HK1234091B - Trifunctional t cell-antigen coupler and methods and uses thereof - Google Patents

Description

Trifunctional T cell antigen conjugate and preparation method and use thereof

Related applications

This application claims priority to U.S. Provisional Patent Application No. 61/936,906, filed February 7, 2014, the contents of which are hereby incorporated by reference in their entirety.

field

The present disclosure relates to methods for treating cancer by engineering T cells with high cytotoxicity against specific target cells and reduced off-target toxicity. Specifically, the present disclosure relates to engineered T cells that express novel biologics that mimic the natural T cell activation process.

Background of the Invention

Cancer is a major health challenge, with over 150,000 cases expected to be diagnosed in Canada in 2013 alone. While patients with early-stage disease can be effectively treated with conventional therapies (surgery, radiation, chemotherapy), patients with advanced disease have fewer options available, and those options are often palliative in nature. Active immunotherapy seeks to harness the patient's immune system to eliminate tumor implants and offers an exciting option for patients for whom conventional therapies have failed (Humphries, 2013). Indeed, several clinical studies have demonstrated that T cell immunotherapy can be effective in patients with advanced melanoma, confirming the utility of this approach (Humphries, 2013). Furthermore, patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) have also been effectively treated and cured with T cell immunotherapy (Fry and Mackall, 2013) (Kochenderfer and Rosenberg, 2013). Although there are several immunotherapy methods, engineered T cells with chimeric receptors enable any patient's immune cells to target any desired target in a major histocompatibility complex (MHC) independent manner. To date, the chimeric receptors used for engineered T cells are composed of: a targeting domain, typically a single-chain variable fragment (scFv); a transmembrane domain; and a cytoplasmic domain containing signal transduction elements and associated proteins from T cell receptors (Dotti et al., 2009). Such chimeric receptors have been referred to as "T bodies (T-body)", "chimeric antigen receptors" (CARs) or "chimeric immune receptors" (CIRs) - currently, most researchers use the term "CAR" (Dotti et al., 2009). These CARs are considered in modular terms and scientists have spent considerable time studying the effects of different cytoplasmic signal transduction domains on CAR function. The first generation of CARs uses a single signal transduction domain from CD3ζ or FcεRIγ. Second-generation CARs combine the signaling domain of CD3ζ with the cytoplasmic domain of a co-stimulatory receptor from the CD28 or TNFR family of receptors (Dotti et al., 2009). Third-generation CARs combine multiple co-stimulatory domains, but there are concerns that third-generation CARs may lose antigen specificity (Han et al., 2013). Most CAR-engineered T cells tested in the clinic use second-generation CARs in which CD3ζ is coupled to the cytoplasmic domain of CD28 or CD137 (Han et al., 2013)(Finney et al., 2004)(Milone et al., 2009).

Although CAR-engineered T cells have shown considerable promise for clinical use, they rely on synthetic methods to replace the activation signals provided by T cell receptors (TCRs). Since the synthetic receptor does not deliver all signal transduction components associated with TCR (such as CD3ε, Lck), it is still unclear whether T cells are activated by CAR in the best way or how CAR activation affects T cell differentiation (such as progression to memory T cells). Moreover, since the CAR signaling domain is separated from their natural regulatory partners by the unique properties of the CAR structure, there is also such an inherent risk: CAR can cause low levels of constitutive activation, which can produce off-target toxicity.

Taking these limitations into account, it is preferred to redirect T cells to attack tumors via their natural TCR. For this reason, a class of recombinant proteins referred to as "bispecific T cell adapters" (BiTE) have been constructed (Chames and Baty, 2009) (Portell et al., 2013). These proteins use bispecific antibody fragments to cross-link T cell TCR receptors with target antigens. This results in effective activation of T cells, thereby triggering cytotoxicity. Similarly, bispecific antibodies that achieve this goal have been produced and some scientists use chemical connections to simply connect anti-CD3 antibodies to tumor-specific antibodies (Chames and Baty, 2009). Although these bispecific proteins have shown some activity in vitro, GMP production, short biological half-life and bioavailability represent the major challenges that these molecules have successfully used in cancer treatment. In addition, these molecules cannot correctly reproduce natural TCR signal transduction because they do not involve TCR co-receptors (CD8 and CD4).

Therefore, there is still a need for T cell antigen conjugates with enhanced activity and safety compared to traditional CARs.

SUMMARY OF THE INVENTION

The present inventors have demonstrated that trifunctional T cell antigen conjugates have enhanced activity and safety compared to traditional chimeric antigen receptors, which better mimic native signaling through the T cell receptor (TCR) while retaining unrestricted targeting of the major histocompatibility complex.

Thus, one aspect of the present disclosure provides a nucleic acid comprising:

a. a first polynucleotide encoding a target-specific ligand;

b. a second polynucleotide encoding a ligand that binds to a protein associated with the TCR complex; and

c. A third polynucleotide encoding a T cell receptor signaling domain polypeptide.

Another aspect of the present disclosure provides a polypeptide encoded by the nucleic acid described above.

Another aspect of the present disclosure provides an expression vector comprising the nucleic acid described above.

Another aspect of the present disclosure provides a T cell expressing the nucleic acid described above. Another aspect of the present disclosure provides a pharmaceutical composition comprising a T cell and a vector.

The present disclosure also provides a use of a T cell for treating cancer in a subject in need thereof, wherein the T cell expresses a nucleic acid comprising:

a. a first polynucleotide encoding a target-specific ligand;

b. a second polynucleotide encoding a ligand that binds to a protein associated with the TCR complex; and

c. A third polynucleotide encoding a T cell receptor signaling domain polypeptide.

In one embodiment, the target-specific ligand binds to an antigen on a cancer cell.

In another embodiment, the target-specific ligand is a designed ankyrin repeat (DARPin) polypeptide or a scFv.

In another embodiment, the protein associated with the TCR complex is CD3.

In another embodiment, the ligand that binds to a protein associated with the TCR complex is a single chain antibody.

In another embodiment, the ligand that binds to a protein associated with the TCR complex is UCHT1 or a variant thereof.

In another embodiment, the T cell receptor signaling domain polypeptide comprises a cytoplasmic domain and a transmembrane domain.

In another embodiment, the cytoplasmic domain is the CD4 cytoplasmic domain and the transmembrane domain is the CD4 transmembrane domain.

In another embodiment, the first polynucleotide and the third polynucleotide are fused to the second polynucleotide.

In another embodiment, the second polynucleotide and the third polynucleotide are fused to the first polynucleotide.

The present disclosure also provides a vector construct comprising:

a. a first polynucleotide encoding a target-specific ligand;

b. a second polynucleotide encoding a ligand that binds to a protein associated with the TCR complex; and

c. a third polynucleotide encoding a T cell receptor signaling domain polypeptide, and

d. Promoters that are functional in mammalian cells.

In one embodiment, the first polynucleotide and the third polynucleotide are fused to the second polynucleotide to provide a fusion T cell antigen conjugate and the coding sequence for the fusion T cell antigen conjugate is operably linked to a promoter.

In another embodiment, the second polynucleotide and the third polynucleotide are fused to the first polynucleotide to provide a fusion T cell antigen conjugate and the coding sequence for the fusion T cell antigen conjugate is operably linked to a promoter.

The present disclosure also provides isolated T cells transfected with the vector construct.

Other features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that this detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a schematic overview of trifunctional T cell antigen conjugates (Tri-TACs) compared to conventional second-generation CARs, including schematic diagrams of the constructs used in this work.

Figure 2 shows surface expression analysis of Tri-TAC variants and classical CAR.

Figure 3 shows analysis of cell activation looking at different markers IFN-γ, TNF-α and CD107a.

Figure 4 Analysis of killing by two different cell lines expressing (D2F2E2) or not expressing (D2F2) the molecular targets of classical CAR and Tri-TAC.

Figure 5 depicts naive T cell priming (A), two currently used artificial methods for T cell activation (B and C), and the TAC activation technique (D).

FIG6 depicts (A) Configuration 1 of a TAC molecule and (B) Configuration 2 of a TAC molecule.

Figure 7 shows the functionality of scFv CD4TAC. (A) is a histogram showing surface expression of scFv CD4TAC receptors relative to empty vector, (B) shows antigen-specific activation of T cells expressing scFv CD4TAC (upper panel) or scFv CAR (lower panel), and (C) shows comparable killing of the MCF-7 human tumor cell line (Her2 positive) by scFv CD4TAC and scFv CAR.

Figure 8 depicts CD4-TAC Configuration 2. (A) is a histogram showing surface expression of DARPin CD4 TAC receptors relative to empty vector, (B) shows cytokine production and cell degranulation following exposure of T cells engineered with DARPin TAC Configuration 2 to Her2 antigen, and (C) shows the growth of CD4 TAC Configuration 2 relative to an empty vector control.

Figure 9 shows functionality of DARPin CD4 TAC configuration 1. (A) shows the increase in surface expression of DARPin CD4 YAC compared to DARPin CAR and NGFR only controls, (B) shows the increase in CD4 TAC configuration 1, and (C) and (D) show the percentage of cells positive for various activation and degradation markers.

Figure 10 shows the cytotoxicity and overall activity of TACs and CARs. Cells engineered with TACs, CARs, or an empty vector control were incubated in various human tumor cell lines.

Figure 11 shows receptor surface expression and activation of various TAC controls. (A) shows cell surface expression (left), cell degranulation (center), and cytokine production (right), and (B) shows that only full-length CD4-TAC is able to elicit a cytotoxic response.

Figure 12 shows the properties of various transmembrane TAC variants. (A) is an overview of various transmembrane domain variants, (B) shows the surface expression of each engineered construct in CD8 purified T cells, and (C) shows the assay of cellular degranulation and cytokine production of each variant.

Figure 13 shows the interaction of Lck with TAC variants. (A) shows that full-length TAC and cytoplasmic deletions can pull down Lck, and (B) is a densitometry analysis of Lck detected in the pellet of (A).

Figure 14 shows CD4 TAC surface expression and activity compared to BiTE-like variants. (A) Depicts surface expression of an NGFR-only control, CD4 TAC, and BiTE-like variants, and (B) compares cytotoxicity in various cell lines.

Figure 15 shows a comparison of a random mutant library of wild-type CD4 TAC and UCHT1. (A) shows a graphic representation of the mutants, (B) a histogram showing surface expression of the library, and (C) shows that the library can activate T cells and produce cytokines.

Figure 16 shows enhanced surface expression of the A85V, T161P mutant. (A) Comparison of the final CD/CD8 population between CD4 TAC and the A85V, T161P mutant, (B) shows enhanced surface expression of the A85V, T161P mutant, and (C) shows decreased cytokine production in the A85V, T161 mutant.

Figure 17 shows the cytotoxicity and growth of the A85V, T161P mutant. (A) shows the cytotoxicity of the A85V, T161P mutant in various cell lines, and (B) shows the cell growth in culture over 2 weeks.

Details

(i) Definition

As used herein, the term "a cell" includes a single cell as well as a plurality of cells.

As used herein, the term "T cell" refers to a type of lymphocyte that plays a key role in cell-mediated immunity. T cells, also known as T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T cell receptor (TCR) on their cell surface. There are several T cell subsets with different functions, including but not limited to helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, and natural killer T cells.

As used herein, the term "T cell antigen conjugate" refers to an engineered nucleic acid construct or polypeptide that, when expressed on a T cell, targets the T cell to a specific antigen.

As used herein, the terms "polynucleotide" and/or "nucleic acid sequence" and/or "nucleic acid" refer to a sequence of nucleoside or nucleotide monomers consisting of bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and can include naturally occurring bases, including adenine, guanine, cytosine, thymine, and uracil. The sequence can also contain modified bases. Examples of such modified bases include aza- and deazaadenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine. The nucleic acids of the present disclosure can be isolated from biological organisms, can be formed by laboratory methods of genetic recombination, or can be obtained by chemical synthesis or other known protocols for producing nucleic acids.

As used herein, the term "isolated polynucleotide" or "isolated nucleic acid sequence" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). The term "nucleic acid" is intended to include DNA and RNA, can be double-stranded or single-stranded, and represent either the sense strand or the antisense strand. Additionally, the term "nucleic acid" includes complementary nucleic acid sequences.

As used herein, the term "recombinant nucleic acid" or "engineered nucleic acid" refers to a nucleic acid or polynucleotide that does not occur in a biological organism. For example, a recombinant nucleic acid can be formed by laboratory methods of genetic recombination (e.g., molecular cloning) to construct a sequence that does not otherwise occur in nature. A recombinant nucleic acid can also be constructed by chemical synthesis or other known methods for producing nucleic acids.

The terms "polypeptide" or "protein" as used herein describe an amino acid chain corresponding to those amino acids encoded by a nucleic acid. The polypeptide or protein of the present disclosure can be a peptide, which generally describes an amino acid chain of 2 to about 30 amino acids. The term "protein" as used herein also describes an amino acid chain with more than 30 amino acids, and can be a fragment or domain or full-length protein of a protein. Moreover, as used herein, the term protein can refer to a linear chain of amino acids or it can refer to an amino acid chain that has been processed and folded into a functional protein. However, it should be understood that 30 is an arbitrary number for distinguishing between peptides and proteins, and that the terms are used interchangeably for amino acid chains. The protein of the present disclosure can be obtained by isolating and purifying the protein from cells that naturally produce the protein, by enzymatic (e.g., proteolytic) cleavage, and/or by expressing nucleic acid recombinants encoding the protein of the present disclosure or fragments. The protein and/or fragment of the present disclosure can also be obtained by chemical synthesis or other known schemes for producing proteins and fragments.

The term "isolated polypeptide" refers to a polypeptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, single-chain antibodies, chimeric antibodies, and antibody fusions. Antibodies can be derived from recombinant sources and/or produced in transgenic animals. The term "antibody fragment" as used herein is intended to include, but is not limited to, Fab, Fab', F(ab') ₂ , scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments, and domain antibodies.

The term "vector" as used herein refers to a polynucleotide that can be used to deliver a nucleic acid into a cell. In one embodiment, the vector is an expression vector that comprises an expression control sequence (e.g., a promoter) operably linked to a nucleic acid to be expressed in a cell. Vectors known in the art include, but are not limited to, plasmids, phages, cosmids, and viruses.

(ii) Composition

The present inventors have developed trifunctional T cell antigen conjugates (Tri-TACs) to better mimic native signaling through the T cell receptor (TCR) while retaining MHC-unrestricted targeting. Specifically, the present inventors constructed a molecule in which the transmembrane and intracellular regions of the CD4 co-receptor (which are localized to lipid rafts and bind to Lck, respectively) were fused to a single-chain antibody that binds CD3. The construct was designed to direct the CD3 molecule and TCR to the lipid raft region and bring Lck into proximity with the TCR, similar to native MHC binding. To target the chimeric receptor, a designed ankyrin repeat (DARPin) polypeptide was linked to the CD4-UCHT1 chimera to generate the trifunctional T cell antigen conjugate (Tri-TAC).

Experimentally, human T cells were engineered to express either a prototype Tri-TAC or a conventional CAR with the same DARPin. It was determined that T cells engineered with Tri-TAC exhibited functionality equivalent to conventional CARs in all respects. For two parameters (TNF-α production and CD107a mobilization), Tri-TAC was observed to be more active than conventional CARs. Furthermore, the data showed that on a per-molecule basis, Tri-TAC exhibited significantly enhanced activity. In addition, Tri-TAC offers enhanced safety compared to conventional CARs because the activation domain is not part of the protein.

Thus, the present disclosure relates to a nucleic acid comprising:

a first polynucleotide encoding a target-specific ligand;

a second polynucleotide encoding a ligand that binds the TCR complex; and

A third polynucleotide encodes a T cell receptor signaling domain polypeptide.

In one embodiment, the nucleic acid is a recombinant or engineered nucleic acid.In another embodiment, the first, second and/or third polynucleotide is a recombinant or engineered polynucleotide.

The present disclosure also relates to polypeptides encoded by the nucleic acids and compositions comprising the nucleic acids.

Nucleic acids comprising each of the first, second, and third polynucleotides, and polypeptides encoded by the nucleic acids, are also referred to herein as trifunctional T-cell antigen conjugates or Tri-TACs.

Target-specific ligands

Target-specific ligands direct the T cell antigen conjugate to the target cell. Thus, a target-specific ligand refers to any substance that directly or indirectly binds to a target cell. The target cell can be any cell associated with a disease state, including but not limited to cancer. In one embodiment, a target-specific ligand binds to an antigen (a protein produced by a cell that can elicit an immune response) on the target cell. A target-specific ligand can also be referred to as an antigen binding domain.

In one embodiment, the target cell is a tumor cell. Here, the target-specific ligand can bind to a tumor antigen or a tumor-associated antigen on a tumor cell. Tumor antigens are well known in the art. The term "tumor antigen" or "tumor-associated antigen" as used herein means any antigenic substance (for example, which can be represented by an MHC complex) produced in a tumor cell that triggers a host immune response. When it is a protein, a tumor antigen can be, for example, a sequence of 8 or more amino acids up to a complete protein, and a sequence of any number of amino acids from 8 to a full-length protein, the sequence comprising at least one antigenic fragment of the full-length protein, which can be represented by an MHC complex. Examples of tumor antigens include, but are not limited to, HER2 (erbB-2), B cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), prostate-specific antigen (PSA), glioma-associated antigen, beta-human chorionic gonadotropin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

Examples of target-specific ligands include antibodies and fragments thereof, such as single-chain antibodies, eg, scFV, or small proteins that bind to target cells and/or antigens.

An example of a target-specific ligand is a designed ankyrin repeat (DARPin) polypeptide that targets a specific cell and/or antigen. In one embodiment, the target-specific ligand is a DARPin polypeptide that targets HER2 (erbB-2). An example of a DARPin polypeptide that targets HER2 (erb-2) is provided herein as SEQ ID NOs: 7 and 8.

Another example of a target-specific ligand is an scFv that targets a specific cell and/or antigen. In one embodiment, the target-specific ligand is an scFv that binds to HER2 (erb-2). An example of an scFv that binds to HER2 (erb-2) is provided herein as SEQ ID NOs: 22 and 23.

Ligands that bind to the TCR complex

T cell antigen conjugates are designed to recruit T cell receptors (TCRs) in combination with co-receptor stimulation. Therefore, T cell antigen conjugates contain a ligand that binds to a protein associated with the T cell receptor complex.

TCR (T cell receptor) is a complex of integral membrane proteins that participates in the activation of T cells in response to the binding of antigens. TCR is a membrane-anchored heterodimer connected by disulfide bonds, which is usually composed of highly variable alpha (α) chain and beta (β) chain that are represented as part of a complex with invariant CD3 (cluster of differentiation 3) chain molecules. T cells expressing this receptor are referred to as α:β (or αβ) T cells, although a small number of T cells express alternative receptors formed by variable gamma (γ) and delta (δ) chains, which are referred to as γδ T cells. CD3 is a protein composed of four different chains. In mammals, the complex includes CD3γ chain, CD3δ chain and two CD3ε chains.

As used herein, the term "ligand that binds to a protein associated with the T cell receptor complex" includes any substance that directly or indirectly binds to a protein of the TCR. Proteins associated with the TCR include, but are not limited to, TCR alpha (α) chain, TCR beta (β) chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3γ chain, CD3δ chain, and CD3ε chain. In one embodiment, the ligand that binds to a protein associated with the T cell receptor complex is an antibody to the TCRεD3δ chain and/or CD3ε chain.

In one embodiment, the ligand is an antibody or fragment thereof that binds to CD3. Examples of CD3 antibodies are known in the art (muromonab, otelixizumab, teplizumab, and visilizumab). In one embodiment, the antibody that binds to CD3 is a single-chain antibody, such as a single-chain variable fragment (scFv).

Another example of a CD3 antibody is UCHT1, which targets CD3ε. The sequence of UCHT1 is provided herein as SEQ ID NOs: 13 and 14.

T cell receptor signaling domain polypeptide

The T cell antigen conjugate comprises a T cell receptor signaling domain polypeptide. As used herein, the term "T cell receptor signaling domain" refers to a polypeptide that (a) localizes to lipid rafts and/or (b) binds to Lck. The T cell receptor signaling domain polypeptide may comprise one or more protein domains, including but not limited to a cytoplasmic domain and/or a transmembrane domain. As used herein, a "protein domain" refers to a given protein sequence structure of a conserved portion that can function and exist independently of the rest of the protein chain. In one embodiment, the T cell receptor signaling domain polypeptide comprises a cytoplasmic domain. In another embodiment, the T cell receptor signaling domain polypeptide comprises a transmembrane domain. In a further embodiment, the T cell receptor signaling domain polypeptide comprises both a cytoplasmic domain and a transmembrane domain.

T cell receptor signaling domain polypeptides comprise TCR co-receptor and co-stimulator and TCR co-receptor and co-stimulator protein domains.

"TCR co-receptors" refer to molecules that help the T cell receptor (TCR) communicate with antigen presenting cells. Examples of TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD5S, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CDt37, and CD154.

"TCR co-stimulator" refers to molecules necessary for T cells to respond to antigens. Examples of TCR co-stimulators include, but are not limited to, PD-1, ICOS, CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

In one embodiment, the T cell receptor signaling domain polypeptide comprises a cytoplasmic domain and a transmembrane domain of a TCR co-receptor or co-stimulatory protein. The cytoplasmic domain and the transmembrane domain can be from the same co-receptor or co-stimulator or from different co-receptors or co-stimulators. The cytoplasmic domain and the transmembrane domain are optionally connected by a linker.

In one embodiment, the T cell receptor signaling domain polypeptide comprises the transmembrane domain and cytoplasmic domain of the CD4 co-receptor (see, e.g., SEQ ID NOs: 17 and 18).

In another embodiment, the T cell receptor signaling domain polypeptide comprises the transmembrane domain and the cytoplasmic domain of the CD8α co-receptor.

In other embodiments, the cytoplasmic and/or transmembrane domains of the T cell receptor signaling domain polypeptide are synthetic. For example, the transmembrane domain is optionally a synthetic, highly hydrophobic membrane domain.

In another example, the transmembrane domain is a glycophorin transmembrane domain. In another example, the T cell receptor signaling domain polypeptide comprises a CD48 GPI signal sequence to attach the T cell antigen conjugate to the membrane using a GPI anchor.

In addition to the three components of the T cell antigen conjugates described herein (target-specific ligand, ligand that binds to the TCR complex, and T cell receptor signaling domain polypeptide), the present application contemplates that other polypeptides may also be included. For example, the T cell antigen conjugates optionally contain additional polypeptides that act directly or indirectly to target or activate T cells.

Linker

The various components of the T cell antigen conjugate can be fused directly to each other, or they can be connected by at least one linker (optionally a peptide linker). The peptide linker can be of any size, provided that the peptide linker does not interfere with the function of the individual connected components. In one embodiment, the length of the peptide linker is from about 1 to about 15 amino acids, more specifically from about 1 to about 10 amino acids, and most specifically from about 1 to about 6 amino acids.

Examples of linkers that can be used in T cell antigen conjugates include G ₄ S ₃ linkers. Other examples of linkers are peptides corresponding to SEQ ID NOs: 11, 12, 15, 16, 19, 20 and 21, and variants and fragments thereof.

Configuration

T cell antigen conjugates can exist in a variety of configurations readily apparent to those skilled in the art.

In one embodiment, both the target-specific ligand and the T cell receptor signaling domain polypeptide are fused to a ligand that binds to the TCR complex. For example, the N-DARPin TAC described herein (also referred to as Configuration 1; SEQ ID NOs: 1 and 2) comprises, in order:

i) N-Darpin Tri TAC leader sequence (secretion signal) (SEQ ID NO: 5 and 6)

ii) DARPin specific for Her2 antigen (SEQ ID NOs: 7 and 8)

iii) Myc tag (SEQ ID NO: 9 and 10)

iv) Linker 1 (SEQ ID NOs: 11 and 12)

v) UCHT1 (SEQ ID NOs: 13 and 14)

vi) Linker 2 (SEQ ID NOs: 15 and 16)

vii) CD4 (SEQ ID NOs: 17 and 18)

In another embodiment, the DARPin is replaced by a scFV ScFv (SEQ ID NOs: 22 and 23) specific for the Her2 antigen.

In another embodiment, both the ligand that binds the TCR complex and the T cell receptor signaling domain polypeptide are fused to the target-specific ligand C-DARPin TAC described herein (also referred to as Configuration 1; SEQ ID NOs: 3 and 4). Alternative configurations will be apparent to those skilled in the art.

Vector constructs

A variety of delivery vectors and expression media can be used to introduce the nucleic acids described herein into cells.Thus, the aforementioned polynucleotides are optionally included in a vector to provide a vector construct, also referred to herein as a vector.

Therefore, the present disclosure also relates to a vector comprising:

a. a first polynucleotide encoding a target-specific ligand;

b. a second polynucleotide encoding an antibody that binds CD3; and

c. a third polynucleotide encoding a T cell receptor signaling domain polypeptide,

and optionally a promoter functional in mammalian cells.

Promoters are well known in the art and are regions of DNA that initiate transcription of a specific nucleic acid sequence. A "promoter functional in mammalian cells" refers to a promoter that drives expression of the associated nucleic acid sequence in mammalian cells. A promoter that drives expression of a nucleic acid sequence can be said to be "operably linked" to the nucleic acid sequence.

In one embodiment, the first polynucleotide and the third polynucleotide are fused to the second polynucleotide to provide a fusion T cell antigen conjugate and the coding sequence for the fusion T cell antigen conjugate is operably linked to a promoter.

In another embodiment, the second polynucleotide and the third polynucleotide are fused to the first polynucleotide to provide a fusion T cell antigen conjugate and the coding sequence for the fusion T cell antigen conjugate is operably linked to a promoter.

Optionally, the vector is designed for expression in mammalian cells, such as T cells. In one embodiment, the vector is a viral vector, optionally a retroviral vector.

Useful vectors include those derived from lentivirus, murine stem cell virus (MSCV), poxvirus, oncoretroviruses, adenovirus, and adeno-associated virus. Other useful delivery vectors include those derived from herpes simplex virus, transposon, vaccinia virus, human papillomavirus, simian immunodeficiency virus, HTLV, human foamy virus, and their derivatives. Other useful vectors include those derived from foamy virus, mammalian type B retrovirus, mammalian type C retrovirus, avian type C retrovirus, mammalian type D retrovirus, and HTLV/BLV type retrovirus. An example of a lentiviral vector useful in the disclosed compositions and methods is the pCCL vector.

Changes in polynucleotides and polypeptides

Many modifications are made to the polynucleotide sequences disclosed in this application (including vector sequences and polypeptide sequences), and these modifications are obvious to those skilled in the art. Modifications include substitutions, insertions or deletions of nucleotides or amino acids or changes in the relative position or order of nucleotides or amino acids.

In one embodiment, the polynucleotides described herein may be modified or mutated to optimize the function of the encoded polypeptide and/or the function, activity and/or expression of the T cell antigen conjugate.

Herein, it is shown that UCHT1 mutants can be generated that result in enhanced surface expression of TACs (Figures 15-17). Thus, in one embodiment, the TAC comprises a modified or mutated ligand that binds to the TCR complex, wherein the TAC comprising the modified or mutated antibody has increased surface expression and/or activity compared to a TAC comprising a wild-type or unmodified or unmutated ligand that binds to the TCR complex. Examples of mutant or modified antibodies that bind to CD3 are the UCHT1 A85V, T161P mutants described herein (SEQ ID NOs: 24 and 25).

Sequence identity

The polynucleotide of the present application also comprises such nucleic acid molecules (or its fragment), and it has at least about 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% or 99.5% identity with the nucleic acid molecules of the present application. The polypeptide of the present application also comprises such polypeptide (or its fragment): it has at least about 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% or 99.5% identity with the polypeptide of the present application. Identity refers to the similarity of two nucleotides or peptide sequences, and the sequences are compared to obtain the highest level match. Identity is calculated according to methods known in the art. For example, if a nucleotide sequence (referred to as "sequence A") is 90% identical to a portion of SEQ ID NO: 1, then sequence A is identical to the reference portion of SEQ ID NO: 1, except that sequence A may contain up to 10 point mutations (e.g., substitutions by other nucleotides) for every 100 nucleotides of the reference portion of SEQ ID NO: 1.

Sequence identity is preferably arranged to have at least about 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or most preferably at least 99% or 99.5% identity with the nucleotide sequence provided herein and/or its complementary sequence. Sequence identity is also preferably arranged to have at least about 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or most preferably at least 99% or 99.5% identity with the peptide sequence provided herein. Sequence identity is preferably calculated using the GCG program from Bioinformatics (University of Wisconsin). Other programs are also available for calculating sequence identity, such as the Clustal W program (preferably used with default parameters; Thompson, JD et al., Nucleic Acid Res. 22:4673-4680).

hybridization

The present application includes DNA having a sequence that is sufficiently identical to the nucleic acid molecules described herein to hybridize under stringent hybridization conditions (hybridization techniques are well known in the art). The present application also includes nucleic acid molecules that hybridize to one or more of the sequences described herein and/or their complements. Such nucleic acid molecules preferably hybridize under high stringency conditions (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). High stringency washes preferably have low salt (preferably about 0.2% SSC), a temperature of about 50-65°C, and are optionally performed for about 15 minutes.

Expression in T cells

The T cell antigen conjugate is designed to be expressed in a T cell. Thus, one aspect of the present disclosure provides a T cell expressing the T cell antigen conjugate. Another aspect of the present disclosure relates to a T cell transduced or transfected with a T cell antigen conjugate or a vector comprising the T cell antigen conjugate. Optionally, the T cell is an isolated T cell.

T cells can be obtained from many sources, including but not limited to blood (e.g., peripheral blood mononuclear cells), bone marrow, thymus tissue, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, spleen tissue, and tumors. In one embodiment, the T cells are autologous T cells. In another embodiment, the T cells are obtained from a cell line of T cells. Methods for culturing and maintaining T cells in vitro are well known in the art.

Once obtained, T cells are optionally enriched in vitro. As is well known in the art, cell populations can be enriched by positive selection or negative selection. In addition, T cells can be optionally frozen or cryopreserved and then thawed at a later date.

Before or after the T cell antigen conjugate is introduced into the T cells, the T cells are optionally activated and/or expanded using methods well known in the art. For example, T cells can be expanded by contacting a surface having attached thereto an agent that stimulates CD3/TCR complex-associated signals and a ligand that stimulates a co-stimulatory molecule on the T cell surface.

Methods for transducing or transfecting T cells with nucleic acid sequences and expressing the transduced nucleic acids in T cells are well known in the art. For example, nucleic acids can be introduced into cells by physical, chemical, or biological means. Physical means include, but are not limited to, microinjection, electroporation, particle bombardment, lipofection, and calcium phosphate precipitation. Biological means include the use of DNA and RNA vectors.

In one embodiment, viral vectors (including retroviral vectors) are used to introduce nucleic acids into T cells and express them in the T cells. Viral vectors include vectors derived from the following: lentivirus, murine stem cell virus (MSCV), poxvirus, herpes simplex virus, type I adenovirus, and adeno-associated virus. The vector optionally contains a promoter that drives the expression of the transduced nucleic acid molecule in the T cell.

A variety of assays can be used to confirm the presence of the transduced nucleic acid sequence and/or the polypeptide encoded by the nucleic acid in the T cells and/or the expression of the transduced nucleic acid sequence and/or the polypeptide encoded by the nucleic acid in the T cells. Assays include, but are not limited to, Southern and Northern blotting, RT-PCR and PCR, ELISA, and Western blotting.

In one embodiment, the T cells expressing T cell antigen conjugates have increased T cell activation in the presence of antigens, compared to T cells that do not express T cell antigen conjugates and/or compared to T cells expressing traditional CARs. Increased T cell activation can be determined by many methods, including but not limited to increased tumor cell line killing, increased cytokine production, increased cell lysis, increased cell degranulation and/or increased activation markers (such as CD107α, IFNγ, IL2 or TNFα). The increase can be measured in single cells or cell populations.

As used herein, the term "increased" or "increasing" refers to an increase of at least 2%, 5%, 10%, 25%, 50%, 100%, or 200% in the number of T cells or T cell populations expressing the T cell antigen conjugate compared to T cells or T cell populations that do not express the T cell antigen conjugate and/or compared to T cells or T cell populations that express traditional CARs.

T cells expressing the T cell antigen conjugate, optionally autologous T cells, can be administered to a subject in need thereof. Thus, T cells transduced with the T cell antigen conjugate and/or T cells expressing the T cell antigen conjugate can be formulated in a pharmaceutical composition. Preferably, the T cells are formulated for intravenous administration.

Pharmaceutical compositions can be prepared by methods known per se for preparing pharmaceutically acceptable compositions that can be administered to a subject, such that an effective amount of T cells is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, Easton, Pa., USA, 2000). On this basis, compositions include, although not excluding others, solutions of substances in combination with one or more pharmaceutically acceptable carriers or diluents and contained in an isotonic solution of a buffer solution having an appropriate pH and a physiological fluid.

Suitable pharmaceutically acceptable carriers essentially include chemically inert and non-toxic components that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound and a suitable amount of carrier to provide a form for direct administration to a patient.

Pharmaceutical composition can also include but is not limited to lyophilized powder or aqueous or non-aqueous sterile injectable solution or suspension, which can further comprise an antioxidant, a buffer, an antibacterial agent and a solute that makes the composition and the tissue or blood of the intended recipient is substantially isotonic. Other components that can exist in such compositions include, for example, water, a surfactant (such as Tween), alcohol, polyols, glycerol and vegetable oil. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, tablets or concentrated solutions or concentrated suspensions.

(iii) Methods and uses

One aspect of the present disclosure provides the use of a trifunctional T cell antigen conjugate to direct T cells to a specific antigen.

Thus, the present disclosure also relates to the use of modified T cells for treating cancer in a subject in need thereof, wherein the modified T cells express a nucleic acid comprising a first polynucleotide encoding a target-specific ligand; a second polynucleotide encoding a ligand that binds to the TCR complex; and a third polynucleotide encoding a T cell receptor signaling domain polypeptide. The present disclosure also relates to a method for treating cancer comprising administering an effective amount of modified T cells to a subject in need thereof. Also disclosed is the use of an effective amount of modified T cells for treating cancer in a subject in need thereof. Further disclosed is the use of modified T cells in the preparation of a medicament for treating cancer in a subject in need thereof. Even further disclosed is the use of modified T cells for treating cancer in a subject in need thereof. In one embodiment, the target-specific ligand binds to an antigen on a cancer cell, thereby targeting the modified T cells to the cancer cell.

Cancers that can be treated include any form of neoplastic disease. Examples of cancers that can be treated include, but are not limited to, breast cancer, lung cancer, and leukemias, such as mixed lineage leukemia (MLL), chronic lymphocytic leukemia (CLL), or acute lymphocytic leukemia (ALL). Other cancers include carcinomas, blastomas, melanomas, sarcomas, hematological cancers, lymphoid malignancies, benign and malignant tumors, and malignant tumors. Cancers can include non-solid tumors or solid tumors. Cancers that can be treated include non-vascularized tumors, or tumors that are not yet vascularized, and vascularized tumors.

The modified T cells and/or pharmaceutical compositions described herein can be administered or used in living organisms, including humans and animals. As used herein, the term "subject" refers to any member of the animal kingdom, preferably a mammal, and more preferably a human.

Procedures for isolating, genetically modifying T cells, and administering the T cells to subjects in need thereof are known in the art. Specifically, T cells are isolated from a mammal (preferably a human), optionally expanded and/or activated, as described herein, and transduced or transfected with the nucleic acid molecules disclosed herein. For a subject, the T cells can be autologous. In another embodiment, for a subject, the cells can be allogeneic, homologous, or xenogeneic.

The modified T cells can be administered alone or in a pharmaceutical composition, as described herein. The compositions of the present disclosure are preferably formulated for intravenous administration.

An "effective amount" of the modified T cells and/or pharmaceutical composition administered is defined as an amount effective at the dosage and time period necessary to achieve the desired result. For example, the effective amount of a substance can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the recombinant protein to elicit the desired response in the individual. The dosing regimen can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

For example, the modified T cells and/or pharmaceutical compositions described herein can be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, optionally 10 ⁵ to 10 ⁸ cells/kg body weight, or 10 ⁶ to 10 ⁷ cells/kg body weight. The dose can be administered in a single or multiple doses.

The modified T cells and/or pharmaceutical compositions can be administered by any method known in the art, including but not limited to aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The modified T cells and/or pharmaceutical compositions can be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intrameduliary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. The modified T cells and/or pharmaceutical compositions thereof can be directly injected into a tumor, lymph node, or site of infection.

As used herein, and as is well understood in the art, "treating" or "treatment" is a method for obtaining a beneficial or desired result (including a clinical result). Beneficial or desired clinical results can include, but are not limited to, alleviating or relieving one or more symptoms or conditions, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, preventing the spread of the disease, delaying or slowing the progression of the disease, improving or alleviating the disease state, and mitigating (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival if not receiving treatment. In one embodiment, "treatment" includes preventing a disease or condition.

Table 1. Sequence Listing

¹ Light chain, nucleotides 1-324; linker, nucleotides 325-387; heavy chain, nucleotides 388-750

² light chain, amino acids 1-108; linker, amino acids 109-128; heavy chain, amino acids 129-250

³ Extracellular linker, nucleotides 1-66; transmembrane domain, nucleotides 67-132; cytoplasmic domain, nucleotides 133-254

⁴ extracellular linker, amino acids 1-22; transmembrane domain, amino acids 23-44; cytoplasmic domain, amino acids 45-84

The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the present application. Where circumstances provide or provide convenience, variations in form and substitutions of equivalents are contemplated. Although specific terms are employed herein, these terms are intended to be descriptive rather than restrictive.

The following non-limiting examples of the present application are illustrative.

Example

Example 1.

Background and Overview

Developed trifunctional T cell antigen conjugate (Tri-TAC) to better reflect natural signal transduction by TCR, while retaining the unrestricted targeting of MHC. T cell activation occurs after connecting MHC by TCR and the co-receptors (CD4 or CD8) on T cells simultaneously bind to the conserved region within the MHC molecule (Yin et al., 2012) (Kuhns and Davis, 2012). Co-receptors are specifically located in "lipid rafts" (Fragoso et al., 2003) (Arcaro et al., 2000), which are membrane microdomains that are particularly important for the formation of TCR signaling complexes (He and Marguet, 2008). In addition to ensuring the correct microdomain positioning of TCR activation complexes, these co-receptors also directly bind to Lck (Kim et al., 2003), which is a protein kinase crucial to T cell activation (Methi et al., 2005) (Acuto and Cantrell, 2000). As previously mentioned, existing chimeric receptors or bifunctional proteins do not engage co-receptor molecules or Lck. A molecule was constructed in which the transmembrane and intracellular regions of the CD4 co-receptor (which localize to lipid rafts and bind Lck, respectively) were fused to a single-chain antibody that binds CD3 (UCHT1; SEQ ID NOs: 13 and 14; the sequence is also in the public domain). This construct is designed to direct CD3 molecules and TCRs to the lipid raft region and bring Lck into proximity with the TCR, similar to natural MHC binding. To target this chimeric receptor, a designed ankyrin repeat (DARPin) polypeptide was linked to the CD4-UCHT1 chimera to generate a trifunctional T cell antigen conjugate (Tri-TAC). In this particular case, the DARPin is specific for the proto-oncogene, erbB-2.

Human T cells were engineered to express either a prototype Tri-TAC or a conventional CAR with the same DARPin. It was determined that in all respects, T cells engineered with Tri-TAC showed functionality equivalent to that of conventional CAR. Interestingly, for two parameters (TNF-α production and CD107a mobilization), Tri-TAC was observed to be more active than conventional CAR. In addition, the data showed that on a per-molecule basis, Tri-TAC showed significantly enhanced activity. In addition, compared to conventional CAR, Tri-TAC provides enhanced safety because the activation domain is not part of the protein.

Traditional CARs are effective in stimulating T cells by combining several signaling domains (Figure 1C). In contrast, Tri-TAC (Figure 1A/B) does not contain any signaling domains of its own. It relies purely on promoting proposed interactions between other key players (shown in gray) in an antigen-dependent manner. To test the hypothesis of this design, several variants of the full-length N-Darpin Tri-TAC were generated (Figure 1D).

Previous work has established consistent and significant cell surface expression of CAR molecules. It was found that DarpinCAR showed robust surface expression (Figure 2). In contrast, Tri-TAC showed much lower surface expression. This was observed in all variants with the UCHT1 domain. However, the Tri-TAC variant without the UCHT1 domain showed similar surface expression to DarpinCAR.

T cells engineered to express Tri-TAC, Tri-TAC variants or DARPin CAR were stimulated with antigens bound on the plate. T cells engineered to express Tri-TAC and DARPin CAR were able to exert all measured functions (TNF-α production, IFN-γ production and CD107a mobilization) (Figures 3A and 3B). It was found that Tri-TAC binding to CD3 and target antigens was critical for T cells to exert their functions. In Figure 3, it was shown that removing UCHT1 (which cancels binding to CD3) eliminated the function of Tri-TAC. In other data, it was determined that removing DARPin from Tri-TAC also eliminated function.

As expected, when these T cells were tested for cytotoxicity, Tri-TAC-UCHT1-Darpin showed no ability to kill cells expressing the antigen (Figure 4). N-Darpin Tri-TAC showed a high level of selective cytotoxicity, which is very similar to the classic DARPin-CAR. Interestingly, T cells expressing DARPin-CAR appeared to show off-target killing at high T cell:target cell ratios (see killing of D2F2 in Figure 4), while T cells expressing Tri-TAC did not show these effects.

experiment

Figure 1 is a schematic overview. (A) N-Darpin Tri-TAC is described. Using a (G ₄ S) ₃ linker, the ankyrin repeat domain targeting Her2 is fused to the single-chain variable fragment (scFv) UCHT1. The scFv is then connected to the CD4 molecule. CD4 contains a connecting region and a transmembrane region as well as a cytoplasmic anchoring region. Possible interactions are shown in faded gray. (B) C-Darpin Tri-TAC is described. In this construct, the scFv UCHT1 is replaced with the Darpin domain. Possible interactions are again described in faded gray. (C) Model of a classic second-generation CAR. The Darpin targeting domain is connected to the CD28 transmembrane domain via a CD8α linker. The CD3ζ domain with three activated ITAM motifs is then connected to the cytoplasmic portion of CD28. (D) Overview of each Tri-TAC control, which has no Darpin targeting domain, the scFv portion that binds CD3, or the cytoplasmic portion of the CD4 domain.

Figure 2 shows phenotypic surface expression analysis of T cells transduced with each Tri-TAC variant shown in the histogram. T cells were incubated with Her2Fc and subsequently analyzed by flow cytometry. The data presented were gated on CD8+ lymphocytes. The gates shown were selected based on untransduced controls.

Fig. 3 is the functional analysis of engineered T cells.In (A), in the culture medium containing GolgiPlug ^TM , the Her2Fc on the cell plate was stimulated for 4 hours.Cells were first dyed for CD8+, then permeabilized and analyzed for TNF-α and IFN-γ production.Initial gating was set for single peak CD8+ lymphocytes.The gating shown was set based on the control of non-transduction.In B), as previously described, the Her2Fc on the cell plate was stimulated.Culture medium includes GolgiPlug ^TM and anti-CD107a antibody.It is expected that the cells of active degranulation have a higher ratio of CD107a recirculation, and subsequently show higher signal for anti-CD107a.

Fig. 4 shows the cytotoxicity of engineered T cells. Before adding T cells, two different adherent mouse tumor cell lines were plated and kept for 24 hours. D2F2/E2 has been engineered to express human Her2, but D2F2 has not been engineered. T cells of a specified ratio are added to the wells containing the tumor. Tumor cells and T cells are incubated for 6 hours. Subsequently, T cells are removed by washing. Culture medium containing 10% Alamar blue is added to each well and kept for 3 hours. Metabolic activity (as an indicator of cell survival) is determined via endpoint analysis. The wells without T cells are defined as maximum viability/metabolic activity and are set to 100%, whereas the culture medium without cell incubation is set to 0% metabolic activity. The data provided are the mean values of 3 repetitions.

discuss

The use of chimeric receptors to redirect T cells to specific targets in an MHC-independent manner is an attractive approach for treating cancer and may be applicable to infectious diseases where antigens from pathogens are present in the plasma membrane. Chimeric receptors will result in: (1) specific cytotoxicity against target cells and (2) minimal off-target toxicity. Conventional CARs are limited in this regard because they rely on synthetic structures and may not receive proper regulation if the signaling domain is located in an abnormal position, so cellular control of specific activity is reduced.

Tri-TACs are designed to redirect the signaling components of native TCRs without the need for ectopic localization of signaling domains. Tri-TACs were designed using the following principles: (1) chimeric receptors should interact with and promote the orderly assembly of important activating protein complexes, (2) chimeric receptors should exploit pre-existing cellular adaptations, such as microdomain environments, and (3) chimeric receptors should not possess any activation domains. Tri-TACs were able to achieve this effectively, as shown in the data, with activation rates equal to (if not better than) those of second-generation CARs.

Therefore, this Tri-TAC is very suitable for further integration with additionally designed co-receptors to further fine-tune T cell activation. Ultimately, this should lead to a substantial reduction in off-target effects without compromising targeted cytotoxicity. Tri-TAC appears to show lower toxicity than existing CARs. Darpin CARs show slight off-target killing at high cell-to-target ratios, which may become problematic when used for therapy. However, Tri-TAC, which functions almost identically to traditional CARs, does not show off-target effects. Because DARPins bind to their targets with high affinity, off-target effects may be more common in cells that express high levels of chimeric receptors that employ DARPins. Therefore, without being bound by theory, the low surface expression of Tri-TAC may be advantageous because it reduces the likelihood of such off-target effects.

Ultimately, the modular nature of Tri-TAC technology allows for more precise tuning of the T cell activation process. For example, recruitment of the TCR complex can be regulated by engineering Tri-TAC molecules with lower CD3 affinity. This can be used to mimic the naturally low affinity of TCRs (Chervin et al., 2009) while retaining high affinity for the targeting domain to detect cancer targets. Unlike classic CARs, Tri-TAC technology can be engineered to more closely mimic the above scenario.

In summary, the presented Tri-TAC technology is a highly potent molecular tool that can (1) effectively trigger T cell activation and cytotoxicity, (2) do so by mimicking natural T cell activation, and (3) does not require its own activation domain.

Example 2. Characterization of Tri-TAC Technology

An overview of the Tri-TAC technology is provided in Figure 5.

Fig. 5 A shows the example of CD8T cell activation based on the co-assembly of different receptors and their related protein partners. Initially, major histocompatibility complex I presents antigen (helix). This is recognized by the T cell receptor (TCR) complex that can bind antigen. The TCR complex comprises several independent subunits. The α/β domain can directly interact with the antigen presented by MHC-I. Then, the α/β domain interacts with several other domains (ε, γ, δ and ζ), and all of the domains participate in T cell activation via various intracellular activation domains. The TCR complex can interact with MHC-I simultaneously with CD8 co-receptors. The CD8 co-receptors bind MHC-I in an antigen-independent manner. CD8 directly interacts with Lck, which is a protein kinase important for activating the TCR receptor complex. CD8 and Lck interact and also ensure that they are combined with lipid rafts (membrane part) microdomains, assuming that the microdomains organize and encapsulate other related signal transduction parts (black spheres). Then, the later stage of activation causes CD28 to raise. If this cascade of interactions occurs several times in parallel, T cells become activated and are able to exert their cytotoxic effect.

Fig. 5B provides an overview of chimeric antigen receptor (CAR).CAR attempts to reproduce the complex mechanism of T cell activation by combining several important activation domains, such as ζ and CD28 in a single synthetic engineered molecule.Then, using the specific binding domain, CAR directly interacts with the selected antigen.Described here is ankyrin repeat protein (DARPin).It is believed that several such interactions occurring in parallel lead to T cell activation.

Figure 5C describes a bispecific T cell adapter (BiTE)-like molecule that engages T cells by directly cross-linking the TCR complex with the selected antigen. The BiTE-like molecule described here comprises two binding domains. The DARPin portion interacts with the target antigen. The single-chain variable fragment domain (scFv) binds to the TCR complex via its ε domain. Several such cross-links occur in parallel, leading to T cell activation.

Figure 5D provides an overview of TAC technology, which mimics the natural activation process. A T cell antigen conjugate (TAC) is capable of binding to the T cell antigen conjugate's antigen via the DARPin binding domain. The DARPin is then linked to a scFv capable of binding to the epsilon domain of the TCR complex. The TAC is then bound to the CD4 transmembrane and cytoplasmic domains. CD4, like CD8, interacts with Lck and is localized in lipid rafts. Thus, TACs combine TCR recruitment with co-receptor stimulation. Without being bound by theory, it is believed that several such interactions occur in parallel, leading to T cell activation.

Different configurations of TAC molecules are possible. Figure 6A shows a model of a TAC molecule in configuration 1. The CD4-tail, transmembrane domain, and linker domain are combined with a TCR-ε-specific scFv (UCHT1). The scFv is then connected to the antigen-binding domain. The domains are interchangeable. In this scheme, the antigen-binding domain used is a scFv or DARPin domain specific for the Her2 antigen. Figure 6B shows a TAC molecule in configuration 2. Here, the CD4 domain first interacts with the antigen-binding domain. This domain is then connected to the scFv (UCHT1) domain that recruits the TCR.

Figure 7 shows the functionality of scFv CD4TAC. Figure 7A is a histogram showing the surface expression of scFv CD4TAC receptors relative to an empty vector. The cells were stained with FcHer2 antigen and then detected with a fluorescently labeled antibody. Figure 7B shows antigen-specific activation of T cells expressing scFv CD4TAC (upper side) or scFV CAR (lower side). T cells expressing scFv CD4TAC (upper side) or scFv CAR (lower side) were incubated with Her2 antigen bound to the plate. The two modified cells showed antigen-specific activation. The DMSO negative control did not show activity (data not shown). Figure 7C shows the comparable killing of MCF-7 human tumor cell line (Her2 positive) by scFv CD4TAC and scFv CAR. scFv CD4TAC and scFv CAR were incubated with MCF-7 human tumor cell line (Her2 positive) and compared with the empty vector control.

Figure 8 is a characterization of CD4-TAC Configuration 2. Figure 8A is a histogram of DARPin CD4-TAC Configuration 2 relative to vector control. Surface expression was probed with antigen modified with FcHer2. Cells expressing CD4-TAC Configuration 2 showed a significant increase in FcHer2 binding, demonstrating high surface expression of the receptor. For clarity, a model of CD4 TAC Configuration 2 is shown. Figure 8B shows T cells engineered with DARPin TAC Configuration 2 exposed to plate-bound Her2 antigen. Cytokine production and cell degranulation were measured. The data show that DARPin TAC Configuration 2 is a functional receptor. Treatment without antigen did not show T cell activation (data not shown). Figure 8C shows the growth of CD4 TAC Configuration 2 relative to the empty vector control. Cells were grown in 100u/ml IL2 10ng/ml IL7. Starting with 100,000 cells, growth was monitored by counting culture samples at predetermined intervals. Configuration 2 had a significantly reduced growth rate relative to the control.

Figure 9 shows the functionality of DARPin CD4 TAC configuration 1. Figure 9A shows the surface expression of DARPin CD4 TAC (red) compared to DARPin CAR (green) and NGFR-only control (blue). Cells were probed with the receptor-specific antigen FcHer2. The histogram shows that DARPin CD4 TAC is well expressed on the surface. However, the maximum surface expression of DARPin CD4 TAC is lower compared to the CAR construct. Figure 9B shows the growth of CD4 TAC configuration 1. For two weeks of culture, growth was monitored by sampling and manually counting the cells. The empty vector showed similar growth to DARPin CAR. However, TAC had reduced growth in comparison. Figures 9C and 9D show the percentage of cells positive for various activation markers and degradation markers. Empty vector, DARPin CD4 and DARPin CAR were incubated with plate-bound antigen Her2 or DMSO control. The results of three separate experiments were summarized using the statistical analysis software SPICE. The scatter plots show the percentage of cells positive for a panel of activation markers. CD4-TAC showed a higher percentage of cells that were positive for cell degranulation markers. DARPinCAR cells were positive for multiple activation markers, but no significant enrichment of cell degranulation markers. The pie chart represents the same data. The pie chart shows that CD4-TAC has a significantly higher concentration of cell populations that are degranulated. CD4-TAC has the ability to degranulate most activated cells while producing various levels of cytokines. However, CAR showed a more randomly distributed pattern of activation and cell degranulation, which constituted less than 50% of the total population. The pattern may indicate less controlled T cell activation by CAR.

Figure 10 shows the cytotoxicity and overall activity of TACs and CARs. Cells engineered with TACs, CARs, or an empty vector control were incubated with various human tumor cell lines. MDA MB 231, SK OV 3, and A549 all express the Her2 antigen. LOXIMVI is Her2-negative. It was observed that in all cases, TACs exhibited enhanced cytotoxicity. The antigen-negative cell line LOXIMVI was not targeted, supporting the idea that the cytotoxicity was antigen-specific.

Figure 11 shows receptor surface expression and activation of various TAC controls. Figure 11A shows cell surface expression (left), cell degranulation (middle) and cytokine production (right). Constructs without specific domains were used to determine the importance of these domains. From top to bottom, the following domains were removed: DARPin antigen binding domain and UCHT1 TCR binding domain, with the full-length TAC at the bottom. Surface expression of TAC without the UCHT1 domain resulted in enhanced surface expression relative to the full-length CD4 TAC. DARPin-negative mutants could not be detected using the FcHer2 antigen. Cell degranulation (middle) was only observed in the full-length TAC. UCHT1 and DARPin deletion resulted in no cell degranulation. Similarly, cytokine production was only observed in the full-length TAC. Figure 11B shows that the mouse cell line D2F2 was engineered to express the human Her2 antigen (D2F2/E2). Both cell lines were incubated with engineered T cells with the full-length CD4-TAC or its deletion variants. The data showed an effector-to-target ratio of 4:1 (endpoint). Only the full-length CD4-TAC was able to elicit a cytotoxic response, demonstrating that the DARPin and UCHT1 domains are involved in receptor function.

Figure 12 shows the properties of various transmembrane TAC variants. Figure 12A is an overview of the various transmembrane constructs. The first group of variants lacked the cytoplasmic domain. CD4TAC-cytoplasmic removed the entire cytoplasmic domain. The synthetic construct replaced CD4TM with a designed, highly hydrophobic membrane domain. The glycophorin variant replaced the CD4 transmembrane domain with the glycophorin transmembrane domain. The GPI anchor variant utilized the CD48 GPI signal sequence to attach the TAC to the membrane using a GPI anchor. The CD8A TAC variant replaced the transmembrane domain and the cytoplasmic CD4 domain with the CD8α counterpart. Figure 12B shows that CD8 purified T cells were engineered with each construct. The surface expression of each receptor relative to the full-length TAC is shown. All data are relative to the median fluorescence intensity of the control. All variants had significantly lower receptor surface expression compared to the full-length CD4-TAC. The GPI anchored TAC variants were not detectable above background levels. Figure 12C describes testing of cell degranulation and cytokine production of the different variants. Cells were incubated with plate-bound Her2 antigen. Activity was expressed as the percentage of cells positive for the cell degranulation marker CD107a (left bar graph) or the percentage of all cytokine-producing cells combined (TNFa, IFNg, and TNFa/IFNg, right bar graph). GPI-anchored or CD8a variants showed background levels of cell degranulation and cytokine production. Glycophorin, synthetic and -cytoplasmic TAC variants showed moderate levels of cell degranulation and low levels of cytokine production. In all cases, activity was much lower than full-length CD4-TAC. In summary, this shows that anchoring TACs without a cytoplasmic domain results in reduced activity of functional receptors.

Figure 13 shows the interaction between Lck and TAC variants. In Figure 13A, Her2 antigen was covalently linked to magnetic beads. 293TM cells were engineered to express TAC and TAC cytoplasmic deletion variants as well as Lck. The beads were incubated with cell lysates overnight, then washed and subjected to Western blot analysis. Lck was detected using Lck antibodies and TAC was detected via Myc antibodies. B-actin was used as a control. B-actin was not pulled down and was only detected in the supernatant (S). However, full-length TAC and cytoplasmic deletion were effectively pulled down and detected in the precipitate portion (B). The vector control and TAC without cytoplasmic domain showed a considerable level of background Lck signal. However, full-length CD4 TAC showed a significant level of Lck relative to the total amount. Figure 13B shows a density determination analysis of Lck detected in the precipitate. The signal was corrected relative to the negative control. The data support that Lck can interact with full-length CD4-TAC.

In Figure 14, CD4TAC surface expression and activity are compared with BiTE-like variants. Figure 14A shows only NGFR control (left side), CD4TAC (middle) and BiTE-like variants (right side). Surface expression was tested using transduction markers NGFR and Her2 antigens. Compared with BiTE, TAC shows much lower surface expression. Most notably, BiTE seems to secrete enough coupled antibodies to enable transduction negative cells (NGFR-) to show strong receptor expression. Compared with TAC engineered cells, cytokine production and cell degranulation are both higher in BiTE-like cells. Figure 14B compares the cytotoxicity in various Her2-positive cell lines (MDAMB 231, SK OV 3, A549). In contrast to cytokine production, TAC engineered cells show significantly enhanced cytotoxic activity.

Figure 15 shows a comparison of a random mutation library of CD4 TAC WT and UCHT1. To test the ability to alter TAC properties, 24 amino acids present on the binding surface of UCHT1 and TCRε were individually mutated. This yields a theoretical number of 480 unique clones, all of which should be represented in this random library. Figure 15A shows a schematic representation of the mutants. The markers indicate the mutations, all of which are at the scFv-ε interface. Figure 15B is a histogram of surface expression. Engineered cells were probed with FcHer2 antigen to detect surface-expressed receptors. The library shows greatly enhanced surface expression of the receptor. Figure 15C shows WT and library CD4 TAC cells incubated with plate-bound antigen. Their ability to activate and produce cytokines is shown. The library has similar activity compared to WT. Without being bound by theory, this supports the view that the expression properties of TACs can be improved by altering the scFv domain while retaining the original functional characteristics.

Figure 16 shows that the surface expression of the A85V, T161P mutant is enhanced. The library is propagated for an extended period of time to select mutants with a growth advantage over WT. The selected mutants (A85V, T161P; numbering is based on the UCHT1 domain fragment) were analyzed. Figure 16A shows that peripheral blood mononuclear cells (PBMCs) were engineered with WT CD4-TAC or A85V, T161P mutants. Comparison of the final CD4/CD8 populations between CD4TAC (left) and A85V, T161P mutants (right). Clearly, WT CD4-TAC leads to a decrease in the CD4-positive cell population. This effect was not observed in mutant cells. Figure 16B shows surface expression, as measured by NGFR transduction markers and FcHer2 positivity, and indicates that the surface expression of the A85V, T161P mutant is enhanced. Figure 16C shows that the A85V, T161P mutant cytokine production is reduced (DMSO control did not show activity, data not shown). Cell degranulation was comparable between WT TAC and the A85V,T161P mutant.

Figure 17 shows the cytotoxicity and growth of the A85V, T161P mutant. In Figure 17A, T cells engineered with the WT CD4 TAC and the A85V, T161P mutant were incubated with the Her2 antigen-positive cell lines SK OV 3, MDA MB 231, and A549. In all cases, the mutants showed reduced levels of cytotoxicity; no cytotoxicity was detected for A549. In Figure 17B, cell growth was monitored over 2 weeks in cultures starting with 100,000 cells. Samples were taken regularly and cells were counted manually. The A85V, T161P mutant showed significantly improved growth compared to the WT variant. Overall, this suggests that the library may contain a variety of mutants that enable modification and optimization of several TAC functions. Therefore, UCHT1 can be used as a functional modulator.

While the present application has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the application is not limited to the disclosed embodiments, but rather is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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Claims (15)

1. Nucleic acids, which include: The first polynucleotide, which encodes the tumor ligand as component a; The second polynucleotide encodes a single-chain antibody that binds to CD3 as component b; and A third polynucleotide encoding the transmembrane and intracellular regions of the CD4 co-receptor as component c, wherein the transmembrane and intracellular regions of the CD4 co-receptor contain amino acid sequences having at least 98% identity with SEQ ID NO: 18. The nucleic acid encodes a T-cell antigen conjugate, wherein all components a, b, and c of the T-cell antigen conjugate are directly fused to each other and/or linked by at least one linker, provided that the nucleic acid does not encode an activation domain, and wherein the order in which components a, b, and c are directly fused to each other and/or linked by at least one linker is a-b-c or b-a-c. 2. The nucleic acid of claim 1, wherein the tumor ligand is a designed ankyrin repeat (DARPin) polypeptide or scFv. 3. The nucleic acid of claim 1 or 2, wherein the CD3-binding single-chain antibody is UCHT1 or a variant thereof. 4. A polypeptide encoded by any one of the nucleic acids claimed in claims 1-3. 5. An expression vector comprising the nucleic acid of any one of claims 1-3. 6. T cells expressing the nucleic acid of any one of claims 1-3. 7. A carrier construct, comprising: The first polynucleotide, which encodes the tumor ligand as component a; The second polynucleotide encodes a single-chain antibody that binds to CD3 as component b; and A third polynucleotide encoding the transmembrane and intracellular regions of the CD4 co-receptor as component c, wherein the transmembrane and intracellular regions of the CD4 co-receptor contain an amino acid sequence having at least 98% identity with SEQ ID NO: 18; and A functional promoter in mammalian cells, wherein all components a, b, and c are directly fused to each other and/or linked by at least one linker, provided that the first polynucleotide, the second polynucleotide, and the third polynucleotide do not encode an activation domain, and wherein the order in which components a, b, and c are directly fused to each other and/or linked by at least one linker is a-b-c or b-a-c, to provide a T-cell antigen conjugate. 8. The vector construct of claim 7, wherein the coding sequence of the T-cell antigen conjugate is operatively linked to the promoter. 9. The vector construct according to any one of claims 7-8, wherein the tumor ligand is a designed ankyrin repeat (DARPin) polypeptide or scFv. 10. The vector construct according to any one of claims 7-8, wherein the CD3-binding single-chain antibody is UCHT1 or a variant thereof. 11. Isolated T cells transfected with the vector construct according to any one of claims 7-10. 12. A pharmaceutical composition comprising a carrier and the T cells of claim 6 or 11. 13. Use of T cells in the preparation of pharmaceutical compositions for treating cancer, wherein said T cells express nucleic acids comprising: The first polynucleotide, which encodes the tumor ligand as component a; The second polynucleotide encodes a single-chain antibody that binds to CD3 as component b; and A third polynucleotide encodes the transmembrane and intracellular regions of a CD4 co-receptor as component c, the transmembrane and intracellular regions of the CD4 co-receptor comprising an amino acid sequence having at least 98% identity with SEQ ID NO: 18, wherein the nucleic acid encodes a T-cell antigen conjugate, all components a, b, and c of the T-cell antigen conjugate being directly fused to each other and/or linked by at least one linker, provided that the first polynucleotide, the second polynucleotide, and the third polynucleotide do not encode an activation domain, and wherein the order in which components a, b, and c are directly fused to each other and/or linked by at least one linker is a-b-c or b-a-c. 14. The use as described in claim 13, wherein the tumor ligand is a designed ankyrin repeat (DARPin) polypeptide or scFv. 15. The use as described in claim 13 or 14, wherein the CD3-binding single-chain antibody is UCHT1 or a variant thereof.