Patent Application
20220048971
The Sequence Listing submitted Sep. 27, 2021, as a text file named “Sequence listing 064466.067CON_ST25” created on Sep. 24, 2021, and having a size of 171,904 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). The sequence information in the “Sequence listing” in this application is identical to the sequence information in the CRF in its priority patent application, U.S. patent application Ser. No. 15/969,211, which was filed on May 2, 2018 and is currently pending. Further, the inventors on U.S. patent application Ser. No. 15/969,211 are identical to the inventors of the instant Application.
The invention is generally directed to immunology, in particular to T cell receptors and methods of their use in treating immune disorders including cancer.
With 800,000 new cases each year, hepatocellular carcinoma (HCC) is the 5th most common cancer in the world. In the US, according to American Cancer Society, the number of liver cancers (the majority of them are HCC) has doubled in last decade in the United States, representing one of the fast-growing malignancies—mainly due to the prevalence of obesity. The HCC incidence will likely remain high due to the large number of existing chronic HBV and HCV patients and the pandemic obesity. To make matters worse, the lack of effective management makes HCC the 2nd leading cause of cancer death in adult men. Thus, there is an urgent need to develop novel therapies. Adoptive transfer of tumor-specific T cells has great potential of controlling tumor growth without significant toxicity. Because of the difficulty of isolating tumor-specific T cells from most solid tumors (other than the melanoma), genetically engineering patient's autologous T cells with tumor antigen-specific TCR genes will likely provide the functional tumor-specific T cells for adoptive cell transfer immunotherapy. HCC frequently re-expresses human glypican 3 (hGPC3) and human alpha fetoprotein (hAFP) as tumor associated antigens (TAAs). These antigens not only serve as biomarkers for diagnosis, but can also be targets for immunotherapy. Recently, hGPC3-specific human TCR genes were cloned and demonstrated antitumor efficacy using a xenografted HCC model in immune compromised mice (Dargel et al., 2015). However, because TAAs are often not expressed at equal levels throughout an HCC, anti-hGPC3 treatment may select hGPC3-negative cells, causing relapse. In theory, using a combination of TCRs toward different epitopes and different tumor antigens can avoid or delay tumor immune escape. Four HLA-A2 restricted hAFP epitopes have been identified (Butterfield et al., 2001). The epitope hAFP158 is frequently presented by HCC tumor cells and the hAFP158-specific immune cells are found in HCC patients (Butterfield et al., 2003) though antitumor effect was weak (Butterfield et al., 2006) possibly because the human hAFP158-specific T cells are low affinity. Thus, finding high affinity hAFP158-specific TCR may increase antitumor efficacy of targeting AFP antigen.
There are two hAFP158-specific TCR genes that have been reported. One recent patent directed to human TCR specific for the hAFP158 epitope (CN 104087592 A) showed limited antitumor effect (Sun et al., 2016). The weaker antitumor effect was further confirmed by another patent application from Adaptimmune Co (US 2016/0137715 A1), in which the wild type human TCR specific for hAFP158 epitope did not produce any effector function when co-cultured with human HCC tumor cells. Thus, hAFP158-specific high affinity TCRs were created and patented via mutating the CDR regions of wild type human TCR to increase the recognition of HCC tumor cells (US 2016/0137715 A1). But, no clinical data is available to show that such TCR modified human T cells (TCR-T) can indeed generate antitumor effect in vivo. Furthermore, in light of the recent reports that adoptive transfer of high affinity TCR-T cells has been associated with severe off-target toxicity (Cameron et al., 2013; Linette et al., 2013; Morgan et al., 2013), it is desirable to have more TCRs available to increase the chance of finding the optimal TCRs with high antitumor efficacy and low toxicity. Thus, there is a true demand for identifying additional TCRs specific for TAAs that can be used to engineer a patient's autologous T cells for immunotherapy.
T cell receptors that specifically recognize hAFP158 are provided. One embodiment provides an engineered murine T cell receptor (mTCR) Vα chain polypeptide having at least 90% sequence identity to SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the mTCR specifically recognizes hAFP158 (SEQ ID NO:1).
Another embodiment provides an engineered murine T cell receptor (mTCR) Vβ chain polypeptide having at least 90% sequence identity to SEQ ID NO:11, 12, 13, or 14, wherein the mTCR specifically recognizes hAFP158 (SEQ ID NO:1).
Still another embodiment provides an engineered murine T cell receptor (mTCR) full length α chain polypeptide having at least 90% sequence identity to SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22, or 23, wherein the mTCR specifically recognizes hAFP158 (SEQ ID NO:1).
Another embodiment provides an engineered murine T cell receptor (mTCR) full length β chain polypeptide having at least 90% sequence identity to SEQ ID NO:24, 25, 26, or 27, wherein the mTCR specifically recognizes hAFP158 (SEQ ID NO:1).
One embodiment provides an engineered murine T cell receptor (mTCR) α chain polypeptide having a CDR1 region with at least 90% sequence identity to DRNVDY (residues 47 to 52 of SEQ ID NO:2), a CDR2 region with at least 90% sequence identity to IFSNGE (residues 70 to 75 of SEQ ID NO:2), and a CDR3 region with at least 90% sequence identity to SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, or 36, wherein the mTCR specifically recognizes hAFP158 (SEQ ID NO:1).
Another embodiment provides an engineered murine T cell receptor (mTCR) β chain polypeptide having a CDR1 region with at least 90% sequence identity to NSHNY (residues 44 to 48 of SEQ ID NO:11), a CDR2 region with at least 90% sequence identity to SYGAGN (residues 66 to 71 of SEQ ID NO:11), and a CDR3 region with at least 90% sequence identity to SEQ ID NO:37, 38, 39, or 40, wherein the mTCR specifically recognizes hAFP158 (SEQ ID NO:1).
The leader sequence of any one of the disclosed polypeptide sequences can be removed.
One embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:2, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:28, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:37.
Another embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:3, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:29, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:37.
Another embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:4, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:30, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:12, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:38.
Still another embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:5, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:31, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:13, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:39.
One embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:6, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:32, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:37.
Some embodiment provide an engineered murine T cell receptor (mTCR) having a Vα domain with at least 90% sequence identity to SEQ ID NO:7, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:33, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:14, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:40.
Another embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:8, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:34, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:37.
Yet another embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:9, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:35, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:37.
One embodiment provides an engineered murine T cell receptor (mTCR) having a Vα domain having at least 90% sequence identity to SEQ ID NO:10, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:36, and a Vβ domain having at least 90% sequence identity to SEQ ID NO:11, wherein the CDR3 region has at least 90% sequence identity to SEQ ID NO:37. Any of the disclosed mTCRs can be humanized.
One embodiment provides a T cell engineered to express any of the disclosed mTCRs. The T cell can be human. The T cell can also be an autologous T cell.
Another embodiment provides a soluble mTCR α chain comprising Q21 to L242 of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
Another embodiment provides the extracellular domain of an engineered mTCR β chain containing SEQ ID NO:50, 51, 52, or 53, or E18 to Y276 of SEQ ID NO:24, 25, 26, or 27.
One embodiment provides a fusion protein having at least 90% sequence identity to SEQ ID NO:54, 55, 56, 57, 58, 59, 60, 61, or 62.
Another embodiment provides a vector encoding any one of the mTCR proteins or polypeptides.
Another embodiment provides a nucleic acid having at least 90% sequence identity to SEQ ID NO:63, 64, 65, 66, 67, 68, 69, 70, or 71.
Another embodiment provides a non-naturally occurring hAFP158 epitope-specific murine T cell receptor including:
Another embodiment provides a fusion protein including a first polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, or 10, linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO:11, 12, 13, or 14.
Another embodiment provides a fusion protein including a first polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO:15, 16, 17, 18, 19, 20, 21, 22, or 23, linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO:24, 25, 26, or 27.
Another embodiment provides a T cell engineered to express the TCR encoded by SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 70, or 71. The T cell can be human.
Another embodiment provides a hybridoma having a CD8+Tet158+ cell fused to a donor cell lacking TCR α and β chains. The hybridoma typically responds to hAFP+ tumor cells. In certain embodiments, the hybridoma secretes IL-2.
Another embodiment provides a fusion protein having a first polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, or 10 linked to a second polypeptide having at least 90% sequence identity to an amino acid sequence according to SEQ ID NO:11, 12, 13, 14, 24, 25, 26, or 27, wherein in the fusion protein is linked to a single chain anti-CD3 antibody.
Another embodiment provides a method for treating tumors in a subject in need thereof by genetically engineering human T cells to express a disclosed mTCR and administering the engineered T cells to the subject in an amount effective to reduce tumor burden in the subject. In certain embodiments the tumors are hepatocellular carcinoma. The T cells can be autologous T cells.
Another embodiment provides a method for detecting heptacellular carcinoma by contacting a disclosed polypeptide or protein to a sample of cells, wherein specific binding of the polypeptide or protein to a cell is indicative of a heptacellular carcinoma cell.
Another embodiment provides an antibody or antigen binding fragment there of that specifically bindes to a disclosed polypeptide or protein.
The phrase “having antigenic specificity” as used herein means that the TCR can specifically bind to and immunologically recognize the cancer antigen, such that binding of the TCR to the cancer antigen elicits an immune response.
The term “Tet158” refers to HLA-A2/hAFP158 tetramers.
The term “hAFP158” refers to human alpha fetoprotein polypeptide having the amino acid sequence of FMNKFIYEI (SEQ ID NO:1).
The initialism “TCR” refers to a T cell receptor which is a specific receptor on the surface of T cells that is responsible for identifying the antigen presented by the major histocompatibility complex (MHC).
As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region includes a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region includes amino acid residues from a “Complementarity Determining Region” or “CDR” (i.e., typically at approximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; International Publication Nos. WO 94/04678 and WO 94/25591; U.S. Pat. No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994)), single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody's Complementarity Determining Regions (“CDRs”) and optionally the framework residues that include the antibody's “variable region” antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab′, F(ab′)2, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins including the antibody's “variable region” antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.).
As used herein, the term “fragment” refers to a peptide or polypeptide including an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.
As used herein the term “modulate” relates to a capacity to alter an effect, result, or activity (e.g, signal transduction). Such modulation can be agonistic or antagonistic. Antagonistic modulation can be partial (i.e., attenuating, but not abolishing) or it can completely abolish such activity (e.g., neutralizing). Modulation can include internalization of a receptor following binding of an antibody or a reduction in expression of a receptor on the target cell. Agonistic modulation can enhance or otherwise increase or enhance an activity (e.g., signal transduction). In a still further embodiment, such modulation can alter the nature of the interaction between a ligand and its cognate receptor so as to alter the nature of the elicited signal transduction. For example, the molecules can, by binding to the ligand or receptor, alter the ability of such molecules to bind to other ligands or receptors and thereby alter their overall activity. Preferably, such modulation will provide at least a 10% change in a measurable immune system activity, more preferably, at least a 50% change in such activity, or at least a 2-fold, 5-fold, 10-fold, or still more preferably, at least a 100-fold change in such activity.
The term “substantially,” as used in the context of binding or exhibited effect, is intended to denote that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is able to substantially block an activity of a ligand or receptor if the extent of blockage is physiologically or therapeutically relevant (for example if such extent is greater than 60% complete, greater than 70% complete, greater than 75% complete, greater than 80% complete, greater than 85% complete, greater than 90% complete, greater than 95% complete, or greater than 97% complete). Similarly, a molecule is said to have substantially the same immunospecificity and/or characteristic as another molecule, if such immunospecificities and characteristics are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical).
As used herein, a “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.
As used herein, the term “humanized antibody” refers to an immunoglobulin including a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-99%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR's, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody including a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human.
As used herein, the term “endogenous concentration” refers to the level at which a molecule is natively expressed (i.e., in the absence of expression vectors or recombinant promoters) by a cell (which cell can be a normal cell, a cancer cell or an infected cell).
As used herein, the terms “treat,” “treating,” “treatment” and “therapeutic use” refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder.
As used herein, a “therapeutically effective amount” refers to that amount of a therapeutic agent sufficient to mediate a clinically relevant elimination, reduction or amelioration of such symptoms. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
As used herein, the term “prophylactic agent” refers to an agent that can be used in the prevention of a disorder or disease prior to the detection of any symptoms of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease.
As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. As used herein, cancer explicitly includes leukemias and lymphomas. The term “cancer” refers to a disease involving cells that have the potential to metastasize to distal sites and exhibit phenotypic traits that differ from those of non-cancer cells, for example, formation of colonies in a three-dimensional substrate such as soft agar or the formation of tubular networks or web-like matrices in a three-dimensional basement membrane or extracellular matrix preparation. Non-cancer cells do not form colonies in soft agar and form distinct sphere-like structures in three-dimensional basement membrane or extracellular matrix preparations.
As used herein, an “immune cell” refers to any cell from the hemopoietic origin including, but not limited to, T cells, B cells, monocytes, dendritic cells, and macrophages.
As used herein, “valency” refers to the number of binding sites available per molecule. As used herein, the terms “immunologic,” “immunological” or “immune” response is the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against a peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II WIC molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, activation or recruitment of neutrophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating antibodies and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.
As used herein, an “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.
As used herein, the terms “individual,” “host,” “subject, and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.
As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). The term polypeptide includes proteins and fragments thereof. The polypeptides can be “exogenous,” meaning that they are “heterologous,” i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
As used herein, the term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.
In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (−0.5±1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Trp: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.
The term “percent (%) sequence identity” is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
For purposes herein, the % sequence identity of a given nucleotide or amino acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:
100 times the fraction W/Z,
where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.
As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
As used herein, the terms “antigenic determinant” and “epitope” are used interchangeably and refer to the structure recognized by an antibody.
As used herein, a “conformational epitope” is an epitope that includes discontinuous sections of the antigen's amino acid sequence. Antibodies bind a conformational epitope based on 3-D surface features, shape, or tertiary structure of the antigen.
As used herein, a “linear epitope” is an epitope that formed by a continuous sequence of amino acids from the antigen. Linear epitopes typically include about 5 to about 10 continuous amino acid residues. Antibodies bind a linear epitope based on the primary sequence of the antigen.
As used herein, a “paratope,” also called an “antigen-binding site,” is a part of an antibody which recognizes and binds to an antigen.
As used herein, “adoptive cell transfer” or ACT is a type of immunotherapy in which a patient's own T cells are collected, expanded ex vivo, and re-infused into the patient. Two types of ACT are chimeric antigen receptor (CAR) and T cell receptor (TCR) T cell therapy. Both techniques improve the ability of T cell receptors to recognize and attack specific antigens. In CAR T-cell therapy, T cells are engineered to produce receptors on their surface called chimeric antigen receptors. The receptors allow the T cells to recognize and attach to antigens on tumor cells. In TCR-T cell therapy, T cells are collected from a patient, modified to express a TCR specific to a tumor antigen, expanded ex vivo, and re-infused into the patient.
T cell receptor genes are provided that encode a receptor that specifically binds to hAFP, preferably to hAFP158 that is expressed on the surface of cancer cells. Nine pairs of murine TCR α and β chain genes specific for the HLA-A2/hAFP158 were identified and synthesized. The amino acid and nucleic acid sequences for the TCR genes are provided below. Vectors that contain one or more of the TCR genes are also provided.
Another embodiment provides hAFP158-specific (Tet158) mouse CD8 T cells that recognize and kill human HepG2 cells in vitro and eradicate large HepG2 tumor xenografts in NSG mice.
Still another embodiment provides T cell hybridomas made from the Tet158 CD8 T cells. The T cell hybridomas enabled the identification of paired TCR α and β chain genes. One embodiment provides healthy donor CD8 T cells transduced with the murine TCR α and β chain genes specific for the HLA-A2/hAFP158. The genetically engineered TCR enables the donor cells to recognize and effectively kill HepG2 tumor cells at very low E/T ratio. These murine TCR genes specific for the HLA-A2/hAFP158 have the potential to modify and redirect the patient's autologous T cells to treat HCC tumors via adoptive cell transfer.
Another embodiment provides soluble T cell receptors. In one embodiment the soluble TCR includes the extracellular domain of the TCR polypeptides.
A. Genetically Engineered T Cell Receptors
The disclosed TCR genes are from the recombinant lv-primed and peptide-boosted AAD mice. Thus, technically, these TCRs are not “naturally occurring”. In addition, after obtaining the Vα and Vβ region of TCRs, full length α and β chains were designed by using the identified V-region of α and β chains, and the constant region (C-region) of α and β chains of HLA-A2 mouse TCR specific for hgp100. Thus, the entire TCRs and their genes are not naturally occurring.
1. Protein Sequences of the mTCR-1, 2, 3, 6, 8, 10, 11, 17 and 38 Vα Domain
One embodiment provides mTCR-1 Vα (
Another embodiment provides mTCR-2 Vα (
Comparing mTCR-1 α chain V-region to mTCR-2 α chain, there is only 2 amino acids difference (bolded and underlined).
Another embodiment provides mTCR-3 Vα (
There are multiple amino acid differences between mTCR-1 α chain and mTCR-3 α chain (bolded and underlined). In fact, the J segment is different (Instead of TRAJ30, the TRAJ12 is used in mTCR-3 Vα).
One embodiment provides mTCR-6 Vα (
Another embodiment provides mTCR-8 Vα (
There are multiple amino acid differences between mTCR-6 α chain and mTCR-8 α chain (bolded and underlined). In fact, the J segment is different (Instead of TRAJ30, the TRAJ12 is used in mTCR-8 Vα).
Another embodiment provides mTCR-10 Vα (
Comparing mTCR-6 α chain V-region to mTCR-8 α chain, there is only 1 amino acids difference (bolded and underlined).
Another embodiment provides mTCR-11 Vα (
Comparing mTCR-6 α chain V-region to mTCR-8 α chain, there are only 2 amino acid differences (bolded and underlined).
One embodiment provides mTCR-17 Vα (
Another embodiment provides mTCR-38 Vα (
In certain embodiments, the leading methionine and/or signal sequence is cleaved in the post-translationally modified protein.
2. Protein sequences of the mTCR-1, 2, 3, 6, 8, 10, 11, 17 and 38 VD domains One embodiment provides mTCR-1, 2, 8, 11, 17 & 38 Vβ domain (
Another embodiment provides mTCR-3 Vβ domain (
There is only one amino acid difference (bold and underlined) between mTCR-1 and mTCR-2 with TCR-3
Another embodiment provides mTCR-6 Vβ domain (
There is only one amino acid difference from mTCR-1, 2, 8, and 11, a 2 amino acid difference from mTCR-3, and a 3 amino acid difference from mTCR-10.
Another embodiment provides mTCR-10 Vβ domain (
There are 3 amino acid differences (bold and underlined) between mTCR-6 Vβ with TCR-10
In certain embodiments, the leading methionine and/or signal sequence is cleaved in the post-translationally modified protein.
3. Protein Sequences of the Full Length mTCR-1, 2, 3, 6, 8, 10, 11, 17 and 38 α Chains
Another embodiment provides mTCR-1 α chain (
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
The constant region (Ca) (bolded) of mTCR-1, 2, 3, 6, 8, 10, and 11 α chains are the same, and they are identical to the hgp100 specific TCR α chain C-region from HLA-A2 Tg mice.
Another embodiment provides mTCR-2 α chain (
Another embodiment provides mTCR-3 α chain (
Another embodiment provides mTCR-6 α chain (
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
Another embodiment provides mTCR-8 α chain (
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
MTLRLWSS
Another embodiment provides mTCR-10 α chain (
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
Another embodiment provides mTCR-11 α chain (
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
One embodiment provides mTCR-17 α chain (
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
Another embodiment provides mTCR-38 α chain (
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
In certain embodiments, the leading methionine and/or signal sequence is cleaved in the post-translationally modified protein.
In one embodiment, based on the nomenclature on IMGT and Uniprot website, the mTCR α chain features are believed to be as follows:
1. M1-S20: Leader Sequences to be removed on maturation of TCR α chain
2. Q21-5268: TCR-1, 2, 3 α chain
3. Q21-N132 (or D132): α chain V-region
4. I133-S268: TCR α chain C-region identical to the HLA-A2 mouse TCR specific for hgp100 (DQ452619)
5. Q21-L242: TCR α chain extracellular domain
6. 5243-L263: transmembrane region of mature TCR α chain
7. R264-5268: intracellular region of mature TCR α chain
4. Protein sequences of full length mTCR-1, 2, 3, 6, 8, 10, 11, 17, and 38 β chains
Another embodiment provides mTCR-1, 2, 8, 11, 17, and 38 β chain (
EIANKRKATL VCLARGFFPD HVELSWWVNG KEVHSGVSTD PQAYKESNYS
YCLSSRLRVS ATFWHNPRNH FRCQVQFHGL SEEDKWPEGS PKPVTQNISA
EAWGRADCGI TSASYQQGVL SATILYEILL GKATLYAVLV STLVVMAMVK
RKNS
The constant region (CD) (bolded) of mTCR-1, 2, 3, 6, 8, 10, 11, 17, and 380 chains are the same, and they are identical to the hgp100 specific TCR chain C-region from HLA-A2 Tg mice.
In certain embodiments, the leading methionine and/or signal sequence is cleaved in the post-translationally modified protein.
Another embodiment provides mTCR-3 β chain (
EIANKRKATL VCLARGFFPD HVELSWWVNG KEVHSGVSTD PQAYKESNYS
YCLSSRLRVS ATFWHNPRNH FRCQVQFHGL SEEDKWPEGS PKPVTQNISA
EAWGRADCGI TSASYQQGVL SATILYEILL GKATLYAVLV STLVVMAMVK
RKNS
Another embodiment provides mTCR-6 β chain (
EIANKRKATL VCLARGFFPD HVELSWWVNG KEVHSGVSTD PQAYKESNYS
YCLSSRLRVS ATFWHNPRNH FRCQVQFHGL SEEDKWPEGS PKPVTQNISA
EAWGRADCGI TSASYQQGVL SATILYEILL GKATLYAVLV STLVVMAMVK
RKNS
Another embodiment provides mTCR-10 β chain (
EIANKRKATL VCLARGFFPD HVELSWWVNG KEVHSGVSTD PQAYKESNYS
YCLSSRLRVS ATFWHNPRNH FRCQVQFHGL SEEDKWPEGS PKPVTQNISA
EAWGRADCGI TSASYQQGVL SATILYEILL GKATLYAVLV STLVVMAMVK
RKNS
In certain embodiments, the leading methionine and/or signal sequence is cleaved in the post-translationally modified protein.
In one embodiment, based on the nomenclature on IMGT and Uniprot website, the mTCR α chain features are as follows:
1. M1-M17: Leader Sequences to be removed on maturation of mTCR β chain
2. E18-5304: mTCR β chain
3. E18-L131: mTCR β chain V-region (TRBV13-1*01/TRBJ2-4*01 with S113 (TCR-1&2) or A113 (TCR-3)-A116 as the D region)
4. E132-5304: mTCR β chain Cl-region of HLA-A mTCR specific for hgp100 (DQ452620)
5. E18-Y276: mTCR β chain extracellular domain
6. E277-M298: transmembrane region of mature mTCR β chain
7. V299-5304: intracellular (topological) region of mature mTCR β chain
5. Protein Sequences of CDR3 Region of mTCR-1, 2, 3, 6, 8, 10, 11, 17, and 38 α Chains
One embodiment provides mTCR-1 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:28):
Another embodiment provides mTCR-2 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:29):
One embodiment provides mTCR-3 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:301:
One embodiment provides mTCR-6 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:31):
Another embodiment provides mTCR-8 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:32):
One embodiment provides mTCR-10 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:33):
One embodiment provides mTCR-11 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:34):
Another embodiment provides mTCR-17 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:35):
One embodiment provides mTCR-38 α chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:36):
6. Protein sequences of CDR3 region of mTCR-1, 3, 6, and 10 β chains
One embodiment provides mTCR-1 β chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:37):
One embodiment provides mTCR-3 β chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:38):
Another embodiment provides mTCR-6 β chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:39):
One embodiment provides mTCR-10 β chain CDR3 region having 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:40):
7. The Extracellular Domain of mTCR-1, 2, 3, 6, 8, 10, 11, 17 and 38 α Chains (for Soluble TCRs) (Underlined are the Vα)
The extracellular domain of TCR α and chains can form soluble TCRs. If they are labeled with detectable labels, for example fluorescent molecules, they can be used to detect the HLA-A2/hAFP158 complex presented on tumor cells. The labeled soluble TCRs have the potential of as diagnosis reagents for detecting circulating tumor cells in the blood and to determine the cognate antigen epitope presentation in tumor tissues, which is important for the success of using such TCR-T cells in the setting of adoptive cell transfer therapy.
One embodiment provides the soluble mTCR-1 α chain (
QQKVQQSPES LIVPEGGMAS LNCTSSDRNV DYFWWYRQHS
GKSPKMLMSI FSNGEKEEGR FTVHLNKASL
HTSLHIRDSQ PSDSALYLCA ASITNAYKVI FGKGTHLHVL
PNIQNPEPAV YQLKDPRSQD STLCLFTDFD
Another embodiment provides soluble mTCR-2 α chain (
QQKVQQSPES LIVPEGGMAS LNCTSSDRNV DYFWWYRQHS
GKSPKMLMSI FSNGEKEEGR FTVHLNKASL
HTSLHIRDSQ PSDSALYLCA ASTVNAYKVI FGKGTHLHVL
PNIQNPEPAV YQLKDPRSQD STLCLFTDFD
Another embodiment provides soluble mTCR-3 α chain (
QQKVQQSPES LIVPEGGMAS LNCTSSDRNV DYFWWYRQHS
GKSPKMLMSI FSNGEKEEGR FTVHLNKASL
HTSLHIRDSQ PSDSALYLCA ASMAGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD STLCLFTDFD
Another embodiment provides soluble mTCR-6 α chain (
QQKVQQSPES LIVPEGGMAS LNCTSSDRNV DYFWWYRQHS
GKSPKMLMSI FSNGEKEEGR FTVHLNKASL
HTSLHIRDSQ PSDSALYLCA ASMINAYKVI FGKGTHLHVL
PNIQNPEPAV YQLKDPRSQD STLCLFTDFD
Another embodiment provides soluble mTCR-8 α chain (
QQKVQQSPES LIVPEGGMAS LNCTSSDRNV DYFWWYRQHS
GKSPKMLMSI FSNGEKEEGR FTVHLNKASL
HTSLHIRDSQ PSDSALYLCA ASISGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD STLCLFTDFD
Another embodiment provides soluble mTCR-10 α chain (
QQKVQQSPES LIVPEGGMAS LNCTSSDRNV DYFWWYRQHS
GKSPKMLMSI FSNGEKEEGR FTVHLNKASL
HTSLHIRDSQ PSDSALYLCA ASIVGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD STLCLFTDFD
Another embodiment provides soluble mTCR-11 α chain (
QQKVQQSPES LIVPEGGMAS LNCTSSDRNV DYFWWYRQHS
GKSPKMLMSI FSNGEKEEGR FTVHLNKASL
HTSLHIRDSQ PSDSALYLCA ASKTGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD STLCLFTDFD
One embodiment provides soluble mTCR-17 α chain (
Another embodiment provides soluble mTCR-38 α chain (
8. The extracellular domain of TCR-1, 2, 3, 6, 8, 10, and 11 β chains (underlined are the Vβ)
One embodiment provides soluble mTCR-1, 2, 8, 11 β chain (
EAA VTQSPRNKVT VTGGNVTLSC RQTNSHNYMY WYRQDTGHGL
RLIHYSYGAG NLQIGDVPDG YKATRTTQED
FFLLLELASP SQTSLYFCAS SDAGTSQNTL YFGAGTRLSV
LEDLRNVTPP KVSLFEPSKA EIANKRKATL
Still another embodiment soluble mTCR-3 β chain (
EAA VTQSPRNKVT VTGGNVTLSC RQTNSHNYMY WYRQDTGHGL
RLIHYSYGAG NLQIGDVPDG YKATRTTQED
FFLLLELASP SQTSLYFCAS SDAGTAQNTL YFGAGTRLSV
LEDLRNVTPP KVSLFEPSKA EIANKRKATL
Another embodiment soluble mTCR-6 β chain (
EAAVTQSPRN KVTVTGGNVT LSCRQTNSHN YMYWYRQDTG
HGLRLIHYSY GAGNLQIGDV PDGYKATRTT
QEDFFLLLEL ASPSQTSLYF CASCDAGVSQ NTLYFGAGTR
LSVLEDLRNV TPPKVSLFEP SKAEIANKRK
Another embodiment soluble mTCR-10 β chain (
EAAVTQSPRN KVTVTGGNVT LSCRQTNSHN YMYWYRQDTG
HGLRLIHYSY GAGNLQIGDV PDGYKATRTT
QEDFFLLLEL ASPSQTSLYF CASSDHGTGQ NTLYFGAGTR
LSVLEDLRNV TPPKVSLFEP SKAEIANKRK
9. Fusion Protein Sequences of Designed mTCRs
Fusion Proteins
In another embodiment, fusion proteins are provided that contain a first polypeptide domain and a second polypeptide. The fusion proteins can optionally contain a targeting domain that targets the fusion protein specific cells or tissues, for example the tumor cells or tumor cell-associated neovasculature.
The fusion proteins also optionally contain a peptide or polypeptide linker domain that separates the first polypeptide domain from the antigen-binding domain.
Fusion proteins disclosed herein are of formula I:
N—R1—R2—R3—C
wherein “N” represents the N-terminus of the fusion protein, “C” represents the C-terminus of the fusion protein, “R1” is one of the disclosed a mTCR chains or a fragment thereof. “R2” is a peptide/polypeptide linker domain, and “R3” is a β mTCR chain or fragment thereof. In an alternative embodiment, R1 is a β mTCR chain or fragment thereof and R3 is an α mTCR chain.
Optionally, the fusion proteins additionally contain a domain that functions to dimerize or multimerize two or more fusion proteins. The domain that functions to dimerize or multimerize the fusion proteins can either be a separate domain, or alternatively can be contained within one of one of the other domains of the fusion protein.
The fusion proteins can be dimerized or multimerized. Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric/homomultimeric or heterodimeric/heteromultimeric.
The modular nature of the fusion proteins and their ability to dimerize or multimerize in different combinations provides a wealth of options for targeting molecules that function to enhance an immune response to the tumor cell microenvironment.
Another embodiment provides a fusion protein according to formula II,
N—R1—R2—R3—R2—R4—C
wherein “N” represents the N-terminus of the fusion protein, “C” represents the C-terminus of the fusion protein, “R1” is one of the disclosed α mTCR chains or a fragment thereof “R2” is a peptide/polypeptide linker domain, and “R3” is a β mTCR chain or fragment thereof and “R4” is an anti-CD3 single chain antibody. Anti-CD3 single chain antibodies are known in the art and are commercially available.
One embodiment provides fusion protein of designed mTCR-1 (α chain is underlined, P2A is bolded, and β chain is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:54):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASITNAYKVI FGKGTHLHVL PNIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT NFSLLKQAGD VEENPGPR
MG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGTSQNTLYF GAGTRLSVLE DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK ATLYAVLVST LVVMAMVKRK
NS
The α chain is underlined, the β chain is double underlined, and the P2A and furin cleavage site (from US2016/0137715 A1) is in bold.
Another embodiment provides designed mTCR-2 (α chain, P2A, and β chain) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:55)
MKSFSISLVV LWLQLNWVNS QQKVQQSPES LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASTVNAYKVI FGKGTHLHVL PNIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT NFSLLKQAGD VEENPGPR
MG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGTSQNTLYF GAGTRLSVLE DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK ATLYAVLVST LVVMAMVKRK
NS
Another embodiment provides designed mTCR-3 (α chain, P2A, and β chain) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:56):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASMAGGYKVV FGSGTRLLVS PDIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT NFSLLKQAGD VEENPGPR
MG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGTAQNTLYF GAGTRLSVLE DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK ATLYAVLVST LVVMAMVKRK
NS
Another embodiment provides designed mTCR-6 (α chain, P2A, and β chain) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:57):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASMINAYKVI FGKGTHLHVL PNIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT NFSLLKQAGD VEENPGPR
MG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGVSQNTLYF GAGTRLSVLE DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK ATLYAVLVST LVVMAMVKRK
NS
Another embodiment provides designed mTCR-8 (α chain, P2A, and β chain) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:58):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES
LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR
FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASISGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV
LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS
FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT
NFSLLKQAGD VEENPGPRMG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT
GGNVTLSCRQ TNSHNYMYWYEE
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK
ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGTSQNTLYF GAGTRLSVLE
DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE
VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE
EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK
ATLYAVLVST LVVMAMVKRK
NS
Another embodiment provides designed mTCR-10 (α chain, P2A, and β chain) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:59):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES
LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR
FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASIVGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV
LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS
FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT
NFSLLKQAGD VEENPGPRMG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT
GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK
ATRTTQEDFF LLLELASPSQ
TSLYFCASSD HGTGQNTLYF GAGTRLSVLE
DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE
VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE
EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK
ATLYAVLVST LVVMAMVKRK
NS
Another embodiment provides designed mTCR-11 (α chain, P2A, and β chain) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:60):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES
LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR
FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASKTGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV
LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS
FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT
NFSLLKQAGD VEENPGPRMG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT
GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK
ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGTSQNTLYF GAGTRLSVLE
DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE
VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE
EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK
ATLYAVLVST LVVMAMVKRK
NS
One embodiment provides designed mTCR-17 (α chain, P2A, and β chain) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:61):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES
LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR
FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ASMTGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV
LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS
FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT
NFSLLKQAGD VEENPGPRMG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT
GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK
ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGTSQNTLYF GAGTRLSVLE
DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE
VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE
EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK
ATLYAVLVST LVVMAMVKRK
NS
One embodiment provides designed mTCR-38 (α chain, P2A, and β chain) having at least 90%, 95%, 99% or 100% sequence identity to (SEQ ID NO:62):
MKSFSISLVV LWLQLNWVNS QQKVQQSPES
LIVPEGGMAS LNCTSSDRNV
DYFWWYRQHS GKSPKMLMSI FSNGEKEEGR
FTVHLNKASL HTSLHIRDSQ
PSDSALYLCA ATLTGGYKVV FGSGTRLLVS
PDIQNPEPAV YQLKDPRSQD
STLCLFTDFD SQINVPKTME SGTFITDKTV
LDMKAMDSKS NGAIAWSNQT
SFTCQDIFKE TNATYPSSDV PCDATLTEKS
FETDMNLNFQ NLSVMGLRIL
LLKVAGFNLL MTLRLWSS
GS RAKRSGSGAT
NFSLLKQAGD VEENPGPRMG
SRLFLVLSLL CTKHMEAAVT QSPRNKVTVT
GGNVTLSCRQ TNSHNYMYWY
RQDTGHGLRL IHYSYGAGNL QIGDVPDGYK
ATRTTQEDFF LLLELASPSQ
TSLYFCASSD AGTSQNTLYF GAGTRLSVLE
DLRNVTPPKV SLFEPSKAEI
ANKRKATLVC LARGFFPDHV ELSWWVNGKE
VHSGVSTDPQ AYKESNYSYC
LSSRLRVSAT FWHNPRNHFR CQVQFHGLSE
EDKWPEGSPK PVTQNISAEA
WGRADCGITS ASYQQGVLSA TILYEILLGK
ATLYAVLVST LVVMAMVKRK
NS
10. Nucleotide Sequences of the Designed TCR-1, TCR-2, TCR-3, TCR-6, TCR-8, TCR-10, TCR-11, TCR-17, and TCR-38:
The sequences are based on the above protein sequences and codon optimized for expression in human cells.
One embodiment provides a designed mTCR-1 (α is underlined, P2A is bolded, and β is double underlined) having a nucleic acid sequence that is at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:63):
ATGAAATCTT TTAGCATCTC CCTGGTCGTC
CTGTGGCTGC AGCTGAATTG GGTGAATAGT
CAGCAGAAGG TCCAGCAGTC CCCCGAGTCC
CTGATCGTGC CTGAGGGCGG CATGGCCTCT
CTGAACTGCA CCAGCTCCGA CCGGAATGTG
GATTATTTCT GGTGGTACAG ACAGCACTCT
GGCAAGAGCC CAAAGATGCT GATGTCCATC
TTCTCTAACG GCGAGAAGGA GGAGGGCCGG
TTTACAGTGC ACCTGAATAA GGCTAGCCTG
CACACCTCCC TGCACATCAG AGACTCCCAG
CCCTCCGATT CTGCCCTGTA TCTGTGCGCC
GCCTCTATCA CAAACGCCTA CAAAGTGATC
TTCGGCAAGG GAACCCACCT GCACGTGCTG
CCTAACATCC AGAATCCAGA GCCCGCCGTG
TATCAGCTGA AGGACCCACG GAGCCAGGAT
AGCACCCTGT GCCTGTTCAC CGACTTTGAT
AGCCAGATCA ATGTGCCTAA GACAATGGAG
TCCGGCACCT TTATCACAGA CAAGACCGTG
CTGGATATGA AGGCCATGGA CAGCAAGTCC
AACGGCGCCA TCGCCTGGTC TAATCAGACA
AGCTTCACCT GCCAGGATAT CTTTAAGGAG
ACAAACGCCA CCTACCCATC TAGCGACGTG
CCCTGTGATG CCACCCTGAC AGAGAAGAGC
TTCGAGACAG ACATGAACCT GAATTTTCAG
AACCTGTCCG TGATGGGCCT GAGAATCCTG
CTGCTGAAGG TGGCCGGCTT CAATCTGCTG
ATGACACTGC GCCTGTGGTC CTCT
GGCTCT
AGGGCAAAGC GGAGCGGCAG CGGAGCAACC
AACTTCAGCC TGCTGAAGCA GGCCGGCGAT
GTGGAGGAGA ATCCTGGCCC ACGGATGGGC
TCTAGACTGT TTCTGGTGCT GAGCCTGCTG
TGCACAAAGC ACATGGAGGC AGCAGTGACC
CAGTCCCCAC GGAACAAGGT GACCGTGACA
GGCGGCAATG TGACACTGAG CTGTAGACAG
ACCAACTCCC ACAATTACAT GTATTGGTAC
CGGCAGGATA CCGGACACGG CCTGAGACTG
ATCCACTATA GCTACGGCGC CGGCAATCTG
CAGATCGGCG ACGTGCCAGA TGGCTATAAG
GCCACAAGGA CCACACAGGA GGACTTCTTT
CTGCTGCTGG AGCTGGCCTC CCCCTCTCAG
ACCTCTCTGT ATTTCTGCGC CAGCTCCGAT
GCCGGCACAA GCCAGAACAC CCTGTACTTT
GGAGCAGGAA CAAGGCTGTC CGTGCTGGAG
GACCTGCGCA ATGTGACCCC CCCTAAGGTG
TCCCTGTTCG AGCCTTCTAA GGCCGAGATC
GCCAACAAGA GGAAGGCCAC CCTGGTGTGC
CTGGCAAGGG GCTTCTTTCC AGATCACGTG
GAGCTGTCCT GGTGGGTGAA TGGCAAGGAG
GTGCACTCTG GCGTGAGCAC AGACCCCCAG
GCCTACAAGG AGTCCAACTA TTCTTACTGC
CTGTCTAGCC GGCTGAGAGT GAGCGCCACC
TTTTGGCACA ACCCCAGGAA TCACTTCCGC
TGTCAGGTGC AGTTTCACGG CCTGTCCGAG
GAGGATAAGT GGCCTGAGGG CTCTCCCAAG
CCTGTGACAC AGAACATCAG CGCCGAGGCA
TGGGGAAGGG CAGACTGTGG CATCACCAGC
GCCTCCTATC AGCAGGGCGT GCTGAGCGCC
ACAATCCTGT ACGAGATCCT GCTGGGCAAG
GCCACCCTGT ATGCTGTGCT GGTGTCAACT
CTGGTGGTCA TGGCTATGGT GAAACGGAAA
AACTCCTAA
Another embodiment provides a designed mTCR-2 (α is underlined, P2A is bolded, and β double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:64):
ATGAAATCTT TTAGCATCTC CCTGGTCGTC
CTGTGGCTGC AGCTGAATTG GGTGAATAGT
CAGCAGAAGG TCCAGCAGTC CCCCGAGTCC
CTGATCGTGC CTGAGGGCGG CATGGCCTCT
CTGAACTGCA CCAGCTCCGA CCGGAATGTG
GATTATTTCT GGTGGTACAG ACAGCACTCT
GGCAAGAGCC CAAAGATGCT GATGTCCATC
TTCTCTAACG GCGAGAAGGA GGAGGGCCGG
TTTACAGTGC ACCTGAATAA GGCTAGCCTG
CACACCTCCC TGCACATCAG AGACTCCCAG
CCCTCCGATT CTGCCCTGTA TCTGTGCGCC
GCCTCTACCG TGAACGCCTA CAAAGTGATC
TTCGGCAAGG GAACCCACCT GCACGTGCTG
CCTAACATCC AGAATCCAGA GCCCGCCGTG
TATCAGCTGA AGGACCCACG GAGCCAGGAT
AGCACCCTGT GCCTGTTCAC CGACTTTGAT
AGCCAGATCA ATGTGCCTAA GACAATGGAG
TCCGGCACCT TTATCACAGA CAAGACCGTG
CTGGATATGA AGGCCATGGA CAGCAAGTCC
AACGGCGCCA TCGCCTGGTC TAATCAGACA
AGCTTCACCT GCCAGGATAT CTTTAAGGAG
ACAAACGCCA CCTACCCATC TAGCGACGTG
CCCTGTGATG CCACCCTGAC AGAGAAGAGC
TTCGAGACAG ACATGAACCT GAATTTTCAG
AACCTGTCCG TGATGGGCCT GAGAATCCTG
CTGCTGAAGG TGGCCGGCTT CAATCTGCTG
ATGACACTGC GCCTGTGGTC CTCT
GGCTCT
AGGGCAAAGC GGAGCGGCAG CGGAGCAACC
AACTTCAGCC TGCTGAAGCA GGCCGGCGAT
GTGGAGGAGA ATCCTGGCCC ACGGATGGGC
TCTAGACTGT TTCTGGTGCT GAGCCTGCTG
TGCACAAAGC ACATGGAGGC AGCAGTGACC
CAGTCCCCAC GGAACAAGGT GACCGTGACA
GGCGGCAATG TGACACTGAG CTGTAGACAG
ACCAACTCCC ACAATTACAT GTATTGGTAC
CGGCAGGATA CCGGACACGG CCTGAGACTG
ATCCACTATA GCTACGGCGC CGGCAATCTG
CAGATCGGCG ACGTGCCAGA TGGCTATAAG
GCCACAAGGA CCACACAGGA GGACTTCTTT
CTGCTGCTGG AGCTGGCCTC CCCCTCTCAG
ACCTCTCTGT ATTTCTGCGC CAGCTCCGAT
GCCGGCACAA GCCAGAACAC CCTGTACTTT
GGAGCAGGAA CAAGGCTGTC CGTGCTGGAG
GACCTGCGCA ATGTGACCCC CCCTAAGGTG
TCCCTGTTCG AGCCTTCTAA GGCCGAGATC
GCCAACAAGA GGAAGGCCAC CCTGGTGTGC
CTGGCAAGGG GCTTCTTTCC AGATCACGTG
GAGCTGTCCT GGTGGGTGAA TGGCAAGGAG
GTGCACTCTG GCGTGAGCAC AGACCCCCAG
GCCTACAAGG AGTCCAACTA TTCTTACTGC
CTGTCTAGCC GGCTGAGAGT GAGCGCCACC
TTTTGGCACA ACCCCAGGAA TCACTTCCGC
TGTCAGGTGC AGTTTCACGG CCTGTCCGAG
GAGGATAAGT GGCCTGAGGG CTCTCCCAAG
CCTGTGACAC AGAACATCAG CGCCGAGGCA
TGGGGAAGGG CAGACTGTGG CATCACCAGC
GCCTCCTATC AGCAGGGCGT GCTGAGCGCC
ACAATCCTGT ACGAGATCCT GCTGGGCAAG
GCCACCCTGT ATGCTGTGCT GGTGTCAACT
CTGGTGGTCA TGGCTATGGT GAAACGGAAA
AACTCCTAA
Another embodiment provides a designed mTCR-3 (α is underlined, P2A is bolded, and β is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:65):
ATGAAATCTT TTAGCATCTC CCTGGTCGTC
CTGTGGCTGC AGCTGAATTG GGTGAATAGT
CAGCAGAAGG TCCAGCAGTC CCCCGAGTCC
CTGATCGTGC CTGAGGGCGG CATGGCCTCT
CTGAACTGCA CCAGCTCCGA CCGGAATGTG
GATTATTTCT GGTGGTACAG ACAGCACTCT
GGCAAGAGCC CAAAGATGCT GATGTCCATC
TTCTCTAACG GCGAGAAGGA GGAGGGCCGG
TTTACAGTGC ACCTGAATAA GGCTAGCCTG
CACACCTCCC TGCACATCAG AGACTCCCAG
CCCTCCGATT CTGCCCTGTA TCTGTGCGCC
GCCTCTATGG CCGGCGGCTA CAAAGTGGTG
TTCGGCAGCG GAACCCGGCT GCTGGTGAGC
CCTGACATCC AGAATCCAGA GCCCGCCGTG
TATCAGCTGA AGGACCCACG GAGCCAGGAT
AGCACCCTGT GCCTGTTCAC CGACTTTGAT
AGCCAGATCA ATGTGCCTAA GACAATGGAG
TCCGGCACCT TTATCACAGA CAAGACCGTG
CTGGATATGA AGGCCATGGA CAGCAAGTCC
AACGGCGCCA TCGCCTGGTC TAATCAGACA
AGCTTCACCT GCCAGGATAT CTTTAAGGAG
ACAAACGCCA CCTACCCATC TAGCGACGTG
CCCTGTGATG CCACCCTGAC AGAGAAGAGC
TTCGAGACAG ACATGAACCT GAATTTTCAG
AACCTGTCCG TGATGGGCCT GAGAATCCTG
CTGCTGAAGG TGGCCGGCTT CAATCTGCTG
ATGACACTGC GCCTGTGGTC CTCT
GGCTCT
AGGGCAAAGC GGAGCGGCAG CGGAGCAACC
AACTTCAGCC TGCTGAAGCA GGCCGGCGAT
GTGGAGGAGA ATCCTGGCCC ACGGATGGGC
TCTAGACTGT TTCTGGTGCT GAGCCTGCTG
TGCACAAAGC ACATGGAGGC AGCAGTGACC
CAGTCCCCAC GGAACAAGGT GACCGTGACA
GGCGGCAATG TGACACTGAG CTGTAGACAG
ACCAACTCCC ACAATTACAT GTATTGGTAC
CGGCAGGATA CCGGACACGG CCTGAGACTG
ATCCACTATA GCTACGGCGC CGGCAATCTG
CAGATCGGCG ACGTGCCAGA TGGCTATAAG
GCCACAAGGA CCACACAGGA GGACTTCTTT
CTGCTGCTGG AGCTGGCCTC CCCCTCTCAG
ACCTCTCTGT ATTTCTGCGC CAGCTCCGAT
GCCGGCACAG CCCAGAACAC CCTGTACTTT
GGAGCAGGAA CAAGGCTGTC CGTGCTGGAG
GACCTGCGCA ATGTGACCCC CCCTAAGGTG
TCCCTGTTCG AGCCTTCTAA GGCCGAGATC
GCCAACAAGA GGAAGGCCAC CCTGGTGTGC
CTGGCAAGGG GCTTCTTTCC AGATCACGTG
GAGCTGTCCT GGTGGGTGAA TGGCAAGGAG
GTGCACTCTG GCGTGAGCAC AGACCCCCAG
GCCTACAAGG AGTCCAACTA TTCTTACTGC
CTGTCTAGCC GGCTGAGAGT GAGCGCCACC
TTTTGGCACA ACCCCAGGAA TCACTTCCGC
TGTCAGGTGC AGTTTCACGG CCTGTCCGAG
GAGGATAAGT GGCCTGAGGG CTCTCCCAAG
CCTGTGACAC AGAACATCAG CGCCGAGGCA
TGGGGAAGGG CAGACTGTGG CATCACCAGC
GCCTCCTATC AGCAGGGCGT GCTGAGCGCC
ACAATCCTGT ACGAGATCCT GCTGGGCAAG
GCCACCCTGT ATGCTGTGCT GGTGTCAACT
CTGGTGGTCA TGGCTATGGT GAAACGGAAA
AACTCCTAA
Another embodiment provides a designed mTCR-6 (α is underlined, P2A is bolded, and β is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:66):
ATGAAGTCCT TCTCTATCAG CCTGGTGGTG
CTGTGGCTGC AGCTGAACTG GGTGAATAGC
CAGCAGAAGG TGCAGCAGTC TCCTGAGAGC
CTGATCGTGC CAGAGGGCGG CATGGCCTCC
CTGAACTGCA CCAGCTCCGA CCGGAATGTG
GATTATTTTT GGTGGTACAG ACAGCACTCC
GGCAAGTCTC CCAAGATGCT GATGAGCATC
TTCTCCAACG GCGAGAAGGA GGAGGGCCGG
TTTACAGTGC ACCTGAATAA GGCCTCTCTG
CACACCAGCC TGCACATCAG AGACTCCCAG
CCTTCCGATT CTGCCCTGTA TCTGTGCGCC
GCCTCTATGA TCAATGCCTA CAAAGTGATC
TTCGGCAAGG GCACACACCT GCACGTGCTG
CCCAACATCC AGAATCCAGA GCCCGCCGTG
TATCAGCTGA AGGACCCTCG GTCTCAGGAT
AGCACCCTGT GCCTGTTCAC CGACTTTGAT
AGCCAGATCA ATGTGCCAAA GACCATGGAG
TCCGGCACCT TTATCACAGA CAAGACCGTG
CTGGATATGA AGGCCATGGA CAGCAAGTCC
AACGGCGCCA TCGCCTGGTC CAATCAGACA
TCTTTCACCT GCCAGGATAT CTTTAAGGAG
ACAAACGCCA CCTACCCATC TAGCGACGTG
CCCTGTGATG CCACCCTGAC AGAGAAGAGC
TTCGAGACCG ACATGAACCT GAATTTTCAG
AACCTGTCCG TGATGGGCCT GAGAATCCTG
CTGCTGAAGG TGGCCGGCTT CAATCTGCTG
ATGACACTGC GCCTGTGGTC CTCT
GGCTCT
AGGGCAAAGC GGAGCGGCAG CGGAGCAACC
AACTTCAGCC TGCTGAAGCA GGCCGGCGAT
GTGGAGGAGA ATCCTGGCCC ACGGATGGGC
TGCACAAAGC ACATGGAGGC AGCAGTGACC
CAGAGCCCAC GGAACAAGGT GACCGTGACA
GGCGGCAATG TGACACTGTC TTGTAGACAG
ACCAACAGCC ACAATTACAT GTATTGGTAC
CGGCAGGATA CCGGACACGG CCTGAGACTG
ATCCACTATT CCTACGGAGC AGGAAACCTG
CAGATCGGCG ACGTGCCTGA TGGCTACAAG
GCCACAAGAA CCACACAGGA GGACTTCTTT
CTGCTGCTGG AGCTGGCCTC CCCATCTCAG
ACCTCTCTGT ATTTCTGCGC AAGCTCCGAT
GCAGGCGTGA GCCAGAACAC ACTGTACTTT
GGAGCAGGAA CCAGGCTGAG CGTGCTGGAG
GACCTGCGCA ATGTGACACC CCCTAAGGTG
AGCCTGTTCG AGCCCTCCAA GGCCGAGATC
GCCAACAAGA GGAAGGCCAC CCTGGTGTGC
CTGGCAAGGG GCTTCTTTCC TGATCACGTG
GAGCTGAGCT GGTGGGTGAA TGGCAAGGAG
GTGCACTCCG GCGTGTCTAC AGACCCACAG
GCCTATAAGG AGAGCAACTA TTCCTACTGC
CTGTCTAGCC GGCTGAGAGT GTCCGCCACC
TTTTGGCACA ACCCAAGGAA TCACTTCCGC
TGTCAGGTGC AGTTTCACGG CCTGAGCGAG
GAGGATAAGT GGCCAGAGGG CTCCCCAAAG
CCTGTGACCC AGAATATCTC TGCCGAGGCA
TGGGGAAGGG CAGACTGTGG AATCACAAGC
GCCTCCTACC AGCAGGGCGT GCTGTCCGCC
ACCATCCTGT ATGAGATCCT GCTGGGCAAG
GCCACACTGT ACGCCGTGCT GGTGTCCACC
CTGGTGGTCA TGGCCATGGT GAAGCGCAAG
AACTCTTGA
Another embodiment provides a designed mTCR-8 (α is underlined, P2A is bolded, and β is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:67):
ATGAAGTCCT TCTCTATCAG CCTGGTGGTG
CTGTGGCTGC AGCTGAACTG GGTGAATTCT
CAGCAGAAGG TGCAGCAGTC CCCTGAGTCT
CTGATCGTGC CAGAGGGCGG CATGGCCTCC
CTGAACTGCA CCAGCTCCGA CCGGAATGTG
GATTATTTTT GGTGGTACAG ACAGCACAGC
GGCAAGTCCC CCAAGATGCT GATGTCTATC
TTCAGCAACG GCGAGAAGGA GGAGGGCCGG
TTTACAGTGC ACCTGAATAA GGCCTCCCTG
CACACCTCTC TGCACATCAG AGACAGCCAG
CCTTCCGATT CTGCCCTGTA TCTGTGCGCA
GCAAGCATCT CCGGAGGATA CAAGGTGGTG
TTCGGCAGCG GAACAAGGCT GCTGGTGTCC
CCCGATATCC AGAATCCAGA GCCCGCCGTG
TATCAGCTGA AGGACCCTCG CTCCCAGGAT
AGCACCCTGT GCCTGTTCAC CGACTTTGAT
TCCCAGATCA ACGTGCCAAA GACCATGGAG
TCTGGCACCT TTATCACAGA CAAGACCGTG
CTGGATATGA AGGCCATGGA CTCTAAGAGC
AACGGCGCCA TCGCCTGGAG CAATCAGACA
TCCTTCACCT GCCAGGATAT CTTTAAGGAG
ACAAATGCCA CCTACCCATC TAGCGACGTG
CCCTGTGATG CCACCCTGAC AGAGAAGTCT
TTCGAGACCG ACATGAACCT GAATTTTCAG
AACCTGAGCG TGATGGGCCT GAGAATCCTG
CTGCTGAAGG TGGCCGGCTT CAATCTGCTG
ATGACACTGA GGCTGTGGTC CTCTGGCTCC
AGGGCAAAGC GGAGCGGCTC TGGAGCCACC
AACTTCTCTC TGCTGAAGCA GGCAGGCGAC
GTGGAGGAGA ATCCTGGACC ACGGATGGGC
TCTAGACTGT TTCTGGTGCT GAGCCTGCTG
TGCACAAAGC ACATGGAGGC AGCAGTGACC
CAGAGCCCAC GGAACAAGGT GACCGTGACA
GGCGGCAATG TGACACTGTC CTGTAGACAG
ACCAACTCTC ACAATTACAT GTATTGGTAC
CGGCAGGATA CCGGCCACGG CCTGAGACTG
ATCCACTATT CCTACGGAGC AGGAAACCTG
CAGATCGGCG ACGTGCCTGA TGGCTACAAG
GCCACAAGGA CCACACAGGA GGACTTCTTT
CTGCTGCTGG AGCTGGCCAG CCCATCCCAG
ACCAGCCTGT ATTTCTGCGC CAGCTCCGAT
GCCGGCACAT CCCAGAACAC CCTGTACTTT
GGAGCAGGAA CAAGGCTGAG CGTGCTGGAG
GACCTGCGCA ATGTGACCCC CCCTAAGGTG
TCTCTGTTCG AGCCCAGCAA GGCCGAGATC
GCCAACAAGA GGAAGGCCAC CCTGGTGTGC
CTGGCAAGGG GCTTCTTTCC TGATCACGTG
GAGCTGAGCT GGTGGGTGAA TGGCAAGGAG
GTGCACAGCG GCGTGTCCAC AGACCCACAG
GCCTATAAGG AGTCTAACTA TAGCTACTGC
CTGTCTAGCC GGCTGAGAGT GTCCGCCACC
TTTTGGCACA ACCCAAGGAA TCACTTCCGC
TGTCAGGTGC AGTTTCACGG CCTGTCCGAG
GAGGATAAGT GGCCAGAGGG CTCTCCAAAG
CCTGTGACCC AGAATATCAG CGCCGAGGCA
TGGGGAAGGG CAGACTGTGG CATCACATCT
GCCAGCTACC AGCAGGGCGT GCTGTCCGCC
ACCATCCTGT ATGAGATCCT GCTGGGCAAG
GCCACACTGT ACGCCGTGCT GGTGAGCACC
CTGGTGGTCA TGGCCATGGT GAAGAGAAAG
AACTCCTGA
Another embodiment provides a designed mTCR-10 (α is underlined, P2A is bolded, and β is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:68):
ATGAAGTCCT TCTCTATCAG CCTGGTGGTG
CTGTGGCTGC AGCTGAACTG GGTGAATAGC
CAGCAGAAGG TGCAGCAGTC TCCTGAGAGC
CTGATCGTGC CAGAGGGCGG CATGGCCTCC
CTGAACTGCA CCAGCTCCGA CCGGAATGTG
GATTATTTTT GGTGGTACAG ACAGCACTCC
GGCAAGTCTC CCAAGATGCT GATGAGCATC
TTCTCCAACG GCGAGAAGGA GGAGGGCCGG
TTTACAGTGC ACCTGAATAA GGCCTCTCTG
CACACCAGCC TGCACATCAG AGACTCCCAG
CCTTCCGATT CTGCCCTGTA TCTGTGCGCC
GCCTCTATCG TGGGCGGCTA CAAGGTGGTG
TTCGGCTCCG GCACAAGGCT GCTGGTGTCT
CCCGATATCC AGAATCCAGA GCCCGCCGTG
TATCAGCTGA AGGACCCTCG CTCTCAGGAT
AGCACCCTGT GCCTGTTCAC CGACTTTGAT
TCTCAGATCA ACGTGCCAAA GACCATGGAG
AGCGGCACCT TTATCACAGA CAAGACCGTG
CTGGATATGA AGGCCATGGA CAGCAAGTCC
AACGGCGCCA TCGCCTGGTC CAATCAGACA
TCTTTCACCT GCCAGGATAT CTTTAAGGAG
ACAAATGCCA CCTACCCATC TAGCGACGTG
CCCTGTGATG CCACCCTGAC AGAGAAGAGC
TTCGAGACCG ACATGAACCT GAATTTTCAG
AACCTGTCCG TGATGGGCCT GAGAATCCTG
CTGCTGAAGG TGGCCGGCTT CAATCTGCTG
ATGACACTGC GCCTGTGGTC CTCTGGCTCT
AGGGCAAAGC GGAGCGGCAG CGGAGCAACC
AACTTCAGCC TGCTGAAGCA GGCAGGCGAC
GTGGAGGAGA ATCCTGGACC ACGGATGGGC
AGCAGACTGT TTCTGGTGCT GTCCCTGCTG
TGCACAAAGC ACATGGAGGC AGCAGTGACC
CAGAGCCCAC GGAACAAGGT GACCGTGACA
GGCGGCAATG TGACACTGTC TTGTAGACAG
ACCAACAGCC ACAATTACAT GTATTGGTAC
CGGCAGGATA CCGGCCACGG CCTGAGACTG
ATCCACTATT CCTACGGAGC AGGAAACCTG
CAGATCGGCG ACGTGCCTGA TGGCTACAAG
GCCACAAGGA CCACACAGGA GGACTTCTTT
CTGCTGCTGG AGCTGGCCTC CCCATCTCAG
ACCAGCCTGT ATTTCTGCGC CAGCTCCGAT
CACGGCACAG GCCAGAACAC CCTGTACTTT
GGAGCAGGAA CAAGGCTGTC CGTGCTGGAG
GACCTGCGCA ATGTGACCCC CCCTAAGGTG
AGCCTGTTCG AGCCCTCCAA GGCCGAGATC
GCCAACAAGA GGAAGGCCAC CCTGGTGTGC
CTGGCAAGGG GCTTCTTTCC TGATCACGTG
GAGCTGAGCT GGTGGGTGAA TGGCAAGGAG
GTGCACTCCG GCGTGTCTAC AGACCCACAG
GCCTATAAGG AGAGCAACTA TTCCTACTGC
CTGTCTAGCC GGCTGAGAGT GTCCGCCACC
TTTTGGCACA ACCCAAGGAA TCACTTCCGC
TGTCAGGTGC AGTTTCACGG CCTGTCTGAG
GAGGATAAGT GGCCAGAGGG CAGCCCAAAG
CCTGTGACCC AGAATATCTC CGCCGAGGCA
TGGGGAAGGG CAGACTGTGG AATCACAAGC
GCCTCCTACC AGCAGGGCGT GCTGAGCGCC
ACCATCCTGT ATGAGATCCT GCTGGGCAAG
GCCACACTGT ACGCCGTGCT GGTGTCCACC
CTGGTGGTCA TGGCCATGGT GAAGAGAAAG
AACTCTTGA
Another embodiment provides a designed mTCR-11 (α is underlined, P2A is bolded, and β is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:69):
ATGAAGTCCT TCTCTATCAG CCTGGTGGTG
CTGTGGCTGC AGCTGAACTG GGTGAATAGC
CAGCAGAAGG TGCAGCAGTC TCCTGAGAGC
CTGATCGTGC CAGAGGGCGG CATGGCCTCC
CTGAACTGCA CCAGCTCCGA CCGGAATGTG
GATTATTTTT GGTGGTACAG ACAGCACTCC
GGCAAGTCTC CCAAGATGCT GATGAGCATC
TTCTCCAACG GCGAGAAGGA GGAGGGCCGG
TTTACAGTGC ACCTGAATAA GGCCTCTCTG
CACACCAGCC TGCACATCAG AGACAGCCAG
CCTTCCGATT CTGCCCTGTA TCTGTGCGCC
GCCTCCAAGA CAGGCGGCTA CAAGGTGGTG
TTCGGCTCCG GAACCAGGCT GCTGGTGTCT
CCCGATATCC AGAATCCAGA GCCCGCCGTG
TATCAGCTGA AGGACCCTCG CTCTCAGGAT
AGCACCCTGT GCCTGTTCAC CGACTTTGAT
TCTCAGATCA ACGTGCCAAA GACAATGGAG
AGCGGCACCT TTATCACAGA CAAGACCGTG
CTGGATATGA AGGCCATGGA CAGCAAGTCC
AACGGCGCCA TCGCCTGGTC CAATCAGACA
TCTTTCACCT GCCAGGATAT CTTTAAGGAG
ACAAATGCCA CCTACCCATC TAGCGACGTG
CCCTGTGATG CCACCCTGAC AGAGAAGTCT
TTCGAGACCG ACATGAACCT GAATTTTCAG
AACCTGAGCG TGATGGGCCT GAGAATCCTG
CTGCTGAAGG TGGCCGGCTT CAATCTGCTG
ATGACACTGA GGCTGTGGTC CTCT
GGCTCC
AGGGCAAAGC GGAGCGGCAG CGGAGCAACC
AACTTCTCTC TGCTGAAGCA GGCAGGCGAC
GTGGAGGAGA ATCCTGGACC ACGGATGGGC
AGCAGACTGT TTCTGGTGCT GTCCCTGCTG
TGCACAAAGC ACATGGAGGC AGCAGTGACC
CAGAGCCCAC GGAACAAGGT GACCGTGACA
GGCGGCAATG TGACACTGTC TTGTAGACAG
ACCAACAGCC ACAATTACAT GTATTGGTAC
CGGCAGGATA CCGGCCACGG CCTGAGACTG
ATCCACTATT CCTACGGAGC AGGAAACCTG
CAGATCGGCG ACGTGCCTGA TGGCTACAAG
GCCACAAGGA CCACACAGGA GGACTTCTTT
CTGCTGCTGG AGCTGGCCTC CCCATCTCAG
ACCTCCCTGT ATTTCTGCGC CAGCTCCGAT
GCCGGCACAT CTCAGAACAC CCTGTACTTT
GGAGCAGGAA CAAGGCTGAG CGTGCTGGAG
GACCTGCGCA ATGTGACCCC CCCTAAGGTG
AGCCTGTTCG AGCCCTCCAA GGCCGAGATC
GCCAACAAGA GGAAGGCCAC CCTGGTGTGC
CTGGCAAGGG GCTTCTTTCC TGATCACGTG
GAGCTGAGCT GGTGGGTGAA TGGCAAGGAG
GTGCACTCCG GCGTGTCTAC AGACCCACAG
GCCTATAAGG AGAGCAACTA TTCCTACTGC
CTGTCTAGCC GGCTGAGAGT GTCCGCCACC
TTTTGGCACA ACCCAAGGAA TCACTTCCGC
TGTCAGGTGC AGTTTCACGG CCTGTCTGAG
GAGGATAAGT GGCCAGAGGG CAGCCCAAAG
CCTGTGACCC AGAATATCTC CGCCGAGGCA
TGGGGAAGGG CAGACTGTGG AATCACAAGC
GCCTCCTACC AGCAGGGCGT GCTGAGCGCC
ACCATCCTGT ATGAGATCCT GCTGGGCAAG
GCCACACTGT ACGCCGTGCT GGTGAGCACC
CTGGTGGTCA TGGCCATGGT GAAGAGAAAG
AACTCCTGA
One embodiment provides a designed mTCR-17 (α is underlined, P2A is bolded, and β is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:70):
Another embodiment provides a designed mTCR-38 (α is underlined, P2A is bolded, and β is double underlined) having at least 90%, 95%, 99%, or 100% sequence identity to (SEQ ID NO:71):
ATGAAATCCT TTAGTATTTC CCTAGTGGTC
CTGTGGCTTC AGCTAAACTG GGTGAACAGC
61
CAACAGAAGG TGCAGCAGAG CCCAGAATCC
CTCATTGTTC CAGAGGGAGG CATGGCCTCT
CTCAACTGCA CTTCCAGTGA TCGTAATGTT
GACTACTTCT GGTGGTACAG ACAGCACTCT
GGGAAAAGCC CCAAGATGCT GATGTCTATC
TTCTCCAATG GTGAAAAGGA AGAAGGCAGA
TTCACAGTTC ACCTCAATAA AGCCAGCCTG
CATACTTCCC TGCACATCAG AGACTCCCAG
CCCAGTGACT CTGCTCTCTA CCTCTGTGCA
GCAACCCTGA CTGGAGGCTA TAAAGTGGTC
TTTGGAAGTG GGACTCGATT GCTGGTAAGC
CCTGACATCC AGAACCCAGA ACCTGCTGTG
TACCAGTTAA AAGATCCTCG GTCTCAGGAC
AGCACCCTCT GCCTGTTCAC CGACTTTGAC
TCCCAAATCA ATGTGCCGAA AACCATGGAA
TCTGGAACGT TCATCACTGA CAAAACTGTG
CTGGACATGA AAGCTATGGA TTCCAAGAGC
AATGGGGCCA TTGCCTGGAG CAACCAGACA
AGCTTCACCT GCCAAGATAT CTTCAAAGAG
ACCAACGCCA CCTACCCCAG TTCAGACGTT
CCCTGTGATG CCACGTTGAC TGAGAAAAGC
TTTGAAACAG ATATGAACCT AAACTTTCAA
AACCTGTCAG TTATGGGACT CCGAATCCTC
CTGCTGAAAG TAGCCGGATT TAACCTGCTC
ATGACGCTGA GGCTGTGGTC CAGT
GGCAGC
AGAGCCAAGA GAAGCGGATC CGGCGCCACC
AACTTCAGCC TGCTGAAGCA GGCCGGCGAC
GTGGAGGAAA ACCCTGGCCC TAGGATGGGC
TCCAGGCTCT TTCTGGTCTT GAGCCTCCTG
TGTACAAAAC ACATGGAGGC TGCAGTCACC
CAAAGCCCTA GAAACAAGGT GACAGTAACA
GGAGGAAACG TGACATTGAG CTGTCGCCAG
ACTAATAGCC ACAACTACAT GTACTGGTAT
CGGCAGGACA CTGGGCATGG GCTGAGGCTG
ATCCATTACT CATATGGTGC TGGCAACCTT
CAAATAGGAG ATGTCCCTGA TGGGTACAAG
GCCACCAGAA CAACGCAAGA AGACTTCTTC
CTCCTGCTGG AATTGGCTTC TCCCTCTCAG
ACATCTTTGT ACTTCTGTGC CAGCAGTGAT
GCTGGGACTA GTCAAAACAC CTTGTACTTT
GGTGCGGGCA CCCGACTATC GGTGCTAGAG
GATCTGAGAA ATGTGACTCC ACCCAAGGTC
TCCTTGTTTG AGCCATCAAA AGCAGAGATT
GCAAACAAAC GAAAGGCTAC CCTCGTGTGC
TTGGCCAGGG GCTTCTTCCC TGACCACGTG
GAGCTGAGCT GGTGGGTGAA TGGCAAGGAG
GTCCACAGTG GGGTCAGCAC GGACCCTCAG
GCCTACAAGG AGAGCAATTA TAGCTACTGC
CTGAGCAGCC GCCTGAGGGT CTCTGCTACC
TTCTGGCACA ATCCTCGAAA CCACTTCCGC
TGCCAAGTGC AGTTCCATGG GCTTTCAGAG
GAGGACAAGT GGCCAGAGGG CTCACCCAAA
CCTGTCACAC AGAACATCAG TGCAGAGGCC
TGGGGCCGAG CAGACTGTGG GATTACCTCA
GCATCCTATC AACAAGGGGT CTTGTCTGCC
ACCATCCTCT ATGAGATCCT GCTAGGGAAA
GCCACCCTGT ATGCTGTGCT TGTCAGTACA
CTGGTGGTGA TGGCTATGGT CAAAAGAAAG
AATTCATGA
B. Genetically Engineered T Cells
Another embodiment provides genetically engineered CD8+ immune cells that express the disclosed mouse TCR genes to produce a TCR that specifically binds to hAFP or a fragment thereof expressed on the surface of a tumor cell. Preferrably, the immune cells are human T cells, for example human cytotoxic T cells. The engineered human cytotoxic T cells can be autologous human cytotoxic T cells. TCR genes can be codon optimized.
Another embodiment provides CD4+ immune cells, preferably human CD4+ immune cells, genetically engineered to express the disclosed mouse TCR genes. CD4+ cells include T helper cells, monocytes, macrophages, and dendritic cells. The engineered human CD4+ immune cells can be autologous human T helper cells. The TCR genes can be codon optimized.
The 9 hAFP158 specific murine TCRs identified in this study are able to render CD4 T cells the capability of binding Tet158 tetramer, suggesting that they are high affinity and independent of CD8 help. The role of CD4 in adoptive cell therapy is not clear. But the production of IL-2 by CD4 TCR-T cells in response to HepG2 tumor cells stimulation may provide the cytokine for maintaining T cell proliferation. In addition, the CD4 TCR-T cells demonstrate a low cytotoxicity against HepG2 tumor cells. Thus, although the CD8 TCR-T may be the major player for killing hAFP+ tumor cells, the CD4 TCR-T can provide the help for T cell proliferation, which can be critical for generating antitumor effect in vivo.
It is believed that murine TCRs may provide increased expression on the surface of a human host cell as compared to human TCR. Murine TCRs can also replace endogenous TCRs on the surface of the human host cell more effectively than exogenous human TCR. However, humanization of the mTCR may be necessary to avoid anti-TCR responses with repeated use of TCR-T cells. Methods of humanizing mTCRs are known in the arts. See for example U.S. Pat. Nos. 5,861,155, and 5,859,205, WO2007/131092, EP0460167 and Davis, et al., Clin Cancer Res., 16:5852-5861 (2010), which are incorporated by reference in their entirety. In one embodiment, the disclosed mTCR genes are humanized before being introduced to a T cell or administered to a human subject.
C. T Cell Hybridomas
Another embodiment provides T cell hybridomas that express the disclosed mTCR polypeptides. For example, the hybridoma can be a result of the fusion of fusing sorted mouse CD8+Tet158+ cells with BW-Lyt2.4 cells that lack TCR α and β chains and are selected as described (He., Y. et al., J Immunol, 174:3808-3817 (2005)). Methods of making the hybridomas are provided in the Examples.
D. Antibodies
One embodiment provides antibodies that specifically bind the disclosed mTCR proteins, for example SEQ ID NO:2-62. Suitable antibodies can be prepared by one of skill in the art. The antibody or antigen binding fragment therefore, can be an agonist or antagonist of mTCR or simply binds specifically to the mTCR or a polypeptide thereof.
In some embodiments, the disclosed antibodies and antigen binding fragments thereof immunospecifically bind to mTCR or a polypeptide thereof (e.g., any one of SEQ ID NO:2-62). In some embodiments, the antibody binds to an extracellular domain of an mTCR (SEQ ID NOs:41-53).
For example, molecules are provided that can immunospecifically bind to the disclosed mTCR polypeptides:
(I) arrayed on the surface of a cell (especially a live cell); or
(II) arrayed on the surface of a cell (especially a live cell) at an endogenous concentration;
Compositions are also provided that can immunospecifically bind to soluble endogenous mTCR polypeptides. In some embodiments the molecules reduce or prevent the soluble mTCR polypeptide from binding or otherwise interacting with its ligand.
The antibodies or antigen binding fragments thereof can be prepared using any suitable methods known in the art such as those discussed in more detail below.
1. Human and Humanized Antibodies
The antibodies that specifically bind to the disclosed mTCR polypeptides can be human or humanized. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
Transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.
Optionally, the antibodies are generated in other species and “humanized” for administration in humans. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all, of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, a humanized form of a nonhuman antibody (or a fragment thereof) is a chimeric antibody or fragment, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity. According to the “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.
It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.
The antibody can be bound to a substrate or labeled with a detectable moiety or both bound and labeled. The detectable moieties contemplated with the present compositions include fluorescent, enzymatic and radioactive markers.
2. Single-Chain Antibodies
The antibodies that specifically bind the disclosed mTCR polypeptides can be single-chain antibodies. Methods for the production of single-chain antibodies are well known to those of skill in the art. A single chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other variable domain via a 15 to 25 amino acid peptide or linker have been developed without significantly disrupting antigen binding or specificity of the binding. The linker is chosen to permit the heavy chain and light chain to bind together in their proper conformational orientation. These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody.
3. Monovalent Antibodies
The antibodies that specifically bind to the disclosed mTCR polypeptides can be monovalent antibodies. In vitro methods can be used for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab′)2 fragment, that has two antigen combining sites and is still capable of cross-linking antigen.
The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab′)2 fragment is a bivalent fragment comprising two Fab′ fragments linked by a disulfide bridge at the 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. Antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
4. Hybrid Antibodies
The antibodies that specifically bind the disclosed mTCR polypeptides can be hybrid antibodies. In hybrid antibodies, one heavy and light chain pair is homologous to that found in an antibody raised against one epitope, while the other heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously. Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques. Such hybrids may, of course, also be formed using chimeric chains.
5. Conjugates or Fusions of Antibody Fragments
The targeting function of the antibody can be used therapeutically by coupling the antibody or a fragment thereof with a therapeutic agent. Such coupling of the antibody or fragment (e.g., at least a portion of an immunoglobulin constant region (Fc)) with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, comprising the antibody or antibody fragment and the therapeutic agent.
Such coupling of the antibody or fragment with the therapeutic agent can be achieved by making an immunoconjugate or by making a fusion protein, or by linking the antibody or fragment to a nucleic acid such as an siRNA, comprising the antibody or antibody fragment and the therapeutic agent.
In some embodiments, the antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it is present in the circulation or at the site of treatment for longer periods of time. For example, it may be desirable to maintain titers of the antibody in the circulation or in the location to be treated for extended periods of time. Antibodies can be engineered with Fc variants that extend half-life, e.g., using Xtend™ antibody half-life prolongation technology (Xencor, Monrovia, Calif.). In other embodiments, the half-life of the anti-DNA antibody is decreased to reduce potential side effects. The conjugates disclosed can be used for modifying a given biological response. The drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin.
The disclosed antibodies and mTCR polypeptides can be conjugated or linked to one or more detectable labels. The disclosed antibodies and mTCR polypeptides can be linked to at least one agent to form a detection conjugate. In order to increase the efficacy of the molecules as diagnostic it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one reporter molecule. A reporter molecule is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies or polypeptides include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin.
E. Formulations
The disclosed antibodies, fusion proteins, and mTCR polypeptides can be formulated into pharmaceutical compositions. Pharmaceutical compositions containing the antibody, fusion protein, or mTCR polypeptides can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.
In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein the term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and/or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.
For the disclosed compositions, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. For the disclosed immunomodulatory agents, generally dosage levels of 0.001 to 20 mg/kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower.
In certain embodiments, the disclosed compositions are administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the composition which is greater than that which can be achieved by systemic administration. The compositions can be combined with a matrix as described above to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.
1. Formulations for Parenteral Administration
In some embodiments, compositions disclosed herein, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a peptide or polypeptide, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved/resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
2. Formulations for Oral Administration
In embodiments the compositions are formulated for oral delivery. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the disclosed. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form. Liposomal or proteinoid encapsulation may be used to formulate the compositions. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulation will include the peptide (or chemically modified forms thereof) and inert ingredients which protect peptide in the stomach environment, and release of the biologically active material in the intestine.
The agents can be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where the moiety permits uptake into the blood stream from the stomach or intestine, or uptake directly into the intestinal mucosa. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. PEGylation is an exemplary chemical modification for pharmaceutical usage. Other moieties that may be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-tioxocane [see, e.g., Abuchowski and Davis (1981) “Soluble Polymer-Enzyme Adducts,” in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem. 4:185-189].
Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.
Controlled release oral formulations may be desirable. The agent can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release is based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.
For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine. In some embodiments, the release will avoid the deleterious effects of the stomach environment, either by protection of the agent (or derivative) or by release of the agent (or derivative) beyond the stomach environment, such as in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L3OD™, Aquateric™ cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may be used as mixed films.
3. Formulations for Topical Administration
The disclosed immunomodulatory agents can be applied topically. Topical administration does not work well for most peptide formulations, although it can be effective especially if applied to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa.
Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.
A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.
Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.
Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations may require the inclusion of penetration enhancers.
4. Controlled Delivery Polymeric Matrices
The fusion proteins, mTCR polypeptides, and antibodies disclosed herein can also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where the agent is dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.
Either non-biodegradable or biodegradable matrices can be used for delivery of fusion polypeptides, antibodies, or mTCR polyepeptidesor nucleic acids encoding them, although in some embodiments biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred in some embodiments due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or “bulk release” may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.
The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).
The devices can be formulated for local release to treat the area of implantation or injection—which will typically deliver a dosage that is much less than the dosage for treatment of an entire body—or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.
In certain embodiments, the disclosed antibodies, mTCR polypeptides and engineered immune cells can be used to detect or treat cancer. A preferred cancer to be detected, diagnosed, or treated includes heptacellular carcinoma or any other cancer the expresses hAFP.
A. Adoptive Transfer
In one embodiment, immune cells are engineered to express the disclosed mTCR. The immune cells are T cells, preferably human T cells. The cells can be autologous or heterologous. The cells are typically transduced in vitro. The transduced cells can optionally be expanded in vitro to obtain a large population transduced cells that can administered to a subject in need thereof. Such subjects typically have or are believed to have a cancer or tumor that expresses hAFP. The T cells can be CD8+ or CD4+. The transduced cells can be administered in one or more doses to the subject.
The adoptive transfer can be combined with other therapies for the treatment of cancer. Such additional therapies include chemotherapy, radiation therapy, and surgery or a combination thereof. The adoptive transfer of TCR-T can be combined with cancer vaccines so that the TCR-T will be further expanded in vivo to reduce the number of transferred TCR-T cells and the associated toxicity.
One embodiment provides a method for reducing tumor burden in a subject in need thereof by administering engineered T cells that express one of the disclosed mTCRs, wherein the engineered mTCR cells inhibit or reduce tumors that express hAFP. The subject can optionally be treated with a cotherapy for cancer. A preferred cancer to be treated is heptacellular carcinoma.
B. Detection or Diagnosis
The disclosed antibodies or mTCR polypeptides can be labeled as discussed above. The labeled polypeptides can be applied to a sample of cancer cells wherein the specific binding of the labeled polypeptides to the sample of cancer cells is indicative of a hAFP specific cancer including heptacellular carcinoma.
The detection of the label can optionally be quantified to determine the number of cancer cells in sample. The sample is typically a liver tissue sample but can include a blood or serum sample.
Also provided are compositions and kits. Such kits can include containers, each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, one or more binding agents (antibodies), already attached to a marker or optionally with reagents for coupling a binding agent to an antibody (as well as the marker itself), buffers, and/or reagents and instrumentation for the isolation (optionally by microdissection) to support the practice of the invention. A label or indicator describing, or a set of instructions for use of, kit components in a ligand detection method of the present invention, will also be typically included, where the instructions may be associated with a package insert and/or the packaging of the kit or the components thereof.
Additional embodiments provide immunodetection kits for use with the immunodetection methods described herein. As the antibodies or mTCR polyeptides are generally used to detect hAFP, the antibodies and mTCR polypeptides will generally be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to an mTCR polypeptide and/or optionally, an immunodetection reagent.
The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with and/or linked to the given antibody. Detectable labels that are associated with and/or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.
Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted herein, a number of exemplary labels are known in the art and/or all such labels may be suitably employed in connection with the present invention.
The kits may further comprise a therapeutic agent for the treatment of cancer, such as an an engineered immune cell expressing the disclosed mTCR polypeptides.
Materials and Methods
Mice: HLA-A2 transgenic AAD mice (Pichard, V., et al, J Immunother, 31:246-253 (2008)) and NSG mice were purchased from Jackson Laboratory and housed in specific pathogen-free animal facility. Animal protocols were approved by the Institutional Animal Care and Use Committee of Augusta University.
Immunization: Mice were immunized with recombinant lentivector (lv) expressing hAFP or influenza virus M1 protein subcutaneously as described (Morgan, R A. et al., J Immunother, 36:133-151 (2013)). Twelve days later, mice were boosted with hAFP158 (FMNKFIYEI) (SEQ ID NO:1) or M158 (GILGFVFTL)(SEQ ID NO:72) peptide together with PolyI:C and anti-CD40 antibody as described (Linette, G. P., et al., Blood, 122:863-871 (2013)).
Results
To induce mouse CD8 T cells capable of recognizing human HLA-A2 presented hAFP epitopes, the HLA-A2 transgenic AAD mice were immunized with recombinant hAFP-lv. The AAD mice express a chimeric MHC I molecule of human HLA-A2 α1-α2 domain and the mouse H-2d α3 domain that bind mouse CD8 molecule to help immune priming (Pichard, V., et al, J Immunother, 31:246-253 (2008)). It was found that hAFP-lv immunization reproducibly induced a modest level of hAFP158 epitope-specific CD8 responses in AAD mice (
Materials and Methods
Fifteen million total splenocytes (1.5 million of hAFP158-specific CD8 T cells) of the naïve or immunized mice were injected into NSG mice, which were then challenged with HepG2 tumor cells. In the therapeutic model, NSG mice were injected with 15 million total splenocytes of the immunized mice when tumor size reaches 2 cm in diameter. The experiment was done twice with similar data.
Results
In this adoptive transfer experiment, it was found that the splenocytes from AAD mice immunized with hAFP-lv and hAFP158 peptide, but not that from naïve mice, could completely prevent HepG2 tumor cell challenge in NSG mice (
Materials and Methods
To further identify the immune cells responsible for eradicating the established HepG2 tumor, CD8 T cells from the AAD mice immunized with hAFP-lv and hAFP158 peptide were isolated using magnetic CD8 beads (
Immunization: Mice were immunized with recombinant lentivector (lv) expressing hAFP or influenza virus M1 protein subcutaneously as described (Morgan, R A. et al., J Immunother, 36:133-151 (2013)). Twelve days later, mice were boosted with hAFP158 (FMNKFIYEI)(SEQ ID NO:1) or M158 (GILGFVFTL)(SEQ ID NO:72) peptide together with PolyI:C and anti-CD40 antibody as described (Linette, G. P., et al., Blood, 122:863-871 (2013)).
Tumor inoculation: Five millions of HepG2 tumor cells were inoculated into the flank of NSG mice. Tumor growth was monitored every other day.
It was found that adoptive transfer of CD8 T cells from the hAFP immunized mice could eradicate HepG2 tumors in NSG mice (
Materials and Methods
T cell hybridoma: T cell hybridomas were created by fusing the sorted mouse CD8+Tet158+ cells with BW-Lyt2.4 cells that is lack of TCR α and β chains and selected as described (He, Y., et al., J Immunol, 174:3808-3817 (2005)).
Immunological and tetramer staining: Splenocytes were stained with surface marker and anti-Vβ antibody panel (BD Biosciences, San Diego, Calif.). HLA-A2/hAFP158tetramers (Tet158) were kindly provided by NIH Tetramer Core Facility at Emory University.
Results
To identify and clone paired TCR α and β chain gene from a single T cells, T cell hybridoma cell lines were established. Prior to generating T cell hybridomas, the TCR Vβ chains were characterized using the anti-Vβ panel antibodies. The data showed that >90% of the Tet158+CD8 T cells could be stained with anti-Vβ8.3 antibody (
Next, a total of 39 T cell hybridoma clones that could be stained by Tet158 tetramer were obtained from two cell fusion experiments using sorted Tet158+CD8 T cells (data not shown). Among them, 5 clones could respond to the stimulation of hAFP+HepG2 tumor cells and produce IL-2 (
Materials and Methods
Identification of the TCRα and β chain genes: Total RNA was isolated from T cell hybridomas. 5-RACE technique (Hong, Y., et al., Hepatology, 59:1448-1458 (2014)) was conducted to amplify the TCR α and β chain genes. The cDNA was made with oligo dT primer and then the PolyC was added to the 5 end of cDNA. PCR was conducted by using the 5′ pGI primer (CACCGGGIIGGGIIGGGIIGG)(SEQ ID NO:73) and the 3′ primers corresponding to the constant (C) region of α chain (GGCATCACAGGGAACG) (SEQ ID NO:74) or β chain (CCAGAAGGTAGCAGAGACCC) (SEQ ID NO:75). Based on the obtained partial sequence of the TCR α and β variable (V) regions, specific primer corresponding to the V region of α (ATGAAATCCTTTAGTATTTCCC) (SEQ ID NO:76) or β (ATGGGCTCCAGGCTCTTTCTG) (SEQ ID NO:77) chains were used together with the internal primer of α C-region (GCACATTGATTTGGGAGTC) (SEQ ID NO:78) or β C-region: (GGGTAGCCTTTTGTTTGTTTG) (SEQ ID NO:79) to amplify and verify the V region. Finally, sequences of the TCR α and β chains were obtained and verified.
Results
In this experiment, the paired TCRα and β chain genes from the 14 hybridomas capable of producing IL-2 after co-culture with HepG2 tumor cells were amplified and sequenced. The result was summarized in Table 1. Out of 14 hybridomas, 9 unique sets of TCR genes were identified. The sequences of TCR2 (Clone 1G8) and TCR4 (Clone 2F6) are identical, so are the TCR3 (Clone 10A3) and TCR5 (Clone 6D9). TCR6 (Clone 5B3), TCR7 (Clone 5B10), and TCR13 (Clone 11B10) are identical, so are TCR38 (Clone 2C3) and TCR39 (Clone 3E4). The amino acid sequences of α and β chains of TCR1-3, 6, 8, 10, 11, 17, and 38 were then compared to the NCBI and IMGT databank. The sequence data showed that all nine TCR α chains had the same variable region (TRAV7D-2), and that the TCR β chains also shared the same variable region (TRBV13-1), which is consistent with the data from anti-V(38.3 antibody staining (
All of the amino acid differences in the 9 TCR genes identified herein are found in the CDR3 region of the α (
Table 1. The 14 paired TCR alpha and beta chains are summarized. TCR4 and TCR2 are identical. TCR5 and TCR3 are identical. TCR6, TCR7 and TCR13 are identical. TCR38 and TCR39 are identical.
1. Protein sequences of TCRA-1 and TCRA-2 have 2AA difference between V-J junction
2. The TCRA-3 is significantly different from TCRA-1 and TCRA-2. In fact, the J region of TCRA-3 is totally different from TCRA-1 and TCRA-2.
3. Protein sequences of TCRB-1 and TCRB-2 are identical.
4. Protein sequences of TCRB-3 and TCRB-1 are identical except one AA from Serine (116S) to Alanine (116A) between V-D Junction.
5. The D region in TCRB between V-J is 9nt without obvious homology to any sequence in the databank.
Materials and Methods
TCR genes and recombinant lv: TCR α and β chains gene were designed and synthesized by using the above identified V-D-J region and C-region. A P2A sequence (Cho, H. I., et al., Cancer Res, 69:9012-9019 (2009)) is inserted between α and β chain to separate α and β chains. The entire genes were codon optimized and cloned into lv.
Transduction of human T cells: Human T cells were isolated from buffy coat by negative selection. CD3/CD28 Tetrameric antibody complex (Stemcell Technologies, Vancouver) was then added to activate the cells for 2 days before they were transduced with recombinant lv.
Results
In this experiment, whether the hAFP-specific TCR genes could engineer human T cells to create TCR redirected T cells (TCR-T) and whether such TCR-T cells would acquire the capability of specifically recognizing and killing hAFP+ tumor cells were investigated. To this end, three TCR genes (TCR1, TCR2, and TCR3) were synthesized and cloned into lv. The P2A sequence was inserted between the TCR α and β genes to generate equal number of TCR α and β chains in target T cells (
Materials and Methods
IFNγ was detected by ELISA after the TCR gene transduced human T cells were co-cultured overnight with HLA-A2+hAFP+HepG2 cells, HLA-A2+hAFP− HepG2 cells, or HLA-A2-hAFP+ tumor cells.
Results
To study whether the human TCR-T cells could be specifically activated by HLA-A2 presented hAFP158 peptide, cytokine production of human TCR-T was measured after stimulation with HLA-A2 mouse splenocytes pulsed with hAFP158 peptides. The data showed that human TCR-T cells could be specifically stimulated to produce IFNγ by HLA-A2 cells pulsed with hAFP158 peptide. In contrast, no IFNγ was detected in the human TCR-T culture after stimulation with influenza virus M1 peptide. The mock transduced human T cells do not respond to hAFP158 peptide stimulation (
But both the CD4 (
Materials and Methods
ELISA of cytokines: ELISA of IFNγ and IL-2 was conducted as instructed by manufacturer (Biolegend, San Diego, Calif.).
Cytotoxic assay: TCR-T cells were added to growing HepG2 tumor cells. Cytotoxicity assay was conducted using LDH assay as instructed (Promega, Madison, Wis.).
Prodidium iodide (P1) stainings The dead cells were stained with PI as instructed (BD Biosciences, San Diego, Calif.).
Results
In this experiment, the cytotoxic activity of human TCR-T cells was studied. Using LDH assay, we first showed that the human TCR-T cells could effectively kill 80% of HepG2 tumor cells within 24 hours of co-culture at E/T ratio of 5. In contrast, the TCR-T cells did not generate significant cytotoxic effect on the AFP− or HLA-A2− tumor cells (
Materials and Methods:
In order to study the in vivo antitumor effect of human TCR-T, NSG mice bearing 4 day HepG2 tumors were adoptively transferred with 20 million human TCR-T cells. Tumor growth was monitored by measuring the length, width, and height. Tumor volume was calculated as ½(length×width×height).
Results:
In this experiment, the in vivo antitumor effect of human TCR-T cells was studied. Primary human T cells were transduced with lv and approximately 40% of the total T cells (25% of CD8 T cells and 54% of CD4 T cells) were Tet158+TCR-T cells before adoptive transfer (
This application is a continuation of U.S. Nonprovisional patent application Ser. No. 15/969,211 filed on May 2, 2018, which claims benefit of and priority to U.S. Provisional Patent Applications 62/505,406 filed on May 12, 2017, 62/609,614 filed on Dec. 22, 2017, and 62/625,051 filed on Feb. 1, 2018, and where permitted all of which are incorporated by reference in their entireties.
This invention was made with government support under R01CA168912 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62625051 | Feb 2018 | US | |
| 62609614 | Dec 2017 | US | |
| 62505406 | May 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15969211 | May 2018 | US |
| Child | 17353409 | US |
