Patent Grant
11305011
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: HMLX_002_02US_2nd_Sub_SeqList_ST25.txt, date recorded: Nov. 4, 2021, file size 63.1 kilobytes).
Multiple myeloma (MM) is a malignancy of bone marrow plasma cells which despite recent advances in therapy, remains incurable. Its clinical course is characterized by an initial response to therapy, followed by repeated relapse with eventual resistance to all forms of treatment. It is also associated with significant morbidity and disability both due to the disease itself and toxicity from available treatments.
Multiple myeloma is characterized by malignant plasma cells which secrete either a kappa or lambda light chain restricted monoclonal paraprotein. Kappa restriction occurs in 60% of myeloma patients and the expression of kappa myeloma antigen (KMA) is highly restricted to multiple myeloma and B-cell malignancies. KappaMab is a KMA-specific monoclonal antibody which has demonstrated safety and efficacy in phase I and II clinical trials.
Treatment with monoclonal antibodies alone is not curative with incomplete eradication of the tumor leading to eventual relapse. This may be due to inadequate penetration of antibody into the tumor (via passive diffusion), heterogeneity of antigen expression on tumor cells or resistance of tumor cells to mechanisms of antibody dependent cytotoxicity. Thus, there is a need for effective therapies with low toxicity which can provide long term disease cure.
Chimeric Antigen Receptor bearing T cells (CAR T-cells) represent a possible solution to this problem. CAR T-cells incorporate the antigen binding domain of monoclonal antibodies with one or more intracellular signaling domain(s) of T cells to produce a localized, tumor specific immune response. CAR T-cells have several advantages over monoclonal antibodies: they actively migrate into the tumor, proliferate in response to antigen bearing tumor cells, secrete factors that recruit other arms of the immune response and can survive long term to provide ongoing protection from relapse. Another benefit of a CAR-T cell over an antibody therapeutic targeting the same antigen is that the CAR T-cell may also be further modified to enhance safety and function. For example, a T cell can be modified to include expression of a homing receptor which enhances T cell specificity and the ability of the T cell(s) to infiltrate cancer cells or tumors or they may include an “off switch” that can function to eliminate cells when toxicity occurs. Furthermore, and importantly for the treatment of multiple myeloma and its related disorders, the T cell may be modified to express additional biologically active or pharmaceutically active molecules that may enhance the anti-tumor response, such as, for example, tumor suppressive cytokines. As described herein, the current inventors have designed novel CAR constructs which are able to specifically bind to a particular conformational KMA epitope expressed only on MM cells and have engineered CAR T-cells to express an extracellular antigen binding domain specific for this epitope and an intracellular T cell signaling domain alone or in combination with the expression of other anti-tumoral immune mediators.
The present invention is drawn to chimeric antigen receptors (CAR) that are specific for kappa myeloma antigen (KMA) but contain intracellular signaling domains capable of triggering an anti-KMA T cell response, T cells containing such CARs and method of treating multiple myeloma and related disorders by administering T cells expressing KMA-specific CARs. The resulting CAR T-cells are able to mediate a targeted immune response against cancer cells while avoiding unwanted side effects associated with systemic delivery of monoclonal antibodies and/or anti-tumoral cytokines.
In one embodiment, the chimeric antigen receptors (CARs) of the present invention comprise one or more intracellular signaling domains and an extracellular antigen binding domain that specifically recognizes kappa myeloma antigen (KMA). In one embodiment, the intracellular signaling domain is one or more co-stimulatory endodomains. In a further embodiment, the one or more co-stimulatory domain is one or more of a CD28 domain, a CD3ζ domain, a 4-1BB domain, or an OX-40 domain or combinations thereof. In one embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain and a CD28 domain. In another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, and an OX-40 domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a CD28 domain and an OX-40 domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain and a 4-1BB domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a CD28 domain and a 4-1BB domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a 4-1BB domain and an OX-40 domain.
In one embodiment, the extracellular binding domain comprises a single chain variable fragment (scFv) that specifically recognizes KMA. In still another embodiment, the scFv comprises the complemetarity determining regions (CDRs) derived from KappaMab. In still another embodiment the scFv comprises the VL CDRs of SEQ ID NOs: 6-8. In yet another embodiment, the scFV comprises the VL region of SEQ ID NO: 21. In still another embodiment the scFv comprises the VH CDRs of SEQ ID NOs: 3-5. In yet another embodiment, the scFV comprises the VH region of SEQ ID NO: 22. In still a further embodiment, the scFv comprises the VL CDRs of SEQ ID NOs: 6-8 and the VH CDRs of SEQ ID NOs: 3-5. In yet a further embodiment, the scFv comprises the VL region of SEQ ID NO: 21 and the VH region of SEQ ID NO: 22. In one embodiment, the VL chain of SEQ ID NO: 2 and VH chain of SEQ ID NO: 1 are attached via a glycine-serine linker. In one embodiment, the VL region of SEQ ID NO: 21 and VH region of SEQ ID NO: 22 are attached via a glycine-serine linker. In still another embodiment, the linker is a (Gly4Ser)x where X is 1-5. In still another embodiment, the glycine-serine linker is a 15-20 amino acid linker. In still another embodiment, the linker is a 15 amino acid glycine serine linker and comprises (Gly4Ser)3. In one embodiment, the (Gly4Ser)3 linker is SEQ ID NO: 23. In one embodiment, the scFv is attached to the one or more intracellular signaling domains with a spacer. In still another embodiment, scFv is attached to the one or more intracellular domains by a spacer that comprises an immunoglobulin constant region. In one embodiment, the immunoglobulin constant region comprises one or more of an IgG hinge, an IgG CH2 and an IgG CH3 domain. In a particular embodiment, the immunoglobulin constant region comprises an immunoglobulin hinge domain. In still another embodiment, the immunoglobulin constant region comprises an immunoglobulin CH3 domain. In still another embodiment, the immunoglobulin constant region comprises an IgG CH2 domain. In still another embodiment, the scFv is attached to the one or more intracellular domains by a spacer that comprises a CD8a domain. In one embodiment, the spacer is attached to the scFV via a glycine-serine linker. In still another embodiment, the linker is a (Gly4Ser)x where X is 1-5. In still another embodiment, the glycine-serine linker is a 15-20 amino acid linker. In still another embodiment, the linker is a 15 amino acid glycine serine linker and comprises (Gly4Ser)3. In one embodiment, the (Gly4Ser)3 linker is SEQ ID NO: 23.
In one embodiment, the invention provides T cells comprising chimeric antigen receptors (CAR T-cells). In one embodiment, the CAR T-cells comprise CARs comprising one or more intracellular signaling domains and an extracellular binding domain. In a particular embodiment, the extracellular binding domain specifically recognizes a kappa myeloma antigen. In one embodiment the CAR T-cells are further engineered to express one or more additional biological molecules. In one embodiment, the additional one or more molecules comprise IL-12 and/or SANT7 and/or Galectin −3C (GAL3C). In one embodiment, the CAR T-cells express a single chain polypeptide comprising one IL-12 p35 subunit and one IL-12 p40 subunit joined by a flexible linker. In one embodiment the IL-12 p35 and IL-12 p40 are joined by a (G45)3 linker. In one embodiment the single chain IL-12 polypeptide forms a bioactive IL-12 p70 heterodimer. In one embodiment, the CAR T-cell expresses IL-12 and a selectable marker. In one embodiment, the one or more biological molecules is SANT7. In one embodiment, the CAR T-cell expresses GAL3C. In one embodiment, the CAR T-cell expresses GAL3C and a selectable marker. In one embodiment, the CAR T-cell expresses SANT7 and GAL3C. In one embodiment, the CAR T-cell expresses SANT7, GAL3C and a selectable marker. In one embodiment, the CAR T-cell expresses IL-12 and GAL3C. In one embodiment, the CAR T-cell expresses IL-12, GAL3C and a selectable marker. In one embodiment, the CAR T-cell expresses SANT7 and a selectable marker. In one embodiment, the CAR T-cell expresses IL-12, and SANT7. In one embodiment, the CAR T-cell expresses IL-12, SANT7 and a selectable marker. In one embodiment, the CAR T-cell expresses IL-12, SANT7 and GAL3C. In one embodiment, the CAR T-cell expresses IL-12, SANT7, GAL3C and a selectable marker.
In one embodiment, the CAR T-cells of the current invention also express a hepatocyte growth factor (HGF) binding protein that is capable of inhibiting HGF signaling and effector function. In one aspect, the HGF binding protein is an antibody or fragment thereof.
In one aspect, the current invention provides a method for producing a genetically modified T cell comprising introducing an expression vector encoding a CAR comprising one or more intracellular signaling domains and an extracellular antigen binding domain into a T cell. In a particular embodiment, the extracellular antigen binding domain specifically recognizes KMA. In one embodiment, the expression vector is a transposable vector expression system. In certain embodiments, the expression vector is a PiggyBac transposon expression vector. In another embodiment the expression vector is a viral vector. In one embodiment the viral vector is a lentiviral vector or a retroviral vector. In one embodiment, the expression vector is introduced into the cells by electroporation. In one embodiment, the one or more intracellular signaling domains in the CAR is one or more co-stimulatory endodomains. In a further embodiment, the one or more co-stimulatory domain is one or more of a CD28 domain, a CD3ζ domain, a 4-1BB domain, or an OX-40 domain or combinations thereof. In one embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain and a CD28 domain. In another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, and an OX-40 domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a CD28 domain and an OX-40 domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain and a 4-1BB domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a CD28 domain and a 4-1BB domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a 4-1BB domain and an OX-40 domain.In one embodiment, the extracellular binding domain comprises a single chain variable fragment (scFv) that specifically recognizes KMA. In still another embodiment, the scFv comprises the complemetarity determining regions (CDRs) derived from KappaMab. In still another embodiment the scFv comprises the VL CDRs of SEQ ID NOs: 6-8. In yet another embodiment, the scFV comprises the VL region of SEQ ID NO: 21. In still another embodiment the scFv comprises the VH CDRs of SEQ ID NOs: 3-5. In yet another embodiment, the scFV comprises the VH region of SEQ ID NO: 22. In still a further embodiment, the scFv comprises the VL CDRs of SEQ ID NOs: 6-8 and the VH CDRs of SEQ ID NOs: 3-5. In yet a further embodiment, the scFv comprises the VL region of SEQ ID NO: 21 and the VH region of SEQ ID NO: 22. In one embodiment, the VL chain of SEQ ID NO: 2 and VH chain of SEQ ID NO: 1 are attached via a glycine-serine linker. In one embodiment, the VL region of SEQ ID NO: 21 and VH region of SEQ ID NO: 22 are attached via a glycine-serine linker. In still another embodiment, the glycine-serine linker is a 15-20 amino acid linker. In still another embodiment, the linker is a 15 amino acid glycine serine linker and comprises (Gly4Ser)3. In one embodiment, the (Gly4Ser)3 linker is SEQ ID NO: 23. In one embodiment, the scFv is attached to the one or more intracellular signaling domains with a spacer. In still another embodiment, scFv is attached to the one or more intracellular domains by a spacer that comprises an immunoglobulin constant region. In one embodiment, the immunoglobulin constant region comprises one or more of an IgG hinge, an IgG CH2 and an IgG CH3 domain. In a particular embodiment, the immunoglobulin constant region comprises an immunoglobulin hinge domain. In still another embodiment, the immunoglobulin constant region comprises an immunoglobulin CH3 domain. In still another embodiment, the immunoglobulin constant region comprises an IgG CH2 domain. In still another embodiment, the scFv is attached to the one or more intracellular domains by a spacer that comprises a CD8a domain. In one embodiment, the spacer is attached to the scFV via a glycine-serine linker. In still another embodiment, the linker is a (Gly4Ser)x where X is 1-5. In still another embodiment, the glycine-serine linker is a 15-20 amino acid linker. In still another embodiment, the linker is a 15 amino acid glycine serine linker and comprises (Gly4Ser)3. In one embodiment, the (Gly4Ser)3 linker is SEQ ID NO: 23. In one embodiment the method further comprises introducing one or more additional expression vectors engineered to express one or more additional biological molecules. In one embodiment, the additional one or more molecules comprise IL-12 and/or SANT7 and/or GAL3C. In one embodiment, the one or more additional expression vectors comprise a sequence encoding a single chain polypeptide comprising one IL-12 p35 subunit and one IL-12 p40 subunit joined by a flexible linker. In one embodiment the IL-12 p35 and IL-12 p40 are joined by a (G45)3 linker. In one embodiment the sequence encoding a single chain IL-12 polypeptide encodes a bioactive IL-12 p70 heterodimer. In one embodiment, the expression vector expressing the one or more biologically active agents also comprises a selectable marker. In one embodiment the expression vector comprises a sequence encoding a single chain IL-12 polypeptide comprising IL-12 p35 and IL-12 p40 joined with a flexible linker and a selectable marker joined to the single chain IL-12 with a 2A ribosomal skip. In one embodiment, the one or more biological molecules is SANT7. In one embodiment, the expression vector expressing one or more biologically active agents comprises SANT7 and a selectable marker. In one embodiment the sequence encoding SANT7 and the selectable marker are joined by a 2A ribosomal skip sequence. In one embodiment, the expression vector expressing one or more biologically active agents comprises GAL3c and a selectable marker. In one embodiment, the sequence encoding GAL3C and the selectable marker are joined by a ribosomal skip sequence. In one embodiment, the CAR T-cell comprises IL-12, SANT7 and a selectable marker. In one embodiment, the sequence encoding the IL-12 is linked to the selectable marker via a 2A ribosomal skip and the sequence encoding SANT7 is connected to the sequence encoding IL-12 by an additional 2A ribosomal skip. In one embodiment, the CAR T-cell comprises GAL3C, SANT7 and a selectable marker. In one embodiment, the sequence encoding the GAL3C is linked to the selectable marker via a 2A ribosomal skip and the sequence encoding SANT7 is connected to the sequence encoding GAL3C by an additional 2A ribosomal skip. In one embodiment, the CAR T-cell comprises IL-12, GAL3C and a selectable marker. In one embodiment, the sequence encoding the IL-12 is linked to the selectable marker via a 2A ribosomal skip and the sequence encoding GAL3C is connected to the sequence encoding IL-12 by an additional 2A ribosomal skip. In one embodiment, the CAR T-cell comprises GAL3C, SANT7, IL-12 and a selectable marker. In one embodiment, the sequence encoding each of GAL3C, SANT7, IL-12 and the selectable marker are connected via 2A ribosomal skip sequences.
In one embodiment, a method for treating a KMA-expressing malignancy is provided. In one embodiment, the KMA-expressing malignancy is a B cell malignancy. In a further embodiment, the B-cell malignancy is multiple myeloma, Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), or amyloidosis. In a particular embodiment, the method includes administering to a subject with multiple myeloma, Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), amyloidosis or another B cell malignancy expressing KMA genetically modified T cells engineered to express one or more intracellular signaling domains and an extracellular antigen binding domain that specifically recognizes KMA. In one embodiment, the one or more intracellular signaling domains in the CAR is one or more co-stimulatory endodomains. In a further embodiment, the one or more co-stimulatory domain is one or more of a CD28 domain, a CD3ζ domain, a 4-1BB domain, or an OX-40 domain or combinations thereof. In one embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain and a CD28 domain. In another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, and an OX-40 domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a CD28 domain and an OX-40 domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain and a 4-1BB domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a CD28 domain and a 4-1BB domain. In still another embodiment, the one or more co-stimulatory endodomains of the CAR comprises a CD3ζ domain, a 4-1BB domain and an OX-40 domain. In one embodiment, the extracellular binding domain comprises a single chain variable fragment (scFv) that specifically recognizes KMA. In still another embodiment, the scFv comprises the complemetarity determining regions (CDRs) derived from KappaMab. In still another embodiment the scFv comprises the VL CDRs of SEQ ID NOs: 6-8. In yet another embodiment, the scFV comprises the VL region of SEQ ID NO: 21. In still another embodiment the scFv comprises the VH CDRs of SEQ ID NOs: 3-5. In yet another embodiment, the scFV comprises the VH region of SEQ ID NO: 22. In still a further embodiment, the scFv comprises the VL CDRs of SEQ ID NOs: 6-8 and the VH CDRs of SEQ ID NOs: 3-5. In yet a further embodiment, the scFv comprises the VL region of SEQ ID NO: 21 and the VH region of SEQ ID NO: 22. In one embodiment, the VL chain of SEQ ID NO: 2 and VH chain of SEQ ID NO: 1 are attached via a glycine-serine linker. In one embodiment, the VL region of SEQ ID NO: 21 and VH region of SEQ ID NO: 22 are attached via a glycine-serine linker. In still another embodiment, the linker is a (Gly4Ser)x where X is 1-5. In still another embodiment, the glycine-serine linker is a 15-20 amino acid linker. In still another embodiment, the linker is a 15 amino acid glycine serine linker and comprises (Gly4Ser)3. In one embodiment, the (Gly4Ser)3 linker is SEQ ID NO: 23. In one embodiment, the scFv is attached to the one or more intracellular signaling domains with a spacer. In still another embodiment, scFv is attached to the one or more intracellular domains by a spacer that comprises an immunoglobulin constant region. In one embodiment, the immunoglobulin constant region comprises one or more of an IgG hinge, an IgG CH2 and an IgG CH3 domain. In a particular embodiment, the immunoglobulin constant region comprises an immunoglobulin hinge domain. In still another embodiment, the immunoglobulin constant region comprises an immunoglobulin CH3 domain. In still another embodiment, the immunoglobulin constant region comprises an IgG CH2 domain. In still another embodiment, the scFv is attached to the one or more intracellular domains by a spacer that comprises a CD8α domain. In one embodiment, the spacer is attached to the scFV via a glycine-serine linker. In still another embodiment, the linker is a (Gly4Ser)x where X is 1-5. In still another embodiment, the glycine-serine linker is a 15-20 amino acid linker. In still another embodiment, the linker is a 15 amino acid glycine serine linker and comprises (Gly4Ser)3. In one embodiment, the (Gly4Ser)3 linker is SEQ ID NO: 23. In another embodiment, the genetically modified T cells are further engineered to express one or more additional biological molecules. In one embodiment, the additional one or more molecules comprise IL-12 and/or SANT7 and or GAL3C. In one embodiment, the CAR T-cells express a single chain polypeptide comprising one IL-12 p35 subunit and one IL-12 p40 subunit joined by a flexible linker. In one embodiment the IL-12 p35 and IL-12 p40 are joined by a (G4S)3 linker. In one embodiment the single chain IL-12 polypeptide forms a bioactive IL-12 p70 heterodimer. In one embodiment, the CAR T-cell expresses IL-12 and a selectable marker. In one embodiment, the one or more biological molecules is SANT7. In one embodiment, the CAR T-cell expresses SANT7 and a selectable marker. In one embodiment the selectable marker is GAL3C. In one embodiment, the CAR T-cell expresses GAL3C and a selectable marker. In one embodiment, the CAR T-cell expresses IL-12, SANT7 and a selectable marker. In one embodiment, the CAR T-cell expresses IL-12, GAL3C and a selectable marker. In one embodiment, the CAR T-cell expresses SANT7, GAL3C and a selectable marker. In one embodiment, the CAR T-cell expresses IL-12, SANT7, GAL3C and a selectable marker.
In a further embodiment, the method includes further administering to a patient with multiple myeloma, Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), amyloidosis or another B cell malignancy expressing KMA an HGF binding protein. In one embodiment, the HGF binding protein is an antibody or fragment thereof. In one embodiment, an expression vector comprising the HGF binding protein is co-transfected with the expression vector encoding the CAR construct into a T cell such that the resulting CAR T-cell also expresses the HGF binding protein.
In another embodiment, the method includes administering one or more additional biologically or pharmaceutically active agents. In one embodiment, the one or more additional pharmaceutically active agent is a chemotherapeutic agent. In another embodiment, the one or more pharmaceutically active agent is an immunomodulatory drug. In a particular embodiment, the immunomodulatory drug is thalidomide or an analog thereof. In still another embodiment, the thalidomide analog is actimid, lenalidomide, or pomalidomide. In still another embodiment, the additional pharmaceutically active agent is a histone deacetylase inhibitor. In still another embodiment, the histone deacetylase inhibitor is panobinostat, vorinostat, trichostatin A, depsipeptides, phenylbutyrate, valproic acid, belinostat, LAQ824, entinostat, CI944, or mocetinostat. In still another embodiment, the one or more additional biological or pharmaceutically active agents is administered before, during or after treatment with said genetically modified T cells. In still another embodiment, the genetically modified T cells are administered intravenously. In still another embodiment, the generically modified T cells are derived from said patient. In still another embodiment, the genetically modified T cells are not derived from said patient.
In one embodiment, the CAR T-cells of the current invention are administered before, during or after an allogenic stem cell transplant. In still another embodiment, the the CAR T-cells of the current invention are administered before during or after an allogenic stem cell transplant.
These above-characterized aspects, as well as other aspects, of the present invention are exemplified in a number of illustrated implementations and applications, some of which are shown in the figures and characterized in the claims section that follows. However, the above summary is not intended to describe each illustrated embodiment or every implementation of the invention.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention is obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and material similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following definitions will be used. It will also be understood that the terminology used herein is not meant to be limiting but rather is used herein for the purpose of describing particular embodiments.
The articles “a” and “an” are used herein to refer to one or more than one (i.e. to at least one or to one or more) of the grammatical object of the article.
The term “expression vector” as used herein refers to a vector comprising a recombinant nucleic acid sequence comprising at least one expression control sequence operatively linked to the nucleic acid sequence to be expressed. An expression vector comprises all necessary cis acting elements required for expression. Examples of expression vectors include, but are not limited to, plasmids, cosmids, and viruses that encode the recombinant polynucleotide to be expressed. In some embodiments, the expression vector comprises transposable elements that are capable of integrating into the genome, for example, the PiggyBac expression system. In some embodiments, the expression vector is a viral vector that allows for integration of the expression vector contents into the host genome, for example retroviral and lentiviral vectors.
By “chimeric antigen receptor” or “CAR” is meant an engineered receptor that includes an extracellular antigen binding domain and an intracellular signaling domain. While the most common type of CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a transmembrane and intracellular domain of a T cell co-receptor, such as the CD3ζ chain, the invention described herein is not limited to these domains. Rather, as used herein “chimeric antigen receptor” or “CAR” refers to any receptor engineered to express and extracellular antigen binding domain fused or linked to any intracellular signaling molecule.
As used herein the term “CAR-T cell” refers to a T lymphocyte that has been genetically engineered to express a CAR. The definition of CAR T-cells encompasses all classes and subclasses of T-lymphocytes including CD4−, CD8+ T cells as well as effector T cells, memory T cells, regulatory T cells, and the like. The T lymphocytes that are genetically modified may be “derived” or “obtained” from the subject who will receive the treatment using the generically modified T cells or they may “derived” or “obtained” from a different subject.
By “intracellular signaling domain” is meant the portion of the CAR that is found or is engineered to be found inside the T cell. The “intracellular signaling domain” may or may not also contain a “transmembrane domain” which anchors the CAR in the plasma membrane of a T cell. In one embodiment, the “transmembrane domain” and the “intracellular signaling domain” are derived from the same protein (e.g. CD3ζ) in other embodiments; the intracellular signaling domain and the transmembrane domain are derived from different proteins (e.g. the transmembrane domain of a CD3ζ and intracellular signaling domain of a CD28 molecule, or vice versa).
By “co-stimulatory endodomain” is meant an intracellular signaling domain or fragment thereof that is derived from a T cell costimulatory molecule. A non-limiting list of T cell costimulatory molecules include CD3, CD28, OX-40, 4-1BB, CD27, CD270, CD30 and ICOS. The co-stimulatory endodomain may or may not include a transmembrane domain from the same or different co-stimulatory endodomain.
By “extracellular antigen binding domain” is meant the portion of the CAR that specifically recognizes and binds to the antigen of interest. The “extracellular binding domain” may be derived from a monoclonal antibody. For example, the “extracellular binding domain” may include all or part of an Fab domain from a monoclonal antibody. In certain embodiments, the “extracellular binding domain” includes the complementarity determining regions of a particular monoclonal antibody. In still another embodiment, the “extracellular binding domain” is a single-chain variable fragment (scFv).
By “single-chain variable fragment” or “scFv” is meant a fusion protein of the variable heavy (VH) and variable light (VL) chains of an antibody with a peptide linker between the VL and VH. The linker length and composition vary depending on the antibody portions used, but generally are between about 10 and about 25 amino acids in length. In some embodiments, the peptide linker is a glycine rich to provide for flexibility. In some embodiments, the linker also includes serine and/or threonine which may, without being bound by theory, aid in solubility. In some embodiments, the linker is an amino acid with SEQ ID NO: 23. ScFvs are designed to retain the antigen binding specificity of the parent antibody from which their variable chains were derived despite lacking the immunoglobulin heavy chain. In some embodiments, only the complementary determining regions (CDRs) from the VH and VL are used in the scFV. In some embodiments, the entire VL and VH chains are used.
The term “antibody” as used herein refers to an immunoglobulin molecule which specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). As used herein the term “antibody” also encompasses antibody fragments.
The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.
An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two major antibody light chain isotypes.
As used herein the term “complementarity determining region” or “CDR” refers to the part of the two variable chains of antibodies (heavy and light chains) that recognize and bind to the particular antigen. The CDRs are the most variable portion of the variable chains and provide the antibody with its specificity. There are three CDRs on each of the variable heavy (VH) and variable light (VL) chains and thus there are a total of six CDRs per antibody molecule.
By “KappaMab” is meant the monoclonal antibody previously termed IST-1097 or MDX-1097. Furthermore, as used herein KappaMab may refer to the full antibody sequence of the KappaMab antibody (See e.g. U.S. Pat. Nos. 7,344,715 and 7,556,803 each of which are hereby incorporated by reference in their entireties.) Additionally, the term “KappaMab” as used herein is used to encompass any polypeptide containing the CDR sequences of SEQ ID NOs: 3-8 and/or the VL sequence of SEQ ID NO: 2 and the VH sequence of SEQ ID NO: 1. The term “KappaMab” as used herein can encompass any polypeptide containing the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 22. In the compositions and methods of the current invention, KappaMab may include the full monoclonal antibody or any antigen binding fragment thereof including Fab and scFv.
The term “antigen” or “Ag” as used herein is defined as a molecule that is recognized by an immune cell receptor (e.g. a T cell receptor, B cell receptor/Immunogloblulin). In some embodiments, an antigen is a molecule that elicits an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, release of cytotoxic mediators or immunostimulatory or regulatory cytokines. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
As used herein the term “specifically binds” or “specifically recognizes” as used in connections with an antibody, antibody fragment or CAR refers to a an antibody, antibody fragment or CAR which recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample.
By “ribosomal skip” is meant an alternative mechanism of translation in which a specific peptide prevents the ribosome of a cell from covalently linking a new inserted amino acid and instead allows it to continue translation thus resulting in a co-translational cleavage of the polyprotein. This process is induced by a “2A ribosomal skip” element or cis-acting hydrolase element (e.g. CHYSEL sequence). In some embodiments, this sequence comprises a non-conserved amino acid sequence with a strong alpha-helical propensity followed by the consensus sequence −D(V/I)ExNPG P, where x=any amino acid. The apparent cleavage occurs between the G and P. In some embodiments, the ribosomal skip element is a 2A ribosomal skip element. The 2A ribosomal skip element can be a 5′ T2A ribosomal skip element.
As used herein “immunomodulatory drug” or “IMiD” is a class of drugs that constitute thalidomide and its analogs. Thalidomide analogs include lenalidomide, pomalidomide and apremilast.
As used herein the term “histone deacetyalse inhibitor” or “HDAC inhibitor” or “HDI” refers to a class of compounds that interferes with the function of histone deacetylase. Examples of HDIs include, but are not limited to, hyroxamic acids including, for example, trichostatin A, vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (LBH589); cyclic tripeptides, including for example, depsipeptides and tapoxin B; benzamides, including for example, entinostat (MS-275), CI994 and mocetinostat (MGCD0103); electrophilic ketones; and aliphatic compounds, such as for example, phenylbutyrate and valproic acid.
Kappa Myeloma Antigen and Antibodies
Kappa myeloma antigen or KMA is a cell membrane antigen that is found on the surface of myeloma cells. Specifically, KMA consists of free kappa light chains expressed in non-covalent association with actin on the cell membrane (Goodnow et al. (1985) J. Immunol. 135:1276). While any antibody that specifically binds to KMA may be used in accordance with the present invention, in a preferred embodiment the KappaMab monoclonal antibody will be used as a basis for the extracellular antigen binding domain of the CARs of the current invention. The monoclonal antibody designated KappaMab (formally designated IST-1097, also known as MDX-1097) binds to a conformational epitope in the switch region of human kappa free light chain that is only available when the kappa chain is not associated with a heavy chain and therefore does not bind to intact kappa-chain containing IgG, IgM, IgE or IgA (Hutchinson et al. (2011) Mol. Immunol.). Typical expression of KMA on primary myeloma cells derived from patient bone marrow biopsies is shown by KappaMab binding in
Chimeric Antigen Receptors
Chimeric antigen receptors (CARs) are artificial receptors consisting of the tumor antigen binding regions of monoclonal antibodies and the intracellular activating portion of the T cell receptor complex in a single polypeptide chain held together by a series of linker(s) and spacer(s) (
I. Extracellular Antigen Binding Domain
In one embodiment, the CAR of the current invention comprises an extracellular antigen binding domain from a monoclonal antibody that is specific for one or more KMA epitopes expressed on MM cells. In one embodiment, the CAR of the current invention comprises an extracellular antigen binding domain from KappaMab. In one embodiment, the extracellular binding domain comprises the VL CDRs of SEQ ID NOs: 6-8 and VH CDRs of SEQ ID NOs: 3-5. In a particular embodiment, the extracellular binding domain is a scFv comprising the VL (SEQ ID NO: 2) and VH (SEQ ID NO: 1) domains of KappaMab. In another embodiment, the extracellular binding domain is a scFv comprising the VL (SEQ ID NO: 21) and VH (SEQ ID NO: 22) domains of KappaMab.
II. Linker Between VL and VH Domains of KappaMab scFv
In a further embodiment, the KappaMab VL is linked to the KappaMab VH via a flexible linker. Specifically, the flexible linker is a glycine/serine linker of about 10-30 amino acids (for example 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids) and comprises the structure (Gly4Ser)3. In a particular embodiment the linker is 15 amino acids in length. Linker length is an important determinant of a CAR. Without being bound by theory, shorter linkers may enhance affinity but can also lead to intracellular multimer formation thus impairing expression of the CAR whereas longer linkers tend to decrease antigen affinity by moving the VL and VH CDRs further apart in space.
III. Spacers Between Extracellular Antigen Binding Domain and Intracellular Signaling Domain
The extracellular antigen binding domain (e.g. KappaMab scFv) is linked to the intracellular signaling domain by the use of a “spacer”. The spacer is designed to be flexible enough to allow for orientation of the antigen binding domain in such a way as facilitates antigen recognition and binding. The spacer may derive from immunoglobulins themselves and can include the IgG1 hinge region or the CH2 and/or CH3 region of an IgG. Alternatively, the hinge may comprise all or part of a CD8a chain. The length and flexibility of the spacer(s) is dependent on both the antigen recognition domain as well as the intracellular binding regions and what may be functional and/or optimal for one CAR construct may not be for another CAR. In certain instances the spacer may be designated herein as “opti” (See
IV. Intracellular Signaling Domain
The intracellular signaling domain comprises all or part of the CD3ζ chain. CD3ζ, also known as CD247, together with either the CD4 or CD8 T cell co-receptor is responsible for coupling extracellular antigen recognition to intracellular signaling cascades. In one embodiment, the CD3ζ used in any of the constructs provided herein is SEQ ID NO: 26.
In addition to the including of the CD3C signaling domain, the inclusion of co-stimulatory molecules has been shown to enhance CAR T-cell activity in murine models and clinical trials. Several have been investigated including CD28, 4-1BB, ICOS, CD27, CD270, CD30 and OX-40. The CAR of the current invention, in addition to including the KappaMab scFv, flexible linker, optimal hinge and CD3C chain also include one or more additional costimulatory domains from CD28, 4-1BB, ICOS, CD27, CD270, CD30 and/or OX-40, for example. These co-stimulatory domains are selected based on the desired functionality of the resulting CAR T-cell. Exemplary combinations are shown, for example, in
Co-Expression of Biologically Active Molecules
The CAR T-cells of the current invention have the added benefit, when compared to the use of the KappaMAb alone to be further modifiable to contain additional biologically active molecules to enhance the anti-tumor function and/or safety of the compositions. In one embodiment, the CAR T-cells may be further genetically modified to produce antitumor cytokines which allow for focused delivery to the tumor microenvironment/cancer cells, while avoiding systemic toxicity. Examples of additional biologically active molecules which may enhance the anti-tumor response of the CAR T-cells of the current invention include, without limitation, IL-12, the carbohydrate binding protein Galectin-3 (GAL3) or it's truncated form, GAL3C, and the cytokine receptor super antagonist SANT7. In another embodiment, CAR T-cells of the current invention may also be co-transduced with a plasmid that expresses a hepatocyte growth factor (HGF) binding protein. In one embodiment, the hepatocyte growth factor protein is an antibody or fragment thereof that is able to bind to and inhibit the function of HGF.
IL-12 is a potent tumor suppressor cytokine, decreasing tumor growth and angiogenesis and enhancing the tumor specific immune response. Multiple myeloma cells retain expression of the IL-12 receptor and administration of IL-12 to myeloma bearing mice decreases tumor progression as a single agent and acts synergistically with the proteasome inhibitor bortezomib (Airoldi, et al. (2008) Blood, 112(3):750-759; Wang, et al. (2014) Anticancer Drugs, 25(3): 282-288). Expression of IL-12 by CAR T-cells dramatically enhances their ability to eradicate solid tumors but this approach has not yet been investigated in multiple myeloma (Pegram, (2012) Blood, 119(180:4133-4141 and Zhang, et al. (2011) Mol. Ther. 19(4):751-759).
SANT7 is a cytokine receptor super-antagonist. It is an analogue of IL-6 that has been genetically modified to enhance its binding to the IL-6 receptor α-subunit 70-fold, with virtually no interaction with the gp130 signaling subunit. SANT7 induces apoptosis in IL-6 dependent myeloma cell lines in-vitro, overcomes stroma mediated resistance to dexamethasone in in-vitro and murine model and combined with NFκB inhibitors, completely overcomes resistance to apoptosis. IL-6 is a cytokine which plays a role in the growth and survival of a variety of tumors including multiple myeloma, lung cancer, colorectal cancer, breast cancer and others. Binding of IL-6 to its receptor activates the JAK-STAT pathway, with subsequent phosphorylation of STAT3 which modulates expression of apoptosis related genes such as BCL-XL and p53, causing resistance to apoptosis. IL-6 also promotes down-regulation of the IL-12 receptor on myeloma cells, decreasing IL-12's tumor suppressive properties. (Airoldi, et al. (2008) Blood, 112(3):750-759). The IL-6 receptor is upregulated in myeloma and elevated systemic levels of IL-6 correlate with a poor prognosis. (Rawstron, et al. (2000) Blood 96(12) 3880-3886; Ludwig, et al. (1991) Blood, 77(12):2794-2795). Monoclonal antibodies to IL-6 have been developed for clinical use, however, although early clinical trials in myeloma showed measurable biological effects, the antibodies appeared to form complexes with circulating IL-6, leading to reduced clearance and potentially limiting their efficacy. (Bataille, et al. (1995) Blood, 86(2): 685-691). Recently, the chimeric IL-6 specific monoclonal antibody Siltuximab has been assessed in phase I and II clinical trials in relapsed and refractory multiple myeloma. There were no responses to Siltuximab alone, but hematological toxicity was common with more than half experiencing therapy related infections.
Galectin-3 is a carbohydrate binding protein which may play a role in tumour adhesion and invasion. A truncated form of Galectin-3, Gal3C, acts as a dominant negative form and can inhibit myeloma cell growth and invasion. A Gal3C construct for activation inducible secretion was designed based on John et al (2003) Clin Cancer Res., 9(6):2374-83 and Mirandola et al. (2011) PLoS One, 6(7):e21811. This consists of the 143 amino acid carboxy terminal which retains its carbohydrate binding properties, but lacks the N-terminal amino acids required for ligand crosslinking. The construct also contains the CD8-alpha leader peptide to direct secretion and a 6× His tag for detection.
In certain embodiments, in addition to expression vectors containing the CAR construct described above, T cells are further modified with one or more expression vectors comprising IL-12, SANT7 and/or GAL3C. Specifically, expression constructs expressing a single chain IL-12 comprising the IL-12 p35 subunit linked to the IL-12 p40 subunit are particularly useful in that the resulting protein is a fully bioactive IL-12 p70 heterodimer, however, expressed as a single polypeptide. In one embodiment, the single chain IL-12 construct, termed Flexi-12, is described, for example in Anderson, et al. (1997) Hum. Gene Ther. 8(9):1125-35 is used. The IL-12 single chain construct may be expressed in the same expression vector as the CAR construct or it may expressed in a separate expression vector and co-transduced into the T cell. Similarly, T cells transduced with the CAR construct described above, may be co-transduced with an additional expression vector comprising SANT7 and/or GAL3C, alternatively, one expression vector may be used to transduce T cells with both of SANT7 and GAL3C either alone or in combination and the CAR construct described above. In another embodiment three expression vectors may be used, one expressing the CAR construct, one expressing the single chain IL-12 construct and one expressing the SANT7 construct. A similar strategy may be used to co-express GAL3C with IL-12 and/or SANT7. Alternatively, the IL-12, GAL3C and/or SANT7 construct may be expressed via a single expression vector while the CAR construct is expressed by its own expression vector. One of skill in the art will appreciate the different combinations and possibilities for expressing these molecules in the same T cell.
HGF Binding Protein
Hepatocyte growth factor (HGF) and its receptor, MET have been implicated in cancer development and progression, in particular in tumor invasion and progression to metastatic disease. Multiple myeloma cells express both HGF and MET, thus creating both an autocrine and paracrine loop whereas normal plasma cells do not express HGF (Zhan et al. (2002); Borset, et al. (1996). Furthermore, HGF concentrations are significantly increased in the blood and bone marrow of plasma patients with multiple myeloma and high serum HGF levels correlate with advanced stage disease and extensive bone lesions (Seidel et al. (1998); Wader, et al. (2008); Alexandrakis, et al. (2003). Furthermore, serum biomarker analysis of patients in a phase I trial with KappaMab shows statistically significant dose related decrease in serum HGF after treatment with KappaMab compared to control. In order to enhance this reduction in serum HGF, in certain embodiments an HGF binding protein will be expressed in the CAR T-cells of the current invention. In a particular embodiment, the HGF binding protein expressed is an antibody or fragment thereof. In a particular embodiment, the anti-HGF binding protein is an antibody, a diabody, a scFv or an Fab. In one embodiment, the HGF binding protein is expressed in the same expression vector as the CAR construct. In a further embodiment, the HGF binding protein is expressed in a separate expression vector but is co-transduced with the CAR construct. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and IL-12. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and SANT7. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and GAL3C. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and IL-12 and GAL3C. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein and SANT7 and GAL3C. In still another embodiment, the CAR-T cell expresses the CAR, an anti-HGF binding protein, IL-12 and SANT7. In still a further embodiment, the CAR-T cell expresses the CAR, an HGF binding protein, IL-12, SANT7 and GAL3C.
Methods of Producing the CAR T-Cells of the Present Invention
In one aspect, methods are provided for generating CAR T-cells expressing the CAR(s) described herein and optionally one or more anti-tumoral cytokine (e.g. IL-12 and/or SANT7) and/or one or more HGF binding protein. One of skill in the art will readily understand that while preferred methods of constructing expression vectors containing the CARs and anti-tumoral cytokines/antibodies of the present invention are described herein, that any methods which are able to transduce T cells to express these constituents may be used.
In one embodiment, T cells are obtained from the blood of a subject by venous puncture, aspiration of bone marrow, steady state leukapheresis or cytokine primed leukapheresis and subsequent isolation of peripheral blood mononuclear cells including T cells using density gradient separation. In certain embodiments, after lysing red blood cells, T cells are sorted by flow cytometry or purified using antibodies to antigens expressed on T cells and magnetic beads to obtain a population of pure T cells. In a particular embodiment, T cells are sorted based on their expression of CD3 to obtain a whole T cell fraction. In another embodiment T cells are sorted based on their expression of CD4 or CD8 to obtain a population of either CD4+ T cells or CD8+ T cells. In a particular embodiment, T cells are obtained from the subject in need of CAR T-cell therapy. In another embodiment, T cells are obtained from a donor subject who is not the intended recipient of CAR T-cell therapy.
In one embodiment, separated T cells are cultured in vivo under conditions suitable for their survival and are transduced with expression vectors containing the sequences necessary for expression of the CARs described herein and/or IL-12, SANT7, GAL3C and/or an HGF binding protein. In one embodiment, the expression vector is a transposable vector expression system. In a particular embodiment, the expression vector is a PiggyBac transposon expression plasmid or a viral vector (e.g. retroviral vector or lentiviral vector). In one embodiment, the PiggyBac transposon expression plasmid is inducible such as, for example, the PiggyBac transposon plasmid described in the Examples provided herein. In one embodiment, the PiggyBac transposon expression plasmid comprises a constitutively active promoter and/or an activation inducible promoter. The constitutively active promoter can be an elongation factor 1 alpha (EF1alpha) promoter. The activation inducible promoter can be a (NFAT pro) promoter. In one aspect, a PiggyBac expression plasmid is used and produces permanent integration of the CAR by cutting and pasting the CAR, IL-12, SANT-7, GAL3C and/or HGF binding protein coding sequences into the T cell's genome. In a particular embodiment, the expression vectors of the current invention further comprise a detectable marker which allows for identification of T cells that have been successfully transduced with the one or more expression vectors. In one embodiment, the detectable marker is chosen from the group consisting of a cell surface marker such as CD34 or CD20 or another surface protein, a fluorophore such as fluorescein isothiocyanate or any other fluorescent dye that emits light when excited to a higher energy state including by a laser, and an antibiotic resistance cassette such as kanamycin resistance, ampicillin resistance or any other cassette that confers resistance to an antibiotic substance contained in medium in which transduced T cells are to be cultured. In one embodiment, the detectable marker is a green fluorescence protein (GFP). The GFP can be an enhanced GFP, such as, for example, the constructs shown in the Examples provided herein. In a particular embodiment, each expression vector used (e.g. one expression vector comprising a CAR, and one comprising an IL-12, GAL3C and/or SANT-7 and one comprising an HGF binding protein) comprises a unique detectable marker. In one embodiment, the expression vectors are transduced into the T cell by a method suitable for the expression vector(s) selected. In one embodiment, the PiggyBac expression vector is transduced into T cells by electroporation.
After introduction of the appropriate expression vectors, T cells may be cultured and expanded in vitro by co-culture with autologous peripheral blood mononuclear cells (PBMCs) and the appropriate growth factors and further screened for the presence of the one or more detectable markers. T cells expressing the appropriate detectable markers for the expression vectors chosen may then be sorted and purified for use in the methods of the current invention.
Methods of Treating KMA-Expressing Malignancies
In one aspect, methods are provided for treating subjects in need thereof with the CAR T-cells provided herein. In a particular aspect, the subject in need thereof is a human subject who has been diagnosed with or is suspected of having a malignancy that expresses KMA, for example a B cell malignancy expressing KMA. In certain embodiments, a patient has or is suspected of having multiple myeloma (MM), Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), or amyloidosis. Methods for diagnosing B cell malignancies expressing KMA, for example, multiple myeloma (MINI) Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), and amyloidosis are known in the art, and as such are not described in detail herein. The CAR T-cells may be used alone or in combination with other therapeutically effective agents for the treatment of multiple myeloma (MM) Waldenstroms macroglobulinemia, diffuse large B cell lymphoma (DLBCL), amyloidosis or another B cell malignancy expressing KMA. In certain aspects, the CAR T-cells of the current invention are administered in a pharmaceutical formulation suitable for intravenous delivery.
In certain aspects, the CAR T-cells of the current invention are administered before, during or after one or more immunomodulatory drugs. In a particular aspect, the one or more immunomodulatory drugs is thalidomide or a thalidomide analog such as, for example, lenolidomide or pomalidomide.
In certain aspects of the invention, the CAR T-cells of the current invention act synergistically when administered with one or more immunomodulatory drugs.
In a further embodiment, the CAR T-cells of the current invention are administered before, during or after treatment with one or more histone deacetylase inhibitors such as panobinostat, vorinostat, trichostatin A, depsipeptides, phenylbutyrate, valproic acid, belinostat, LAQ824, entinostat, CI944 or mocetinostat.
In certain aspects of the invention, the CAR T-cells of the current invention act synergistically when administered in combination with one or more histone deacetylase inhibitors.
In certain aspects of the invention, the CAR T-cells of the current invention act synergistically when administered in combination with intermediate or high dose chemotherapy and following administration of autologous or allogenic human blood stem cells.
In one embodiment, the CAR T-cells of the current invention are administered before, during or after an allogenic stem cell transplant. In still another embodiment, the CAR T-cells of the current invention are administered before during or after an allogenic stem cell transplant. Without being bound by theory, the CAR T-cells of the present invention, when administered in combination with an autologous or allogeneic stem cell transplant prevent the appearance of minimal residual disease that may occur by incomplete ablation of the bone marrow prior to stem cell transplant or by reemergence of malignant B cell clones expressing KMA.
All patents, patent applications, and publications cited herein are expressly incorporated by reference in their entirety for all purposes.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for the purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can using the preceding description and following examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.
Based on the nucleotide sequence coding for the variable regions of KappaMab (SEQ ID NOS: 9 and 10), a scFv was designed and cloned into a CAR construct containing an immunoglobulin heavy chain hinge, a CD28 co-stimulatory domain and the CD3-zeta endodomain (
This amino acid sequence (SEQ ID NO: 27) is encoded by the following DNA sequence:
In terms of constructing this construct, a gene sequence consisting of a 5′ EcoRI restriction enzyme site, a 5′ Kozak sequence, Leader Peptide, single chain variable fragment and a portion of the IgG1 constant region incorporating an AleI restriction enzyme site, was synthesised by GeneArt (ThermoFisher Scientific), sequence verified and then cloned into the pIRII-CAR.CD19-28z PiggyBac transposon expression plasmid. This was then introduced into donor T-cells from 2 normal donors by co-electroporation with the PiggyBac Transposase plasmid to mediate stable integration. The PiggyBac transposon/transposase system produces permanent integration of the CAR by cutting and pasting the gene of interest into the target cell genome. The PiggyBac expression system was chosen because it is capable of producing high levels of permanent genetic modification at a fraction of the cost of retroviral vectors. However, one of skill in the art will understand that other expression systems, including retroviral vectors could also be used in accordance with the current invention.
The KM.CAR-hCH2CH3-28z expressing T-cells were expanded according to our optimised protocols by co-culturing with autologous peripheral blood mononuclear (PBMC) feeder cells supplemented with interleukin-15 (IL-15) 10 ng/ml. After culturing for 3 weeks with replacement of PBMCs on a weekly basis and replenishment of IL-15 two to three times per week, T-cells were harvested and assessed for phenotype and CAR expression by flow cytometry, KMA-specific function by interferon gamma intracellular cytokine flow cytometry on stimulation with KMA+ and KMA− cell lines (
At the end of 3 weeks, the cultures were predominantly CAR expressing CD3+ T-cells (55% and 70% of live cells), expressed interferon-gamma in response to KMA+ myeloma and B-cell lines (
A human myeloma to mouse xenotransplant model of multiple myeloma was established. RPMI8226 or alternative myeloma cell lines were inoculated i.v. into Rag2−/− γc−/− (BALB/c) mice to form the Rag MM model (
Based on the construct described in Example 1, 6 CAR constructs containing the KM scFv described in Example 1 with variable length spacer regions and co-stimulatory endodomains (e.g., CD28 or 4-1BB (CD137-Uniport Q07011)) with the CD3 zeta endodomain were constructed (
3 of the 6 KM.CAR constructs contained aCD28 Costimulatory Endodomain and were as follows:
The first construct of this group was the KM.CAR_hCH3 28z construct, which contains only the hinge and CH3 domains of IgG1 heavy chain constant region as the spacer and whose nucleic acid sequence is as follows:
CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTG
GGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGC
ACTGATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCA
CAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAG
TCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTA
CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAG
GGTGGCGGTGGCTCGG
GCGGTGGTGGGTCGGGTGGCGGCGGATCT
GAGGTGCAGCTGCAGCAGTCA
GGGGCGGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGC
TTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGGC
CTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAAC
ACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAATAGCAGACAC
ATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACA
CTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGAC
TACTGGGGCCAAGGGACCACGCTCACCGTCTCCTCC
GGTGGAGGCGGGTC
TGGGGGCGGAGGTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACA
AAACTCACACATGCCCAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG
GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
AGAGTGAAGTTCAGCAGGAG
CGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAG
CTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG
GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGA
GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC
TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA
CATGCAGGCCCTGCCCCCTCGC
.
From 5′ to 3′, this construct (SEQ ID NO: 29) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker, a KappaMab heavy chain variable region, a second (G4S)3 linker, an IgG1 hinge & CH3 constant region domains, a CD28 transmembrane and intracellular domains, and a CD3 zeta intracellular domain. A diagram of this construct is shown in
The second construct of this group is the KM.CAR_h_28z construct, which contains only the hinge domain of IgG1 heavy chain constant region as the spacer, and whose nucleic acid sequence is as follows:
CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTG
GGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGC
ACTGATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCA
CAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAG
TCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTA
CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAG
GGTGGCGGTGGCTCGG
GCGGTGGTGGGTCGGGTGGCGGCGGATCT
GAGGTGCAGCTGCAGCAGTCA
GGGGCGGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGTACAGC
TTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGGC
CTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAAC
ACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAATAGCAGACAC
ATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACA
CTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGGGAC
TACTGGGGCCAAGGGACCACGCTCACCGTCTCCTCC
GGTGGAGGCGGGTC
TGGGGGCGGAGGTTCAGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACA
AAACTCACACATGC
AGAGTGAAGT
TCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCT
CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC
AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA
ACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGA
GGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG
CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG
ACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
.
From 5′ to 3′, this construct (SEQ ID NO: 30) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker, a KappaMab heavy chain variable region, a second (G45)3 linker, an IgG1 hinge constant region domain, a CD28 transmembrane and intracellular domains, and a CD3 zeta intracellular domain. A diagram of this construct is shown in
The third construct of this group was the KM. CAR_CD8a_28z construct, which contains a CD8 alpha stalk (Uniprot P01732, amino acids 138-182) as the spacer, and whose nucleic sequence is as follows:
CCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAAT
GTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAA
AGCACTGATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCT
TCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTG
CAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCC
GTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAG
GGTGGCGGTGGCT
CGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT
GAGGTGCAGCTGCAGCAG
TCAGGGGCGGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGTAC
AGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGA
GGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGT
AACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAATAGCAGA
CACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG
ACACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGG
GACTACTGGGGCCAAGGGACCACGCTCACCGTCTCCTCC
ACCACGACGCC
AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGT
CCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACG
AGGGGGCTGGACTTCGCCTGTG
AGAGTGAAGTTCAGCAGG
AGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACG
AGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACG
TGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAG
GAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA
GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGG
CCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTT
CACATGCAGGCCCTGCCCCCTCGC
.
From 5′ to 3′, this construct (SEQ ID NO: 31) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker, a KappaMab heavy chain variable region, a CD8 alpha stalk, a CD28 transmembrane and intracellular domains, and a CD3 zeta intracellular domain.
The remaining 3 constructs of the 6 KM.CAR constructs described in this example contained a 4-1BB (CD137) Costimulatory Endodomain and were as follows:
The first construct of this group is KM.CAR_h_28TM_41BBz, which contains only the hinge domain of IgG1 heavy chain constant region as the spacer and replaces the intracellular domain of CD28 with the intracellular domain of the 4-1BB co-stimulatory molecule, and whose nucleic acid sequence is as follows:
TCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGA
ATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCC
TAAAGCACTGATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGAT
CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA
ATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAG
CTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAG
GGTGGC
GGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT
GAGGTGCAGC
TGCAGCAGTCAGGGGCGGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTT
GTCCTGTACAGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGG
GTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATC
CTGCGAATGGTAACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCAC
TATAATAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGC
CTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGGGGGGTCTACC
ATGATTACGACGGGGACTACTGGGGCCAAGGGACCACGCTCACCGTCTC
CTCC
GGTGGAGGCGGGTCTGGGGGCGGAGGTTCAGGCGGGGGTGGTTCC
GAGCCCAAATCTCCTGACAAAACTCACACATGCCCA
AGAGTGAAGTTCAGCAGGAGCGCA
GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCT
CAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG
GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAG
GAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA
CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG
ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC
GCCCTTCACATGCAGGCCCTGCCCCCTCGC
.
From 5′ to 3′, this construct (SEQ ID NO: 32) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker, a KappaMab heavy chain variable region, a second (G4S)3 linker, an IgG hinge constant region domain, a CD28 transmembrane domain, a 4-IBB intracellular domain, and a CD3 zeta intracellular domain.
The second construct of this group was KM.CAR_8a_28TM_41BBz, which contains the CD8 alpha stalk (Uniprot P01732, amino acids 138-182) as the spacer and replaces the intracellular domain of CD28 with the intracellular domain of the 4-1BB co-stimulatory molecule, and whose nucleic sequence is as follows:
CCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAAT
GTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAA
AGCACTGATTTACTCGACATCCTACCGGTACAGTGGAGTCCCTGATCGCT
TCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTG
CAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCC
GTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAG
GGTGGCGGTGGCT
CGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT
GAGGTGCAGCTGCAGCAG
TCAGGGGCGGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGTAC
AGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGA
GGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGT
AACACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAATAGCAGA
CACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGG
ACACTGCCGTCTATTACTGTGCTAGGGGGGTCTACCATGATTACGACGGG
GACTACTGGGGCCAAGGGACCACGCTCACCGTCTCCTCC
ACCACGACGCC
AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGT
CCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACG
AGGGGGCTGGACTTCGCCTGTGAT
GAAGTTCAGCAGGAGCGCAGA
CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAA
TCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCG
GGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG
CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCT
TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCA
CATGCAGGCCCTGCCCCCTCGC
.
From 5′ to 3′, this construct (SEQ ID NO: 33) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker, a KappaMab heavy chain variable region, a CD8 alpha stalk, a CD28 transmembrane domain, a 4-IBB intracellular domain, and a CD3 zeta intracellular domain.
The third construct of this group is KM. CAR_hCH2CH3mut_28TM_41BBz, which contains the hinge, CH2 and CH3 domains of IgG1 heavy chain constant region as the spacer, with mutations introduced at amino acids important for CH2 interaction with Fc-receptors (3-6) which may mediate reduced CAR T-cell survival in-vivo (3, 6, 7) by clearance of CAR T-cells in the reticuloendothelial system. The nucleic acid sequence is as follows:
GTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCA
GAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATC
TCCTAAAGCACTGATTTACTCGACATCCTACCGGTACAGTGGAGTCCC
TGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT
CAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATA
TAACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA
G
GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT
GA
GGTGCAGCTGCAGCAGTCAGGGGCGGAGCTTGTGAAGCCAGGGGCCTC
AGTCAAGTTGTCCTGTACAGCTTCTGGCTTCAACATTAAAGACACCTA
TATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGG
AAGGATTGATCCTGCGAATGGTAACACTAAATATGACCCGAAGTTCCA
GGGCAAGGCCACTATAATAGCAGACACATCCTCCAACACAGCCTACCT
GCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGC
TAGGGGGGTCTACCATGATTACGACGGGGACTACTGGGGCCAAGGGAC
CACGCTCACCGTCTCCTCC
GGTGGAGGCGGGTCTGGGGGCGGAGGTTC
AGGCGGGGGTGGTTCCGAGCCCAAATCTCCTGACAAAACTCACACATG
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT
CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGC
CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA
ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCC
GGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG
GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTC
AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC
TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTC
ACATGCAGGCCCTGCCCCCTCGC
.
From 5′ to 3′, this construct (SEQ ID NO: 34) has a leader peptide, a KappaMab light chain variable region, a (G4S)3 linker, a KappaMab heavy chain variable region, a second (G4S)3 linker, a mutated IgG1 hinge, CH2 and CH3 constant region domains, a CD28 transmembrane domain, a 4-IBB intracellular domain, and a CD3 zeta intracellular domain. The mutated IgG1 hinge domain has, from 5′ to 3′, E233P, L234V, L235A, G236-, S254A, D265N, and N297A mutations highlighted within the shaded boxes of this construct (SEQ ID NO: 34). Mutations at these sites (E233P, L234V, L235A, G236-, S254A, D265N, N297A) may decrease Fc interaction with CAR T-cells, allowing improved survival post-infusion.
Addition of 2A ribosomal skip element and eGFP to KM.CARs
For ease of detection of T-cells expressing each of the CARs described above, an eGFP with a 5′ T2A ribosomal skip element with overlapping sequences was synthesized with the CAR-CD3 zeta endodomain and the plasmid backbone. This was then cloned by restriction enzyme digestion and ligation into the CAR containing pVAX1 PB transposon plasmids to create the following—
28z Endodomain_2A_GFP Containing Constructs:
1. pVAX1PB KM.CAR_hCH2CH3_28z_2A_GFP
2. pVAX1PB KM.CAR_hCH3_28z_2A_GFP
3. pVAX1PB KM.CAR_h_28z_2A_GFP
4. pVAX1PB KM.CAR_8a_28z_2A_GFP
41BBz Endodomain_2A_GFP containing constructs:
1. pVAX1PB KM.CAR_h_28TM_41BBz_2A_GFP
2. pVAX1PB KM. CAR_8a_28TM_41BBz_2A_GFP
3. pVAX1PB KM.CAR_hCH2CH3mut_28TM_41BBz_2A_GFP
Generation of KM.CAR T-cells with 4-1BB costimulatory domain.
Comparison was made between the preliminary KM.CAR_hCH2CH3_28z and the 4-1BB containing CARs. KM.CAR T-cells were generated by electroporation using the PiggyBac system as previously described herein and in the art (2). Four million peripheral blood mononuclear cells (PBMCs) from healthy donors were electroporated with the Neon electroporation system at 2400V for 20 ms, single pulse, in the presence of 5 ug each of PiggyBac transposase and PiggyBac Transposon plasmids. KMA.CAR constructs tested included KM.CAR_hCH2CH3_28z_2A_GFP; KM.CAR_h_28TM_41BBz_2A_GFP; KM.CAR_8a_28TM_41BBz_2A_GFP; or KM.CAR_hCH2CH3mut_28TM_41BBz_2A_GFP.
Electroporated PBMCs (CAR-PBMCs) were rested overnight in AIMV with 10% Fetal calf serum (AIM-V CM), harvested, washed and resuspended in AIM-V CM at 1×106/ml. CAR-PBMCs were cocultured with autologous irradiated PBMC feeder cells with or without irradiated KMA expressing JJN3 cells at a CAR-PBMC:JJN3 ratio of 5:1. Interleukin-15 (IL-15) was added at 10 ng/m1 every 3 days. Cells were enumerated by trypan blue exclusion and fresh irradiated stimulator/feeder cells were added every 7 days.
Assessment of KM.CAR Expression
KM.CAR expression was assessed by flow cytometry at initiation of culture (Day 1), Day 15 and Day 21 (
KM.CAR T-Cells Require Kappa Myeloma Antigen to Persist In-Vitro
Cultures containing the KMA expressing JJN3 cell line showed either greater total expansion, increased KM.CAR expression or both, compared to cultures with PBMC alone (
KM.CAR_hCH2CH3mut_28TM_41BBz expressing T-cells showed only low level CAR expression (6%) and expansion (6-fold) with PBMC alone compared to co-culture with JJN3 (26% CAR expression and 17-fold expansion). The KM.CAR T-cells containing the IgG1 hinge only spacer had similar expansion (5-fold with JJN3, 6 fold without JJN3) but increased CAR expression (17% with JJN3, 9% without). Only the KM.CAR T-cells containing the CD8alpha chain spacer did not show any enhanced expansion or enrichment in the presence of JJN3 cells (8-fold expansion and 5% CAR expression in the presence of JJN3, compared to 5-fold expansion and 5% CAR expression without JJN3).
Functional Assessment of KM.CAR T-Cells
KMA-specific interferon-gamma production and cytotoxicity of KM.CAR T-cells were assessed by intracellular cytokine flow cytometry and standard chromium release assay with KMA+ and KMA− cell lines using protocols previously described (2). KMA positive cell lines used included JJN3, Pfeiffer, NCI-H929. KMA negative cell lines included Nalm-6 and Molt (
For cytokine flow cytometry, 2×105 KM.CAR T-cells were stimulated with target cells at a ratio of 1:1 for 5 hours. Monensin (2 μM) (BD Biosciences) and Brefeldin A (1 μg/mL) (BD Biosciences) were added after 1 hour. CAR T-cells activated non-specifically with 50 ng/ml phorbol myristate acetate (PMA: Sigma-Aldrich) and 1 ug/ml ionomycin (Sigma-Aldrich) and unstimulated cells were used as positive and negative controls. CAR T-cells were then harvested, washed, surface stained for CD3, CD4 and CD8. CAR T-cells were fixed and permeabilised with cytofix and perm/wash buffer (BD Biosciences) and stained with anti-interferon gamma antibody (BD Biosciences) followed by further washing with perm/wash buffer. Stained cells were analysed using a FACSCanto™ II flow cytometer with acquisition of at least 30,000 events.
KMA-specific cytotoxicity was assessed using a standard chromium (51Cr) release assay. Target cells were labelled with Sodium chromate (Na251CrO4) (Perkin-Elmer, Waltham, Mass., USA). KM.CAR T-cells were preincubated with the K562 cell line at a 1:1 ratio to absorb NK cell activity. Chromium labelled target cells were added to the KM.CAR T-cells in triplicate at effector:target ratios ranging from 40:1 to 1.25:1 and incubated at 37° C., 5% CO2 for 4 hours. Triplicate targets were lysed with 10% sodium dodecyl sulphate to determine maximal release and triplicate targets with no effectors were used to assess spontaneous release. Supernatants were aspirated and read using a MicroBeta2 Plate Counter (PerkinElmer). Percentage specific lysis was calculated using the standard formula-% Specific lysis=(test release−spontaneous release)/(maximal release−spontaneous release)×100]
A single transposon cassette containing a constitutively active promoter (EF1alpha) and an activation inducible promoter (NFATpro) was designed and cloned. The activation inducible gene expression cassette was produced by designing the NFATpro using Clone Manage 9 (Sci-Ed Software), based on Fiering et al(8). This includes 6 copies of the 30 base pair DNA sequence (response element-RE) bound by the Nuclear Factor of Activated T-cells (NFAT-RE)-GGAGGAAAAACTGTTTCATACAGAAGGCGT (SEQ ID NO: 35) followed by the minimal IL-2 promoter-ACATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCT TGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCC TG (SEQ ID NO: 36) found on chromosome 4 (NCBI Reference Sequence: NG_016779.1).
To enable detection of activation induced gene expression, the enhanced green fluorescent protein (eGFP) DNA sequence followed by the bovine growth hormone (BGH) polyadenylation signal (9-11) was placed 3′ of the NFATpro. The DNA sequence of this gene cassette is as follows
GGAGGAAAAACTGTTTCATACAGAAGGCGTCAATTAGGAGGAAAA
ACTGTTTCATACAGAAGGCGTCAATTAGGAGGAAAAACTGTTTCATACAG
AAGGCGTCAATTGTCCCATCGAATTAGGAGGAAAAACTGTTTCATACAGA
AGGCGTCAATTAGGAGGAAAAACTGTTTCATACAGAAGGCGTCAATTAGG
AGGAAAAACTGTTTCATACAGAAGGCGTCAATTGTCCCGGGACATTTTGA
CACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGT
TCCTGAACTCCATGGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT
GGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA
GCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTG
AAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGT
GACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACA
TGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAG
GAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGA
GGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA
TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC
TACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCAT
CAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC
TCGCCGACCACTACCAGCAGAACACCCCCATCGGATCCGGAGCCACGAAC
TCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTC
CTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA
TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAC
AATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGC.
From 5′ to 3′, this constructs contains the NFAT-RE, the IL-2 Minimal Promoter, the eGFP, and the BGH polyadenylation signal.
This cassette was synthesised commercially by Genscript and cloned into the pVAX1PB transposon plasmid between the 5′ cHS4 Insulator (GenBank: U78775.2)(12) and the human elongation factor 1 promoter. To identify transduced T-cells in initial experiments, the chimeric RQR8 marker consisting of the epitope of CD34 recognised by the QBEnd10 monoclonal antibody and mimotopes of the CD20-specific monoclonal antibody Rituximab(13) was cloned into the transposon multicloning site to produce the transposon gene insert shown in
Demonstration of Function of the Activation Inducible Gene Containing Transposon
To demonstrate the function of the pVAX1PB NFATGFP-RQR8 transposon from Example 4 (see
Expression plasmids containing IL-12 and/or the interleukin-6 receptor antagonist SANT7and also containing the optimized chimeric antigen receptor with the expression of IL-12 and/or SANT7 under control of an activation inducible promoter (Hooijberg et al. 2000) were also constructed. The SANT-7 sequence was provided by Prof Rocco Savino and was based on mutating the wildtype IL-6 gene sequence (NCBI Reference Sequence: NM_000600.4) provided as per Savino et al 1994 and Sporeno et al 1996(14-17). The sequence was imported into Clone Manage 9 (Sci-Ed Software) and a 6× His tag added for detection in supernatants by ELISA.
The nucleotide sequence of SANT-7 is provided with amino acid substitutions highlighted, underlined and listed below:
The nucleotide substitutions correspond to Y31D, G35F, L57D, E59F, N60W, Q75Y, S76K, S118R, V121D. The sequence provided also contained a Q211A substitution not listed in the published sequence.
The DNA sequence corresponding to this amino acid sequence (i.e., SEQ ID NO: 38) is as follows:
A single chain interleukin-12 (Flexi-IL-12) construct was designed by joining the IL-12 p40 and p35 subunits (Uniprot P29459 and P29460) with a flexible (G4S)3 linker similar to Zhang et al and Chinnasamy et al (18, 19), which allows both subunits to be expressed as a single peptide chain that readily forms the bioactive p70 heterodimer was used. The Flexi-IL-12 construct was synthesized and constructs containing IL-12 and SANT7 were cloned into the activation inducible transposon cassette described herein and shown in
Additionally, the Flexi-IL-12 construct could be synthesized and constructs containing IL-12 and SANT7 separated by 2A ribosomal skip elements could be cloned into the PiggyBac plasmid described herein and shown in
The amino acid sequence of Flexi-IL-12 is as follows:
VVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGE
VLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWL
TTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQE
DSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKP
LKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKT
SATVICRKNASISVRAQDRYYSSSWSEWASVPCS
GGGGSGGGGSGGGGS
From 5′ to 3′, the Flexi-IL-12 construct contains a leader peptide, the IL-12 p40 subunit, the (G4S)3Linker, and the IL-12 p35 subunit.
The DNA sequence corresponding to the amino acid sequence above (i.e., SEQ ID NO: 40) is as follows:
Additionally, expression plasmids containing the truncated dominant negative form of Galectin-3, GAL3C is also constructed. The construct contains a CD8-alpha leader peptide to direct secretion as well as a 6× His tag for detection. The amino acid sequence of GAL3C is listed here:
MEFGLSWLFLVAILKGVQCSR
HHHHHHGAPAGPLIVPYNLPLPGGV
The corresponding DNA sequence for the GAL 3C construct is provided here:
The CAR and ‘biologicals’ transposon plasmids will be nucleofected to generate CAR T-cells expressing either IL-12 alone, SANT7 alone, GAL3C alone or both IL-12 and SANT7 or both of IL12 and GAL3C or both of SANT7 and GAL3C or all three of IL-12. SANT7 and GAL3C. Cells successfully transduced with ‘biologicals’ constructs may be identified by selectable marker expression for example by flow cytometry. Levels of IL-12, SANT7. And or GAL3C will be measured intracellularly by cytokine flow cytometry and in supernatants of CAR T-cell cultures by ELISA using commercial kits and reagents and compared to control T-cells expressing CAR alone. CAR T-cells will be assessed for function by cytokine flow cytometry and cytotoxicity assays as above as well as co-culture assays with myeloma cell lines to assess inhibition of tumour growth. Experiments will be performed in triplicate and the 2 optimal CAR constructs identified will be chosen to be assessed in a murine model with and without IL-12, GAL3C and/or SANT7 expression.
Based on the previously established RPMI-Rag human myeloma murine xenograft model, RPMI-Rag-Luc (KMA-) and JJN3-Rag-Luc (KMA+) models will be developed to assess the function of our CAR T-cells in-vivo. JJN3 and RPMI8226 cells will be transfected with Luc-1 and then inoculated i.v. into Rag2−/−γc−/− (BALB/c) mice to form the JJN3-Rag-Luc and RPMI-Rag-Luc MM models. Engraftment and disease levels will be monitored by optical imaging following IP injection with luciferin and correlated with levels of levels of serum human kappa (JJN3) and lambda (RPMI) light chain. Optimal time for inoculation with candidate CAR T-cells will be established using Optical Imaging prior to the development of hind limb paralysis, usually from weeks 5-8. Cohorts of 6 JJN3-Rag-Luc and RPMI-Rag-Luc mice will be inoculated IV with increasing doses of CAR T-Cells (with and without IL-12/SANT7 expression) to establish the therapeutic dose starting at 1×106 total cells. Mice will be imaged on day 0, +1, +3, +8 and weekly thereafter until the development disease progression as determined by the development of hind limb paralysis, increasing serum free light chains (SFLC) or other institutional guidelines. Marrow and extramedullary tumors will be collected and examined histologically for distribution of MM cells and CAR T-cells. Efficacy will be determined by imaging response and survival compared with controls.
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This application is a national phase of International Application No. PCT/US16/29203, filed Apr. 25, 2016, which claims priority from U.S. Provisional Application Ser. No. 62/151,968, filed Apr. 23, 2015, and U.S. Provisional Application Ser. No. 62/158,407, filed May 7, 2015 each of which is incorporated by reference herein in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/029203 | 4/25/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/172703 | 10/27/2016 | WO | A |
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| 20090068199 | Chessler | Mar 2009 | A1 |
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| 20120114555 | Hutchinson et al. | May 2012 | A1 |
| 20130280285 | Schönfeld et al. | Oct 2013 | A1 |
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| Number | Date | Country | |
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| 20180228892 A1 | Aug 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62158407 | May 2015 | US | |
| 62151968 | Apr 2015 | US |
