptor polypeptide binds the p19 subunit of IL23.

In some embodiments, the intracellular portion comprises an amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto.

In some embodiments, the transmembrane domain is selected from the transmembrane domains of IL12Rb2, IL23Ra, CD8, and CD28.

In some embodiments, the transmembrane domain comprises an amino acid sequence selected from SEQ ID No: 3, 4, 22, 33, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto.

In some embodiments, the chimeric receptor polypeptide further comprises a signaling sequence.

In some embodiments, the message sequence comprises the amino acid sequence of SEQ ID NO: 1 or 16.

In some embodiments, the chimeric receptor polypeptide further comprises one or more linkers.

In some embodiments, the linker is a GS linker, a 2A linker, an α-helix linker, a glycine-alanine polymer linker, an alanine-serine polymer linker, or an IgG4-Fc linker.

In some embodiments, the chimeric receptor polypeptide further comprises a tag polypeptide, such as a Flag tag.

In some embodiments, the chimeric receptor polypeptide further comprises a label, such as a fluorescent polypeptide, such as GFP or eGFP.

In some embodiments, the chimeric receptor polypeptide comprises from N-terminus to C-terminus: Optional signaling peptide – Optional tag – Optional linker – IL23 binding domain – Transmembrane domain – Intracellular portion containing the intracellular domain of IL12Rb2 – Optional linker – Optional tag.

In some embodiments, the IL23 binding domain comprises the extracellular domain of IL23Rα (also known as IL23R).

In some embodiments, the IL23 binding domain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto.

In some embodiments, the IL23 binding domain comprises the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto.

In some embodiments, the IL23 binding domain comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto.

In some embodiments, the chimeric receptor polypeptide further comprises an amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto between the IL23 binding domain and the transmembrane domain.

In some embodiments, the chimeric receptor polypeptide further comprises an amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto between the IL23 binding domain and the transmembrane domain.

In some embodiments, the chimeric receptor polypeptide further comprises an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity thereto between the IL23 binding domain and the transmembrane domain.

In some embodiments, the IL23 binding domain comprises Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, crossFab, scFab, single domain antibody (sdAb), or designer anchor protein repeat protein (DARPin).

In some embodiments, the chimeric receptor polypeptide comprises an amino acid sequence having at least 80% (preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%) sequence identity with an amino acid sequence selected from SEQ ID Nos: 36 to 44.

In a second aspect, the disclosure provides a nucleic acid or a plurality of nucleic acids encoding a chimeric receptor polypeptide disclosed herein (e.g., in the first aspect and embodiments thereof), and optionally further comprising a nucleic acid sequence encoding a chimeric antigen receptor.

In a third aspect, the disclosure provides an expression vector or a plurality of expression vectors comprising a nucleic acid or a plurality of nucleic acids disclosed herein (e.g., in the second aspect).

In a fourth aspect, the disclosure provides a cell comprising a chimeric receptor polypeptide according to the first aspect, a nucleic acid or a plurality of nucleic acids according to the second aspect, or an expression vector or a plurality of expression vectors according to the third aspect.

In some embodiments, the cell expresses IL12Rb1 (interleukin 12 receptor β1 chain).

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell.

In some embodiments, the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

In some embodiments, the cell is an immune cell.

In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL), a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a natural killer T (NKT) cell, or other T cells. In some embodiments, the immune cell further comprises a chimeric antigen receptor (CAR). In some embodiments, the immune cell further comprises an engineered TCR.

In some embodiments, the cells exhibit dose-dependent phosphorylation of STAT4 but not STAT3 when treated with IL23.

In some embodiments, the cell is a transduced T cell capable of expressing the chimeric receptor polypeptide according to the first aspect and embodiments thereof.

In some embodiments, the cell expresses an antigen binding receptor comprising an anchored transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen binding portion, the antigen binding portion comprising (i) a heavy chain variable domain (VH) comprising a heavy chain complementation determining region (HCDR) 1 of SEQ ID NO:49, HCDR 2 of SEQ ID NO:50 or SEQ ID NO:51, and HCDR 3 of SEQ ID NO:52, and (ii) a light chain variable domain (VL) comprising a light chain complementation determining region (LCDR) 1 of SEQ ID NO:53, LCDR 2 of SEQ ID NO:54, and LCDR 3 of SEQ ID NO:55.

In some embodiments, the VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59.

In some embodiments, the VL domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:61.

In some embodiments, the anchor transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domains or fragments thereof, in particular, wherein the anchor transmembrane domain is a CD8 transmembrane domain or a fragment thereof.

In some embodiments, the antigen binding receptor further comprises at least one stimulatory signaling domain and/or at least one co-stimulatory signaling domain.

In some embodiments, at least one stimulatory signaling domain in the antigen binding receptor is individually selected from the group consisting of a CD3z intracellular domain, a FCGR3A intracellular domain and a NKG2D intracellular domain or fragments thereof that retain stimulatory signaling activity. Specifically, the at least one stimulatory signaling domain is a CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity.

In some embodiments, at least one costimulatory signaling domain in the antigen binding receptor is individually selected from the group consisting of CD27 intracellular domain, CD28 intracellular domain, CD137 intracellular domain, OX40 intracellular domain, ICOS intracellular domain, DAP10 intracellular domain and DAP12 intracellular domain or fragments thereof that retain costimulatory signaling activity.

In some embodiments, the antigen binding receptor comprises at least one CD28 costimulatory domain or a fragment thereof that retains CD28 costimulatory activity and/or at least one CD137 costimulatory domain or a fragment thereof that retains CD137 costimulatory activity.

In some embodiments, the antigen binding receptor comprises a stimulatory signaling domain, which comprises a CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity, and wherein the antigen binding receptor comprises a co-stimulatory signaling domain, which comprises a CD28 intracellular domain or a fragment thereof that retains CD28 co-stimulatory signaling activity.

In some embodiments, the antigen binding receptor comprises a stimulatory signaling domain, which comprises a CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity, and wherein the antigen binding receptor comprises a co-stimulatory signaling domain, which comprises a CD137 intracellular domain or a fragment thereof that retains CD137 co-stimulatory signaling activity.

In some embodiments, the antigen binding portion is linked at the C-terminus to the N-terminus of the anchor transmembrane domain, optionally via a peptide linker.

In some embodiments, the light chain variable domain (VL) of the antigen binding portion of the antigen binding receptor is linked at the C-terminus to the N-terminus of the anchor transmembrane domain, optionally via a peptide linker, and/or wherein the heavy chain variable domain (VH) is linked at the C-terminus to the N-terminus of the light chain variable domain (VL), optionally via a peptide linker.

In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising the cell according to the fourth aspect and a pharmaceutically acceptable formulation selected as appropriate.

In a fifth aspect, the disclosure provides a chimeric receptor polypeptide according to the first aspect, or a nucleic acid according to the second aspect, or a cell according to the third aspect, or a pharmaceutical composition according to the fourth aspect, for use in a method for medical treatment of a disease or prevention of a disease. In some embodiments, the method is cell therapy, such as transfer cell therapy. The disease is cancer, an autoimmune disease, or an infection. In some embodiments, the cancer is selected from the group consisting of acute leukemia (including but not limited to acute myeloid leukemia (AML), B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL) and acute lymphoid leukemia (ALL)), chronic leukemia (including but not limited to chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL)), multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), chronic myeloid leukemia (CML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN).

In some embodiments, the chimeric receptor polypeptide according to the first aspect, or the nucleic acid according to the second aspect, or the cell according to the third aspect, or the pharmaceutical composition according to the fourth aspect is administered intravenously, intratumorally, or subcutaneously.

In a sixth aspect, the disclosure provides a method for regulating the activity of an immune cell, the method comprising: administering to the immune cell a nucleic acid or a plurality of nucleic acids according to the second aspect, or an expression vector or a plurality of expression vectors according to the third aspect. In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL), a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a natural killer T (NKT) cell, or other T cells.

Sequence Listing

This application contains a sequence listing that has been submitted electronically in XML format compliant with WIPO Standard ST.26 and is incorporated herein by reference in its entirety. The XML file was created on May 7, 2024, is named P38561-WO-Seq.xml, and is 88,941 bytes in size.

T cells expressing chimeric antigen receptors (CARs) have demonstrated remarkable clinical efficacy, particularly in improving survival in patients with advanced hematological malignancies. However, inhibitory factors in the tumor microenvironment, such as checkpoint molecules and immunosuppressive cytokines, have limited the function of CAR-T cells, particularly in targeting solid cancers to date. One approach to manipulating the tumor microenvironment used in the present invention is to develop switch receptors that translate inhibitory messages into beneficial messages.

We found that several tumors (mainly lung and colon) showed transcriptomic evidence for overexpression of IL23A in tumors compared to normal tissues (Figure 1A). Here, we exploited the evolutionary relationship of the IL23 and IL12 pathways and their shared receptor chain IL12Rb1. Indeed, as indicated in Figure 1B, both complexes share IL12Rb1 via their common p40 interleukin subunit. IL12Rb1 is constitutively expressed on the surface of human immune cells. IL12 specifically interacts with IL12Rb2 via its p35 subunit to promote TYK2- and JAK2-mediated STAT4 phosphorylation. IL23 specifically interacts with IL23R to promote TYK2- and JAK2-mediated phosphorylation of STAT4 and STAT3. The chimeric IL23-IL12 switch receptor is composed of the extracellular domain (ECD) of IL23R and the intracellular domain (ICD) of IL12Rb2. Together with IL12Rb1, the IL23R-IL12Rb2 switch receptor induces IL12-like signaling via STAT4 phosphorylation following stimulation with IL23.

The chimeric switch receptors of the present invention translate the cancer-promoting message of IL23 into the cytotoxic IL12 message. Surprisingly, T cells, such as CAR-T cells engineered with these switch receptors, differentially regulate important surface markers and secreted factors in response to stimulation and show increased cytotoxicity against cancer cell lines in vitro.

Alternative IL23-IL12sR (sR: switch receptor) formats are indicated in Figure 1C. IL23 binds to the IL23R_ECD1 domain. All chimeric IL23R-IL12Rb1 switch receptors contain IL23R_ECD1 with the C-terminal IL23R ECD replaced by the corresponding amount of IL12Rb2 ECD, resulting in different chimeric IL23R-IL12Rb2 designs.

An overview of the expression cassettes for screening the IL23-IL12sR version in plastids coupled to a downstream GFP reporter gene is shown in Figure 1D. Constructs (1) and (2) correspond to SEQ ID Nos: 36 and 37, respectively.

An overview of the IL23-IL12sR version of the expression cassette (including variants with a chimeric ECD) as downstream genetic elements of the different CAR constructs is indicated in Figure 1E. Constructs (3), (4), (5), (6) and (8) correspond to SEQ ID Nos: 38, 39, 40, 41 and 42, respectively.

The mechanism of action of IL23-IL12sR engineered immune cells is shown in Figure 1F. IL23 is released by cells in the vicinity of the engineered immune cells (e.g., in the tumor microenvironment). IL23 is recognized by the chimeric IL23-IL12sR and endogenous IL12Rb1, leading to the formation of the chimeric IL23-IL12sR complex and phosphorylation of STAT4. STAT4 signaling leads to upregulation of IL18Ra, IL12Rb2, CD25, and IFNγ secretion. These effects combined can lead to improved cytotoxicity of IL23-IL12sR engineered immune cells against target cells in vitro.

In the detailed description below, the illustrative alternatives described in the detailed description and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the various aspects as generally described herein may be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are expressly contemplated and constitute a part of this application.

Definition

Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural references. For example, the term "a cell" includes one or more cells, including mixtures thereof. As used herein, "A and/or B" is intended to include all of the following alternatives: "A," "B," "A or B," and "A and B."

As used herein, the term "administration" or "administering" refers to the delivery of a composition or formulation as disclosed herein by an administration route, including but not limited to intravenous, intraarterial, intracranial, intramuscular, intraperitoneal, subcutaneous, intramuscular, or a combination thereof. The term includes but is not limited to administration by a healthcare professional and self-administration.

"Cancer" refers to the presence of cells with a variety of typical characteristics of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological characteristics. Cancer cells can be aggregated into clumps, such as tumors, or can exist individually in an individual's body. Tumors can be solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "cancer" also encompasses other types of non-tumor cancers. Non-limiting examples include blood cancers or blood cancers, such as leukemia. Cancer can include precancerous lesions as well as malignant cancers.

The terms "cell," "cell culture," and "cell line" refer not only to a specific target cell or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It is understood that not all progeny are exactly the same as the parent cell. This is because certain modifications may occur in subsequent generations due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that the progeny may not actually be the same as the parent cell, but are still included in the scope of the terms as used herein, as long as the progeny retain the same functions as the original cell, cell culture, or cell line.

As used herein, the term "percent identity" in the context of two or more nucleic acids or proteins refers to sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (e.g., about 80% sequence identity, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when maximally aligned and aligned over a comparison window or specified region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm (default parameters described below) or by manual alignment and visual inspection. See, e.g., the NCBI website at ncbi.nlm.nih.gov/BLAST. Such sequences are then referred to as "substantially identical." This definition also relates to or can be used for the supplement of sequences. This definition also includes sequences with deletions and/or additions and sequences with substitutions. Sequence identity can be calculated over a region of at least about 20 amino acids or nucleotides in length, or over a region of 10 to 100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) with its default parameters.

As used herein, "subject" or "individual" includes animals, such as humans (e.g., human subjects) and non-human animals. In some embodiments, a "subject" or "individual" is a patient under the care of a physician. Thus, the subject may be a human patient or an individual who has, is at risk of, or is suspected of having a disease of interest (e.g., cancer) and/or one or more symptoms of the disease. The subject may also be an individual who is diagnosed at risk for the condition of interest at the time of diagnosis or thereafter. The term "non-human animals" includes all vertebrates, such as mammals, such as rodents, such as mice, non-human primates and other mammals, such as sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

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

All ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range may be considered to fully describe the same range and enable the range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein may be easily decomposed into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all language such as "at most," "at least," "greater than," "less than," and the like includes the listed numbers and refers to a range that can subsequently be decomposed into subranges as discussed above. Finally, as will be understood by those skilled in the art to which the present invention pertains, a range includes each individual member. Thus, for example, a group with 1 to 3 items refers to groups with 1, 2, or 3 items. Similarly, a group with 1 to 5 items refers to groups with 1, 2, 3, 4, or 5 items, and so on.

It should be understood that certain features of the present disclosure described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present disclosure are specifically contemplated by the present disclosure and are disclosed herein as if each combination were individually and expressly disclosed. In addition, all subcombinations of various embodiments and elements thereof are also specifically contemplated by the present disclosure and are disclosed herein as if each such subcombination were individually and expressly disclosed herein.

Chimeric receptor polypeptide

The present disclosure is based, inter alia, on chimeric receptor polypeptides (also referred to as chimeric receptors or switch receptors) comprising: an extracellular portion comprising an IL23 binding domain, an intracellular portion comprising an intracellular domain of IL12Rb2, and a transmembrane domain connecting the extracellular portion and the intracellular portion. Immune cells expressing such chimeric receptors can be used in the context of modulating immune cell activity. In some embodiments, the ligand can be added exogenously and is not limited to being produced intracellularly.

The present disclosure also relates to nucleic acid molecules encoding the chimeric interleukin receptors of the present invention, vectors, cells, pharmaceutical compositions, and uses and methods as described herein, for example, in the claims.

Antigen binding part

In some embodiments, cells expressing a chimeric receptor polypeptide of the invention also express an antigen binding receptor comprising an anchored transmembrane domain and an extracellular domain.

As used herein, "antigen binding moiety" refers to a moiety that binds to a given target antigen.

Antigen binding moieties include antibodies (i.e., immunoglobulins (Ig)) and antigen binding fragments and derivatives thereof. In some embodiments, the antigen binding moieties according to the present disclosure include or consist of: monoclonal antibodies, monospecific antibodies, multispecific (e.g., bispecific, trispecific, etc.) antibodies, variable fragment (Fv) moieties, single-chain Fv (scFv) moieties, fragment antigen binding (Fab) moieties, single-chain Fab moieties (scFab), crossFab moieties, Fab' moieties, Fab'-SH moieties, F(ab') ₂ moieties, bispecific moieties, trispecific moieties, scFv-Fc moieties, miniantibodies moieties, heavy chain antibodies only (HCAb) moieties, or single domain antibodies (dAb, VHH) moieties.

Antigen binding moieties according to the present disclosure also include other target antigen binding peptides/polypeptides, such as peptide aptamers, thioredoxins, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodys, affilins, armadillo repeat proteins (ArmRP), OBodys and adnectins (e.g., as summarized in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082–1101, which is incorporated herein by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48)). Antigen binding moieties according to the present disclosure also include target antigen binding nucleic acids, such as nucleic acid aptamers (e.g., as summarized in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). Antigen binding moieties according to the present disclosure also include target antigen binding small molecules (e.g., low molecular weight (<1000 Daltons, usually between about 300 and 700 Daltons) organic compounds).

In some embodiments, the antigen binding portion is capable of binding to a variant Fc domain, such as a variant Fc domain as disclosed in WO2022029051A1, the contents of which are incorporated herein by reference in their entirety. The antigen binding portion described herein preferably exhibits specific binding to a variant Fc domain, such as disclosed in WO2022029051A1. As used herein, "specific binding" refers to selective binding to a target antigen, and it can be distinguished from non-specific binding to non-target antigens. An antigen binding portion that specifically binds to a given target antigen preferably binds to the target antigen with greater affinity and/or longer duration than it binds to other non-target antigens.

The ability of a given moiety to specifically bind to a given target antigen can be determined by assaying according to methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (BLI; see, e.g., Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. By such assays, binding to a given target antigen can be measured and quantified. In some embodiments, the level of binding can be the response detected in a given assay.

An antigen-binding moiety that "does not bind" or "shows substantially no binding" to a given antigen exhibits a level of binding to the given antigen that is similar to the level of binding to an antigen that the antigen-binding moiety is known not to bind to or is known to bind non-specifically, such as a non-target antigen. In some embodiments, the level of binding of an antigen-binding moiety that does not bind to or shows substantially no binding to a given antigen is ≥0.5 times and ≤2 times, such as ≥0.75 times and ≤1.5 times, ≥0.8 times and ≤1.4 times, ≥0.85 times and ≤1.3 times, ≥0.9 times and ≤1.2 times, ≥0.95 times and ≤1.1 times, the level of binding exhibited by the antigen-binding moiety to an antigen that the antigen-binding moiety is known not to bind to or is known to bind non-specifically (e.g., a non-target antigen).

Connector

The polypeptide disclosed herein (e.g., chimeric receptor polypeptide) may further comprise other amino acids or sequences of amino acids.

The polypeptide may comprise one or more linker sequences between the sequences of amino acids. For example, linker sequences may be provided between different domains of a chimeric receptor polypeptide (e.g., an extracellular portion and a transmembrane domain, or between a transmembrane domain and an intracellular portion).

Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, the linker sequence can be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those skilled in the art and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences typically contain a high proportion of glycine and/or serine residues.

In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments, the linker sequence comprises or consists of: glycine and serine residues. In some embodiments, the linker sequence has the following structure: (GxS)n or (GxS)nGm; wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1, 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5 or 6) copies (e.g., in tandem) of the sequence motif G ₄ S. In some embodiments, the linker sequence comprises or consists of (G ₄ S) ₃ or (G ₄ S) ₄ . In some embodiments, the linker sequence has a length of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids. In some embodiments, the linker sequence is one or more repeats of GGSG.

In some embodiments, the linker sequence comprises one or more copies of an amino acid sequence according to SEQ ID NO: 92. In some embodiments, the linker sequence comprises at least 1, 2, 3, or 4 copies of an amino acid sequence according to SEQ ID NO: 20.

In some embodiments, the linker sequence comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO: 18.

In some embodiments, the linker sequence comprises a cleavage site, e.g., a cleavage site as described below.

The polypeptides disclosed herein may comprise an amino acid sequence to facilitate the expression, folding, transport, processing, purification or detection of the polypeptides. For example, the chimeric receptor polypeptides disclosed herein may further comprise an amino acid sequence that forms a detectable portion, for example, as described below.

The polypeptide may additionally comprise a signaling peptide (also called leader sequence or signaling sequence). Signaling peptides usually consist of a sequence of 5 to 30 hydrophobic amino acids that form a single alpha helix. Secreted proteins and proteins expressed at the cell surface usually comprise signaling peptides. Signaling peptides are known for many proteins and are recorded in databases such as GenBank, UniProt and Ensembl and/or can be identified/predicted, for example, using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

The signal peptide may be present at the N-terminus of the chimeric receptor polypeptide and may be present in a newly synthesized polypeptide. The signal peptide provides for efficient transport of the chimeric receptor polypeptide. The signal peptide is typically removed by cleavage and is therefore not included in the mature chimeric receptor polypeptide.

Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl and InterPro, and/or can be identified/predicted using, for example, amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 16.

In some embodiments, the chimeric receptor polypeptides disclosed herein include a detectable portion, also referred to as a tag. In some embodiments, the detectable portion is provided at the N-terminus and/or C-terminus of the polypeptide.

In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical, a nucleic acid, or an enzyme label. The chimeric receptor polypeptide can be covalently or non-covalently labeled with the detectable moiety.

Fluorescent labels include, for example, fluorescein, rose bengal, allophycocyanin, eosin and NDB, green fluorescent protein (GFP), enhanced GFP (eGFP), chelates of rare earths such as eutectic (Eu), thallium (Tb) and sammonium (Sm), tetramethyl rose bengal, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3 and Cy5. Radioactive labels include radioactive isotopes such as hydrogen ^-3 , sulfur ^-35 , carbon ^-14 , phosphorus- ³² , iodine- ¹²³ , iodine- ¹²⁵ , iodine- ¹²⁶ , iodine ^{-131 ,} iodine- ¹³³ , bromine-77, technetium- ^99m , indium ^-111 , indium ^-113m , gallium- ⁶⁷ , gallium- ⁶⁸ , ruthenium- ⁹⁵ , ruthenium ^{-97 , ruthenium-103 ,} ruthenium ^-105 , mercury ^-207 , mercury ^-203 , rhenium- ^99m , rhenium-101, rhenium ^-105 , plutonium- ⁴⁷ , tellurium ^-121m , tellurium- ^122m , tellurium ^-125m , thulium-165, thulium- ¹⁶⁷ , thulium ^{- 168} , copper ^-67 , fluorine ^-18 , yttrium ^-90 , palladium ^-100 , bismuth ^-217 , and antimony ^-211 . Luminescent labels include radioluminescent, chemiluminescent labels (e.g., acridinium esters, luminol, isoluminol), and bioluminescent labels. Immunodetectable labels include haptens, peptides/polypeptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or focitin. Nucleic acid labels include aptamers.

In some embodiments, the chimeric receptor polypeptide comprises an epitope tag, such as His (e.g., 6XHis), FLAG, c-Myc, Strep tag (StrepTag), hemagglutinin E, calcium binding protein (CBP), glutathione-s-transferase (GST), maltose binding protein (MBP), thioredoxin, S peptide, T7 peptide, SH2 domain, avidin, streptavidin and hapten (e.g., biotin, focitin, dinitrophenol) at the N- or C-terminus of the chimeric receptor polypeptide, as appropriate.

In some embodiments, the chimeric receptor polypeptide comprises a portion having a detectable activity, e.g., an enzyme portion. Enzyme portions include, e.g., luciferase, glucose oxidase, β-galactosidase (e.g., β-galactosidase), glucosidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

In some embodiments, the polypeptide of the present disclosure comprises a fluorescent marker. In some embodiments, the polypeptide comprises an eGFP portion. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with SEQ ID NO:7.

The chimeric receptor polypeptide disclosed herein may also comprise one or more cleavage sites. A cleavage site refers to an amino acid sequence that serves as a substrate for an enzyme capable of cleaving a peptide bond.

Many such cleavage sites are known to and can be used by those skilled in the art of molecular biology. In some embodiments, the cleavage sequence comprises an auto-cleavage site. Auto-cleavage sites include the 2A cleavage sequence from the picornavirus "NPGP", which is cleaved at "G/P". Auto-cleavage sites are further described, for example, in Kim et al., PLoS ONE (2011) 6: e18556 (incorporated by reference in its entirety), and include, for example, T2A, P2A, E2A, and F2A cleavage sites. The amino acid sequences of the T2A and E2A cleavage sites are shown in SEQ ID NOs: 6 and 9, respectively.

A cleavage site may be included in a polypeptide according to the present disclosure to provide for removal of a portion or domain. It may be desirable to remove a given portion or domain so that it is not included in a polypeptide complex formed by the polypeptide. For example, in an embodiment of a chimeric receptor polypeptide disclosed herein, a cleavage site (specifically, a T2A cleavage site) is provided upstream of the eGFP portion to remove the portion so that the eGFP portion is not included in the final chimeric receptor polypeptide. Accordingly, in some embodiments, a chimeric receptor polypeptide according to the present disclosure comprises a cleavage site adjacent to a detectable portion according to the present disclosure (i.e., in the amino acid sequence of the polypeptide, e.g., immediately upstream or downstream thereof).

In some embodiments, the cleavage site according to the present disclosure is a 2A cleavage site, for example selected from T2A, P2A, E2A and F2A cleavage sites. In some embodiments, the cleavage site is a T2A cleavage site.

Specific Exemplary Chimeric Receptor Polypeptides

In some embodiments, the chimeric receptor polypeptide according to the present disclosure comprises or consists of one of the following structures: Optionally selected signal peptide – Optionally selected tag – Optionally selected linker – IL23 binding domain – Transmembrane domain – Intracellular portion comprising the intracellular domain of IL12Rb2 – Optionally selected linker – Optionally selected tag.

Schematic diagrams of some chimeric receptor polypeptides of the present invention are provided in Figures 1B, C, D, E.

In some embodiments, a chimeric receptor polypeptide according to the present disclosure comprises an amino acid sequence having at least 60%, preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to SEQ ID NO:36, 37, 62, 63, 64, 65, 66, 38, 39, 40, 41, 42, 43 or 44.

Nucleic Acids and Vectors

The disclosure provides a nucleic acid or a plurality of nucleic acids encoding a chimeric receptor polypeptide according to the disclosure. In some embodiments, the nucleic acid comprises or consists of: DNA and/or RNA.

The chimeric receptor polypeptide according to the present disclosure can be produced in a cell by the translation of RNA encoding the chimeric receptor polypeptide. The chimeric receptor polypeptide according to the present disclosure can be produced in a cell by the transcription of a nucleic acid encoding the chimeric receptor polypeptide and the subsequent translation of the transcribed RNA.

In some embodiments, the nucleic acid may be a vector or a plurality of vectors, or may be contained in a vector or a plurality of vectors. As used herein, a "vector" is a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acid into a cell.

Accordingly, the present disclosure also provides a vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate the delivery of one or more nucleic acids encoding a chimeric receptor polypeptide according to the present disclosure to a cell. The vector can be an expression vector comprising elements required for the expression of a chimeric receptor polypeptide, optionally together with a CAR molecule or linked to a CAR molecule. The vector can comprise elements that facilitate the integration of the nucleic acid into the genomic DNA of a cell into which the vector is introduced.

Nucleic acids and vectors according to the present disclosure can be provided in a purified or isolated form, ie, separated from other nucleic acids or naturally occurring biological materials.

The vector may be a vector for expressing nucleic acid in a cell (i.e., an expression vector). Such a vector may include a promoter sequence operably linked to a nucleotide sequence encoding a chimeric receptor polypeptide according to the present disclosure. The vector may also include a stop codon (i.e., the nucleotide sequence of the vector 3' to the nucleotide sequence encoding the chimeric receptor polypeptide) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

The term "operably linked" may include situations in which a nucleic acid encoding a chimeric receptor polypeptide according to the present disclosure and a regulatory nucleic acid sequence (e.g., a promoter and/or enhancer) are covalently linked in such a manner as to place the expression of the nucleic acid encoding the chimeric receptor polypeptide under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of affecting the transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

Vectors contemplated for use with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g., binding plasmids (e.g., F plasmids), non-binding plasmids, R plasmids, col plasmids, factor plasmids), viral vectors (e.g., retroviral vectors, e.g., γ-retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated herein by reference in their entirety. In some embodiments, the vector according to the present disclosure is a lentiviral vector.

In some embodiments, the vector can be a eukaryotic cell vector, that is, a vector that contains the elements necessary for protein expression from the vector in a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, for example, containing a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

In some embodiments, a nucleic acid/a plurality of nucleic acids or a vector/a plurality of vectors according to the present disclosure comprises an EF1α promoter.

In some embodiments, a nucleic acid/a plurality of nucleic acids or a vector/a plurality of vectors according to the present disclosure encodes a chimeric receptor polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to one of the following: SEQ ID NO: 36, 37, 62, 63, 64, 65, 66, 38, 39, 40, 41, 42, 43 or 44.

The constituent polypeptides of the chimeric receptor polypeptide according to the present disclosure may be encoded by different nucleic acids among the plurality of nucleic acids according to the present disclosure, or encoded by different vectors among the plurality of nucleic acids according to the present disclosure.

In the aspects and embodiments of the present disclosure, a nucleic acid or a plurality of nucleic acids according to the present disclosure encodes two or more (e.g., 2, 3, 4 or more) chimeric receptor polypeptides according to the present disclosure.

In some embodiments, wherein a nucleic acid/a plurality of nucleic acids or a vector/a plurality of vectors encodes two or more (e.g., 2, 3, 4 or more) chimeric receptor polypeptides according to the present disclosure, transcription of the nucleic acids encoding the two or more chimeric receptor polypeptides is under the control of the same promoter.

In some embodiments, transcription of nucleic acids encoding two or more chimeric receptor polypeptides is under the control of different promoters.

In some embodiments, the nucleic acid/nucleic acids or vector/vectors are polycistronic (e.g., bicistronic, tricistronic, etc.). In other words, in some embodiments, the nucleic acid/nucleic acids or vector/vectors contain multiple nucleotide sequences encoding polypeptides. In some embodiments, nucleic acids encoding two or more chimeric receptor polypeptides are provided in different cistrons.

Include / Cells expressing the chimeric receptor polypeptide disclosed herein

The present disclosure also provides a cell comprising a chimeric receptor polypeptide according to the present disclosure, or a nucleic acid/multiple nucleic acids or a vector/multiple vectors according to the present disclosure.

It should be understood that when a cell is mentioned in the singular form herein (i.e. "a/the cell"), the plural/group of such cells is also encompassed.

The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus macaque, cynomolgus macaque, non-human primate or human) or a non-human mammal (such as rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodeoda), cat, dog, pig, sheep, goat, cattle (including cattle, such as dairy cows, or any animal in the order Bovis), horse (including any animal in the family Equidae), donkey and non-human primate). In a preferred embodiment, the cell is a human cell.

In some embodiments, the cells are immune cells. The immune cells may be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes or monocytes. Lymphocytes may be, for example, T cells, B cells, NK cells, NKT cells or innate lymphoid cells (ILC) or their precursors. The immune cells may express CD27, CD28, CD4 and/or CD8. In some embodiments, the immune cells are T cells, such as CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cell is a T helper cell ( _TH cell). In some embodiments, the T cell is a cytotoxic T cell (eg, a cytotoxic T lymphocyte (CTL)). In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL), a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a natural killer T (NKT) cell, or other T cells.

Aspects and embodiments of the present disclosure particularly relate to T cells comprising/expressing a chimeric receptor polypeptide according to the present disclosure.

In some aspects and embodiments, a cell according to the disclosure expresses/presents a chimeric receptor polypeptide according to the disclosure at the cell surface. That is, the chimeric receptor polypeptide may be present in or at the cell membrane. The surface expression of the chimeric receptor polypeptide by the cell may be assessed, for example, using antibody-based methods, such as flow cytometry techniques (e.g., as described in the examples of the disclosure) .

In the aspects and embodiments of the present disclosure, the cell according to the present disclosure comprises or expresses the chimeric receptor polypeptide according to the present disclosure. In some aspects and embodiments, the cell according to the present disclosure comprises a nucleic acid encoding the chimeric receptor polypeptide according to the present disclosure. In some aspects and embodiments, the cell according to the present disclosure comprises a nucleic acid/multiple nucleic acids or a vector/multiple vectors according to the present disclosure.

In aspects and embodiments of the present disclosure, the cell according to the present disclosure comprises or expresses the polypeptide complex according to the present disclosure, which is bound to the variant Fc domain as described herein.

Dose-dependent IL12 signaling mediated by exposure of the cells of the invention to IL23 can be studied as described in the examples herein, for example, by detecting, quantifying and analyzing downstream molecules in the pathway, such as STAT4 and its phosphorylation state or level.

The level of chimeric receptor polypeptide-mediated signaling can also be analyzed using reporter gene-based methods, such as methods that quantify the activity of a transcription factor or gene whose expression/activity is upregulated in response to signaling via IL12.

As used herein, "expression" can be gene or protein expression. Gene expression encompasses transcription of DNA to RNA and can be measured by various methods known to those skilled in the art, such as measuring the level of mRNA by quantitative real-time PCR (qRT-PCR), or using reporter gene-based methods. Similarly, protein expression can be measured by various methods known in the art, such as antibody-based methods, such as by Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT or reporter gene-based methods.

The immune cells (e.g., T cells) disclosed herein can exhibit cytotoxicity to cells containing/expressing the variant Fc domain disclosed herein. In other words, the immune cells (e.g., T cells) disclosed herein have the ability to kill cells containing/expressing the variant Fc domain disclosed herein.

Cytotoxicity and cytotoxicity can be studied, for example, using any of the methods summarized in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro assays for cytotoxicity/cytotoxicity assays include release assays such as ^51Cr release assays, lactate dehydrogenase (LDH) release assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyltetrazolium bromide (MTT) release assays, and calcein-acetyloxymethyl (calcein-AM) release assays. These assays measure cytotoxicity based on the detection of factors released from lysed cells. Cytotoxicity by a given test cell type (e.g., an immune cell according to the present disclosure (e.g., a T cell)) can be analyzed, for example, by co-culturing the test cell with a given target cell type (e.g., a cell comprising a variant Fc domain according to the present disclosure) and measuring the number of live (i.e., unlysed)/dead (e.g., lysed) target cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay, described in Cerignoli et al., PLoS One. (2018) 13(3):e0193498 (incorporated herein by reference in its entirety), and the Incucyte immunocytotoxicity assay employed in the experimental examples of the present disclosure.

The disclosure also provides methods for producing cells according to the disclosure, and cells obtained or obtainable by such methods.

Methods for producing cells containing/expressing a polypeptide/polypeptide complex of interest are known to the skilled person and generally comprise introducing into the cell a nucleic acid/vector encoding the polypeptide of interest.

Such methods may include nucleic acid transfer for permanent (i.e., stable) or transient expression of the transferred nucleic acid. In some embodiments, after introduction into a cell, the nucleic acid encoding the polypeptide of interest may be integrated into or form part of the genomic DNA of the cell. In some embodiments, after introduction into a cell, the nucleic acid encoding the polypeptide of interest may be maintained extrachromosomally.

Any suitable genetic engineering platform may be used, and includes gamma retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, for example, as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, which is incorporated herein by reference in its entirety. Methods also include those described, for example, in Wang and Rivière Mol Ther Oncolytics. (2016) 3:16015, which is incorporated herein by reference in its entirety. Suitable methods for introducing nucleic acids/vectors into cells include transduction, transfection, and electroporation.

Methods for producing/expanding cell populations containing/expressing a polypeptide of interest in vitro/in vivo are known to those skilled in the art. Suitable culture conditions (i.e., cell culture medium, additives, stimulation, temperature, atmosphere), cell number, culture cycle, and methods for introducing nucleic acids/vectors encoding the polypeptide of interest into cells, etc., can be determined by reference to, for example, WO 2018/177966 A1 . In some embodiments, cells/cell populations according to the present disclosure are prepared under GMP (Good Manufacturing Practice; for example, as set out in the Good Manufacturing Practice Guidelines published by the European Commission (Volume 4 of the "EU Medicines Regulation" containing guidance on the interpretation of Council Directive 91/356/EEC (as amended by Directives 2003/94/EC and 91/412/EEC, respectively) on good manufacturing practices for medicinal products for human and veterinary use) conditions.

Conveniently, cell cultures according to the present disclosure can be maintained at 37°C in a humidified atmosphere containing 5% _CO2 . The cells of the cell culture can be established and/or maintained at any suitable density, as can be readily determined by the skilled artisan. The culture can be carried out in any container suitable for the volume of the culture, such as a well of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, the cells are cultured in a bioreactor, such as the bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, which is incorporated herein by reference in its entirety. Immune cells (eg, T cells) may be activated prior to the introduction of nucleic acids encoding the polypeptide of interest. For example, T cells within a PBMC population may be non-specifically activated by in vitro stimulation with agonist anti-CD3 antibodies and agonist anti-CD28 antibodies in the presence of IL-2.

Introducing the nucleic acid into the cell may comprise transduction, such as lentiviral transduction. Transduction of immune cells with viral vectors is described, for example, in Simmons and Alberola-Ila, Methods Mol Biol. (2016) 1323:99-108, which is incorporated herein by reference in its entirety.

Agents can be used to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer that is often used to improve transduction by neutralizing the charge repulsion between viral particles and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include, for example, poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech), and SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs). In some embodiments, the methods comprise centrifuging cells into which one wishes to introduce a nucleic acid encoding a polypeptide of interest in the presence of a cell culture medium comprising a viral vector containing the nucleic acid (referred to in the art as "spinfection").

The method generally comprises introducing a nucleic acid encoding a polypeptide of interest into a cell and culturing the cell under conditions suitable for the cell to express the polypeptide of interest. In some embodiments, the methods comprise culturing immune cells into which a nucleic acid encoding a polypeptide of interest has been introduced to expand their number.

In some embodiments, the methods include analyzing the cells to confirm that the nucleic acid was successfully introduced into the cells. In some embodiments, the methods include analyzing the cells to confirm that the cells express the polypeptide of interest (e.g., via assessment of a detectable entity).

In some embodiments, the methods further comprise cells expressing the polypeptide of interest, e.g., from other cells (e.g., cells that do not express the polypeptide of interest). Methods for purifying/isolating immune cells from a heterogeneous population of cells are known in the art, and methods based on, e.g., FACS or MACS, can be used to select cell populations based on the expression of immune cell markers. In some embodiments, the methods purify/isolate specific types of cells, e.g., CD8+ T cells or CTLs that express the polypeptide of interest.

Modification of a given target nucleic acid can be achieved in a variety of ways known to the skilled artisan, including modification of the target nucleic acid by homologous recombination and editing of the target nucleic acid using site-specific nucleases (SSNs).

Suitable methods may employ targeting by homologous recombination, which is reviewed, for example, in Mortensen Curr Protoc Neurosci. (2007) Chapter 4: Unit 4.29 and Vasquez et al., PNAS 2001, 98(15): 8403-8410, both of which are incorporated herein by reference in their entirety. Targeting by homologous recombination involves exchanging nucleic acid sequences through crossover events directed by homologous sequences. Other suitable techniques include nucleic acid editing using SSNs. Gene editing using SSNs is reviewed, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is incorporated herein by reference in its entirety. Enzymes capable of creating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into target nucleic acid sequences of interest. DSBs can be repaired by error-prone nonhomologous end joining (NHEJ), in which the two ends of the break are rejoined, usually with insertions or deletions of nucleotides. Alternatively, DSBs can be repaired by homology-directed repair (HDR), a high-fidelity mechanism in which a DNA template with ends homologous to the break site is supplied and introduced into the DSB site.

SSNs that can be engineered to generate target nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcript activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) systems. ZFN systems are summarized, for example, in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is incorporated herein by reference in its entirety. ZFNs comprise a programmable zinc finger DNA binding domain and a DNA cleavage domain (e.g., a Fok I endonuclease domain). The DNA binding domain can be identified by screening zinc finger arrays that are able to bind to a target nucleic acid sequence. The TALEN system is described, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is incorporated herein by reference in its entirety. TALENs comprise a programmable zinc finger DNA binding TALE domain and a DNA cleavage domain (e.g., a Fok I endonuclease domain). TALEs comprise a repeat domain consisting of a repeat sequence of 33 to 39 amino acids, which are identical except that the two residues at positions 12 and 13 of each repeat sequence are repeat variable diresidues (RVDs). Each RVD determines the binding of the repeat sequence to a nucleotide in the target DNA sequence according to the following relationship: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501.). CRISPR/Cas9 and related systems, such as CRISPR/Cpf1, CRISPR/C2c1, CRISPR/C2c2, and CRISPR/C2c3 are described in Nakade et al., Bioengineered (2017) 8(3):265-273, which is incorporated herein by reference in its entirety. Such systems include nucleases (e.g., Cas9, Cpf1, etc.) and single guide RNA (sgRNA) molecules. sgRNAs can be engineered to target endonuclease activity to nucleic acid sequences of interest.

In some embodiments, modification of a nucleic acid encoding a chimeric receptor polypeptide according to the present disclosure (e.g., an endogenous nucleic acid) employs a site-specific nuclease (SSN) system that targets the nucleic acid encoding the chimeric receptor polypeptide. The SSN system can be a ZFN system, a TALEN system, a CRISPR/Cas9 system, a CRISPR/Cpf1 system, a CRISPR/C2c1 system, a CRISPR/C2c2 system, or a CRISPR/C2c3 system.

Composition

The present disclosure also provides compositions, such as pharmaceutical compositions, comprising the polypeptides, nucleic acids, expression vectors and cells described herein.

The polypeptides, nucleic acids, expression vectors and cells described herein (and in particular the nucleic acids, expression vectors and cells described herein) can be formulated as pharmaceutical compositions or drugs for clinical use, and may contain pharmaceutically acceptable carriers, diluents, excipients or adjuvants. In preferred aspects and embodiments, the present disclosure provides a pharmaceutical composition or drug comprising a cell according to the present disclosure. Therefore, the present disclosure also provides a pharmaceutical composition/drug comprising the polypeptides, nucleic acids/multiple nucleic acids, expression vectors/multiple expression vectors or cells described herein. In preferred embodiments, the pharmaceutical composition/drug according to the present disclosure comprises the nucleic acids/multiple nucleic acids, expression vectors/multiple expression vectors or cells described herein.

The pharmaceutical composition/drug disclosed herein may include one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents/formulators (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl parahydroxybenzoate, propyl parahydroxybenzoate), antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silicon dioxide, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g. wetting agents), masking agents or coloring agents (e.g. titanium dioxide).

The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are suitable, within the scope of sound medical judgment, for use in contact with the tissues of the subject in question (e.g., human subjects) without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring agent or sweetener of the composition according to the present disclosure must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavoring agents or sweeteners can be found, for example, in standard medical textbooks such as Remington's "The Science and Practice of Pharmacy (ed. A. Adejare) 23rd edition (2020), Academic Press).

The pharmaceutical compositions and drugs disclosed herein can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal administration. In some embodiments, the pharmaceutical compositions/drugs can be formulated for administration by injection or infusion, or by ingestion.

Suitable formulations may include cells provided in a sterile or isotonic medium. Drugs and pharmaceutical compositions may be formulated in fluid form, including gel form. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) into a selected area of the human or animal body.

In some embodiments, the pharmaceutical composition/drug is formulated for injection or infusion into, for example, a blood vessel, a tissue/organ of interest, or a tumor.

The present disclosure also provides methods for producing pharmaceutically useful compositions, such production methods may include one or more steps selected from the following: Producing the cells described herein; Isolating/purifying the cells described herein; and/or Mixing the cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

For example, another aspect of the present disclosure relates to a method of formulating or producing a drug or pharmaceutical composition for treating a disease/condition (e.g., a disease/condition described herein), the method comprising formulating the pharmaceutical composition or drug by mixing the cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

Therapeutic and preventive applications

The articles disclosed herein can be used in therapeutic and preventive methods. In particular, cells according to the disclosure, such as cells comprising/expressing chimeric receptor polypeptides according to the disclosure, can be used in therapeutic and preventive methods. Similarly, compositions according to the disclosure, such as pharmaceutical compositions comprising cells according to the disclosure (such as cells comprising/expressing chimeric receptor polypeptides according to the disclosure) can be used in such methods.

Accordingly, the present disclosure provides a cell or composition as described herein for use in a method of medical treatment or prevention. Also provided is a cell or composition as described herein for use in a method of treating or preventing a disease or condition as described herein. Also provided is the use of a cell or composition as described herein in the manufacture of a medicament for use in a method of treating or preventing a disease or condition as described herein. Also provided is a method of treating or preventing a disease or condition as described herein, comprising administering to an individual a therapeutically or prophylactically effective amount of a cell or composition as described herein.

The interventions described in the previous paragraph may be effective in reducing the development or progression of the disease/condition, reducing the symptoms of the disease/condition, or reducing the pathology of the disease/condition. The intervention may be effective in preventing the progression of the disease/condition, such as preventing the disease/condition from worsening or slowing the rate of progression of the disease/condition. In some embodiments, the intervention may result in an improvement in the disease/condition, such as reducing the symptoms of the disease/condition or reducing some other correlation of the severity/activity of the disease/condition. In some embodiments, the intervention may prevent the progression/development of a later stage of the disease/condition (e.g., a chronic stage or metastasis).

Therapeutic or preventive intervention according to the present disclosure generally comprises administering a cell or pharmaceutical composition according to the present disclosure to an individual, wherein the individual has been or is to be administered an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain according to the present disclosure.

In particular, cells and compositions according to the present disclosure are contemplated for use in methods of treating/preventing diseases/conditions by acquired cell transfusion (ACT).

Transfusion generally refers to the process of obtaining cells (e.g., immune cells) from an individual, usually by drawing a blood sample and isolating the cells therefrom. The cells are then typically modified and/or expanded and then administered to the same individual (in the case of autologous/autologous cell transfusion) or to a different individual (in the case of allogeneic cell transfusion). Treatment is typically aimed at providing an individual with a population of cells that have certain desired characteristics, or at increasing the frequency of such cells in that individual that have such characteristics. Transfusion may be performed for the purpose of introducing a cell or cell population into an individual and/or increasing the frequency of a cell or cell population in an individual.

For example, immune cell transfection is described in, for example, Kalos and June (2013), Immunity 39(1): 49-60, and Davis et al. (2015), Cancer J. 21(6): 486–491, both of which are incorporated herein by reference in their entirety. A skilled artisan will be able to determine appropriate reagents and procedures for cell transfection based on this disclosure, for example by referring to Dai et al., 2016 J Nat Cancer Inst 108(7): djv439, which is incorporated herein by reference in its entirety.

The present disclosure provides methods comprising administering cells and compositions according to the present disclosure to a subject.

Administration of the articles of the present disclosure is preferably carried out in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to show a therapeutic or preventive benefit to an individual. The actual amount administered, as well as the rate and schedule of administration, will depend on the nature and severity of the disease/condition and the specific article being administered. Decisions on treatment prescriptions, such as dosage, are the responsibility of general practitioners and other physicians, and generally take into account the disease/condition to be treated, the condition of the individual individual, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above-mentioned techniques and protocols can be found in Remington's "The Science and Practice of Pharmacy" (Ed. A. Adejare), 23rd edition (2020), Academic Press.

Administration of the articles of the present disclosure may be parenteral, systemic, intravenous, intraarterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, supracortial, subcutaneous, intradermal, intrathecal, oral, intranasal, topical or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral. In some cases, the articles of the present disclosure may be formulated for targeted delivery to specific cells, tissues, organs and/or tumors.

Multiple doses of the articles of the present disclosure may be provided. The multiple doses may be separated by predetermined time intervals, which predetermined time intervals may be selected as one of the following: 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 or 31 days, or 1, 2, 3, 4, 5 or 6 months.

According to the therapeutic and preventive interventions described herein, the cells or compositions according to the present disclosure and the antigen-binding molecules described herein can be administered to a subject simultaneously or sequentially.

Concurrent administration refers to administering (i) a cell or composition according to the present disclosure and (ii) an antigen-binding molecule described herein together, for example as a pharmaceutical composition comprising both agents (i.e., a combination preparation), or administering them immediately one after the other and, as appropriate, via the same administration route, for example, into the same artery, vein, or other blood vessel.

Sequential administration refers to administration of one of (i) a cell or composition according to the present disclosure and (ii) an antigen-binding molecule described herein, followed by administration of the other agent separately after a given time interval. It is not required that the two agents be administered by the same route, although this is the case in some embodiments. The time interval can be any time interval.

Individual

The subject according to the various aspects of the present disclosure can be any animal or human. Therapeutic and preventive applications can be in humans or animals (veterinary use).

The subject to whom the article of the present disclosure is to be administered (e.g., pursuant to a therapeutic or preventive intervention) may be a subject in need of such intervention. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject may be male or female. The subject may be a patient.

An individual may have (e.g., may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease/condition, or may be at risk of developing/infecting such a disease/condition. In embodiments according to the present disclosure, an individual may be selected for treatment based on a method of characterizing one or more markers for such a disease/condition.

In some embodiments, individuals may be selected for therapeutic or preventive intervention as described herein based on the detection of, for example, expression of a target antigen in a sample obtained from the individual (i.e., a target antigen of an antigen binding molecule used in conjunction with a cell or composition according to the present disclosure), or detection of cells/tissues expressing the target antigen.

For interventions according to the present disclosure, an individual may be an allogeneic or non-autologous individual. As used herein, when an individual is referred to herein as "allogeneic" or "non-autologous" with respect to an intervention, the individual is an individual other than the individual from which the intervention cell (i.e., the cell to be administered, or the cell in the pharmaceutical composition/drug to be administered) is derived. An individual to be treated/prevented according to the present disclosure may be genetically different from the individual from which the cell to be administered to the individual (e.g., the cell in the pharmaceutical composition/drug) is derived. An individual to be treated/prevented according to the present disclosure may comprise MHC/HLA genes encoding MHC/HLA molecules (e.g., MHC I α and/or MHC II class molecules) that are different from the MHC/HLA molecules (e.g., MHC I α and/or MHC II class molecules) encoded by the cells to be administered to the individual (e.g., cells in the pharmaceutical composition/drug). An individual to be treated/prevented according to the present disclosure may be HLA mismatched relative to the individual from whom the cells to be administered to the individual (e.g., cells in the pharmaceutical composition/drug) were derived.

The individual to whom cells are administered according to the present disclosure may be allogeneic/non-autologous relative to the source from which the cells to be administered to the individual are derived (e.g., cells in a pharmaceutical composition/drug). The individual to whom cells are administered may be a different individual from the individual from whom the cells are obtained to produce the cells to be administered to the individual (e.g., cells in a pharmaceutical composition/drug). The individual to whom cells are administered may be genetically different from the individual from whom the cells to be administered to the individual are obtained to produce the cells (e.g., cells in a pharmaceutical composition/drug).

For interventions according to the present disclosure, the individual may be an autologous/autologous individual. As used herein, when an individual is referred to herein as "autologous" or "autologous" with respect to an intervention, the individual is the same individual from which the intervention cell (i.e., the cell to be administered, or the cell in the pharmaceutical composition/drug to be administered) is derived. The individual to be treated/prevented according to the present disclosure may be genetically identical to the individual from which the cell to be administered to the individual (e.g., the cell in the pharmaceutical composition/drug) is derived. An individual to be treated/prevented according to the present disclosure may comprise MHC/HLA genes encoding MHC/HLA molecules (e.g., MHC I α and/or MHC II class molecules) identical to MHC/HLA molecules (e.g., MHC I α and/or MHC II class molecules) encoded by cells to be administered to the individual (e.g., cells in a pharmaceutical composition/drug). An individual to be treated/prevented according to the present disclosure may be HLA-matched relative to the individual from whom the cells to be administered to the individual (e.g., cells in a pharmaceutical composition/drug) are derived.

The individual to whom cells are administered according to the present disclosure may be autologous/autologous relative to the source from which the cells to be administered to the individual are derived (e.g., cells in a pharmaceutical composition/drug). The individual to whom cells are administered may be the same individual from whom cells are obtained to produce the cells to be administered to the individual (e.g., cells in a pharmaceutical composition/drug). The individual to whom cells are administered may be genetically identical to the individual from whom cells to be administered to the individual are obtained (e.g., cells in a pharmaceutical composition/drug) to produce the cells.

It must also be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Ranges may be expressed herein as "about" one particular value and/or to "about" another particular value. When such a range is expressed, another embodiment includes the one particular value and/or to the other particular value. Similarly, when a value is expressed as an approximation by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

When a nucleic acid sequence is disclosed herein, its reverse complement sequence is also expressly contemplated.

The methods described herein may preferably be performed in vitro. The term "in vitro" is intended to encompass procedures performed with cells in culture, while the term "in vivo" is intended to encompass procedures performed with/on intact multicellular organisms.

Examples

The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be implemented in view of the general description given above.

Examples 1 Materials and methods

1.1 Recombinant DNA / RNA technology

Manipulate DNA using standard methods, such as Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Use molecular biology reagents according to the manufacturer's instructions. For general information on human immunoglobulin light and heavy chain nucleotide sequences, see: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242.

1.2 DNA sequencing

DNA sequences were determined by double-strand Sanger sequencing.

1.3 Gene synthesis

The desired gene fragments were generated by PCR using appropriate templates or synthesized by automated gene synthesis from synthetic oligonucleotides and PCR products by GeneArt AG (Regensburg, Germany). The gene fragments flanked by single restriction endonuclease cleavage sites were cloned into second generation lentiviral vectors. Plasmid DNA was purified from transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene fragments were designed with appropriate restriction sites to allow subcloning into the respective expression vectors. All constructs were designed with a 5’ end DNA sequence coding for a leader peptide that targets the protein for secretion in eukaryotic cells. When more than one protein chain is expressed, the coding sequences are separated by DNA encoding the P2A/T2A/E2A self-splicing peptides. E2A is derived from equine rhinitis virus; P2A is derived from swine timothy virus-1 2A; and T2A is derived from sphaerotheca mori virus 2A.

1.4 Isolation of T cells from fresh healthy donor blood

Bio-One LeucoSEP™ polypropylene tubes (Greiner, #10349081) were prepared with 15 mL of Histopaque®-1077 density gradient medium (Merck, #10771-500ML) and centrifuged at 400 g for 5 minutes until the liquid was below the filter unit. Fresh blood from an anonymous healthy donor was mixed with Dulbecco's phosphate-buffered saline (DPBS, Merck #D8537-500ML) in a 1:1 ratio and transferred to the LeuSEP™ tubes without disturbing the prepared solution. After centrifugation at 1200 g for 20 min (acceleration: 1; rest: 0), the buffy coat visible as a white layer was separated into a new 50 mL falcon tube using a 10 mL serological pipette. After washing three times with 40 mL DPBS and centrifugation at 780 > 450 > 280 g, the enriched cell population was further manually purified according to the Human Pan T Cell Isolation Kit (Miltenyi, #130-094-535). Briefly, cell pellets were labeled with Pan T Cell Biotin-Antibody, incubated at 4°C for 5 min, and further labeled with Pan T Cell Magnetic MicroBead Cocktail. After negative selection on a MACS® manual separator, the purified Pan T cell population was cultured in Advanced RPMI 1640 (Fisher Scientific, #12633012) + 20% FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) + 20% DMSO (Sigma-Aldrich, #D2650-100ML) or frozen in aliquots of 5×10 ⁶ cells mL ^-1 , placed in a dedicated freezing container at -80°C for at least 24 hours, and then transferred to liquid nitrogen.

1.5 Production of IgG-like proteins in Expi293F cells

Antibodies and antibody-like proteins were produced by transient transfection of Expi293F cells. Cells were seeded at a density of 2.5 × 10 ⁶ mL ^-1 in Expi293 medium (Gibco, #1435101). Expression vector and ExpiFectamine (Gibco, ExpiFectamine Transfection Kit, #13385544) were mixed separately in OptiMEM (Gibco, #11520386). After 5 minutes, the two solutions were combined, mixed by pipetting and incubated at room temperature for 25 minutes. Cells were added to the vector/ExpiFectamine solution and cultured in a shaking incubator at 37°C and 5% CO ₂ for 24 hours. One day after transfection, supplements (Enhancer 1+2, ExpiFectamine Transfection Kit) were added. After 4 to 5 days, cell supernatants were harvested by centrifugation and subsequent filtration (0.2 μm filter), and proteins were purified from the harvested supernatants by standard methods as described below.

1.6 Purification of IgG-like protein

Proteins were purified from filtered cell culture supernatants following standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was performed at pH 3.0, and the pH of the samples was immediately neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated proteins were separated from monomeric proteins by size-selective chromatography in 20 mM histidine, 140 mM NaCl, pH 6.0.

1.7 Production of IgG-like proteins in CHO K1 cells

Alternatively, the antibodies and antibody-like proteins described herein are produced by Evitria using its proprietary vector system and customary (non-PCR-based) cloning techniques and using suspension-adapted CHO K1 cells (originally obtained from ATCC and adapted for serum-free growth in suspension culture at Evitria). For the production, Evitria uses its proprietary animal component-free and serum-free media (eviGrow and eviMake2) and its proprietary transfection reagent (eviFect). The supernatant is harvested by centrifugation and subsequent filtration (0.2 μm filter), followed by purification from the harvested supernatant by standard methods.

1.8 Analysis of IgG-like proteins

The concentration of the purified protein was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. The purity and molecular weight of the protein were analyzed by CE-SDS in the presence and absence of reducing agents using LabChip GXII or LabChip GX Touch (Perkin Elmer). Determination of aggregate content was performed by HPLC chromatography using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (200 mM KH ₂ PO ₄ , 250 mM KCl pH 6.2, 0.02 % NaN ₃ ) at 25°C.

1.9 Preparation of virus-like particles

Approximately 70% confluent Lenti-X™ 293T cells (Takara, #632180) were transfected with CAR/CCR encoding transfer vector and packaging vector pCAG-VSVG and psPAX2 at a molar ratio of 2:1:2 (Giry-Laterriere M et al., Methods Mol Biol. 2011;737:183-209, Myburgh R et al., Mol Ther Nucleic Acids. 2014). As a control for each experiment, mock virus-like particles (VLPs) were generated using packaging vector alone without transfer vector. After 48 h, the supernatant was collected and centrifuged at 500 g for 10 min to remove residual cells and concentrated 10-fold by centrifugation and resuspension according to the manufacturer's protocol (Lenti-x-Concentrator, Takara, #631231).

1.10 Transduction of healthy donor T cells

T cells were quickly thawed in a 37°C water bath and washed with 10x (V:V) Advanced RPMI 1640 (Fisher Scientific, #12633012) + 10% FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) + 50 IU×mL ^-1 interleukin-2 (Miltenyi, #130-097-748) + 25ng×mL ^-1 interleukin-7 (Miltenyi, #130-095-364) + 50ng×mL ^-1 interleukin-15 (Miltenyi, #130-095-766). The cells were then seeded at 10 ⁶ × mL ^-1 in a 12-well plate (Sigma Aldrich, # Z707791-126EA) and activated for 24 hours using Immunocult CD3/CD28/CD2 T cell activator mix (Stemcell Technologies, #10990).

After a brief incubation with 8 µg × mL ^-1 polybrene (Sigma Aldrich) and Lentiboost P (1:100) (Sirion Biotech, #SB-P-LV-101-12), previously purified virus-like particles were added to the activated T cells. After at least 72 hours of incubation, transduction efficiency was assessed by flow cytometry using fluorescently labeled anti-CAR antigen or commercial fluorescently labeled anti-tag antibodies.

1.11 T cell culture

Engineered T cells or wild-type T cells were cultured in G-Rex® 24 multiwell cell culture plates (Wilson Wolf, #80192M) at a density of 0.2×10 ⁶ mL ^-1 to 4×10 ⁶ ML ^-1 in advanced RPMI 1640 (Fisher Scientific, #12633012) + 10% FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) + 50 IU×mL ^-1 interleukin-2 (Miltenyi, #130-097-748) + 25 ng×mL ^-1 interleukin-7 (Miltenyi, #130-095-364) + 50 ng×mL ^-1 interleukin-15 (Miltenyi, #130-095-766) in culture.

1.12 Flow cytometry surface staining

Transduction efficiency or cell signaling events were assessed by flow cytometry. To control for transduction efficiency, constructs were tagged with a FLAG epitope, either by co-expression of GFP via a 2A self-cleaving peptide, or by using fluorescently labeled antigens. Signaling events were assessed by alternate modulation of surface markers. 100,000 cells were prepared in 96-well plates and stimulated with dilutions of interleukin IL12 (recombinant human IL12 (conjugated heterodimer) protein, R&D Systems #10018-IL-050) or IL23 (recombinant human IL23 protein, Fisher Scientific #PHC9321). After incubation at 37°C in an incubator with a 5% CO ₂ atmosphere for 24 hours, the cells were washed with DPBS, centrifuged at 300g, and stained with 50 µL of pre-diluted antibody solution at 4°C for 30 minutes. The cells were washed twice more and then fixed with 4% PFA solution (fixation buffer, BD Biosciences, #554655). The cells were washed once more before finally resuspending in FACS buffer (PBS containing 2% FBS, 10% 0.5 M EDTA (pH 8) and 0.5 g×L ^-1 NaN ₃ ) and analyzed on a BD LSRFortessa™ cell analyzer.

1.13 Phospho-STAT staining

In preparation for the assay, 200,000 cells were washed with Advanced RPMI 1640 (Fisher Scientific, #12633012) + 10% FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) without the addition of interleukins and serum starved for 4 to 12 hours in the same medium in a 96-well plate. Cells were stimulated with serial dilutions of interleukins and incubated at 37°C in an incubator with a 5% _CO2 atmosphere for 40 minutes. Immediately thereafter, cells were fixed and permeabilized (BD Phosflow™ Fixation Buffer I, BD Biosciences, #557870) at 37°C for 15 minutes. After centrifugation at 300 g for 2 minutes, the cells were resuspended in Phosflow Perm Buffer III (BD Biosciences, 558050) and incubated at 4°C for 30 minutes. The cells were washed twice more in DPBS and then stained with mouse anti-pSTAT4 antibody conjugated to Alexa Fluor™ 647 (BD, #558137) and mouse anti-pSTAT3 antibody conjugated to PE (BD, #562072) using pre-diluted antibody solution for 60 minutes at 4°C. After washing the cells once more in FACS buffer (PBS containing 2% FBS, 10% 0.5 M EDTA (pH 8) and 0.5 g × L ^-1 NaN ₃ ), the cells were collected on a BD LSRFortessa™ cell analyzer.

1.14 Homogeneous Time-of-Flight Fluorescence

Homogeneous time-lapse fluorescence assay was used as an alternative to ELISA to assess secreted interleukin concentrations. Secreted interferon-γ concentrations in cell culture supernatants were determined using the human IFN-γ kit (Perkin Elmer CisBio, #62HIIFNGPET) according to the manufacturer's protocol. Briefly, 100,000 cells were washed with Advanced RPMI 1640 (Fisher Scientific, #12633012) + 10% FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) + 50 IU mL ^-1 interleukin-2 (Miltenyi, #130-097-748) + 25 ng×mL-1 interleukin-7 (Miltenyi, #130-095-364) + 50 ng×mL-1 interleukin-15 (Miltenyi, #130-095-766). Cells were stimulated with serial dilutions of IL12 and IL23 and incubated at 37°C in an incubator with a 5% _CO2 atmosphere for 24 hours. IFNγ EU Cryptate Antibody and IFNγ XL Antibody were diluted 20x in assay buffer. Next, the supernatant was combined with a 4:1 dilution of pre-mixed donor and acceptor antibodies in a 1:1 ratio and incubated for 4 to 12 hours at room temperature. Standards were obtained in serial 1:3 dilutions from 4000 pg×mL ^-1 to 0 pg×mL ^-1 . FRET-fluorescence emission was measured at 665 nm (acceptor) and 620 nm (donor). Data were normalized to the donor signal, background subtracted and fitted to the IFNγ standard curve obtained in the same assay.

1.15 Incucyte poisoning assay

Target cells (MKN-45-NLR or HPAF-II-NLR) were plated in RPMI 1640 (Fisher Scientific, #11875093) + 2% FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) in a 96-well plate with rimmed reservoirs (Fisher Scientific, #167425) and incubated for at least 2 hours. Engineered cells were washed twice in Dulbecco's Phosphate Buffered Saline (DPBS, Merck #D8537-500ML) and resuspended in RPMI 1640 (Fisher Scientific, #11875093) + 2% FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061). Cell density was normalized to an equal percentage of CAR-positive cells before addition to target cells. Engineered T cells were used at varying effector to target cell ratios (E:T) for direct CARs or at a fixed E:T with dilutions of targeting IgG for adapter-based CARs. Target cell cytotoxicity was monitored for 7 days in the Incucyte® Live Cell Assay System. The assay was terminated when growth of target cells alone reached plateau. Data were normalized to the initial cell concentration and growth of target cells in the absence of effector cells.

In the case of a replicate assay setup (e.g., as in Example 9), target cell cytotoxicity was monitored for 2 days per round in the Incucyte® Live Cell Assay System, as indicated by the dashed line. RPMI1640 (Fisher Scientific, #11875093) supplemented with 1x GlutaMAX™ (Fisher Scientific, #35050061) and 10% FBS (Sigma-Aldrich) was selected as the culture medium. Engineered T cells were washed twice in PBS and normalized to the number of CAR-positive cells by adding wild-type cells, and then added to the target cells at a 1:1 E:T ratio. Finally, IL12 or IL23 was added as indicated. Two days later, corresponding to one round later, new target cells were seeded in new dishes. Engineered T cells were also separated from cancer cells by gentle resuspension and aspiration. Supernatant was removed and fresh medium was added before combining engineered T cells with new target cells. Finally, IL12 or IL23 was added again as indicated. Analysis was continued in the same Incucyte® Live Cell Assay System. This process was repeated 4 times for a total of five rounds of replicate killing. Data were normalized to the initial cell concentration in each round.

Examples 2 ：Human immune cells IL23 – IL12 Detection of chimeric interleukin receptors

T cells isolated from 50 mL of healthy donor blood were transduced with VLPs containing transgenes encoding different IL23-IL12 chimeric interleukin receptors (IL23-IL12sR). Peripheral blood mononuclear cells (PBMCs) were first isolated from fresh human blood donations by density gradient centrifugation. Untouched human Pan T cells were further enriched in the cell population by negative selection using the Human Pan T Cell Isolation Kit (Miltenyi, #130-096-535). The cells were then cultured in Advanced RPMI 1640 + 10% FBS + 1x GlutaMAX + 50 IU×mL ^-1 interleukin-2 + 25 ng×mL ^-1 interleukin-7 + 50 ng×mL ^-1 interleukin-15 for 24 hours and then stimulated with Immunocult CD3/CD28/CD2 T Cell Activator Mix (StemCELL technologies, #10990) to induce clonal expansion. Previously prepared 10x concentrated VLPs encoding the desired constructs were added to the cells along with polybrene (Sigma, #TR-1003-G) and LentiBoostP (Sirion Biotech, #SB-A-LF-901-01) for subsequent incubation for at least 72 hours. Expression of full-length constructs was assessed by eGFP reporter gene fluorescence, fluorescently labeled anti-CAR antigens (e.g., Biot-Avi-tag_Fc_huCEACAM5(A3B3) or Fc_(PGLALA)_AF647). Where applicable, surface expression of IL23-IL12sR was assessed directly by flow cytometry using an N-terminal FLAG tag (DYKDDDDK) and a commercially available fluorescently labeled anti-FLAG tag antibody (Biolegend, #637322). More specifically, 100,000 cells were harvested per condition, washed with PBS and plated in 96-well V-bottom plates. After staining with 20 nM Fc_(PGLALA)_AF647 and a 1:200 dilution of BV421 anti-FLAG-tag antibody in PBS at 4°C in the dark for 30 min, the samples were washed twice more and then fixed with 4% PFA solution (fixation buffer, BD Biosciences, #554655). The cells were washed once more before resuspending in FACS buffer (PBS containing 2% FBS, 10% 0.5 M EDTA (pH 8) and 0.5 g×L ^-1 NaN ₃ ) and analyzed on a BD LSRFortessa™ cell analyzer.

All tested constructs were expressed after transduction of primary T cells from healthy human donors, as detected indirectly by the fluorescent signal of the eGFP reporter protein (Fig. 2A). In addition, all recombinant receptors could be directly detected by staining for the N-terminal FLAG-tag (Fig. 2B). Chimeric antigen receptors encoded on the same construct were expressed in comparable ratios, as indicated by the detection of compatible fluorescently labeled CAR antigens (Fig. 2B).

Examples 3 :by IL23-IL12sR Analysis of phosphorylation of signal transducers and transcriptional activators in engineered human immune cells

Analysis of phosphorylation of signal transducers and activators of transcription (STAT) utilizing IL23-IL12sR signaling was performed by flow cytometry.

T cells transduced with IL23–IL12sR were stimulated with serial dilutions of recombinant human interleukin 23 (huIL23) (Thermo Fisher Scientific, #PHC9321) for 40 min and immediately fixed (BD, #557870), permeabilized (BD, #558050), and stained intracellularly with mouse anti-pSTAT4 antibody conjugated to Alexa Fluor™ 647 (BD, #558137) and mouse anti-pSTAT3 antibody conjugated to PE (BD, #562072) for flow cytometric analysis. Cell gates were set for lymphocytes, monocytes (FSC-A and FSC-H), monocytes (SSC-A and SSC-H), and GFP-positive cells (if applicable). IL23–IL12sR-modified cells showed dose-dependent phosphorylation of STAT4 (Jacobson et al., 1995, Kaplan et al., 1998) but not STAT3 (Chen et al., 2003) when stimulated with IL23, similar to the endogenous IL12 receptor complex stimulated with IL12 (Figure 3A, Figure 3B). Notably, the EC50 for upregulation of pSTAT4 via IL23-IL12sR was higher and the maximal message appeared to be increased. Co-phosphorylation of STAT4 and STAT3 was evident only in cells overexpressing IL23R (Fig. 3C, D).

Examples 4 : Engineered human immune cells IL23-IL12sR Functional analysis of mitochondrial upregulation of characteristic cell surface markers

Functional analysis of IL23-IL12sR was accomplished by flow cytometric analysis of cell surface markers known to be expressed upon IL12 signaling. These markers include IL18Ra, the most prominent (Yoshimoto et al., 1998), IL12Rb2, which is mediated by STAT4 (Valenzuela et al., 2002), as well as PD-1 (Gerner et al., 2013) and CD25 (Valenzuela et al., 2002), although PD-1 and CD25 are irreproducible and donor-dependent, respectively. T cells transduced with IL23-IL12sR carrying an eGFP reporter gene were stimulated for 24 hours with serial dilutions of huIL23 (Thermo Fisher Scientific, #PHC9321). After incubation, cells were stained (constructs no. 1, 2: panel 1, constructs no. 3, 4, 5, 6, 7: panel 2, constructs no. 8, 9: panel 3) and fixed for flow cytometric analysis. The panels are described in Tables 1 to 3 below. Cells were gated for lymphocytes, monomorphic (FSC-A and FSC-H), and live cells expressing CD3 and GFP reporter or anti-CAR antigen (if applicable). IL23–IL12sR showed dose-dependent upregulation, with the most notable being IL18Ra (Figure 4A). Here, the EC50 of IL23-IL12sR_(IL12Rb2-TMD) was reduced by approximately 100-fold, and the EC50 of IL23IL12sR_(IL23R-TMD) was reduced by 1000-fold compared to the message of IL12 and endogenous IL12 receptor complex. Similarly, IL12Rb2 was upregulated by stimulation of IL23-IL12 chimeric interleukin receptor with IL23 (Fig. 4B), which is consistent with the previously reported feed-forward mechanism triggered by STAT4 signaling (e.g., Valenzuela et al. 2002). In this example, IL23-IL12sR (IL12Rb2-TMD) T cells displayed a lower IL12Rb2 baseline, more similar to wild-type cells, when compared to IL23-IL12sR (IL23R-TMD). Specifically, in wild-type cells, this feeding mechanism cannot be visualized in this way because the selected anti-IL12Rb2 antibody (Biolegend, #394205) competes with IL12 and is therefore either eliminated or high concentrations of IL12 lead to increased internalization of IL12Rb2. In T cells engineered with the 41BB-based P329G CAR plus IL23-IL12sR, upregulation of IL18Ra (Figure 5A) showed similar dose-responsive behavior with a higher EC50 and higher maximum message. Feed-forward upregulation of IL12Rb2 was also observed (Figure 5B). Table 1. Panel 1. Target molecular original Catalog Number GFP Not applicable Not applicable Not applicable IL18Ra APC Biolegend 313814 IL12Rb2 PE BD Biosciences 550723 PD1 BV421 Biolegend 329920 CD25 BV605 Biolegend 302631 CD8 BV711 Biolegend 301043 CD3 BUV395 BD Biosciences 740283 Live/DEAD ^™ Aqua Aqua405 Thermo Fisher Scientific L34957 Table 2. Panel 2. Target molecular original Catalog Number PG-CAR Fc_(PGLALA)_AF647 Interior IL18Ra FITC Biolegend 313810 IL12Rb2 PE BD Biosciences 550723 PD1 BV421 Biolegend 329920 CD25 BV605 Biolegend 302631 CD8 BV711 Biolegend 301043 CD3 BUV395 BD Biosciences 740283 Live/DEAD ^™ Aqua Aqua405 Thermo Fisher Scientific L34957 Table 3. Panel 3. Target Molecules / Fluorophores original Catalog Number PG-CAR Biot-Avi-Tag_Fc_huCEACAM5(A3B3) Interior Avi-Tag Streptavidin-AF647 Biolegend 405237 IL18Ra FITC Biolegend 313810 IL12Rb2 PE BD Biosciences 550723 PD1 BV421 Biolegend 329920 CD25 BV605 Biolegend 302631 CD8 BV711 Biolegend 301043 CD3 BUV395 BD Biosciences 740283 Live/DEAD ^™ Aqua Aqua405 Thermo Fisher Scientific L34957

Examples 5 : Human immune cells engineered to respond to IL23-IL12sR Interferon - γ secretion

Upregulation of interferon-γ (IFNγ) in primary T cells from human healthy donors transduced with IL23-IL12sR (Yoshimoto et al., 1998) was assessed by stimulation with serial dilutions of huIL23 (Thermo Fisher Scientific, #PHC9321) for 24 hours. After incubation, cell supernatants were homogenized and transferred to new containers. The presence of IFNγ was detected using the HTRF kit (Cis-Bio, #62HIFNGPEG). Briefly, supernatants were mixed with IFNγ EU Cryptate antibody and IFNγ XL antibody, sealed and incubated at room temperature for >4 hours. Readout was performed by measuring FRET-induced fluorescence emission at 665 nm (acceptor) and fluorescence at 620 nm (donor). Background was subtracted and relative signal was fit to a standard curve of recombinant IFNγ. When stimulated with huIL23, IL23-IL12sR showed a dose-dependent upregulation of IFNγ (Figure 5C). Notably, IL23-IL12sR stimulation resulted in IFNγ release only when a co-stimulatory CAR was co-present (Figure 5D).

Examples 6 : Use IL23-IL12sR Engineered human immune cells for cytotoxicity assays

Evaluation of IL23-IL12sR-mediated target cell cytotoxicity in the context of adapter-CAR. Initially, healthy donor human T cells were purified, activated and transduced with VLPs carrying the respective CAR and IL23-IL12sR versions. Cells were expanded for >5 days at 37°C in a 5% _CO2 atmosphere. Prior to preparing the cytotoxicity assay, the transduction efficiency of immune cells was assessed by flow cytometry as described in Example 1. Target cells MKN-45-NLR or HPAF-II-NLR were seeded in 96-well plates with rimmed reservoirs and incubated for at least 2 hours to adhere to the plates. Engineered cells were washed twice in PBS and normalized to the number of CAR-positive cells by adding wild-type cells before adding to target cells. For transferrin-based CAR-T cells, engineered T cells were inoculated with a dilution of targeting IgG at a fixed E:T. Target cytotoxicity was observed for 5 to 7 days in the Incucyte® Live Cell Assay System. Data were normalized to the initial cell concentration and growth of target cells without effector cells. Under suboptimal cytotoxic conditions (e.g., non-saturating concentrations of targeting IgG), the addition of 20 nM huIL23 to human immune cells engineered with CAR and IL23-IL12sR resulted in a 20% to 40% increase in target cell cytotoxicity (Fig. 6A,B).

Examples 7 : IL23-IL12sR In direct CAR Function in engineered human immune cells

The detection of chimeric constructs, analysis of signaling, functional analysis, and cytotoxicity of engineered human immune cells were evaluated similarly to Examples 2 to 6. Direct CEA-CAR was detected in flow cytometry using Biot-Avi-Tag_Fc_hu_CEACAM5(A3B3) and streptavidin-AF647 (Figure 7A). To show that the FLAG-epitope used in other examples does not affect the function of the chimeric receptor, it was omitted in the design of this example. Therefore, only IL23-IL12sR was functionally tested by upregulation of IL18Ra, IL12Rb2, CD25, and IFNγ, consistent with previous assays (Figure 7B). Under suboptimal cytotoxic conditions (e.g., low effector to target (E:T) ratios), addition of 20 nM huIL23 to human immune cells engineered with CEA-CAR and IL23-IL12sR resulted in a 10% to 50% increase in target cell cytotoxicity (Fig. 8A, 8B).

Examples 8 ：When transduced in human immune cells, it has chimerism ECD Of IL23-IL12sR Designed to have similar or improved sensitivity and performance

Detection of chimeric constructs, analysis of signaling, and/or functional analysis are evaluated similarly to Examples 2 to 7.

These constructs were expressed after transduction of primary T cells from healthy human donors, as detected indirectly by expression of the P329G CAR by staining with Fc_(PGLALA)_AF647, and directly by staining the FLAG-tag with a fluorescently labeled anti-FLAG antibody (Figure 9A). This suggests that the IL23-IL12sR design with an intact IL23R-ECD and either IL12Rb2-TMD or IL23R-TMD is superior to designs with a chimeric ECD in influencing the expression of the upstream CAR as well as the expression of the receptor itself. Functionally, when stimulated with serial dilutions of IL23 in a dose-dependent manner, stimulation with only the IL23-IL12sR variants with chimeric ECD1 and intact ECD2-3 interface showed improved sensitivity and maximal signal across readout (IL18Ra, IL12Rb2, IFNγ) (Figure 9B). In target cytotoxicity experiments with MKN-45 cells, all T cells engineered with P329G CAR-28z and IL23-IL12sR variants, except for the variant with IL23-TMD, showed increased CAR-T cell cytotoxicity, especially at low E:T ratios and when stimulated with IL23 (Figure 10).

Examples 9 : Use IL23-IL12sR Engineered human immune cells for repeated cytotoxicity assays

In preparation for the assay, engineered human immune cells were generated and cultured as previously outlined (e.g., according to Example 1). Target cells HPAF-II-NLR were seeded in 96-well plates. Engineered T cells (transfected with construct #12 [SEQ ID NO: 66 and 67] or construct #13 [SEQ ID NO: 68 and 6]) were co-cultured with target cells at a 1:1 E:T ratio with or without the addition of cytokines at 2-day intervals and then transferred to new target cells for five rounds of repetitive killing. In the absence of added IL12 or IL23, CAR-T cells can control cancer cell growth for 2 rounds (Figure 11). The cytotoxicity of the two CAR-T cell populations was similar regardless of whether they were co-engineered with IL23-IL12sR. The cytotoxicity of CAR-T cells could be prolonged by continuously adding high doses of IL23 to the CAR-T cells. However, only when IL12 or IL23 was added to the CAR IL23-IL12sR T cells did they effectively kill cancer cells in five rounds (Figure 11). Sequence Listing SEQ ID NO: 1 (huIL23R_SP) MNQVTIQWDAVIALYILFSWCHG SEQ ID NO: 2 (huIL23R_ECD) GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGG KKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIG SEQ ID NO: 3 (huIL12Rb2_TMD) WMAFVAPSICIAIIMVGIFST SEQ ID NO: 4 (huIL23R_TMD) LLLGMIVFAVMLSILSLIGIF SEQ ID NO: 5 (huIL12Rb2_ICD) HYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPP PRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 6 (T2A) GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 7 (EGFP) VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 8 (FLAG-tag) DYKDDDDK SEQ ID NO: 9 (E2A) GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 10 (huIL23R_ECD1) GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPHFQETLICGKDISSGY SEQ ID NO: 11 (huIL23R_ECD2) SEQ ID NO: 12 (huIL23R_ECD3) SAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTP SEQ ID NO: 13 (huIL12Rb2_ECD2) APEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRP SEQ ID NO: 14 (huIL12Rb2_ECD3) LPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTP SEQ ID NO: 15 (huIL12RB2_ECD4-6) EEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQV PLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKAN SEQ ID NO: 16 (SP) MGWSCIILFLVATATGVHS SEQ ID NO: 17 (huP329G_VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSS SEQ ID NO: 18 ((G4S)4) GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 19 (huP329G_VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL SEQ ID NO: 20 (G4S) GGGGS SEQ ID NO: 21 (huCD8_stalk) AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 22 (huCD8_TMD) IYIWAPLAGTCGVLLLSLVIT SEQ ID NO: 23 (hu4-1BB ICD) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 24 (huCD3z_ICD) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 25 (huIL23R_ICD) NRSFRTGIKRRILLLIPKWLYEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKKENTGPLETRDYPQNSLFDNTTVVYIPDLNTGYKPQISNFLPEGSHLSNNN EITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHFNRISLLEK SEQ ID NO: 26 (anti-huCEACAM5_T84.66_VH) EVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWGQGTSVTVSS SEQ ID NO: 27 (anti-huCEACAM5_T84.66_VL) DIVLTQSPASLAVSLGQRATMSCRAGESVDIFGVGFLHWYQQKPGQPPKLLIYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIK SEQ ID NO: 28 (anti-CEACAM5 IgG light chain) DIVLTQSPASLAVSLGQRATMSCRAGESVDIFGVGFLHWYQQKPGQPPKLLIYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 29 (anti-CEACAM5 IgG heavy chain) EVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWG QGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 30 (Fc_(P329G L9A L10A) CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 31 (Biot-Avi-tag_Fc_huCEACAM5(A3B3) heavy chain) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 32 (Biot-Avi-tag_Fc_huCEACAM5(A3B3) light chain) GLNDIFEAQKIEWHEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGGGSQLTTESMPFNVAEGKEV LLLVHNLPQQLFGYSWYKGERVDGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLP VSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSAS SEQ ID NO: 33 (huCD28_TMD) FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 34 (huCD28_ICD) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 35 (IL23R ECD1+2) GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISS GYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIP SEQ ID NO: 36 (Construct #1) MNQVTIQWDAVIALYILFSWCHGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNA GKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQ ETGKRYWQPWSSLFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQ VTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 37 (Construct #2) MNQVTIQWDAVIALYILFSWCHGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECP KHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIH LDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPETVP QVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGLLLGMIVFAVMLSILSLIGIFHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLI DWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGY LPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLMLGSGERGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNG HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK EDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 38 (Construct #3) MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGG TVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPM NQVTIQWDAVIALYILFSWCHGDYKDDDDKGGSGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSG PKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYS RQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSI LGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQL PLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 39 (Construct #4) MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGG TVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPG PMNQVTIQWDAVIALYILFSWCHGDYKDDDDKGGSGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVH VKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQ QISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSIL GNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQL PLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 40 (Construct #5) MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGG TVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGP MNQVTIQWDAVIALYILFSWCHGDYKDDDDKGGSGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVK SLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPEEEPTGMLDVWYMKRHIDYSR QQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSIL GNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQL PLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 41 (Construct #6) MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGGGGSAKPTTTPAP RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPMNQVTIQWDAVIALYILFSWCHGDYKDDDDKGGSGGITNINCSG HIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQ YLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSPFFH KTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHP PCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 42 (Construct #8) MGWSCIILFLVATATGVHSEVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWGQGTSVTVS SGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATMSCRAGESVDIFGVGFLHWYQQKPGQPPKLLIYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIKGGGGSAKPTTTP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPMNQVTIQWDAVIALYILFSWCHGGITNINCSGHIWVEPA TIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSY INISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSPFFHKTPE TVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPC SNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 43 (Construct #10) MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGGGGSAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPMNQVTIQWDAVIALYILFSWCHGDYKDDDDKGGSGGITNINC SGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQ QYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSPFF HKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRH PPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 44 (Construct #11) MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGGGGSAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPMNQVTIQWDAVIALYILFSWCHGDYKDDDDKGGSGGITNINC SGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQ QYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSPFF HKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGLLLGMIVFAVMLSILSLIGIFHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRH PPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 45 (Construct #7) MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGGGGSG GGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVLGGGGSAKPTTTPAPRPPTPAPT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPMNQVTIQWDAVIALYILFSWCHGDYKDDDDKGGSGGITNINCSGHIWVEPATIFKMGMNIS IYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLV WVQAANALGMEESKQLQIHLDDIVIPSSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSPFFHKTPETVPQVTSKAFQHDTWNSGLTVA SISTGHLTSDNRGDIGLLLGMIVFAVMLSILSLIGIFNRSFRTGIKRRILLLIPKWLYEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKKENTGPLETRDYPQNSLFDNTTVVYIPDLNTGY KPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHFNRISLLEK SEQ ID NO: 46 (Construct #9) MGWSCIILFLVATATGVHSEVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATMSCRAGESVDIFG VGFLHWYQQKPGQPPKLLIYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIKGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 47 (IL12Rb2 ECD2-6) APEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWR DEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKA MTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEI RVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKAN SEQ ID NO: 48 (IL12Rb2 ECD3-6) LPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTG MLDVWYMKRHIDYSRQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLA PRQVSANSEGMDNILVTWQPPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPL SGPHINAITEEKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKAN SEQ ID NO: 49 (VH3 CDR H1) RYWMN SEQ ID NO: 50 (VH3 CDR H2) EITPDSSTINYAPSLKG SEQ ID NO: 51 (HCDR2) EITPDSSTINYTPSLKG SEQ ID NO: 52 (VH3 CDR H3) PYDYGAWFAS SEQ ID NO: 53 (VL1 CDR L1) RSSTGAVTTSNYAN SEQ ID NO: 54 (VL1 CDR L2) GTNKRAP SEQ ID NO: 55 (VL1 CDR L3) ALWYSNHWV SEQ ID NO: 56 (VH3 VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSS SEQ ID NO: 57 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYTPSLKGRFTISSRDNAKNSLYLQMNSLRAEDTAVYYCVRPYDYGAWFASWGQGTLVTVSS SEQ ID NO: 58 (VH) EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMNWVRQAPGKGLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRPYDYGAWFASWGQGTLVTVSS SEQ ID NO: 59 (VH3VL1 (ds) scFv) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMNWVRQAPGKCLEWVGEITPDSSTINYAPSLKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPYDYGAWFASWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGCGTKLTVL SEQ ID NO: 60 (VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL SEQ ID NO: 61 (VL1 (ds) VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGCGTKLTVL SEQ ID NO: 62 (Exemplary chimeric receptor polypeptide) GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLE TEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSS PFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFR HPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 63 (Exemplary chimeric receptor polypeptide) GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDI PDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETIN ATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKTPEEEPTGMLDVWYMKRHIDYSR QQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQP PRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQ EQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFV LLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQ AESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 64 (Exemplary chimeric receptor polypeptide) GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDI PDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPLPPWDIRIKFQ KASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYSRQ QISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQPP RKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQE QMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFVL LAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 65 (Exemplary chimeric receptor polypeptide) GITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYAPEQP QNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRI KFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHIDYS RQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWTTVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGMDNILVTWQ PPRKDPSAVQEYVVEWRELHPGGDTQVPLNWLRSRPYNVSALISENIKSYICYEIRVYALSGDQGGCSSILGNSKHKAPLSGPHINAITEEKGSILISWNSIPVQ EQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNSHPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKANWMAFVAPSICIAIIMVGIFSTHYFQQKVFV LLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQ AESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 66 (Construct #12 [CEA-CAR-28z_IL23-IL12sR (IL12Rb2_TMD)] – protein sequence) MGWSCIILFLVATATGVHSEVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWGQGTSVTVS SGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATMSCRAGESVDIFGVGFLHWYQQKPGQPPKLLIYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIKGGGGSAKPTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPMNQVTIQWDAVIALYILFSWCHGGITNINCSGHIWV EPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNAGKLTYIDTKYVVHVKSLETEEEQQYLT SSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSLFFHKT PETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPP CSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML SEQ ID NO: 67 (Construct #12 [CEA-CAR-28z_IL23-IL12sR (IL12Rb2_TMD)] – DNA sequence) ATGGGCTGGAGCTGCATCATTCTGTTTCTGGTGGCCACAGCCACAGGCGTGCATTCTGAAGTTCAGCTGCAGCAGTCTGGCGCCGAACTTGTTGAACCTGGCGCCTCTGTGAAGCTGTCTTGTACCGCCAGCGGCTTTCAACATCAAGGACACCTACATGCACTGGGTCAAGCAGAGGCCTGAGCAGGGACTCGAATGGATCGGAAGAAT CGACCCCGCCAACGGCACAGCAAATACGTGCCCAAGTTCCAGGGCAAAGCCACCATCACAGCCGACACAAGCAGCAACACAGCCTACCTGCAGCTCACCAGCCTGACATCTGAGGACACCGCCGTGTACTACTGCGCCCCTTTTGGCTACTACGTGTCCGACTACGCCATGGCCTATTGGGGCCAGGGCACATCTGTGACAGTTTCTAG CGGAGGCGGAGGAAGTGGTGGCGGAGGTTCTGGCGGCGGAGGATCTGATATTGTGCTGACACAGAGCCCTGCCAGCCTGGCTGTTTCTTGGACAGAGGGCCACCATGTCTTGTAGAGCCGGCGAGTCCGTGGATATCTTCGGCGTGGGATTCCTGCACTGGTATCAGCAGAAACCCGGCCAGCCTCCTAAGCTGCTGATCTACAGAGC CAGCAACCTGGAATCTGGCATCCCTGTGCGGTTTAGCGGCACCGGCAGCAGAACCGATTTCACCCTGATCATCGACCCTGTGGAAGCCGATGACGTGGCCACCTACTACTGCCAGCAGACCAATGAGGACCCCTACACCTTTGGCGGAGGCACCAAGCTGGAAATCAAAGGCGGCGGAGGATCCGCCAAGCCTACAACAACACCAGCTCC TAGACCTCCAACTCCTGCTCCTACAATCGCCAGCCAGCCTGTCTCTGAGGCCTGAAGCTTGTAGACCAGCAGCTGGCGGAGCCGTGCATACCAGAGGACTGGATTTCGCCTGCGACTTCTGGGTGCTCGTGGTTGTTGGCGGAGTGCTGGCCTGTTACTCTCTGCTGGTCACCGTGGCCTTCATCATCTTTTGGGTCAGAAGCAAGC GGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGGAGCAGAGTGAAGTTTAGCAGATCAGCCGACGCTCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGGCGCAGAGAAGAGTACG ACGTGCTGGATAAGAGAAGAGGCAGAGATCCCGAGATGGGCGGCAAGCCCCAGAGAAGAAAGAATCCCCAAGAGGGCCTCTACAACGAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAACGCAGAAGAGGAAAGGGCCACGACGGACTGTATCAGGGCCTGTCTACAGCCACCAAGGACACCTATG ATGCCCTGCACATGCAGGCCCTGCCTCCAAGAGGATCTGGCCAGTGTACCAACTATGCCCTGCTGAAGCTAGCCGGCGACGTTGAATCTAATCCCGGGCCTATGAATCAAGTGACCATCCAGTGGGATGCCGTGATCGCCCTGTATATCCTGTTCAGCTGGTGCCACGGCGGCATCACCAACATCAATTGCTCTGGCCACATCTGGGTCG AGCCCGCCACAATCTTCAAGATGGGCATGAACATCAGCATCTACTGCCAGGCCGCCATCAAGAACTGCCAGCCTAGAAAGCTGCACTTCTACAAGAACGGCATCAAAGAGCGGTTCCAGATCACCCGGATCAACAAGACCACCGCCAGACTGTGGTACAAGAACTTCCTGGAACCTCACGCCAGCATGTACTGCACCGCCGAGTGTCCC AAGCACTTCCAAGAGACACTGATCTGCGGCAAGGACATCAGCAGCGGCTACCCTCCAGACATCCCTGATGAAGTGACCTGCGTGATCTACGAGTACAGCGGCAACATGACCTGCACCTGGAATGCCGGCAAGCTGACCTACATCGACACCAAATACGTGGTGCACGTGAAGTCCCTGGAAACCGAGGAAGAACAGCAGTACCTGACCAGC AGCTACATCAACATCTCCACCGATAGCCTGCAAGGCGGCAAGAAATACCTCGTGTGGGTGCAAGCCGCCAACGCTCTCGGAATGGAAGAGAGCAAGCAGCTGCAGATCCACCTGGACGACATCGTGATTCCATCTGCCGCCGTGATCAGCAGAGCCGAGACAATCAATGCCACCGTGCCTAAGACCATCATCTACTGGGACAGCCAGACC ACCATCGAGAAGGTGTCCTGCGAGATGAGATACAAGGCCACCACCAACCAGACCTGGAACGTGAAAGAGTTCGATACCAACTTCACCTACGTGCAGCAGAGCGAGTTCTACCTCGAACCGAACATCAAATATGTGTTCCAAGTGCGGTGCCAAGAAACCGGCAAGAGATACTGGCAGCCTTGGAGCAGCCTCTTCTTCCACAAGACCCCT GAGACAGTGCCCCAAGTGACAAGCAAGGCCTTCCAGCACGATACCTGGAATAGCGGCCTGACAGTGGCCAGCATTAGCACCGGTCACCTGACCTCCGACAACAGAGGCGATATCGGATGGATGGCCTTCGTGGCCCCTAGCATCTGTATCGCCATCATGGTCGGAATCTTCAGCACCCACTACTTCCAGCAGAAAGTGTTCGTGCTG TTGGCTGCTCTGCGGCCGCAGTGGTGTAGCAGAGAAATCCCCGATCCTGCCAACTCTACCTGCGCCAAGAAGTACCCTATCGCCGAGGAAAAGACCCAGCTGCCTCTGGACAGACTGCTGATCGATTGGCCTACACCTGAGGACCCTGAGCCTCTGGTTATCAGCGAAGTGCTGCACCAAGTGACCCCTGTGTTCAGACACCCTCCTTGC AGCAACTGGCCTCAGAGAGAGAAGGGAATCCAGGGACACCAGGCCAGCGAGAAGGATATGATGCACTCTGCCAGCTCTCCACCTCCTCCAAGAGCACTTCAGGCCGAAAGCAGACAGCTGGTGGACCTGTATAAGGTGCTGGAAAGCAGGGGCAGCGACCCCAAGCCTGAAAATCCAGCTTGTCCTTGGACTGTGCTGCCTGCCGGCGAT CTGCCTACACACGATGGCTACCTGCCTAGCAACATCGACGATCTGCCATCTCATGAGGCCCCTCTGCCCGACTCTCTGGAAGAACTGGAACCACAGCACATCAGCCTGAGCGTGTTCCCCAGTAGCTCTCTGCACCCTCTGACCTTCAGCTGCGGCGATAAGCTGACCCTGGATCAGCTGAAGATGAGATGCGACAGCCTGATGCTGTGA SEQ ID NO: 68 (Construct #13 [CEA-CAR-28z_GFP] – protein sequence) MGWSCIILFLVATATGVHSEVQLQQSGAELVEPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKATITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYYVSDYAMAYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATMSCRAGESVDIFGVG FLHWYQQKPGQPPKLLIYRASNLESGIPVRFSGTGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTFGGGTKLEIKGGGGSAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGQCTNYALLKLAGDVESNPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 69 (Construct #13 [CEA-CAR-28z_GFP] – DNA sequence)

Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures. FIG1A . Scientific rationale for the design of IL23-IL12sR for relay cell therapy. A) Heat map depicting the distribution of IL23 in tumor and normal tissues, compiled from >11,000 RNA-seq samples from The Cancer Genome Atlas (TCGA) database. Several tumors (primarily lung and colon) showed transcriptomic evidence of overexpression of IL23A in tumors compared to normal tissues. B) Immunohistochemical staining of HPAF-II (left) and MKN-45 (right) tumors in xenograft mouse models shows protein presence in the epithelial portion of the tumor. Figure 1B . Schematic representation of wild-type IL12 and IL23 receptor complexes and chimeric IL23-IL12 switched receptor (IL23-IL12sR). Figure 1C . Depiction of alternative IL23-IL12sR forms. Figure 1D . Overview of the expression cassettes for the IL23-IL12sR forms combined with a downstream GFP reporter gene in screening plasmids. Figure 1E . Overview of the expression cassettes for the IL23-IL12sR forms (including variants with a chimeric ECD) as downstream genetic elements of different CAR constructs. AB-SP: human IgG1 antibody signaling peptide; Tag: FLAG epitope tag. E2A can be replaced by another 2A self-cleaving peptide (e.g. T2A). Fig. 1F . Depiction of the mechanism of action of immune cells engineered with IL23-IL12sR. Fig. 2. Detection of IL23-IL12sR on engineered immune cells. A) Expression of engineered IL23-IL12sR was assessed indirectly by the expression of eGFP, which is located downstream of the IL23-IL12sR expression cassette and separated from it by the self-cleaving 2A peptide. B) Anti-P329G-CAR detection by AF647-tagged Fc molecules with P329G mutation and FLAG-tagged IL23-IL12sR variants Figure 3. T cells engineered with IL23-IL12sR show dose-dependent phosphorylation of A) STAT4 but not B) STAT3 in response to IL23 stimulation. Data in flow cytometry analysis were gated on lymphocytes and monocytes (both FSC and SSC). IL23-IL12sR T cells were additionally gated on GFP reporter fluorescence. C) Upregulation of STAT4 was also observed in T cells engineered with P329G CAR and IL23-IL12sR. D) STAT3 upregulation in response to IL23 stimulation is only observed in T cells overexpressing recombinant wild-type IL23R in vitro. Figure 4. Functional analysis of IL23-IL12sR engineered T cells by detecting alternative surface expression of markers associated with IL12 signaling in flow cytometry. A) Most notably, IL18Ra is upregulated in response to IL23 stimulation only in the engineered cells. The response of the IL23-IL12sR variant with the IL12Rb2-TMD appears to be 10-fold more sensitive. Either response showed a lower EC50 than wild-type cells stimulated by IL12. B) Upregulation of IL12Rb2 via a feed-forward mechanism triggered by STAT4 phosphorylation is observed only in IL23-IL12sR T cells. Here, IL23-IL12sR (IL12Rb2-TMD) T cells show a lower IL12Rb2 baseline, more similar to wild-type cells, when compared to IL23-IL12sR (IL23R-TMD). Figure 5. Functional analysis of T cells engineered with P329G-CAR and IL23-IL12sR with 41BB co-stimulation by detecting alternative surface expression of markers associated with IL12 signaling in flow cytometry. A) In contrast to previous results without co-expressing the P329G CAR, IL18Ra was upregulated 2-fold compared to wild-type T cells stimulated with IL12. B) Upregulation of IL12Rb2 via a feed-forward mechanism triggered by STAT4 phosphorylation was also observed. C) IFNγ secretion in a dose-dependent manner in response to IL23 or IL12 was observed. D) IL12-mediated IFNγ release was increased compared to wild-type T cells. Similar responses were also triggered by stimulation with IL23 in cells with the P329G-CAR and IL23-IL12sR, but not in cells engineered with only IL23-IL12sR. Figure 6. Immune cells engineered with P329G-CAR and FLAG-tagged IL23-IL12sR show increased cytotoxicity against MKN-45 and HPAF-II target cells when stimulated with IL23. A) Engineered cells titrated with CEA-CAM5-targeted IgG antibodies carrying a P329G mutation in the Fc portion (e.g., as disclosed in WO2022029051A1) show dose-dependent in vitro cytotoxicity. At suboptimal IgG adapter concentrations, the addition of IL23 triggering IL23-IL12sR enhances target cell growth inhibition, as evidenced by a 20%-40% increase in in vitro MKN-45 cytotoxicity. B) Similarly, CEACAM5 enhanced targeted cytotoxicity of HPAF-II target cells by 10%-20%. Figure 7. Detection and functional characterization of immune cells engineered with CEA-CAM5 CAR linked to a self-cleaving 2A peptide and IL23-IL12sR without a FLAG tag. A) Anti-CEA-CAM5 CAR expression was assessed by secondary staining with biotinylated CEA-Fc fusion molecules and streptavidin labeled with AF647. IL23-IL12sR was not detectable but is expected to be similar to the CAR, similar to previous results. B) T cells engineered with direct CEA-CAR and IL23-IL12sR showed dose-dependent upregulation of surface markers IL18Ra and IL12Rb2, CD25, and IFNg secretion. Baseline CD25 levels were increased compared to wild-type T cells. Figure 8. Immune cells engineered with CEA-CAR and IL23-IL12sR showed increased cytotoxicity against MKN-45 target cells when stimulated with IL23. A) The effect was most pronounced under conditions of low effector to target (E:T) ratios, as evidenced by increases in overall growth inhibition of cytotoxicity ranging from 10%-40%. Notably, the increase in killing capacity induced by 100 ng/mL IL12 or 100 or 1000 ng/mL IL23 was very similar. B) Summary of killing capacity of extracts plotted as a function of E:T ratio on day 2 of the experiment. Figure 9. Alternative IL23-IL12sR forms and their functional characteristics. A) Expression of alternative IL23-IL12sR forms can be detected indirectly by their effect on P329G-CAR expression and directly by detecting the N-terminal FLAG tag. Notably, the intact IL23R-ECD IL23-IL12sR variant performed best, and all variants with a chimeric ECD negatively affected the performance of the P329G-CAR. B) All IL23-IL12sR forms resulted in similar upregulation of IL18Ra, IL12Rb2, and IFNg release. In all readouts, the dose response was left-shifted and the overall message was increased in the IL23-IL12sR (1+5) and (3+3) variants. Figure 10. Alternative IL23-IL12sR forms and the resulting increased CAR-T cell cytotoxicity. All variants except IL23-IL12sR with IL23R-TMD showed increased T cell cytotoxicity upon stimulation with IL23. Notably, variants with IL12Rb2 TMD and variants with (1+5) chimeric ECD also showed the best cytotoxicity in the absence of IL23 stimulation. Variants with (1+5) or (3+3) chimeric ECD showed the highest response to IL23 at different E:T ratios. Figure 11. Immune cells engineered with CEA-CAR and IL23-IL12sR and stimulated with IL23 showed sustained cytotoxicity against HPAF-II target cells in repeated cytotoxicity assays. Effector cells were inoculated with target cells at a ratio of 1:1. Effector cells were transferred to fresh target cells every 2 days with or without the addition of cytokines. Both CEA-CAR T cells and CEA-CAR IL23-IL12sR T cells controlled the growth of HPAF-II cells. CEA-CAR IL23-IL12sR T cells showed repeated cytotoxicity against target cells in 5 rounds.

TW202506711A_113119507_SEQL.xml

Claims (56)

A chimeric receptor polypeptide comprising: (i) an extracellular portion comprising an IL23 binding domain; (ii) an intracellular portion comprising an intracellular domain of IL12Rb2 (interleukin 12 receptor β2); and (iii) a transmembrane domain connecting the extracellular portion and the intracellular portion. The chimeric receptor polypeptide of claim 1, wherein the IL23 binding domain binds to the p19 subunit of IL23. The chimeric receptor polypeptide of claim 1 or 2, wherein the intracellular portion comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 80% sequence identity thereto. The chimeric receptor polypeptide of any one of claims 1 to 3, wherein the transmembrane domain is selected from the transmembrane domains of IL12Rb2, IL23Ra, CD8 and CD28. The chimeric receptor polypeptide of any one of claims 1 to 4, wherein the transmembrane domain comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 22, 33, or an amino acid sequence having at least 80% sequence identity thereto. The chimeric receptor polypeptide of any one of claims 1 to 5, further comprising a message sequence. The chimeric receptor polypeptide of claim 6, wherein the message sequence comprises the amino acid sequence of SEQ ID NO: 1 or 16. The chimeric receptor polypeptide of any one of claims 1 to 7, further comprising one or more linkers. The chimeric receptor polypeptide of claim 8, wherein the linker is a GS linker, a 2A linker, an α-helix linker, a glycine-alanine polymer linker, an alanine-serine polymer linker, or an IgG4-Fc linker. The chimeric receptor polypeptide of any one of claims 1 to 9, further comprising a tag polypeptide, such as a Flag tag. The chimeric receptor polypeptide of any one of claims 1 to 10, further comprising a tag, such as a fluorescent polypeptide, such as GFP or eGFP. The chimeric receptor polypeptide of any one of claim items 1 to 11, which comprises from N-terminus to C-terminus: Optional signaling peptide – Optional tag – Optional linker – IL23 binding domain – Transmembrane domain – Intracellular portion containing the intracellular domain of IL12Rb2 – Optional linker – Optional tag. The chimeric receptor polypeptide of any one of claims 1 to 12, wherein the IL23 binding domain comprises the extracellular domain of IL23Rα. The chimeric receptor polypeptide of any one of claims 1 to 13, wherein the IL23 binding domain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity thereto. The chimeric receptor polypeptide of any one of claims 1 to 14, wherein the IL23 binding domain comprises the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 80% sequence identity thereto. The chimeric receptor polypeptide of any one of claims 1 to 15, wherein the IL23 binding domain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 80% sequence identity thereto. The chimeric receptor polypeptide of claim 14, further comprising an amino acid sequence of SEQ ID NO: 47 or an amino acid sequence having at least 80% sequence identity thereto between the IL23 binding domain and the transmembrane domain. The chimeric receptor polypeptide of claim 15, further comprising an amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having at least 80% sequence identity thereto between the IL23 binding domain and the transmembrane domain. The chimeric receptor polypeptide of claim 16, further comprising an amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80% sequence identity thereto between the IL23 binding domain and the transmembrane domain. The chimeric receptor polypeptide of any one of claims 1 to 12, wherein the IL23 binding domain comprises Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, crossFab, scFab, single domain antibody (sdAb) or designer anchor protein repeat protein (DARPin). The chimeric receptor polypeptide of any one of claims 1 to 19, wherein the chimeric receptor polypeptide comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NOs: 36 to 44 and 62 to 66. A nucleic acid or a plurality of nucleic acids encoding a chimeric receptor polypeptide as claimed in any one of claims 1 to 21, and optionally further comprising a nucleic acid sequence encoding a chimeric antigen receptor. An expression vector or a plurality of expression vectors comprising the nucleic acid or a plurality of nucleic acids of claim 22. A cell comprising the chimeric receptor polypeptide of any one of claims 1 to 21, the nucleic acid or multiple nucleic acids of claim 22, or the expression vector or multiple expression vectors of claim 23. The cell of claim 24, wherein the cell expresses IL12Rb1. The cell of claim 25, wherein the cell is a eukaryotic cell. The cell of claim 26, wherein the eukaryotic cell is an animal cell. The cell of claim 27, wherein the animal cell is a mammalian cell. The cell of claim 28, wherein the mammalian cell is a human cell. The cell of claim 28 or 29, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. The cell of claim 30, wherein the cell is an immune cell. The immune cell of claim 31, wherein the immune cell is a tumor infiltrating lymphocyte (TIL), a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a natural killer T (NKT) cell, or other T cells. The cell of claim 31 or 32, further comprising a chimeric antigen receptor (CAR). The cell of any one of claims 31 to 33, further comprising an engineered TCR. The cell of any one of claims 24 to 34, wherein the cell shows dose-dependent phosphorylation of STAT4 but not STAT3 when treated with IL23. The cell of any one of claims 24 to 35, wherein the cell is a transduced T cell capable of expressing the chimeric receptor polypeptide of any one of claims 1 to 21. A cell as claimed in any one of claims 24 to 36, wherein the cell expresses an antigen binding receptor comprising an anchored transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen binding portion, the antigen binding portion comprising (i) a heavy chain variable domain (VH) comprising a heavy chain complementation determining region (HCDR) 1 of SEQ ID NO:49, HCDR 2 of SEQ ID NO:50 or SEQ ID NO:51, and HCDR 3 of SEQ ID NO:52, and (ii) a light chain variable domain (VL) comprising a light chain complementation determining region (LCDR) 1 of SEQ ID NO:53, LCDR 2 of SEQ ID NO:54, and LCDR 3 of SEQ ID NO:55. The cell of claim 37, wherein the VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59. The cell of claim 37 or 38, wherein the VL domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:61. The cell of any one of claims 37 to 39, wherein the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of CD8, CD4, CD3z, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domains or fragments thereof, in particular, wherein the anchoring transmembrane domain is a CD8 transmembrane domain or a fragment thereof. A cell as in any one of claims 37 to 40, wherein the antigen binding receptor further comprises at least one stimulatory signaling domain and/or at least one co-stimulatory signaling domain. A cell as claimed in any one of claims 37 to 41, wherein at least one stimulatory signaling domain in the antigen binding receptor is individually selected from the group consisting of a CD3z intracellular domain, a FCGR3A intracellular domain and a NKG2D intracellular domain or fragments thereof that retain stimulatory signaling activity, specifically, wherein the at least one stimulatory signaling domain is a CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity. The cell of any one of claims 37 to 42, wherein at least one co-stimulatory signaling domain in the antigen binding receptor is individually selected from the group consisting of CD27 intracellular domain, CD28 intracellular domain, CD137 intracellular domain, OX40 intracellular domain, ICOS intracellular domain, DAP10 intracellular domain and DAP12 intracellular domain or fragments thereof that retain co-stimulatory signaling activity. A cell as claimed in any one of claims 37 to 43, wherein the antigen binding receptor comprises at least one CD28 costimulatory domain or a fragment thereof that retains CD28 costimulatory activity, and/or at least one CD137 costimulatory domain or a fragment thereof that retains CD137 costimulatory activity. A cell as claimed in any one of claims 37 to 44, wherein the antigen binding receptor comprises a stimulatory signaling domain, the stimulatory signaling domain comprises a CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity, and wherein the antigen binding receptor comprises a co-stimulatory signaling domain, the co-stimulatory signaling domain comprises a CD28 intracellular domain or a fragment thereof that retains CD28 co-stimulatory signaling activity. A cell as claimed in any one of claims 37 to 45, wherein the antigen binding receptor comprises a stimulatory signaling domain, the stimulatory signaling domain comprises a CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity, and wherein the antigen binding receptor comprises a co-stimulatory signaling domain, the co-stimulatory signaling domain comprises a CD137 intracellular domain or a fragment thereof that retains CD137 co-stimulatory signaling activity. A cell as claimed in any one of claims 37 to 46, wherein the antigen binding portion is linked to the N-terminus of the anchor transmembrane domain at the C-terminus, optionally via a peptide linker. A cell as claimed in any one of claims 37 to 47, wherein in the antigen binding receptor the light chain variable domain (VL) of the antigen binding portion is linked at the C-terminus to the N-terminus of the anchor transmembrane domain, optionally via a peptide linker, and/or wherein the heavy chain variable domain (VH) is linked at the C-terminus to the N-terminus of the light chain variable domain (VL), optionally via a peptide linker. A pharmaceutical composition comprising the cell of any one of claims 24 to 36 and, if appropriate, a pharmaceutically acceptable formulation. A chimeric receptor polypeptide as claimed in claim 1 to 21, or a nucleic acid as claimed in claim 22, or a vector as claimed in claim 22, or a cell as claimed in any one of claim 34 to 36, or a pharmaceutical composition as claimed in claim 49, for use in a method for medical treatment of a disease or prevention of a disease. A chimeric receptor polypeptide, nucleic acid, vector, cell or pharmaceutical composition for use as claimed in claim 50, wherein the method is cell therapy, such as relay cell therapy. A chimeric receptor polypeptide, nucleic acid, vector, cell or pharmaceutical composition for use as claimed in claim 50 or 51, wherein the chimeric receptor polypeptide, nucleic acid, vector, cell or pharmaceutical composition is administered intravenously, intratumorally or subcutaneously. A chimeric receptor polypeptide, nucleic acid, vector, cell or pharmaceutical composition for use as claimed in any one of claims 50 to 52, wherein the disease is cancer, autoimmune disease or infection. A chimeric receptor polypeptide, nucleic acid, vector, cell or pharmaceutical composition for use as claimed in claim 53, wherein the cancer is selected from the group consisting of acute leukemia (including but not limited to acute myeloid leukemia (AML), B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL) and acute lymphoid leukemia (ALL)), chronic leukemia (including but not limited to chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL)), multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), chronic myeloid leukemia (CML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). A method for regulating the activity of an immune cell, the method comprising: administering the nucleic acid or multiple nucleic acids of claim 22, or the expression vector or multiple expression vectors of claim 23 to the immune cell. The method of claim 55, wherein the immune cell is a tumor infiltrating lymphocyte (TIL), a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a natural killer T (NKT) cell, or other T cells.