Patent Application
20230272040
The present application relates to the field of biopharmaceuticals, and particularly relates to stealth chimeric antigen receptor (CAR), and related polynucleotides, vectors, compositions, preparation methods and uses thereof.
The initial success of CAR T cell therapy in the treatment of hematological malignancies has heralded rapid growth in a new area of immunotherapeutic treatment strategies. CARs are designer receptors typically with modular activation domains derived from the CD3ζ ITAM motifs and a costimulatory domain, for example, from 4-1BB or CD28. Target recognition by these receptors is usually provided through an scFv domain on the extracellular terminal of the CAR, though alternative approaches have also been designed.
Safety is a major hurdle hindering the development of CAR-T therapeutics. CAR molecules are usually constitutively expressed on the T-cell surface. Thus, when CAR-T cells are injected into patients, they will migrate to different tissues expressing the target antigen often with unintended toxicity and damage to normal tissues alongside tumor cells. In addition, constitutive expression of the CAR molecule promotes tonic signaling and T cell exhaustion.
To date, several types of switches have been developed to control CAR-T activity. Some of these are based on the incorporation of “suicide genes” into the CAR construct. For example, the HSV-TK transgene has been used in adoptive cell therapies to allow for inducible apoptosis of the modified cells on treatment with Ganciclovir. Another approach relies on the use of an inducible Caspase 9 (iCasp9). In these strategies, the therapeutic response is terminated through targeted elimination of the CAR-T cells themselves.
Other switch systems rely on modification of the receptor, for example, adapter-mediated CARs, also known as “universal CARs”. These CAR T cells share a common receptor that can be modified to recognize a variety of tumor targets through linking of the receptor to an adapter molecule that binds both the CAR and the tumor target. Similarly, recruitment of the intracellular signaling domain to the transmembrane component of the CAR via drug-induced dimerization has been proposed as a means of controlling dosing and activation. While current clinically approved CARs are designed to be constitutively active, adapter-dependent CAR T cells can only recognize and kill when the adapter is administered, allowing for titratable and reversible control of the CAR-T cells.
For these types of switches, the CAR expression and functionality is regulated externally through application of small molecule drugs or other therapeutics. There are major challenges for these types of switches including: the availability of existing FDA approved switch molecules that can be applied, the safety profile of the switch molecule, bioavailability, biodistribution, and the complexity of regulation in vivo due to the complex dynamics and distribution of the switch molecules and the CAR-T cells.
Selective pressure on the target cell population brought on through the targeting of a single tumor associated antigen (TAA) can eventually result in downregulation of the target antigen and evasion of the CAR T cell immune response1 (Ruella & Maus, 2016). Loss of target antigen expression results in unmitigated tumor cell expansion and, ultimately, disease relapse. Studies have reported that on downregulation of the primary TAA, target cells may upregulate a secondary TAA, allowing for a second round of therapeutic intervention2 (
The high-affinity IL2 Receptor is a transmembrane receptor comprised of three distinct, noncovalently associated components: the α, β, and γ chains. It has been shown that in the absence of the α and γ subunits, the β chain is constitutively endocytosed and degraded3 (Hémar et al., 1994).
Analysis of the C-terminal residues of LDLR indicates the presence of the endosomal sorting motif NDXY. Studies indicate that this mediates an indirect endosomal sorting mechanism through interactions with ARH and binding to the AP-2 complex4-5 (Tao et al., 2016; Fasano et al., 2009). SEZ6L2 is characterized by the presence of two endosomal-targeting consensus sequences in its c-terminal region6 (Bonifacino et al., 2003).
The present application aims to solve the CAR-T safety problem by providing stealth CAR for reducing cytotoxicity towards normal cells.
In the novel CAR system of the present application, expression/stabilization of the CAR molecule is dependent on target antigen availability. When there is no antigen available, there is no detectable CAR on the T cell surface and no CAR function. When the engineered T cell recognizes its target antigen, the CAR is stabilized and signaling can be initiated. This is a target dependent on-switch. By modulating CAR expression in response to antigen, the present application can reduce CAR T cell toxicity and exhaustion. Since the CAR is “invisible” when there is no antigen, the inventors of the present application have elected to name it, “Stealth CAR”. Stealth CAR can be applied in mono- and dual-specific CAR settings, as well as in autologous and allogeneic settings. It can be applied in the context of T cells, NK cells, gamma delta T cells, and iPSC-derived T cells.
The endocytic stealth CAR system can also be applied to address a particular challenge facing CAR T therapies, that is, “on-target, off-tumor” effects. As TAAs are often upregulated on tumor tissues, but also expressed at lower levels on normal tissues, CAR T therapy can have the unwanted effect of targeting non-cancerous cells. By using a CAR that is stabilized by antigen expression, cells expressing high levels of the target antigen (e.g., tumor cells) provide a stronger activation for our CAR system. Thus, the present application can promote tumor-specific killing by CAR T cells, while minimizing the effect on normal tissue (
By applying stealth CAR in a dual-specific setting, the present application expects the system could be used to address the issue of antigen escape. By expressing a CAR variant that is only transiently stable at the cell surface, the present application provides a mechanism to reduce collateral targeting of, and damage to, normal tissue. Upregulation of the secondary target antigen on the tumor cells provides a stronger signal and promotes CAR surface stability and downstream activation. By targeting the primary TAA with one CAR and the secondary TAA with a second, stealth CAR, the present application can elicit a strong initial tumor response and targeted secondary response only once the secondary TAA has been upregulated.
The inventors of the present application have designed and prepared several CAR variants with modified transmembrane and juxtamembrane sequences which facilitate the intracellular trafficking of receptors. These are derived from the IL2 Receptor β chain (IL2Rβ or IL2Rb), the Low-Density Lipoprotein Receptor (LDLR), and the human Seizure 6-like Protein 2 (SEZ6L2). The amino acid motifs in the transmembrane and intracellular sequences of these receptors promote intracellular trafficking via the endosome pathway.
The present application provides novel stealth CAR for reducing cytotoxicity towards normal cells and improving CAR-T safety, as well as reducing inflammatory cytokine and tonic signaling caused by constitutive CAR surface expression. The present application also provides dual CARs comprising the stealth CAR and a second CAR, and related nucleic acids, vectors, host cells and pharmaceutical compositions which comprise a polynucleotide encoding the stealth CAR or the dual CAR. The present application further provides a method of treating disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition, a method of reducing the cytotoxicity of a CAR-T cell towards normal cells, and a method of producing a CAR-T cell with reduced cytotoxicity towards normal cells.
In one aspect, the application provides a chimeric antigen receptor (CAR) comprising:
In a further aspect, the CAR comprises from N-terminal to C-terminal: TAA scFv-CD8Hinge-IL2Rβ tm jm DT-4-1BB-CD3ζ, TAA scFv-CD8Hinge-IL2Rβ tm jm DT-CD28-CD3ζ, TAA scFv-CD8Hinge-LDLR tm jm-4-1BB-CD3ζ, or TAA scFv-CD8Hinge-SEZ6L2 tm jm-4-1BB-CD3ζ
In a further aspect, the TAA scFv is selected from one or more of CEA scFv, Claudin 18.2 scFv and HER2 scFv; more preferably, CEA scFv is MN14op CEA scFv, or Claudin 18.2 scFv is 841 Claudin 18.2 scFv; most preferably, MN14op CEA scFv, 841 Claudin 18.2 scFv, or HER2 scFv comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 5, 30, or 31 respectively.
In a further aspect, the N-terminal of the CAR further contains a Leader Sequence and/or a HA Sequence.
In a further aspect, the CAR comprises from N-terminal to C-terminal:
In a further aspect, the CAR comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 9, 14, 15, 17, 18, 19, 21, 22 or 24 respectively, optionally the amino acid sequence of CAR does not include the amino acid sequence of P2A-GFP, Leader Sequence, and/or HA.
In a further aspect, the CAR comprises hinge domain; preferably the hinge domain comprises CD8 or CD28 hinge derived from the extracellular region, or IgG hinge
In another aspect, the application also provides a chimeric antigen receptor (CAR) comprising:
In a further aspect, the CAR comprises from N-terminal to C-terminal: TAA scFv-CD8Hinge-CD8 tm-4-1BB-CD3ζ-IL2R DT.
In a further aspect, the N-terminal of the CAR further contains Leader Sequence and/or HA Sequence.
In a further aspect, the TAA scFv is CEA scFv; more preferably, the TAA scFv is MN14op CEA scFv; most preferably, MN14op CEA scFv comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 5; most preferably, the CAR comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 8 or 16 respectively, optionally the amino acid sequence of CAR does not include the amino acid sequence of P2A-GFP, Leader Sequence, and/or HA.
In a further aspect, the transmembrane (tm) of the IL2Rβ, LDLR, or SEZ6L2 comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 1, 38 or 40 respectively;
In a further aspect, the application provides a chimeric antigen receptor (CAR) comprising:
In a further aspect, the application also provides a chimeric antigen receptor (CAR) comprising:
In a specific embodiment, the CAR comprising IL2Rβ transmembrane (tm), juxtamembrane (jm), and degradation sequence (DT), from N-terminal to C-terminal, comprises:
In a specific embodiment, the CAR comprising LDLR transmembrane (tm) and juxtamembrane (jm), from N-terminal to C-terminal, comprises:
In a specific embodiment, the CAR comprising SEZ6L2 transmembrane (tm) and juxtamembrane (jm), from N-terminal to C-terminal, comprises:
In a specific embodiment, the CAR comprising cytoplasmic segment comprising an IL2Rβ degradation sequence (DT) and at least one signaling domain, from N-terminal to C-terminal, comprises:
In another aspect, the application also provides a dual CAR comprising: a first CAR according to any of the above-described embodiments, and a second CAR comprising:
In a further aspect, the first CAR and the second CAR is linked by P2A.
In a further aspect, the P2A comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 43 or 44.
In a further aspect, the dual CAR comprises N-terminal to C-terminal:
TAA scFv-CD8Hinge-IL2Rβ tm jm DT-4-1BB-CD3ζ-P2A-another TAA scFv-CD8Hinge-CD8 tm-CD28-CD3ζ, TAA scFv-CD8Hinge-CD8 tm-4-1BB-CD3ζ-P2A-another TAA scFv-CD8Hinge-IL2Rβ tm jm DT-CD28-CD3ζ, TAA scFv-CD8Hinge-LDLR tm jm-4-1BB-CD3ζ-P2A-another TAA scFv-CD8Hinge-CD8 tm-CD28-CD3ζ, TAA scFv-CD8Hinge-CD8 tm-4-1BB-CD3ζ-P2A-another TAA scFv-CD8Hinge-LDLR tm jm-CD28-CD3ζ, TAA scFv-CD8Hinge-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-another TAA scFv-CD8Hinge-CD8 tm-CD28, or TAA scFv-CD8Hinge-CD8 tm-4-1BB-CD3ζ-P2A-another TAA scFv-CD8Hinge-SEZ6L2 tm jm-CD3ζ.
In a further aspect, the dual CAR comprises N-terminal to C-terminal: 841 Claudin 18.2 scFv-CD8Hinge-SEZ6L2 tm jm-4-1BB-CD3ζ-P2A-PD-L1 scFv-CD8Hinge-CD8 tm-CD28-(G4S)2-GFP, 841 Claudin18.2 scFv-CD8Hinge-CD8 tm-4-1BB-CD3ζ-P2A-HER2 scFv-IL2Rβ tm jm DT-CD28-CD3ζ, or 841 Claudin18.2 scFv-CD8Hinge-CD8 tm-4-1BB-CD3ζ-P2A-HER2 scFv-SEZ6L2 tm jm-CD3ζ respectively, optionally the dual CAR does not include (G4S)2-GFP; more preferably, 841 Claudin 18.2 scFv, HER2 scFv, or PD-L1 scFv comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 30, 31, or 32 respectively; most preferably, the CAR comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO. 20, 25 or 26 respectively, optionally the amino acid sequence of CAR does not include the amino acid sequence of (G4S)2-GFP and/or Leader Sequence.
In a specific embodiment, the dual CAR, from N-terminal to C-terminal, comprises:
In another aspect, the application also provides the nucleic acid comprising a polynucleotide encoding the above-mentioned CAR or dual CAR.
In another aspect, the application also provides the vector comprising the above-mentioned polynucleotide encoding the above-mentioned CAR or dual CAR.
In another aspect, the application also provides the composition comprising at least one of the above-mentioned nucleic acids or at least one of the above-mentioned vectors.
In another aspect, the application also provides a host cell comprising one or more nucleic acids or vectors or a composition of the present application.
In a further aspect, the host cell is an autologous cell or an allogeneic cell.
In a further aspect, the host cell is a mammalian cell, preferably a primate cell, more preferably a human cell.
In a further aspect, the host cell is selected from a T cell, a NK cell, an iNKT cell, a cord blood NK cell, a gamma delta T cell (γδ T-cell), a TCR knockout T cell, a virus-specific T cell, a monocyte, a macrophage, or an iPSC-derived T cell.
In another aspect, the application also provides a pharmaceutical composition comprising the CAR or dual CAR, one or more nucleic acids, one or more vectors, or a host cell of the present application.
In a further aspect, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
In another aspect, the application relates to a method of treating disease in a subject in need thereof, comprising administering to the subject (e.g. a therapeutically effective amount of) the pharmaceutical composition, the CAR, the dual CAR of the present application.
In a further aspect, the disease is a cancer comprising hematological malignancy or one or more solid tumors.
In a further aspect, the cancer is ovarian cancer, pancreatic cancer, colon cancer, colorectal cancer, lymphoma, esophageal cancer, lung cancer, hepatic cancer, head-neck cancer, or cancer of the gallbladder.
In another aspect, the application relates to a method of reducing the cytotoxicity of a CAR-T cell towards normal cells, using the CAR or dual CAR, the nucleic acid, or the vector, or the composition of the present application.
In one aspect, the application relates to a method of producing a CAR-T cell with reduced cytotoxicity towards normal cells comprising:
In a further aspect, the host cell is a mammalian cell, preferably a primate cell, more preferably a human cell.
In a further aspect, the host cell is selected from a T cell, a NK cell, an iNKT cell, a cord blood NK cell, a gamma delta T cell (γδ T-cell), a TCR knockout T cell, a virus-specific T cell, a monocyte, a macrophage, or an iPSC-derived T cell.
The technical solutions of the application have at least the following advantages:
The novel features of the application are set forth with particularity in the appended claims.
Some of the features and advantages of the present application are explained in the following detailed description in the embodiments and in the examples.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as generally used in the art to which this disclosure belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” also refer to the plural forms unless the context clearly dictates otherwise, e.g., reference to “a host cell” includes a plurality of such host cells.
As used herein, the term a “chimeric antigen receptor (CAR)” means a fused protein comprising an extracellular domain capable of binding to a predetermined antigen, an intracellular domain comprising a signaling domain, and a transmembrane domain. The phrase “binding to a predetermined antigen” means any proteinaceous molecule or part thereof that can specifically bind to the predetermined antigen. The “signaling domain” means any oligopeptide or polypeptide domain known to function to transmit a signal causing activation or inhibition of a biological process in a cell, for example, activation of an immune cell such as a T cell or a NK cell. Examples include 4-1BB, CD28 and/or CD3ζ signaling domain.
As used herein, the term a “IL2R” or “IL2Rb” means the IL2 Receptor β chain. The high-affinity IL2 Receptor is a transmembrane receptor comprised of three distinct, noncovalently associated components: the α, β, and γ chains. It has been shown that in the absence of the α and γ subunits, the β chain is constitutively endocytosed and degraded.
As used herein, the term a “Stealth CAR” or “Endocytic CAR” refers in its broadest sense to a chimeric antigen receptor (CAR) comprising: (1) an extracellular ligand-binding domain comprising a single chain variable fragment (scFv) specifically binding to a predetermined antigen; (2) a transmembrane (tm) linking juxtamembrane (jm) domain; wherein the transmembrane linking juxtamembrane domain comprises an IL2 Receptor 3 chain (IL2Rβ) transmembrane domain and an IL2Rβ juxtamembrane domain, and the transmembrane linking juxtamembrane domain is adjacent to IL2Rβ degradation sequence (DT); wherein preferably, the IL2R degradation sequence is at the C-terminal of the transmembrane linking juxtamembrane domain; wherein preferably, the IL2R degradation sequence comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO: 2; wherein the transmembrane linking juxtamembrane domain comprises a Low-Density Lipoprotein Receptor (LDLR) transmembrane domain and an LDLR juxtamembrane domain; or wherein the transmembrane linking juxtamembrane domain comprises a Seizure 6-like Protein 2 (SEZ6L2) transmembrane domain and a SEZ6L2 juxtamembrane domain; (3) an intracellular domain; wherein preferably, the intracellular domain comprises a signaling domain; more preferably, the signaling domain comprises one or more signaling domains selected from the group consisting of a 4-1BB signaling domain, a CD28 signaling domain and a CD3ζ signaling domain. Or a chimeric antigen receptor (CAR) comprising: (1) an extracellular ligand-binding domain comprising single chain variable fragment (scFv) specifically binding to a predetermined antigen; (2) a transmembrane domain, and (3) a cytoplasmic segment comprising an IL2Rβ degradation sequence (DT) and at least one signaling domain; wherein preferably, the IL2Rβ degradation sequence comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence represented by SEQ ID NO: 2; wherein preferably, the IL2Rβ degradation sequence is at the C-terminal of the cytoplasmic segment. Or a dual CAR comprising a first CAR according to any of the above-described embodiments, and a second CAR.
As used herein, the term an “antigen binding fragment” or “antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins. The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
As used herein, the term a “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. In addition, antibody fragments comprising single chain polypeptides have the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site, thereby providing the antigen binding properties of full length antibodies.
As used herein, the term a “therapeutically effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
As used herein, the term an “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Particularly, the individual or subject is a human. The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable excipient” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable excipient includes, but is not limited to, a buffer, a stabilizer, or a preservative.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the molecules of the application are used to delay development of a disease or to slow the progression of a disease.
As used herein, the term “tumor-associated antigens (TAAs)” or “tumor antigens” means a biological molecule having antigenicity, the expression of which comes to be recognized in association with malignant alteration of a cell. The tumor antigen in the present disclosure includes a tumor specific antigen (an antigen which is present only in tumor cells and is not found in other normal cells), and a tumor-associated antigen (an antigen which is also present in other organs and tissues or heterogeneous and allogeneic normal cells, or an antigen which is expressed during development and/or differentiation).
The term “amino acid” as used within this application denotes the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: Ala, one letter code: 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). “Percent (%) amino acid sequence identity” with respect to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid 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. SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
The terms “host cell”, “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.
The term “cancer” or “tumor” as used herein refers to proliferative diseases, such as ovarian cancer, pancreatic cancer, colon cancer, colorectal cancer, lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.
Preparation of CAR constructs is a common technical method in the art. For example, first, CAR gene fragments are prepared through gene synthesis technology, then CAR PiggyBac transposon expression vectors are constructed, CAR constructs are electroporated into a target cell, and construct expression is assessed by flow cytometry or total protein analysis.
In this Example 1, two novel CAR constructs based on the IL2Rβ chain transmembrane and juxtamembrane motifs were designed (
HA-MN14op CAR (MLB001: HA-MN14op CEA scFv-CD8Hinge-CD8tm-4-1BB-CD3).
The pMAX-GFP is a control plasmid used to determine electroporation efficiency, particularly when a fluorescent marker is not available in the test construct.
Functional efficacy was evaluated by T cell activation assays, including but not limited to NFAT reporter assays and cytotoxicity assays.
MALPVTALLLPLALLLHAARPEVQLVESGGGVVKPGGSLRLSCSA
APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IYIWAPL
MALPVTALLLPLALLLHAARP
YPYDVPDYAEVQLVESGGGVVKPG
D
IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
MALPVTALLLPLALLLHAARP
YPYDVPDYAEVQLVESGGGVVKPG
D
IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
MALPVTALLLPLALLLHAARP
YPYDVPDYAEVQLVESGGGVVKPG
D
IPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNT
PDPSKFFSQLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IYIW
FSQL*
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
IPW
LGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPS
KFFSQLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG
Transposon-mediated delivery of endocytic CAR constructs in Example 1: The EF1α promoter was used to drive expression of our CAR constructs in a transposon vector.
Transduction of primary T cells and Jurkat E6.1 T cells (Jurkat E6.1 cells) with CAR constructs: PBMC-derived, CD3+ purified T cells (primary T cells) or Jurkat E6.1 T cells were transduced with transposon vectors carrying our CAR in Example 1 constructs using electroporation.
Transduced CAR-T cells were analyzed by flow cytometry to measure construct integration into the host cell. Cells were stained using anti-human Fab′ conjugated to AlexaFluor 488 (AF488). The endocytic CARs were also engineered with an extracellular, N-terminal HA tag to aid in detection of the receptor and perform pulse-chase experiments. CAR surface expression was detectable following electroporation of the constructs into Jurkat E6.1 cells (
Jurkat E6.1 NFAT-Luciferase reporter cells expressing CAR Constructs were mixed 1:1 with antigen-expressing target cells (LoVo) and luciferase activity was measured after 6-hours (
Donor-derived primary CD3+ cells (primary cells) transduced to express our CAR constructs were mixed 1:1 with antigen-expressing luciferase+ target cells (Kato-III, which express target antigen CEA) and co-cultured for 24 hours. Cytotoxicity was measured by increased luciferase activity (
In this Example 6, CAR constructs were designed as follows:
The pMAX-GFP is a control plasmid used to determine electroporation efficiency, particularly when a fluorescent marker is not available in the test construct.
PSKFFSQL
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE
The T cell line, Jurkat E6.1, expressing an NFAT-driven luciferase reporter, and donor-derived T cells were induced to express our CAR constructs by electroporation of a Piggy Bac transposon plasmid with the CAR construct under control of the EF1α promoter and with transposase mRNA.
CAR surface expression was evaluated by flow cytometry through staining with AF647-conjugated αhuman Fab′ antibody; overall CAR construct expression was determined by co-expression of GFP under a P2A sequence (
LoVo cells expressing the CEACAM-5 target antigen were cultured in white, opaque flat-bottom 96-well plates in the presence of 1 μg/mL of Mitomycin C to inhibit cell proliferation. 25,000 LoVo target cells were seeded per well 6-8 hours prior to the initiation of the time course assay to allow for adherence. The assay was performed at an E:T ratio of 1:1 with CAR+ Jurkat T cells. After 8 hours of incubation, the wells were washed, and media replenished. 24, 6, 3, and 1 hour prior to luminescence readout, the wells were washed and Jurkat T cells expressing the CAR constructs were added to each well. At the end of the 24-hour period, Bio-Glo™ reagent (Promega) was added to each well to assess overall Jurkat cell activation (
Observation of the luminescent signal indicated that the activation kinetics of all of the CAR variants were similar and comparable to the original CAR construct, with NFAT-driven gene expression occurring strongly around 6 hours of activation. Although the kinetics of activation were comparable, the degree of cellular activation was strongest in the original (MLB010) and c-terminal (MLB025) tagged versions of the CARs. Both the LDLR- and SEZ6L2-based variants (MLB047 and MLB048) showed much lower levels of overall activation, and the IL2Rβ-based construct (MLB020) showed the lowest levels of expression (
To further determine the effect of the various transmembrane domains on the overall activation state of the Jurkat T cells, we performed a second co-culture assay to determine the extent of CD69 upregulation following stimulation. 50,000 LoVo Target cells were co-cultured with Jurkat CAR-positive T cells at a 1:1 ratio for 3, 6, or 24 hours and CD69 expression was evaluated by flow cytometry. By gating the cells on GFP expression, we were able to determine the fraction of CD69-expressing cells in the CAR-positive population and the overall degree of CD69 upregulation by assessing fluorescence intensity (
To assess the effect of the IL2Rβ (MLB013), SEZ6L2 (MLB047), and LDLR (MLB048) domains on downstream T cell activity, cytotoxicity assays against CEACAM-5-positive target cell lines LoVo, A549, and HT-29 were set up. These target cells lines were selected as they have high, intermediate, and low levels of CEA expression, respectively. Given that CAR target antigens often have variable expression between the tumor tissue and noncancerous tissue, this assay allows us to assess the potential for on-target, off-tumor CAR activity, which can cause severe side effects in a clinical setting.
Donor-derived CD3+ PBMCs were transduced to express CAR constructs using the PiggyBac Expression system. Following electroporation, CAR expression was evaluated via flow cytometry by co-expression of GFP on the CAR expression vector and used to normalize cell numbers to directly compare CAR-T cell activity (
As expected, cytotoxic activity was directly related to the amount of antigen expression on the target cell lines, with the highest amount of cytotoxicity detected against LoVo Cells, and the lowest amount of cytotoxicity detected against HT-29 cells (
Having shown that the novel transmembrane CAR constructs were able to retain cytotoxic activity against antigen-expressing target cells, we wanted to measure additional readouts of CAR T cell activity. Two common markers of activation are IL-2 and IFN-7. To assay for cytokine secretion, media was harvested following overnight cytotoxicity assays and soluble cytokine expression was determined via ELISA.
The trends that were observed in cytokine secretion mirrored those that we observed in cytotoxicity, with the highest levels of cytokine secretion detected against CEA-high target cells, and greatly reduced cytokine expression detected against HT-29 cells (
These findings are significant as CAR T cell therapies are known to promote potentially fatal Cytokine Release Syndrome (CRS). For this reason, the results are of clinical interest and indicate that the cytokine profile of CAR T cells can be tuned through the use of alternative transmembrane and juxtamembrane domains. By using the transmembrane domains and associated trafficking motifs of receptors that are known to be targeted towards the endosomal pathway, the results have shown that CAR T cell activity can be tuned, expanding the therapeutic window.
Jurkat E6.1 T cells expressing an NFAT-driven luciferase reporter were electroporated with the Piggy Bac vector expressing Claudin18.2-specific CAR constructs: MLB026, MLB054, MLB055; and Piggy Bac transposase mRNA. As before, CAR expression was evaluated by surface staining with AF647-conjugated αhuman-Fab′ antibody and co-expression of GFP on the CAR vector via a P2A sequence (
To screen for CAR T cell activity, CLDN18.2(Claudin 18.2)-specific CAR T cells were co-cultured with antigen-negative HEK293T cells or HEK293T and NUGC4 cells transduced to express CLDN18.2(Claudin 18.2). As before, 50,000 target cells were cultured at a 1:1 ratio with CAR-expressing Jurkats. As with the αCEA CAR variants (
PBMCs were isolated via Ficoll-Paque separation and purified by CD3 negative selection to enrich the T cell population. Isolated T cells were stimulated with StemCell T cell activator (CD2/CD3/CD28) in the presence of IL-2, IL-7, and IL-15 for three days and then electroporated with hyperactive PiggyBac transposase mRNA and PiggyBac vectors MLB026 and MLB054 to induce the expression of CARs based on either the CD8a transmembrane domain or the SEZ6L2 transmembrane and juxtamembrane domain, respectively. These CAR constructs utilize the 841 scFv, which is specific to Claudin18.2.
CAR surface expression was verified by flow cytometry by staining with a fluorescently-conjugated anti-human F(ab′)2 antibody. Transduced cells were identified by the joint expression of GFP on the CAR construct via a P2A sequence. Results indicate strong surface staining in T cells expressing the CD8a transmembrane sequence (MLB026) while T cells expressing the SEZ6L2 modified CAR (MLB054) had negligible surface staining (
To test the functional capabilities of the anti-Claudin18.2, SEZ6L2-modified Stealth CAR, a luciferase-based cytotoxicity assay was performed against HEK293T or NUGC4 cells transduced to overexpress the Claudin18.2 isoform as well as luciferase. The number of CAR positive cells was evaluated by flow cytometry and counts were normalized to the number of CAR positive cells (
The co-culture supernatants were analyzed for expression of IFN-γ produced by the CAR T cells via sandwich ELISA. Supernatant was diluted 1-in-3 in ELISA diluent and colorimetric activity of the HRP detection substrate indicated that the SEZ6L2-modified Stealth CAR (MLB054) had a reduced cytokine secretion profile against both the HEK-cldn18.2 (
In this Example 12, CAR constructs were designed as follows:
SQL
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR
SQL
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR
PLY
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR
PBMCs were isolated via Ficoll-Paque separation and purified by CD3 negative selection to enrich the T cell population. Isolated T cells were stimulated with StemCell T cell activator (CD2/CD3/CD28) in the presence of IL-2, IL-7, and IL-15 for three days and then electroporated with hyperactive PiggyBac transposase mRNA and PiggyBac vectors MLB038, MLB039, MLB079, and MLB080. All four CAR variants were engineered with the 4D5V8 scFv derived from the Herceptin antibody, which recognizes HER2 (the amino acid sequence of 4D5V8 Her2 scFv is SEQ ID NO. 31). The MLB038 and MLB039 CARs were designed based on the IL2 Receptor β transmembrane and juxtamembrane sequence. MLB038 and MLB039 are distinguishable by their coreceptor signaling domains, MLB038 was designed using the CD28 coreceptor domain and MLB039 was designed with the 4-11BB signaling domain. Both formats also have the CD3ζ intracellular ITAM signaling motifs. MLB079 was designed as a control and based on the CD8α transmembrane domain, while MLB080 was engineered with the SEZ6L2 transmembrane and juxtamembrane sequences.
CAR surface expression was verified by flow cytometry by staining with a fluorescently-conjugated anti-human F(ab′)2 antibody. Transduced cells were identified by the joint expression of GFP on the CAR construct via a P2A sequence. Results indicate strong surface staining in T cells expressing the CD8a transmembrane sequence while T cells expressing the IL2Rβ- or SEZ6L2-modified CARs (MLB038, MLB039, MLB080) had greatly reduced surface staining indicative of their predominantly intracellular localization (
To test the functional capacity of the anti-HER2 stealth CAR variants, luciferase-based cytotoxicity assays were performed against the following cell lines SK-BR-3, LoVo, and MDA-MB-231—which is clinically described as a triple-negative breast cancer cell line (
The co-culture supernatants were analyzed for expression of IFN-γ produced by the CAR T cells via sandwich ELISA. Supernatant was diluted 1-in-3 in ELISA diluent and colorimetric activity of the HRP detection substrate indicated that the Stealth CAR variants MLB038, MLB039, and MLB080 had reduced cytokine secretion relative to the CD8ca transmembrane control CAR, MLB079, against all of the cell lines tested: MDA-MB-231, LoVo, SKBR3 (
HEKcldn18.2 Cells were profiled for HER2 and Claudin18.2 expression using either a commercially available HER2 antibody or the Claudin18.2 antibody clones developed in-house, hCLDN18.2-808 antibody (the amino acid sequence of VH is SEQ ID NO:47; the amino acid sequence of VL is SEQ ID NO:48) and hCLDN18.2-841 antibody (the amino acid sequence of VH is SEQ ID NO:49; the amino acid sequence of VL is SEQ ID NO:50) (
PBMCs were isolated via Ficoll-Paque separation and purified by CD3 negative selection to enrich the T cell population. Isolated T cells were stimulated with StemCell T cell activator (CD2/CD3/CD28) in the presence of IL-2, IL-7, and IL-15 for three days and then electroporated with hyperactive PiggyBac transposase mRNA and PiggyBac vectors MLB026 and MLB108. CAR surface expression was verified by flow cytometry by staining with a fluorescently-conjugated anti-human F(ab′)2 antibody (
Functional assays indicated that Dual CAR MLB108 was able to elicit antigen specific killing of HEKcldn18.2 cells in a dose-dependent manner (
In additional dual CAR assays, the MLB040 Dual CAR format was compared with both the Claudin18.2-specific dominant CAR (MLB026) and the HER2 stealth CARs (MLB038 and MLB039). PBMCs were isolated via Ficoll-Paque separation and purified by CD3 negative selection to enrich the T cell population. Isolated T cells were stimulated with StemCell T cell activator (CD2/CD3/CD28) in the presence of IL-2, IL-7, and IL-15 for three days and then electroporated with hyperactive PiggyBac transposase mRNA and PiggyBac vectors MLB026, MLB038, MLB039, and MLB040. CAR surface expression was verified by flow cytometry by staining with a fluorescently-conjugated anti-human F(ab′)2 antibody (
Functional assays indicated a slight enhancement of cytotoxicity against HEKcldn18.2 cells, which express high levels of Claudin18.2 and low levels of HER2 (
It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
This application claims the benefit of U.S. Provisional Patent Application No. 63/067,870, entitled “Target-Dependent On-Switch Chimeric Antigen Receptors”, filed on Aug. 19, 2020; the contents of which are herein incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/113420 | 8/19/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63067870 | Aug 2020 | US |
