Patent Application
20200331999
The present application relates to antibodies that specifically bind to TIGIT (T cell immunoreceptor with Ig and ITIM domains) and uses of the same.
Tigit (T cell immunoglobulin and ITIM domain) is a type I transmembrane protein, a member of the CD28 family of proteins that plays an important role in inhibiting T- and NK cell-mediated functional activities in anti-tumor immunity [Boles K S, et al., 2009 Eur J Immunol, 39:695-703; Stanietsky N, et al., 2009 PNAS 106:17858-63; Yu X, et al. 2009 Nat. Immunol, 10:48-57].
The genes and cDNAs coding for TIGIT were cloned and characterized in mouse and human. Full length human TIGIT has a sequence of 244 amino acids (SEQ ID NO: 1) in length, in which the first 21 amino acids consist a signal peptide. The amino acid sequence of the mature human TIGIT contains 223 amino acid (aa) residues (NCBI accession number: NM_173799). The extracellular domain (ECD) of mature human TIGIT consists of 120 amino acid residues (SEQ ID NO: 2, corresponding to amino acids 22-141 of SEQ ID NO: 1) with a V-type Ig-like domain (corresponding to amino acids 39-127 of SEQ ID NO: 1), followed by a 21 aa transmembrane sequence, and an 82 aa cytoplasmic domain with an immunoreceptor tyrosine-based inhibitory motif (ITIM) [Yu X, et al. 2009 Nat. Immunol, 10:48-57; Stengel K F et al. 2012 PNAS 109:5399-04]. Within the ECD, human TIGIT shares only 59% and 87% aa sequence identity with mouse and cynomolgus monkey, respectively.
TIGIT is expressed on T cells (including activated T cells, memory T cells, regulatory T (Treg) cells, and follicular T helper (Th) cells), and NK cells [Boles K S, et al., 2009 Eur J Immunol, 39:695-703; Joller N, et al., 2014 Immunity 40:569-81; Levin S D, et al., 2011 Eur J Immunol, 41:902-15; Stanietsky N, et al., 2009 PNAS 106:17858-63; Yu X et al. 2009 Nat. Immunol, 10:48-57].
So far, two Tigit ligands, CD155 (also known as poliovirus receptor or PVR) and CDll2 (also known as poliovirus receptor-related 2, PVRL2, nectin-2), have been identified. These ligands are primarily expressed on APCs (such as dendritic cells and macrophages) and tumor cells [Casado J G, et al., 2009 Cancer Immunol Immunother 58:1517-26; Levin S D, et al., 2011 Eur J Immunol, 41:902-15; Mendelsohn C L et al., 1989 56:855-65; Stanietsky N, et al., 2009 PNAS 106:17858-63; Yu X et al. 2009 Nat. Immunol, 10:48-57]. As an immune “checkpoint” molecule, Tigit initiates inhibitory signaling in immune cells when engaged by its ligands, CD155 and CD112. The binding affinity of Tigit to CD155 (Kd: ˜1 nM) is much higher than to CD112 and whether the TIGIT: CD112 interaction is functionally relevant in mediating inhibitory signals yet remain to be determined. A co-stimulatory receptor, CD226 (DNAM-1), binds to the same ligands with lower affinity (Kd:˜100 nM), but delivers a positive signal [Bottino C, et al., 2003 J Exp Med 198:557-67]. In addition, CD96 (Tactile), a “Tigit-like” receptor, also plays a similarly inhibitory role in the same pathway [Chan C J, et al., 2014 Nat. Immunol 15:431-8].
Tigit can inhibit immune responses through different mechanisms. First, interaction between TIGIT and PVR on dendritic cells (DCs) could deliver a “reverse signaling” in DCs, leading to up-regulation of IL-10 and decrease of IL-12 secretion, thereby inhibiting T-cell activation [Yu X, et al. Nat Immunol. 2009 10:48-57]. Second. TIGIT binds to CD155 with higher affinity, thereby competing off DNAM-1-CD155 interaction. Third, direct ligation of TIGIT on T cells could down-regulate TCR-mediated activation and subsequent proliferation and engagement of TIGIT on NK cells block NK cell cytotoxicity [Joller N, et al. 2011 186: 1338-42; Stanietsky N. et al., 2009 PNAS 106:17858-63]. Fourth, Tigit expression on Tregs has been associated with a highly activated and suppressive phenotype in tumor tissue and TIGIT signaling in Tregs may favor Treg stability [Joller N. et al. Immunity 2014 40:569-81; Kurtulus S, et al. J Clin Invest. 2015125: 4053-4062].
TIGIT has an immunoglobulin tail tyrosine (ITT)-like motif followed by an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic tail [Yu X, et al. Nat Immunol. 2009 10:48-57; Engels N, et al. Curr Opin Immunol 2011 23: 324-329]. These motifs could mediate recruitment of the phosphatase SHIP-1 and β-arrestin 2 [Li M, et al. J Biol Chem. 2014 289:17647-17657; Liu S, et al. Cell death and differentiation 2013 20: 456-464], thus providing a mechanism by which TIGIT can intrinsically deliver inhibitory signals to dampen activating signals.
Up-regulation of Tigit expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) has been reported in many types of cancers such as lung [Tassi, et al., Cancer Res. 2017 77: 851-861], esophageal [Xie J, et al., Oncotarget 2016 7.63669-63678], breast [Gil Del Alcazar C R, et al. 2017 Cancer Discov.], acute myeloid leukemia (AML) [Kong Y et al., Clin Cancer Res. 2016 22:3057-66] and melanoma [Chauvin J M, et al., J Clin Invest. 2015 125:2046-2058]. The increased expression of Tigit in AML is associated with poor prognosis of patient survival outcome [Kong Y et al., Clin Cancer Res. 2016 22:3057-66]. Not only does up-regulation of Tigit signaling play important roles in immune tolerance to cancer, but also to chronic viral infection. During HIV infection, expression of Tigit on T cells was significantly higher and positively correlated with viral loads and disease progression [Chew G M, et al., 2016 PLoS Pathog. 12:e1005349]. In addition, blockade of Tigit receptor alone or in combination with other blockade could rescue functionally “exhausted” T cells both in vitro and in vivo [Chauvin J M, et al., J Clin Invest. 2015125:2046-2058; Chew G M, et al., 2016 PLoS Pathog. 12:e1005349; Johnston R J, et al. Cancer Cell 2014 26:923-937]. In the cases of cancer and viral infections, activation of Tigit signaling promotes immune cell dysfunction, leading to the cancer outgrowth or extended viral infection. Inhibition of Tigit-mediated inhibitory signaling by therapeutic agents may restore the functional activities of immune cells including T cells, NK cells and dendritic cells (DCs), therefore enhancing immunity against cancer or chronic viral infection.
Therefore, modulation of Tigit signaling by antagonistic molecules may rescue immune cells from tolerance, inducing efficient immune responses to eradicate tumors or chronic viral infections.
The present invention is at least in part based on the discovery of a set of monoclonal antibodies (mAbs) which inhibit Tigit-mediated cellular signaling in immune cells, re-activate the immune cells and enhance immunity by specifically binding to Tigit. Accordingly, in the first aspect, the present application relates to an anti-Tigit antibody and antigen-binding fragment thereof which is capable of binding to human Tigit (SEQ ID NO: 1). The present invention also relates to the humanized version of the anti-Tigit mAbs of the first aspect.
In particular embodiments, the antibody of the present application comprises a heavy chain variable region (VH) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NOs: 3, 4, 5 or 13, or variants thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions in the amino acid sequences of SEQ ID NOs 3, 4, 5 or 13; and/or a light chain variable region (VL) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NOs: 6, 7, or 8, or variants thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions in the amino acid sequences of SEQ ID NOs: 6, 7, or 8.
In a more specific embodiment, the antibody of the present application comprises a heavy chain variable region (VH) comprising a VH-CDR1 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions, a VH-CDR2 having an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 13 or a variant thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions, and a VH-CDR3 having an amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions; and/or a light chain variable region (VL) comprising a VL-CDR1 having an amino acid sequence of SEQ 1D NO: 6 or a variant thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions, a VL-CDR2 having an amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions, and a VL-CDR3 having an amino acid sequence of SEQ 1D NO: 8 or a variant thereof comprising one or more conservative substitutions, e.g. one or two conservative substitutions.
The antibody or the antigen-binding fragment thereof of the present application is capable of binding to human Tigit and comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 9, 14, 19, or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 9, 14, 19. In one embodiment, the difference in sequence lies in the framework region. In one embodiment, the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region encoded by an nucleotide sequence selected from SEQ 1D NO: 10, 15 or 20, or a variant thereof.
The antibody or the antigen-binding fragment thereof of the present application is capable of binding to human Tigit and comprises a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 11, 16, 21, or 24, or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 11, 16, 21 or 24. In one embodiment, the difference in sequence lies in the framework region. In one embodiment, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region encoded by an nucleotide sequence selected from SEQ ID NO: 12, 17 or 22, or a variant thereof.
In one embodiment, the antibody or antigen-binding fragment thereof is capable of binding to human Tigit with a Kd value of about 1×10−9 M to about 1×10−12 M. For example, the antibody or the antigen-binding fragment thereof is capable of binding to human Tigit with a Kd value less than about 1×10−9 M, less than about 1×10−10 M, less than about 1×10−11 M, or less than about 1×10−12 M.
In one embodiment, the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region of the subclass of IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and a light chain constant region of the type of kappa or lambda or a variant thereof. In a more specific embodiment, the Fc region of the antibody is human IgG1 Fc or a variant thereof, e.g. a Fc region of SEQ ID NO: 18.
In one embodiment, the antibody or antigen-binding fragment thereof promotes the production of IFN-γ by antigen-specific T cells. In a more specific embodiment, the antibody or antigen-binding fragment thereof promotes the production of IFN-γ by antigen-specific T cells in a dose-dependent manner.
In a more specific embodiment, the antibody of the present application reduces the surface expression of Tigit receptor via FcγR-mediated trogocytosis, particularly FcγRIIB-mediated trogocytosis.
In one embodiment, the antibody of the present application shows a pH-dependent antigen binding such that the antibody exhibits a stronger binding to human TIGIT at a mild acidic pH in tumor microenvironment (e.g. pH 6.0) as compared to the binding to human TIGIT at a physiologic pH (e.g. pH 7.4). In a more specific embodiment, the antibody of the present application has (1) a KD ratio at pH 7.4/pH 6.0 of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more, and/or (2) a Rmax (RU) value at pH 6.0 which is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold higher than the Rmax at pH 7.4, as measured by surface plasmon resonance (Biacore) or similar technology.
The anti-Tigit mAbs disclosed herein have potential therapeutic uses in treating cancer, controlling viral infections and other human diseases that are mechanistically involved in immune tolerance or “exhaustion”. Accordingly, in further embodiments, the anti-Tigit antibody of the present application is for use in treatment of cancer. In another specific embodiment, the anti-Tigit antibody of the present application is for use in treating an infection, treating an infectious disease and/or controlling viral infections. In another specific embodiment, the anti-Tigit antibody of the present application is for use in the treatment of other human diseases related to or caused by immune tolerance, or the treatment of a disease that can be improved by increasing immune cell activation.
In a further aspect, the present application relates to a composition comprising the anti-Tigit antibody or antigen-binding fragment thereof and a therapeutically acceptable excipient.
Conservative amino acid substitutions of amino acids are commonly known in the art and exemplarily shown in the table below. Generally, a conservative amino acid substitution means that an amino acid residue is replaced by another amino acid residue having a similar side chain.
Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, including the appended claims, the singular forms of words such as “a”, “an”, and “the”, include their corresponding plural references unless the context clearly dictates otherwise.
The term “or” is used to mean, and is used interchangeably with, the term “and/or” unless the context clearly dictates otherwise.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated amino acid sequence, DNA sequence, step or group thereof, but not the exclusion of any other amino acid sequence, DNA sequence, step. When used herein the term “comprising” can be substituted with the term “containing”, “including” or sometimes “having”.
The term “Tigit” includes various mammalian isoforms, e.g., human Tigit, orthologs of human Tigit, and analogs comprising at least one epitope within Tigit. The amino acid sequence of Tigit, e.g., human Tigit, and the nucleotide sequence encoding the same, is known in the art.
The terms “administration”, “administering”, “treating” and “treatment” as used herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. The term “administration” or “treatment” also includes in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term “subject” herein refers to any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.
Disclosed herein are antibody molecules that bind to Tigit with high affinity and specificity.
In some embodiments, the anti-Tigit antibody binds to human Tigit and includes at least one, two, three, four, five or six complementarity determining regions (CDR's) comprising an amino acid sequence SEQ ID NOs 3, 4, 5, 13 or SEQ ID NOs: 6, 7, 8. In particular embodiments, the antibody of the present application comprises a heavy chain variable region (VH) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NOs: 3, 4, 5 or 13, or a variant thereof comprising one or more conservative substitutions; and/or a light chain variable region (VL) comprising one, two or three CDRs having an amino acid sequence selected from SEQ ID NOs: 6, 7, or 8, or a variant thereof comprising one or more conservative substitutions.
In some embodiments, the anti-Tigit antibody is an isolated antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.
In some embodiments, the anti-Tigit antibody comprises at least one antigen-binding site, or at least a variable region. In some embodiments, the anti-Tigit antibody comprises an antigen-binding fragment derived from an antibody described herein.
The term “antibody” herein is used in the broadest sense and specifically covers antibodies (including full length monoclonal antibodies) and antibody fragments so long as they recognize antigen, e.g., Tigit. An antibody is usually monospecific, but may also be described as idiospecific, heterospecific, or polyspecific. Antibody molecules bind by means of specific binding sites to specific antigenic determinants or epitopes on antigens.
The term “monocional antibody” or “mAb” or “Mab” herein means a population of substantially homogeneous antibodies, i.e., the antibody molecules comprised in the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their complementarity determining regions (CDRs), which are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) may be obtained by methods known to those skilled in the art. See, for example Kohler G et al., Nature 1975 256:495-497; U.S. Pat. No. 4,376,110; Ausubel F M et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow E et al., ANTIBODIES: A LABORATORY MANUAL, Cold spring Harbor Laboratory 1988; and Colligan J E et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The mAbs disclosed herein may be of any immunoglobulin class including IgG, IgM, IgD, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained by in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as α, δ, ε, γ, or μ, and define the antibody's isotypes as IgA, IgD, IgE, IgG, and IgM, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.
The variable regions of each light/heavy chain (VL/VH) pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.
Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called “complementarity determining regions (CDRs)”, which are located between relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chain variable domains sequentially comprise FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md.; 5<m> ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al, (1987) J Mol. Biol. 196:901-917 or Chothia, et al, (1989) Nature 342:878-883.
The term “hypervariable region” means the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “CDR” (i.e., VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable domain). See, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service. National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure). The term “framework” or “FR” residues mean those variable domain residues other than the hypervariable region residues defined herein as CDR residues.
Unless otherwise indicated, “antibody fragment” or “antigen-binding fragment” means antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antigen binding fragments include, but not limited to, Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., single chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.
An antibody that binds to a specified target protein with specificity is also described as specifically binding to a specified target protein. This means the antibody exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered “specific” for its intended target if its binding is determinative of the presence of the target protein in a sample, e.g. without producing undesired results such as false positives. Antibodies or binding fragments thereof, useful in the present invention will bind to the target protein with an affinity that is at least two fold greater, preferably at least 10-times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with non-target proteins. An antibody herein is said to bind specifically to a polypeptide comprising a given amino acid sequence, e.g. the amino acid sequence of a mature human Tigit molecule, if it binds to polypeptides comprising that sequence but does not bind to proteins lacking that sequence.
The expressions “pH-dependent binding”, “pH-dependent target binding” and “pH-dependent antigen binding” are interchangeable in the present disclosure, indicating that the antibody of the present application binds to its target/antigen, namely human TIGIT, in a pH-dependent manner. Specifically, the antibody of the present application shows a higher binding affinity and/or binding signal to its antigen at a mild acidic pH, e.g. pH 6.0, which is usually found in tumor microenvironment, as compared to the binding affinity and/or binding signal at physiologic pH, e.g. pH 7.4. The methods for determining the binding affinity and/or the intensity of binding signal of the antibody of the present application are well known in the art and include but not limited to surface plasmon resonance (Biacore) or similar technology. More specifically, the antibody of the present application has a KD ratio at pH 7.4/pH 6.0 of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more, as measured by surface plasmon resonance (Biacore) or similar technology. Alternatively or additionally, the antibody of the present application has a Rmax (RU) value at pH 6.0 which is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold higher than the Rmax at pH 7.4 as measured by surface plasmon resonance (Biacore) or similar technology. The binding affinity of the antibody can be measured at 25° C. or 37° C. Tumor microenvironment has been found to show a relatively more acidic pH than physiological condition or normal tissues (Zhang et al. Focus on molecular Imaging 2010; Tannock and Rotin et al. Cancer Res 1989). Therefore, the antibody of the present application having above-mentioned pH-dependent binding is advantageous as an anti-TIGIT therapeutic agent for targeting TIGIT-positive lymphocytes in the tumor microenvironment with selectivity and having lower toxicity associated with periphery activation of lymphocytes.
The term “human antibody” herein means an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” means an antibody that comprises only mouse or rat immunoglobulin protein sequences, respectively.
The term “humanized antibody” means forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The prefix “hum”, “hu”, “Hu” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.
The antibody of the present application has potential therapeutic uses in treating cancer. The term “cancer” or “tumor” herein means or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, lung cancer (including small-cell lung cancer, or non-small cell lung cancer), adrenal cancer, liver cancer, stomach cancer, cervical cancer, melanoma, renal cancer, breast cancer, colorectal cancer, leukemia, bladder cancer, bone cancer, brain cancer, an endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or metastatic lesion of the cancer.
Further, the antibody of the present application has potential therapeutic uses in controlling viral infections and other human diseases that are mechanistically involved in immune tolerance or “exhaustion”. In the context of the present application, the term “exhaustion” refers to a process which leads to a depleted ability of immune cells to respond to the infecting virus during a prolonged period of chronic viral infection.
In some aspects, this disclosure provides compositions, e.g., pharmaceutically acceptable compositions, which include an anti-Tigit-3 antibody described herein, formulated together with at least one pharmaceutically acceptable excipient. As used herein, the term “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The excipient can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g. by injection or infusion).
The compositions herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, liposomes, and suppositories. A suitable form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusion solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.
The term “therapeutically effective amount” as herein used, refers to the amount of an antibody that, when administered to a subject for treating a disease or a disorder, or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment for the disease, disorder, or symptom. The “therapeutically effective amount” can vary with the antibody, the disease, disorder, and/or symptoms of the disease or disorder, severity of the disease, disorder, and/or symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated. An appropriate amount in any given instance can be apparent to those skilled in the art or can be determined by routine experiments. In the case of combination therapy, the “therapeutically effective amount” refers to the total amount of the active agents comprised in the combination for the effective treatment of a disease, a disorder or a condition.
The “subject” as used herein is a mammal, e.g., a rodent or a primate, preferably a higher primate, e.g., a human (e.g., a patient having, or at risk of having, a disorder described herein).
Anti-Tigit monoclonal antibodies (mAbs) were generated based on conventional hybridoma fusion technology [de StGroth and Sheidegger; 1980 J Immunol Methods 35:1; Mechetner, 2007 Methods Mol Biol 378:1] with minor modifications. The mAbs with high binding activity in enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) assay were selected for further characterization.
Tigit Recombinant Protein for Immunization and Binding Assays
The cDNA coding for the full-length human Tigit (SEQ ID NO:1) was synthesized by and purchased from Sino Biological (Beijing, China) based on its GenBank sequence (Accession No: NM_173799). The coding region of extracellular domain (ECD) of the full-length human Tigit corresponding to the amino acid (AA) 1-141 of SEQ ID NO: 1 was PCR-amplified, and cloned into pcDNA3.1-based expression vector (Invitrogen, Carlsbad, Calif., USA) with C-terminus fused either to the Fc domain of mouse IgG2a or to the Fc domain of human IgG1 heavy chain, which resulted in two recombinant fusion protein expression plasmids, Tigit-mIgG2a and Tigit-huIgG1, respectively. The schematic presentation of Tigit fusion proteins were shown in
Stable Expression Cell Lines
To establish stable cell lines that express full-length human Tigit (huTigit) or monkey Tigit (mkTigit, accession #: XM_005548101.2), Tigit genes (synthesized by Genescript, Nanjing, China) were cloned into a retroviral vector pFB-Neo (Cat.: 217561, Agilent, USA). Dual-tropic retroviral vectors were generated according to a previous protocol [Zhang T, et al. 2005, Blood]. Vectors containing huTigit and mkTigit were transduced into Jurkat and NK92MI cells (ATCC, Manassas, Va., USA), respectively, to generate the cell lines, Jurkat/huTigit and NK92MI/mkTigit. The high expression cell lines were selected by cultivation in medium with G418 and FACS binding assay.
Immunization, Hybridoma Fusion and Cloning
Eight to twelve week-old Balb/c mice (from HFK BIOSCIENCE CO., LTD, Beijing, China) were immunized intraperitoneally (i.p.) with 100 μL of antigen mixture containing 10 μg of Tigit-mIgG2a and a water-soluble adjuvant (Cat.: KX0210041, KangBiQuan, Beijing, China). The procedure was repeated three weeks later. Two weeks after the 2nd immunization, mouse sera were evaluated for Tigit binding by ELISA and FACS. Ten days after serum screening, the mice with the highest anti-Tigit antibody serum titers were boosted via i.p. injection with 50 μg of Tigit-mIgG2a. Three days after boosting, the splenocytes were isolated and fused to the murine myeloma cell line, SP2/0 cells (ATCC), using the standard techniques [1977 Somat Cell Genet, 3:231].
Assessment of Tigit Binding Activity of Antibodies by ELISA and FACS
The supernatants of hybridoma clones were initially screened by ELISA as described in “Methods in Molecular Biology (2007) 378:33-52” with some modifications. Briefly, Tigit-huIgG1 protein was coated in 96-well plates. The HRP-linked anti-mouse IgG antibody (Cat.: 7076S, Cell Signaling Technology, USA) and substrate (Cat.: 00-4201-56, eBioscience, USA) were used to develop color absorbance signal at the wavelength of 450 nm, which was measured by using a plate reader (SpectraMax Paradigm, Molecular Devices, USA). The ELISA-positive clones were further verified by FACS using either NK92MI/huTigit or NK92mi/mkTigit cells described above. Tigit-expressing cells (105 cells/well) were incubated with ELISA-positive hybridoma supernatants, followed by binding with Alexa Fluro-647 labeled goat anti-mouse IgG antibody (Cat.: A0473, Beyotime Biotechnology, China). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte 8HT, Merck-Millipore, USA).
The conditioned media from the hybridomas that showed positive signals in both ELISA and FACS screening were subjected to functional assays to identify antibodies with good functional activity in human immune cell-based assays (see following sections). The antibodies with desired functional activities were further sub-cloned and characterized.
Subcloning and Adaptation of Hybridomas to Serum-Fee or Low Serum Medium
After primary screening by ELISA, FACS and functional assays as described above, the positive hybridoma clones were sub-cloned by the limiting dilution. Three positive subclones based on ELISA and FACS screening from each plate were selected and characterized by functional assays. The top antibody subclones verified through functional assays were adapted for growth in the CDM4MAb medium (Cat.: SH30801.02, Hyclone, USA) with 3% FBS.
Expression and Purification of Monoclonal Antibodies
Hybridoma cells or 293G cells transiently transfected with an antibody expression plasmid (Cat. No. R79007, Invitrogen) was cultured either in CDM4MAb medium (Cat.: SH30801.02, Hyclone) or in Freestyle™ 293 Expression medium (Cat.: 12338018, Invitrogen), and incubated in a CO2 incubator for 5 to 7 days at 37° C. The conditioned medium was collected through centrifugation and filtrated by passing a 0.22 μm membrane before purification. Murine or recombinant antibodies containing supernatants were applied and bound to a Protein A column (Cat.: 17127901, GE Life Sciences) following the manufacturer's guide. The procedure usually yielded antibodies at purity above 90%. The Protein A-affinity purified antibodies were either dialyzed against PBS or further purified using a HiLoad 16/60 Superdex200 column (Cat.: 17531801, GE Life Sciences) to remove aggregates. Protein concentrations were determined by measuring absorbance at 280 nm. The final antibody preparations were stored in aliquots in −80° C. freezer.
Murine hybridoma clones were harvested to prepare total cellular RNAs using Ultrapure RNA kit (Cat.: 74104, QIAGEN, Germany) based on the manufacturer's protocol. The 1st strand cDNAs were synthesized using a cDNA synthesis kit from Invitrogen (Cat.: 18080-051) and PCR amplification of the nucleotide sequences coding for heavy chain variable region (Vh) and kappa chain variable region (VA) of murine mAbs was performed using a PCR kit (Cat.: CW0686, CWBio, Beijing, China). The oligo primers used for antibody cDNAs cloning of Vh and Vk were synthesized by Invitrogen (Beijing, China) based on the sequences reported previously (Brocks et al. 2001 Mol Med 7:461). PCR products were then subcloned into the pEASY-Blunt cloning vector (Cat.:C B101-02, TransGen, China) and sequenced by Genewiz (Beijing, China). The amino acid sequences of Vh and Vk regions were deduced from the DNA sequencing results.
The murine mAbs were analyzed by comparing sequence homology and grouped based on sequence similarity (
The Tigit antibodies with high binding activities in ELISA and FACS, as well as with potent functional activities in the cell-based assays (described in Examples 1 and 2) were characterized for their binding kinetics by SPR assays using BIAcore™ T-200 (GE Life Sciences). Briefly, anti-human IgG antibody was immobilized on an activated CM5 biosensor chip (Cat. No.: BR100530, GE Life Sciences). Human Fc-tagged Tigit was flowed over the chip surface and captured by anti-human IgG antibody. Then a serial dilution (0.12 nM to 10 nM) of purified murine antibodies were flowed over the chip surface and changes in surface plasmon resonance signals were analyzed to calculate the association rates (kon) and dissociation rates (koff) by using the one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (K)) was calculated as the ratio koff/kon. The binding affinity profiles of top mAbs including mu1217, mu1257, mu1226 and mu242, were shown in
mAb Humanization and Engineering
For humanization of the mu1217, human germline IgG genes were searched for sequences that share high degrees of homology to the cDNA sequences of mu1217 variable regions by blasting the human immunoglobulin gene database in IMGT (http://www.imgt.org/IMGT_vquest/share/textes/index.html) and NCBI (http://www.nebi.nlm.nih.gov/igblast/) websites. The human IGVH and IGVK genes that are present in human antibody repertoires with high frequencies (Glanville 2009 PNAS 106:20216-20221) and are highly homologous to mu1217 were selected as the templates for humanization.
Humanization was carried out by CDR-grafting (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols, Humana Press) and the humanization antibodies (hu1217s) were engineered as the human IgG1mf format using an in-house developed expression vector. In the initial round of humanization, mutations from murine to human amino acid residues in framework regions were guided by the simulated 3D structure, and the murine framework residues of structural importance for maintaining the canonical structures of CDRs were retained in the 14 version of humanization antibody 1217 (hu1217-1-1, with six CDRs having amino acid sequences of SEQ ID NOs: 3, 13, 5 (heavy chain CDRs) and SEQ ID NOs: 6, 7, 8 (light chain CDRs), a heavy chain variable region having an amino acid sequence of SEQ ID NO: 14 and encoded by a nucleotide sequence of SEQ ID NO: 15, and a light chain variable region having an amino acid sequence of SEQ ID No:16 and encoded by a nucleotide sequence of SEQ ID NO: 17). Specifically, CDRs of mu1217 Vκ (SEQ ID NO: 6-8) were grafted into the framework of human germline variable gene IGVκ3-15 with 1 murine framework residue (V58) retained, resulting in the humanized Vκ sequence of Hu1217-1-1 (SEQ ID NO: 16 for amino acid sequence and SEQ ID NO: 17 for nucleotide sequence). N-terminal of H-CDR2 (SEQ ID NO: 4), H-CDR1 and H-CDR3 (SEQ ID NOs: 3 and 5) of mu1217 Vh were grafted into the framework of human germline variable gene IGVH3-7 with two murine framework (T24 and 137 of SEQ ID NO: 10) residues retained. In the hu1217 humanization variants, only the N-terminal half of Kabat H-CDR2 was grafted, as only the N-terminal half was predicted to be important for antigen binding according to the simulated 3D structure. The amino acid sequence and nucleotide sequence of the resultant humanized Vh sequence of Hu1217-1-1 are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.
Hu1217-1-1 were constructed as human full-length antibody format using in-house developed expression vectors that contain constant regions of a human IgG1 variant termed as IgG1mf (SEQ ID NO: 18) and kappa chain, respectively, with easy adapting sub-cloning sites. Expression and preparation of hu1217-1-1 antibody was achieved by co-transfection of the above two constructs into 293G cells and by purification using a protein A column (Cat.: 17543802, GE Life Sciences). The purified antibodies were concentrated to 0.5-5 mg/mL in PBS and stored in aliquots in −80° C. freezer.
Based on hu1217-1-1 template, we made several single-mutations converting the retained murine residues in framework region of Vκ to corresponding human germline residues, which include V58I in Vκ and in T24A and 137V Vh. The resulted hu1217-2A-1 (T24A), hu1217-2B-1 (I37V), and hu1217-1-2a (V58I) all had similar binding and functional activities to hu1217-1-1. All humanization mutations were made using primers containing mutations at specific positions and a site directed mutagenesis kit (Cat. No. FM111-02, TransGen, Beijing, China). The desired mutations were verified by sequencing analysis. These hu1217-derived variant antibodies were tested in binding and functional assays as described previously.
Hu1217 antibodies were further engineered by introducing mutations in CDRs and framework regions to improve molecular and biophysical properties for therapeutic use in human. The considerations include amino acid compositions, heat stability (Tm), surface hydrophobicity and isoelectronic points (pIs) while maintaining functional activities.
Taken together, a well-engineered version of humanized monoclonal antibody, hu1217-2-2 (SEQ ID NOs:3, 5-8, 13, and 19-21), was derived from the mutation process described as above, and characterized in detail. The results showed both hu1217-2-2 and hu1217-1-1 were very similar in binding affinity and functional activities such as inhibiting the Tigit-mediated downstream signaling.
For affinity determination, antibodies were captured by anti-human Fc surface, and used in the affinity-assay based on surface plasmon resonance (SPR) technology. The results of SPR-determined binding profiles of anti-Tigit antibodies were summarized in Table 4. Hu217-2-2 and hu1217-1-1 showed very similar binding profiles with average dissociation constant at 0.415 nM and 0.266 nM, respectively, which are close to that of ch1217.
All the humanization antibodies shown above were also confirmed for functional activities on primary human immune cells isolated from healthy donors (described in Example 7).
To evaluate the binding activity of anti-Tigit antibodies to native Tigit on living cells, NK92mi cells were engineered to over-express human Tigit. Living NK92mi/Tigit cells were seeded in 96-well plate, and were incubated with a series of dilutions of anti-Tigit antibodies. Goat anti-Human IgG was used as secondary antibody to detect antibody binding to the cell surface. EC50 values for dose-dependent binding to human native Tigit were determined by fitting the dose-response data to the four-parameter logistic model with GraphPad Prism. As show in
Tigit binds to PVR with a high affinity (Kd: ˜1 nM), which can compete against CD266-PVR interaction [Yu et al., 2009].
To determine whether anti-Tigit antibodies could block Tigit-PVR and Tigit-PVR-L2 interactions, HEK293 cells were engineered to express high levels of PVR or PVR-L2. The resultant cell lines were named HEK293/PVR and HEK293/PVR-L2, respectively. The binding of soluble Tigit (Tigit-mIgG2a fusion protein) to PVR or PVR-L2 was determined by flow cytometry (
The functional activity of the Tigit antibodies were further assessed using naturally derived T-cells that recognized human CMV PP65 peptide (NLVPMVATV, 495-503, HLA-A2.1-restricted) [Boeckh M, Boeckh M and Geballe A P 2011 J Clin Invest. 121:1673-80]. Briefly, PBMCs from HLA-A2.1+ healthy donors were simulated with PP65 peptide (>98% purity, synthesized by GL Biochem, Shanghai) in the complete RPMI with 10% FBS for a week. The pp65-primed PBMCs were used as effector cells. Prior to assay, target cells, HCT116 cells (HLA-A2.1+, 104), were pulsed with pp65 peptide (5 μg/mL) for 30 mins and co-cultured with equal numbers of pp65-sensitized PBMCs in 96-well plates overnight in the presence or absence of anti-Tigit antibodies or a blank control (medium only). As shown in
Tigit is known to be constitutively expressed on natural killer (NK) cells at relatively higher levels and the interaction between Tigit and its ligands inhibits NK cell-mediated cytotoxicity [Wang F et al. 2015 Eur. J. Immunology 45:2886-97, Stanietsky N et al., 2009 Proc Natl Acad Sci USA 106:17858-63].
To confirm whether humanized anti-Tigit antibodies could promote NK-mediated cytotoxicity, an NK cell line NK92M1 was engineered to co-express both Tigit and DNAM-1 receptors (NK92M1/Tigit-DNAM-1) as an effector cell by retroviral transduction, according to the protocols described previously [Zhang et al, 2006 Cancer Res. 66: 5927-5933]. A PVR-expressing lung cancer cell line SK-MES-1/PVR was established similarly as a target.
Cytotoxicity of NK92MI/Tigit-DNAM-1 cells against SK-MES-1/PVR cells was determined by an LDH release assay using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, Wis.). In brief, NK92MI/Tigit-DNAM-1 cells (8×105) were co-cultured with SK-MES-1/PVR cells (2×104) in the presence of anti-Tigit Abs (0.007-30 μg/mL) for 5 hr in 96-well V-bottom plates. LDH-release assay Specific lysis was determined using the following equation: percentage of specific lysis=[(experimental-effector spontaneous-target spontaneous)/(target maximum−target spontaneous)]×100. The results showed that anti-Tigit antibodies hu1217-2-2/IgG1mf enhances NK cell killing in a dose-dependent manner (EC50: 0.185 g/mL) (
Trogocytosis is a phenomenon, in which cell surface molecules are transferred from donor cells to acceptor cells [Joly E, et al. 2003 Nat. Immunol; Machlenkin A. et al. 2008 Cancer Res.; Beum P V et al. 2008 J. Immunol; Rossi E A, et al. 2013 Blood]. Antibody-induced trogocytosis via Fe γ receptors (FcγRs) leads to down-modulation of receptors on the cell surface [Carlsten M, et al. 2016 Clin Cancer Res; Beum P V et al. 2011 J. Immunology]. Therefore, down-regulation of target receptor by trogocytosis may cause dampened signaling. In view of these observations, it would be possible that hu1217-2-2/IgG1 might induce trogocytosis of Tigit receptor in the presence of FcγR+ cells, resulting in lower surface expression. To address this possibility, Jurkat/Tigit cells were incubated with HEK cells expressing various FcγRs (including FcγRIIAH131, FcγRIIB, FcγRIIIAV158) with biotin-labeled hu1217-2-2/IgG1wt (a humanized antibody comprising the same VL and VH sequences as hu1217-2-2/IgG1mf and a wild-type IgG1 Fc region) or hu1217-2-2/IgG1mf overnight. Surface expression of Tigit receptor was determined by with SA-APC (Biolegend). As shown in
The abilities of anti-Tigit antibodies to induce ADCC and CDC in human primary PBMCs were determined using in vitro assay as described below.
ADCC Using Human PBMCs as Target Cells
A flow cytometry-based ADCC assay was set up to determine whether Tigit antibodies could induce ADCC in Tigit+ T cells. The assay effector cell line, NK92MI/CD16V cells, was generated from NK92MI cells (ATCC) by co-transducing expression plasmids containing CD16r se (V158 allele) and FcRγ cDNAs. Human PBMCs from healthy donors were stimulated with PHA (1 μg/ml) to up-regulate Tigit expression. As shown in
CDC Using Human PBMCs as Target Cells
Whether hu1217-2-2/IgG1mf and hu1217-2-2/IgG1wt would trigger CDC was determined by using pre-activated human PBMCs and fresh autologous sera from healthy donors. Cell lysis by CDC was determined by a Celltiter glo assay kit (Promega, Beijing, China). In brief, PBMCs were pre-activated with PHA (10 μg/mL) for 3 days, and then were incubated in RPMI640 plus autologous serum (15%) and anti-Tigit or control antibodies (0.01-100 μg/mL) for overnight at 37° C. The cell death due to CDC was assayed by the decrease of ATP released from viable cells after cell lysis at the end of reaction. Anti-MHC-I A, B, C was used as a positive control. The fluorescence readout was conducted using a 96-well fluorometer (PHERA Star FS, BMG LABTECH), and the CDC activities were calculated from the relative fluorescence unit (RFU) readout as follows: % CDC activity=[(RFU test−RFU background)/(RFU at total cell lysis−RFU background)]×100. The experimental results demonstrated that both hu1217-2-2/IgG1mf and hu1217-2-2/IgG1wt had no detactable CDC with PBMCs isolated from two different donors. In contrast, the positive control antibody, anti-MHC-1, induces significant CDC activity (
To investigate whether pH would influence the binding property of hu1217-2-2/IgG1, target binding SPR tests were performed in running buffers at pH 7.4 and at pH 6.0 for comparison. The antibody hu1217-2-2/IgG1 was immobilized to a CM5 chip (GE). Serial dilutions of TIGIT-his were flown over the immobilized hu1217-2-2/IgG1 in running buffer HBS at pH 7.4 or pH 6.0.
As shown by the results listed in Table 7 below, hu1217-2-2/IgG1 showed higher binding affinity (KD) and stronger binding signal (Rmax) against human TIGIT at pH 6.0 (an acidic pH which is similar to the pH of tumor microenvironment) as compared to the data obtained at pH 7.4 (physiologic pH). These results indicate a potential advantage of the antibody as a therapeutic agent targeting TIGIT-positive lymphocytes in the tumor environment, since hu1217-2-2/IgG1 might more selectively target the TIGIT-positive lymphocytes in the tumor microenvironment while have lower potential toxicity associated with activation of periphery lymphocytes.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/120392 | Dec 2017 | CN | national |
This application claims priority to patent application number PCT/CN2017/120392, filed on Dec. 30, 2017. The entire content of aforementioned application is incorporated herein by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/125375 | 12/29/2018 | WO | 00 |
