Patent Application
20240034776
This application claims priorities to Chinese Patent Application No. 202011191447.3, entitled “USE OF REGULATOR OF ITPRIPL1 IN PREPARATION OF DRUG THAT REGULATES IMMUNE RESPONSES OR FIGHTS TUMORS”, filed to China National Intellectual Property Administration on Oct. 30, 2020, and Chinese Patent Application No. 202110566040.2, entitled “ISOLATED ANTIGENIC ITPRIPL1-BINDING PROTEIN AND USE THEREOF”, filed to China National Intellectual Property Administration on May 24, 2021, the entire contents of which are incorporated herein by reference.
The present disclosure relates to the field of biomedicine, and specifically to a use of a regulator of ITPRIPL1 in the preparation of a drug that regulates immune responses or resists tumors.
ITPRIPL1-encoded protein is Inositol 1,4,5-trisphosphate receptor-interacting protein-like 1, for which the function of ITPRIPL1 has never been reported. Based on the annotation of UniProtKB database, ITPRIPL1 of human includes 555 amino acids, which are divided into an extracellular domain (1-103 amino acids), a transmembrane domain (104-124 amino acids), and an intracellular domain (125-555 amino acids).
T cells are key effector cells of adaptive immune responses, which have many important roles in eliminating pathogens and autoimmune diseases. There are several subpopulations of T cells, each with a different function. TCRs (T cell receptors) found on the surface of T cells are heterodimers composed of α and β polypeptide chains, which constitute about 95% of the TCR population, or they are composed of γ and δ polypeptide chains (Pitcher and van Oers, 2003). Each kind of polypeptide includes constant (C) and variable (V) regions. The constant regions are anchored in the cell membrane, while the variable regions extend outside the cells and are responsible for binding the antigen. The cytoplasmic short tails of TCRs are lack of the ability to transduce signals. Intracellular signaling is initiated by the CD3 protein complex, which includes intracellular immunoreceptor tyrosine-based activation motifs (ITAMs).
CD3 (Cluster of Differentiation 3) T cell co-receptors are a kind of protein complex consisting of four different chains. In mammals, a complex includes one CD3γ (γ) chain, one CD3δ (δ) chain and two CD3ε (ε) chains. These chains are correlated to TCRs and ξ chain (zeta chain), and produce activation signals in T lymphocytes. The TCRs, ξ chain and CD3 molecules together form the TCR complex. CD3γ, CD3δ and CD3ε chains are highly related cell surface protein of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. TCRs are not capable of binding free epitopes/antigens. In contrast, TCRs can bind cleaved fragments of larger polypeptides associated with a major histocompatibility complex (MHC), which is synonymous with the human leukocyte antigen (HLA) system in humans. Such an interaction occurs in a space known as the immune synapse. Class I MHC molecules are expressed on all nucleated cells of human, and present antigens to cytotoxic T cells, on which CD8 stabilizes the MHC/TCR interaction. The activation of cytotoxic T cells then leads to the destruction of target cells. Class II MHCs are found on macrophages, B cells and dendritic cells. These immune cells present antigens to helper T cells with CD4 that stabilizes the MHC/TCR interaction. The interaction between Class II MHC and TCR finally leads to antibody-mediated immune responses. Other costimulatory molecules, such as CD45, CD28 and CD2, contribute to the activation of T cells in the immune synapse and initiate the formation of TCR signalosomes which are macromolecular protein complexes responsible for intracellular signaling.
Several antibodies that bind to human CD3ε have ever been reported, for example, antibody OKT3 (see, e.g., Kung, P. et, al, Science 206 (1979) 347-349; Salmeron, A. et, al, J Immunol 147 (1991) 3047-3052), antibody UCHT1 (see, e.g., Callard, R E et, al, Clin Exp Immunol 43 (1981) 497-505) or antibody SP34 (see, e.g., Pessano, S. et, al, EMBO J 4 (1985) 337-344). Among the currently known antibodies, SP34 is cross-reactive in human and cynomolgus monkey (Conrad M. L. et, al, Cytometry A 71 (2007)925-933). It has been known that proteins directly binding to the CD3ε extracellular domain are antibody molecules, and there has never reported that natural non-antibody proteins (e.g., transmembrane proteins, secretory proteins and other typical ligands) bind to the CD3ε extracellular domain.
Neuropilin-2 (i.e., NRP-2) is a kind of receptor capable of regulating the function of immune cells (Am J Physiol Lung Cell Mol Physiol. 2018), which is reported to regulate the function of antigen-presenting cells and promote the immune evasion of tumors (Sohini Roy et, al, Cancer Res. 2018); NRP2 may affect the migration and phagocytic function of immune cells as well as the contact among immune cells (S Schellenburg et, al, Mol Immunol. 2017). Co-receptors formed from NRP2 and Plexin have negative chemotactic effects on the migration of lymphatic endothelial cells (Liu X et, al, Cell Rep. 2016). NRP2 can also regulate the NFKB signaling in cells, which is seen in the report (Rizzolio, S. et, al. Cancer Research.2017). NRP2 ligands that have been found include Semaphorin family members, but other types of ligands have not been reported.
The present disclosure is intended to provide a method of regulating immune responses and suppressing tumors, as well as a use of a regulator of ITPRIPL1 in the preparation of a drug that regulates immune responses or resists tumors.
To achieve the above objectives, the present disclosure provides a use of a regulator of ITPRIPL1 in the preparation of a drug that regulates immune responses or resists tumors, in which the regulator is used to increase or decrease the expression or function of the ITPRIPL1 gene or protein in an organism.
Preferably, the regulator includes any one of the following:
Preferably, the gene editing system is a CRISPR/Cas9 gene editing system; a target sequence used in the CRISPR/Cas9 gene editing system is selected from any one sequence as set forth in SEQ ID NOs: 11-13, and an oligomeric DNA sequence for encoding sgRNA is selected from SEQ ID NOs:14-19;
Preferably, the sequence of the functional fragment is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 4, or a derivative sequence thereof. The derivative sequence includes DRMDLDTLARSRQLEKRMSEEMRxLEMEFEERxxxAExxQKxENxWxGxTSxDQ (“x” is any amino acid). The derivation method includes: substituting, deleting or inserting more than one amino acid without changing the function of the sequence.
Preferably, the ITPRIPL1 recombinant protein forms a fusion protein (as set forth in SEQ ID NOs: 5-7) with an antibody constant region, or forms a fusion protein with a coagulation factor; alternatively, the ITPRIPL1 recombinant protein is modified by means of: polyethylene glycol modification, glycosylation modification, polysialic acid modification, fatty acid modification, KLH modification, biotin modification.
Preferably, the nucleic acid molecule is introduced into the cell through a drug delivery system which includes recombinant expression vectors, viruses, lipidosome or nanomaterials.
Preferably, the regulation of immune responses includes: regulating the functions of antigen presenting cells and T lymphocytes during the processes of autoimmune responses, transplant rejection-suppressing immune responses, allergies, anti-infection immune responses, and anti-tumor immune responses.
Preferably, the immune responses include: type I diabetes, immunologic infertility, rejection after organ transplantation, allergies, systemic inflammation or cytokine storm, and infection.
Preferably, the tumors are solid tumors or hematological tumors; the solid tumors include: glioma, lung cancer, head and neck cancer, gastric cancer, colorectal cancer, thyroid cancer, esophagus cancer, urothelial carcinoma, testicular cancer, breast cancer, cervical cancer, endometrial cancer, melanoma, pancreatic cancer or liver cancer; the hematological tumors include: leukemia or lymphoma.
The present disclosure further provides a pharmaceutical composition, which includes a regulator used in the above use, and a pharmaceutically acceptable carrier.
The present disclosure further provides an isolated ITPRIPL1 recombinant protein, and the recombinant protein is a functional fragment capable of binding to CD3ε or NRP2 protein in the extracellular domain of the ITPRIPL1 protein.
The present disclosure further provides an antibody that recognizes and binds to the ITPRIPL1, and the antibody recognizes and binds to the extracellular domain of the ITPRIPL1 protein.
The present disclosure further provides an application of the isolated ITPRIPL1 recombinant protein for detecting the presence of its own anti-ITPRIPL1 antibody, wherein the main steps of detection include directly contacting the ITPRIPL1 recombinant protein with the blood sample from a subject, and washing to remove nonspecific binding.
The present disclosure further provides applications of the above antibody for detecting the content of ITPRIPL1 in a sample, and for judging the expression of ITPRIPL1 in cells, tissues, organs or individuals by the antibody that recognizes and binds to the ITPRIPL1, or for judging whether it is suitable to apply the method of the present disclosure to regulate immune responses and suppress tumors by targeting ITPRIPL1. In some embodiments, provided are applications of an antibody that specifically recognizes ITPRIPL1 for marking boundaries between cancer tissues and para-cancerous tissues in primary lesions, boundaries between cancer cells that have metastasized to lymph nodes and normal lymphatic tissues, boundaries between cancer cells with distant metastases and normal tissues of the metastatic organ, as well as marking living cancer tissue cells in other biological samples.
The present disclosure provides a use of an isolated antigenic ITPRIPL1-binding protein in the preparation of a drug that regulates immune responses or resists tumors as well as for detecting the expression of ITPRIPL1 in an individual, where, the isolated antigenic ITPRIPL1-binding protein is capable of binding to an amino acid sequence as set forth in SEQ ID NO: 49 or SEQ ID NO: 1 in the antigenic ITPRIPL1.
In some embodiments, a heavy chain variable region and a light chain variable region are included, where, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH as set forth in any one of amino acid sequence SEQ ID NO: 24 or SEQ ID NO: 34; the light chain variable region includes LCDR1, LCDR2 and LCDR3 in the light chain variable region VL as set forth in any one of amino acid sequence SEQ ID NO: 25 or SEQ ID NO: 35.
In some embodiments, in the heavy chain variable region VH in the amino acid sequence SEQ ID NO: 24, the amino acid sequence of the HCDR1 is as set forth in SEQ ID NO: 26, the amino acid sequence of the HCDR2 is as set forth in SEQ ID NO: 27, and the amino acid sequence of the HCDR3 is as set forth in SEQ ID NO: 28.
In some embodiments, in the amino acid sequence SEQ ID NO: 34, the amino acid sequence of the HCDR1 is as set forth in SEQ ID NO: 36, the amino acid sequence of the HCDR2 is as set forth in SEQ ID NO: 37, and the amino acid sequence of the HCDR3 is as set forth in SEQ ID NO: 38.
In some embodiments, in the amino acid sequence SEQ ID NO: 25, the amino acid sequence of the LCDR1 is as set forth in SEQ ID NO: 29, the amino acid sequence of the LCDR2 is KV, and the amino acid sequence of the LCDR3 is as set forth in SEQ ID NO: 31, or is more than 80% similar to an amino acid sequence as set forth in SEQ ID NO: 31.
In some embodiments, in the amino acid sequence SEQ ID NO: 35, the amino acid sequence of the LCDR1 is as set forth in SEQ ID NO: 39, the amino acid sequence of the LCDR2 is KV, and the amino acid sequence of the LCDR3 is as set forth in SEQ ID NO: 41.
In some embodiments, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH as set forth in the amino acid sequence SEQ ID NO: 24; the light chain variable region includes LCDR1, LCDR2 and LCDR3 in the light chain variable region VL as set forth in the amino acid sequence SEQ ID NO: 25.
In some embodiments, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH as set forth in the amino acid sequence SEQ ID NO: 34; the light chain variable region includes LCDR1, LCDR2 and LCDR3 in the light chain variable region VL as set forth in the amino acid sequence SEQ ID NO: 35.
In some embodiments, an antibody heavy chain constant region is included, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
In some embodiments, an antibody light chain constant region is included, and the antibody light chain constant region includes a human Igx constant region.
In some embodiments, an antibody heavy chain HC is included, and the HC includes an amino acid sequence as set forth in any one of SEQ ID NO: 22 or 32.
In some embodiments, an antibody light chain LC is included, and the LC includes an amino acid sequence as set forth in any one of SEQ ID NO: 23 or 33.
In some embodiments, an antibody or an antigen binding fragment thereof is included, wherein the antigen binding fragment includes Fab, Fab′, F(ab)2, Fv fragment, F(ab′)2, scFv and/or di-scFv.
In some embodiments, the ITPRIPL1 protein includes human ITPRIPL1 or the ITPRIPL1 protein of cynomolgus monkey, rat, mouse, gorilla, grivet, golden snub-nosed monkey, black snub-nosed monkey, Amazon squirrel monkey.
In some embodiments, the human ITPRIPL1 protein includes an amino acid sequence as set forth in SEQ ID NO: 20.
In another aspect, the present disclosure further provides a chimeric antigen receptor (CAR), which includes the isolated antigenic ITPRIPL1-binding protein of the present disclosure.
In another aspect, the present disclosure further provides an immunoconjugate, which includes the isolated antigenic ITPRIPL1-binding protein of the present disclosure.
In another aspect, the present disclosure further provides one or more isolated nucleic acid molecules, which encode the isolated antigenic ITPRIPL1-binding protein or the chimeric antigen receptor of the present disclosure.
In another aspect, the present disclosure further provides a vector, which includes the nucleic acid molecules of the present disclosure.
In another aspect, the present disclosure further provides a cell, which includes the nucleic acid molecules or the vector of the present disclosure.
In another aspect, the present disclosure further provides a pharmaceutical composition, which includes the isolated antigenic ITPRIPL1-binding protein, the chimeric antigen receptor, the immunoconjugate of the present disclosure, and optionally a pharmaceutically acceptable adjuvant.
In another aspect, the present disclosure further provides a method for preparing the isolated antigenic ITPRIPL1-binding protein of the present disclosure, which includes culturing the cell of the present disclosure under a condition enabling the expression of the isolated antigenic ITPRIPL1-binding protein of the present disclosure.
In another aspect, the present disclosure further provides uses of the isolated antigenic ITPRIPL1-binding protein, the chimeric antigen receptor, the immunoconjugate, and/or the pharmaceutical composition of the present disclosure in the preparation of a drug which is used for preventing, alleviating and/or treating tumors. In some embodiments, the tumors include solid tumors and lymphoma.
In another aspect, the present disclosure further provides a linear epitope polypeptide that can be used for efficiently screening and preparing an ITPRIPL1 function-regulating antibody, which is characterized in that, the peptide includes: (i) an amino acid sequence of SEQ ID NO: 49, i.e., RLLEMEFEERKRAAE; (ii) or, an amino acid sequence of xxLxxxFxxRxxx (x is any amino acid), in which 1-3 amino acids at both ends can be deleted; (iii) or, an amino acid sequence obtained by substituting, inserting or deleting 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ ID NO: 49.
The above linear epitope peptide has outstanding advantages in many aspects: it can be synthesized at low cost; the function of the ITPRIPL1 specific antibody can be determined by analyzing the binding ability of the epitope peptide with advantages of simple operation and stable and reliable detection results; the linear epitope peptide can be directly injected to animals as immunogen or be screened, thereby obtaining more effective functional antibodies than those obtained by using full-length proteins, thus improving the discovery efficiency of related drugs.
The present disclosure further provides a method for searching and identifying the function regulator of ITPRIPL1, which is characterized in that, one or more of the following properties of the test molecules are detected: ability of specifically binding to ITPRIPL1 expressed on the cell surface; effect on the binding of ITPRIPL1 to CD3ε; effect on the binding of ITPRIPL1 to NRP2; effect on the binding of ITPRIPL1 to SEMA3G; effect on the binding of ITPRIPL1 to EBI2; and effect on the function of immune cells or tumor cells.
The present disclosure creatively develops a targeting antibody that can bind to ITPRIPL1, which can bind the above target protein with high affinity and neutralize its function, and inhibit its binding to one or more ligands, thereby disabling the immune evasion function of the tumor cells and promoting the killing of tumor cells by immune cells in vitro and in vivo. The antibody provided in the present disclosure can be used as the active ingredient to prepare a drug for treating tumors, providing a new effective solution for the treatment of tumors. At the same time, the present disclosure also provides linear epitopes corresponding to the antibody with excellent neutralizing functions, and biomarkers for administering the antibody.
The conception, specific structure and technical effects produced therefrom will be further illustrated below in conjunction with the accompanying drawings, so as to fully understand the purposes, features and effects of the present disclosure.
Compared with the prior art, the present disclosure has the following beneficial effects:
The present disclosure discloses a method of regulating immune responses and suppressing tumors, which is achieved by regulating the expression or function of the ITPRIPL1 gene. The present disclosure is based on a new scientific discovery that ITPRIPL1 binds to proteins such as CD3ε, so as to regulate the functions of different immune cells, and then participate in the regulation of immune responses and the immune evasion process of tumors. The present disclosure has confirmed that the regulator of ITPRIPL1 can be used to prepare drugs or pharmaceutical compositions, with promising applications in the suppression of diseases such as tumors, autoimmune diseases, transplant rejection, allergies and infections.
Although the present invention can be implemented in many different forms, disclosed herein are specific illustrative examples thereof that verify the principles of the invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated by the examples given herein. In addition, any section headings used herein are only for organizational purposes and are not to be construed as limiting the subject matter described. Experimental methods for which the specific conditions are not indicated in the following examples are usually conducted according to conventional conditions, for example, the conditions as described in (Sambrook and Russell et, al, Molecular Cloning-A Laboratory Manual (Third Edition) (2001) CSHL Press), or the conditions as recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, materials and reagents used in the examples of the present invention are all commercial products.
Unless otherwise defined below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by those skilled in the art. While the following terms are believed to be easily understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
The term “include”, “comprise”, “have”, “contain” or “involve” is inclusive or open-ended, and does not exclude other unenumerated elements or process steps. The term “composed of . . . ” is considered as the preferable implementation of the term “comprise”. If one group is defined below as comprising at least a certain number of examples, this should also be understood as disclosing a group which preferably consists only of these examples.
The indefinite or definite article used when referring to a noun in the singular form, such as “a” or “a”, “the”, it also includes the plural form of the noun.
Furthermore, the terms first, second, third, (a), (b), (c) and the like in the specification and claims are used to distinguish similar elements and are not necessary for the descriptive order or the chronological order. It should be understood that the terms so applied are interchangeable in appropriate circumstances, and that the examples described herein can be implemented in other sequences different from those described or exemplified herein.
The term “and/or” is considered to be a specific disclosure of each of the two specified features or components with or without the other. Therefore, the term “and/or” used in the phrase, e.g., “A and/or B” as used herein is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” used in the phrase, e.g., “A, B and/or C” is intended to cover each of the followings: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
The term “e.g.” and “i.e.” are only used as examples, with no intention of limiting, and should not be interpreted as only relating to items explicitly enumerated in the description.
The terms “or more”, “at least”, “more than” and the like, e.g., “at least one” should be understood to include, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or those more than the value. Any larger numbers or fractions therebetween are also included.
On the contrary, the term “not more than” includes each value less than that value. For example, “not more than 100 nucleotides” include 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. Any smaller numbers or fractions therebetween are also included.
The term “multiple”, “at least two”, “two or more”, “at least a second”, and the like should be understood to include, but not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Any larger numbers or fractions therebetween are also included.
The term “approximately” or “substantially” indicates a range of accuracy that can be understood by those skilled in the art and can still ensure the technical effect of the feature in question. The term generally indicates a deviation of ±10%, preferably ±5%, from the indicated value.
The term “derivation” or “mutation” refers to the formation of a new sequence through substitution, deletion, insertion, or other changes of a nucleic acid or amino acid sequence, the amino acid sequence in the group which is composed of the new sequence may be at least 70%, 80%, 90%, 95% or 99% identical to the sequence in the group;
As used herein, unless otherwise specified, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the indicated range, and, if appropriate, include fractions thereof (e.g., one-tenth and one-hundredth of the integer).
In order to make the purposes, technical solutions, and advantages of the present disclosure clearer, some of the terms involved in the present invention will be explained below:
In the present disclosure, the term “isolated” generally refers to artificially obtained from the natural state or synthesized artificially. If a certain “isolated” substance or component occurs in nature, it may be due to a change in its natural environment, or the substance may be isolated from its natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with a high purity isolated from this natural state is called isolated. The term “isolated” does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
As used herein, the term “antibody” (Ab) includes, but not limited to, glycoprotein immunoglobulin that specifically binds to an antigen. In general, an antibody can include at least two heavy (H) chains and two light (L) chains which are connected to each other through disulfide bonds, or antigen binding molecules thereof. Each H chain includes a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region includes three constant domains: CH1, CH2 and CH3. Each light chain includes a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region includes one constant domain, CL. VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR) interspersed with more conservative regions called framework regions (FR). Each of VH and VL includes three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of heavy chain and light chain contain binding domains interacting with antigens. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).
The light chain variable region and the heavy chain variable region include a “framework” region interspersed with three hypervariable regions (also known as “complementarity determining region” or “CDR”), respectively. The “complementarity determining region” or “CDR region” or “CDR” or “hypervariable region” (that can be used interchangeably with hypervariable region “HVR” herein) is a region in the variable domain of an antibody, which is hypervariable in sequence and forms a structurally defined loop (“a hypervariable loop”) and/or contains antigen-contacting residues (“antigen contact points”). CDRs are mainly responsible for binding to antigenic epitopes. The CDRs of the heavy chain and the light chain are generally referred to as CDR1, CDR2 and CDR3, which are numbered sequentially from the N-terminus. The CDRs within the heavy chain variable domain of an antibody are referred to as HCDR1, HCDR2 and HCDR3, and the CDRs within the light chain variable domain of an antibody are referred to as LCDR1, LCDR2 and LCDR3. In the amino acid sequence of a given light chain variable region or heavy chain variable region, the precise amino acid sequence boundary of each CDR can be determined by any one of many well-known antibody CDR assignment systems or a combination thereof, which include, for example: Chothia based on the three-dimensional structure of an antibody and the topology of CDR loops (Chothia et, al. (1989) Nature 342:877-883, Al-Lazikani et, al, “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of antibody sequence (Kabat et, al, Sequences of Protein of Immunological Interest, the 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT), as well as North CDR Definition based on the affinity propagation clustering using a large number of crystal structures.
However, it should be noted that, the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems might differ. That is, the CDR sequences of the variable regions of the same antibody as defined by different assignment systems are different. For example, the residue ranges defined by different assignment systems for CDR regions using Kabat and Chothia numbering are shown in Table A below.
Therefore, when referring to instances where an antibody is defined by specific CDR sequences as defined in the present invention, the scope of the antibody also encompasses antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, different assignment system rules or combination thereof), the claimed CDR boundaries may be different from the specific CDR boundaries as defined in the present invention.
The CDRs of the antibodies of the present invention can be evaluated manually to determine the boundaries according to any protocols in the art or a combination thereof. Unless otherwise stated, in the present invention, the term “CDR” or “CDR sequence” encompasses CDR sequences determined in any one of the above ways.
Antibodies can include, for example, a monoclonal antibody, a recombinantly produced antibody, a monospecific antibody, a multi-specific antibody (including a bispecific antibody), a human antibody, an engineered antibody, a humanized antibody, a chimeric antibody, immunoglobulin, a synthetic antibody, a tetrameric antibody comprising two heavy chain and two light chain molecules, an antibody light-chain monomer, an antibody heavy-chain monomer, an antibody light-chain dimer, an antibody heavy-chain dimer, an antibody light chain-antibody heavy chain pair, an intracellular antibody, an antibody fusion (herein sometimes referred to as “antibody conjugate”), a heteroconjugate antibody, a single-domain antibody, a monovalent antibody, a single-chain antibody or a single-chain Fv (scFv), a camelid antibody, an affibody, a Fab fragment, a F(ab′)2 fragment, Fv(sdFv) linked through a disulfide bond, an anti-idiotype (anti-Id) antibody (including, for example, anti-anti-Id antibody), a minibody, a domain antibody, a synthetic antibody (herein sometimes referred to as “antibody mimic”) and antigen binding fragments of any of the above.
The term “humanized antibody” is intended to refer to an antibody obtained by grafting a CDR sequence derived from the germline of another mammalian species, such as mouse, onto the human framework sequence. Other framework region modifications can be made in human framework sequences.
As used herein, “antigen binding molecules”, “antigen binding fragments” or “antibody fragments” refer to any molecules including antigen binding fragments (for example, CDR) of an antibody from which the molecules are derived. The antigen binding molecules may include antigen complementarity determining regions (CDRs). Examples of antibody fragments include, but not limited to, Fab, Fab′, F(ab′)2 and Fv fragments formed from antigen binding molecules, dAb, linear antibodies, scFv antibodies and multi-specific antibodies. In some embodiments, antigen binding molecules bind to ITPRIPL1 protein. In some embodiments, antigen binding molecules have neutralizing activities so that they can inhibit the binding of ITPRIPL1 to the receptor CD3E or EBI2.
The term “chimeric antigen receptors” (i.e., CARs) includes extracellular domains, transmembrane domains and possible intracellular domains, the extracellular domains being composed of protein domains that recognize and bind to specific antigens. The chimeric antigen receptors can be expressed in immune cells and regulate their ability to interact with target cells.
The term “immunoconjugate” may include antibody immunoconjugate (that is, antibody-drug conjugate, ADC), in which biologically active small-molecule drugs are linked to antibodies through chemical linkage. Similarly, the immunoconjugate also includes protein-drug conjugates, nucleic acid-drug conjugates.
As used herein, the term “antigen” refers to any molecules that induce immune responses or can be bound by antibody or antigen binding molecules. Immune responses may involve the production of antibodies or the activation of specific immunocompetent cells or both. Those skilled in the art will readily understand that any macromolecules (including almost all the proteins or peptides) can serve as antigens. Antigens can be expressed endogenously, i.e., they can be expressed by genomic DNA or can be expressed recombinantly. Antigens may be specific to certain tissues, e.g., cancer cells, or they may be expressed widely. Furthermore, fragments of larger molecules can serve as antigens. In some embodiments, the antigens are ITPRIPL1 protein antigens.
As used herein, in some embodiments, antigen binding molecules, scFv, antibodies or fragments thereof block the binding sites on the ligands directly or change the binding ability of the ligands indirectly (for example, by changing the structure or energy of the ligands). In some embodiments, antigen binding molecules, scFv, antibodies or fragments thereof prevent the proteins to which they bind from performing their biological functions.
As used herein, the terms “peptide”, “polypeptide” and “protein” can be used interchangeably and refer to compounds comprising amino acids residues covalently linked through peptide bonds. Proteins or peptides contain at least two amino acids and there is no limitation to the maximum number of amino acids that can include the sequence of the protein or peptide. Polypeptides include any peptides or proteins that comprise two or more amino acids linked to each other via peptide bonds. As used herein, this term refers to both short chains (in the art, they are also generally referred to as, for example, peptides, oligopeptides and oligomers) and longer chains (in the art, they are generally referred to as proteins, of many types). “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homologous dimers, heterologous dimers, variants of the polypeptides, modified polypeptides, derivatives, analogues, fusion proteins, etc. Polypeptides include native peptides, recombinant peptides, synthetic peptides or a combination thereof.
As used herein, the term “specific binding” or “specifically binding to” refers to a non-random binding reaction between two molecules, for example, between an antibody and an antigen.
As used herein, the ability of “inhibiting the binding”, “blocking the binding” or “competing for the same epitope” refers to the ability of an antibody to inhibit the binding of two molecules to any detectable degree. In some embodiments, the antibody blocking the binding of two molecules inhibits the binding interaction between the two molecules by at least 50%. In some embodiments, the inhibition may be greater than 20%, 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90%.
As used herein, the term “Ka” is intended to indicate the association rate of the specific antibody-antigen interaction, while the term “Kd” used herein is intended to indicate the dissociation rate of the specific antibody-antigen interaction. As used herein, the term “KD” or “KD value” is intended to indicate the dissociation constant of the specific antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and represented as molar concentration (M). The KD value of an antibody can be determined using a well-established method in the art.
As used herein, the term an antibody of “high affinity” refers to an antibody with a KD value of 1×10−7 M or lower, more preferably 5×10−8 M or lower, even more preferably 1×10−8 M or lower, even more preferably 5×10−9 M or lower, and even more preferably 1×10−9 M or lower, against the target antigen.
As used herein, the term “epitope” refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. The “epitope” is also referred to as “antigenic determinant”. The epitope or antigenic determinant is usually composed of chemically active surface groups on the side chain of molecules such as amino acids, carbohydrates, or sugar, and usually has a specific three-dimensional structure and specific charge features. For example, an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or discontinuous amino acids in a unique stereoscopic conformation, which can be a “linear epitope” or “conformational epitope”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In the linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are arranged linearly along the primary amino acid sequence of the protein. In the conformational epitope, interaction sites span amino acid residues in the protein that are separated from each other. Depending on the competition of binding identical epitopes as detected by conventional techniques known to those skilled in the art, the antibodies can be screened. For example, competition or cross-competition studies can be conducted to obtain antibodies that compete or cross-compete with each other to bind antigens (e.g., CLDN18.2). International Patent Application WO 03/048731 describes a high-throughput method for obtaining antibodies that bind identical epitopes, which is based on their cross-competition.
The term “nucleic acid” or “nucleic acid sequence” in the present invention refers to any molecules, preferably polymeric molecules, comprising units of ribonucleic acid, deoxyribonucleic acid or analogues thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be the nucleic acid of one strand of denatured double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid not deriving from any double-stranded DNA.
The term “complementary” as used herein relates to the hydrogen-bonded base pairing between nucleotide bases G, A, T, C and U, such that when two given polynucleotides or polynucleotide sequences anneal to each other, A pairs with T and G pairs with C in DNA, and G pairs with C and A pairs with U in RNA.
As used herein, the term “cancer” refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth result in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distal parts of the body through the lymphatic system or bloodstream. “Cancers” or “cancerous tissues” can include tumors, such as: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastrointestinal cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, neoplasms/tumors of central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and combinations of these cancers.
As used herein, the term “ITPRIPL1-associated cancers” refers to any cancers caused by, exacerbated by, or otherwise associated with, increased or decreased expression or activity of ITPRIPL1. In some embodiments, the method disclosed herein can be used in the treatment of a cancer selected from colorectal cancer, lung cancer, breast cancer, melanoma, lymphoma, liver cancer, head and neck cancer, gastric cancer, kidney cancer, bladder cancer, prostatic cancer, testicular cancer, endometrial cancer, breast cancer, and ovarian cancer.
As used herein, an “effective dose”, “effective amount” or “therapeutically effective dose” is any amount that, when used alone or in combination with another therapeutic agent, protects the subject from the onset of a disease or promotes the disease regression. Evidences of the disease regression include a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease or prevention of injury or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled practitioner, such as by assessing in human subjects during clinical trials, by assessing in animal model systems used to predict efficacy in humans, or by determining the activity of the reagent in an in vitro assay.
As used herein, an “individual” or “subject” is a mammal. Mammals include primates (for example, human and non-human primates, such as monkey) and rodents (for example, mice and rats). In some embodiments, an individual or subject is human. The “subject” can be a “patient”—the patient is a human subject in need of treatment, which may be an individual with ITPRIPL1-associated cancer such as breast cancer, or a subject with the risk of having an ITPRIPL1-associated cancer such as breast cancer.
As used herein, the term “in vitro cells” refers to any cells cultured ex vivo. Particularly, in vitro cells may include T cells.
As used herein, the term “pharmaceutically acceptable” refers to that the vector, diluent, excipient and/or salts thereof are chemically and/or physically compatible with other components in the preparation, and physiologically compatible with the recipient.
As used herein, the term “pharmaceutically acceptable carrier and/or excipient” refers to the carrier and/or excipient that are pharmacologically and/or physiologically compatible with the subject and the activating agent, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro A R, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but not limited to, a pH regulator, a surfactant, an adjuvant and an ionic strength enhancer. For example, the pH regulator includes, but not limited to, phosphate buffer; the surfactant includes, but not limited to, cationic, anionic or nonionic surfactant, for example, Tween-80; and the ionic strength enhancer includes, but not limited to, sodium chloride.
As used herein, the term “modulate” or “regulate” generally includes the meaning of upward or downward regulation in two different directions, which in some cases can be understood as inhibition or enhancement, in some cases can be understood as reduction or improvement, in some cases can be understood as decreasing or increasing, etc. Its specific interpretation is not limited, and it should be understood and interpreted according to the actual application context. Exemplarily, in some embodiments, “modulating” the tumor cell growth can be understood as inhibiting or enhancing the tumor cell growth.
As used herein, the term “decreasing” and “reduction” are used interchangeably and indicate any changes that are less than the original. “Decreasing” and “reduction” are relative terms, and it requires a comparison before and after measurement. “Decreasing” and “reduction” include complete consumption; likewise, the terms “increasing” and “improvement” are interpreted on the contrary.
The “treatment” or “treating” of a subject means any type of intervention or process performed on the subject, or the administration of an activating agent to the subject, for the purpose of reversing, alleviating, ameliorating, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition or biochemical indicators associated with a disease. In some embodiments, “treatment” or “treating” includes partial remission. In another embodiment, “treatment” or “treating” includes complete remission.
The term “the expression of ITPRIPL1 in an individual” refers to the expression level of the protein or mRNA of ITPRIPL1 in normal human and patients, including the contents in diseased tissues such as tumor tissues or autoimmune disease tissues, blood samples, urine, feces and other biological samples. This expression level can be used as a diagnostic biomarker for a disease, or as a companion diagnostic marker for a related drug.
The technical solutions of the present disclosure will be further illustrated below in conjunction with the attached drawings and examples.
The present disclosure firstly discloses that, the ITPRIPL1 extracellular domain binds to CD3ε extracellular domain and NRP2 extracellular domain, and also discloses the regulation roles of ITPRIPL1 on T cells and antigen presenting cells respectively after binding to CD3ε and NRP2. Based on new scientific findings, the present disclosure discloses a method for regulating immune responses and suppressing tumors by targeting ITPRIPL1 and discloses a use of a regulator of ITPRIPL1 in the preparation of a drug that regulates immune responses or resists tumors. Specifically, the present disclosure provides a method for editing the ITPRIPL1 gene, and a method for introducing genetic materials into cells to regulate the expression of ITPRIPL1. The present disclosure further discloses the receptor-binding domain (RBD) of ITPRIPL1 and provides a protocol for preparing ITPRIPL1-RBD isolated protein, and demonstrates a method for binding ITPRIPL1-RBD to CD3ε and NRP2 so as to regulate the function of immune cells. Furthermore, the present disclosure exemplifies a method for preparing an ITPRIPL1 antibody, and demonstrates that the antibody can enhance the anti-tumor effect of immune cells. The method for regulating immune responses and suppressing tumors by targeting ITPRIPL1 as disclosed in the present disclosure shows the value of ITPRIPL1 as a target in the treatment of tumors, autoimmune diseases, transplant rejection, allergies, infections and other diseases.
There are no previous reports on the function of ITPRIPL1 gene. The present disclosure discloses ITPRIPL1 as a newly identified ligand for CD3ε (Example 1) and elucidates the receptor-binding domain (RBD) of the ITPRIPL1 extracellular domain as amino acids 25-103 (Example 2), on the basis of which isolated recombinant proteins with the function of regulating CD3ε and T cells are prepared (Example 3). The present disclosure also discloses the preparation, use and administration method of the above isolated proteins.
Further, the present disclosure discloses the ability of the isolated ITPRIPL1-RBD protein to bind CD3ε (Example 4), and the ability of the ITPRIPL1-RBD isolated protein to bind to CD3ε extracellular domain has been demonstrated by using enzyme-linked immunosorbent assay (ELISA), flow cytometry or any other experimental methods, respectively.
Since ITPRIPL1-RBD (seen SEQ ID NO: 1 for its sequence) is the key region for binding CD3ε, the isolated ITPRIPL1-RBD protein can be prepared by means of recombinant expression and can be used to bind the CD3ε protein on the cell surface and transmit inhibitory signals into T cells (Example 5), so the isolated ITPRIPL1-RBD protein can be used to regulate the function of T cells. Although the present disclosure has provided the roles of the isolated protein of sequence SEQ ID NO: 1 in binding to CD3ε, regulating T cells and preparing neutralizing antibodies, other protein sequences with similar functions obtained by non-creative derivation methods (such as, truncation, insertion, mutation or fusion, etc.) that are easy to implement by those skilled in the art also fall within the scope as claimed in the present disclosure.
The present disclosure discloses an application of the isolated ITPRIPL1-RBD as immunogen in the preparation of neutralizing antibodies (Example 6). In the case that ITPRIPL1 has not been disclosed as a ligand for CD3ε, those skilled in the art cannot purposefully prepare antibodies for blocking the binding of ITPRIPL1 to CD3. The present disclosure discloses the existence of the pair of receptor ligands CD3ε and ITPRIPL1, defines the key structure for the binding of ITPRIPL1 to CD3, and provides a preparation method of the ITPRIPL1-RBD isolated protein, making the preparation of antibodies for blocking the binding of ITPRIPL1 to CD3 become an easily achievable goal by those of skills in the art through conventional techniques (e.g., hybridoma, phage display, etc.). Therefore, the blocking antibodies prepared by using the fragment from ITPRIPL1 extracellular domain as the immunogen also fall within the scope of the claims of the present disclosure.
The present disclosure also discloses that, the activation of proliferation signaling pathway of T cell-derived cell lines is regulated by regulating the binding of ITPRIPL1-RBD to the CD3 extracellular domain (Example 7).
The present disclosure discloses that, ITPRIPL1-RBD recombinant protein can reduce the killing of kidney-derived H3K293 cells by human peripheral blood mononuclear cells (PBMCs) (Example 8), on the basis of which the application of the targeting ITPRIPL1 in inhibiting autoimmunity, as well as its application value in the preparation of pharmaceutical compositions for treating autoimmune diseases, transplant rejection, allergies, infections and other diseases are proposed.
The present disclosure discloses that, the ITPRIPL1 that reduces the expression of tumor cells can significantly increase the killing of tumor cells by human peripheral blood mononuclear cells (Example 9), supporting the role of ITPRIPL1 in the immune evasion of tumors, on the basis of which the important value of ITPRIPL1 as the target of the immunotherapy of tumors is proposed.
The present disclosure also discloses that, the antibodies prepared by using the ITPRIPL1-RBD protein as the immunogen effectively promote the killing of tumor cells by immune cells (Example 10). This verifies the important value of ITPRIPL1 as the target for immunotherapy of tumors, and establishes a method for blocking the binding of ITPRIPL1 to CD3 with the antibodies. The antibodies represent a class of antibodies with completely new functions, i.e., antibodies capable of recognizing ITPRIPL1 and blocking its binding to CD3c, thereby regulating the function of T cells.
Furthermore, by comparing the fragments from the ITPRIPL1 extracellular domain of different length, the present disclosure demonstrates that the ability of the ITPRIPL1-RBD2 sequence to bind CD3ε is slightly reduced, while the ability of the ITPRIPL1-RBD3 to bind CD3 is significantly reduced, thereby disclosing the positive correlation between the length of the ITPRIPL1 extracellular domain and the ability to bind CD3ε and characterizing the essential properties of a functional ITPRIPL1 extracellular domain sequence (Example 11). The present disclosure discloses that the ITPRIPL1-RBD protein has the ability of binding NRP2 (Example 12). And the isolated ITPRIPL1-RBD protein has the ability of transmitting inhibitory signals to differentiated THP1 macrophages that express the NRP2 protein (Example 13).
Modification methods are often used in the development of drugs to improve their pharmacokinetic properties, in which polypeptides or proteins can be linked to other compounds or functional groups, for example, antibody constant regions (Fc), polyethylene glycol modification, glycosylation modification, polysialic acid modification, fatty acid modification, KLH modification, biotin modification, etc. The present disclosure exemplifies the effect of modification on the function of the ITPRIPL1 recombinant protein by means of Fc modification. The ITPRIPL1-RBD-Fc modified protein obtained after purification has the functions of inhibiting T cell pathway signaling and killing (Example 14). Furthermore, the ITPRIPL1-RBD-Fc protein can inhibit the phosphorylation pathway of T cells, and can be blocked by CD3ε (Example 14).
The present disclosure prepares Jurkat CD3 mutants and further discloses the related mechanism of affecting the phosphorylation pathway (Example 15). At the same time, the effect of ITPRIPL1-Fc on Jurkat cells with different CD3 expression is explored by detecting the intracellular calcium ion flux (Example 15), and find that ITPRIPL1-Fc regulates the pathway by increasing the binding of CD3 to Nck (Example 16).
Furthermore, the present disclosure is applied to an in vivo animal model to construct a MC38 subcutaneous xenograft tumor model in a humanized CD3ε mouse, so as to detect the tumor growth, the function of T cells in PBMCs, and the infiltration of T cells in tumor cells (Example 17). At the same time, an ITPRIPL1 knockout mouse model is established to detect the function of T cells in PBMCs, the changes of cytokine, and the infiltration of testicular T cells, thereby verifying that the present disclosure can be applied in vivo (Example 17).
The present disclosure discloses the expression of ITPRIPL1 in common multi-organ cancers and compares it with the corresponding para-cancerous tissues, confirming that ITPRIPL1 is increased in carcinomas (Example 18).
1. Construction of expression plasmid
Based on the published sequence (NCBI reference sequence NM_001008949.3), a Flag-tagged ITPRIPL1 plasmid was produced by synthesizing the cDNA of a full-length human ITPRIPL1 with pcDNA3.1 as the vector, in which the C terminus was fused to the Flag tag. The results of plasmid construction were shown in
2. The analysis results of co-immunoprecipitation and Western Blot demonstrate that ITPRIPL1 binds to CD3.
The pcDNA3.1 plasmids containing Flag-tagged ITPRIPL1 (or empty) and HA-tagged CD3ε were co-transfected into HCT116 cells (ATCC, VA, USA), and cultured in a 6-well plate (Corning, NY, USA) for 48-72 hours until the protein was fully expressed, and then lysed with a hybrid lysate of immunoprecipitation lysate (Thermo Fisher, MA, USA) mixed with a triple of protease-phosphatase-PMSF (Consun, Shanghai, China) at 1:100, and the cells were scraped. A portion of the cell samples were centrifuged, mixed with loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain an input level of protein samples; the remaining cell samples were immunoprecipitated with Flag-tagged specific mouse antibodies (CST, MA, USA), washed with PBS, mixed with the loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain immunoprecipitated protein samples. And then, 12.5% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of the ITPRIPL1-Flag and CD3ε-HA protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding ITPRIPL1-Flag and CD3&-HA strips were respectively incubated with Flag-tagged specific rabbit antibodies (Abcam, MA, USA) and HA-tagged specific rabbit antibodies (CST, MA, USA) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-rabbit secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
The results of the co-immunoprecipitation experiment were shown in
3. Immunofluorescence and colocalization analysis demonstrates that ITPRIPL1 has significant colocalization with CD3.
The pcDNA3.1 plasmid containing Flag-tagged ITPRIPL1 (or empty) and HA-tagged CD3ε was co-transfected into HCT116 cells (ATCC, VA, USA), cultured in an 8-well glass slide (Thermo Fisher, MA, USA) for 30 hours until the protein was fully expressed, and then the culture medium was discarded. After washing with PBS, it was fixed with 4% paraformaldehyde for 20 minutes, washed with PBS again and blocked with a membrane-permeable blocking buffer for 1 hour, and then Flag-tagged specific mouse antibodies (CST, MA, USA) and HA-tagged specific rabbit antibodies (CST, MA, USA) which had been diluted with the membrane-permeable blocking buffer were added and incubated at 4° C. overnight. After washing the 8-well glass slides with PBS overnight, Alexa Fluor 488 fluorescence specific anti-mouse antibodies (Invitrogen, CA, USA) and Alexa Fluor 594 fluorescence specific anti-rabbit antibodies (Invitrogen, CA, USA) which had been diluted with PBS were added and incubated at room temperature for 20 minutes. After washing with PBS, the slides were mounted with DAPI, and observed under a fluorescence microscope after the mounting medium was dried.
1. Construction of Expression Plasmid
Based on the published sequence (NCBI reference sequence NM_001008949.3), from which an extracellular domain (25-103 amino acids), an extracellular domain-and-transmembrane domain (25-124 amino acids), a transmembrane domain-and-intracellular domain (104-555 amino acids) were respectively selected as three different target sequences to synthesize a full-length human ITPRIPL1 with pcDNA3.1 as the vector, wherein the C termini of the extracellular domain and the extracellular domain-and-transmembrane domain were fused to Flag tags and green fluorescent proteins (GFPs), the N terminus of the transmembrane domain-and-intracellular domain was fuse to Flag tags and green fluorescent proteins (GFPs), thereby producing ITPRIPL1 extracellular domain, extracellular domain-and-transmembrane domain, transmembrane domain-and-intracellular domain plasmids containing Flag tags and green fluorescent proteins (GFPs). Wherein, the construction results of the expression vector of ITPRIPL1 (25-103), i.e., the CD3 binding domain, were shown in
2. The results of the co-immunoprecipitation experiment demonstrate that the ITPRIPL1 extracellular domain (rather than the intracellular domain or the transmembrane domain) binds to CD3ε.
The pcDNA3.1 plasmids containing the Flag-tagged ITPRIPL1 extracellular domain, the extracellular domain-and-transmembrane domain, the intracellular domain-and-transmembrane domain and HA-tagged CD3ε were respectively co-transfected into HCT116 cells (ATCC, VA, USA), and cultured in a 6-well plate (Corning, NY, USA) for 48-72 hours until the protein was fully expressed, and then lysed with a hybrid lysate of immunoprecipitation lysate (Thermo Fisher, MA, USA) mixed with a triple of protease-phosphatase-PMSF (Consun, Shanghai, China) at 1:100, and the cells were scraped. A portion of the cell samples were centrifuged, mixed with loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain an input level of protein samples; the remaining cell samples were immunoprecipitated with Flag-tagged specific mouse antibodies (CST, MA, USA), washed with PBS, mixed with the loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain immunoprecipitated protein samples. And then, 12.5% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of the ITPRIPL1 extracellular domain, the extracellular domain-and-transmembrane domain, the intracellular domain-and-transmembrane domain-Flag and the CD3&-HA protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding ITPRIPL1-Flag and CD3&-HA strips were respectively incubated with Flag-tagged specific rabbit antibodies (Abcam, MA, USA) and HA-tagged specific rabbit antibodies (CST, MA, USA) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-rabbit secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
3. Immunofluorescence and Colocalization Analysis Demonstrates that the ITPRIPL1 Extracellular Domain and the CD3 Extracellular Domain are in Trans-Binding.
HA-tagged CD3ε was transfected into HCT116 cells; additionally, the pcDNA3.1 plasmids of Flag-tagged ITPRIPL1 (respectively the ITPRIPL1 extracellular domains of human, mouse, gorilla, grivet, golden snub-nosed monkey and Amazon squirrel monkey, and the ITPRIPL1 transmembrane domain of human)-green fluorescent protein were separately transfected into another batch of HCT116; finally, the two separately transfected cells were co-cultured and the localization patterns of the two proteins were determined by an immunofluorescence double staining process. Wherein, the sequences of the ITPRIPL1 extracellular domains of rat, mouse, gorilla, grivet, golden snub-nosed monkey, black snub-nosed monkey, and Amazon squirrel monkey were seen in
The specific steps were as below: the pcDNA3.1 plasmids containing HA-tagged CD3ε were transfected into one batch of HCT116 cells (ATCC, VA, USA); additionally, the pcDNA3.1 plasmids containing Flag-tagged ITPRIPL1 (the extracellular domain-and-transmembrane domain)-green fluorescent protein were separately transfected into another batch of HCT116 cells (ATCC, VA, USA); 20 hours after transfection, the cells were individually digested with trypsin, mixed and resuspended well, and spread onto 8-well glass slides (Thermo Fisher, MA, USA) and continued to culture for 10 hours. The culture medium was discarded. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes, washed with PBS again and blocked with a membrane-permeable blocking buffer for 1 hour, and then HA-tagged specific rabbit antibodies (CST, MA, USA) which had been diluted with the membrane-permeable blocking buffer were added and incubated at 4° C. overnight. After washing the 8-well glass slides with PBS overnight, Alexa Fluor 594 fluorescence specific anti-rabbit antibodies (Invitrogen, CA, USA) which had been diluted with PBS were added and incubated at room temperature for 20 minutes. After washing with PBS, the slides were mounted with DAPI, and observed under a fluorescence microscope after the mounting medium was dried. The localization patterns of the two proteins were determined by an immunofluorescence double staining process.
As shown in
Based on the published sequence (NCBI reference sequence NP_001008949.1), human ITPRIPL1 extracellular domain (25-103 amino acids) recombinant protein was synthesized by Cusabio (Wuhan, China), expressed and purified by yeast, wherein the C terminus was fused to 6×-His and Myc tags, so as to produce ITPRIPL1-RBD recombinant proteins containing 6×-His and Myc-tags.
As shown in
Enzyme-linked immunosorbent assay (ELISA) shows that there is direct concentration-dependent binding of Concanavalin A (ConA) and the isolated fragments from the ITPRIPL1 extracellular domain to the CD3ε extracellular domain, respectively. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with 0.5/1/2/4 μg/ml of ConA or 0.03125/0.0625/0.125/0.25/0.5/1/2 μg/ml of ITPRIPL1-RBD recombinant protein each dissolved in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of coating buffer free of proteins. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, they were incubated with 1 μg/ml of the protein fragments from the CD3ε extracellular domain (Sino Biological Inc., Beijing, China) in an incubator at 37° C. for 60 minutes for binding, in which the CD3ε (Met1-Asp126) proteins contained hFc tags. After washing with PBST, they were incubated with PBS-diluted specific anti-human Fc segment antibodies (Abcam, MA, USA) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Sangon, Shanghai, China) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
1. Construction of HCT116-ITPRIPL1 and HCT116-CD3ε Stably Transfected Cell Lines
The constructed full-length ITPRIPL1-Flag plasmid and CD3ε-HA plasmid as well as empty pcDNA3.1 plasmid was respectively transfected into HCT116 cells (ATCC, VA, USA), cultured in an incubator for 24-48 hours, and screened by adding 1000 μg/ml of Geneticin (G418) (Gibco, CA, USA). 10−14 days later, after the empty pcDNA3.1 plasmid-transfected group of cells all died, HCT116-ITPRIPL1 and HCT116-CD3ε stably transfected cell lines were obtained.
2. Flow Cytometry Demonstrates that the Purified Protein Fragments from the ITPRIPL1 Extracellular Domain Bind to Jurkat Cells with High Expression of CD3.
The cultured Jurkat cells (ATCC, VA, USA) were counted, and the cell number was adjusted to 2×105/ml. 200 μl of them was respectively added into six EP tubes of 1.5 ml (Axygen, CA, USA). Into four of the EP tubes were respectively added 0.1 μg, 0.2 μg, 0.4 μg, 0.8 μg ITPRIPL1-RBD-6×-His recombinant protein (IT1-6×-His protein) so that the concentrations were 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, respectively. All the EP tubes were placed in a cell incubator, standing for 30 minutes. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. The 6×-His-FITC antibodies (Abcam, MA, USA) were diluted with the cell staining buffer at 1:500. In addition to the negative control, 200 μl of antibody diluent was added into each EP tube and incubated at room temperature for 30 minutes at 40 rpm on a shaker. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 1000 μl of the cell staining buffer and centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. After the completion of washing, 300 μl of the cell staining buffer was added into each EP tube for resuspension, and transferred into flow tubes (Falcon, NY, USA) for on-board analysis (Miltenyi Biotec, Cologne, Germany).
3. Flow Cytometry Demonstrates that ITPRIPL1 Protein Binds to Cells Overexpressing CD3 with Higher Efficiency
The cultured HCT116 wild-type cells (ATCC, VA, USA) and HCT116-CD3ε stably transfected cell lines were digested and then counted, and the cell number was respectively adjusted to 2×105/ml. 200 μl of them were respectively added into five and three EP tubes of 1.5 ml (Axygen, CA, USA). Into three EP tubes of HCT116 wild-type cells and three EP tubes of HCT116-CD3ε stably transfected cell lines were respectively added 0.2 μg, 0.4 μg, 0.8 μg ITPRIPL1-RBD-6×-His recombinant protein so that the concentrations were 1 μg/ml, 2 μg/ml, 4 μg/ml, respectively. All the EP tubes were placed in a cell incubator, standing for 30 minutes. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. The 6×-His-FITC antibodies (Abcam, MA, USA) were diluted with the cell staining buffer at 1:500. In addition to the negative control, 200 μl of antibody diluent was added into each EP tube and incubated at room temperature for 30 minutes at 40 rpm on a shaker. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 1000 μl of the cell staining buffer and centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. After the completion of washing, 300 μl of the cell staining buffer was added into each EP tube for resuspension, and transferred into flow tubes (Falcon, NY, USA) for on-board analysis (Miltenyi Biotec, Cologne, Germany).
4. Flow Cytometry Demonstrates that CD3 Binds to Cells Overexpressing ITPRIPL1 with Higher Efficiency.
The cultured HCT116 wild-type cells (ATCC, VA, USA) and HCT116-ITPRIPL1 stably transfected cell lines were digested and then counted, and the cell number was respectively adjusted to 2×105/ml. 200 μl of them were respectively added into three and one EP tubes of 1.5 ml (Axygen, CA, USA). Into one EP tube of HCT116 wild-type cells and the EP tube of HCT116-ITPRIPL1 stably transfected cell lines were respectively added 0.4 μg CD3E-human Fc protein (Sino Biological Inc., Beijing, China) so that the concentration was 2 μg/ml. All the EP tubes were placed in a cell incubator, standing for 30 minutes. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. The anti-human IgG Alexa Fluor 647 antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:1000. In addition to the negative control, 200 μl of antibody diluent was added into each EP tube and incubated at room temperature for 30 minutes at 40 rpm on a shaker. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 1000 μl of the cell staining buffer and centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. After the completion of washing, 300 μl of the cell staining buffer was added into each EP tube for resuspension, and transferred into flow tubes (Falcon, NY, USA) for on-board analysis (Miltenyi Biotec, Cologne, Germany).
5. Luciferin Reporter Assay Demonstrates that the Purified Protein Fragments from the ITPRIPL1 Extracellular Domain Inhibit the ConA-Activated NFKB Proliferation Signaling in Jurkat-Dual Cells.
The cultured Jurkat-dual cells (Invivogen, CA, USA) were counted, centrifuged at 1000 rpm for 5 minutes, and then resuspended with antibiotic-free IMDM medium (Gibco, CA, USA) to adjust the cell number to 2×106/ml. 200 μl of the cells were added into each well of a transparent 96-well plate (Thermo Fisher, MA, USA). Into each well was respectively added 0.2 μg, 0.4 μg, 0.8 μg ITPRIPL1-6×-His recombinant protein so that the concentrations were 1 μg/ml, 2 μg/ml, 4 μg/ml, respectively. After reaction for two hours in a cell incubator, 10 μg/ml of Concanavalin A (ConA) (Aladdin, Shanghai, China) was added into each well and reacted in the cell incubator for 18-24 hours. After the completion of reaction, a non-transparent 96-well plate (costar, ME, USA) was taken, into each well of which were added 50 μl of Quanti-luc reagent (Invivogen, CA, USA) and 20 μl of the reaction mixture and mixed well, and then the signals were detected immediately with a multimode microplate reader (Thermo Fisher, MA, USA).
As shown in
6. Luciferin Reporter Assay Demonstrates that the Purified Protein Fragments from the ITPRIPL1 Extracellular Domain Immobilized on the Surface of Microspheres Inhibit the ConA-Activated NFKB Proliferation Signaling in Jurkat-Dual Cells.
Protein G magnetic microspheres (Thermo Fisher, MA, USA) and His antibodies (Abcam, MA, USA) were placed in four EP tubes (Axygen, CA, USA), and revolved in a DNA mixer (Scientz, Ningbo, China) at the lowest speed at room temperature for 1 hour. Into three of the EP tubes were added 0.2 μg, 0.4 μg, 0.8 μg ITPRIPL1-6×-His recombinant protein, respectively, and revolved at the lowest speed at room temperature for another 1 hour. The cultured Jurkat-dual cells (Invivogen, CA, USA) were counted, centrifuged at 1000 rpm for 5 minutes and then resuspended with antibiotic-free IMDM medium (Gibco, CA, USA) to adjust the cell number to 2×106/ml. 200 μl of the cells were added into each well of a transparent 96-well plate (Thermo Fisher, MA, USA). Into each well was added the content of each EP tube respectively so that the concentrations of the coated proteins were 1 μg/ml, 2 μg/ml, 4 μg/ml, respectively. After reaction for two hours in a cell incubator, 50 μg/ml Concanavalin A (ConA) (Aladdin, Shanghai, China) was added into each well and reacted in the cell incubator for 18-24 hours. After the completion of reaction, a non-transparent 96-well plate (costar, ME, USA) was taken, into each well of which were added 50 μl of Quanti-luc reagent (Invivogen, CA, USA) and 20 μl of the reaction mixture and mixed well, and then the signals were detected immediately with a multimode microplate reader (Thermo Fisher, MA, USA).
As shown in
7. Detection of the Expression Level of ITPRIPL1 in Different Types of Tumor Cells
Since the ITPRIPL1 extracellular domain has the function of inhibiting immune cells (for example, T cells), tumor cells may express ITPRIPL1 to evade surveillance and killing by the immune system. If a variety of tumor cells abnormally express ITPRIPL1, it suggests that ITPRIPL1 may contribute to tumorigenesis and its progression, including immune evasion. The present invention reveals that ITPRIPL1 is abnormally expressed in cell lines from different types of tumors. The specific experimental procedures were: the cultured cell lines were counted, from which 2×106 cells were taken into a centrifuged tube of 15 ml and centrifuged at 800 rpm for 4 minutes, and the supernatants were then discarded. The cells were resuspended and washed with PBS, centrifuged at 800 rpm for 4 minutes, and resuspended and washed with PBS again. After the completion of centrifugation, the supernatants were discarded. The RIPA lysate and a triple of protease-phosphatase-PMSF were formulated at a ratio of 1:100. 120 μl of the hybrid lysate was added into each tube of cells, and transferred into EP tubes. Each EP tube was frozen and thawed on liquid nitrogen-ice for three cycles, and centrifuged at 12000 rpm at 4° C. for 15 minutes after the last thawing. After the completion of centrifugation, the supernatants were taken and formulated into a variety of cell samples at a ratio of 4:1 of the supernatant to 5×loading buffer, and denatured in a metal bath at 100° C. for 10 minutes. After the completion of denaturation, the endogenous expression of ITPRIPL1 in the variety of tumor cell lines was determined through gel electrophoresis and Western Blot assay, with GAPDH as the internal reference.
Correspondingly, Protein Atlas database indicated, based on the results extracted by analyzing the high-throughput mRNA expression profile, that ITPRIPL1 may have significantly elevated expression in a variety of tumors. There have not been any reports of ITPRIPL1 function in the past. Therefore, before the disclosure of the contents of the present invention, those skilled in the art could not have predicted any functions of ITPRIPL1 in tumorigenesis and its progression as well as immune evasion. The present invention has disclosed the abnormal elevated expression level of ITPRIPL1 tumor cell proteins, the ITPRIPL1 extracellular domain binding to CD3ε and causing inhibition of T cell, as well as other experimental data, which firstly disclosed the important value of ITPRIPL1 as the target in tumor immunotherapy.
As shown in
8. OCTET Molecular Interaction Experiments Demonstrate that the Purified Protein Fragments from the ITPRIPL1 Extracellular Domain can Directly Bind to CD3ε Protein.
The OCTET instrument was initiated. 100 μg of CD3E-Fc protein (Sino Biological Inc., Beijing, China) was adsorbed to saturation by Fc probes, and then 50 μg of recombinant protein from the ITPRIPL1 extracellular domain was correspondingly bound at concentrations of 800 nM/1600 nM/3200 nM, thereby plotting the binding curves and calculating the dissociation constants.
As shown in
1. Preparation of Mouse Polyclonal Antibodies and Polyclonal Antibodies with ITPRIPL1-RBD as an Immunogen
By using the human ITPRIPL1-RBD recombinant protein as the immunogen, and after verifying the purity and biological activity of the samples in the above examples, C57BL/6 mice were subjected to multiple immunizations to enhance the effect: (1) primary immunization, with the antigen at 50 μg per mouse, subcutaneous multi-point injection with Freund's complete adjuvant, at an interval of 3 weeks; (2) secondary immunization, with the dose and route the same as above but using Freund's incomplete adjuvant, at an interval of 3 weeks; (3) third immunization, with the dose the same as above, no addition of adjuvant, intraperitoneal injection at an interval of 3 weeks; (4) booster immunization, at a dose of 50 μg, intraperitoneal injection. Three days after the last injection, blood was taken and tested for its potency. After the immunization effect was detected to meet the requirements, the blood was taken and the polyclonal antibodies were isolated in a cumulative manner for multiple times, and the polyclonal antibodies were purified, for which the specific experimental procedures included: (1) Preparation of a protein G sepharose CL-4B affinity column. 10 mL of protein G sepharose CL-4B packings were prepared, and equal volumes of the packings and TBS buffer solution were mixed in a vacuum flask and stirred. Evacuation was performed for 15 minutes to remove air bubbles in the packings. The protein G sepharose CL-4B packings were slowly added into a glass column while controlling the filling rate at 1 mL/min-2 mL/min with a pump. To avoid column dryness, the column was equilibrated with a pre-cooled TBS buffer solution that was 10 times the bed volume. (2) Preparation of polyclonal antibodies. The polyclonal antibodies were slowly thawed in ice water or in a 4° C. refrigerator to avoid the aggregation of protein. The aggregation that occurs during the protein thawing can be dissolved by preheating at 37° C. Solid sodium azide was added to a concentration of 0.05%, and centrifuged at 15,000×g for 5 min at 4° C. The clarified polyclonal antibodies were removed out and filtered through a filter to remove excess lipid. (3) Affinity chromatography. The antibodies were diluted with TBS buffer solution at a ratio of 1:5, and filtered through a filter. The polyclonal antibodies were loaded onto the column at a rate of 0.5 mL/min. To ensure the binding of the polyclonal antibodies to the packings, the column should be loaded twice in succession and the loading effluent should be retained. After washing the column with TBS buffer solution until AX 280 nm was <0.008, an elution buffer solution at Ph 2.7 was added to elute at a rate of 0.5 mL/min until all protein flew down. The eluent was collected into EP tubes of 1.5 ml which had been added with 100 μL of neutralizing buffer solution, mixed well and detected with pH test paper for the pH of the eluent. If the pH was lower than 7, it could be adjusted to about pH 7.4 with a neutralizing buffer so as to avoid the denaturation of the antibodies. Into the column was added 10 mL of elution buffer solution at pH 1.9 to collect the eluent according to the above method until AX 280 nm was <0.008. The protein content in each tube was determined by a spectrophotometer.
2. Enzyme-Linked Immunosorbent Assay (ELISA) Demonstrates that Polyclonal Antibodies with ITPRIPL1-RBD as the Immunogen can Bind to Cells Expressing ITPRIPL1
An ELISA special plate (costar, ME, USA) was used. Firstly, B16 cells not expressing ITPRIPL1 (ATCC, VA, USA), LoVo cells moderately expressing ITPRIPL1 (ATCC, VA, USA) and HCT116-ITPRIPL1 stably transfected cell lines were digested with trypsin and counted, and the cell number was adjusted to 2×106/ml. Each well was plated with 100 μl of cells and coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% skim milk powder dissolved in PBS (Sangon, Shanghai, China) in an incubator at 37° C. for 90 minutes. After washing with PBST, the polyclonal antibodies at a total IgG concentration of 10 mg/ml were then diluted at a gradient of 1:1000/1:500/1:250/1:125, incubated with plated cells in an incubator at 37° C. for 60 minutes for binding. After washing with PBST, the PBS-diluted specific anti-mouse Fc segment antibodies (Consun, Shanghai, China) were incubated in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Sangon, Shanghai, China) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
3. Enzyme-Linked Immunosorbent Assay (ELISA) Demonstrates that Polyclonal Antibodies can Block the Binding of ITPRIPL1 to CD3ε.
An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with 0.1 μg of CD3ε protein fragments (Sino Biological Inc., Beijing, China) dissolved in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), in which the CD3ε (Met 1-Asp 117) proteins contained hFc tags, while for the negative control, the plate was coated with 100 μl of coating buffer free of proteins. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% skim milk powder dissolved in PBS (Sangon, Shanghai, China) in an incubator at 37° C. for 90 minutes. After washing with PBST, 2 μg/ml of ITPRIPL1-RBD-6×-His protein was mixed with polyclonal antibodies at a total IgG concentration of 10 mg/ml at ratios of 1:1000/1:500/1:250/1:125, respectively, and then co-incubated in an incubator at 37° C. for 60 minutes for binding. After washing with PBST, they were incubated with PBS-diluted specific anti-6×-His-tagged horseradish peroxidase antibodies (Abcam, MA, USA) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Sangon, Shanghai, China) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
As shown in
4. Flow Cytometry Demonstrates that Polyclonal Antibodies can Block the Binding of ITPRIPL1 to Cells Overexpressing CD3ε.
HCT116-CD3ε stably transfected cell lines were digested and then counted, and the cell number was respectively adjusted to 2×105/ml. 200 μl of them were respectively added into eight EP tubes of 1.5 ml (Axygen, CA, USA). Into each EP tube was added 0.8 μg IT 1-RBD protein so that the concentrations were 4 μg/ml respectively. Into five EP tubes of them were respectively added polyclonal antibodies at a total IgG concentration of 10 mg/ml which had been diluted at 1:1000/1:500/1:250/1:125/1:67.5, and left in a cell incubator for 30 minutes. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. The 6×-His FITC antibodies (Abcam, MA, USA) were diluted with the cell staining buffer at 1:500. In addition to the negative control, 200 μl of antibody diluent was added into each EP tube and incubated at room temperature for 30 minutes at 40 rpm on a shaker. The EP tubes were taken out and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 1000 μl of the cell staining buffer and centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. After the completion of washing, 300 μl of the cell staining buffer was added into each EP tube for resuspension, and transferred into flow tubes (Falcon, NY, USA) for on-board analysis (Miltenyi Biotec, Cologne, Germany).
5. Preparing Monoclonal Antibodies on the Basis of Polyclonal Antibodies, and Further Verifying the Ability of the Antibodies to Inhibit the Tumor Growth.
Since the expression of ITRPRIPL1 is up-regulated in a variety of tumors, the antibody that specifically binds to ITPRIPL1 can recognize tumor cells in the body, and kill tumor cells by exerting ADCC, ADCP and CDC effects through the Fc segment of the constant region of the antibody. ADCC, i.e., Antibody-Dependent Cell-mediated Cytotoxicity, refers to that the Fab segment of an antibody binds to the antigenic epitopes of virus-infected cells or tumor cells, and its Fc segment binds to the FcR on the surface of killer cells (NK cells, macrophages, neutrophils, etc.), which mediate the direct killing of target cells by killer cells, and this is an important mechanism for the action of anti-tumor therapeutic antibody drugs. ADCP, i.e., Antibody-Dependent Cellular Phagocytosis, is also an important mechanism for recognizing and mediating the effect of therapeutic antibodies on tumor cells. CDC, i.e., Complement Dependent Cytotoxicity, refers to complement-involved cytotoxicity, which means that through the binding of specific antibodies to the corresponding antigen on the surface of the cell membrane, a complex is formed to activate the classical pathway of complement, and the resulting membrane-attacking complex has a lytic effect on target cells. This example will confirm the inhibition of in vivo tumor growth by an antibody that specifically recognizes the ITPRIPL1 extracellular domain. The specific implementation steps are as below:
The above results show that, antibodies that specifically recognize the ITPRIPL1 extracellular domain, i.e., RBD1, can significantly inhibit the tumor cell growth in vivo. Since ITPRIPL1 is expressed in primary cancer, lymph metastatic carcinoma, distant metastatic carcinoma simultaneously, this allows the antibody drug to act on tumors at different sites and stages of development, exert a more sustained and widespread anti-tumor effect through ADCC, ADCP and CDC effects, and activate the local immune response in the tumor. At the same time, since ITPRIPL1 is expressed very low in the control normal tissues of the above tumors, the toxic side effects that may be produced from such an antibody against ITPRIPL1 on normal tissues and cells are mor limited. The above properties highlight the outstanding advantages and application prospects of the ITPRIPL1 specific antibody as a new anti-cancer therapy.
Since previous studies have shown that, CD3ε binding may cause alterations in T cell proliferation and function, and since T cells are primary cells that are not conducive to intervention and detection, the most widely used model for such studies is Jurkat cells, that is, a cell line derived from T cells, and NF-KB signaling is a widely used assay that reflects the degree of activation of Jurkat or T cells. Therefore, we constructed a Jurkat-NFKB reporter cell line (by transducing firefly luciferase downstream of the NF-KB promoter into Jurkat cells by lentivirus, which was tested to be activated with Concanavalin A), in order to test the effect of ITPRIPL1 on the functional status of co-cultured T cells in the presence of tumor cell expression.
Firstly, the luciferin reporter assay demonstrates that HCT116 cells overexpressing ITPRIPL1 can reduce the NFKB proliferation signaling in Jurkat-dual cells more, as shown in
Furthermore, the luciferin reporter assay demonstrates that CD3ε protein can block the inhibition of NFKB proliferation signaling in Jurkat-dual cells by the ITPRIPL1 protein, as shown in
It was demonstrated by using flow cytometry that ITPRIPL1-RBD recombinant protein can reduce the killing of kidney-derived H3K293 cells by human peripheral blood mononuclear cells (PBMCs). CD3 and CD28 antibodies (Invitrogen, CA, USA) were diluted by mixing with PBMCs (ATCC, VA, USA) to a final concentration of 1 μg/ml so as to activate T cells, and then cultured overnight. The next day, the PBMCs and 293E (ATCC, VA, USA) cells were counted, and the cell number was respectively adjusted to 1×106/ml. Into the control group were added 100 μl of 293E cells and 100 μl of the culture medium. Each 100 μl of the two cells in the experimental groups were added into a 96-well plate (Thermo Fisher, MA, USA). Into 4 of the experimental groups was respectively added 1, 2, 4, 8 μg/ml of the ITPRIPL1-RBD recombinant protein, and incubated in an incubator for 6 hours. The 96-well plate was taken out. Each well of cells were placed in an EP tube (Axygen, CA, USA), centrifuged at 400 ref for 5 minutes and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. The CD45-APC antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:20. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature while shaking slowly for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Meilun Biotech, Shanghai, China); and centrifuged and washed again. An unstained group without the addition of PBMCs, a double-stained group without the addition of PBMCs, an unstained group with the addition of PBMCs, a single-stained Annexin V-FITC group with the addition of PBMCs, a single-stained PI group with the addition of PBMCs, a double-stained group with the addition of PBMCs, and a double-stained group each with the addition of PBMCs were set, into which were respectively added 100 μl of the binding buffer, 5 μl of Annexin V-FITC (Meilun Biotech, Shanghai, China) and 10 μl of PI (Meilun Biotech, Shanghai, China) according to the conditions, and incubated at room temperature while shaking slowly for 15 minutes. 400 μl of the binding buffer was respectively further added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany).
1. Construction of CRISPR/Cas9 Gene Editing System, i.e., a Lentivirus Containing a Puromycin-Resistant sgRNA for Specific Cleavage of ITPRIPL1.
General experimental procedures: Firstly synthesizing single-stranded DNA oligo of the gRNA sequence, then annealing and pairing to produce double-stranded DNA oligo, and then linking it directly into the enzymatically cleaved CRISPR/Cas9 vector through the enzymatic cleavage sites contained in its both ends; transferring the ligation product into the prepared bacterial competent cells, and sending the grown monoclonal colonies to a sequencing company for sequencing identification, wherein the clones that were correct through comparison were successfully constructed CRISPR/Cas9 vectors. With regard to the target gene sequence of the target gene, multiple target site sequences were designed using the design principles of gRNA sequences as provided in the public website, which were shown in SEQ ID NOs:11-13. 3 pairs of gRNA oligomeric single-stranded DNA were respectively designed and synthesized according to the gene sequence, with the oligo sequences shown in SEQ ID NOs: 14-19, wherein SEQ ID NOs: 14, 15 are gRNA oligomeric single-stranded DNA sequences corresponding to the target sequence SEQ ID NO: 11; SEQ ID NOs: 16, 17 are gRNA oligomeric single-stranded DNA sequences corresponding to the target sequence SEQ ID NO: 12; and SEQ ID NOs: 18, 19 are gRNA oligomeric single-stranded DNA sequences corresponding to the target sequence SEQ ID NO: 13. The oligomeric single-stranded DNA was annealed to double-stranded, and the double-stranded gRNA oligo was respectively inserted into CRISPR/Cas9 vectors to construct CRISPR/Cas9 recombinant plasmids, which were transformed to competent cells Stbl3. The construction of vectors includes the following specific steps:
2. Gene Editing with CRISPR/Cas9 Gene Editing System to Construct HCT116-ITPRIPL1-Knockout Cell Lines.
HCT116 cells (ATCC, VA, USA) were plated in a 24-well plate (Corning, NY, USA) at an appropriate density (growing to a density of about 30-40% the next day), and digested and counted the next day. The cells were first infected with a quantitative gradient of GFP control lentivirus (Genomeditech, Shanghai, China). After 48 hours, the medium was changed. 72 hours later, the GFP fluorescence was observed under a microscope to determine the optimal virus-to-cell infection ratio so as to explore the MOI value. After the MOI value was determined, the plate was re-plated to infect HCT116 cells with Cas9 system lentivirus (Genomeditech, Shanghai, China) containing blasticidin-resistance at the MOI value, and the medium was changed after 48 hours. 72 hours later, blasticidin (Invivogen, CA, USA) was added at a concentration gradient for screening for 10−14 days, and the cell lines obtained from the final screening were HCT116 cells containing the Cas9 system. After then, the HCT116 cells containing the Cas9 system were plated in a 24-well plate and counted, and infected with the lentivirus containing puromycin-resistant sgRNA that specifically cleaves ITPRIPL1 at the MOI value, and the medium was changed after 48 hours. 72 hours later, puromycin (Invivogen, CA, USA) was added at a concentration gradient for screening for 10−14 days, resulting in the HCT116-ITPRIPL1-knockout cell lines.
3. Flow Cytometry Demonstrates that the Knockout of ITPRIPL1 Expressed in Tumor Cells can Significantly Increase the Killing of Tumor Cells by Human Peripheral Blood Mononuclear Cells.
CD3 and CD28 antibodies (Invitrogen, CA, USA) were diluted by mixing with PBMCs (ATCC, VA, USA) to a final concentration of 1 μg/ml so as to activate the T cells, and then cultured overnight. The next day, the PBMCs and HCT116 wild-type (ATCC, VA, USA)/ITPRIPL1-overexpressed/ITPRIPL1-knockout cells were counted, and the cell number was respectively adjusted to 1×106/ml. Into the control group were added 100 μl of tumor cells and 100 μl of the culture medium. Each 100 μl of the tumor cells and PBMCs in the experimental groups were added into a 96-well plate (Thermo Fisher, MA, USA), and incubated in an incubator for 6 hours. The 96-well plate was taken out and rinsed with PBS (Meilun Biotech, Dalian, China), and then the cells were digested with EDTA-free trypsin (Beyotime, Shanghai, China), placed in EP tubes (Axygen, CA, USA), and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. The CD45-APC antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:20. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature while shaking slowly for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Meilun Biotech, Dalian, China); and centrifuged and washed again. An unstained group without the addition of PBMCs, a double-stained group without the addition of PBMCs, an unstained group with the addition of PBMCs, a single-stained Annexin V-FITC group with the addition of PBMCs, a single-stained PI group with the addition of PBMCs, a double-stained group with the addition of PBMCs, and a double-stained group each with the addition of IT 1 protein were set, into which were respectively added 100 μl of the binding buffer, 5 μl of Annexin V-FITC (Meilun Biotech, Dalian, China) and 10 μl of PI (Meilun Biotech, Dalian, China) according to the conditions, and incubated at room temperature while shaking slowly for 15 minutes. 400 μl of the binding buffer was respectively further added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany).
CD3 and CD28 antibodies (Invitrogen, CA, USA) were diluted by mixing with PBMCs (ATCC, VA, USA) to a final concentration of 1 μg/ml so as to activate T cells, and then cultured overnight. The next day, the PBMCs and HCT116 cells (ATCC, VA, USA) were counted, and the cell number was respectively adjusted to 1×106/ml. Into the control group were added 100 μl of HCT116 cells and 100 μl of the culture medium. Each 100 μl of the two cells of the experimental groups were added into a 96-well plate (Thermo Fisher, MA, USA). Into 4 of the experimental groups were respectively added ITPRIPL1 polyclonal antibodies of 1:500/250/125/62.5, and incubated in an incubator for 6 hours. The 96-well plate was taken out and rinsed with PBS (Meilun Biotech, Dalian, China), and then the cells were digested with EDTA-free trypsin (Beyotime, Shanghai, China), placed in EP tubes (Axygen, CA, USA), and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. The CD45-APC antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:20. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature while shaking slowly for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Meilun Biotech, Shanghai, China); and centrifuged and washed again. An unstained group without the addition of PBMCs, a double-stained group without the addition of PBMCs, an unstained group with the addition of PBMCs, a single-stained Annexin V-FITC group with the addition of PBMCs, a single-stained PI group with the addition of PBMCs, a double-stained group with the addition of PBMCs, and a double-stained group each with the addition of ITPRIPL1 protein were set, into which were respectively added 100 μl of the binding buffer, 5 μl of Annexin V-FITC (Meilun Biotech, Shanghai, China) and 10 μl of PI (Meilun Biotech, Shanghai, China) according to the conditions, and incubated at room temperature while shaking slowly for 15 minutes. 400 μl of the binding buffer was respectively further added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany).
1. Construction of HCT116-RBD2 and RBD3 Sequence Mutants
Based on the published sequence (NCBI reference sequence NM_001008949.3), from the extracellular domain (25-103 amino acids) of which DRMDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAENFWTGDTSSDQ (ITPRIPL1-RBD2, i.e., SEQ ID NO: 2) and MDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAEN (ITPRIPL1-RBD3, i.e., SEQ ID NO: 3) were respectively selected as two different target sequences to synthesize a specific ITPRIPL1 sequence mutant with pcDNA3.1 as the vector, wherein the C termini of the extracellular domain and the extracellular domain-and-transmembrane domain were fused to Flag tags and green fluorescent proteins (GFPs), thereby producing ITPRIPL1 extracellular domain, ITPRIPL1-RBD2, ITPRIPL1-RBD3 plasmids containing Flag tags and green fluorescent proteins (GFPs).
2. It is Analyzed by Co-Immunoprecipitation Experiment that ITPRIPL1-RBD2 is the Shortest Sequence that Binds to CD3E.
The pcDNA3.1 plasmids respectively containing Flag-tagged ITPRIPL1 extracellular domain, ITPRIPL1-RBD2, ITPRIPL1-RBD3 and HA-tagged CD3E were co-transfected into HCT116 cells (ATCC, VA, USA), cultured in a 6-well plate (Corning, NY, USA) for 48-72 hours until the protein was fully expressed, and then lysed with a hybrid lysate of immunoprecipitation lysate (Thermo Fisher, MA, USA) mixed with a triple of protease-phosphatase-PMSF (Consun, Shanghai, China) at 1:100, and the cells were scraped. A portion of the cell samples were centrifuged, mixed with loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain an input level of protein samples; the remaining cell samples were immunoprecipitated with HA-tagged specific mouse antibodies (Biolegend, CA, USA) or Flag-tagged specific mouse antibodies (CST, MA, USA), washed with PBS, mixed with the loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain immunoprecipitated protein samples. And then, 12.5% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of the ITPRIPL1 extracellular domain, ITPRIPL1-RBD2, ITPRIPL1-RBD3 and CD3E-HA protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding ITPRIPL1 extracellular domain, ITPRIPL1-RBD2, ITPRIPL1-RBD3 and CD3E-HA strips were respectively incubated with Flag-tagged specific rabbit antibodies (Abcam, MA, USA) and HA-tagged specific rabbit antibodies (CST, MA, USA) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-rabbit secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
1. Enzyme-Linked Immunosorbent Assay (ELISA) Shows that there is Direct Concentration-Dependent Binding of the Isolated Fragments from the ITPRIPL1 (IT1) Extracellular Domain to the NRP2 Extracellular Domain.
An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with 0.03125/0.0625/0.125/0.25/0.5/1/2 μg/ml of ITPRIPL1-RBD recombinant protein each dissolved in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of coating buffer free of proteins. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, they were incubated with 1 μg/ml of protein fragments from the NRP2 extracellular domain (Sino Biological Inc., Beijing, China) in an incubator at 37° C. for 60 minutes for binding, in which the NRP2 proteins contained hFc (N2-Fc). After washing with PBST, they were incubated with PBS-diluted specific anti-human Fc segment antibodies (Abcam, MA, USA) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Sangon, Shanghai, China) was added at 100 μl per well for color development, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
As shown in
2. Flow Cytometry Demonstrates that NRP2 Binds to Cells Overexpressing ITPRIPL1 with Higher Efficiency.
It was found after testing that, HEK293 cells endogenously expressed a certain level of ITPRIPL1, and the expression level was further increased after stable transfection. The cultured 293E wild-type cells (ATCC, VA, USA) and 293E-ITPRIPL1 stably transfected cell lines were digested and then counted, and the cell number was respectively adjusted to 2×106/ml. 200 μl of them were respectively added into the wells of a 96-well plate (costar, ME, USA). Into the wells of each experimental group were respectively added 0/0.5/1/2/4 μg/ml of NRP2 protein (Sino Biological Inc., Beijing, China). The 96-well plate was placed in a cell incubator, standing for 30 minutes, and then taken out and transferred into EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA) and centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. Anti-human IgG Alexa Fluor 647 antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:1000. In addition to the negative control, 200 μl of antibody diluent was added into each EP tube and incubated at room temperature for 30 minutes at 40 rpm on a shaker. After then, the EP tubes were taken out and centrifuged at 400 rcf for 5 minutes and the supernatants were then discarded. The cells were resuspended and washed with 1000 μl of the cell staining buffer and centrifuged at 400 rcf for 5 minutes, the supernatants were then discarded again and the washing was repeated. After the completion of washing, 300 μl of the cell staining buffer was added into each EP tube for resuspension, and transferred into flow tubes (Falcon, NY, USA) for on-board analysis (Miltenyi Biotec, Cologne, Germany).
The luciferin reporter assay demonstrates that the free isolated purified protein from the ITPRIPL1 extracellular domain (IT1-RBD) can inhibit the IFN proliferation signaling of THP1-dual cells completed by PMA-induced differentiation under the condition of inactivation.
The cultured THP1-dual cells (Invivogen, CA, USA) were counted, centrifuged at 1000 rpm for 5 minutes, and then the cells were resuspended with antibiotic-free RPMI-1640 medium (Gibco, CA, USA) to adjust the cell number to 2×106/ml. 200 μl of the cells and 20 ng/ml of PMA reagent (Invivogen, CA, USA) were added into each well of a transparent 96-well plate (Thermo Fisher, MA, USA) for induction, cultured in an incubator for 3 hours, and then washed with PBS and the medium was exchanged. 72 hours later, the medium was exchanged again, 0/1/2/4/8 μg/ml of free isolated purified protein from the ITPRIPL1 extracellular domain were respectively added and cultured in a cell incubator for 18-24 hours. After the completion of reaction, a non-transparent 96-well plate was taken, into each well of which were added 50 μl of Quanti-luc reagent (Invivogen, CA, USA) and 10 μl of the cell mixture and mixed well, and then the signals were detected immediately with a multimode microplate reader.
As shown in
1. Separation and Purification of IT1-RBD-Fc Protein.
Based on the published sequence (NCBI reference sequence NM_001008949.3), from which an extracellular domain (HPLMVSDRMDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAENFWTGDTSSD QLVLGKKDMGWPFQADGQEG) was selected as IT1-RBD1, i.e., all parts in the extracellular domain with signal peptides removed; DRMDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAENFWTGDTSSDQ was selected as IT1-RBD2, i.e., multiple conserved sequence amino acids at the N-terminus and multiple conserved and non-conserved sequence amino acids at the C-terminus were removed on the basis of RBD1 (for verifying the importance of conserved amino acid sites of mammals on the function); MDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAEN was selected as IT1-RBD3 (only the sequence predicted to be the Alpha helical secondary structure was reserved, verifying whether the secondary structure was the minimal sequence necessary for the function), followed by a tandem Fc sequence that did not contain the CH1 region (PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), corresponding plasmids were construct with pcDNA3.1 as the vector. The mutated derivative sequences of the above several ITPRIPL1 extracellular domains are shown in
The sequence in which the ITPRIPL1 extracellular domain exerted its function was characterized through this example. Protein-protein docking using the AlphaFold predicted structure of the functional fragment of the ITPRIPL1 extracellular domain and the x-ray diffraction crystal structure (1XIW.pdb) of CD3E was analyzed, indicating that the ITPRIPL1 of multiple species such as human, mouse, rat, grivet, golden monkey, black snub-nosed monkey, Bolivian squirrel monkey, Ma's night monkey, chimpanzee, etc. as shown in
Therefore, according to the different sites of different species, an RBD1 derivative sequence with CD3E binding function was obtained: DRMDLDTLARSRQLEKRMSEEMRxLEMEFEERxxxAExxQKxENxWxGxTSxDQ (wherein x represents an amino acid that can be substituted).
Functional detection and analysis of RBD1, RBD2, RBD3 were conducted in this example.
After plasmids were acquired, they were transfected with a PEI reagent (Life-iLab, Shanghai, China) into 293E cells (ATCC, VA, USA), and 120 hours later, the cells were lysed with RIPA lysate (Beyotime, Shanghai, China) and a triple of protease inhibitor-phosphatase inhibitor-PMSF (Consun, Shanghai, China) on ice for 10 minutes, and centrifuged at 12000 rpm for 15 minutes. The supernatants were then aspirated and shaken with 1:100 of protein A magnetic bead (Smart-Lifesciences, Changzhou, China) at 130 rpm for 2 hours at room temperature. The mixture solution was aspirated into a filter column (Millipore, MA, USA) pre-equilibrated with equilibrium liquid (20 mM disodium hydrogen phosphate, 0.15 M sodium chloride, pH 7.0) for washing with the equilibrium liquid for two times, and then eluted with an eluent (0.1 M glycine, pH 3.0): neutralization solution (1 M Tris-HCl, pH 8.5) of 16:1, resulting in the purified IT1-RBD-Fc protein. The concentration of the protein was determined with a Nanodrop spectrophotometer, and it was stored at −20° C.
2. Luciferin Reporter Assay Demonstrates that the Purified IT1-RBD-Fc Protein can Inhibit the NFKB Proliferation Signaling in Jurkat-Dual Cells Under Activation and Inactivation Conditions of ConA.
The cultured Jurkat-dual cells (Invivogen, CA, USA) were counted, centrifuged at 1000 rpm for 5 minutes and then resuspended with antibiotic-free IMDM medium (Gibco, CA, USA) to adjust the cell number to 2×106/ml. 200 μl of the cells were added into each well of a transparent 96-well plate. Into each well was respectively added 0/4 μg/ml of the purified IT1-RBD1/RBD2/RBD3-Fc protein. After reaction in a cell incubator for two hours, into each well was added 50 μg/ml of Concanavalin A (ConA) (Aladdin, Shanghai, China) as the activation group or added an equal volume of endotoxin-free water as the inactivation group and reacted in the cell incubator for 18-24 hours. After the completion of reaction, a non-transparent 96-well plate (costar, ME, USA) was taken, into each well of which were added 50 μl of Quanti-luc reagent (Invivogen, CA, USA) and 20 μl of the reaction mixture and mixed well, and then the signals were detected immediately with a multimode microplate reader.
As shown in
3. Flow Cytometry Demonstrates that the Purified IT1-RBD1-Fc Recombinant Protein can Reduce the Killing of Kidney-Derived H3K293 Cells by Human Peripheral Blood Mononuclear Cells (PBMCs).
HEK293 cells have been widely used to construct autoimmune disease models for the studies on the disease progression, molecular and cellular biological mechanisms, and pharmacology of kidney autoimmune diseases, autoimmune diseases of the nervous system, systemic lupus erythematosus (SLE), etc. (Stepanenko AA, Gene. 2015 Sep. 15; 569(2):182-90, Hira S. J Physiol Sci. 2019 September; 69(5):723-732., Keskitalo S, Front Immunol. 2019 Dec. 5; 10:2770). Furthermore, HEK293 cells have also been used to construct post-transplant rejection disease models (Yi Gao. Int J Clin Exp Med. 2014; 7(11): 4572-4583, Lorber, Marc I, Transplantation: 1999-67(δ)-p 897-903, Pabois A, Biochem Pharmacol. 2016 Mar. 15; 104:95-107), antiviral infection immune responses (Ismail Cem Yilmaz, Allergy. 2021 Sep. 14. doi: 10.1111/all.15091, Elizabeth A Reap, Vaccine. 2007 Oct. 16; 25(42):7441-9), and tumor immune responses, etc. (A A Stepanenko, Gene. 2015 Sep. 15; 569(2):182-90).
Furthermore, peripheral blood mononuclear cells (PBMCs) derived from autoimmune diseases or normal people have also been used in many studies to construct autoimmune disease models (Yoshikawa N. Horm Metab Res. 1994 September; 26(9):419-23.), post-transplant rejection (Transplant Proc. 2016 October; 48(8):2840-2844.), anti-infective immune responses (Har-Noy M, J Transl Med. 2020 May 12; 18(1):196, Nakamura-Hoshi M, Sci Rep. 2020 Jul. 9; 10(1):11394.), anti-tumor immune responses, etc. (Zhuang X, Cancer Immunol Res. 2019 June; 7(δ):939-951), in which the target cells involved also include HEK293 cells. In fact, the effect of PBMC-based humanized NSG mouse model is highly correlated with the effect of PBMCs on target cells in vitro. Therefore, an in vitro model based on PBMCs and target cells plays an important role in the disease process and the manifestation of drug efficacy.
By using HEK293 cells as target cells for autoimmunity, transplant rejection, allergies, and anti-tumor reactions, and by using human peripheral blood mononuclear cells (PBMCs) as the effector cells, this example exemplifies the effect of ITPRIPL1-based regulators on the interaction of antigen-presenting cells and T cells as well as the intervention effect on the above different diseases through a representative disease model.
The implementation process is detailed below: CD3 and CD28 antibodies (Invitrogen, CA, USA) were diluted by mixing with PBMCs (ATCC, VA, USA) to a final concentration of 1 μg/ml so as to activate T cells, and then cultured overnight. The next day, the PBMCs and 293E wild-type (ATCC, VA, USA)/ITPRIPL1-overexpressed cells were counted, and the cell number was respectively adjusted to 1×106/ml. Into the control group were added 100 μl of 293E wild-type/ITPRIPL1-overexpressed cells and 100 μl of culture medium. For the experimental groups, each 100 μl of PBMCs and another kind of cells were added into a 96-well plate (costar, ME, USA). Among the experimental groups, into 3 groups of wild-type 293E cells was further respectively added 2 μg/ml of IT1-RBD1/RBD2/RBD3-Fc protein, and incubated in an incubator for 6 hours. The 96-well plate was taken out, from which the cells were aspirated, placed in an EP tube (Axygen, CA, USA), and centrifuged at 400 rcf for 5 minutes, and the supernatant was then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. The CD45-APC antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:20. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature while shaking slowly for 30 minutes. After centrifugation at 400 ref for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Beyotime, Shanghai, China); and centrifuged and washed again. An unstained group without the addition of PBMCs, a double-stained group without the addition of PBMCs, an unstained group with the addition of PBMCs, a single-stained Annexin V-FITC group with the addition of PBMCs, a single-stained PI group with the addition of PBMCs, a double-stained group with the addition of PBMCs, and a double-stained group each with the addition of IT1 protein were set, into which were respectively added 100 μl of the binding buffer, 5 μl of Annexin V-FITC (Beyotime, Shanghai, China) and 10 μl of PI (Beyotime, Shanghai, China) according to the conditions, and incubated at room temperature while shaking slowly for 15 minutes. 400 μl of the binding buffer was respectively further added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany).
4. Western Blot Experiment Demonstrates that the ITPRIPL1-RBD Recombinant Protein and the Purified IT1-RBD1-Fc Recombinant Protein have the Function of Inhibiting the ZAP70 and Akt Pathways Downstream of the CD3E.
The cultured Jurkat cells (ATCC, VA, USA) were counted. 4×106 of the cells were taken into a centrifugal tube of 15 ml (Corning, NY, USA) and centrifuged at 800 rpm for 4 minutes, and the supernatant was then discarded. The cells were resuspended in serum-free RPMI1640 medium (Meilun Biotech, Dalian, China), cultured in an incubator for 3 hours of starvation, centrifuged at 800 rpm for 4 minutes, and resuspended in 4 ml of complete medium (Meilun Biotech, Dalian, China). Each 1 ml of them was taken and added into each well of a 12-well plate (Corning, NY, USA). Into the each well was respectively added: no treatment; 4 μg/ml of IT1-RBD protein; 4 μg/ml of RBD1-Fc protein; 4 μg/ml of RBD2-Fc protein. After mixing well and culturing in the incubator for 10 min, the plate was taken out, and the cells were transferred into an EP tube of 1.5 ml (Axygen, CA, USA), centrifuged at 800 rpm for 4 minutes, resuspended and washed with PBS and centrifuged, and washed again. After the completion of centrifugation, the supernatant was discarded. The RIPA lysate (Beyotime, Shanghai, China) and a triple of protease inhibitor-phosphatase inhibitor-PMSF (Consun, Shanghai, China) were formulated at 1:100, and 80 μl of the hybrid lysate was added into each tube of cells. Each EP tube was frozen and thawed on liquid nitrogen-ice for three cycles, and centrifuged at 12000 rpm at 4° C. for 15 minutes after the last thawing. After the completion of centrifugation, the supernatants were taken and formulated into a variety of cell samples at a ratio of 4:1 of the supernatant to 5×loading buffer (Beyotime, Shanghai, China), and denatured in a metal bath at 100° C. for 10 minutes to produce protein samples. And then, 10% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of Akt, ZAP70 and GAPDH protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding strips were respectively incubated with pAkt specific rabbit antibodies (CST, MA, USA), Akt specific rabbit antibodies (CST, MA, USA), pZAP70 specific rabbit antibodies (CST, MA, USA), GAPDH antibodies (Consun, Shanghai, China) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-rabbit secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
5. Western Blot Experiment Demonstrates that CD3E Protein has the Function of Blocking the Effect of ITPRIPL1-RBD1-Fc Recombinant Protein on the Phosphorylation of ZAP70 and Akt Pathways.
The cultured Jurkat cells (ATCC, VA, USA) were counted. 4×106 of the cells were taken into a centrifugal tube of 15 ml (Corning, NY, USA) and centrifuged at 800 rpm for 4 minutes, and the supernatant was then discarded. The cells were resuspended in serum-free RPMI1640 medium (Meilun Biotech, Dalian, China), cultured in an incubator for 3 hours of starvation, centrifuged at 800 rpm for 4 minutes, and resuspended in 4 ml of complete medium (Meilun Biotech, Dalian, China). Each 1 ml of them was taken and added into each well of a 12-well plate (Corning, NY, USA). Into the each well was respectively added: no treatment; 4 μg/ml of RBD1-Fc protein; 4 μg/ml of RBD1-Fc protein+4 μg/ml of CD3E protein. After mixing well and culturing in the incubator for 10 min, the plate was taken out, and the cells were transferred into an EP tube of 1.5 ml (Axygen, CA, USA), centrifuged at 800 rpm for 4 minutes, resuspended and washed with PBS and centrifuged, and washed again. After the completion of centrifugation, the supernatant was discarded. The RIPA lysate (Beyotime, Shanghai, China) and a triple of protease inhibitor-phosphatase inhibitor-PMSF (Consun, Shanghai, China) were formulated at 1:100, and 80 μl of the hybrid lysate was added into each tube of cells. Each EP tube was frozen and thawed on liquid nitrogen-ice for three cycles, and centrifuged at 12000 rpm at 4° C. for 15 minutes after the last thawing. After the completion of centrifugation, the supernatants were taken and formulated into a variety of cell samples at a ratio of 4:1 of the supernatant to 5×loading buffer (Beyotime, Shanghai, China), and denatured in a metal bath at 100° C. for 10 minutes to produce protein samples. And then, 10% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of Akt, ZAP70, ERK and GAPDH protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding strips were respectively incubated with pAkt specific rabbit antibodies (CST, MA, USA), Akt specific rabbit antibodies (CST, MA, USA), pZAP70 specific rabbit antibodies (CST, MA, USA), ZAP70 specific rabbit antibodies (CST, MA, USA), pERK specific rabbit antibodies (CST, MA, USA), ERK specific rabbit antibodies (CST, MA, USA), GAPDH antibodies (Consun, Shanghai, China) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-rabbit secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
1. Construction of CD3 Mutant Jurkat Cells
The cultured Jurkat cells (ATCC, VA, USA) were counted. 2×105 of the cells were taken into each well of a 24-well plate (Corning, NY, USA), and plated overnight. 500 μl of complete medium (Meilun Biotech, Dalian, China) was mixed with knockout lentivirus (GenePharma, Shanghai, China) against CD3 at a MOI value of 100 for viral infection. 48 hours later, the medium was changed. After continuing the incubation for 24 hours, 1 μg/ml of puromycin was added for screening for a period of one week. At the end of screening, CD3-knockout Jurkat cell lines were obtained.
The CD3-knockout Jurkat cells were counted. 2×105 of the cells were taken into each well of a 24-well plate, and plated overnight. 500 μl of complete medium was mixed with CD3-K76T (unable to make correct conformational changes) and CD3-APRS (with changes in the PRS segments)-overexpressed viruses at a MOI value of 100 for viral infection. 48 hours later, the medium was changed. After continuing the incubation for 24 hours, 600 μg/ml of Geneticin (G418) (Gibco, CA, USA) was added for screening for a period of one week. At the end of screening, CD3-mutated Jurkat cell lines were obtained.
2. Western Blot Experiment Demonstrates that the Modulation on the Phosphorylation Pathway of T Cells by ITPRIPL1 Requires the Conformational Changes in CD3 and PRS Segments
Wild-type Jurkat cells, CD3-knockout Jurkat cells, CD3-K76T Jurkat cells, and CD3-APRS cells were counted. 1×106 of the cells were taken into each well of a 12-well plate (Corning, NY, USA). After activation by adding 10 μg/ml of OKT3, the wells were respectively subjected to no treatment or treated with 4 μg/ml of ITPRIPL1-RBD1-Fc, and cultured for 10 minutes to harvest the cells. The cells were transferred into EP tubes of 1.5 ml (Axygen, CA, USA), centrifuged at 800 rpm for 4 minutes, resuspended and washed with PBS and centrifuged, and washed again. After the completion of centrifugation, the supernatants were discarded. The RIPA lysate (Beyotime, Shanghai, China) and a triple of protease inhibitor-phosphatase inhibitor-PMSF (Consun, Shanghai, China) were formulated at 1:100, and 80 μl of the hybrid lysate was added into each tube of cells. Each EP tube was frozen and thawed on liquid nitrogen-ice for three cycles, and centrifuged at 12000 rpm at 4° C. for 15 minutes after the last thawing. After the completion of centrifugation, the supernatants were taken and formulated into a variety of cell samples at a ratio of 4:1 of the supernatant to 5×loading buffer (Beyotime, Shanghai, China), and denatured in a metal bath at 100° C. for 10 minutes to produce protein samples. And then, 10% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of Akt, ZAP70, ERK and GAPDH protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding strips were respectively incubated with pAkt specific rabbit antibodies (CST, MA, USA), Akt specific rabbit antibodies (CST, MA, USA), pZAP70 specific rabbit antibodies (CST, MA, USA), ZAP70 specific rabbit antibodies (CST, MA, USA), pERK specific rabbit antibodies (CST, MA, USA), ERK specific rabbit antibodies (CST, MA, USA), GAPDH antibodies (Consun, Shanghai, China) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-rabbit secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
3. Fluorescence Staining Experiment of Intracellular Calcium Ion Flux Demonstrates that ITPRIPL1 has the Effect of Reducing the Activity of T Cells
The cultured Jurkat cells (ATCC, VA, USA) were counted. 5×105 of the cells were taken into each well of a 12-well plate (Corning, NY, USA), and treated with PBS or 2 μg/ml of ITPRIPL1-RBD protein for 24 hours. After the treatment was completed, 4 μmol of Fluo-8 AM (Abcam, MA, USA) intracellular calcium ion indicator was given for staining, and incubated in an incubator in dark for 1 hour. After washing with HHBS buffer (Solarbio, Beijing, China) twice, the plate was observed under a fluorescence microscope.
4. Fluorescence Staining Experiment of Intracellular Calcium Ion Flux Demonstrates that the Effect of ITPRIPL1 to Reduce the Activity of T Cells Relies on the Conformational Changes of CD3 and PRS Segments
Wild-type Jurkat cells, CD3-knockout Jurkat cells, CD3-K76T Jurkat cells, CD3-APRS cells were counted. 5×105 of cells were taken into each well of a 12-well plate (Corning, NY, USA), and treated with PBS or 4 μg/ml of ITPRIPL1-RBD1-Fc protein for 24 hours. After the treatment was completed, 4 μmol of Fluo-8 AM (Abcam, MA, USA) intracellular calcium ion indicator was given for staining, and incubated in an incubator in dark for 1 hour. After washing with HHBS buffer (Solarbio, Beijing, China) twice, the plate was observed under a fluorescence microscope.
1. Western Blot Experiment Demonstrates that ITPRIPL1-RBD1-Fc Protein can Increase the Binding Signal of CD3 to Nck in a Concentration-Dependent Manner.
The cultured Jurkat cells (ATCC, VA, USA) were counted. 5×106 of the cells were taken into a centrifugal tube of 15 ml (Corning, NY, USA) and centrifuged at 800 rpm for 4 minutes and the supernatant was then discarded. The cells were resuspended in serum-free RPMI1640 medium (Meilun Biotech, Dalian, China), cultured in an incubator for 3 hours of starvation, centrifuged at 800 rpm for 4 minutes, and resuspended in 5 ml of complete medium (Meilun Biotech, Dalian, China). Each 1 ml of them was taken and added into each well of a 12-well plate (Corning, NY, USA), into which was firstly added 10 μg/ml of OKT3 for activation. And at the same time, into the each well was respectively added: no treatment; 0.5/1/2/4 μg/ml of RBD1-Fc protein. After mixing well and culturing in the incubator for 5 min, the plate was taken out, and the cells were transferred into an EP tube of 1.5 ml (Axygen, CA, USA), centrifuged at 800 rpm for 4 minutes, resuspended and washed with PBS and centrifuged, and washed again. After the completion of centrifugation, the supernatant was discarded. The immunoprecipitation lysate (Thermo Fisher, MA, USA) and a triple of protease inhibitor-phosphatase inhibitor-PMSF (Consun, Shanghai, China) were formulated at 1:100, and 200 μl of the hybrid lysate was added into each tube of cells. A portion of the cell samples were centrifuged, mixed with loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain an input level of protein samples; the remaining cell samples were immunoprecipitated with CD3E specific mouse antibodies (Santa cruz biotechnology, CA, USA), washed with PBS, mixed with the loading buffer (Beyotime, Shanghai, China) and denatured in a metal bath at 100° C. to obtain immunoprecipitated protein samples. The supernatant after centrifugation was formulated with 5×loading buffer (Beyotime, Shanghai, China) at a ratio of 4:1 into various cell samples, and denatured in a metal bath at 100° C. for 10 minutes to obtain protein samples. 10% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of CD3E and Nck protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding strips were respectively incubated with CD3E specific rabbit antibodies (CST, MA, USA) and Nck specific rabbit antibodies (CST, MA, USA) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-rabbit secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
As shown in
2. Proximity Ligation Assay Demonstrates that ITPRIPL1-RBD1-Fc Protein can Increase the Binding Signal of CD3 to Nck.
The cultured Jurkat cells (ATCC, VA, USA) were counted. 2×106 of the cells were taken into a centrifugal tube of 15 ml (Corning, NY, USA), centrifuged at 800 rpm for 4 minutes and the supernatant was then discarded. The cells were resuspended in serum-free RPMI1640 medium (Meilun Biotech, Dalian, China), cultured in an incubator for 3 hours of starvation, centrifuged at 800 rpm for 4 minutes, and resuspended in 2 ml of complete medium (Meilun Biotech, Dalian, China). Each 1 ml of them was taken and added into each well of a 12-well plate (Corning, NY, USA), into which was firstly added 10 μg/ml of OKT3 for activation. And at the same time, into the each well was respectively added: no treatment; 4 μg/ml of RBD1-Fc protein. After culturing in the incubator for 5 min, Jurkat cells were taken out, and subjected to relative operations with the reagents in the proximity ligation assay kit (Merck, NJ, USA) as well as CD3E specific mouse antibodies (Santa cruz biotechnology, CA, USA) and Nck specific rabbit antibodies (CST, MA, USA) according to the instructions provided with the kit. After mounting was completed, the slide was observed under a fluorescence microscope.
As shown in
1. Construction of Humanized CD3ε Mouse MC38 Subcutaneous Xenograft Tumor In Vivo Model
The constructed full-length ITPRIPL1-Flag plasmid and the empty pcDNA3.1 plasmid was respectively transfected into MC38 cells (Kerafast, MA, USA), cultured in an incubator for 24-48 hours, and screened by adding 200 μg/ml of Geneticin (G418) (Gibco, CA, USA). 10−14 days later, after the empty pcDNA3.1 plasmid-transfected group of cells all died, MC38-ITPRIPL1 stably transfected cell lines were obtained.
6-8-week-old humanized CD3ε mice (Model Organisms Center, Shanghai, China) were selected and randomly grouped according to the weight, with 6 mice in each group. MC38 wild-type and MC38-ITPRIPL1 stably transfected cell lines were counted, and resuspended with PBS to a cell density of 1.5×107/ml. The mice were shaved, and 1.5×106 wild-type or ITPRIPL1-overexpressed MC38 cells were subcutaneously inoculated into the right armpit to construct in vivo models of humanized CD3ε mouse MC38 subcutaneous xenograft tumor.
2. Overexpression of ITPRIPL1 Significantly Increases the Tumor Growth in Mice
After completing the construction of humanized CD3ε mouse MC38 subcutaneous xenograft tumor in vivo models, the tumor size was measured with a vernier caliper from the fifth day after inoculation. The long diameter and short diameter of the tumor were measured each time, and the tumor size was calculated following the formula of ½*A*a*a. Measurement was conducted every three days, and the tumor sizes were recorded. On day 23 after inoculation, all mice were sacrificed. After removing the tumors, the tumor weight was weighed and statistically analyzed.
As shown in
3. Flow Cytometry Demonstrates that the Overexpression of ITPRIPL1 Inhibits the Activity of T Cells in Mice
At least 1 ml of peripheral blood was obtained from mice by means of cardiac blood sampling. Mouse PBMC cells were obtained by using a mouse peripheral blood PBMC separation kit (Solarbio, Beijing, China) according to the corresponding instruction of the kit.
The mouse PBMCs were placed into EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. Mouse CD8-APC antibodies (Biolegend, CA, USA), mouse CD69-APC antibodies (Biolegend, CA, USA), and mouse CD137-APC antibodies (Biolegend, CA, USA) were diluted with the cell staining buffer at 1:20. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature while shaking slowly for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 500 μl of the cell staining buffer; and centrifuged and washed again. 200 μl of the cell staining buffer was respectively added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany).
As shown in
As shown in
4. Immunohistochemical Staining Demonstrates that the Overexpression of ITPRIPL1 Reduces the Infiltration of T Cells in Tumors
MC38 tumor tissues were stripped for sectioning and paraffin embedding treatment (Biossci, Wuhan, China). The resulting paraffin sections were subjected to dewaxing, hydration, antigen retrieval or other treatment, and then incubated with mouse CD8 antibodies (CST, MA, USA) in a wet box overnight. The next day, after rewarming, secondary antibodies were incubated according to the reagent instructions, stained with DAB (Solarbio, Beijing, China) and hematoxylin (Solarbio, Beijing, China), and then air-dried in a reverse alcohol concentration gradient and mounted. After the mounting was completed, the slide was observed under a fluorescence microscope and photographed under natural light.
As shown in
5. Construction of ITPRIPL1 Heterozygous/Homozygous Knockout Mouse In Vivo Models
By using the CRISPR/Cas9 technique, Itpripl1 gene protein reading frame shift and function deletion were caused by introducing mutations through non-homologous recombination repairment. The brief process was as below: Cas9 mRNA and gRNA were obtained by means of in vitro transcription; Cas9 mRNA and gRNA were microinjected into the fertilized eggs of C57BL/6J mice to obtain mice of F0 generation. The positive mice of F0 generation identified by PCR amplification and sequencing were mated with C57BL/6J mice to obtain six positive mice of F1 generation, that were, heterozygous knockout and homozygous knockout ITPRIPL1 mouse.
6. Flow Cytometry Demonstrates that ITPRIPL1-Knockout Enhances the Activity of T Cells
Blood was sampled from mouse models, from which PBMCs were separated. The cells in the cell suspension were counted. After counting, the cell suspension was diluted with PBS solution to adjust the concentration of the test cells to 106/mL. 200 μl of the cell suspension was taken and centrifuged at 1000 rpm for 5 min (at 4° C.). The cells were resuspended in 100 μl of PBS; the test tubes were set as below:
Incubation at 4° C. in dark for 30 minutes. At the end of incubation, testing with a flow cytometer.
As shown in
7. ELISA Assay Demonstrates that the Knockout of ITPRIPL1 Increases the Secretion of Immuno-Activated Cytokines
Mouse PBMCs were operated and treated according to the instructions of ELISA kits for cytokines such as granzyme A, granzyme B, IL-2, TNF-alpha, IL-21 (Abnova, CA, USA), obtaining the corresponding experimental results.
As shown in
8. Immunohistochemical Staining Demonstrates that the Knockout of ITPRIPL1 Increases the Infiltration of T Cells in Testicular Tissues
The testicular tissues of mice were stripped for staining and sectioning (JRDUN, Shanghai, China), and observed under a fluorescence microscope for the corresponding results.
As shown in
The present disclosure innovatively reveals that ITPRIPL1 μlays an important role in the immune evasion of tumors, and tumors with elevated expression of ITPRIPL1 are suitable to be treated with the inhibitor of ITPRIPL1 to inhibit the tumor growth. Therefore, it is very important to detect the expression of ITPRIPL1. The present disclosure exemplifies a method of detecting the expression of ITPRIPL1 using its specific antibodies, and reveals that the high expression of ITPRIPL1 can be used to indicate the presence of tumor cells in the body, and can precisely mark the boundaries between tumor tissues and normal tissues. Furthermore, ITPRIPL1 is also significantly correlated with the progression and stages of tumors. In particular:
The procedures for producing ITPRIPL1 polyclonal antibodies were as previously described. ITPRIPL1 monoclonal antibodies were further prepared by hybridoma screening and isolation and culture. Immunohistochemical staining was performed on microarrays of a variety of tumor tissues (Shanghai Outdo Biotech) with the antibodies of ITPRIPL1, and the images were scanned and the expression level and distribution of ITPRIPL1 were statistically analyzed. As shown in
The staining of ITPRIPL1 in tumor tissues showed a very prominent consistency. Firstly, ITPRIPL1 was generally significantly up-regulated in many of the above tumors, with exceptions in only a few tumors. Secondly, there was strong uniformity in the expression of ITPRIPL1 in tumor tissues, i.e., the expression was relatively consistent in different sites of the primary lesions and in different cells of the metastases. This characteristic can be seen in
1. Preparation of ITPRIPL1 Mouse Hybridoma Antibody
(1) By using human ITPRIPL1-Fc recombinant protein (as set forth in SEQ ID NO: 21) as the immunogen, C57BL/6 mice were immunized for multiple times to enhance the effect:
(2) After the immune effect had been tested to meet the requirements, blood was taken, polyclonal antibodies were separated and purified in a cumulative manner for several times. Specific experimental steps include:
2. Enzyme-Linked Immunosorbent Assay (ELISA) Verifying the Binding of Each Hybridoma Antibody to ITPRIPL1
In order to verify the binding capacity of each hybridoma antibody to ITPRIPL1, an enzyme-linked immunosorbent assay (ELISA) was performed to confirm the direct binding of each antibody to ITPRIPL1 protein. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with 100 μl (1 μg/ml) of ITPRIPL1 recombinant protein (cusabio, Wuhan, China) in ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of coating buffer free of ITPRIPL1 recombinant protein. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, they were incubated in an incubator at 37° C. for 60 minutes for binding. After washing with PBST, they were incubated with PBS-diluted specific anti-mouse Fc segment antibodies (Consun, Shanghai, China) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
2. Flow Cytometry (FACS) Verifying the Binding of Each Hybridoma Antibody to ITPRIPL1
In order to further verify the binding capacity of each hybridoma antibody to ITPRIPL1, flow cytometry (FACS) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. Jurkat cells (ATCC, VA, USA) with high endogenous expression of ITPRIPL1 were counted, and the cell number was adjusted to 1×106/ml. Each 100 μl of the cells were added into the wells of a 96-well plate (Thermo Fisher, MA, USA). Into each well was respectively added hybridoma antibody or control serum at a final concentration of 1 μg/ml, and incubated in an incubator for 30 minutes. The 96-well plate was taken out, and the cells of each well were placed in EP tubes (Axygen, CA, USA) and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. Anti-mouse Fc segment-Alexa Fluor 488 antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:500. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature for 30 minutes. After centrifugation at 400 ref for 5 minutes, the supernatants were discarded, and the cells were resuspended and washed with 1 ml of the cell staining buffer; and centrifuged and washed again. 300 μl of the cell staining buffer was respectively added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany) to test.
1. Flow Cytometry (FACS) Verifying the Binding of 13B7 Antibody to Cells with Different Expression Levels of ITPRIPL1
In order to further verify the binding capacity of 13B7 to ITPRIPL1, flow cytometry (FACS) was used to confirm the direct binding of 13B7 antibody to various cells with different expression levels of ITPRIPL1. The cells with different endogenous expression levels of ITPRIPL1, i.e., HCT116 (ATCC, VA, USA), A549 (ATCC, VA, USA), MC38 (Kerafast, MA, USA), Jurkat (ATCC, VA, USA), Raji cells (ATCC, VA, USA) as well as MC38-ITPRIPL1 stably transfected cell lines, were counted and the cell number was adjusted to 1×106/ml. Each 100 μl of the cells were added into the wells of a 96-well plate (Thermo Fisher, MA, USA). Into each well was respectively added the 13B7 antibody at a final concentration of 1 μg/ml, and incubated in an incubator for 30 minutes. The 96-well plate was taken out, and the cells of each well were placed in EP tubes (Axygen, CA, USA) and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. Anti-mouse Fc segment-Alexa Fluor 488 antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:500. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatants were discarded, and the cells were resuspended and washed with 1 ml of the cell staining buffer; and centrifuged and washed again. 300 μl of the cell staining buffer was respectively added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany) to test.
2. Flow Cytometry (FACS) Verifies that the 13B7 Antibody can Bind to Jurkat Cells in a Concentration-Dependent Manner
In order to verify the binding capacity of 13B7 to ITPRIPL1, flow cytometry (FACS) was used to confirm the concentration-dependent binding of the 13B7 antibody to Jurkat cells. The Jurkat cells with high endogenous expression of ITPRIPL1 were counted, and the cell number was adjusted to 1×106/ml. Each 100 μl of the cells were added into the wells of a 96-well plate (Thermo Fisher, MA, USA). Into each well was respectively added the 13B7 antibody at a final concentration of 0.0625/0.125/0.25/0.5/1/2 μg/ml, and incubated in an incubator for 30 minutes. The 96-well plate was taken out, and the cells of each well were placed in EP tubes (Axygen, CA, USA) and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. Anti-mouse Fc segment-Alexa Fluor 488 antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:500. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatants were discarded, and the cells were resuspended and washed with 1 ml of the cell staining buffer; and centrifuged and washed again. 300 μl of the cell staining buffer was respectively added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany) to test.
3. Protein Western Blot Verifies that the 13B7 Antibody can Specifically Bind to the ITPRIPL1 Protein of Cells with Different Expression Levels of ITPRIPL1
Jurkat, HCT116, MC38 cells with different endogenous expression profiles of ITPRIPL1 were counted. 1×106 cells were respectively taken, washed with PBS and centrifuged, and then lysed with 60 μl of RIPA lysate (Beyotime, Shanghai, China) and a triple of protease inhibitor-phosphatase inhibitor-PMSF (Consun, Shanghai, China) at a ratio of 1:100 on ice for 15 minutes. They were frozen and thawed with liquid nitrogen for three cycles. After centrifugation, mixing with loading buffer (Beyotime, Shanghai, China) and denaturation in a metal bath at 100° C., an input level of protein sample was produced. And then, 12.5% of PAGE gel (Epizyme, Shanghai, China) was formulated in a gel plate (Bio-Rad, CA, USA) according to the instructions. The formulated gel was placed in an electrophoresis cell (Bio-Rad, CA, USA), the power (Bio-Rad, CA, USA) was turned on to let the strips run through the stacking gel at a constant voltage of 80 V and run through the separating gel at a constant voltage of 120 V. When the strips run to the bottom of the separating gel, the film was transferred in an electrophoretic transfer cell (Bio-Rad, CA, USA) by a method of tank blot at a constant current of 350 mA for 90 minutes. After the film transfer was completed, the film was sheared according to the mass of the ITPRIPL1 protein and the internal reference GAPDH protein. After blocking with rapid blocking buffer (Epizyme, Shanghai, China) for 10 minutes, the corresponding ITPRIPL1 and GAPDH strips were respectively diluted with the 13B7 antibody at a final concentration of 1 μg/ml and incubated with GAPDH-HRP antibodies (Consun, Shanghai, China) at 4° C. overnight. The next day, after washing with TBST, the strips were incubated with specific anti-mouse secondary antibodies (Consun, Shanghai, China) that were diluted with 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS at room temperature for 1 hour, then washed with TBST, placed in a hybrid luminescent fluid (Share-Bio, Shanghai, China) for 1 minute, and exposed under a Gel-Imager (Bio-Rad, CA, USA).
In order to verify whether there is a blocking effect of each hybridoma antibody on the binding of ITPRIPL1 to CD3E or SEMA3G, an enzyme-linked immunosorbent assay (ELISA) was used to verify the blocking effect of the antibodies. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with 1 μg/ml of ITPRIPL1-Fc-tagged recombinant protein (Abclonal, Wuhan, China) or 1 μg/ml of CD3E-Fc-tagged protein (Acro, Beijing, China) dissolved in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of coating buffer free of proteins. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, into the ITPRIPL1-Fc protein-coated wells were added SEMA3G-His-tagged protein (cusabio, Wuhan, China) at a final concentration of 1 μg/ml and hybridoma antibodies at a final concentration of ½ μg/ml at the same time; into the CD3E-Fc protein-coated wells were added ITPRIPL1-His-tagged protein (cusabio, Wuhan, China) at a final concentration of 1 μg/ml and hybridoma antibodies at a final concentration of ½ μg/ml at the same time; they were bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-His segment antibodies (Abcam, MA, USA) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
1. Immunosorbent Assay (ELISA) Verifies the Binding Capacity of Each Monoclonal Antibody to ITPRIPL1
After monoclonal purification of each hybridoma antibody, 16 monoclonal antibodies with better growth were obtained. In order to verify the binding capacity of each monoclonal antibody to ITPRIPL1, an enzyme-linked immunosorbent assay (ELISA) was used to verify the direct binding of each antibody to the ITPRIPL1 protein. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with 100 μl (1 μg/ml) of ITPRIPL1 recombinant protein (cusabio, Wuhan, China) in ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of coating buffer free of protein. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, different monoclonal antibodies at a final concentration of 1 μg/ml were added and bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-mouse Fc segment antibodies (Consun, Shanghai, China) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
2. Flow Cytometry (FACS) Verifying the Binding of Each Monoclonal Antibody to ITPRIPL1
In order to further verify the binding capacity of each monoclonal antibody to ITPRIPL1, flow cytometry (FACS) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. Jurkat cells with high endogenous expression of ITPRIPL1 were counted, and the cell number was adjusted to 1×106/ml. Each 100 μl was added into a 96-well plate (Thermo Fisher, MA, USA). Into each well was respectively added monoclonal antibody or control serum at a final concentration of 1 μg/ml, and incubated in an incubator for 30 minutes. The 96-well plate was taken out, and each well of cells was placed in EP tubes (Axygen, CA, USA) and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. Anti-mouse Fc segment-Alexa Fluor 488 antibodies (Invitrogen, CA, USA) were diluted with the cell staining buffer at 1:500. 200 μl of the mixture solution was added into each EP tube for resuspension, and incubated at room temperature for 30 minutes. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of the cell staining buffer; and centrifuged and washed again. 300 μl of the cell staining buffer was respectively added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany) to test.
The results were shown in
In order to verify whether there is a blocking effect of the resulting monoclonal antibodies on the binding of ITPRIPL1 to CD3E or SEMA3G, an enzyme-linked immunosorbent assay (ELISA) was used to verify the blocking effect of the antibodies. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with ITPRIPL1-Fc-tagged recombinant protein (Abclonal, Wuhan, China) at a final concentration of 1 μg/ml or CD3E-Fc-tagged protein (Acro, Beijing, China) at a final concentration of 1 μg/ml which was dissolved in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of coating buffer free of proteins. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, into the ITPRIPL1-Fc protein-coated wells were added SEMA3G-His-tagged protein (cusabio, Wuhan, China) at a final concentration of 1 μg/ml and monoclonal antibodies at a final concentration of 1 μg/ml at the same time; into the CD3E-Fc protein-coated wells were added ITPRIPL1-His-tagged protein (cusabio, Wuhan, China) at a final concentration of 1 μg/ml and monoclonal antibodies at a final concentration of ½ μg/ml at the same time; they were bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-His segment antibodies (Abcam, MA, USA) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
Based on the protein sequence of the extracellular segment of ITPRIPL1, polypeptide segments with 1/3 overlapping sequence were designed, with a total of 17 segments (Genscript, Nanjing, China). Their sequences were shown in SEQ ID NOs: 42-58. The resulting polypeptide segments were dissolved in DMSO at a final concentration of 400 μg/ml. An enzyme-linked immunosorbent assay (ELISA) was used to verify each hybridoma antibody, and 4 strains of monoclonal antibodies were selected to verify the binding of polypeptide segments. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with polypeptide segments at a final concentration of 1 μg/ml in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of the coating buffer free of protein. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, different hybridoma antibodies or monoclonal antibodies at a final concentration of 1 μg/ml were added and bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-mouse Fc segment antibodies (Consun, Shanghai, China) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
Flow cytometry demonstrates that monoclonal antibodies with the effect of blocking the binding of ITPRIPL1 to CD3E/SEMA3G can promote the killing of tumor cells by PBMCs.
Resuscitated PBMC cells were activated with 1 μg/mL of Anti-CD3/CD28 (Invitrogen, CA, USA) 24 hours in advance. PBMC cells and Raji cells were counted, and the cell number was respectively adjusted to 4×106/ml or 1×106/ml. Each 100 μl was added into a 96-well plate (Thermo Fisher, MA, USA). At the same time, two concentrations of different monoclonal antibodies or control serum were added, mixed and then co-incubated in an incubator for 6 hours. The 96-well plate was taken out, and each well of cells was placed in EP tubes (Axygen, CA, USA) and centrifuged at 400 rcf for 5 minutes, and the supernatants were then discarded. The cells were resuspended and washed with 500 μl of cell staining buffer (Invitrogen, CA, USA), and centrifuged and washed again. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Beyotime, Shanghai, China); and centrifuged and washed again. Experimental groups and a control group were set, into which were optionally added 100 μl of the binding buffer, 5 μl of Annexin V-FITC (Beyotime, Shanghai, China) and 10 μl of PI (Beyotime, Shanghai, China) respectively, and incubated at room temperature for 15 minutes. 400 μl of the binding buffer was further respectively added, and transferred into flow tubes (Falcon, NY, USA) for loading (Miltenyi Biotec, Cologne, Germany) to test, with the results shown in
It can be known from sequencing that, for the monoclonal antibody 13B7A6H3, the heavy chain sequence is as set forth in SEQ ID NO: 22, the light chain sequence is as set forth in SEQ ID NO: 23, the sequence of the heavy chain variable region VH is as set forth in SEQ ID NO: 24, the sequence of the light chain variable region VL is as set forth in SEQ ID NO: 25, the sequence of the heavy chain complementarity determining region HCDR1 is as set forth in SEQ ID NO: 26, the sequence of the heavy chain complementarity determining region HCDR2 is as set forth in SEQ ID NO: 27, the sequence of the heavy chain complementarity determining region HCDR3 is as set forth in SEQ ID NO: 28, the sequence of the light chain complementarity determining region LCDR1 is as set forth in SEQ ID NO: 29, the sequence of the light chain complementarity determining region LCDR2 is KV, and the sequence of the light chain complementarity determining region LCDR3 is as set forth in SEQ ID NO: 31; for the monoclonal antibody 18B12D1A6, the heavy chain sequence is as set forth in SEQ ID NO: 32, the light chain sequence is as set forth in SEQ ID NO: 33, the sequence of the heavy chain variable region VH is as set forth in SEQ ID NO: 34, the sequence of the light chain variable region VL is as set forth in SEQ ID NO: 35, the sequence of the heavy chain complementarity determining region HCDR1 is as set forth in SEQ ID NO: 36, the sequence of the heavy chain complementarity determining region HCDR2 is as set forth in SEQ ID NO: 37, the sequence of the heavy chain complementarity determining region HCDR3 is as set forth in SEQ ID NO: 38, the sequence of the light chain complementarity determining region LCDR1 is as set forth in SEQ ID NO: 39, the sequence of the light chain complementarity determining region LCDR2 is KV, and the sequence of the light chain complementarity determining region LCDR3 is as set forth in SEQ ID NO: 41.
By analyzing the similarity of the antibody sequences (
Immunosorbent assay (ELISA) verifying the effect of point mutation of polypeptide segments on monoclonal antibodies
The specific amino acid sequences of the previously obtained polypeptide segment P8 was mutated to alanine (A) one by one, and the original alanine sites were not mutated, and the resulting sequences were as set forth in SEQ ID NOs: 59-71. The resulting polypeptide segments and non-mutated polypeptide segments were dissolved in DMSO at a final concentration of 400 μg/ml, and the binding to the 13B7A6H3 monoclonal antibody was verified by enzyme-linked immunosorbent assay (ELISA). An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with polypeptide segments at a final concentration of 1 μg/ml in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of the coating buffer free of protein. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, 13B7A6H3 monoclonal antibody at a final concentration of 1 μg/ml was added and bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-mouse Fc segment antibodies (Consun, Shanghai, China) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
The cluster analysis of ITPRIPL1 monoclonal antibodies was performed by epitope mapping.
The resulting ITPRIPL1 monoclonal antibodies were analyzed by epitope mapping for the competition among the currently obtained ITPRIPL1 monoclonal antibodies and clustered. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with ITPRIPL1 protein at a final concentration of 0.5 μg/ml in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of the coating buffer free of protein. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, 0.5 μg/ml of each ITPRIPL1 monoclonal antibody was added, and at the same time each ITPRIPL1 monoclonal antibody at a final concentration of 0.5 μg/ml was added crossover, and bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-mouse Fc segment antibodies (Consun, Shanghai, China) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
1. Construction of Humanized ITPRIPL1 Antibody
According to the results of the previous antibody sequencing, the paired sequences SEQ ID NO: 73-82 corresponding to the 13B7A6H3 monoclonal antibody were optimized and re-encoded by using humanized gene expression to obtain a corresponding humanized sequence. The obtained sequence was inserted into the corresponding sequence vector of human Fc segments to obtain a humanized ITPRIPL1 antibody sequence plasmid. The humanized ITPRIPL1 antibody sequence plasmid was transfected into HEK293 cells and cultured. 72 hours later, the supernatant was harvested, mixed with the equilibrated protein A magnetic beads at room temperature for 2 hours, and then flowed through a chromatographic column to be equilibrated/washed and eluted, and the product obtained after neutralization was the humanized ITPRIPL1 antibody.
2. Determination of Binding Activity and Site of Humanized ITPRIPL1 Antibody
An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with ITPRIPL1 P8 polypeptide at a final concentration of 1 μg/ml in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of the coating buffer free of protein, and for the positive control, the plate was coated with human ITPRIPL1 P8 polypeptide. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, chimeric and humanized 13B7A6H3 monoclonal antibodies at a final concentration of 0.5 μg/ml were added and bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-human Fc segment antibodies (Abcam, MA, USA) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
Immunosorbent assay (ELISA) verifies that 13B7A6H3 monoclonal antibodies can bind to the corresponding polypeptide segments of cynomolgus monkey ITPRIPL1.
According to NCBI protein database, it can be known that the sequence of cynomolgus monkey ITPRIPL1 protein is SEQ ID NO: 72; wherein the sequence of the cynomolgus monkey corresponding polypeptide segment to human ITPRIPL1 P8 is SEQ ID NO: Corresponding polypeptide was constructed according to the cynomolgus monkey corresponding polypeptide segment. An ELISA special plate (costar, ME, USA) was used. Firstly, the plate was coated with cynomolgus monkey ITPRIPL1 polypeptide at a final concentration of 1 μg/ml in 100 μl of ELISA coating buffer (Solarbio, Beijing, China), while for the negative control, the plate was coated with 100 μl of the coating buffer free of protein, and for the positive control, the plate was coated with human ITPRIPL1 P8 polypeptide. The plate was coated at 4° C. overnight. After washing with PBST, they were blocked with 100 μl of 5% BSA dissolved in PBS (VWR, PA, USA) in an incubator at 37° C. for 90 minutes. After washing with PBST, 13B7A6H3 monoclonal antibodies at a final concentration of 0.5 μg/ml were added and bound in an incubator at 37° C. for 60 minutes. After washing with PBST, they were incubated with PBS-diluted specific anti-mouse Fc segment antibodies (Consun, Shanghai, China) in an incubator at 37° C. for 30 minutes for binding. After washing with PBST, a color developing solution (Invitrogen, CA, USA) was added at 100 μl per well, and the plate was placed in the incubator to react for 5-30 minutes, then 50 μl of stop solution (Sangon, Shanghai, China) was further added, and the plate was placed under a microplate reader (Thermo Fisher, MA, USA) for color development reading at 450 nm.
In summary, the present application discloses a newly identified native membrane protein ITPRIPL1 that binds the CD3ε extracellular domain, and also discloses the altered signaling that occurs following the binding of ITPRIPL1 to CD3ε and controls T cell activation. This indicates that, antibodies that have been identified in the past to bind CD3ε may mimic or affect the natural ligand function of ITPRIPL1 to some extent. The discovery of CD3ε and ITPRIPL1, a pair of immune checkpoint receptor ligands, brings a new insight to the in-depth understanding of the maintenance of the body's immune homeostasis and the immune evasion mechanism of tumor cells.
Although the content of the present application has been described in detail through the preferred examples described above, it should be appreciated that the above description should not be construed as limiting the present application. Various modifications and substitutions to the present application will be apparent to those skilled in the art after reading the foregoing. Therefore, the protection scope of the present application shall be defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011191447.3 | Oct 2020 | CN | national |
| 202110566040.2 | May 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/127234 | 10/29/2021 | WO |
