CHIMERIC TRANSMEMBRANE RECEPTORS AND USES THEREOF

TECHNICAL FIELD

The present invention relates to molecular biology, and particularly to methods and compositions for regulating selective gene expression in cells (e.g., cells of the monocyte/macrophage lineage), and applications thereof.

BACKGROUND

A variety of new tools for treating cancer have been developed in recent years. For example, expression vectors encoding chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) that target certain cancer antigens present on cancer cells have been introduced into immune cells, which engineered immune cells are then administered to a subject having cancer. Regulating the expression, activity, or both, of such engineered immune cells remains an active area of endeavor. A variety of regulatory mechanisms to control the expression, activity, or both, of chimeric antigen receptors, for example, are known in the art. See, e.g., Roybal et al, “Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits”, Cell 2016; Wu et al, “Remote control of therapeutic T cells through a small molecule-gated chimeric receptor”, Science 2015; Sakemura et al, “A Tet-On Inducible System for Controlling CD19-Chimeric Antigen Receptor Expression upon Drug Administration”, Cancer Immunol Res 2016; Rakhit et al, “Chemical biology strategies for posttranslational control of protein function”, Chem Biol 2014; Navarro et al, “A Novel Destabilizing Domain Based on a Small-Molecule Dependent Fluorophore”, ACS Chem Biol 2016, each of which is incorporated herein by reference in its entirety. Improved compositions and methods for regulating engineered immune cells expressing, for example, chimeric antigen receptors or T cell receptors are needed.

SUMMARY OF THE INVENTION

Provided herein are methods and compositions for regulating selective expression of a protein (e.g., a therapeutic protein, e.g., a chimeric antigen receptor or T-cell receptor) in cells (e.g., immune cells), and applications thereof.

In some embodiments, provided herein are chimeric transmembrane receptors that include: an extracellular antigen-binding domain that is capable of specifically binding to a target antigen; an extracellular integrin ligand-binding domain that includes an S2 protease cleavage site; a transmembrane domain; an intracellular regulatory domain that includes a gamma-secretase protease cleavage site; and an intracellular transcriptional regulatory domain; wherein, when the chimeric transmembrane receptor is expressed in a mammalian cell, binding of the extracellular antigen-binding domain to the target antigen induces (1) cleavage of the extracellular integrin-ligand binding domain at the S2 protease cleavage site and (2) cleavage of the intracellular regulatory domain at the gamma-secretase protease cleavage site, thereby releasing the intracellular transcriptional regulatory domain from the transmembrane domain.

In some embodiments, chimeric transmembrane receptors provided herein include an antigen-binding domain that is an antibody or an antibody fragment. In some embodiments, a chimeric transmembrane receptor includes an antigen-binding domain that an antibody, wherein the antibody is selected from the group consisting of: a Fab fragment, an Fv fragment, a scFv fragment, an Fd fragment, a chimeric antibody, a humanized antibody, a fully-human antibody, a single-chain antibody (scAb), a single domain antibody (dAb), a single domain heavy chain antibody, a single domain light chain antibody, a nanobody, a bi-specific antibody, and a multi-specific antibody.

In some embodiments, chimeric transmembrane receptors provided herein include an antigen-binding domain that binds a target antigen selected from the group consisting of: BCMA, MAGE, MUC16, CD19, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD20, CD125 CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

In some embodiments, chimeric transmembrane receptors provided herein include an extracellular integrin ligand-binding domain that is a human fibronectin III domain or a mouse fibronectin III domain. In some embodiments, chimeric transmembrane receptors provided herein include an extracellular integrin ligand-binding domain that includes a sequence at least 80% identical to a sequence of a wild type human fibronectin III domain or a sequence of a wild type mouse fibronectin III domain. In some embodiments, chimeric transmembrane receptors provided herein include an additional extracellular integrin ligand-binding domain. In some embodiments, an additional extracellular integrin ligand-binding domain of a chimeric transmembrane receptor provided herein includes a wild type human fibronectin type III domain or a wild type mouse fibronectin type III domain. In some embodiments, an additional extracellular integrin ligand-binding domain of a chimeric transmembrane receptor provided herein includes comprises a sequence at least 80% identical to a sequence of a wild type human fibronectin III domain or a sequence of a wild type mouse fibronectin III domain.

In some embodiments, chimeric transmembrane receptors provided herein include a transmembrane domain that is present in a receptor-like tyrosine phosphatase. In some embodiments, chimeric transmembrane receptors provided herein include a transmembrane domain that is at least 80% identical to a sequence of a transmembrane domain present in a receptor-like tyrosine phosphatase. In some embodiments, chimeric transmembrane receptors provided herein include a transmembrane domain that is present in a polypeptide selected from the group consisting of: CD28, CD3 epsilon, CD4, CD5, CD6, CD8a, CD9, CD16, CD22, CD33, CD37, CD 45, CD64, CD80, CD86, CD134, 4-1BB, GITR, NGFR, and CD154. In some embodiments, chimeric transmembrane receptors provided herein include a transmembrane domain that includes a sequence that is at least 80% identical to the sequence of a transmembrane domain present in a polypeptide selected from the group consisting of: CD28, CD3 epsilon, CD4, CD5, CD6, CD8a, CD9, CD16, CD22, CD33, CD37, CD 45, CD64, CD80, CD86, CD134, 4-1BB, GITR, NGFR, and CD154.

In some embodiments, chimeric transmembrane receptors provided herein include a gamma-secretase cleavage site that includes a Gly-Val dipeptide amino acid sequence.

In some embodiments, chimeric transmembrane receptors provided herein include an intracellular transcriptional regulatory domain is a transcriptional activator. In some embodiments, chimeric transmembrane receptors provided herein include an intracellular transcriptional regulatory domain is a transcriptional repressor. In some embodiments, chimeric transmembrane receptors provided herein include an intracellular transcriptional regulatory domain that is present in a polypeptide selected from the group consisting of: VP64, RelA (p65), YAP, WWTR1(TAZ), CREB3(LZIP), and MyoD. In some embodiments, chimeric transmembrane receptors provided herein include an intracellular transcriptional regulatory domain that includes a sequence that is at least 80% identical to a sequence of a transcriptional activation domain present in a polypeptide selected from the group consisting of: VP64, RelA (p65), YAP, WWTR1(TAZ), CREB3(LZIP), and MyoD.

Also provided herein are nucleic acids that encode any of the chimeric transmembrane receptors described herein. Also provided herein are vectors that include any of the nucleic acids encoding any of the chimeric transmembrane receptors described herein. Also provided herein are mammalian cells that include any of the nucleic acids encoding any of the chimeric transmembrane receptors described herein described herein or any of the vectors described herein. In some embodiments, the mammalian cell is an immune cell. For example, the immune cell can be selected from the group consisting of: a CD4+ T cell, a CD8+ T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a regulatory T cell, and a helper T cell. In some embodiments, the mammalian cell further includes a heterologous target gene that includes (i) a transcription regulatory sequence that is capable of being specifically recognized by the intracellular transcriptional regulatory domain and (ii) a nucleic acid sequence that encodes a recombinant protein, wherein the nucleic acid sequence that encodes the recombinant protein is operably linked to the transcription regulatory sequence.

In some embodiments, a recombinant protein encoded by a heterologous target gene is a secreted polypeptide.

In some embodiments, a recombinant protein encoded by a heterologous target gene is a chimeric antigen receptor (CAR). For example, a CAR can include an antigen-binding domain capable of specifically binding to an antigen selected from the group consisting of: BCMA, MAGE, MUC16, CD19, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD20, CD125 CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-0, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

In some embodiments, a recombinant protein encoded by a heterologous target gene is a T cell receptor (TCR). For example, a TCR can include an antigen-binding domain capable of specifically binding to an antigen selected from the group consisting of: BCMA, CD11a, CD19, CD20, CD22, CD30, CD38, CD52, Her2/neu, ENPP3, EGFR, MAGE-A1, IL-13R-a2, GD2, alpha-integrin, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), or high molecular weight-melanoma associated antigen (HMW-MAA).

Also provided herein are pharmaceutical compositions that include any of the mammalian cells described herein. Also provided herein are pharmaceutical compositions that include any of the nucleic acids or vectors described herein. In some embodiments, pharmaceutical compositions that include any of the nucleic acids or vectors described herein can further include a heterologous target gene that includes (i) a transcription regulatory sequence that is capable of being specifically recognized by the intracellular transcriptional regulatory domain and (ii) a nucleic acid sequence that encodes a recombinant protein, wherein the nucleic acid sequence that encodes the recombinant protein is operably linked to the transcription regulatory sequence.

Also provided herein are methods of treating disease in a subject in need thereof that include administering a therapeutically effective amount of any of the pharmaceutical compositions described herein to the subject. In some embodiments, the disease is cancer. In some embodiments, the pharmaceutical composition includes a mammalian cell that is autologous to the subject. In some embodiments, the pharmaceutical composition includes a mammalian cell that is allogenic to the subject.

Also provided herein are nucleic acids encoding a chimeric transmembrane receptor that include: a first nucleic acid segment that encodes an extracellular antigen-binding domain that is capable of specifically binding to a target antigen; a second nucleic acid segment that encodes an extracellular integrin ligand-binding domain that includes an S1 protease cleavage site, an S2 protease cleavage site, or both; a third nucleic acid segment that encodes a transmembrane domain; a fourth nucleic acid segment that encodes an intracellular regulatory domain that includes a gamma-secretase protease cleavage site; and a fifth nucleic acid segment that encodes an intracellular transcriptional regulatory domain; wherein, when the chimeric transmembrane receptor is expressed in a mammalian cell, binding of the extracellular antigen-binding domain to the target antigen induces (1) cleavage of the extracellular integrin-ligand binding domain at the S2 protease cleavage site and (2) cleavage of the intracellular regulatory domain at the gamma-secretase protease cleavage site, thereby releasing the intracellular transcriptional regulatory domain from the transmembrane domain. Also provided herein are vectors that include any of the nucleic acids encoding a chimeric transmembrane receptor described herein. In some embodiments of vectors that include a nucleic acid encoding a chimeric transmembrane receptor, the nucleic acid encoding a chimeric transmembrane receptor is operably linked to a transcription regulatory sequence.

Also provided herein are mammalian cells that include any of the nucleic acids encoding a chimeric transmembrane receptor or any of the vectors described herein. In some embodiments, the mammalian cell is an immune cell. For example, the mammalian cell can be selected from the group consisting of: a CD4+ T cell, a CD8+ T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a regulatory T cell, and a helper T cell. In some embodiments, the mammalian cell further includes a heterologous target gene that includes (i) a transcription regulatory sequence that is capable of being specifically recognized by the intracellular transcriptional regulatory domain and (ii) a nucleic acid sequence that encodes a recombinant protein, wherein the nucleic acid sequence that encodes the recombinant protein is operably linked to the transcription regulatory sequence. In some embodiments, a recombinant protein encoded by a heterologous target gene is a secreted polypeptide. In some embodiments, a recombinant protein encoded by a heterologous target gene is a chimeric antigen receptor (CAR). For example, a CAR can include an antigen-binding domain capable of specifically binding to an antigen selected from the group consisting of: BCMA, MAGE, MUC16, CD19, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD20, CD125 CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin. In some embodiments, a recombinant protein encoded by a heterologous target gene is a T cell receptor (TCR). For example, a TCR can include an antigen-binding domain capable of specifically binding to an antigen selected from the group consisting of: BCMA, CD11a, CD19, CD20, CD22, CD30, CD38, CD52, Her2/neu, ENPP3, EGFR, MAGE-A1, IL-13R-a2, GD2, alpha-integrin, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), or high molecular weight-melanoma associated antigen (HMW-MAA).

Other features and advantages of the invention will be apparent from the following Detailed Description of the Invention, and from the claims. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. All publications mentioned herein, including patents, patent application publications, and scientific papers, are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 is a schematic diagram of an exemplary synPTPR based on the receptor-like protein tyrosine phosphatase type-K (PTPRK). PTPRK is composed of a MAM domain, Ig domain, four fibronectin type-III (FN-III) domains, and two intracellular phosphatase domains. In the embodiment of a synPTPR shown, only the transmembrane region and the first two most membrane-proximal fibronectin domains were kept from PTPR. An anti-CD19 scFv was fused to the N-terminus of the PTPR-core, and a transcription factor was fused to the intracellular, C-terminus of the PTPR-core. Without wishing to be bound by theory, association of the anti-CD19 scFv with its cognate ligand is hypothesized to cause a protease from the ADAM family (e.g., ADAM10 or ADAM17) to cleave the S2 cleavage site and gamma-secretase processing of the PTPR-core, releasing the intracellular transcription factor to shuttle to the nucleus and affect transcription of a nucleic acid sequence encoding a protein (e.g., a therapeutic protein, e.g., a chimeric antigen receptor or a T-cell receptor).

FIG. 2 is a schematic diagram of the constructs used to assess the functionality of exemplary synPTPRs as an antigen-sensing platform. An exemplary synPTPR (FIG. 2A) is composed of an aCD19 scFv, the PTPR-core, and a gal4-vp64 transcription factor. The reporter construct (FIG. 2B) includes a constitutive mCherry marker, and an inducible promoter driving GFP with multiple gal4 binding sites. In the presence of gal4-vp64, the reporter will upregulate the production of GFP. As a positive control, a synthetic Notch protein with the same aCD19 scFv and gal4-vp64 transcription factor was used (FIG. 2C). The synthetic Notch protein also used the same reporter (FIG. 2D).

FIG. 3 is a graph showing GFP expression in cells expressing a Notch1 positive control and synPTPR in the presence of CD19-expressing cells. The exemplary synPTPR used in this experiment upregulated GFP expression in the presence of both low and high antigen levels of CD19, with minimal basal expression in the absence of CD19.

FIG. 4 is a schematic showing wildtype PTPR proteins and the different chimeric transmembrane receptor that each include a portion of one of the wildtype PTPR proteins that were tested in Example 3.

FIG. 5 is a schematic showing the pairs of nucleic acid constructs encoding different chimeric transmembrane receptors and reporter nucleic acids that were tested in Example 3.

FIG. 6 is a graph showing the percentage of GFP-positive cells in a population of CD3⁺ cells transduced with pCDL1932, pCDL1933, pCDL1934, pCDL1935, pCDL1936, pCDL1937, or pCDL1541, and their corresponding reporter nucleic acid (as depicted in FIG. 5) upon co-culture with CD19⁻ K562 cells (un-stimulated) or CD19⁺ Raji cells (stimulated).

FIG. 7 is a graph showing the mean fluorescence intensity in GFP⁺ and mCherry⁺ cells in a population of CD3⁺ cells transduced with pCDL1932, pCDL1933, pCDL1934, pCDL1935, pCDL1936, pCDL1937, or pCDL1541, and their corresponding reporter nucleic acid (as depicted in FIG. 5) upon co-culture with CD19− K562 cells (un-stimulated) or CD19+ Raji cells (stimulated).

FIG. 8 is a graph showing the percentage of myc-positive cells in a population of CD3⁺ cells transduced with pCDL1933, pCDL2243, pCDL2244, pCDL2246, or pCDL2244, and their corresponding reporter nucleic acid (as depicted in FIG. 5) upon co-culture with CD19⁻K562 cells (un-stimulated) or CD19⁺ Raji cells (stimulated).

FIG. 9 is a graph showing the mean fluorescence intensity in GFP⁺ and mCherry⁺ cells in a population of CD3⁺ cells transduced with pCDL1933, pCDL2243, pCDL2244, pCDL2246, or pCDL2244, and their corresponding reporter nucleic acid (as depicted in FIG. 5) upon co-culture with CD19− K562 cells (un-stimulated) or CD19+ Raji cells (stimulated).

FIG. 10 shows the percentage of myc⁺ positive cells in a population of CD3⁺ cells transduced with pCDL2762, pCDL2763, pCDL2764, pCDL2765, or pCDL1933.

FIG. 11 is a graph showing the mean fluorescence intensity in GFP⁺ and mCherry⁺ cells in a population of CD3⁺ cells transduced with pCDL2762, pCDL2763, pCDL2764, pCDL2765, or pCDL1933, and their corresponding reporter nucleic acid (as depicted in FIG. 5) upon co-culture with CD19− K562 cells (un-stimulated) or CD19+ Raji cells (stimulated).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are chimeric transmembrane receptors that include an extracellular antigen-binding domain that is capable of specifically binding to a target antigen, an extracellular integrin ligand-binding domain comprising an S2 protease cleavage site, a transmembrane domain, an intracellular regulatory domain comprising a gamma-secretase protease cleavage site, and an intracellular transcriptional regulatory domain. In some embodiments, chimeric transmembrane receptors provided herein include one or more linkers between their various domains. In some embodiments, when the chimeric transmembrane receptor is expressed in a mammalian cell, binding of the extracellular antigen-binding domain to the target antigen induces (1) cleavage of the extracellular integrin-ligand binding domain at the S2 protease cleavage site and (2) cleavage of the intracellular regulatory domain at the gamma-secretase protease cleavage site, thereby releasing the intracellular transcriptional regulatory domain from the transmembrane domain. In some embodiments, release of the intracellular regulatory domain modulates an activity of a cell. For example, an intracellular regulatory domain can include a DNA-binding domain (e.g., any of the DNA-binding domains described herein or known in the art) and a transcriptional activation domain. (e.g., any of the transcriptional activation domains described herein or known in the art) When the intracellular regulatory domain is released upon binding of the extracellular antigen-binding domain to the target antigen, it can translocate to the nucleus of the cell where it can regulate the transcription of an mRNA encoding a polypeptide (e.g., a recombinant polypeptide, e.g., a chimeric antigen receptor or a T-cell receptor) under control of a regulatory element that is regulated by the intracellular regulatory domain (e.g., a promoter that is bound by the DNA-binding domain of the intracellular regulatory domain).

Chimeric transmembrane receptors provided herein exhibit a number of advantages over existing technology. For example, chimeric transmembrane receptors provided herein are more sensitive to activation (e.g., resulting in stronger gene regulation in the presence of a lower concentration of antigen) than other engineered receptors that are designed to regulate gene transcription upon binding a target antigen. Moreover, chimeric transmembrane receptors provided herein are smaller in size than other engineered receptors. For example, synNotch receptors such as those described in U.S. Pat. Nos. 9,670,281 and 9,834,608, each of which is incorporated herein by reference in its entirety, are limited both by sensitivity of response which is a function of the mechanism by which they occlude their S2 cleavage sites, as well as by their size. Chimeric transmembrane receptors provided herein are smaller than other engineered receptors that are designed to regulate gene transcription upon binding a target antigen, thus providing a variety of benefits over existing technology.

Various non-limiting aspects of chimeric transmembrane receptors are described herein, and can be used in any combination without limitation. Additional aspects of various components of chimeric transmembrane receptors are known in the art.

As used herein, the word “a” before a noun refers to one or more of the particular noun.

As used herein, the term “antigen” refers generally to a binding partner specifically recognized by an extracellular antigen-binding domain described herein. Exemplary antigens include different classes of molecules, such as, but not limited to, polypeptides and peptide fragments thereof, small molecules, lipids, carbohydrates, and nucleic acids. Non-limiting examples of antigen or antigens that can be specifically bound by any of the extracellular antigen-binding domains are described herein. Additional examples of antigen or antigens that can be specifically bound by any of the extracellular antigen-binding domains are known in the art.

The terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell, which generally but not exclusively include an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants, i.e., CAR variants are described, e.g., in PCT Application No. US2014/016527; Fedorov et al., Sci Transl. Med. 5(215):215ra172, 2013; Glienke et al., Front. Pharmacol. 6:21, 2015; Kakarla & Gottschalk, Cancer J. 20(2):151-155, 2014; Riddell et al., Cancer J. 20(2):141-144, 2014; Pegram et al., Cancer J. 20(2):127-33, 2014; Cheadle et al., Immunol Rev. 257(1):91-106, 2014; Barrett et al., Ann. Rev. Med. 65:333-347, 2014; Sadelain et al., Cancer Discov. 3(4):388-98, 2013; and Cartellieri et al., J. Biomed. Biotechnol. 956304, 2010; the disclosures of which are incorporated herein by reference in their entirety.

The term “extracellular antigen-binding domain” means a domain that is present on the extracellular side of the plasma membrane and binds specifically to a target antigen. In some examples, an extracellular antigen-binding domain can be formed from the amino acids present within a single-chain polypeptide. In other examples, an extracellular antigen-binding domain can be formed from amino acids present within a first single-chain polypeptide and the amino acids present in one or more additional single-chain polypeptides (e.g., a second single-chain polypeptide). Non-limiting examples of extracellular antigen-binding domains are described in more detail herein, including, without limitation, scFvs, or LBDs (Ligand Binding Domains) of growth factors. Additional examples of extracellular antigen-binding domains are known in the art.

The phrase “extracellular side of the plasma membrane” when used to describe the location of a transmembrane polypeptide means that the polypeptide includes at least one transmembrane domain that traverses the plasma membrane and at least one domain (e.g., at least one extracellular antigen-binding domain) that is located in the extracellular space.

“GFP” or green fluorescent protein (GFP) is a commonly used reporter of gene expression. Arun et al., J. Pharmacol. Toxicol. Methods 51(1):1-23, 2005.

An “isolated” polypeptide is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the polypeptide will be purified to greater than 90%, greater than 95%, or greater than 98%.

“Linkers” are amino acid sequences that separate multiple domains in a single protein, and, generally, can be classified into three groups: flexible, rigid and cleavable. Chen, X., et al., 2013, Adv. Drug Deliv. Rev., 65, 1357-1369. Linkers can be natural or synthetic. A number of linkers are employed to realize the subject invention including “flexible linkers.” The latter are rich in glycine. Klein et al., Protein Engineering, Design & Selection Vol. 27, No. 10, pp. 325-330, 2014; Priyanka et al., Protein Sci., 2013 February; 22(2): 153-167. In some embodiments, the linker is a synthetic linker. A synthetic linker can have a length of from about 10 amino acids to about 200 amino acids, e.g., from 10 to 25 amino acids, from 25 to 50 amino acids, from 50 to 75 amino acids, from 75 to 100 amino acids, from 100 to 125 amino acids, from 125 to 150 amino acids, from 150 to 175 amino acids, or from 175 to 200 amino acids. A synthetic linker can have a length of from 10 to 30 amino acids, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. A synthetic linker can have a length of from 30 to 50 amino acids, e.g., from 30 to 35 amino acids, from 35 to 40 amino acids, from 40 to 45 amino acids, or from 45 to 50 amino acids. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 1) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 2) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG, SEQ ID NO: 3) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences. In some embodiments, the linker is or comprises GSAAAGGSGGSGGS (SEQ ID NO: 4). In some embodiments, the linker is or comprises GGGSGGGS (SEQ ID NO: 5).

In some examples, a Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 2) linker can be encoded by the nucleic acid sequence of: GGTGGAGGAGGCTCT (SEQ ID NO: 47), GGTGGTGGGGGCTCC (SEQ ID NO: 48), GGAGGTGGTGGGAGT (SEQ ID NO: 49), GGCGGAGGCGGGAGC (SEQ ID NO: 50), GGCGGTGGAGGTTCC (SEQ ID NO: 51), GGGGGAGGTGGGAGT (SEQ ID NO: 52), or GGCGGGGGAGGGAGC (SEQ ID NO: 53).

In some examples, the GGGSGGGS (SEQ ID NO: 5) linker is encoded by the nucleic acid sequence of GGCGGTGGAAGCGGAGGAGGTTCC (SEQ ID NO: 29).

The terms “polypeptide,” “peptide,” and “protein,” used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

As used herein, a “portion” of a polypeptide or protein refers at least 10 amino acids of the reference sequence, e.g., 10 to 200, 25 to 300, 50 to 400, 100 to 500, 200 to 600, 300 to 700, 400 to 800, 500 to 900, or 600 to 1000 or more amino acids of the reference sequence. In some embodiments, the portion of a polypeptide or protein is functional.

The term “subject” refers to any mammal. In some embodiments, the subject or “subject suitable for treatment” may be a canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), ovine, bovine, porcine, caprine, primate, e.g., a simian (e.g., a monkey (e.g., marmoset, baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon) or a human; or rodent (e.g., a mouse, a guinea pig, a hamster, or a rat). In some embodiments, the subject or “subject suitable for treatment” may be a non-human mammal, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, lapine, porcine, canine or primate animals) may be employed.

The term “synNotch” refers to any of the variety of synthetic receptor-like polypeptides that use endogenous or modified Notch domains to effect intracellular signaling. Exemplary synNotch polypeptides are described in U.S. Pat. Nos. 9,670,281 and 9,834,608, and generally comprise, from N-terminal to C-terminal an extracellular antigen-binding domain, one or more ligand-inducible proteolytic cleavage sites, and an intracellular domain, wherein binding of extracellular antigen-binding domain to its target induces cleavage of the Notch receptor polypeptide at the one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain. As will be clear to one of ordinary skill in the art upon reading the present disclosure, “synPTPR” constructs provided herein exhibit certain advantages over synNotch constructs.

The term “synPTPR” refers to any of the variety of chimeric transmembrane receptor described herein. In general, synPTPRs described herein have had a substantial part of their wild type extracellular domains replaced with an extracellular antigen-binding domain. In some embodiments, synPTPRs described herein have an extracellular antigen-binding domain in place of the MAM domain, the Ig domain, and one or more FN-III domains that are endogenously present in a PTPR. In some embodiments, synPTPRs described herein have an intracellular regulatory domain comprising a gamma-secretase protease cleavage site in place of the phosphatase domains that are endogenously present in a PTPR. In some embodiments, synPTPRs described herein have one or more (e.g., one or two) extracellular integrin ligand-binding domain(s), which integrin ligand-binding domain(s) are cleaved upon the extracellular antigen-binding domain of the binding of the chimeric transmembrane receptor to its target ligand. In some embodiments, such cleavage results in cleavage of the gamma-secretase protease cleavage site, resulting in release of the intracellular transcriptional regulatory domain from the transmembrane domain.

The term “TCR” refers to a T cell receptor, a multi-module molecule capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively includes an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. Wild type TCRs are heterodimers, the majority of which include an alpha and a beta chain. A smaller portion of TCRs include a gamma and a delta chain. TCRs as used herein refer to both TCRs having wild type nucleic acid and/or amino acid sequences, as well as engineered TCRs having one or more modifications in their nucleic acid and/or amino acid sequence as compared to a nucleic acid and/or amino acid sequence of a wild type TCR.

Extracellular Antigen-Binding Domains

In some embodiments, chimeric transmembrane receptors provided herein include at least one extracellular antigen-binding domain that specifically binds to a target antigen. In some embodiments, the extracellular antigen-binding domain is selected from the group consisting of: a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab′)2, a diabody, a crossMab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a Fcab, a κλ-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)—IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2 scFv-IgG, IgG-2 scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, a nanobody, scFv-CH3 KIH, Fab-scFv, a F(ab′)₂-scFv₂, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, a dock and lock, a ImmTAC, an IgG-IgG conjugate, a Cov-X-Body, and a scFv1-PEG-scFv₂. See, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, incorporated in its entirety herewith, for a description of these elements. In some embodiments, the extracellular antigen-binding domain is selected from the group consisting of: a Fab fragment, an Fv fragment, a scFv fragment, an Fd fragment, a chimeric antibody, a humanized antibody, a fully-human antibody, a single-chain antibody (scAb), a single domain antibody (dAb), a single domain heavy chain antibody, a single domain light chain antibody, a nanobody, a bi-specific antibody, and a multi-specific antibody.

In some embodiments, chimeric transmembrane receptors provided herein include at least one extracellular antigen-binding domain that includes an antibody, an antibody fragment, or an antibody derivative. Such antibodies, antibody fragments, and antibody derivatives can be of any antibody isotype or subtype, or can be derived from any antibody isotype or subtype. For example, the light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, e.g., IgG2a and IgG2b. Without limitation, chimeric transmembrane receptors provided herein can include at least one extracellular antigen-binding domain that includes an antibody, an antibody fragment, or an antibody derivative, wherein the antibody, antibody fragment, or antibody derivative is of any of the light and heavy chain types or classes described herein.

In some embodiments, an extracellular antigen-binding domain is humanized or fully human. “Humanized” as used herein refers to an antibody comprising portions of antibodies of different origin, wherein at least one portion comprises amino acid sequences of human origin.

For example, a humanized antibody can comprise portions derived from an antibody of nonhuman origin with the requisite specificity, such as a mouse, and from antibody sequences of human origin (e.g., chimeric antibody), joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized antibody is an antibody containing one or more immunoglobulin chains comprising a complementarity-determining region (CDR) derived from an antibody of nonhuman origin and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized antibody. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Neuberger, M. S. et al., WO 86/01533; Winter, U.S. Pat. No. 5,225,539; See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)), regarding single chain antibodies.

Antibody fragments that can be used as extracellular antigen-binding domains in chimeric transmembrane receptors provided herein include a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody, which portion retains the capability of specifically binding to an antigen. Non-limiting examples of antibody fragments that can be used as an extracellular antigen-binding domain of an chimeric transmembrane receptor include an Fv fragment, a Fab fragment, a F(ab′)₂fragment, and a Fab′ fragment. Additional examples of an antigen-binding fragment of an antibody include an antigen-binding fragment of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM). Additional examples of antibody fragments that can be used in antigen-binding domains of chimeric transmembrane receptors provided herein are known in the art.

A Fv fragment is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRS of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

A scFv (also referred to as a “single-chain Fv” or a “sFv”) is an antibody fragment that includes the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

A Fab fragment includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain, in addition to the heavy and light chain variable domains of the Fv fragment. Papain digestion of antibodies produces two identical Fab antigen-binding fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Fab fragments differ from Fab′ fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

A F(ab′)₂fragment includes two Fab fragments joined, near the hinge region, by disulfide bonds. Pepsin treatment yields an F(ab′)₂fragment that has two antigen combining sites and is still capable of cross-linking antigen.

A nanobody (Nb) is the smallest antigen binding fragment or single variable domain (V.sub.HH) derived from naturally occurring heavy chain antibody. They are derived from heavy chain only antibodies, seen in camelids. In the family of “camelids” immunoglobulins devoid of light polypeptide chains are found. “Camelids” comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Llama paccos, Llama glama, Llama guanicoe and Llama vicugna). A single variable domain heavy chain antibody is referred to herein as a nanobody or a VHH antibody.

A VHH domain is a single monomeric variable antibody domain that can be found in camelids. A VNAR domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non-limiting aspects of VHH domains and VNAR domains are described in, e.g., Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

In some embodiments, an engineered immune cell includes a single antigen-binding domain. In some embodiments, a single antigen-binding domain is a “dual variable domain immunoglobulin” or “DVD-Ig”. A dual variable domain immunoglobulin is a multivalent and multispecific binding protein as described, e.g., in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Pat. Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety. In some embodiments, a single antigen-binding domain present in an engineered immune cell is a DART. DARTs are described in, e.g., Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). Diabodies are described in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, 1993.

In some embodiments, an extracellular antigen-binding domain of a chimeric transmembrane receptor provided herein binds to a target antigen selected from the group consisting of: BCMA, MAGE, MUC16, CD19, WT-1, CD22, LI-CAM, ROR-1, CEA, 4-1BB, ETA, 5T4, adenocarcinoma antigen, alpha-fetoprotein (AFP), BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD20, CD125 CD200, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin ανβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostatic carcinoma cells, RANKL, RON, SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

In some embodiments, an extracellular antigen-binding domain of a chimeric transmembrane receptor provided herein is bi-specific or multi-specific in that it binds (e.g., is capable of binding) to more than one different target antigen. In some embodiments, a chimeric transmembrane receptor provided herein includes two or more extracellular antigen-binding domains, each of which binds (e.g., is capable of binding) to two or more different target antigens. For example, a chimeric transmembrane receptor can include two or more scFv domains, wherein each scFv domain binds or is capable of binding to different target antigens (e.g., CD19 and CD20).

The amino acid of an exemplary extracellular antigen-binding domain that binds specifically to human CD19 is shown below. Also shown below is the cDNA sequence that encodes this exemplary antigen-binding domain. In some embodiments, an extracellular antigen-binding domain can include a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 45. In some embodiments, an extracellular antigen-binding domain can be encoded by a nucleic acid including a sequence that is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% to SEQ ID NO: 45.

Exemplary Anti-Human CD19 scFv

(SEQ ID NO: 45)

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYH

TSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG

GTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVS

LPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQV

FLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

cDNA Sequence Encoding an Exemplary Anti-Human

CD19 scFv

(SEQ ID NO: 46)

gacatccagatgacccagaccaccagcagcctgagcgccagcctgggcga

tagagtgaccatcagctgcagagccagccaggacatcagcaagtacctga

actggtatcagcagaaacccgacggcaccgtgaagctgctgatctaccac

accagcagactgcacagcggcgtgcccagcagattttctggcagcggctc

cggcaccgactacagcctgaccatctccaacctggaacaggaagatatcg

ctacctacttctgtcagcaaggcaacaccctgccctacaccttcggcgga

ggcaccaagctggaaatcacaggcggcggaggatctggcggaggcggaag

tggcggagggggatctgaagtgaaactgcaggaaagcggccctggcctgg

tggccccatctcagtctctgagcgtgacctgtaccgtgtccggcgtgtcc

ctgcctgactatggcgtgtcctggatcagacagccccccagaaagggcct

ggaatggctgggagtgatctggggcagcgagacaacctactacaacagcg

ccctgaagtcccggctgaccatcatcaaggacaactccaagagccaggtg

ttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactg

cgccaagcactactactacggcggcagctacgccatggactactggggcc

agggcacaagcgtgaccgtgtctagc

Those of ordinary skill in the art will be able to select appropriate target antigens for use in chimeric transmembrane receptors described herein, including chimeric transmembrane receptors that bind more than one target antigen.

Integrin Ligand-Binding Domains

In some embodiments, chimeric transmembrane receptors provided herein include at least one integrin ligand-binding domain. For example, certain chimeric transmembrane receptors provided herein include a single integrin ligand-binding domain or include (at most) a single integrin ligand-binding domain. Alternatively, certain chimeric transmembrane receptors provided herein include more than one integrin ligand-binding domain (e.g., at most two integrin ligand-binding domains). In some embodiments, one or more integrin ligand-binding domains in a chimeric transmembrane receptor provided herein is cleaved (e.g., at an S2 protease cleavage site) upon binding of the extracellular antigen-binding domain of the chimeric transmembrane receptor to its target ligand. In some embodiments, such cleavage of the integrin ligand-binding domain results in cleavage of a gamma-secretase protease cleavage site, resulting in release of the intracellular transcriptional regulatory domain from the remainder of the chimeric transmembrane receptor (e.g., intracellular transcriptional regulatory domain is liberated from the transmembrane domain, permitting it to travel to the nucleus to regulate transcription of a heterologous target gene). In some embodiments, an integrin ligand-binding domain of a chimeric transmembrane receptor includes a S2 proteolytic cleavage site, which S2 proteolytic cleavage site includes an Ala-Val dipeptide sequence. In some embodiments, an integrin ligand-binding domain of a chimeric transmembrane receptor includes a S2 proteolytic cleavage site, which S2 proteolytic cleavage site is capable of being cleaved by a protease from the ADAM family (e.g., ADAM10 or ADAM17).

Integrins are transmembrane proteins that play a role in cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrins activate signal transduction pathways that mediate a variety of cellular signals, such as, e.g., regulation of the cell cycle, organization of the intracellular cytoskeleton, and movement of new receptors to the cell membrane. Examples of integrin ligands include, without limitation, fibronectin, vitronectin, collagen, and laminin. Those of ordinary skill in the art will be aware of other integrin ligands and their corresponding integrin ligand-binding domains that can be used in accordance with the chimeric transmembrane receptors provided herein.

Any of a variety of integrin ligand-binding domains can be used in accordance with the chimeric transmembrane receptors and methods described herein. In some embodiments, chimeric transmembrane receptors provided herein include at least one (e.g., only one or only two) integrin ligand-binding domain that is present in a protein in receptor-like protein tyrosine phosphatase, including without limitation, a receptor-like protein tyrosine phosphatase in the Type IIa or Type IIb sub-families. For example, chimeric transmembrane receptors provided herein can include at least one (e.g., only one or only two) integrin ligand-binding domain that is present in RPTP(mu), RPTP(delta), RPTP(kappa), LAR, or RPTP(gamma). Exemplary RPTP(mu) polypeptide sequences are shown in NCBI Reference Sequence: NP_001098714.1 and NCBI Reference Sequence: NP 002836.3 (found at URLs www.ncbi.nlm.nih.gov/protein/NP_001098714 and www.ncbi.nlm.nih.gov/protein/NP_002836, respectively). Exemplary RPTP(delta) polypeptide sequences are shown in NCBI Reference Sequence: NP 001035802.1 and NCBI Reference Sequence: NP 001164496.1 (found at URLs www.ncbi.nlm.nih.gov/protein/NP_001035802 and www.ncbi.nlm.nih.gov/protein/NP_001164496, respectively). Exemplary RPTP(kappa) polypeptide sequences are shown in NCBI Reference Sequence: NP 001129120.1 and NCBI Reference Sequence: NP 001278910.1 (found at URLs www.ncbi.nlm.nih.gov/protein/NP_001129120 and www.ncbi.nlm.nih.gov/protein/NP_001278910, respectively). Exemplary LAR polypeptide sequences are shown in NCBI Reference Sequence: NP_001316066.1 and NCBI Reference Sequence: NP_001316067.1 (found at URLs www.ncbi.nlm.nih.gov/protein/NP_001316066 and www.ncbi.nlm.nih.gov/protein/NP_001316067, respectively). An exemplary RPTP(gamma) polypeptide sequences is shown in NCBI Reference Sequence: NP 002832.3 (found at URL www.ncbi.nlm.nih.gov/protein/NP_002832). Those of ordinary skill in the art will be aware of other suitable RPTP(mu), RPTP(delta), RPTP(kappa), LAR, or RPTP(gamma) polypeptide sequences that can be used in accordance with materials and methods disclosed herein, as well as nucleic acid sequences encoding them.

Full-length RPTP(kappa) (also known as PTPRK) is composed of a MAM domain, Ig domain, four fibronectin type-III (FN-III) domains, and two intracellular phosphatase domains. The full-length PTPRK protein gets processed by Furin cleavage at an 51 cleavage site during production and maturation of the PTPRK, giving rise to the mature transmembrane protein that is composed of the E-subunit and the P-subunit, which mature transmembrane protein is expressed on the surface as a bipartite molecule. Exemplary 51 cleavage sites include the amino acid sequences RXRR (SEQ ID NO: 6) or RXKR (SEQ ID NO: 7), where X is any amino acid. After ligand interaction, presumably caused by high cell-density, a protease from the ADAM family (e.g., ADAM10 or ADAM17) is recruited to cleave at the S2 site releasing the E-subunit and the extracellular stalk of the P-subunit. The membrane bound P-subunit is then processed by gamma-secretase and is shuttled to the nucleus where it can regulate gene transcription.

In some embodiments, chimeric transmembrane receptors provided herein include a “core” portion of a receptor-like protein tyrosine phosphatase (e.g., PTPRK), which core portion includes at least one (e.g., only one or only two) integrin ligand-binding domain (e.g., at least one fibronectin domain (e.g., a fibronectin type-III (FN-III) domain)) comprising an S2 cleavage site, a transmembrane domain, and/or an intracellular regulatory domain comprising a gamma-secretase protease cleavage site.

In some embodiments, an integrin ligand-binding domain is a fibronectin domain (e.g., a fibronectin type-III (FN-III) domain). Fibronectin domains are found in a wide variety of extracellular proteins including other extracellular-matrix molecules, cell-surface receptors, enzymes, and muscle proteins. The FN-III domain is an evolutionary conserved protein domain that is found in a variety of proteins. The FN-III domain is approximately 100 amino acids long and possesses a conserved beta sandwich fold with one beta sheet containing four strands and the other sheet containing three strands. In contrast to the two other fibronectin-type domains, the FN-III domain is the only one without disulfide bonding present. Sites of interaction with other molecules, including integrins, have been mapped to short stretch of amino acids such as the Arg-Gly-Asp (RGD) sequence found in various FN-III domains.

In some embodiments, chimeric transmembrane receptors provided herein include at least one FN-III domain (e.g., one or two FN-III domains) as the integrin ligand-binding domain. In some embodiments, one or more FN-III domains in a chimeric transmembrane receptor provided herein are cleaved upon the extracellular antigen-binding domain of the binding of the chimeric transmembrane receptor to its target ligand. In some embodiments, such cleavage results in cleavage of the S2 protease cleavage site and subsequent cleavage of the gamma-secretase cleavage site, resulting in release of the intracellular transcriptional regulatory domain from remainder of the chimeric transmembrane receptor (e.g., release from the transmembrane domain).

In some embodiments, an integrin ligand-binding domain for use in chimeric transmembrane receptors provided herein comprises portions of integrin ligand-binding domains present in two or more endogenous proteins, such that the integrin ligand-binding domain retains the ability to be cleaved at the S2 cleavage site. In some embodiments, chimeric transmembrane receptors provided herein include an integrin ligand-binding domain that differs from an integrin ligand-binding domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

In some embodiments, chimeric transmembrane receptors provided herein include an integrin ligand-binding domain that shares a degree of amino acid sequence identity to an integrin ligand-binding domain present in an endogenous protein. For example, an integrin ligand-binding domain for use in an chimeric transmembrane receptor provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with an integrin ligand-binding domain present in an endogenous protein. As will be appreciated by those of ordinary skill the art, an integrin ligand-binding domain that differs from an integrin ligand-binding domain present in an endogenous protein by one or more amino acids should still retain the ability to be cleaved at the S2 cleavage site. Methods of identifying and/or testing such modified integrin ligand-binding domains are known in the art.

In some embodiments, an integrin ligand-binding domain (e.g., a fibronectin-type III domain) is encoded by a nucleic acid sequence of SEQ ID NO: 35, shown below:

[SEQ ID NO: 35]

gatgtgcctggtcccgtaccagtaaaatctcttcaaggaacatcctttga

aaataagatcttcttgaactggaaagaacctttggatccaaatggaatca

tcactcaatatgagatcagctatagcagtataagatcatttgatcctgca

gttccagtggctggacctccccagactgtatcaaatttatggaacagtac

acaccatgtctttatgcatctccaccctggaaccacgtaccagtttttca

taagagccagcacggtcaaaggctttggtccagccacagccatcaatgtc

acc.

In some embodiments, an integrin ligand-binding domain (e.g., a fibronectin type III domain) includes a polypeptide sequence of SEQ ID NO: 36, shown below:

[SEQ ID NO: 36]

DVPGPVPVKSLQGTSFENKIFLNWKEPLDPNGIITQYEISYSSIRSFDPA

VPVAGPPQTVSNLWNSTHHVFMHLHPGTTYQFFIRASTVKGFGPATAINV

T.

In some embodiments, an integrin ligand-binding domain (e.g., a fibronectin type III domain) is encoded by a nucleic acid sequence of SEQ ID NO: 37, shown below:

[SEQ ID NO: 37]

cctgactatgaaggagttgatgcctctctcaatgaaactgccaccacaat

aactgtattgttgagaccagcacaagccaaaggtgctcctatcagtgctt

atcagattgttgtggaagaactgcacccacaccgaaccaagagagaagcc

ggagccatggaatgctaccaggttcctgtcacataccaaaatgccatgag

tgggggtgcaccgtattactttgctgcagaactacccccgggaaacctac

ct.

In some embodiments, an integrin ligand-binding domain (e.g., a fibronectin type III domain) includes a polypeptide sequence of SEQ ID NO: 38, shown below:

PDYEGVDASLNETATTITVLLRPAQAKGAPISAYQIVVEELHPHRTKREAGAME CYQVPVTYQNAMSGGAPYYFAAELPPGNLP [SEQ ID NO: 38]. In some embodiments, an integrin ligand-binding domain (e.g., a fibronectin-type III domain) is encoded by a nucleic acid sequence of:

(SEQ ID NO: 55)

AAAAATTTCCACGTGAAGGCTGTTATGAAAACATCCGTTCTCCTGTCATG

GGAAATCCCGGAAAACTATAATTCTGCTATGCCTTTCAAGATATTGTATG

ATGATGGCAAGATGGTTGAAGAGGTCGACGGTCGGGCGACACAAAAACTG

ATCGTTAACCTCAAACCTGAGAAATCATATTCATTCGTCCTCACCAATCG

CGGTAATAGTGCTGGTGGCCTCCAGCACCGGGTAACCGCAAAAACTGCGC

CTGAT.

In some embodiments, an integrin ligand-binding domain (e.g., a fibronectin type III domain) includes an amino acid sequence of:

(SEQ ID NO: 54)

KNFHVKAVMKTSVLLSWEIPENYNSAMPFKILYDDGKMVEEVDGRATQKL

IVNLKPEKSYSFVLTNRGNSAGGLQHRVTAKTAPD.