Macrophage CAR (MOTO-CAR) In Immunotherapy

Abstract

Modified macrophage immune cells are provided for treatment of cancer and other diseases. In particular said macrophages express chimeric antigen receptors (CAR). The single chain variable fragment (scFv) may be directed against thymidine kinase 1 (TK1) or hypoxanthine guanine phosphoribosyltransferase (HPRT). The signaling domain may be derived from a Toll-like receptor (TLR).

Claims

1. A chimeric antigen receptor (CAR) comprising a fusion of an antigen binding antibody fragment and a TLR-4 cytoplasmic signaling domain.

2. The CAR of claim 1, wherein the CAR is comprised in a mammalian cell and the cytoplasmic signaling domain activates the NE-KB pathway upon binding of the antibody fragment to its antigen.

3. The CAR of claim 2, wherein the mammalian cell is a monocyte or a macrophage.

4. The CAR of claim 1, wherein the CAR is comprised in a mammalian monocyte or a macrophage cell and cytoplasmic signaling domain polarizes the cell to M1 phenotype macrophages upon binding of the antibody fragment to its antigen.

5. The CAR of claim 1, wherein the antibody fragment is a single chain variable fragment (scFv).

6. The CAR of claim 5, wherein the scFv is derived from a monoclonal antibody specific for the antigen expressed by cells of a cancer.

7. The CAR of claim 6, wherein the monoclonal antibody is a human or mouse monoclonal antibody.

8. The CAR of claim 1, wherein the antigen is present on cells of a cancer.

9. The method according to claim 1, wherein the antigen is a salvage pathway enzyme.

10. The method according to claim 9, wherein the one or more salvage pathway enzymes is selected from the group consisting of TK1, HGPRT, DCK, and APRT.

11. A vector comprising a sequence encoding the chimeric antigen receptor (CAR) of claim 1.

12. The vector of claim 11, wherein the sequence encoding the CAR is operably linked to a macrophage specific promoter.

13. A cell comprising the vector of claim 11.

14. The cell of claim 13, wherein the cell is a mammalian cell.

15. The cell of claim 13, wherein the cell is a monocyte or a macrophage.

16. The cell of claim 13, wherein the cell is a human cell.

17. The cell of claim 14, wherein the cytoplasmic signaling domain activates the NE-KB pathway upon binding of the antibody fragment to its antigen.

18. The cell of claim 15, wherein the cytoplasmic signaling domain polarizes the cell to M1 phenotype macrophages upon binding of the antibody fragment to its antigen.

19. The cell of claim 17, wherein the cell is a human cell.

20. The cell of claim 18, wherein the cell is a human cell.

Description

DESCRIPTION OF THE DRAWINGS

[0094] FIG. 1 is a schematic illustrating a macrophage chimeric antigen receptor

[0095] FIG. 2 is a schematic showing a Macrophage Toll-like receptor CAR. (MOTO CAR). The intracellular domain and transmembrane domain of Toll like receptors, FC-gamma III receptor, IL-1 or the IFN-gamma receptors can be fused to a suitable hinge and a ScFv against a tumor antigen to activate Macrophages upon binding to a specific tumor antigen.

[0096] FIG. 3a is a schematic showing different macrophage receptors that could be utilized to build a macrophage CAR.

[0097] FIG. 3b is a schematic showing signaling of Fc Gamma Receptor III

[0098] FIG. 4 is a schematic showing where Bispecific Macrophage Engager.sub.IFN-Y (BIME.sub.IFN-Y). M2 tumor resident macrophages can be polarized and anchored to tumor cells using a molecule of IFN-γ linked by a aminoacid spacer to a ScFv against a tumor antigen.

[0099] FIG. 5 is a schematic showing where Bispecific Macrophage Engager (BIME). M2 macrophages can be polarized towards M1 phenotype and directed to tumor cells. A bispecific antibody can block CSF-1 receptor blocking CSF-1 a receptor that leads to an M2 profile At the same time, the macrophage can be anchored with a ScFv against a tumor antigen. The patient then can receive IFN-γ and the macrophage can be polarized towards a M1 phenotype for tumor elimination.

[0100] FIG. 6 is a schematic showing Macrophage Activator.sub.MD2 (BIME.sub.MD2)- Toll-like receptor 4 dimerization can be triggered by using a ScFv against the hydrophobic pocket of the MD2 protein. Then a BIME can be added to anchor macrophages to the tumor cells.

[0101] FIG. 7 is a schematic illustrating Toll Like Receptor Signaling

[0102] FIG. 8 is shown the HGPRT biochemical pathway

[0103] FIG. 9 is a graph illustrating HGPRT protein surface expression compared with APRT nad dCK two other salvage pathway enzymes. (b) confirms using flow cytometry the presence of HGPRT on the cell surface.

DETAILED DESCRIPTION

[0104] TK1 and HPRT are exclusively expressed on the surface membrane of tumor cells and have led to the development of a range of monoclonal antibodies against human TK1 and HPRT. The specific binding capacity of these specific monoclonal antibodies could be used in macrophages transfected with a modified macrophage-specific chimeric antigen receptor to treat cancer patients. A method for modifying a monocyte/macrophage to have receptors against human TK1 (MOTO CAR) might include producing human/humanized monoclonal antibodies that are TK1 and HPRT specific (FIG. 1). These TK1 and HPRT specific monoclonal antibodies would be used to create chimeric antigen receptors (CARs) by fusion of the single-chain variable fragments to macrophage (MO) signaling domains (FIG. 2) (such as the cytoplasmic domain portion from a toll-like receptor (TO), the FC gamma III, IL-1 or INF-gamma receptors) (FIG. 7) that would be transduced into the macrophage (FIG. 3a, b). The premise is that monocytes/macrophages would be removed from the patient and transfected ex vivo with a macrophage-specific chimeric antigen receptor lentiviral vector. This will allow the macrophage to recognize and bind to cells expressing TK1, HPRT or any other tumor antigen on their surface membrane, stimulating macrophage activation and cancer cell death. This could be used to treat many different types of cancer as TK1 is present on the surface of many different tumors and is not found on the surface of normal cells.

[0105] MOTO-CAR Construction

[0106] cDNA was purified from a monoclonal antibody hybridoma cell (CB1) with an antibody specific to human TK1 and used to amplify the heavy and light chains of the CB1 variable region via polymerase chain reaction (PCR) Sequences from the heavy and light chain were confirmed using NCBI Blast. CB1 heavy and light chains were fused together via site overlap extension (SOE) PCR to make a single chain fragment variable (scFv) using a G4S linker. The G4S linker was codon optimized for yeast and humans using the Codon Optimization tool provided by IDT (https://www.idtdna.com/CodonOpt) in order to maximize protein expression. The CB1 scFv was cut using restriction enzymes and inserted into a pMP71 CAR vector.

[0107] TK-1 and HPRT-specific human scFv antibodies were isolated from a yeast antibody library. TK-1 and HPRT protein was isolated, His-tagged, and purified. TK1 and HPRT protein was labeled with an anti-His biotinylated antibody and added to the library to select for TK-1 and HPRT-specific antibody clones. TK-1 and HPRT antibody clones were alternately stained with streptavidin or anti-biotin microbeads and enriched using a magnetic column. Two additional rounds of sorting and selection were performed to isolate TK-1 and HPRT specific antibodies. For the final selection, possible TK-1 and HPRT antibody clones and their respective protein were sorted by fluorescence-activated cell sorting (FACS) by alternately labeling with fluorescently-conjugated anti-HA or anti-c-myc antibodies to isolate TK-1 and HPRT specific antibodies. High affinity clones were selected for CAR construction. Other human antibodies or humanized antibodies from other animals could be selected or altered to be TK-1 or HPRT specific by using phage display or other recombination methods.

[0108] Selected scFv clones were then combined with human IgG1 constant domains to create an antibody for use in applications such as Western blot or ELIZA in order to confirm the binding specificity of the scFv. The antibody construct was inserted into the pPNL9 yeast secretion vector and YVH10 yeast were transformed with the construct and induced to produce the antibody. Other expression systems such as E. coli or mammalian systems could also be used to secrete antibodies.

[0109] Isolation and Characterization of Protein-Specific Antibody Fragments.

[0110] Referring to FIG. 9, 10.sup.5 yeast were incubated with 2.5 ug of protein of interest labeled with the fluorescent tag APC. The higher left (red) peak indicates yeast population that was not binding to the protein of interest (our negative control). The lower left (blue) peak on the left illustrates yeast not expressing their surface protein while the high (blue) peak on the right indicates binding of the expressed antibody fragment to the protein of interest.

[0111] Structural Consensus among Antibodies Defines the Antigen Binding Site. PLoS Comput Biol 8(2): e1002388. doi:10.1371/journal.pcbi.1002388. Kunik V, Ashkenazi S, Ofran Y (2012). Paratome: An online tool for systematic identification of antigen binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue): W521-4. doi:10.1093/nar/gks480. Epub 2012 Jun. 6

[0112] Discovery

[0113] As aspect is the use of a CAR or BiTE produced with a scFv from a humanized or non-human mammal (such as mouse) monoclonal antibody to HPRT and TK1, that could be used with appropriate genetic engineering to manipulate macrophage lymphocytes ultimately from a patient but not limited to such, to treat a disease such as cancer. The fact that HPRT and TK1 are on the surface of cancer cells and not on the surface of any normal cell is a major part of the discovery, as this knowledge can be used to allow lymphocytes to be directed specifically to the tumor cells.

[0114] An aspect of the present system lies in the fact that using specifically generated antibodies to human HPRT or TK1 it has been discovered that HPRT and TK1 are expressed on the surface of human cancer cells and are believed not to be on the surface of normal cells and thereby can be used to target CARs and BiTEs to the tumors. While T cells have been used extensively in CAR therapy with varying results it is also proposed to use genetically modified macrophages using scFv from unique antibodies attached to a cytoplasmic domain of a Toll Like receptor such as Toll like receptor 4 to activate macrophages against tumors. This unique approach overcomes many of the inherent problems associated with the current T cell CAR technology. Utilizing the killing power of macrophages directed at specific unique targets on tumor cells allows for the enhanced response without the major drawbacks such as cytokine storm, memory activation, and on target off target problems.

[0115] An aspect is to couple the potential of a specific monoclonal antibody against a human tumor antigen to that activation receptor of patient macrophages to ensure a localized M1 response directed specifically towards the tumor. This application is to protect the technology that would allow the use of a CAR or BiTE produced with a scFv from a humanized or mouse monoclonal antibody to HPRT, TK1 or other tumor antigen, that could be used with appropriate genetic engineering to manipulate macrophages, neutrophils or other immune cells ultimately from a patient but not limited to such, to treat a disease such as cancer. The scFv from the humanized mouse monoclonal would be engineered to attach to the transmembrane and cytoplasmic domain of the TLR4, resulting in a TLR4 macrophage chimeric antigen receptor. That fact that HPRT is on the surface of cancer cells and not on the surface of any normal cell is a major part of the discovery, as this knowledge and these techniques can be used to allow the macrophages to be directed, (using the HPRT monoclonal portion) and activated (using the TLR4 cytoplasmic domain portion), specifically against the tumor cells.

[0116] It is clear that macrophages play a significant role in cancer progression, and immunotherapies involving macrophages should be included in the treatment of this disease. The polarization of macrophages towards an M1 response with minimal side effects can be a powerful therapy against solid tumors. Inflammatory signals such as LPS or TNF-α can easily polarize macrophages towards an M1 phenotype in vitro. In vivo, substances such an LPS and TNF-α exacerbate a whole-body inflammatory response involving cells in the innate and adaptive immune system, however. They can cause fever and inflammation in several tissues including the mucosal surfaces and the lungs. These inflammatory signals are highly cytotoxic as well (Apostolaki, Armaka, Victoratos, & Kollias, 2010; Kolb & Granger, 1968; Michel & Nagy, 1997). Immunotherapy requires the activation of the immune system however it is difficult to find a cytokine, chemokine, compound, or biomaterial that will not produce some side effects. Macrophages belong to the innate immune system and exhibit pro-inflammatory and anti-inflammatory properties, they are the ideal immunotherapy candidates.

[0117] While this invention has been described with reference to certain specific embodiments and examples, it will be recognized by those skilled in the art that many variations are possible without departing from the scope and spirit of this invention, and that the invention, as described by the claims, is intended to cover all changes and modifications of the invention which do not depart from the spirit of the invention.

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