Patent Application
20230031629
The present application claims priority from Australian provisional application no. 2019904614 filed 5 Dec. 2019, the entirety of which is incorporated herein by reference.
The present invention relates to a method for antigen loading an antigen presenting cell or a precursor thereof, to use of the antigen-loaded antigen presenting cell or a precursor thereof for promoting or increasing a T cell response to a target antigen, to compositions for antigen loading antigen presenting cells or a precursor thereof, and to a CD300f binding protein immunoconjugate for antigen loading antigen presenting cells or a precursor thereof, or for promoting or increasing an immune response to a target antigen in a subject.
Immunotherapy is a form of treatment that is designed to activate or suppress the immune system to treat a condition or disease. Immunotherapy has become of particular interest in the treatment of cancer.
Cell-based immunotherapies are effective for some cancers. Immune effector cells such as T lymphocytes (e.g., CD4+ helper T cells and cytotoxic CD8+ T cells), can be targeted to specific target antigens, such as cancer antigens, if the target antigen is presented to the effector cells on the surface of antigen presenting cells. Presentation of the target antigen on the surface of antigen presenting cells to T cells initiates an antigen specific T cell response against cells expressing the target antigen.
Dendritic cells (DCs) are antigen presenting cells for the induction of antigen specific T cell responses. Recent approaches in cell-based immunotherapy involves producing dendritic cell vaccines by loading antigen presenting cells such as dendritic cells with a target antigen. Loading of target antigen into the dendritic cell is usually achieved by isolating dendritic cells from the subject and introducing the target antigen into the dendritic cell. The target antigen is then processed by the dendritic cell and presented on the surface of the dendritic cell, typically complexed with MHC I, to produce a dendritic cell vaccine (DC vaccine). The dendritic cell vaccine can then be administered to the subject to induce an immune response to the target antigen.
Despite recent advances in immunotherapy to treat cancer, DC vaccination therapy has yet to become widely used. DC vaccination has been shown to be safe and effective but cost and deliverability of current ex vivo loading strategies makes access difficult.
What is needed is alternative approaches for antigen loading antigen presenting cells, for DC vaccination and other therapies.
CD300f is a member of the CD300 family of immunoregulatory molecules encoded by a gene complex on chromosome 17q25. It is a transmembrane glycoprotein with a cytoplasmic region and an extracellular domain. The cytoplasmic region contains both inhibitory ITIMs and PI3K phosphorylation sites. There are a number of isoforms of the CD300f molecule, but they all share the same Ig-like extracellular domain.
The inventors have found that CD300f is expressed on antigen presenting cells and precursors thereof, is able to be internalized, and has an expression profile that is more restricted than that of DEC-205. The inventors have found that CD300f monoclonal antibodies are able to deliver a target antigen and induce a target antigen specific T cell response in vitro. The inventors have also shown that an anti-CD300f antibody activates dendritic cells without adjuvant and promotes migration of the dendritic cells.
A first aspect provides a method of antigen loading an antigen presenting cell or precursor thereof with a target antigen for presentation of the target antigen to a T cell, the method comprising contacting the antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen.
A second aspect provides a method of antigen loading an antigen presenting cell or precursor thereof with a target antigen for presentation of the target antigen to a T cell, and promoting activation of the antigen presenting cell or precursor thereof, the method comprising contacting the antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen.
A third aspect provides a method of presenting a target antigen to a T cell, the method comprising:
antigen loading an antigen presenting cell or precursor thereof with a target antigen by contacting the antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen to produce an antigen-loaded antigen presenting cell or precursor thereof; and
culturing the antigen-loaded antigen presenting cell or precursor thereof with a population comprising T cells under conditions which permit presentation of the target antigen to the T cells of the population.
A fourth aspect provides a method of promoting or increasing a T cell response to a target antigen, comprising:
antigen loading an antigen presenting cell or precursor thereof with a target antigen by contacting an antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen to produce an antigen-loaded antigen presenting cell or precursor thereof; and
culturing the antigen-loaded antigen presenting cell or precursor thereof with a population comprising T cells under conditions which permit presentation of the target antigen to the T cells of the population to stimulate a target antigen specific T cell response.
A fifth aspect provides a method of promoting or increasing a T cell response to a target antigen in a subject, the method comprising administering to the subject an effective amount of a CD300f binding protein and the target antigen.
An alternative fifth aspect provides a CD300f binding protein and a target antigen for use in promoting or increasing a T cell response to the target antigen in a subject; or use of a CD300f binding protein and a target antigen in the manufacture of a medicament for promoting or increasing a T cell response to the target antigen in a subject.
A sixth aspect provides a method of treating a disease or condition requiring a T cell response to a target antigen, the method comprising administering an effective amount of a CD300f binding protein and the target antigen.
An alternative sixth aspect provides a CD300f binding protein and a target antigen for use in treating a disease or condition requiring a T cell response to the target antigen; or use of a CD300f binding protein and a target antigen in the manufacture of a medicament for treating a disease or condition requiring a T cell response to the target antigen.
A seventh aspect provides a composition for antigen loading an antigen presenting cell or precursor thereof with a target antigen, the composition comprising a CD300f binding protein and the target antigen.
An eighth aspect provides a pharmaceutical composition for promoting or increasing a T cell response to a target antigen, the composition comprising a CD300f binding protein and the target antigen.
A ninth aspect provides immunoconjugate for antigen loading an antigen presenting cell or precursor thereof with a target antigen, the immunoconjugate comprising a CD300f binding protein coupled to the target antigen.
A tenth aspect provides a method of producing an antigen-loaded antigen presenting cell or a precursor thereof which is capable of presenting a target antigen to a T cell, the method comprising contacting an antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen for sufficient time to permit uptake of the target antigen.
An eleventh aspect provides an antigen-loaded antigen presenting cell or precursor thereof which is capable of presenting a target antigen to a T cell, wherein the antigen-loaded antigen presenting cell or precursor thereof is produced by contacting an antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen for sufficient time to permit uptake of the target antigen.
A twelfth aspect provides a method of promoting a T cell response to a target antigen, comprising incubating the antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect with a population comprising T cells under conditions which promote presentation of the target antigen to the T cells.
A thirteenth aspect provides a method of promoting or increasing a T cell response to a target antigen in a subject, the method comprising administering to the subject an effective amount of the antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect.
An alternative thirteenth aspect provides an antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect for use in promoting or increasing a T cell response to a target antigen in a subject; or use of an antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect in the manufacture of a medicament for promoting or increasing a T cell response to a target antigen in a subject.
A fourteenth aspect provides a method of treating a disease or condition requiring a T cell response to a target antigen, the method comprising administering an effective amount of the antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect.
An alternative fourteenth aspect provides an antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect for use in treating a disease or condition requiring a T cell response to a target antigen; or use of an antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect in the manufacture of a medicament for treating a disease or condition requiring a T cell response to a target antigen.
A fifteenth aspect provides a method of promoting or increasing an immune response to a target antigen in a subject, the method comprising administering to the subject an effective amount of the antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect.
An alternative fifteenth aspect provides an antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect for use in promoting or increasing an immune response to a target antigen in a subject; or use of an antigen-loaded antigen presenting cell or precursor thereof of the eleventh aspect in the manufacture of a medicament for promoting or increasing an immune response to a target antigen in a subject.
A sixteenth aspect provides a method of promoting or increasing a T cell response to a target antigen in a subject, the method comprising administering to the subject an effective amount of the immunoconjugate of the ninth aspect.
An alternative sixteenth aspect provides an immunoconjugate of the ninth aspect for use in promoting or increasing a T cell response to a target antigen in a subject; or use of an immunoconjugate of the ninth aspect in the manufacture of a medicament for promoting or increasing a T cell response to a target antigen in a subject.
A seventeenth aspect provides a method of treating a disease or condition requiring a T cell response to a target antigen, the method comprising administering an effective amount of the immunoconjugate of the ninth aspect.
An alternative seventeenth aspect provides an immunoconjugate of the ninth aspect for use in treating a disease or condition requiring a T cell response to a target antigen; or use of an immunoconjugate of the ninth aspect in the manufacture of a medicament for treating a disease or condition requiring a T cell response to a target antigen.
An eighteenth aspect provides a method of promoting activation of an antigen presenting cell or precursor thereof, comprising contacting the antigen presenting cell or precursor thereof with a CD300f binding protein.
An alternative eighteenth aspect provides a CD300f binding protein for use in promoting activation of an antigen presenting cell or precursor thereof; or use of a CD300f binding protein in the manufacture of a medicament for promoting activation of an antigen presenting cell or precursor thereof.
A nineteenth aspect provides a method of promoting migration of an antigen presenting cell or precursor thereof towards CCL19 and/or CCL21, comprising contacting the antigen presenting cell or precursor thereof with a CD300f binding protein.
A twentieth aspect provides a method of increasing CCR7 expression in an antigen presenting cell or precursor thereof, comprising contacting the antigen presenting cell or precursor thereof with a CD300f binding protein.
A twenty first aspect provides a fusion protein comprising a CD300f binding protein and a target antigen.
A twenty second aspect provides a CD300f binding protein which comprise a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16.
A twenty third aspect provides a composition comprising a CD300f binding protein which comprise a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16.
A twenty fourth aspect provides a nucleic acid encoding a CD300f binding protein which comprise a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16.
The present disclosure relates in one form to antigen loading of an antigen presenting cell or precursor thereof with a target antigen for presentation of the target antigen to a T cell.
As used herein, the phrase “antigen loading an antigen presenting cell or precursor thereof with a target antigen” refers to introducing a target antigen into an antigen presenting cell or a precursor thereof such that the target antigen or a portion thereof is presented on the surface of the cell in a manner that is capable of promoting an antigen specific T cell response against the target antigen.
The inventors have found that contacting an antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of a target antigen results in uptake of the binding protein and the target antigen, and presentation of the target antigen on the cell surface of the antigen presenting cell in a manner that is capable of promoting an antigen specific T cell response to the target antigen. The inventors therefore envisage that CD300f binding proteins can be used to prepare, for example, DC vaccines in which a target antigen is loaded into antigen presenting cells or precursors thereof using the CD300f binding protein.
Accordingly, one aspect provides a method for antigen loading an antigen presenting cell or precursor thereof with a target antigen for presentation of the target antigen to a T cell, the method comprising contacting the antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen.
Another aspect provides a method of producing an antigen-loaded antigen presenting cell or precursor thereof which is capable of presenting a target antigen to a T cell, the method comprising contacting an antigen presenting cell or precursor thereof with a CD300f binding protein in the presence of the target antigen for sufficient time to allow uptake of the target antigen by the antigen presenting cell.
In one embodiment, the antigen presenting cell or precursor thereof is contacted with a CD300f binding protein in the presence of the target antigen by contacting the antigen presenting cell or precursor thereof with an immunoconjugate comprising the CD300f binding protein and the target antigen.
In one embodiment, the immunoconjugate is a fusion protein comprising the CD300f binding protein and the target antigen.
In one embodiment, the antigen presenting cell is a dendritic cell (DC). In one embodiment, the dendritic cell is a myeloid dendritic cell (Mo-DC). In one embodiment, the myeloid DC expresses one or more of the markers selected from CD11c, CD1c and CD16. In one embodiment, the myeloid DC expresses CD11c.
In one embodiment, the myeloid DC expresses CD1c. In one embodiment, the myeloid DC expresses CD16. In one embodiment, the myeloid DC expresses CD11c, CD1c and CD16.
In one embodiment, the antigen-loaded antigen presenting cell or precursor thereof is a DC vaccine.
In one embodiment, the precursor of an antigen presenting cell is a monocyte.
In one embodiment, contacting of the antigen presenting cell or precursor thereof with the CD300f binding protein promotes activation of the antigen presenting cell or precursor thereof.
In one embodiment, activation of the antigen presenting cell or precursor thereof increases expression of one or more activation markers.
In one embodiment, the activation marker is one or more proteins selected from CD80, CD83, CD86, and HLA-DR.
In one embodiment, the antigen presenting cell is a dendritic cell and the activation markers are CD80, CD83 and CD86.
The inventors have shown that antigen presenting cells that have been loaded with a target antigen using CD300f binding protein produce an antigen specific T cell response to the target antigen.
Accordingly, in one aspect, there is provided a method of presenting a target antigen to a T cell, the method comprising:
Another aspect provides a method of promoting or increasing a T cell response to a target antigen, the method comprising:
Methods for culturing T cell populations for antigen presentation and T cell stimulation are known in the art.
In one embodiment, the population comprising T cells comprises a lymphocyte population comprising CD3+ T cells. In one embodiment, the population comprising T cells comprises a lymphocyte population comprising CD3+, CD8+ T cells. In one embodiment, the population comprising T cells comprises a lymphocyte population comprising CD3+, CD4+ and CD8+ T cells. In one embodiment, the population comprising T cells comprises cytotoxic T cells. Methods for the preparation of T cell populations are known in the art, and described in, for example, Hsu et al., (2018) A blood dendritic cell vaccine for acute myeloid leukemia expands anti-tumor T cell responses at remission, Oncolmmunology, 7:4, e1419114.
One aspect provides a T cell population comprising T cells that have been activated by culturing with an antigen-loaded antigen presenting cell or precursor thereof described herein.
Antigen-loaded antigen presenting cells or precursors thereof may be administered to a subject to promote or increase a T cell response in a subject.
Accordingly, in one aspect, there is provided a method of presenting a target antigen to a T cell in a subject, the method comprising:
Another aspect provides a method of promoting or increasing a T cell response to a target antigen in a subject, the method comprising:
The CD300f binding protein may be any protein which specifically binds CD300f and is internalized.
CD300f is a member of the CD300 family of immunoregulatory molecules encoded by a gene complex on chromosome 17q25. It is a transmembrane glycoprotein with a cytoplasmic region and an extracellular domain. The cytoplasmic region contains both inhibitory ITIMs and PI3K phosphorylation sites. There are a number of isoforms of the CD300f molecule, but they all share the same Ig-like extracellular domain.
In one embodiment, the CD300f binding protein is an antibody or antigen binding fragment thereof, which specifically binds to CD300f. In one embodiment, the antibody or antigen binding fragment thereof specifically binds to an extracellular domain of CD300f.
The inventors have previously produced a monoclonal antibody, referred to herein as DCR-2, which specifically binds to the extracellular domain of CD300f. DCR-2 is described in WO 2018/094460, the entirety of which is incorporated herein by reference. A hybridoma producing DCR-2 was deposited at CellBank Australia, 214 Hawkesbury Rd., Westmead, NSW 2145, Australia, under the Budapest Treaty on 27 Sep. 2016 and designated accession number CBA20160029.
In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region which comprises:
(b) a light chain variable region which comprises:
In one embodiment, the CD300f binding protein comprises:
In one embodiment, the CD300f binding protein comprises: (a) a heavy chain variable region which comprises an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, to the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1, and a complementarity determining region 1 (CDR1) that comprises the amino acid sequence represented by SEQ ID NO: 2, a complementarity determining region 2 (CDR2) that comprises an amino acid sequence that is represented by SEQ ID NO: 3, and a complementarity determining region 3 (CDR3) that comprises an amino acid sequence that is represented by SEQ ID NO: 4; and (b) a light chain variable region which comprises an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, to the amino acid sequence represented by SEQ ID NO: 5, and comprises a complementarity determining region 1 (CDR1) that comprises an amino acid sequence represented by SEQ ID NO: 6, a complementarity determining region 2 (CDR2) that comprises an amino acid sequence represented by SEQ ID NO: 7, and a complementarity determining region 3 (CDR3) that comprises an amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises a heavy chain variable region which comprises the amino acid sequence represented by SEQ ID NO: 1, and a light chain variable region which comprises the amino acid sequence represented by SEQ ID NO: 5.
The amino acid sequence of the heavy chain variable region (VH) of DCR-2 is represented by the amino acid sequence:
The amino acid sequence of CDR1 of the heavy chain variable region of DCR-2 is represented by the amino acid sequence GFGFSGSW (SEQ ID NO: 2).
The amino acid sequence of CDR2 of the heavy chain variable region of DCR-2 is represented by the amino acid sequence INPDSSTI (SEQ ID NO: 3).
The amino acid sequence of CDR3 of the heavy chain variable region of DCR-2 is represented by the amino acid sequence ARRGFFEGYSAWFAY (SEQ ID NO: 4).
The amino acid sequence of the light chain variable region (VL) of DCR-2 is represented by the amino acid sequence:
The amino acid sequence of CDR1 of the light chain variable region of DCR-2 is represented by the amino acid sequence QSVSND (SEQ ID NO: 6).
The amino acid sequence of CDR2 of the light chain variable region of DCR-2 is represented by the amino acid sequence YAS (SEQ ID NO: 7).
The amino acid sequence of CDR3 of the light chain variable region of DCR-2 is represented by the amino acid sequence QQDYTSPWT (SEQ ID NO: 8).
In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region which comprises:
(i) an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence represented by SEQ ID NO: 1; or
(ii) a complementarity determining region 1 (CDR1) that comprises the amino acid sequence represented by SEQ ID NO: 2, a complementarity determining region 2 (CDR2) that comprises the amino acid sequence represented by SEQ ID NO: 3, and/or a complementarity determining region 3 (CDR3) that comprises the amino acid sequence represented by SEQ ID NO: 4; and
(b) a light chain variable region which comprises:
(i) an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence represented by SEQ ID NO: 5; or
(ii) a complementarity determining region 1 (CDR1) that comprises the amino acid sequence represented by SEQ ID NO: 6, a complementarity determining region 2 (CDR2) that comprises the amino acid sequence represented by SEQ ID NO: 7, and/or a complementarity determining region 3 (CDR3) that comprises the amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region which comprises:
(i) an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence represented by SEQ ID NO: 1; and/or
(b) a light chain variable region which comprises:
(i) an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence represented by SEQ ID NO: 5.
In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region which comprises:
(i) a complementarity determining region 1 (CDR1) that comprises the amino acid sequence represented by SEQ ID NO: 2, a complementarity determining region 2 (CDR2) that comprises the amino acid sequence represented by SEQ ID NO: 3, and a complementarity determining region 3 (CDR3) that comprises the amino acid sequence represented by SEQ ID NO: 4; or
(b) a light chain variable region which comprises:
(i) a complementarity determining region 1 (CDR1) that comprises the amino acid sequence represented by SEQ ID NO: 6, a complementarity determining region 2 (CDR2) that comprises the amino acid sequence represented by SEQ ID NO: 7, and a complementarity determining region 3 (CDR3) that comprises the amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region which comprises:
(i) a complementarity determining region 1 (CDR1) that comprises the amino acid sequence represented by SEQ ID NO: 2, a complementarity determining region 2 (CDR2) that comprises the amino acid sequence represented by SEQ ID NO: 3, and a complementarity determining region 3 (CDR3) that comprises the amino acid sequence represented by SEQ ID NO: 4; and
(b) a light chain variable region which comprises:
(i) a complementarity determining region 1 (CDR1) that comprises the amino acid sequence represented by SEQ ID NO: 6, a complementarity determining region 2 (CDR2) that is identical to the amino acid sequence represented by SEQ ID NO: 7, and a complementarity determining region 3 (CDR3) that comprises the amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises a heavy chain variable region which comprises an amino acid sequence that is 100% identical to the amino acid sequence represented by SEQ ID NO: 1.
In one embodiment, the CD300f binding protein comprises a light chain variable region which is 100% identical to the amino acid sequence represented by SEQ ID NO: 5.
The inventors have further humanized the heavy and light chain variable regions of DCR-2. The amino acid sequence of the humanized heavy chain variable region (VL) of DCR-2 is represented by the amino acid sequence:
The amino acid sequence of the humanized light chain variable region (VL) of DCR-2 is represented by the amino acid sequence:
In one embodiment, the CD300f binding protein comprises a heavy chain variable region which comprises the amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region which comprises the amino acid sequence represented by SEQ ID NO: 16.
In various embodiments, the CD300f binding protein comprises:
In one embodiment, the CD300f binding protein comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 13.
In one embodiment, the CD300f binding protein comprises a light chain comprising the amino acid sequence represented by SEQ ID NO: 14.
In one embodiment, the CD300f binding protein comprises a heavy chain variable region which comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 1 or 15, and a light chain variable region which comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 5 or 16.
In one embodiment, the CD300f binding protein comprises a heavy chain variable region which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 1 or 15, and a light chain variable region which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 5 or 16.
In one embodiment, the CD300f binding protein comprises: a heavy chain variable region which comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 2, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 4; and a light chain variable region which comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises: a heavy chain variable region which comprises an amino acid sequence that is at least 70% identical to the amino acid sequence represented by SEQ ID NO: 1, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 2, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 4; and a light chain variable region which comprises an amino acid sequence that is at least 70% identical to the amino acid sequence represented by SEQ ID NO: 5, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises: a heavy chain variable region which comprises an amino acid sequence that is at least 75% identical to the amino acid sequence represented by SEQ ID NO: 1, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 2, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 4; and a light chain variable region which comprises an amino acid sequence that is at least 75% identical to the amino acid sequence represented by SEQ ID NO: 5, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8.
The percent identity between two amino acid sequences can be determined using any alignment algorithms known in the art, including for example, the FASTA package of sequence analysis programs (Lipman & Pearson, (1985) Science 227(4693): 1435-1441); BLAST (Altschul et al. J. Mol. Biol. 215(3):403-410.
In one embodiment, the antibody, or antigen binding fragment thereof, binds to human CD300f with an equilibrium dissociation constant (KD) of less than 10−7M, typically less than 10−8M, less than 10−9M, less than 9×10−10M, less than 8×10−10, less than 7×10−10, less than 6×10−10, less than 5×10−10, or less than 4×10−10M.
In one embodiment, the antibody, or antigen binding fragment thereof, binds to human CD300f with an equilibrium dissociation constant (KD) that is in the range of from 1×10−10 to 1×10−7M, typically in the range of from 1×10−10 to 1×10−7 M, 1×10−10 to 1×10−8 M, 1×10−10 to 1×10−9 M, 1×10−10 to 9×10−10 M, 1×10−11 to 8×10−10 M or 1×10−10 to 7×10−10 M, 2×10−11 to 6×10−10M, or 3×10−10 to 5×10−10M.
In one embodiment, the antibody, or antigen binding fragment thereof, binds to human CD300f with a KA of about 1×106 M−1s−1 or less. In various embodiments, the antibody or antigen binding fragment thereof binds to human CD300f with a KA in the range of from 1×105 to 5×106 M−1s−1, typically 1×105 to 1×106 M−1s−1.
In one embodiment, the antibody, or antigen binding fragment thereof, binds to human CD300f with an off rate of 5×10−2 s−1 or less, typically 1×10−2 s−1 or less, 5×10−3 s−1 or less, 1×10−3 s−1 or less, or 5×10−4 s−1 or less, 4×10−4 s−1 or less, 3×10−4 s−1 or less, or 2×10−4 s−1 or less.
In one embodiment, the antibody, or antigen binding fragment thereof, binds to human CD300f with an off rate in the range of from 5×10−2 s−1 to 1×10−5 s−1, 1×10−2 to 5×10−5 s−1, 5×10−3 to 5×10−4 s−1, 1×10−3 to 5×10−4 s−1, or 1×10−3 to 1×10−4 s−1.
An antibody is an immunoglobulin molecule capable of specifically binding to an antigen. The antibody may be recombinant or modified, including chimeric, humanised, deimmunised, CDR-grafted, synhumanised, bi-specific, or human. A full-length antibody typically comprises two light chains covalently linked to two heavy chains. Each heavy chain of the full-length antibody comprises a heavy chain variable region and a heavy chain constant region. Each light chain of a full-length antibody comprises a light chain variable region and a light chain constant region. Full length antibodies may be any of the following type: IgG, IgM, IgE, IgD, or IgA. In one embodiment, the antibody is IgG.
As used herein, an “antigen binding fragment” of an antibody comprises an antigen binding domain of the antibody, and typically comprises a portion of the antibody that specifically binds the same epitope as the full-length antibody. Typically, the antibody fragment of an antibody comprises portions of the variable region of the heavy and/or the light chain of the antibody. Typically, the antigen binding fragment comprises the CDR1, 2 and/or 3 region of the heavy chain variable region and/or the CDR1, 2 and/or 3 region of the light chain variable region. More typically, the antigen binding fragment comprises the CDR1, 2 and 3 region of the heavy chain variable region and/or the CDR1, 2 and 3 region of the light chain variable region. Still more typically, the antigen binding fragment comprises the CDR1, 2 and 3 region of the heavy chain variable region, and the CDR1, 2 and 3 region of the light chain variable region. In some embodiments, the antigen binding fragment of an antibody comprises the heavy chain variable region and the light chain variable region of an antibody. The portions of the heavy and light chain variable regions may be on separate polypeptide chains, such as Fv fragments, or in a single polypeptide chain in which the light chain and heavy chain variable regions are connected by a peptide linker (“scFv proteins”). Examples of antigen binding fragments of an antibody may include F(ab′)2, Fab′, Fab, Fv, sFv, scFv, and the like.
As used herein, an antigen binding fragment of an antibody encompasses one or more polypeptides which comprise an antigen binding domain of the antibody, such as an F(ab′)2, Fab′, Fab, Fv, sFv, or scFv.
An “antigen binding domain” refers to a region of an antibody that is capable of specifically binding to an antigen. Typically, the antigen binding domain comprises CDR1, CDR2 and/or CDR3 from the light chain variable region, and/or CDR1, CDR2 and/or CDR3 from the heavy chain variable region, of an antibody. More typically, the antigen binding domain comprises CDR1, CDR2 and CDR3 from the light chain variable region, and/or CDR1, CDR2 and/or CDR3 from the heavy chain variable region, of an antibody. Still more typically, the antigen binding domain comprises CDR1, CDR2 and CDR3 from the light chain variable region, and CDR1, CDR2 and CDR3 from the heavy chain variable region, of an antibody.
The term “variable region” refers to the portion of the light and/or heavy chain of an antibody that is capable of specifically binding to an antigen. The variable region comprises the complementarity determining regions (CDRs) and the framework regions (FRs). Framework regions are those variable regions other than the complementarity determining regions.
The term “complementarity determining region” refers to one of three amino acid sequences of the variable region of the light chain variable region and/or heavy chain variable region of an antibody that is largely responsible for the ability of the antibody to bind specifically to an antigen. The three complementarity determining regions of the variable region of the light and heavy chain are referred to as CDR1, CDR2 and CDR3.
Methods for determining the CDR regions and the framework regions (FR) of the variable region of the light and heavy chain are known in the art. For example, the amino acid positions assigned to CDRs and FRs may be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md., 1987 and 1991; Enhanced Clothia Numbering Scheme; Clothia and Lesk J. Mol. Biol. 196:901-917; Clothia et al. Nature 342: 877-883; Honnegher and Plukthun, J. Mol. Biol. 309: 657-670. The antibody, or antigen binding fragment thereof, specifically binds to the extracellular domain of CD300f. As used herein, “an antibody, or antigen binding fragment thereof, that specifically binds to an extracellular domain of CD300f” is an antibody or antigen binding fragment thereof that associates with the extracellular domain of CD300f more frequently, more rapidly, for greater length of time, or with greater affinity, that with other antigens.
The variable domains from antibodies may be cloned using conventional techniques that are known in the art and described in, for example, Sambrook and Russell, Eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001. In general, the light chain variable region and heavy chain variable region sequences for antibodies, such as murine antibodies, can be obtained by a variety of molecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA library screening.
As used herein, a chimeric antibody is an antibody protein that comprises the complementarity determining regions (CDRs), typically the variable regions, of an antibody derived from one species, typically a mouse antibody, while the constant domains of the antibody molecule, and in some embodiments, the framework regions (FR), are derived from another species, such as a human. A humanised antibody is a form of chimeric antibody in which the amino acid sequence of the CDRs is from an antibody from one species; e.g., a mouse antibody, and the amino acid sequence of the constant regions, and typically the framework regions, is from a human antibody.
In one embodiment, the antibody or antigen binding fragment thereof is a chimeric antibody. The chimeric antibody comprises the complementarity-determining regions (CDRs), and typically FR, of DCR-2. The chimeric antibody may comprise the light and heavy chain constant regions of a human antibody. The use of antibody components derived from chimerized monoclonal antibodies reduces potential problems associated with the immunogenicity of murine constant regions. Typically, the antibody is a humanised antibody. Humanization of murine antibodies and antibody fragments is known to those skilled in the art, and described in, for example, U.S. Pat. Nos. 5,225,539; 6,054,297; and 7,566,771. For example, humanized monoclonal antibodies may be produced by transferring murine complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts. The use of human framework region sequences, in addition to human constant region sequences, further reduces the chance of inducing HAMA reactions.
Antibodies can be isolated and purified from serum and hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Baines et al., “Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992).
In some embodiments, an antigen binding fragment of an antibody includes portions of the variable region of the heavy and/or light chain of the antibody. The portions of the heavy chain variable region and/or light chain variable region may be on separate polypeptide chains, such as Fv fragments, or in a single polypeptide chain in which light and heavy variable regions are connected by a peptide linker (e.g. scFv proteins). Examples of antibody fragments include F(ab′)2, Fab′, Fab, Fv, sFv, scFv, and the like. Typically, the antibody fragment comprises the CDR1, 2 and 3 region of the heavy chain variable region and/or the CDR1, 2 and 3 region of the light chain variable region. Antibody fragments which recognize specific epitopes can be generated by known techniques. F(ab′)2 fragments, for example, can be produced by pepsin digestion of the antibody molecule. These and other methods are described, for example, by Harlow & Lane (Eds.) Antibodies: A Laboratory Manual, CSHL Press, 1988. Alternatively, Fab′ expression libraries can be constructed to allow rapid and easy identification of Fab′ fragments with the desired specificity.
In some embodiments, an antigen binding fragment of an antibody may be a single chain Fv molecule (scFv). A single chain Fv molecule (scFv) typically comprises a light chain variable region and a heavy chain variable region. The light chain variable region and heavy chain variable region are typically covalently linked by a peptide linker (L) and fold to form an antigen binding site. While the heavy chain variable region and light chain variable region may be directly joined together, those skilled in the art will appreciate that the regions may be separated by a peptide linker consisting of one or more amino acids. Peptide linkers and their use are known in the art. Generally, the peptide linker will have no specific biological activity other than to join the regions or to preserve some minimum distance or other spatial relationship between the heavy chain variable region and light chain variable region. However, the constituent amino acids of the peptide linker may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. Single chain Fv (scFv) antibodies optionally include a peptide linker of no more than 50 amino acids, generally no more than 40 amino acids, preferably no more than 30 amino acids, and more preferably no more than 20 amino acids in length.
Methods of making scFv antibodies are known in the art, and have been described in, for example, U.S. Pat. No. 5,260,203; Lo(Ed), Antibody Engineering: Methods and Protocols (Methods in Molecular Biology, v. 248): p 117-134 and 161-190. In brief, mRNA from B-cells from an immunized animal is isolated and cDNA is prepared. The cDNA is amplified using primers specific for the variable regions of heavy and light chains of immunoglobulins. The PCR products are purified, and the nucleic acid sequences are joined. If a linker peptide is desired, nucleic acid sequences that encode the peptide are inserted between the heavy and light chain nucleic acid sequences. The nucleic acid which encodes the scFv is inserted into a vector and expressed in the appropriate host cell. The scFv that specifically bind to the desired antigen are typically found by panning of a phage display library. Panning can be performed by any of several methods. Panning can conveniently be performed using cells expressing the desired antigen on their surface or using a solid surface coated with the desired antigen. Conveniently, the surface can be a magnetic bead. The unbound phage are washed off the solid surface and the bound phage are eluted.
Methods for preparing other antigen binding fragments of antibodies are known in the art. For example, antigen binding fragments can also be prepared by proteolytic hydrolysis of a full-length antibody or by expression in E. coli or another host of the DNA coding for the fragment. An antibody fragment can be obtained by pepsin or papain digestion of full-length antibodies by conventional methods. For example, an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide an approximate 100 Kd fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce an approximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly.
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the epitope that is recognized by the intact antibody.
One aspect provides a CD300f binding protein which comprise a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16.
In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 15, and
(b) a light chain variable region which comprises an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence represented by SEQ ID NO: 5, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region which comprises an amino acid sequence that is at least 70% identical, typically at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence represented by SEQ ID NO: 1, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 2, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 4, and
(b) a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 16.
In one embodiment, the CD300f binding protein comprises a heavy chain variable region which comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 15 and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 2, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 4, and a light chain variable region which comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 16 and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8.
In one embodiment, the CD300f binding protein comprises a heavy chain variable region which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16.
In one embodiment, the CD300f binding protein comprises: a heavy chain variable region which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 15, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 2, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 4; and a light chain variable region which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16, and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8.
The antigen presenting cell or precursor thereof is loaded with a target antigen by contacting the antigen presenting cell or precursor thereof with the CD300f binding protein in the presence of the target antigen.
The CD300f binding protein is in the presence of the target antigen if internalisation of the CD300f binding protein by the antigen presenting cell or precursor thereof results in internalisation of the target antigen.
In various embodiments, the CD300f binding protein may be in the presence of the target antigen when the CD300f binding protein is:
in the presence of a polypeptide comprising the target antigen;
in the presence of a polypeptide consisting essentially of the target antigen; or
in the presence of a polypeptide consisting of the target antigen.
In one embodiment, the CD300f binding protein is not coupled to the target antigen. In such embodiments, the target antigen is a separate molecule from the CD300f binding protein, but during contacting of the antigen presenting cell or precursor thereof with the CD300f binding protein, the molecule comprising the target antigen is sufficiently close to the CD300f binding molecule to allow internalising of the target antigen when the CD300f binding protein is internalised.
In one embodiment, the CD300f binding protein is coupled to the target antigen. In such embodiments, the CD300f binding protein and target antigen form an immunoconjugate. Thus, in one embodiment, the antigen presenting cell or precursor thereof is contacted with a CD300f binding protein in the presence of the target antigen by contacting the antigen presenting cell or precursor thereof with an immunoconjugate comprising the CD300f binding protein and the target antigen. In one embodiment, the immunoconjugate is a fusion protein comprising the CD300f binding protein and the target antigen.
Accordingly, in one embodiment, there is provided an immunoconjugate comprising a CD300f binding protein coupled to a target antigen. In one embodiment, the CD300f binding protein comprises:
(a) a heavy chain variable region which comprises:
(b) a light chain variable region which comprises:
In one embodiment, there is provided an immunoconjugate comprising a CD300f binding protein comprising a heavy chain variable region which comprises an amino acid sequence that is at least 70%, typically 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 1, and a light chain variable region which comprises an amino acid sequence that is at least 70%, typically 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 5, wherein the CD300f binding protein is coupled to a target antigen.
In one embodiment, there is provided an immunoconjugate comprising a CD300f binding protein comprising a heavy chain variable region which comprises an amino acid sequence represented by SEQ ID NO: 15, and a light chain variable region which comprises an amino acid sequence that is represented by SEQ ID NO: 16, wherein the CD300f binding protein is coupled to a target antigen.
In one embodiment, there is provided an immunoconjugate comprising a CD300f binding protein comprising a heavy chain variable region which comprises an amino acid sequence that is at least 90%, typically at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 15 and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 2, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 3, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 4, and a light chain variable region which comprises an amino acid sequence that is at least 90%, typically at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 16 and comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 6, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8, wherein the CD300f binding protein is coupled to a target antigen.
The term “coupled” is used to describe the association of the CD300f binding protein with the target antigen. Any mode of attachment of the CD300f binding protein to the target antigen is suitable, provided the CD300f binding protein is able to bind to CD300f and be internalised with the attached target antigen as intended. The coupling methods according to the present invention include, for example, expression of the immunoconjugate as a fusion peptide comprising the CD300f binding protein or part thereof directly or indirectly fused to the target antigen, direct or indirect attachment of the target antigen to the CD300f binding protein, with or without prior modification of the target antigen and/or the CD300f binding protein, and/or attachment via linkers. Linkers can be categorized functionally into, for example, acid labile, photosensitive, enzyme cleavable linkers, non-cleavable linkers etc.
In one embodiment, the immunoconjugate is a fusion protein comprising the CD300f binding protein and the target antigen. The CD300f binding protein may be fused directly to the target antigen or may be fused indirectly to the target antigen. The CD300f binding protein is fused directly to the target antigen when there is no intervening amino acid sequence between the CD300f binding protein and the target antigen. The CD300f binding protein is fused indirectly to the target antigen when there is an intervening amino acid sequence between the CD300f binding protein and the target antigen. In embodiments where the CD300f binding protein comprises a separate heavy chain and a light chain, the target antigen may be coupled to the heavy chain or the light chain. Typically, the target antigen is coupled to the heavy chain. In such embodiments, the heavy chain variable region or full-length heavy chain can be expressed as a fusion protein comprising the target antigen, and the fusion combined with the CD300f light chain. In other embodiments, the immunoconjugate may be a fusion protein comprising a CD300f scFv and a target antigen.
In one embodiment, the CD300f binding protein is coupled to the target antigen through a linker.
Examples of methods for producing fusion proteins with antibodies are described in, for example, Tsuji, et al. (2011) J Immunol. 186:1218-1227.
Methods for producing fusion proteins are known in the art. Methods for coupling proteins and other molecules to binding proteins such as antibodies are also known in the art.
In some embodiments, the CD300f binding protein is coupled to a polypeptide comprising the target antigen. In such embodiments, the target antigen is a contiguous amino acid sequence that is part of a polypeptide, and the polypeptide comprising the target antigen is processed following internalisation to present the target antigen on the surface of the antigen present cell or precursor thereof. The polypeptide comprising the target antigen may be any length. Typically, the target antigen comprises an amino acid sequence of a length that can be presented on the surface of the antigen presenting cell in the MHC I complex. Typically, the target antigen comprises an amino acid sequence of 8, 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids.
In some embodiments, the CD300f binding protein is coupled to a full-length protein comprising the target antigen. In some embodiments, the CD300f binding protein is coupled to a polypeptide consisting essentially of the target antigen. In such embodiments, the polypeptide comprises the target antigen and may be flanked on either side by 1, 2, 3, 4, or 5 amino acids that are not amino acid sequence that is presented on the surface of the antigen presenting cell or precursor thereof.
In some embodiments the CD300f binding protein is coupled to a polypeptide consisting of the target antigen.
One aspect provides a fusion protein comprising a CD300f binding protein and a target antigen.
The target antigen may be any antigen to which a T cell response is required, and typically comprises an amino acid sequence which can be presented by an antigen presenting cell to a T cell in a manner that will promote an antigen specific T cell response. The target antigen comprises an amino acid sequence that is capable of promoting a T cell response. Typically, the target antigen comprises an amino acid sequence that can be loaded into the MHC class I molecules of the antigen presenting cell. In some embodiments, the target antigen is a full-length protein. In some embodiments, the target antigen is a fragment of a full-length protein comprising an amino acid sequence that can be presented by an antigen presenting cell to a T cell in a manner that will promote an antigen specific T cell response.
In one embodiment, the target antigen is a cancer antigen. A cancer antigen as used herein is a compound, such as a peptide or protein, associated with a tumor or cancer cell surface and which comprises an amino acid sequence which is capable of promoting an immune response when presented on the surface of an antigen presenting cell. Cancer antigens include but are not limited to antigens that are recombinantly expressed, an immunogenic portion of, or a whole tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Examples of cancer antigens are described in Cheever et al. (2009), The Prioritization of Cancer Antigens: A National Cancer Institute Pilot Project for the Acceleration of Translational Research, Clin. Cancer Res. 15(17):5323-5337, and include, for example, WT-1 or an immunogenic fragment thereof, MUC-1 or an immunogenic fragment thereof, LMP2 or an immunogenic fragment thereof, HPV E6 E7 or an immunogenic fragment thereof, EGFRvIII or an immunogenic fragment thereof, HER-2 or an immunogenic fragment thereof, Idiotype or an immunogenic fragment thereof, MAGE A3 or an immunogenic fragment thereof, p53 non-mutant or an immunogenic fragment thereof, NY-ESO-1 or an immunogenic fragment thereof, PSMA, GD2 or an immunogenic fragment thereof, CEA or an immunogenic fragment thereof, MelanA/MART1 or an immunogenic fragment thereof, Ras mutant or an immunogenic fragment thereof, gp100 or an immunogenic fragment thereof, p53 mutant or an immunogenic fragment thereof, Proteinase3 (PR1) or an immunogenic fragment thereof, Bcr-abl or an immunogenic fragment thereof, Tyrosinase or an immunogenic fragment thereof, survivin or an immunogenic fragment thereof, PSA or an immunogenic fragment thereof, hTERT or an immunogenic fragment thereof, Sarcoma translocation breakpoints or an immunogenic fragment thereof, EphA2 or an immunogenic fragment thereof, PAP or an immunogenic fragment thereof, ML-IAP or an immunogenic fragment thereof, AFP or an immunogenic fragment thereof, EpCAM or an immunogenic fragment thereof, ERG or an immunogenic fragment thereof, NA17 or an immunogenic fragment thereof, PAX3 or an immunogenic fragment thereof, ALK or an immunogenic fragment thereof, Androgen receptor or an immunogenic fragment thereof, Cyclin B1 or an immunogenic fragment thereof, Polysialic Acid or an immunogenic fragment thereof, MYCH or an immunogenic fragment thereof, TRP-2 or an immunogenic fragment thereof, RhoC or an immunogenic fragment thereof, GD3 or an immunogenic fragment thereof, Fucosyl GM1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, PSCA (prostate stem cell antigen) or an immunogenic fragment thereof, MAGE Al or an immunogenic fragment thereof, sLe(a) or an immunogenic fragment thereof, CYP1B1 or an immunogenic fragment thereof, PLAC1 or an immunogenic fragment thereof, GM3 ganglioside or an immunogenic fragment thereof, BORIS (brother of regulator of imprinted sites) or an immunogenic fragment thereof, Tn or an immunogenic fragment thereof, GloboH or an immunogenic fragment thereof, ETV6-AML or an immunogenic fragment thereof, NY-BR-1 or an immunogenic fragment thereof, RGS5 or an immunogenic fragment thereof, SART3 or an immunogenic fragment thereof, STn or an immunogenic fragment thereof, Carbonic anhydrase or an immunogenic fragment thereof, PAX5 or an immunogenic fragment thereof, LCK or an immunogenic fragment thereof, HMWMAA or an immunogenic fragment thereof, AKAP-4 or an immunogenic fragment thereof, SSX-2 or an immunogenic fragment thereof, sperm fibrous sheath proteins or an immunogenic fragments thereof, XAGW 1 or an immunogenic fragment thereof, B7H3 or an immunogenic fragment thereof, Legumain or an immunogenic fragment thereof, Tie 2 or an immunogenic fragment thereof, Page 4 or an immunogenic fragment thereof, VEGFR2 or an immunogenic fragment thereof, MAD-CT-1 or an immunogenic fragment thereof, FAP or an immunogenic fragment thereof, PDGFR-beta or an immunogenic fragment thereof, or Fos-related antigen or an immunogenic fragment thereof. An immunogenic fragment is a fragment of a protein which can elicit a T cell response. Typically, the immunogenic fragment is at least 8 amino acids in length, more typically at least 9, 10, or at least 11 amino acids in length. Examples of immunogenic fragments are described in, for example, the Cancer Antigenic Peptide Database (https://caped.icp.ucl.ac.be/Peptide/list); Wei et al. 2019, Cancer-Testis Antigen Peptide Vaccine for Cancer Immunotherapy: Progress and Prospects, Transl. Oncol. 12(5):733-738.
Cancer antigens for promoting or increasing a T cell response towards a cancer cell are known in the art and described in, for example, “A listing of human tumor antigens recognized by T cells,” Renkvist N, Castelli C, Robbins P F, Parmiani G. Cancer Immunology Immunotherapy 50: (1) 3-15 Mar. 2001.
In one embodiment, the target antigen is a microbial antigen. A microbial antigen as used herein is an antigen of a microorganism and includes but is not limited to virus, bacteria, parasites, and fungi. Examples of infectious viruses that have been found in humans and from which a microbial antigen may be obtained include but are not limited to: Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus); Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis, hepatitis C; Norwalk and related viruses, and astroviruses).
Antigen presenting cells (APC), such as dendritic cells (DC), can be isolated from any tissue of a subject in which such cells are found. APC (e.g., DC) can be isolated from, for example, bone marrow, blood, peripheral blood mononuclear cells (PBMC), or spleen of a subject.
Antigen presenting cells or precursors thereof may be isolated using any methods know in the art. Any of a number of methods are known in the art for isolation of antigen presenting cells (such as dendritic cells), including repetitive density gradient separation, fluorescence activated cell sorting techniques, positive selection, negative selection, or a combination thereof. Methods for antigen cell isolation are described in, for example, Fromm et al., (2016) CMRF-56+ blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses, OncoImmunology, 5:6, e1168555, DOI: 10.1080/2162402X.2016.1168555; Prue et al., Peptides for Immunotherapy of Metastatic Hormone Refractory Prostate Cancer. J Immunother 2015, 38(2):71-76; Sallusto, F. and A. Lanzavecchia, J Exp Med, 1994. 179(4): p. 1109-18.
For example, antigen presenting cells may be isolated by positively selecting CD14+ cells from PBMC obtained from healthy donors. CD14+ cells can be obtained by incubating the PBMC with, for example, CD14 microbeads (Miltenyi Biotec, 130-050-201) and separating the microbeads bound to CD14+ cells. CD14+ cells can be differentiated into myeloid dendritic cells (MoDC) by incubating CD14+ cells in, for example, complete AB media supplemented with GM-CSF and IL-4 for 5 days. MoDC can be loaded with target antigen by incubating with CD300 antibody and target antigen.
As CD300 is restricted in its expression, isolation of dendritic cells is not necessary. In this regard, the CD300f binding protein can be used to target dendritic cells in a mixed population of cells. Accordingly, in some embodiments, dendritic cells may be loaded ex vivo by incubating any tissue, typically PBMC or blood, with CD300f binding protein and the target antigen. In some embodiments, the CD300f binding protein and target antigen, typically in the form of a conjugate, may be administered to the subject without isolation of any tissue or cells from the subject.
The CD300f binding proteins, target antigens, CD300f binding immunoconjugates and dendritic cell vaccines described herein may be formulated as compositions, including pharmaceutical compositions.
Accordingly, a further aspect provides a composition, typically a pharmaceutical composition, comprising:
(a) a CD300f binding protein and a target antigen as described herein; or
(b) an immunoconjugate comprising a CD300f binding protein coupled to a target antigen as described herein; or
(c) an antigen-loaded antigen presenting cell or precursor thereof (e.g., a DC vaccine) as described herein,
and typically, a pharmaceutically acceptable carrier.
A “pharmaceutically acceptable carrier” means that it is compatible with the other ingredients of the composition and is not deleterious to a subject. The compositions may contain other therapeutic agents as described below, and may be formulated, for example, by employing conventional liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavours, etc.) according to techniques such as those well known in the art of pharmaceutical formulation (See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).
The pharmaceutical compositions are typically in the form of a sterile injectable aqueous suspension. This suspension may be formulated according to the known art and contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may include suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable formulations.
The inventors envisage that the methods described herein can be used to prepare a dendritic cell vaccine which can be used to raise an immune response to a target antigen or antigens.
Accordingly, in one embodiment, there is provided a dendritic cell vaccine for promoting a T cell response to a target antigen, the vaccine comprising an antigen-loaded dendritic cell or precursor thereof, wherein the antigen-loaded dendritic cell or precursor thereof is produced by contacting a dendritic cell or precursor thereof with a CD300f binding protein in the presence of a target antigen or antigens.
In one embodiment, the dendritic cell or precursor thereof is contacted with a CD300f binding protein in the presence of a target antigen by contacting the dendritic cell or precursor thereof with a CD300f binding protein conjugate comprising a CD300f binding protein coupled to a target antigen.
Also provided is a method of promoting a T cell response to a target antigen in a subject, the method comprising administering to the subject an effective amount of the dendritic cell vaccine described herein.
Another aspect provides a method of treating a disease or condition requiring a T cell response to a target antigen, the method comprising administering an effective amount of the dendritic cell vaccine described herein.
It is also envisaged that administration of the immunoconjugate described herein can be used to promote an antigen specific immune response to target antigen. In this regard, the target antigen can be delivered or loaded into the DC in vivo. This can be achieved by administering an immunoconjugate in which the CD300f binding protein is coupled to the target antigen. The feasibility of this approach has been demonstrated in Phase 1 clinical trials targeting DEC-205. In this regard, administration of ant-DEC-205 antibodies coupled to a cancer antigen have been shown to produce an anti-tumour immune response. However, DEC-205 has a broad expression profile, and targeting DEC-205 causes tolerance unless co-administered with adjuvant.
One aspect provides a method of promoting or increasing a T cell response to a target antigen in a subject, the method comprising administering an effective amount of an immunoconjugate comprising a CD300f binding protein and the target antigen.
Another aspect provides a method of treating a disease or condition requiring a T cell response to a target antigen in a subject, the method comprising administering an effective amount of a CD300f binding protein conjugate comprising a CD300f binding protein and the target antigen.
As used herein, a disease or condition requiring a T cell response to a target antigen is a disease or condition for which a T cell response to the target antigen may be beneficial.
The methods described herein can be used to treat any disease or condition for which a T cell response to a target antigen may be beneficial.
In one embodiment, the disease or condition is cancer. In such embodiments, the target antigen will be a cancer antigen. Examples of cancer antigens are described in Cheever et al. (2009), The Prioritization of Cancer Antigens: A National Cancer Institute Pilot Project for the Acceleration of Translational Research, Clin. Cancer Res. 15(17):5323-5337, and include, for example, WT-1, MUC-1, LMP2, HPV E6 E7, EGFRvIII, HER-2, Idiotype, MAGE A3, p53 non-mutant, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, Proteinase3(PR1), Bcr-abl, Tyrosinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, Androgen receptor, Cyclin B1, Polysialic Acid, MYCH, TRP-2, RhoC, GD3, Fucosyl GM1, mesothelin, PSCA (prostate stem cell antigen), MAGE Al, sLe(a), CYP1B1, PLAC1, GM3 ganglioside, BORIS (brother of regulator of imprinted sites), Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase, PAX5, LCK, HMWMAA, AKAP-4, SSX-2, sperm fibrous sheath proteins, XAGW 1, B7H3, Legumain, Tie 2, Page 4, VEGFR2, MAD-CT-1, FAP, PDGFR-beta, Fos-related antigen, or an immunogenic fragment thereof. An immunogenic fragment is a fragment of a protein which can elicit a T cell response. Typically, the immunogenic fragment is at least 8 amino acids in length, more typically at least 9, 10, or at least 11 amino acids in length. Examples of immunogenic fragments are described in, for example, the Cancer Antigenic Peptide Database (https://caped.icp.ucl.ac.be/Peptide/list); Wei et al. 2019, Cancer-Testis Antigen Peptide Vaccine for Cancer Immunotherapy: Progress and Prospects, Transl. Oncol. 12(5):733-738.
The pharmaceutical compositions described herein may be administered by any suitable means, typically, parenterally, such as by subcutaneous, intravenous, intramuscular, intra(trans)dermal, or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous solutions or suspensions); in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents.
The pharmaceutical compositions for the administration may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the compound into association with a liquid carrier. In the pharmaceutical composition the active compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
Generally, the term “treating” means affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect and include: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease. In one embodiment, treatment achieves the result of reducing the number of cancer cells in the recipient subject.
The term “subject” refers to any animal having a disease which requires treatment by the present method. In addition to primates, such as humans, a variety of other mammals can be treated using the methods of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated. Dogs in particular are known to experience multiple myeloma.
The term “effective amount” refers to the amount of the antibody, antigen binding fragment or immunoconjugate that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
In the treatment or prevention of cancer, an appropriate dosage level of CD300f binding protein conjugate will generally be about 0.01 to 50 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 25 mg/kg per day; more preferably about 0.5 to about 10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 15 mg/kg per day.
An appropriate dosage level of antigen-loaded dendritic cells per kg patient body weight per day which can be administered in single or multiple doses.
It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
Also disclosed herein is a kit comprising the CD300f binding protein and target antigen described herein, CD300f binding protein conjugate described herein, or the dendritic cell vaccine described herein. In various embodiments, the kit comprises:
(a) a CD300f binding protein and a target antigen as described herein; or
(b) a CD300f binding protein conjugate comprising a CD300f binding protein coupled to a target antigen as described herein; or
(c) an antigen-loaded antigen presenting cell (e.g., a DC vaccine) as described herein,
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an antibody” includes a plurality of such antibodies. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, preferred materials and methods are described herein.
All publications mentioned herein are cited for the purpose of describing and disclosing the protocols and reagents which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
All publications mentioned in this specification are herein incorporated by reference. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
In order to exemplify the nature of the present invention such that it may be more clearly understood, the following non-limiting examples are provided.
The inventors have demonstrated that blood DC are present in localised and advanced disease and assessed the functional contribution of CD300f.
By FACS analysis the inventors assessed three markers that are expressed on DC and other antigen presenting cells for their ability to internalize on cell lines compared to DEC 205. The three chosen markers and their antibodies are DCR-2 (a mouse anti-human CD300f monoclonal antibody), MMRI-20 (a mouse anti-human CD302 monoclonal antibody) and CMRF-56 (a mouse monoclonal antibody that selects for activated DC and monocytes). Internalisation of surface antibody at 37° C. was assessed by comparing total antibody over time (Total, closed symbol) measured using PE labelled MMRI20 and DCR-2 on HL60, a monocytic cell line, and FITC labelled anti-CMRF56 and MMRI-7 on KMH2. This was compared with remaining surface Ig detected with goat anti-mouse IgG AF647 (Invitrogen, A21237). The results are shown in
Using confocal imaging, the internalization of a polyclonal anti-CD300f antibody, gLMIR3, was compared in fixed cells (and therefore no internalization) and live HL-60 and CD14+ monocytes following 30 minutes incubation at 37° C. The results indicated that the CD300f polyclonal antibody gLMIR3 is internalized in live HL-60 and CD14+ cells, but not in fixed cells.
The inventors then compared the expression of CD300f, CD302 and DEC 205 (
In a target for immunotherapy, high expression in lymphoid tissues such as the spleen and low expression elsewhere is desirable. mRNA expression of CD300f, CD302 and DEC-205 was therefore assessed across normal tissue by RNA sequencing. The results are shown in
The inventors then assessed whether antibodies against DEC-205 (MMRI7), CD300f (DCR-2) and CD302 (MMRI-20) produce an antigen specific response. In this regard, CD14+ cells were positively selected from PBMC from 3 healthy donors by staining PBMC with CD14 microbead (Miltenyi Biotec, 130-050-201) for 15 min at 4° C. and magnetic separation using an autoMACS separator. CD14+ cells where differentiated into MoDC by incubating CD14+ cells at 3.33×106/mL in complete AB media supplemented with GM-CSF 800 U/mL and IL-4 1000 u/mL for 5 days. CD3 positive cells were isolated from PBMC using EasySep Human T cell Isolation Kit (Stemcell, 17951) and frozen on day 1. MoDC were loaded with CMV antigen on day 5 by staining with primary biotinylated antibody for 20 min on ice and then stained with HCMV-pp65 delivery reagent (Miltenyi Biotec 130-095-406) for 10 mins, washed and then incubated overnight with LPS 100 ng/ml. Autologous T cells were then thawed, rested for 2 hours and stained with CSFE before being added to the Mo-DC at a ratio of 1:10 for 5 days. At day 5 cells were harvested and ran on FACs. The results are shown in
CD300f looked like a good candidate for antigen loading of DC. It was previously shown that the binding of DCR-2 to CD300f enhances the binding of UPD2 (WO 2018/094460) suggesting it is an active antibody that leads to a structural change in CD300f, opening the epitope up for UPD2 to bind. Thus, the inventors assessed whether DCR2 lead to phenotypic changes in myeloid DC. Initially the inventors did this with crosslinking experiments, looking at the effect of crosslinking DCR-2 on myeloid DC and monocytes. 96 well flat bottom tissue culture plates (Fal 353072) were coated with 10 ug/ml of DCR-2 (anti-CD300f), UPH2 (anti-CD300e, Biolegend, 339702), control CMRF81 antibody and CMRF35 (anti CD300a/c) supernatant overnight at 4° C. then washed with PBS. Myeloid DC isolated using EasySep Human Myeloid DC Enrichment Kit (Stem Cell Technologies, 19061) and 1×105 myeloid DC were added in 200 μl of RPMI 10% AB complete media incubated at 37° C. and 5% CO2 for 18 hours. Supernatant was collected for cytokine analysis and cells harvested for flow cytometry analysis using CD80 Pe-Cy7 (L307.4, BD561135), CD83 FITC (Hb15a, IM2410U), CD86 BV650 (IT2.2, Biolegend) and HLADR APC-H7 (1243, Biolegend). The results are shown in
Crosslinking DC with DCR-2 leads to upregulation of activation markers. The inventors also assessed whether DCR-2 changed how myeloid DC responded to LPS and did not find any difference. If DCR-2 leads to increase in activation markers, the inventors asked the questions are these DC more stimulatory. Using an allogenic MLR the inventors show a trend towards these DC being more stimulatory. Myeloid DC from 3 healthy donors were crosslinked overnight as described above in relation to
The inventors also measured the concentration of cytokines IFN-γ, IL-10 and IL-17a in supernatants of T cells incubated with myeloid DC crosslinked with DCR-2 or CMRF-81. Supernatant was collected at day 6 and cytokine concentration analysed (n=3) using LEGENDplex Human Inflammation Panel Kit (Cat. No. 740409). The results are shown in
The inventors then asked the question is crosslinking required for DCR-2 to activate DC. The inventors looked at the effect of crosslinking with DCR2 and CMRFI-81 on expression of activation markers on myeloid DC and monocytes. HLA-DR, CD80, PD-L1, CD83, CD86 or TIM-3 on myeloid DC was assessed using flow cytometry following isolation of myeloid DC. Myeloid DC were isolated using EasySep Human Myeloid DC Enrichment Kit (Stem Cell Technologies, 19061) and 1×105 myeloid DC were added in 200 μl of RPMI 10% AB complete media incubated at 37° C. and CO2 for 18 hours with 10 pg/ml of DCR-2 or control CMRF-81 antibody. Supernatant was collected for cytokine analysis and cells harvested for flow cytometry analysis using CD80 Pe-Cy7 (L307.4, BD561135), CD83 FITC (Hb15a, IM2410U), CD86 BV650 (IT2.2, Biolegend) and HLA-DR APC-H7 (1243, Biolegend). The results are shown in
The inventors next tested whether crosslinking with DCR-2 affects migration. First, the inventors assessed expression of the chemokine receptor CCR7. Crosslinked myeloid DC were harvested and stained with CCR7 PE (R&D, FAB197P) for 15 min at 37° C. Migration of crosslinked DC or monocytes was assessed by determining number of DC or monocytes that migrated across transwells (Corning, 3421) towards CCL19 at a concentration of 0.1 ug/ml in 4 hours or CCL21 at 100 ng/ml in 2 hours, or towards CCL2 in 2 hours, all in RPMI (1% BSA, PSG) compared to no chemokine. DC were harvested from the bottom of the transwell stained with Lin2 FITC (CD3/14/19/20/56, BD 643397) and HLA-DR APC-H7 (I243, Biolegend) and resuspended in 200 ul FACS buffer with Count bright Absolute Count Beads 2500/ml. Number of cells migrated was calculated as number of (lineage-DR+ cells/counted beads)×5000 beads. Migration index=# cells migrated chemokine/# cells migrated no chemokine. The results of CCR7 expression in myeloid DC and migration of myeloid DC toward CCL21 and CCL19 following crosslinking with DCR-2 or CMRF-81 are shown in
The inventors also assessed the effect of crosslinking on expression of activation markers on monocytes. In this regard, 96 well flat bottom tissue culture plates (Fal 353072) were coated with 10 μg/ml of DCR 2 (anti-CD300f), control CMRF-81 antibody and PBS control at 4° C. then washed with PBS. CD14+ cells were positively selected from N=4 healthy donors using CD14+ microbeads (Miltenyi Biotec 130-050-201) and magnetic separation using AutoMacs. 1×105 CD14+ cells were added in 200 μl of RPMI 10% AB complete media incubated at 37° C. and 5% CO2 for 18 hours. Supernatant was collected for cytokine analysis and cells harvested for flow cytometry analysis using CD80 Pe-Cy7 (L307.4, BD561135), CD83 FITC (Hb15a, IM2410U), CD86 BV650 (IT2.2, Biolegend) and HLADR APC-H7 (1243, Biolegend). Results are shown in
Humanised DCR-2
The amino acid sequence of the mouse VH and mouse VL of DCR-2 was aligned through an IgBLAST to human Ig sequences. The amino acids in the framework region of the VH and VL sequences were changed from mouse to human residues (underlined in
In summary:
| Number | Date | Country | Kind |
|---|---|---|---|
| AU2019904614 | Dec 2019 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2020/051329 | 12/4/2020 | WO |
