Patent Application
20220347213
The content of the ASCII text file of the sequence listing named 102719.0013PCT_ST25, which is 8 KB in size was created on Jul. 19, 2019 and electronically submitted via EFS-Web along with the present application, and is incorporated by reference in its entirety.
The field of the invention is cell-based immunotherapy, especially as it relates to use of genetically modified T cells that express at least a portion of a CD1b restricted T cell receptor.
The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
CD1b presents wide variety of Mycobacterium tuberculosis-derived lipids, including mycolic acid, glucose monomycolate, glycerol monomycolate, diacylated sulfoglycolipids, lipoarabinomannan and phosphatidylinositol mannoside to corresponding CD1b restricted T cells. Mycolic acid constitutes a major lipid constituent of the Mycobacterium tuberculosis cell envelope and is typically considered Mycobacterium tuberculosis virulence factor.
Notably, mycolic acid-specific CD1b-restricted T cells have been found in blood as well as disease sites of Mycobacterium tuberculosis-infected individuals, and some CD1-restricted T cell lines from healthy or mycobacteria-infected individuals exhibited cytotoxicity and produced IFN-gamma and TNF-alpha, which are important for establishing protective immunity against Mycobacterium tuberculosis. More recently, various CD1b restricted T cell receptors have been isolated and characterized with respect to their interaction with CD1b and the bound lipid components (see Nature Communications 2017, DOI: 10.1038/ncomms13257).
In an effort to create a therapeutic composition to treat Mycobacterium tuberculosis infection, CD1 presented antigens can be prepared as a vaccine as described in CA2202680 or CA 2323733. Similarly, transgenic mice were generated that expressed human group 1 CD1 molecules (hCD1Tg) and a CD1b-restricted, mycolic-acid specific TCR in an effort to establish that CD1-targeting vaccines could be developed (eLife 2015; DOI:10.7554/eLife.08525.001). Despite these conceptually simple and attractive approaches, effective vaccines have remained elusive. In yet another known approach, CD1b restricted T cells from various donors can be expanded to relatively large numbers (see e.g., US 2003/0153073), but such expansion generally requires elaborate processes and is typically not suitable in clinical settings.
Thus, there remains a need for improved compositions, methods and uses of modified T cells with desired specificity against bacterial antigens, and especially against antigens related to Mycobacterium tuberculosis.
The inventive subject matter is directed to various compositions of, methods for, and use of a recombinant T cells, and especially primary T cells, that are transfected with a nucleic acid that encodes alpha and beta chains of a CD1b restricted T cell receptor. Advantageously, such recombinant T cells are suitable as a cell-based therapeutic for treatment of tuberculosis.
In one aspect of the inventive subject matter, the inventors contemplate a genetically modified T cell that includes a recombinant RNA molecule that encodes at least one of an alpha chain and a beta chain of a CD1b restricted T cell receptor. Preferably, the recombinant RNA molecule encodes both of the alpha chain and the beta chain of the CD1b restricted T cell receptor, and particularly preferred CD1b restricted T cell receptors are clone 18 CD1b restricted T cell receptors. Therefore, preferred CD1b restricted T cell receptors will include at least one of the sequences according to SEQ ID NO: 1-4. Furthermore, preferred recombinant RNA molecules are bi- or polycistronic mRNA molecules that include at least one IRES sequence. Suitable T cells especially include autologous T cells and/or primary T cells.
In further contemplated aspects, the recombinant RNA molecule is at least temporarily coupled to a polymer particle that is optionally degradable within the T cell. For example, contemplated polymers of the polymer particle include a poly(β-amino ester) or a chitosan. Where desired, the polymer particle may further be coupled to a second mRNA that encodes an enzyme (e.g., chitosanase) capable of degrading the polymer of the polymeric particle.
In a further aspect of the inventive subject matter, the inventors also contemplate a pharmaceutical composition that comprises a liquid carrier suitable for injection and a plurality of genetically modified T cells as described above suspended in the carrier. For example, contemplated pharmaceutical compositions may have at least 107 genetically modified T cells per transfusion unit, and/or the liquid carrier has a pH of at least 7.0. Consequently, in still another aspect of the inventive subject matter, the inventors also contemplate method of treating an individual diagnosed with tuberculosis. Such methods will typically include a step of administering to the individual a plurality of genetically modified T cells as described above in an amount effective to treat tuberculosis (e.g., at least 107 genetically modified T cells).
Therefore, uses of a genetically modified T cell in the treatment of tuberculosis are also contemplated where the genetically modified T cell comprises a recombinant RNA molecule that encodes at least one of an alpha chain and a beta chain of a CD1b restricted T cell receptor. Preferably, but not necessarily, the CD1b restricted T cell receptor is a clone 18 CD1b restricted T cell receptor, and/or the genetically modified T cell is an autologous T cell.
In still another aspect of the inventive subject matter, the inventors contemplate a method of generating a genetically modified T cell. Such methods will typically include a step of obtaining or providing a plurality of T cells, and stimulating the plurality of T cells; and a further step of transfecting the stimulated T cells with a recombinant RNA molecule that encodes at least one of an alpha chain and a beta chain of a CD1b restricted T cell receptor.
Preferably, the plurality of T cells are primary T cells, which may or may not be autologous cells. It is further generally preferred that the plurality of T cells are stimulated prior to transfection (e.g., with CD3-CD28 modified beads). Moreover, it is contemplated that transfection of the stimulated T cells uses a plurality of polymeric particles that are coupled to the recombinant RNA molecule. It should be noted that the polymeric particles may be all of the same type, or comprise at least two different types of polymeric particles.
Additionally, it is contemplated that the plurality of polymeric particles are degradable in the stimulated T cells, and/or that the polymeric particles lack targeting moieties that specifically target the particles to a component of the T cell. Suitable polymer materials for contemplated particles include poly(β-amino esters) and a chitosan. Where desired, the polymer particle may be further coupled to (e.g., coated with) a second and distinct mRNA, which may encode an enzyme capable of degrading the polymer of the polymeric particle.
Preferably, transfection is performed such that at least 40%, or at least 50%, or at least 60% of all cells expressing the CD1b restricted T cell receptor are viable cells. Likewise, transfection is preferably performed such that at least 20% or at least 30% of all cells express the CD1b restricted T cell receptor. As noted earlier, the recombinant RNA molecule encodes the alpha chain and the beta chain of the CD1b restricted T cell receptor, and is preferably (but not necessarily) a clone 18 CD1b restricted T cell receptor. Therefore, the recombinant RNA may be configured as a bi- or polycistronic RNA and includes at least one IRES sequence. Among other suitable sequences, the CD1b restricted T cell receptor may comprise at least one of the sequences according to SEQ ID NO:1-4.
Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing.
The inventors have now discovered that expression of a functional CD1b restricted T cell receptor in T cells, and especially human primary T cells, can be used to generate a cell-based therapeutic for the treatment of tuberculosis. Notably, using various protocols as described in more detail below, T cells (particularly primary T cells or autologous T cells) can be transfected at relatively high rate and viability.
Most typically, but not necessarily, T cells for transfection are obtained from a patient that is acutely infected with Mycobacterium tuberculosis. T cells are in most cases isolated from whole blood following protocols using magnetic bead separation (e.g., coated with antibodies against CD2 and/or CD3), and such isolated T cells can be further cultivated to expand the cell population. Transfection is preferably performed using particle associated mRNA that encodes both the a and p chains of a CD1b restricted T cell receptor (TCR) with high specificity to mycolic acid. For example an especially suitable TCR is clone 18 (e.g., Nature Communications 2016, DOI: 10.1038/ncomms13257) T cell receptor. Upon transfection with the RNA, the T cells are further cultured (and optionally further stimulated) and then administered to the same patient.
Of course, it should be appreciated that the particular nature of the infection of the patient with Mycobacterium tuberculosis is not limiting to the inventive subject matter, and all forms of infection, including pulmonary tuberculosis (e.g., latent/dormant, acute, and chronic) as well as extrapulmonary tuberculosis (e.g., pleural infection, meningeal infection, genitourinary infection, and infection of the lymphatic system) are expressly contemplated herein. Moreover, it should be noted that the patient need not be a human, but indeed may be any mammal that is subject to infection with Mycobacterium tuberculosis.
Additionally, it is contemplated that the CD1b receptor need not necessarily be limited to binding of mycolic acid, but that all other ligands (and especially microbial lipids) for CD1b presentation are also deemed suitable. For example, suitable sequence variations and changes in specificity are described elsewhere (e.g., Nature Communications 2017, DOI: 10.1038/ncomms 13257). Therefore, numerous other microorganisms can also be targeted with the recombinant T cells as described herein. Likewise, it should be appreciated that numerous CD1 restricted TCR other than CD1b can be expressed in the T cells as noted herein, and especially contemplated subtypes include CD1a, CD1c, CD1 d, and CD1e. Therefore, it should be noted that all restricted TCRs can be expressed in the T cells in a manner as described herein to so add/expand the pool of ‘innate immunity’ in a patient.
In especially preferred examples, the recombinant TCR will have or include a sequence portion of the clone 18 CD1b restricted T cell receptor. Where the TCR will only include a portion of that sequence, any one or more of the Vα, Jα, Cα segments, and/or CDR1 and 2 in Vα and/or CDR3 for alpha chain are deemed suitable for use herein. Likewise, any one or more of the Vβ, Jβ, Dβ, Cβ segments, and/or CDR1 and 2 in Vβ and/or CDR3 for beta chain are deemed suitable for use herein. For example, particularly preferred sequences include those shown in
Furthermore, it should be noted that the nature of the T cells may also vary considerably, and suitable T cells for transfection include autologous (with respect to the patient) T cells, primary T cells, cultivated T cells, T cells from T cell culture (which will typically be irradiated before administration), CD4+ T cells, CD8+ T cells, and T cells with tissue compatibility to a recipient. Therefore, the manner of T cell isolation may vary considerably and suitable isolation protocols will include isolations via magnetic beads or FACS using antibodies against CD23, CD3, CD4, and/or CD25. Further known protocols are described elsewhere (see e.g., J Vis Exp. 2010; (40): 2017).
In less preferred aspects, it is also contemplated that the host cells are cells other than T cells, and especially preferred alternative cells include various cytotoxic immune competent cells such as NK cells or iNK cells. However, in such case, these cells will require further genetic modification (via transfection of other genetic engineering) to enable co-expression of one or more of the CD3 gamma, delta, epsilon, and zeta chain.
Regardless of the particular type of cell for transfection, it is typically preferred that the recombinant nucleic acid is an RNA, preferably constructed as a bicistronic or polycistronic RNA that includes one or more IRES sequences for coordinated expression of the alpha and beta chain. However, monocistronic RNA is also deemed suitable, albeit less preferred. Also less preferred transfections include those with DNA (whether as gene edited modification, or as extrachromosomal). Still further contemplated transfection nucleic acids include viral vectors.
Preferably, transfection of the T cells is performed on stimulated (e.g., using CD3/CD28 dynabeads) T cells using one or more types of nanoparticles that are coupled to one or more RNA molecules. In particularly preferred aspects, the nanoparticles are degradable within the T cells, typically in an enzymatic manner. For example, especially preferred polymers of the nanoparticles are poly(β-amino esters) (see e.g., Methods Mol Biol. 2009; 480: 53-63) and chitosan (see e.g., Mol. Pharmaceutics 2017, 14, 2422-2436; or International Journal of Nanomedicine 2010:5 473-481). As will be readily appreciated, these nanoparticles can be targeted (e.g., with antibodies or fragments of antibodies binding CD3) or ‘naked’ (untargeted) as is discussed in more detail below. Alternatively, transfection of the T cells can also be performed using electroporation (see e.g., Molecular Therapy Vol. 13, No. 1, p 151-159, January 2006), or chemical transfection such as Nucleofactor or Amaxa kits (commercially available from Lonza, Allendale, N.J., USA).
Once transfected T cells are prepared, such cells are preferably formulated for transfusion to a patient, typically in an aqueous carrier suitable for injection. For example, a typical solution for transfusion will include at least 106, or at least 107, or at least 108, or at least 109 transfected cells. Thus, pharmaceutical compositions comprising transfected T cells and use of such cells in pharmaceutical formulations (especially for treatment of (myco)bacterial infection) are deemed appropriate.
The following description provides the person of ordinary skill in the art exemplary guidance for preparation and use of the recombinant cells presented herein. However, the examples should not be construed as limiting the inventive subject matter.
To investigate the influence of T cell stimulation and type of transfection on the T cell response to various antigens, T cells were separately transfected with ‘naked’ non-targeting PBAE (poly(β-amino ester)) nanoparticles that were associated with two types of RNA: RNA encoding a clone 18 TCR (bicistronic construct with clone 18 TCR alpha and beta chain) and RNA encoding GFP (green fluorescent protein). Stimulation, in this case, was achieved by culturing isolated T cells with a cocktail of anti-CD3 and anti-CD28 antibodies (Immunocult Human CD3/CD28 T Cell Activator, Stemcell Technologies) for 24 hours followed by another 24 hours in unsupplemented culture medium prior to transfection. PBAE-RNA nanoparticles were prepared by first dissolving both PBAE and RNA separately in an acidic sodium acetate buffer, then adding the PBAE solution to the RNA solution at the appropriate ratio and rapidly mixing them by vortex. After allowing 5 minutes for nanoparticle formation they were added to the T cells in culture for 24 hours. These transfected T cells were then, in some cases, co-cultured with human peripheral blood monocyte-derived dendritic cells with the addition, in some additional cases, of ligands for the clone 18 TCR. These were either mycolic acid or an extract from Mycobacterium tuberculosis. Following approximately 24 hours of culture the amount of interferon gamma present in the culture media was measured by enzyme-linked immunosorbent assay (ELISA). As can be readily seen from
The inventors also investigated whether targeting of the nanoparticles to the T cells (here CD3 targeting) had any effect of the ligand dependent interferon gamma secretion. Notably, as can be taken from
In yet further experiments, the inventors investigated effects of PBAE nanoparticle targeting where the T cells were stimulated or not stimulated.
Stimulation of T cells was performed using CD3-CD28 dynabeads and resulted in similar responses for all types of transfections as can be seen in
Functional expression of the recombinant RNA was also confirmed using RNA encoding GFP in a set of experiments in which transfection was done using stimulated and non-stimulated T cells. Representative results are seen in
To determine the influence of the type of nanoparticles, the inventors also tested chitosan nanoparticles with associated RNA, where the chitosan particles were further optionally coated with RNA encoding chitosanase. An exemplary schematic workflow for preparation of such particles is depicted in
Exemplary results for nanoparticle RNA delivery using pure chitosan with single RNA (encoding GFP) and chitosan core/shell particles with GFP-encoding RNA comprising both the core and shell RNA component are shown in
Chitosanase RNA was prepared by in vitro transcription reactions from a commercially available plasmid (pcDNA3.1+/c-(k)dyk) containing the chitosanase gene from Colletotrichum higginsianum IMI349063 (Gene Symbol: CH63R_01899) as is shown in
Exemplary results for co-delivery of chitosanase mRNA with GFP mRNA via core/shell chitosan complex and separate delivery are illustrated in
It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.
This application claims priority to our co-pending US provisional patent application with the Ser. No. 62/716,292, which was filed Aug. 8, 2018, and which is incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/045241 | 8/6/2019 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62716292 | Aug 2018 | US |
