HK40060707B - Mrna vaccine - Google Patents

Description

mRNA vaccine

Invention Field

This invention generally relates to combinations of mRNA molecules encoding functional immunostimulatory proteins and PD-1 pathway inhibitors. In particular, this invention generally relates to combinations of mRNA molecules encoding functional immunostimulatory proteins and PD-1 pathway inhibitors. Specifically, this invention relates to combinations of one or more mRNA molecules and PD-1 pathway inhibitors, wherein the mRNA molecule encodes at least one functional immunostimulatory protein selected from the list comprising CD40L, CD70, and caTLR4; and the PD-1 pathway inhibitor is optionally also in the form of an mRNA molecule. This invention also relates to vaccines comprising such combinations, and the use of the combinations and vaccines of this invention in humans or veterinary medicine, particularly in the prevention and/or treatment of proliferative diseases.

Background of the Invention

Inducing an effective cytotoxic CD8 T cell response capable of recognizing and killing cancer cells constitutes a key target of any therapeutic cancer vaccine. The ability of a vaccine to elicit such a cytotoxic T cell response is largely determined by the early interaction between the vaccine and dendritic cells (DCs), the most efficient antigen-presenting cells and stimulants of T cell immunity. Unlike protein-based vaccines, mRNA vaccines are able to express mRNA-encoded antigens in the cytoplasm of DCs, which is the natural pathway for antigen processing and presentation to CD8 T cells.

Furthermore, the mRNA produced by viral polymerases such as T7 through in vitro transcription is partially similar to viral RNA and is therefore recognized by innate immune sensors, endowing the mRNA with intrinsic adjuvant properties (Kariko et al., 2005; Yoneyama et al., 2010). Nevertheless, the activation of dendritic cells (DCs) by IVT mRNA is not optimal and can be further enhanced by co-delivery of TriMix mRNA, a mixture of three mRNAs encoding the immunostimulatory proteins CD40L, CD70, and caTLR4 (Bonehill et al., 2008; Van Lint et al., 2012; Van Lint et al., 2016). Adding TriMix mRNA to mRNA encoding tumor antigens has been shown to strongly enhance the magnitude of T cell responses and its antitumor effects in preclinical models, and is currently being explored in clinical trials. Therefore, the inventors have previously determined that, both in vivo and in vitro, the T-cell stimulation capacity of APCs can be greatly enhanced by providing APCs with a specific molecular adjuvant in the form of a mixture of mRNA or DNA molecules encoding one or more immunostimulatory factors CD40L, CD70, and caTLR4. The stimulation with the immunostimulatory factors can be performed in vivo (in situ) by intravenous, intratumoral, intradermal, intraperitoneal, intramuscular, or intranodal administration of mRNA or DNA molecules encoding one or more of the aforementioned immunostimulatory factors and optionally tumor antigen mRNA, DNA, or protein. The mRNA or DNA can be naked or can be protected as described below. When administered, for example, intravenously, the mRNA or DNA can be protected.

The PD-1 gene belongs to the immunoglobulin superfamily and encodes a 55 kDa type I transmembrane protein. Both mouse PD-1 and human PD-1 consist of 288 amino acids and have a signal peptide (20 amino acids) at the N-terminus and a hydrophobic region in the middle, which is the transmembrane region.

In the thymus, PD-1 is expressed on thymocytes during the CD4-/CD8- to CD4+/CD8+ transition phase. Peripherally, PD-1 is expressed on antigen receptor-activated T cells and B cells, as well as activated myeloid cells such as macrophages.

PD-1 possesses an ITIM (immunoreceptor tyrosine inhibitory motif) in its intracellular region; therefore, PD-1 is considered a negative regulator of the immune response.

PD-1 inhibitors, such as nivolumab and pembrolizumab, have been approved and block inhibitory signaling between PD-1 and PD-L1. While these drugs enhance durable responses in some patients, their response rates as monotherapy are low. In particular, these drugs face the problem of lacking clinical benefit in most patients because treatment success largely depends on the presence of pre-existing anti-tumor T cells in the tumor bed. Furthermore, T cell exhaustion or paralysis of these anti-tumor T cells often occurs in the context of therapeutic cancer vaccines. Specifically, intratumoral immunomodulators can initiate/amplify tumor-specific T cell responses, but these T cells are often “paralyzed” by the immunosuppressive tumor microenvironment; one of these paralysis occurs through PD-1 signaling. Therefore, one object of the present invention is to provide a more effective anticancer composition based on PD-1 inhibitors. We have now surprisingly discovered that the combination of PD-1 inhibitors and the immunostimulatory factors of the present invention (CD40L, CD70 and/or caTLR4) produces a significant immunostimulatory effect, making the combination highly suitable for anticancer vaccination and treatment.

Invention Overview

This invention is defined by the following numbers:

1. Combinations, which include:

- One or more mRNA molecules, said mRNA molecules encoding at least one functional immunostimulatory protein selected from the list including CD40L, CD70, and caTLR4; and

- PD-1 pathway inhibitors, which specifically prevent or block the signaling of PD-1 activation.

2. The combination according to statement 1; wherein the one or more mRNA molecules encode all functional immunostimulatory proteins selected from the list including CD40L, CD70 and caTLR4.

3. The combination according to statement 1; wherein the PD-1 pathway inhibitor is in the form of mRNA encoding the PD-1 pathway inhibitor.

4. The combination according to statement 1; wherein the PD-1 pathway inhibitor is selected from the list including: nanobodies against PD-1, antagonistic antibodies against PD-1; nanobodies against PDL1, antagonistic antibodies against PDL1; or derivatives thereof; more particularly antagonistic antibodies against PD-1.

5. The combination according to statement 4; wherein the antagonistic antibody against PD-1 is selected from the list including: nivolumab (BMS-936558/MDX1106), pidilizumab (CT-011), and pembrolizumab (MK-3475).

6. A combination according to any one of statements 1-5; further comprising one or more mRNA molecules encoding a target-specific antigen; more particularly a tumor-associated antigen.

7. The combination according to statement 6; wherein the (mRNA molecule encoding) target-specific antigen is selected from the list including: total mRNA isolated from one or more target cells, one or more target-specific mRNA molecules, protein lysates from one or more target cells, specific proteins from one or more target cells, synthetic target-specific peptides or proteins, and synthetic mRNA or DNA encoding the target-specific antigen or one or more peptides derived therefrom.

8. The combination according to statement 7; wherein the target-specific antigen is a tumor antigen.

9. A combination according to any one of statements 1-3; wherein the one or more mRNA molecules are formulated for parenteral administration; more particularly for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

10. A combination according to any one of statements 1-3; wherein the mRNA molecule is formulated for intravenous administration and is contained in nanoparticles, such as polymeric or lipid nanoparticles.

11. A combination according to any one of statements 1-3; wherein the mRNA molecule is formulated for intratumoral or intranodal administration and is in the form of a naked mRNA molecule in a suitable injection buffer such as Ringer's lactate buffer.

12. A combination according to any one of statements 1-3; wherein the PD-1 pathway inhibitor is formulated for parenteral administration; more particularly for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

13. A vaccine comprising any combination of any one of statements 1-12.

14. The vaccine according to statement 12; wherein the vaccine is formulated for parenteral administration; and more particularly for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular or intranodal administration.

15. A combination of any one of statements 1-12 or a vaccine according to any one of statements 13-14, for use in humans or veterinary medicine.

16. A combination of any one of statements 1-12 or a vaccine according to any one of statements 13-14, for the treatment of proliferative diseases.

17. A combination of any one of statements 1-12 or a vaccine according to any one of statements 13-14, used to induce an immune response against a tumor in a subject.

Attached Figure Description

Referring now specifically to the accompanying drawings, it is emphasized that the detailed descriptions shown are merely illustrative and are intended only to discuss different embodiments of the invention in a illustrative manner. They are presented to provide what is considered most useful and readily described in terms of the principles and concepts of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than necessary for a basic understanding of the invention. The accompanying drawings enable those skilled in the art to readily recognize how several forms of the invention can be embodied in practice.

Figure 1: Illustration of possible application routes in the environment of the present invention.

Figure 2: The effect of the combination of Trimix and antigen administered in IN with anti-PD-1 administered in IP on tumor growth.

Invention Details

As detailed above, the present invention relates to an assembly comprising:

- One or more mRNA molecules, said mRNA molecules encoding at least one functional immunostimulatory protein selected from the list including CD40L, CD70, and caTLR4; and

- PD-1 pathway inhibitors, which specifically prevent or block the signaling of PD-1 activation.

In this invention, the term "TriMix" refers to a mixture of mRNA molecules encoding CD40L, CD70, and caTLR4 immunostimulatory proteins.

The mRNA or DNA used or mentioned herein may be naked mRNA or DNA, or protected mRNA or DNA. Protection of DNA or mRNA increases its stability while preserving the ability to use the mRNA or DNA for vaccination purposes. Non-limiting examples of mRNA and DNA protection may include: liposome encapsulation, protamine protection, (cationic) lipid-lipoplexation, lipid, cationic or polycationic compositions, mannosylated lipid complexes, bubble liposomes, polyethyleneimine (PEI) protection, liposome-loaded microbubble protection, etc.

The term "target" as used throughout this specification is not limited to the specific instances that may be described herein. It can target any infectious agent, such as a virus, bacteria, or fungus. Furthermore, it can target any tumor or cancer cell.

The term "target-specific antigen" as used throughout this specification is not limited to the specific instances that may be described herein. Those skilled in the art will understand that this invention relates to the induction of immune stimulation in APCs, and is unrelated to the target-specific antigen presented. The antigen to be presented will depend on the type of target to which one intends to elicit an immune response in a subject. Typical examples of target-specific antigens are markers of specific expression or secretion of tumor, bacterial, and fungal cells, or specific viral proteins or viral structures. Without intending to limit the scope of the invention, some examples of possible markers are listed below.

The term "antigen-presenting cell" as used throughout this instruction manual includes all APCs. Specific, non-limiting examples are dendritic cells (DCs), dendritic cell lines, B cells, or B cell lines. DCs or B cells may be isolated from or generated from the blood of a patient or healthy subject. The patient or subject may or may not be a previously vaccinated subject.

The terms “vesicle,” “cancer,” and/or “tumor” as used throughout this specification are not intended to be limited to the types of cancer or tumors that may have been exemplified. Therefore, the term encompasses all proliferative conditions, such as vegetations, developmental abnormalities, precancerous or malignant lesions, abnormal cell growth, benign tumors, malignant tumors, cancers, or metastases, wherein the cancers are selected from: leukemia, non-small cell lung cancer, small cell lung cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, glioma, colon cancer, bladder cancer, sarcoma, pancreatic cancer, colorectal cancer, head and neck cancer, liver cancer, bone cancer, bone marrow cancer, stomach cancer, duodenal cancer, esophageal cancer, thyroid cancer, hematologic malignancies, and lymphoma. Cancer-specific antigens may include, for example, MelanA/MART1, cancer-germline antigens, gp100, tyrosinase, CEA, PSA, Her-2/neu, survival proteins, and telomerase.

The combined use of CD40L and caTLR4 produces mature, secretory cytokine/chemokine DCs, as shown by linking CD40 and TLR4 with the addition of soluble CD40L and LPS.

Introducing CD70 into DCs provides co-stimulatory signals to CD27 ⁺ naive T cells by inhibiting activated T cell apoptosis and supporting T cell proliferation.

Other Toll-like receptors (TLRs) can be used as alternatives to caTLR4. For each TLR, the constitutively active form is known and can potentially be introduced into dendritic cells (DCs) to elicit a host immune response. However, caTLR4 is considered the most potent activating molecule and is therefore preferred.

In a preferred embodiment of the vaccine of the present invention, the mRNA or DNA molecule encodes CD40L and CD70 immunostimulatory proteins. In a particularly preferred embodiment of the vaccine of the present invention, the mRNA or DNA molecule encodes CD40L, CD70, and caTLR4 immunostimulatory proteins.

The mRNA or DNA molecule encoding the immunostimulatory protein may be part of a single mRNA or DNA molecule. Preferably, the single mRNA or DNA molecule is capable of simultaneously expressing two or more proteins. In one embodiment, the mRNA or DNA molecule encoding the immunostimulatory protein is separated within a single mRNA or DNA molecule by an internal ribosome entry site (IRES) or by a self-cleaving 2a peptide coding sequence.

In one specific embodiment, one or more mRNA molecules described in this invention may further contain translation enhancers and/or nuclear retention elements. Suitable translation enhancers and nuclear retention elements are those described in WO2015071295.

As used herein, the term "PD-1 inhibitor" includes any compound that can directly or indirectly affect PD-1 regulation by reducing, for example, the expression (i.e., transcription and/or translation) of PD-1 or its natural ligands PD-L1/PD-L2, or the activity of PD-1. It includes intracellular (e.g., agents that block PD-1-related signaling molecules or pathways, such as SHP-1 and SHP-2) and extracellular PD-1 inhibitors. Without this limitation, such inhibitors include siRNAs, antisense molecules, proteins, peptides, small molecules, antibodies, etc.

Therefore, in one specific embodiment, the PD-1 pathway inhibitor is selected from the following list: nanobodies targeting PD-1, antagonistic antibodies against PD-1; nanobodies targeting PDL1, antagonistic antibodies against PDL1; or derivatives thereof. Antibody derivatives may include, for example, scFV, bispecific antibodies, etc.

In one embodiment, the aforementioned PD-1 inhibitor blocks/inhibits the interaction between PD-1 and PD-1 ligands (e.g., PD-L1, PD-L2). Such inhibitors may target, for example, the IgV domains of PD-1 and/or PD-L1 and/or PD-L2, such as one or more residues involved in the interaction, as described above.

In one embodiment, the aforementioned PD-1 inhibitor is a blocking antibody, such as an anti-PD-1 or anti-PD-L1/PD-L2 antibody. Blocking anti-PD-1 and/or anti-PD-L1/PD-L2 antibodies are well known in the art. As described above, those skilled in the art can readily identify and prepare other blocking antibodies based on known interaction domains between PD-1 and PD-L1/PD-L2. For example, peptides corresponding to the IgV region (or a portion thereof) of PD-1 or PD-L1/PD-L2 can be used as antigens and blocking antibodies can be developed using methods well known in the art.

In the context of this invention, "anti-PD-1 antibody," "anti-PD-L1," or "anti-PD-L2" refers to an antibody capable of detecting/recognizing (i.e., binding) PD-1, PD-L1, or PD-L2 proteins or fragments of PD-1, PD-L1, or PD-L2 proteins. In one embodiment, the antibody inhibits the biological activity of PD-1, such as PD-1-PD-L1/PD-L2 interactions or PD-1-mediated T-cell suppression. In another embodiment, the PD-1 or PD-L1/PD-L2 protein fragment is an extracellular domain (e.g., an IgV domain) of PD-1 or PD-L1/PD-L2.

As used herein, the term "PD-1 level" refers to the expression level of PD-1 on T cells or the percentage of PD-1-positive T cells that are also CD4 ⁺ or CD8 ⁺ , expressed as a percentage of the total number of CD4 ⁺ or CD8 ⁺ cells. The expression level of PD-1 on T cells can be determined by any standard method. For example, it can be determined and measured as mean fluorescence intensity (MFI) by flow cytometry.

In one embodiment, this disclosure provides a method of combination therapy for an individual, comprising administering one or more immunostimulatory factors and a PD-1 inhibitor to the individual. The one or more immunostimulatory factors and the PD-1 inhibitor may be administered simultaneously or concurrently as a single composition or separate compositions, or the one or more immunostimulatory factors and the PD-1 inhibitor may be administered at different times.

The compositions of the present invention typically comprise a combination of a PD-1 inhibitor and one or more immunostimulatory factors. PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds to two ligands, PD-L1 and PD-L2. PD-1 plays an important role in downregulating the immune system by preventing T cell activation, thereby reducing autoimmunity and promoting self-tolerance. The inhibitory effect of PD-1 is achieved through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in Tregs. Suitable PD-1 inhibitors for use in the compositions of this disclosure include nivolumab (BMS-936558/MDX1106), pildizumab (CT-011), pembrolizumab (MK-3475), and combinations thereof; alternatively, the PD-L1 inhibitor atezolizumab may also be suitable in the context of the present invention.

The appropriate dosage of a PD-1 inhibitor will depend on many factors, including an individual's age and weight, at least one specific condition requiring treatment, the severity of the condition, the nature of the composition, the route of administration, and the combination thereof. Ultimately, those skilled in the art (e.g., physicians, veterinarians, scientists, and other medical and research professionals) can readily determine the appropriate dosage. For example, those skilled in the art may start with a low dose and increase it until the desired therapeutic effect or outcome is achieved. Alternatively, those skilled in the art may start with a high dose and decrease it until the minimum dose required to achieve the desired therapeutic effect or outcome is reached.

This invention also provides combinations as defined herein; wherein the mRNA molecule is formulated for intravenous administration and is contained in (lipid) nanoparticles. Lipid nanoparticles (LNPs) are generally known as nanoscale particles composed of combinations of 25 different lipids. Although such LNPs may include many different types of lipids, the LNPs of this invention may, for example, consist of combinations of ionizable lipids, phospholipids, sterols, and PEG lipids.

As used herein, the term “nanoparticle” refers to any particle having a diameter suitable for whole-body, particularly intravenous (especially for nucleic acid) administration, typically having a diameter of less than 1000 nanometers (nm).

In an alternative embodiment, the invention provides combinations as defined herein; wherein the mRNA molecule is formulated for intratumoral or intranodal administration and is in the form of naked mRNA molecules in a suitable injection buffer (e.g., Ringer's lactate buffer). The invention also provides combinations and vaccines as defined herein for use in humans or veterinary medicine, particularly for the treatment of proliferative diseases, and more particularly for inducing an immune response against tumors in subjects.

Finally, the present invention provides a method for treating a cell proliferation disorder, comprising the step of administering the combination or vaccine of the present invention to a subject in need.

Example

Example 1: A combination of Trimix, antigen, and anti-PD-1.

The scope of this embodiment is to elicit a T-cell immune response against a predefined TAA when using Trimix as a monotherapy or in combination with anti-PD-1. The advantages of this setup include:

-Ability to join TAA

Trimix mRNA as an adjuvant to enhance T cell responses against TAA.

- Two routes of administration (intra-nodal (IN) and intravenous (IV)) were explored, as shown in Figure 1.

-IV administration shows greater immunogenicity; however, additional formulations are required, such as LNP.

On day 0, female Balb/C mice were injected with 1 × ^10⁶ CT26 tumor cells. On days 5, 10, and 15, GP70-Trimix mRNA (10 μg of each component) was administered intranodally. On day 5, anti-PD-1 monoclonal antibody (10 mg/kg) was administered intraperitoneally, repeated 5 times at 3-day intervals. Mice without tumors by day 40 were considered complete responders (CR).

The results of this embodiment are shown in Figure 2; this clearly demonstrates that when anti-PD-1 (2 out of 6 complete responders) and GP70-Trimix (4 out of 8 complete responders) are used alone, they show a beneficial effect on tumor growth. Furthermore, the combination of GP70-Trimix and anti-PD-1 showed an even further increase in tumor growth, with 7 out of 8 mice being completely tumor-free by day 40.

Claims (10)

1. Combinations, which include: - One or more mRNA molecules that encode all functional immunostimulatory proteins CD40L, CD70, and caTLR4 (constitutive active TLR4); - One or more mRNA molecules encoding colorectal cancer-specific antigens; and - PD-1 pathway inhibitors, wherein the PD-1 pathway inhibitors are selected from the list including: nanobodies against PD-1, antagonistic antibodies against PD-1; nanobodies against PDL1, antagonistic antibodies against PDL1; and It is used to prepare drugs for treating colorectal cancer. The one or more mRNA molecules are formulated for intravenous administration. 2. The combination according to claim 1, wherein the antagonistic antibody against PD-1 is selected from the list including: nivolumab (BMS-936558/MDX1106), pildizumab (CT-011), and pembrolizumab (MK-3475). 3. The combination according to any one of claims 1-2, wherein the mRNA molecule is formulated for intravenous administration and is contained in nanoparticles. 4. The combination according to claim 3, wherein the nanoparticles are lipid nanoparticles. 5. The combination according to claim 4, wherein the lipid nanoparticles comprise a combination of ionizable lipids, phospholipids, sterols, and PEG lipids. 6. The combination according to any one of claims 1-2, wherein the PD-1 pathway inhibitor is formulated for parenteral administration. 7. The combination according to any one of claims 1-2, wherein the PD-1 pathway inhibitor is formulated for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular, or intranodal administration. 8. A vaccine comprising the combination of any one of claims 1-7. 9. The vaccine of claim 8, wherein the vaccine is formulated for parenteral administration. 10. The vaccine of claim 8, wherein the vaccine is formulated for intravenous, intratumoral, intradermal, subcutaneous, intraperitoneal, intramuscular, or intranodal administration.