WO2017201635A1 - Cellular expression of hyaluronidase and use thereof in solid tumour cell therapy - Google Patents

Abstract

Discosed is a free or transmembrane fusion protein hyaluronidase expressed by CAR-T cells or other cells. While maintaining the anti-tumour activity of CAR-T cells or other cells, the cell-expressed hyaluronidase thereof can degrade the hyaluronic acid in the extracellular matrix (ECM), reduce the viscosity of the hyaluronic acid, enhance the tissue permeability of ECM, and facilitate penetration of CAR-T cells or other cells into solid tumours and infiltration thereof in solid tumours, thereby enhancing the efficacy of CAR-T cells or other cells in the treatment of solid tumours.

Description

Cellular expression of hyaluronidase and its application in the treatment of solid tumor cells Technical field

The present invention discloses a cell expression of hyaluronidase and a method of use thereof in the treatment of solid tumor cells.

Background technique

Cell therapy is the treatment of disease with cells. Compared with organic small molecule drugs and biomacromolecular drugs, cells as a drug have significant curative effects and a wide range of indications. Cell therapy is increasingly becoming an important means of treating and curing diseases in humans.

Cellular therapy can be used to treat many diseases, such as diabetes, infectious diseases, neurodegenerative diseases, and a wide variety of cancers. Cells that can act as drugs mainly include different types of immune cells and stem cells. These cells, through amplification, activation, and genetic modification in various ways, have become important weapons for treating and curing diseases.

One of the fastest growing and most successful areas of cell therapy is cancer treatment. There are many types of immune cells that can be used for cancer treatment, including (1) T lymphocytes (T), (2) natural killers (Natural killers, NK), and (3) NK-T cells (Natural killers). T), (4) dendritic cells (DC), (5) cytokine-induced killers (CIK), (6) Cytotoxic lymphocytes (CTL), (7) Tumor infiltrating lymphocytes (TIL), (8) Chimeric Antigen Receptor (CAR) modified T cells (CAR-T), (9) T cell receptors , TCR) modified T cells (TCR-T), (10) genetically modified NK cells (Genetic modified NK).

Its own immune cell T cells, genetically engineered with the chimeric antigen receptor (CAR), this CAR-T cell treatment of leukemia is very effective (N Eng1 J Med.2011Aug 25;365(8):725-33.doi :10.1056/NEJMoa1103849). Now using the most advanced CAR-T technology to treat leukemia, the treatment efficiency is as high as 90%, the patient's survival time is greatly extended, and some patients' leukemia is even cured.

The therapeutic effect of cell therapy such as CAR-T on hematological tumors is extremely significant, but the efficacy in the treatment of malignant solid tumors (including advanced or metastatic malignant solid tumors) is still limited (J Immunol Res. 2016; 2016: 3850839. Doi:10.1155/2016/3850839; Mol Ther Oncolytics.2016 Apr 13;3:16006.doi:10.1038/mto.2016.6).

Many scientists around the world are working hard to adopt different strategies to enhance the therapeutic efficacy of cell therapy such as CAR-T in malignant solid tumors, including advanced or metastatic malignant solid tumors. These strategies can be divided into four directions.

(1) Joint use. CAR-T cell therapy is used in combination with other therapeutic drugs, for example, in combination with chemotherapeutic drugs, in combination with cancer therapeutic antibody drugs such as immunological checkpoint antibodies (Clin Cancer Res. 2013 Oct15; 19(20): 5636-46. Doi:10.1158/1078-0432.CCR-13-0458).

(B) Optimization of CAR-T cell culture. In T cells with cancer cell therapy, Central Memory T cells (Tcm) last longer in vivo and have better clinical efficacy (J Immunother.2012Nov-Dec;35(9):651-60.doi :10.1097/CJI.0b013e31827806e6; J Clin Invest. 2008 Jan; 118(1): 294-305). Therefore, in the expansion culture of T cells, by optimizing the cell culture conditions, in the conventional culture A certain amount of added ingredients are used in the nutrient solution to enhance the ratio and amount of Tcm to achieve a better therapeutic effect (J Clin Invest.2016 Apr 25.pii:85309.doi:10.1172/JCI85309).

(3) Optimize the CAR-T design. On the one hand, different CAR-Ts are designed to recognize more specific antigen targets of different malignant solid tumors (including advanced or metastatic malignant solid tumors); on the other hand, design a CAR-T to recognize two or Specific antigenic targets for multiple malignant solid tumors (including advanced or metastatic malignant solid tumors) (Nat Rev Clin Oncol. 2016 Mar 22.doi:10.1038/nrclinonc.2016.36); and, in the CAR-T design Regulatory elements, regulation of CAR-T activity, strength and weakness with other drugs or other methods (Science.2015 Oct 16; 350 (6258): aab4077.doi: 10.1126/science.aab4077).

(4) Co-expression. CAR-T cells express IL-12 or other cytokines while expressing CAR to further enhance CAR-T anti-tumor activity, so-called "Trucks" (T cells redirected for universal cytokine-mediated killing) (Expert 0pin Biol Ther .2015;15(8):1145-54.doi:10.1517/14712598.2015.1046430); or co-expressing catalase to protect T cells from reactive oxygen species damage to T cell structure and function (J Immunol.2016Jan 15;196(2):759-66.doi:10.4049/jimmunol.1401710); alternatively, co-expressing heparanase to enhance T cell entry into the tumor stroma and enhance T cell infiltration (Nat Med) .2015May; 21(5): 524-9.doi: 10.1038/nm.3833; PCT/US2014/020936).

Despite the various optimizations and developments of CAR-T technology, although individual CAR-T treatments for malignant solid tumors (including advanced or metastatic malignant solid tumors) have been successfully reported, in general, CAR-T treats malignant solid tumors ( There are still significant obstacles including advanced or metastatic malignant solid tumors (J Immunol Res. 2016; 2016: 3850839. doi: 10.1155/2016/3850839; Mol Ther Oncolytics. 2016 Apr 13; 3: 16006. doi: 10.1038/mto. 2016.6).

The main obstacle to the treatment of malignant solid tumors (including advanced or metastatic malignant solid tumors) by CAR-T is that CAR-T is difficult to enter into malignant solid tumors (including advanced or metastatic malignant solid tumors), and anti-tumor of CAR-T The function cannot be fully utilized.

The present invention expresses the hyaluronidase on the surface of CAR-T or other cells, promotes the entry of cells such as CAR-T into malignant solid tumors (including advanced or metastatic malignant solid tumors), and enhances the malignancy of cells such as CAR-T. Efficacy in the treatment of solid tumors, including advanced or metastatic malignant solid tumors.

Summary of the invention

Hyaluronidases (EC 3.2.1.35) are a family of enzymes that are capable of decomposing hyaluronic acid. Human hyaluronidase is encoded by six genes in which expression of hyaluronidase activity includes HYAL1 (NP_149349), HYAL2 (NP_003764), and PH-20/SPAM1 (NP_001167515). Hyaluronidase catalyzes the hydrolysis of hyaluronic acid. Hyaluronic acid is a component of the extracellular matrix (ECM). Hyaluronidase reduces the viscosity of hyaluronic acid by hydrolyzing hyaluronic acid and enhances tissue permeability. Hyaluronidase, in combination with other drugs, promotes drug diffusion and delivery (Chem Rev. 2006 Mar; 106(3): 818-839).

Hyaluronidase has been approved by the FDA as a drug. U.S. FDA approved animal derived hyaluronidase drugs, including Hydase (Prima Pharm), Vitrase (Bausch+Lomb and Valeant Pharmaceuticals), Amphadase (Amphastar Pharmaceuticals), and Wydase.

The US FDA approved Halozyme Therapeutics and Baxter Healthcare on December 2, 2005. The human hyaluronidase PH-20 drug from the genetic recombination source is sold under the trade name Hylenex.

Moreover, on September 12, 2014, the US FDA approved Baxter's drug HyQvia, a sub-cutaneous immunoglobulin (SCIG) using recombinant human hyaluronidase Hylenex. HyQvia contains hyaluronidase Hylenex to allow subcutaneous injection of large volumes of SCIG.

Roche has combined its cancer therapeutic antibody drug Herceptin with Halozyme's recombinant human hyaluronidase Hylenex as a subcutaneous injection. This dosage form has been approved by the European Union on September 2, 2013 and is in Phase III clinical trials in the United States. The Herceptin subcutaneous injection form containing hyaluronidase takes only 3-5 minutes, while the standard intravenous injection takes 30-90 minutes.

The present invention expresses the hyaluronidase on the surface of CAR-T or other cells, promotes the entry of cells such as CAR-T into malignant solid tumors (including advanced or metastatic malignant solid tumors), and enhances the malignancy of cells such as CAR-T. Efficacy in the treatment of solid tumors, including advanced or metastatic malignant solid tumors.

According to the present invention, hyaluronidase can be expressed as a secreted protein, CAR-T cells or other cells express hyaluronidase, hyaluronidase is secreted outside the cell, and free hyaluronidase acts outside the cell.

According to the invention, hyaluronidase can be expressed as a transmembrane fusion protein. In the natural state, hyaluronidase is expressed in the form of a secreted protein rather than a transmembrane protein. The hyaluronidase protein coding sequence is included as an extracellular domain sequence, with a signal peptide sequence, a hinge sequence, a transmembrane sequence, and a cytoplasmic domain. Sequence fusion to form a fusion gene [see Figure (1)]. This fusion protein gene is transferred to CAR-T or other immune cells and can express hyaluronidase protein on the cell surface [see Figure (2)] for malignant solid tumors (including advanced or metastatic malignant solid tumors). Cell therapy.

According to the present invention, the hyaluronidase coding sequence in the secretory hyaluronidase or hyaluronidase transmembrane fusion protein gene sequence can be derived from human HYAL1 (NP_149349), HYAL2 (NP_003764) and PH-20/. SPAM1 (NP_001167515) can also be derived from other coding sequences containing hyaluronidase activity.

According to the invention, the hyaluronidase coding sequence in the secretory hyaluronidase or hyaluronidase transmembrane fusion protein gene sequence may be all protein coding sequences or may contain hyaluronidase-encoding activity. Partial sequence.

According to the present invention, the signal peptide sequence, the hinge region sequence, the transmembrane region sequence and the intracellular region sequence in the hyaluronidase transmembrane fusion protein gene sequence can be derived from any fusion protein.

According to the present invention, the secretory hyaluronidase or hyaluronidase transmembrane fusion protein gene can be expressed not only on the surface of CAR-T cells but also on the surface of other immune cells, including but not limited to T cells. NK cells, NK-T cells, DC cells, CIK cells, CTL cells, TIL cells, TCR-T cells, genetically modified NK cells.

According to the present invention, a CAR-T cell or other immune cell expressing a secretory hyaluronidase or a hyaluronidase transmembrane fusion protein gene maintains a specific anti-tumor function determined by CAR-T while passing through it. The hyaluronidase expressed by the cell breaks down hyaluronic acid in the tissues of malignant solid tumors (including advanced or metastatic malignant solid tumors), and enhances the entry of immune cells such as CAR-T into malignant solid tumors (including advanced or metastatic malignant solid tumors). Enhance the entry and infiltration of immune cells such as CAR-T in malignant solid tumors (including advanced or metastatic malignant solid tumors), and enhance the treatment of malignant solid tumors (including advanced or metastatic malignant solid tumors) by immunological cells such as CAR-T. Efficacy. At the same time, hyaluronidase activity is limited to specific local malignant solid tumors (including late or metastatic malignant solid tumors) that are specifically defined by other genetic elements of CAR-T cells (eg, CAR).

According to the present invention, the transmembrane hyaluronidase activity is more concentrated in the specific tumor region determined by CAR-T than the free-type diffusing hyaluronidase, and has a stronger decomposition of hyaluronic acid and a decrease in hyalurance. The viscosity of the acid and the enhancement of the permeability of the ECM tissue are more conducive to the penetration of CAR-T cells into solid tumors and infiltration in solid tumors, thereby enhancing the efficacy of CAR-T cells in the treatment of solid tumors.

According to the invention, the secretory hyaluronidase or hyaluronidase transmembrane fusion protein gene can be used as an expression unit alone or in combination with other expression elements to form an expression unit; the secretory hyaluronidase Alternatively, the hyaluronidase transmembrane fusion protein gene can be expressed on the cell surface alone or in combination with other gene expression elements on the cell surface.

According to the invention, the CAR-T and other immune cells expressing a secretory hyaluronidase or hyaluronidase transmembrane fusion protein gene are used for the treatment of malignant solid tumors (including advanced or metastatic malignant solid tumors), Including but not limited to originating from human and animal brain, central nervous system, kidney, liver, gallbladder, head and neck, mouth, thyroid, skin, mucous membrane, gland, blood vessels, bone tissue, lymph nodes, lung, esophagus, stomach, breast Primary or secondary cancer, sarcoma or carcinosarcoma of the pancreas, eyes, nasopharynx, uterus, ovary, endometrium, cervix, prostate, bladder, colon or rectum.

DRAWINGS

Figure One

Diagram of expression cassette of hyaluronidase

Figure (1) Schematic diagram of hyaluronidase expression elements

A: Schematic diagram of free hyaluronidase expression element

B: Schematic diagram of hyaluronidase transmembrane fusion protein expression element

There are two types of hyaluronidase gene expression, free form (A) and transmembrane fusion protein type (B). The free hyaluronidase gene expression element consists of a signal peptide sequence (SP) and a hyaluronidase coding sequence (HYAL). The transmembrane fusion protein type hyaluronidase gene expression element consists of a signal peptide sequence (SP), an extracellular domain hyaluronidase coding sequence (HYAL), and a hinge sequence (HIN). , transmembrane sequence (TM), cytoplasmic domain sequence (CYTO) composition. Both expression elements contain a promoter sequence (PRO) and 5' and 3' expression control sequences.

Figure Two

Diagram of CAR-T cell co-expressing CAR and hyaluronidase

Figure (2) Schematic diagram of CAR-T cells co-expressing hyaluronidase transmembrane fusion protein

The CAR expressed in T cells consists of CD3XX signal sequence (CD3ζ), costimulatory signal 1 sequence (co-stim 1), costimulatory signal 2 sequence (co-stim 2), transmembrane sequence (TM), hinge region sequence ( HIN) A single-chain variable region (scFv) sequence composition of an antibody that recognizes a cancer cell surface-specific antigen (antigen). The hyaluronidase transmembrane fusion protein co-expressed by this CAR-T cell is composed of a cytoplasmic domian, a transmembrane sequence (TM), a hinge region (HIN) sequence, and a hyaluronidase sequence. . CAR-T cells co-expressing the hyaluronidase transmembrane fusion protein, degrading hyaluronic acid in the extracellular matrix (ECM) by hyaluronidase on the cell surface, reducing the viscosity of hyaluronic acid and enhancing ECM Tissue permeability, which facilitates the penetration of CAR-T cells into solid tumors And infiltration in solid tumors, thereby enhancing the efficacy of CAR-T cells in the treatment of solid tumors. At the same time, since hyaluronidase is located on the surface of CAR-T cells, hyaluronidase activity is limited to the specific tumor area identified by CAR-T, thereby avoiding the damage of hyaluronidase to healthy tissues.

detailed description

1. Preparation of hyaluronidase transmembrane fusion protein gene

Synthetic hyaluronidase transmembrane fusion protein gene DNA molecule is synthesized by a synthetic method: human hyaluronidase PH-20 coding sequence (NM_001174044.1) including 5'-end signal peptide sequence, human IgG4 hinge sequence, CD8 The transmembrane sequence, the CD28 intracellular sequence, the 4-1BB intracellular sequence, the CD3zeta domain (NM_000734.3) intracellular sequence, and the appropriate restriction endonuclease sites are added to the 5' and 3' ends of the DNA molecule. The synthetic hyaluronidase transmembrane fusion gene was cloned into a lentivirus vector and transfected into a 293FT cell together with a packaging plasmid to prepare a hyaluronidase transmembrane fusion virus.

2. Hyaluronidase transmembrane fusion virus transduction of CAR-T cells

After 3 days of CAR-T cell culture, hyaluronidase transmembrane fusion virus was added to CAR-T cells, and after 6 days of culture, hyaluronidase-positive expression was screened by hyaluronidase antibody immobilized on magnetic beads. CAR-T cells, continue to culture CAR-T cells to the required amount, collect hyaluronidase-positive CAR-T cells, and apply to cell therapy of solid tumors (including advanced or metastatic malignant solid tumors).

Claims (10)

A method in which a cell expresses hyaluronidase and is used in cells for the treatment of malignant solid tumors, including advanced or metastatic malignant solid tumors. According to claim 1, the hyaluronidase comprises an episomal and transmembrane fusion protein. According to claim 1, the episomal hyaluronidase gene consists of a signal peptide sequence, a coding region sequence and a secretion signal sequence. According to claim 1, the hyaluronidase transmembrane fusion protein gene consists of a signal peptide sequence, an extracellular region hyaluronidase coding sequence, a hinge region sequence, a transmembrane region sequence and an intracellular region sequence. The hyaluronidase transmembrane fusion protein gene according to claim 1, wherein the extracellular region hyaluronidase coding sequence comprises a human hyaluronidase HYAL1, HYAL2, and/or PH-20/SPAM1 gene sequence, and comprises Gene sequences of other proteins of human, animal, plant, and microbial hyaluronidase activity. According to claim 1, the hyaluronidase transmembrane fusion protein gene, the extracellular region hyaluronidase coding sequence includes a full-length molecule of hyaluronidase and a gene coding sequence of a partial molecule containing an enzyme function. According to claim 1, the hyaluronidase transmembrane fusion protein gene, the signal sequence, the hinge region, the transmembrane region and the intracellular region can be derived from the gene coding sequence of any transmembrane protein. According to claim 1, the hyaluronidase gene can be used as a single expression unit; or it can be fused to other gene sequences to form an expression unit. According to claim 1, cells expressing the hyaluronidase gene include CAR-T cells, TCR-T cells, T cells, NK cells, genetically modified NK cells, NK-T cells, DC cells, and CIK cells. , CTL cells, TIL cells. According to claim 1, the hyaluronidase gene-expressing cells are used for the treatment of malignant solid tumors (including advanced or metastatic malignant solid tumors), including human and animal brain, central nervous system, kidney, liver, gallbladder , head and neck, mouth, thyroid, skin, mucous membranes, glands, blood vessels, bone tissue, lymph nodes, lungs, esophagus, stomach, breast, pancreas, eyes, nasopharynx, uterus, ovaries, endometrium, cervix, Primary or secondary cancer, sarcoma or carcinosarcoma of the prostate, bladder, colon or rectum.

Images