Patent Application
20240092865
This application contains a sequence listing submitted in Computer Readable Form (CRF). The CFR file containing the sequence listing entitled “SequenctListing.xml”, which was created on Sep. 19, 2023, and is 4321 bytes in size. The information in the sequence listing is incorporated herein by reference in its entirety.
The immune system normally recognizes and defends against foreign pathogens but does not respond to self-components of tissues, such intrinsic state is established and maintained precisely by the process of immune tolerance. The inflammatory damage of one or more organ systems caused by inappropriate activation of self-reactive both or either T cells and B cells is due to the loss of balance between effectors (B cells and T cells) [1, 2] and regulatory components (Treg cells and dendritic cells) of the immune system [3]. Therefore, restoration of immune tolerant state is the ultimate goal in the treatment of autoimmune diseases [4, 5].
Cell surface molecules such as CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, or CD152) and PD-1 (Programmed Death-1, also known as CD279) deliver inhibitory immunoregulatory signals that are thought to be crucial to the maintenance of normal immune tolerance [6, 7] through its recognizing and binding to CD80 (B-7.1) or CD86 (B-7.2). Based on the discoveries, Bristol-Myers Squibb, a pharmaceutical company, developed a chimeric protein composed of the Fc region of the immunoglobulin IgG1 and the extracellular domain of CTLA-4 named as Abatacept (CTLA-4/Fc), thereafter, it was approved for clinic application by FDA in 2011. However, the pharmacological mechanism of Abatacept is to serve as antagonist of CD28 to interrupt the second signal, CD28 binding to B-7 for TCR activation, instead of the delivery of checkpoint signal through CTLA-4. Therefore, Abatacept interrupts the systemic cellular immune response, which, as a side effect, may cause serious infectious diseases, even cancer.
Second cell surface molecule responsible for immune checkpoint is PD-1 (Programmed cell death protein 1, also known as CD279), which is strictly expressed on thymocytes, activated T cells and pro-B cells. It has been believed that PD-1 is related biasedly to the maintenance of peripheral immune tolerance when it is associated with either of its two identified ligands: 1), Programmed Death-1 ligand 1 (PD-L1), also known as CD274 or B7-H1, broadly expressed on the hematopoietic and nonhematopoietic cells including tumor cells; 2), Programmed Death-1 ligand 2 (PD-L2), also known as CD273, or B7-DC strictly expressed on the dendric cells and macrophages. The PD-1 engagement with PD-Ls results in suppressing the activation of T cell or differentiation of mature B cell. Thus far three mechanisms have been elaborated for the pivotal role of PD-1/PD-L1 pathway in immune tolerance: (1) Induction of T cell anergy [8] through PD-L1 binding to PD-1 then activating the SHP2 to suppress the TCR/CD28 signaling; (2) suppression of B cell maturation through PD-L1 binding to PD-1 expressed on pre-B cells then activating the SHP2 to suppress the BCR signaling; [9]; (3) generation of regulatory T cells (especially type 1 (Tr1)) [10] and conversion of human TH1 into Treg cell (iTreg cell) [11]. In addition, soluble forms of PD-1 (sPD-1) and PD-L1 (sPD-L1) in peripheral blood may also be a source for the regulation of PD-1/PD-L1 pathway in immunity [12, 13]. A perfect manifestation of such mechanisms is that certain tumor cells use PD-L1 overexpression on their cell membranes and simultaneously shed into the circulation to successfully escape immune attacks [14].
In contrary with in tumor, according to the latest findings in autoimmune diseases, the weakening or deletion of the PD-1/PD-L1 pathway in immune cells is directly related to the instability or destruction of peripheral immune tolerance [10]. Mounting evidence demonstrate that impaired PD-1/PD-L1 function plays an important role in a variety of autoimmune diseases, such as SLE (systemic lupus erythematosus) and RA (Rheumatoid Arthritis), etc. [15]. The animal model demonstrates that loss of signaling of PD-1/PD-L1 (PD-1 knockout mice) develops lupus-like autoimmune disease [16], or autoimmune dilated cardiomyopathy depending upon the genetic background [17]. PD-L1 deficiency enhances disease progression in the experimental autoimmune encephalomyelitis [18], nonobese diabetic (NOD) model of autoimmune diabetes [19, 20] and the murine model of multiple sclerosis (MS) [21]. Tissue expression of PD-L1 mediates peripheral T cell tolerance [22].
The third cell surface molecule responsible for immune tolerance is Fc-gamma receptor type IIB (one of Fc-gammaRIIA/B, collectively known as CD32). Human Fc-gamma receptors have been identified so far are hFc-gammaRI (CD64), hFc-gammaRII (types A and B, collectively known as CD32) and hFc-gammaRIII (types A and B, collectively known as CD16). Each type of receptor exhibits distinctive tissue distribution, structure and binding specificity towards various IgG subclasses (7,8). Fc-gammaRIIB is restrict expressed on B cell, monocyte/macrophage and dendritic cell in human, while basophil, eosinophil and mast cell as well in mice. Fc-gammaRII displays low affinity for monomeric IgG but high-avidity for aggregated multimeric IgG, which are particularly important in the recognition and binding of antibody-antigen complexes during an immune response [23]. The B cell stage(s) at which Fc-gammaRIIB exerts its function as a gatekeeper of self-tolerance has recently been defined [24, 25]. The main function of Fc-gammaRIIB is to inhibit activating signals, which is achieved through co-ligation of Fc-gammaRIIB with either activating Fc-gammaRs or with the BCR by immune complexes ([26]). This leads to phosphorylation of the cytoplasmic domain ITIM of Fc-gammaRIIB by the Src-family kinase IYN. This phosphorylation event is thought to require access of Fc-gammaRIIB to sphingolipid rafts in which activating Fc-gammaRs and the BCR reside following cross-linking. Subsequent binding of SH2-domain-containing inositol phosphatases (SHIPs), in particular SHIP1, result in the dephosphorization of downstream targets and inhibition of the activating signaling cascade. There is accumulating evidence that Fc-gammaRIIB mediates its function during late stages of B cell maturation, thus representing a distal checkpoint [25]. Through the analysis of an anti-DNA knock-in model, it was established that the absence of Fc-gammaRIIB resulted in the expansion of IgG-positive plasma cells secreting autoreactive antibodies (Fukuyama et al., 2005). Here, Fc-gammaRIIIB might serve as the final barrier to prevent these B cells with potentially harmful BCR specificities from maturing into plasma cells that would otherwise induce tissue pathology by secretion of large amounts of self-reactive antibodies. Another cell type where Fc-gamagRIIB may play an important role in regulating immunity and tolerance are the dendritic cells [27-29]. A number of chronic inflammatory diseases have been shown to be associated with Fc-gamma receptor genetic variants and include (but are not limited to) autoimmune pathologies, such as systemic lupus erythematosus (SLE) [30], rheumatoid arthritis [31], acute allograft rejection and vascular inflammatory and thrombotic disorders, such as coronary artery stenosis, peripheral atherosclerosis and vasculitis [33, 34].
Since PD-1 is expressed in activated T cells and B cells, it is expected that the PD-L1 protein can be effectively used as a therapeutic agent that specifically targets activated immune cells to suppress the inflammatory reaction not only in an autoimmune disease but also in organ transplantation. However, Fc-gammaRIIB is expressed in dendritic cells and monocytes/macrophages, it is expected also that aggregated form of Fc-gamma1 can be used as a therapeutic agent that specifically targets these immune cells to suppress the inflammatory reaction during the autoimmunity. Because the induction and maintenance of peripheral immune tolerance are dependent on the orchestra playing by all type of those cell populations. To date a peripheral immune tolerance-based immunotherapeutic agent using an agonist to rebuild the tolerance through both pathways of PD-1/PD-L1 and Fc-gamma/Fc-gamma receptors has not yet been developed. Therefore, there is a need to develop a therapeutic reagent that the therapeutic effect not only targeting the autoreactive T cells and B cells but also targeting the inappropriately activated monocytes/macrophages and dendritic cells in inflammatory tissues, meanwhile, such reagent will avoid the causes of antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) in vivo due to its feature of biding to the activation of Fc receptor of Fc-gammaRI. Thus, when Ig chimeric proteins are used as an agent for treating an autoimmune disease or as an agent for inducing immune tolerance in organ transplantation, they cannot play the role of inhibiting inflammatory responses, and may rather aggravate inflammation.
In first aspect, the invention provides a bispecific biologic (chimeric protein of PD-L1/FC-gamma1) named BY-001, which is a composition of homomultimeric forms of dimer, tetramer and Hexamer, wherein the ratio of the forms is at 25% of dimer, 30% of tetramer and 45% of Hexamer respectively.
In second aspect, current invention provides the demonstrations of bispecific function of BY-001 that aggregated PD-L1 can trigger the pathway of PD-1 to decrease the cytokine production from activated T cell line (jurkate cell expressing PD-1), and also the aggregated Fc-gamma1 shows the suppressive effect on monocyte/macrophage cell line (THP-1 cell expressing Fc-gamma1RI & II) and erythroid cell line (K562 cell expressing Fc-gamma1RII).
In third aspect, the present invention also demonstrates that the functional features of both the domains PD-L1 and Fc-gamma1 in BY-001 fully inherit their characteristics in their parental proteins that have been confirmed in vitro and in vivo that published in highly recognized scientific journals. Therefore, an obvious perspective emerges that the BY-001 of the present invention is potential for a pharmaceutical composition for treating an autoimmune disease caused by abnormal regulation of an immune response or preventing the rejection of allografts.
An object of the present invention is to provide BY-001 (a composition of bifunctional homomultimeric forms chimeric protein PD-L1/Fc-gamma1) comprises the forms of dimer, tetramer and Hexamer at the ratio of 25%, 30% and 45%, wherein monomer form of chimeric protein PD-L1/Fc-gamma1 consists of two distinct domains. The amino acids of cysteine in the region of immunoglobulin Fc-gamma1 are kept in order to form the multimeric PD-L1/Fc-gamma1, such homomultimeric form provides the aggregated Fc-gamma1 (BY-001) for binding to Fc-gammaRIIB instead of Fc-gammaRI. Another object of the present invention is to provide the aggregated form (BY-001) of extracellular domain of PD-L1 protein, which shows an excellent regulating effect on activity of T cells. Third object of the present invention is to provide the aggregated form of Fc-gamma1 region, which binds only to Fc-gamma1RIIA and B and suppresses the production of pro-inflammatory factors in monocyte/macrophages and pre-erythrocytes.
BY-001 of the present invention is a composition of chimeric protein PD-L1/Fc-gamma1 in the forms of dimer, tetramer and Hexamer. Such aggregated state of the chimeric protein PD-L1/Fc-gamma1 displays higher regulatory efficacy to PD1 and Fc-gammaRII. Therefore, it is more potential to regulate the activities of T cells including Th cells, cytotoxic-T cells and B cells during the active stage of autoimmunity, as well as to regulate the activities of monocytes/macrophages, dendritic cells and iTreg cells to restore and maintain the peripheral immune tolerance. Thereafter, the BY-001 of the present invention is potential for a pharmaceutical composition for treating an autoimmune disease caused by abnormal regulation of an immune response or preventing the rejection of allografts.
A. Schematic diagram for the design of chimeric protein PD-L1/Fc-gamma1 consisting of extracellular portion of human PD-L1 and constant region of human IgG1 Fc. Please be noted: 1). Cysteines in V-set, C-set and IgG1Fc regions are kept for the ——S——S—— “disulfide” bond to form the advanced structure forms. 2). PD-L1/FC1 is 449 amino acid in total 3). The signal peptide of PD-L1 comprises amino acids 1-18, which will be digested when the protein secrets into culture medium. Therefore, the purified PD-L1/Fc-gamma1 of current invention contains 431 amino acids in total. 4). immunoglobulin V like region (V-set, 19˜127 amino-acids, exon-2) 5). immunoglobulin constant like region (C-set, 133˜220 amino-acids, exon 3) 6). human IgG1 heavy chain constant domain (C region, 221-449) is the region of Fc-gamma1.
B. The AA sequence of PD-L1 contained in chimeric protein PD-L1/Fc-gamma1. The sequence highlighted in yellow is the part of signal sequence for leading the protein toward the secretory pathway, which is cut away as the protein released to medium. The sequence highlighted in shadow is the region of Fc-gamma1. For the detail about the sequence of SEQ ID NO:2, please refer to the information of file “SequenctListing.xml”.
A. SDS-page gel, Coommassie blue staining. Lane M1: Protein Marker Cat. No. 3452, Lane 1: BY-001 at Reducing condition (monomer), Lane 2: BY-001 at non-reducing condition (no monomer, but dimer, tetramer and Hexamer).
B. Western blot probed with Primary antibody: Goat Anti-Human IgG-HRP. Lane M2: Protein Marker, GenScript, Cat. No. M00521, Lane P: Human IgG1, Kappa (Sigma, Cat. No. 15154) as a positive control. Lane 1, monomer of PD-11/Fc-gamma1 band at nonreducing condition; Lane 2, multimeric forms of BY-001, dimer, tetramer and hexamer.
C. Western blot probed with Human PD-L1/B7-H1 Antibody (R&D, Cat. No.AF156). Lane M2: Protein Marker, GenScript, Cat. No. M00521; Lane 1, monomer of PD-11/Fc-gamma1 band at nonreducing condition; Lane 2, multimeric forms of BY-001, dimer, tetramer and hexamer.
D. Diagrams of the BY-001, cartoons of D, E and F are the graphic view for illustration of BY-001 at forms of dimer, tetramer and hexamer.
A. 100 ul of Cell suspension jurkate at 3 million/ml was added into each well hereafter, BY-001 was added into wells according to designed. The plate was kept in incubator at 37° C. and 5% CO2 overnight. The culture supernatants were harvested from wells for the measurement of IL-2 production. Human IL-2 High Sensitivity ELISA Kit (eBioscience, 1030 Vienna, Austria) was used for the measurement of IL-2. The results indicate that BY-001 inhibits the IL-2 production effectively at the starting concentration (0.1 ug/ml) of BY-001. CD3 signal along (without the CD28 second signal) the inhibition was 87.5% at concentration of 0.1 ug/ml of BY-001, such inhibition was persistent from the concentrations of 0.1 ug/ml to 1 ug/ml.
B. Anti-human CD28 (BD bioscience, Clone CD28.2) mixed at 2 ug/ml into same jurkate cell suspension solution as used in (A). The plate was incubated at 37° C. and 5% CO2 for overnight. The culture supernatants were harvested from wells for the measurement of IL-2 production by following the method in (A). The results indicate that TCR signal with the help of co-stimulator signal make Jurkate cell produces higher level of IL-2 (from 30 pg/ml at OKT3 along to 60 pg/ml at OKT3+anti-CD28), however it makes BY-001 inhibit IL-2 production as a gradient decent pattern at the concentration (0.5 to 2 ug/ml), the inhibitory rate become to be 13.1% at 0.5 ug/ml, 43% at 1.0 ug/ml to 67.6% at 2 ug/ml of BY-001.
The 96-well plate was coated with 3 ug/ml of OKT3 at 4° C. for overnight. 100 ul of Jurkate cell solution at 3 million/ml in the presence or no presence of Anti-human CD28 at 2 ug/ml were added into wells. The plate was kept in incubator at 37° C. and 5% CO2 for overnight, supernatants were collected from the wells for IL-2 detection with Human IL-2 High Sensitivity ELISA Kit.
A. CD3 signal along (without the CD28 second signal) the inhibition was 90.8% at concentration of 0.25 ug/ml of BY-001, the inhibition is persistent at concentrations of BY-001 from 0.25 to 4 ug/ml.
B. CD3 signal together with the CD28 second signal the inhibition pattern became to be gradient decent from the inhibitory rate 17.5% at 0.25 ug/ml to 74.1% at 4 ug/ml of BY-001.
A. The basal level of IL-4 production in K562 is high (84 pg/ml). Addition of BY-00-1 according to designed into the cells culture decreases the IL-4 level. The inhibition is sufficient at concentration of 4 ug/ml of BY-001, which indicates that aggregated Fc-gamma1 bind to Fc-gamma1/Fc-Gamma1RII and trigger the inhibitory pathway.
B. The basal level of TGF-betta production in K562 is high (1250 pg/ml). Addition of BY-00-1 according to designed into the cells culture decreases the TGF-betta level. The inhibition is sufficient at concentration of 2 and 4 ug/ml of BY-001, which indicates that aggregated Fc-gamma1 bind to Fc-gamma1/Fc-Gamma1RII and trigger the inhibitory pathway.
C. The diagram for BY-001 binding to and triggering the Fc-gammaRII, thereafter to deliver the suppressive signal. ITAM stands for immunoreceptor tyrosine-based activation motif in which the tyrosine residues are phosphorylated upon the binding between Fc-gamma1 and Fc-gamma1RII. While ITIM stands for the immunoreceptor tyrosine-based inhibitory motif in which the tyrosine residues are phosphorylated, thereafter the phosphatase SHIP1 or 2 is activated to dephosphorylate the phosphorylated tyrosine in ITAM of Fc-gammaRIIA or C via the crosslinking of bound Fc-gamma1s of BY-001.
A. The basal level of IL-4 production in THP-1 is high (86 pg/ml). Addition of BY-00-1 according to designed into the cells culture decreases the IL-4 level. The inhibitions are sufficient at concentration of 2 and 4 ug/ml of BY-001, which indicates that aggregated Fc-gamma1 bind to Fc-gamma1/Fc-Gamma1RII and trigger the inhibitory pathway only.
B. The basal level of TGF-betta production in THP-1 is high (485 pg/ml). Addition of BY-00-1 according to designed into the cells culture decreases the TGF-betta level. The inhibition is observed at concentration of 2 and 4 ug/ml of BY-001, which indicates that aggregated Fc-gamma1 bind to Fc-gamma1/Fc-Gamma1RII and trigger the inhibitory pathway only.
C. The diagram for BY-001 binding to and triggering the Fc-gammaRII, thereafter to deliver the suppressive signal. ITAM stands for immunoreceptor tyrosine-based activation motif in which the tyrosine residues are phosphorylated upon the binding between Fc-gamma1 and Fc-gamma1RII. While ITIM stands for the immunoreceptor tyrosine-based inhibitory motif in which the tyrosine residues are phosphorylated, thereafter the phosphatase SHIP1 or 2 is activated to dephosphorylate the phosphorylated tyrosine in ITAM of Fc-gammaRIIA or C via the crosslinking of bound Fc-gamma1s of BY-001. Apparently, the Fc-gammaRI has no effect on the inhibition, which is benefited for BY-001, hereinafter, in dealing with autoimmunity.
A. The diagram is the dimer of BY-001 bound together by S——S bound
B. The diagram is the tetramer of BY-001 bound together by S——S bound
C. the diagram is the hexamer of BY-001 bound together by S——S bound
D. the diagram is the antigen-antibody complex
Definitions: Prior to describing the invention in more detail the following definitions are provided. Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials with similar or equivalent function to those described herein can be used in the practice or testing of the present invention. Methods, devices, and materials suitable for such uses are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention.
In accordance with the object of the present invention, in one aspect, there is provided a fusion protein comprising the extracellular domain of PD-L1 protein or a fragment thereof and a human immunoglobulin Fc-gamma1 region (“PD-L1/Fc-gamma1 chimeric protein,” “PD-L1/Fc-gamma1 protein,” “PD-L1 chimeric protein” or “chimeric protein”). The extracellular domain of PD-L1 may be a polypeptide comprising an immunoglobulin V (V-set) like domain of PD-L1 and an immunoglobulin C (C-set) like domain of PD-L1. The extracellular domain of PD-L1 is a domain exposed outside a cell membrane, and may be a polypeptide comprising the amino acids at positions 19 to 220 of sequence of SEQ ID NO:2 . In addition, the extracellular domain of PD-L1 or a fragment thereof may be of human origin. In addition, the extracellular domain of PD-L1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the polypeptide sequence consisting of the amino acids at positions 19 to 220 of sequence of SEQ ID NO:2. In addition, Human IgG1 heavy chain constant domain (C region, 221˜449) of the sequence of SEQ ID NO:2 is the region of Fc-gamma1. All the Cysteines in V-set and C-set and human immunoglobulin Fc-gamma1 region of PD-L1/Fc-gamma1 thereof are kept in current invention for the ——S——S—— “disulfide” bond to form the advanced structure.
As used herein, the term “BY-001” refers to a composition of homomultimeric forms of bifunctional chimeric protein PD-L1/Fc-gamma1 at the ratio of 25% of dimer, 30% of tetramer and 45% of Hexamer.
As used herein, the term “immune cell” includes cells that are of hematopoietic origin and that play a role in the immune response, such as B cells, T cells, dendritic cells, natural killer cells, monocytes and macrophages, etc.
As used herein, the term “T cell” includes CD4+ Th1 and Th2 cells, CD3+ and CD8+ cytotoxic T cells including self-reactive T cells, CD25+ and FOX3+ Treg cells and inducible Treg cells (iTreg).
As used herein, the term “B cells” refers to the B cell potential to producing the antibodies including the self-reactive antibodies.
As used herein, the term “aggregated PD-L1” refers to the extracellular portion of PD-L1 of human or mouse origin at the aggregated state thereof.
As used herein, the term “aggregated Fc-gamma1” refers to the regions of hinge, CH2 and CH3 of human IgG1 heavy chain of human or mouse origin at the aggregated state thereof, in addition, any peptide combined to the regions of hinge, CH2 and CH3 of human IgG1 heavy chain of human or mouse origin at the aggregated state thereof is include.
As used herein, the term “inhibitory signal” refers to a signal transmitted via an inhibitory receptor molecule on an immune cell. Such as CTLA-4, PD-1 and Fc-gammaRIIB.
Hereinafter, the invention is explained in accordance with the examples to further illustrate the application without limiting its scope.
In accordance with the object of the present invention, first structure of protein in one aspect, there is provided bifunctional chimeric protein in first structure of protein comprising a first function domain, the extracellular domain of PD-L1 protein or a fragment thereof and a second function domain, the human immunoglobulin Fc-gamma1 region. The bifunctional chimeric protein of current invention is stated as “PD-L1/Fc-gamma1 chimeric protein” or “PD-L1/Fc-gamma1 protein” or “PD-L1 chimeric protein” or “chimeric protein”. The extracellular domain of PD-L1 may be a polypeptide comprising an immunoglobulin V (V-set) like domain of PD-L1 and an immunoglobulin C (C-set) like domain of PD-L1. The extracellular domain of PD-L1 is a domain exposed outside a cell membrane, and may be a polypeptide comprising the amino acids at positions 19 to 220 of sequence of SEQ ID NO:2. In addition, the extracellular domain of PD-L1 or a fragment thereof may be of human origin. In addition, the extracellular domain of PD-L1 may have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the polypeptide sequence consisting of the amino acids at positions 19 to 220 of sequence of SEQ ID NO:2. In addition, Human IgG1 heavy chain constant domain (C region, 221˜449) of the sequence of SEQ ID NO:2 is the region of Fc-gamma1.
The present invention provided, advanced structure of protein in another aspect, is bifunctional chimeric protein in advanced structure of protein comprising a composition of homomultimer of dimer, tetramer and hexamer of PD-L1/Fc-gamma1 chimeric protein (BY-001). All the Cysteines in V-set and C-set and human immunoglobulin Fc-gamma1 region of PD-L1/Fc-gamma1 thereof are kept in current invention for the ——S——S—— “disulfide” bond to form the advanced structure. Hence, once the PD-L1 is fused to second domain components Fc-gamma1, it is a complex naturally composed in dimer, tetramer and Hexamer, in fact PD-L1 dimer, tetramer and Hexamer are formed, on the other hand, Fc-gamma1 dimer, tetramer and Hexamer are formed also, for example as presented in
To date, the subsets of immune cells of activated T cells, B cells and dendritic cells are related to the autoimmunity and rejection of allografts, while monocytes/macrophages are one of the key subsets related to inflammatory reaction, particularly to the chronic inflammation. It has highly recognized that the cell signaling pathways PD-1/PD-L1 and Fc-gammaRII/Fc-gamma are crucial in the regulation of functions of the subsets of immune cells mentioned above.
Under physiological condition, PD-1 is expressed in activated T cells and B cells and is bound and triggered by monomeric PD-L1, thereafter, the PD-1/PD-L1 pathway delivers the cell signaling of immune checkpoint. Thus PD-1/PD-L1 axis is one of the pathways that exert the key roles in the establishing and maintenance of immune tolerance. The provided in current invention is the PD-L1 in chimeric protein at its aggregated state and is expected to act on the PD-1 signaling axis. To validate the function of PD-L1 at its aggregated forms as stated (
According to the ability to activate or suppress the immune response Fc-gamma receptors are divided in two classes: hFc-gammaRI, hFc-gammaRIIA and hFc-gammaRIIIA are activating receptors via the cytoplasmic ITAM (immunoreceptor tyrosine-based activation motif), whereas hFc-gammaRIIB is suppressing receptor via the cytoplasmic ITIM (immunoreceptor tyrosine-based inhibitory motif) [37]. Fc-gammaRI is a high-affinity receptor that binds to only monomeric IgG and is expressed constitutively in dendritic cells, monocytes and macrophages in both human and mice [38]. In contrast to Fc-gammaRI, the other three types express in most of the immune cell populations, except the B cells, and exhibit affinity to Fc-gamma of IgGs in antigen-antibody complex, among them Fc-gammaRIIB exerts the suppressive signaling through its binding with one Fc-gamma and crosslinking others Fc-gammaRIIA with others Fc-gamma in antigen-antibody complex. Furthermore, Fc-gammaRIIB is expressed on B cell and appears to function in a B cell-autonomous manner to regulate autoreactive cells in the periphery. There have been extensively reported that Fc-gammaRII plays key role in autoimmunity and inflammatory disorders. Fc-gammaRII/Fc-gamma axis dominates the activation and chemo-cytokines production profile of dendritic cells and monocytes/macrophages, which is critical for the process of inflammation, and also is the key role in maintenance of immune tolerance. The provided in current invention is the Fc-gamma1 in chimeric protein at its aggregated state and is expected to act on the Fc-gammaRII signaling axis.
To validate the function of Fc-gamma1 at its aggregated forms as stated (
Collectively, BY-001 exerts dual functions that in one hand binds to and triggers the PD-1 pathway through multimeric PD-L1, and on the other hand binds to Fc-gamma1RIIB through multimeric Fc-gamma1 to trigger the suppressive signaling into the cells. Thereafter, BY-001 is capable to regulate the subsets of immune cells activated T cells and B cells, meanwhile to regulate the subsets of dendritic cell and monocytes/macrophages, the immune cell populations tightly relate to autoimmunity and protection of allografts. Therefore, BY-001 is potential to treat certain immune and inflammatory disorders. In the setting of autoimmune, alloimmune and inflammatory diseases, the homomultimeric forms of chimeric protein of this invention can reduce autoimmune, alloimmune and inflammatory manifestations by one or more mechanisms. For example, the PD-L1 extracellular domain in homomultimeric forms of chimeric protein of this invention can bind to the PD-1 on immune cell, such as an activated T cell or pre-B cell; while the second domain in homomultimeric forms of chimeric protein binds to and cross-links the FC receptors that express on dendritic cell and macrophage and other immune cell bearing Fc-gammaRII or RIII, etc. Through these binding and cross-linking events, the receptors PD-1 for the first domain trigger the T cell anergy, repress the B cell maturation, differentiation of Treg cells and regulatory dendritic cells; the FC receptors for second domain of the chimeric protein may inhibit the activated dendritic cell and macrophage in the inflammatory location. All these events are critical to induce and maintain the immune tolerance of the body.
In addition, once the FC-gamma1 contained in chimeric protein anchored to FC receptor on cell surface of dendritic cell and macrophage, now membrane-anchored, therefore, the chimeric protein of the present invention may mediate its activity by spanning two neighboring cells, and thereby establish an immune tolerant microenvironment at inflammatory site. Alternatively, for example, a PD-L1 containing chimeric protein can bind to the B7-1 costimulator on an antigen-presenting cell, thereby interfering with its costimulatory, immune-activating function.
As indicated in schematic diagram of
The present invention relates to the advanced structure of chimeric protein PD-L1/Fc-gamma1 composed of homomultimeric forms (BY-001), wherein the first domain, such as PD-L1, at the aggregated state binds to and triggers the PD-1 and the second domain, such as Fc-gamma1, at the aggregated state binds to and triggers Fc-gamma1 receptor type II.
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This application is a continuation of co-pending U.S. patent application Ser. No. 16/190,641, filed on Nov. 14, 2018, which claims the benefit from the priority of U.S. patent application Ser. No. 62/689,214, filed on Jun. 24, 2018, the content of each is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62689214 | Jun 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16190641 | Nov 2018 | US |
| Child | 18476132 | US |
