WO2019246142A1

WO2019246142A1 - Soluble bispecific fusion proteins for cancer immunotherapy

Info

Publication number: WO2019246142A1
Application number: PCT/US2019/037792
Authority: WO
Inventors: Haiming Chen; Ou ZHU; Zongcen XIE
Original assignee: Haiming Chen; Zhu ou; Xie Zongcen
Priority date: 2018-06-18
Filing date: 2019-06-18
Publication date: 2019-12-26

Abstract

A bispecific fusion protein agent may include at least one binding site with human IgG1 hinge that specifically binds to a function protein and at least another one binding site with human IgG1 hinge that specifically binds to a different function protein. For example, the bispecific fusion protein agent can be a bispecific fusion protein agent PD-1/LAG-3 that includes a biding site of the human IgGl hinge that specifically crosslinks to a LAG-3 molecule, and another binding site of the human IgG1 hinge that specifically crosslinks to a PD-1 molecule.

Description

SOLUBLE BISPECIFIC FUSION PROTEINS FOR CANCER

IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 (e) to U.S. Provisional Patent Application Ser. No. 62/763,448, filed on June 18, 2018, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention constructs bispecific fusion proteins PD-1 and another immune checkpoint molecule without a specific antibody. The bispecific fusion proteins target ligands of specific immune checkpoints such as PD-1 and LAG-3 fusion protein targets PD-1/PD-L1 and LAG-3/MHC-II immune checkpoint pathways. PD-1 and TIM-3 fusion protein targets PD-1/PD-L1 and TIM-3/Gal-9 immune checkpoint pathways. Therefore, the bispecific fusion proteins activate endogens immune reaction to attack cancer cells.

BACKGROUND OF THE INVENTION

[0003] Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and negative regulate immune reaction with its ligand, programmed death ligand 1 (PD-L1). (Jin HT, et al. Curr Top Microbiol Immunol 2011; 350:17- 37; Hutloff et al. Nature 1999; 397:263-266). Target PD-1 is the broader context of cancer immunotherapy or and autoimmune diseases. PD-L1 is expressed on cancer cells of human lung, breast, ovarian, and colon carcinoma as well as various myelomas. When PD-1 and PD-L1 are binding together, the PD1/PD-L1 complex forms a biochemical molecule protecting tumor cells being attacked by the immune system. Clinical studies have been demonstrated using monoclonal antibody against either PD-1 or PD-L1 increased T cell destroying tumor cells. It has validated targeting the PD-1/PD-L1 pathway to activate anti-tumor immunity. It will be an important target for cancer immunotherapy (Pardoll DM Nature Reviews Cancer 2012; 12, 252-264) Lymphocyte-activation gene 3 (LAG- 3; CD223) is a cell surface molecule expressed on activated T cells, NK cells, B cells and dendritic cells that plays an important role in immunotherapies. LAG-3 negatively regulates cellular proliferation, activation, and homeostasis of T cells. LAG-3's ligand is MHC class II. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), another immune checkpoint, could inhibit cancer immunity. TIM-3 has four ligands included galectin-9 (Gal-9), high mobility group protein B1 (HMGB1), carcinoembryonic antigen cell adhesion molecule 1 (Ceacam-1), and phosphatidylserine (PtdSer). Study demonstrated that TIM-3 has an important role to play in T-cell exhaustion and correlates with the outcome of anti-PD-1 therapy. In anti-cancer immunity, it is not only PD1/PD-L1 plays an inhibitory immune reaction but also some other groups of molecules are negative regulation of anti-cancer in human. LAG-3 (CD223) is expressed on cell membrane of activated T cells, B cells, and dendritic cells that plays an important role in anti-tumor immunity.

[0004] Immunotherapeutic monoclonal antibodies have revolutionized cancer treatment over the past several years, demonstrating superior tolerability and improvements in long-term survival. However, majority (50-75%) of patients do not respond monoclonal antibody immunotherapy and immune-related adverse events such as colitis, diarrhea, dermatological toxicity, endocrinopathy, hepatotoxicity and pneumonitis limit the clinical use of immunotherapeutic antibodies. Bispecific immunotherapies have the potential to improve clinical efficacy as well as safety and can be seen as the next generation of immunotherapies. This invention is to clone and express a fusion protein of two checkpoint molecules. The fusion protein is soluble and specific targets two ligands of checkpoints. PD-1 and LAG-3 fusion protein blocks PD-1/PD-L1 and LAG-3/MHC-II immune checkpoint pathways. PD-1 and TIM-3 fusion protein blocks PD-1/PD-L1 and TIM-3/Gal-9 immune checkpoint pathways.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 is a schematic view of the bi-specific fusion proteins in the present invention.

[0006] FIG. 2A illustrates a schematic view of the bi-specific fusion proteins of LAG-3-Fc-PD-l in the present invention.

[0007] FIG. 2B illustrates the nucleic acid sequence of LAG-3-Fc-PD-l (SLFP) in the present invention.

[0008] FIG. 2C illustrates the amino acid sequence of LAG-3-Fc-PD-l (SLFP) in the present invention.

[0009] FIG. 2D illustrates the Western Blot analysis of LAG-3-Fc-PD-l (SLFP) in the present invention.

[0010] FIG. 3A illustrates the IL-2 secretion in human mixed lymphocyte reaction (MLR) analysis of LAG-3/PD-1 fusion protein (LPFP) in the present invention.

[0011] FIG. 3B illustrates the IFN-g secretion in human mixed lymphocyte reaction (MLR) analysis of LAG-3/PD-1 fusion protein (LPFP).

[0012] FIG. 4A shows LPFP inhibited tumor growth in NSCLC-bearing mice. Experimental design and treatment schedule for LPFP against NSCLC (HCC-827) tumors. Treatments started when tumors reached 50 mm³ in volume. LPFP treatment groups have significantly smaller tumor size compared with the vehicle control group. [0013] FIG. 4B shows LPFP inhibited breast cancer growth in breast cancer-bearing mice. MDA-MB-231 human breast cancer cells (5 ^c 10⁶) in matrigel were injected into mammary fat pads of breast cancer bearing mice. Experimental design and treatment schedule for LPFP against human breast cancer cells. Treatments started when tumors reached 50 mm³ in volume. LPFP treatment groups have significantly smaller tumor size compared with the vehicle control PBS group.

[0014] FIG. 4C shows percentage of breast cancer volume inhibited by LPFP treatment. LPFP treatment groups have significantly smaller tumor size compared with the vehicle control PBS group.

[0015] FIG. 4D shows mean % inhibition of tumor volume.

[0016] FIG. 5A is a schematic view of bi-specific fusion protein of PD- l/TIM-3 in the present invention.

[0017] FIG. 5B illustrates the amino acid sequence of PD-l/TIM-3 in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The detailed description set forth below is intended as a description of the presently exemplary device provided in accordance with aspects of the present invention and is not intended to represent the only forms in which the present invention may be prepared or utilized. It is to be understood, rather, that the same or equivalent functions and components may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of the invention.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described can be used in the practice or testing of the invention, the exemplary methods, devices and materials are now described.

[0020] All publications mentioned are incorporated by reference for the purpose of describing and disclosing, for example, the designs and methodologies that are described in the publications that might be used in connection with the presently described invention. The publications listed or discussed above, below and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

[0021] As used in the description herein and throughout the claims that follow, the meaning of "a", "an", and "the" includes reference to the plural unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the terms "comprise or comprising", "include or including", "have or having", "contain or containing" and the like are to be understood to be open-ended, i.e., to mean including but not limited to. As used in the description herein and throughout the claims that follow, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0022] It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element could be termed a second element, and, similarly, a second element could be termed a first element, without departing from the scope of the embodiments. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. [0023] Human immune checkpoints are regulators of the immune system. These checkpoint pathways are crucial for prevents immune system attacking cancer cells. Programmed cell death 1 (PD-1), as an immune checkpoint plays an inhibitory immune receptor. Blockade of PD-l/programmed death ligand 1 (PD- Ll) axis activates the immune system to attack cancer cells. Bispecific antibodies are a rapidly growing class of therapeutic molecules for the treatment of cancer. Here we describe the generation of a soluble bispecific fusion proteins by fusion PD-1 with LAG-3 or fusion the PD-1 with Tim3 to block immune checkpoint pathway for treatment of cancers. As a novel immune checkpoint blockade, PD-1 and LAG-3 or PD-1 and Tim3 fusion protein blocks immune checkpoint pathway by neutralizing targeted checkpoint ligand. The fusion protein is soluble and different from bispecific antibodies that binding to immune checkpoint directly. The invention additionally concerns the uses of such immune checkpoint blockade molecules in the treatment of cancers and other diseases.

[0024] Tumor-induced immune suppression is a major obstacle for chemotherapy. Stimulate an immune response and activating an individual's immune system to against tumor cells is our new approach. The present invention is to provide a fusion protein, wherein a new form of immune therapy, which is based on a fusion protein comprised of two different inhibitory immune receptors by endogens human IgG Fc hinge without any other chemical linkers (Figure 1). The soluble fusion protein (PD-1) that interacts with PD-L1 and another inhibitory immune receptor interaction with its ligand, thus, minimizing PD-L1 and another ligand mediated immune suppression.

[0025] A major objective of the present invention is to provide bispecific fusion proteins, wherein a new form of immune therapy, which is based on a fusion protein comprised of the human two different function proteins connected by two human IgGl endogens hinge without any other chemical linkers. This fusion protein is for treatment of cancer and autoimmune disease.

[0026] One aspect of the present disclosure provides fusions that can act as immune checkpoint blockade molecules in the treatment of cancers and other diseases. In one embodiment, the fusion can be novel antigen-binding fragments. Fusion protein disclosed herein is able to bind to human PD-L1 from cancer cells blocking the contact of PD-land PD-L1 and bind to human MHC-II blocking LAG-3/ MHC-II immune checkpoint pathways on immune cells in human, thereby inhibiting the PD-1/ PD-L1 pathway. Exemplary of fusion protein for use in the context of this disclosure is included, but are not limited to fusion produced by clone P-L. This application of invent provide a bi-specific fusion proteins that co-express LAG-3 and PD-1 (Figure 2 A). The sequence of the LAG- 3/f PD-1 fusion protein is linked by two human IgG hinges. The LAG-3/Fc-PD-l (SLFP) nucleic acid sequence is 1971 base pairs (BPs) (Figure 2B) and protein sequence is 656 amino acids (Figure 2C). Western blot analysis demonstrated the fusion protein is a complete molecule with human PD1, LAG-3, Ig-Fc and Ig- hinge (Figure 2D). LAG-3 of the fusion protein prevents MHC class II, a ligand of LAG-3, binding to immune cells and the PD-1 of the fusion protein reduces PD-L1 of cancer cell crosslink to PD-1 membrane protein of T cell. By blocking or inhibiting the interaction of these inhibitory receptors with their ligand by binding specifically to one or more ligand interaction sites, these compositions and methods prevent and/or inhibit PD-1 and LAG-3 inhibitory signals, and thus permit the restoration of or increase in the immune response in cancer patients. IL-2 (in human mixed lymphocyte reaction, MLR) production is as an indication of early stages of T cell activation. We here describe a shorter assay in which IL-2 secretion during MLR was assessed by adding the LAG-3/PD-1 fusion protein (LPFP) MLR supernatants or directly to the original MLR cultures. The result showed IL-2 secretion was increased in human mixed lymphocyte treated with LPFP (Figure 3A) by concentration dependent fashion. IFN-y secreted by innate immune cells and plays a critical role in Th-1 polarization and stimulates dendritic cells (DCs) s and macrophages, up-regulating pro-inflammatory factors such as IL-12 and IL-15. MLR also demonstrated IFN-g secretion was upregulated in human mixed lymphocyte by LPFP (Figure 3B). DCs are central players in immunity, bridging the innate to the adaptive arms of the immune system. To evaluate LPFP effect on human cancers, mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems. Tumor-bearing HuNSG mice are an important experimental model for preclinical immunotherapy research. Growth curves of non-small cell lung cancer (NSCLC) or breast cancer treated with LPFP were compared with control groups. We demonstrated that LPFP treatment significantly delayed NSCLC (HCC-827) growth compared with the vehicle control-treated group (P < 0.01; Figure 4A). To test the LPFP effects on another cancer, we studied treatment responses in the breast cancer tumor-bearing mice generated from human (h)CD34+ hematopoietic progenitor and stem cells transplanted. LPFP significantly inhibited breast cancer (MDA-MB-231) tumor growth (Figure 4B). Percentage of inhibition tumor volume was from 20% to 50% (Figure 4C). Mean %L inhibition of tumor volume is from 90% to 20%.

[0027] During the experiment, the animals were in good mental state and the animals remained stable. Until the end of the experiment, there was no animal death in each group. Treatment with LPFP which targets PD1/PDL-1 and LAG-3/MHC-II immune checkpoint pathways, produced significant growth inhibition in both NSCLC and breast cancer in human (h)CD34+ mice. In various embodiments, the bispecific fusion proteins may be administered to a subject for treatment of a condition. The skilled artisan will realize that any condition that may be treated with bispecific fusion proteins may be treated with the subject compositions and methods. Exemplary conditions include, but are not limited to cancer: adrenal cancer, anal cancer, bile duct cancer, bladder cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gestational trophoblastic disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancers, malignant mesothelioma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancers, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.

[0028] TIM-3 is a type I trans-membrane protein and plays an important role in inhibiting Thl responses and the expression of cytokines such as TNF and INF-y. Dysregulation of TIM-3 expression has been associated with autoimmune diseases. TIM-3 on tumor-infiltrating dendritic cell has been showed a critical role in suppressing innate anti-tumor immune responses through the recognition of tumor-derived nucleic acids. Therefore, TIM-3 plays a negative regulatory checkpoint role associated with both T cell exhaustion and suppression of innate immune responses. In this embodiment, PD-l/TIM-3 fusion protein for targeting immune cells, that co-express the inhibitory the binding site that specifically binds to the PD-Lland TIM-3 signaling pathway interaction (Figure 5A). The amino acid sequence of the PD1/ Tim-3 fusion protein is crosslinked by two human IgGl hinges, and the amino acid sequence of PD-l/TIM-3 is shown in Figure 5B. MLR result showed IL-2 secretion was increased in human mixed lymphocyte treated by adding the TIM3/PD1 fusion protein.

[0029] The present invention is advantageous because the bispecific fusion protein's ingredients originate from human immunoglobulin. Therefore, this protein enters the human body as a medicine; it does not have any foreign antigens (foreign body protein immunity source). Bispecific fusion protein two connection sites (hinge) obtains characteristics such as being nimble flexible rotation and able to connect two target ligands to strongly prohibit inhibitory signal transduction pathway of immune cells. All these results from the experiment prove that the bispecific fusion protein can effectively target specific ligands from cancer cells to prevent immune cell silence. Therefore, the bispecific fusion proteins activate endogens immune reaction to attack cancer cells. This is a novel bio-drugs for cancer therapy and combination with other anti-cancer agents to increase the effect of treatment. It is noted that the use of a fusion protein in the present invention to manufacture a medicament for treating cancer or other diseases also includes an agent to activate T-cells in the patient, e.g. IL-2.

[0030] Having described the invention by the description and illustrations above, it should be understood that these are exemplary of the invention and are not to be considered as limiting. Accordingly, the invention is not to be considered as limited by the foregoing description, but includes any equivalent.

Claims

WHAT IS CLAIMED IS:

1. A bispecific fusion protein agent comprising at least one binding site with human IgGl hinge that specifically binds to a function protein and at least another one binding site with human IgGl hinge that specifically binds to a different function protein.

2. The bispecific fusion protein agent of claim 1, wherein a bispecific fusion protein agent PD-l/LAG-3 has the LAG-3-Fc-PD-l (SLFP) nucleic acid sequence, including a biding site of the human IgGl hinge that specifically crosslinks to a LAG-3 molecule, and another binding site of the human IgGl hinge that specifically crosslinks to a PD-1 molecule.

3. The bispecific fusion protein agent of claim 2, wherein the bispecific fusion protein PD-l/LAG-3 can be used for treatment of cancers including non small cell lung cancer (NSCLC), breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/CNS tumors, breast cancer, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gestational trophoblastic disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancers, malignant mesothelioma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancers, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.

4. The bispecific fusion protein agent of claim 2, wherein the bispecific agent PD-l/LAG-3 fusion protein can be used to combine with other anti-cancer agents such as chemotherapeutic, radioactive, immunomodulatory drugs for treatment of cancers including non-small cell lung cancer (NSCLC), breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/CNS tumors, breast cancer, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gestational trophoblastic disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancers, malignant mesothelioma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancers, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.

5. The bispecific fusion protein agent of claim 1, wherein the bispecific fusion protein agent PD-l/TIM-3 has the amino acid sequence of PD-l/TIM-3, including a biding site of the human IgGl hinge that specifically crosslinks to a TIM-3 molecule, and another binding site of the human IgGl hinge that specifically crosslinks to a PD-1 molecule.

6. The bispecific fusion protein agent of claim 5, wherein the bispecific fusion protein PD-l/TIM-3 can be used for treatment of cancers including non small cell lung cancer (NSCLC), breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/CNS tumors, breast cancer, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gestational trophoblastic disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancers, malignant mesothelioma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancers, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.

7. The bispecific fusion protein agent of claim 5, wherein the bispecific agent PD-l/TIM-3 fusion protein can be used to combine with with other anti-cancer agents such as chemotherapeutic, radioactive, immunomodulatory drugs for treating cancers including non-small cell lung cancer (NSCLC), breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain/CNS tumors, breast cancer, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gestational trophoblastic disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancers, malignant mesothelioma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancers, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.