Patent Application
20230233701
Approximately 10% of the human population suffers from an auto-immune condition, accompanied by mild to life-threatening symptoms. Current treatments for autoimmune diseases include general immunosuppression, which blunts responses across the entire spectrum of antigens. This exposes patients to an increased risk of infection and possibly even malignancies.
The present disclosure, in some aspects, provides compositions comprising one or more conjugates comprising a single domain antibody fragments (nanobodies/VHHs) conjugated to an antigen and/or an agent (e.g., an anti-inflammatory agent or a proinflammatory agent), wherein the VHH binds to a surface protein on an antigen presenting cell (APC). In some embodiments, the antigen and the agent (e.g., an anti-inflammatory agent or a proinflammatory agent) are conjugated to the same VHH. In some embodiments, the antigen and the agent (e.g., an anti-inflammatory agent or a proinflammatory agent) are conjugated to two VHHs.
The conjugates described herein engage antigen presenting cells (APCs), which under non-inflammatory conditions can lead to tolerance, whereas engagement of APCs under inflammatory conditions can elicit a strong immune response against foreign antigens. In was found surprisingly herein that, the composition of the present disclosure, when the antigen is a self-antigen and the agent is an anti-inflammatory agent, is significantly more effective in inducing immune tolerance and alleviate the symptoms of an autoimmune disease in a subject, compared to when a VHH-antigen is administered alone. Similarly, the composition of the present disclosure, when the antigen is from a pathogen and when the agent is a proinflammatory agent is significantly more effective in inducing immune response against the antigen and/or the pathogen, compared to when a VHH-antigen is administered alone.
Some aspects of the present disclosure provide compositions comprising:
(i) a conjugate comprising a single domain antibody (VHH) conjugated to an antigen and an anti-inflammatory agent, wherein the VHH binds to a surface protein on an antigen presenting cell (APC); or
(ii) a first conjugate comprising a VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen presenting cell (APC). In some embodiments, the surface protein on the APC is selected from the group consisting of MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, and F4/80. In some embodiments, the targeting moieties may be replaced with a natural or synthetic polypeptide, including but not limited to peptide fragments, single-chain fragment variable (scFv), diabody, Fab, or similar formats.
In some embodiments, the composition comprises a conjugate comprising a VHH to conjugated to an antigen and an anti-inflammatory agent, wherein the VHH binds to MHCII. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH both bind to MHCII. In some embodiments, the VHH comprises the amino acid sequences of SEQ ID NO: 1. In further embodiments, a VHH conjugated to an antigen or anti-inflammatory agent may have the format of a DNA or RNA molecule encoding the specified conjugate.
In some embodiments, the VHH binding to MHCII further comprises a sortase recognition sequence at the N-terminus or C-terminus. In some embodiments the sortase recognition sequence comprises the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the sortase recognition sequence comprises the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments an anti-inflammatory agent or an antigen is conjugated to the VHH via the sortase recognition sequence. In some embodiments, the anti-inflammatory agent further comprises a hydrolysable or non-hydrolysable linker. In further embodiments, conjugates are produced by means of genetic fusion, other ligation enzymes (e.g., butelase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).
In some embodiments, the composition comprises a conjugate comprising a single domain antibody (VHH) conjugated to an antigen and an anti-inflammatory agent, wherein the VHH binds to CD11c. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH both bind to CD11c. In some embodiments, the VHH comprises the amino acid sequences of SEQ ID NO: 2. In further embodiments, a VHH conjugated to an antigen or anti-inflammatory agent may have the format of a DNA or RNA molecule encoding the specified adduct.
In some embodiments, the VHH binding to CD11c further comprises a sortase recognition sequence at the N-terminus or C-terminus. In some embodiments the sortase recognition sequence comprises the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the sortase recognition sequence comprises the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments an anti-inflammatory agent or an antigen is conjugated to the VHH via the sortase recognition sequence. In some embodiments, the anti-inflammatory agent further comprises a hydrolysable or non-hydrolysable linker. In further embodiments, conjugates are produced by means of genetic fusion, other ligation enzymes (e.g., butelase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH bind to different surface proteins on the APC. In some embodiments, the first VHH binds to MHCII and the second VHH binds to CD11c. In some embodiments, the first VHH binds to DEC205 and the second VHH binds to MHCII.
In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent selected from the group consisting of: dexamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, and bethamethasone. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent selected from the group consisting of: aspirin, celecoxib, diclofenac, ibuprofen, ketoprofen, naproxen, oxaprozin, piroxicam, cyclosporin A, and calcitriol. In some embodiments, the anti-inflammatory agent is an anti-inflammatory cytokine selected from the group consisting of IL-10, IL-35, IL-4, IL-11, IL-13, and TGFβ.
In some embodiments, the antigen comprises a polypeptide, a polysaccharide, a carbohydrate, a lipid, a nucleic acid, or combination thereof. In some embodiments, the antigen is a self-antigen. In some embodiments, the self-antigen is selected from myelin oligodendrocyte glycoprotein, myelin proteolipid protein, citrullinated fibrinogen, insulin, chromogranin A, glutamic acid decarboxylase 65-kilodalton isoform (GAD65), desmoglein 1 (DSG1), desmoglein 3 (DSG3), acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), ribonucleoproteins. In some embodiments, the antigen comprises a protein used in a protein replacement therapy or a gene therapy. In some embodiments, the antigen is selected from Factor IX, Factor VIII, insulin, and AAV-derived proteins.
Other aspects of the present disclosure provide methods comprising administering to a subject in need thereof the compositions described herein. In some embodiments, the composition administered comprises (i) a conjugate comprising a single domain antibody (VHH) conjugated to an antigen and an anti-inflammatory agent, wherein the VHH binds to a surface protein on an antigen presenting cell (APC); or (ii) a first conjugate comprising a VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen presenting cell (APC). In some embodiments, the method is for inducing immune tolerance to an antigen. In some embodiments, the method is for treating an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of autoimmune encephalomyelitis, multiple sclerosis, type I diabetes, Pemphigus vulgaris, myasthenia gravis, lupus, celiac diseases, and inflammatory bowel disease (IBD). In some embodiments, the administration is intravenous. In some embodiments, the subject is human.
Other aspects of the present disclosure provide compositions comprising:
(i) a conjugate comprising a single domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, wherein the VHH binds to a surface protein on an antigen presenting cell (APC); or
(ii) a first conjugate comprising a VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to a pro-inflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen presenting cell (APC). In some embodiments, the surface protein on the APC is selected from the group consisting of MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, and F4/80. In some embodiments, the targeting moieties may be replaced with a natural or synthetic polypeptide, including but not limited to peptide fragments, single-chain fragment variable (scFv), diabody, Fab, or similar formats.
In some embodiments, the composition comprises a conjugate comprising a single domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, wherein the VHH binds to MHCII. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to a pro-inflammatory agent, wherein the first VHH and the second VHH both bind to MHCII. In some embodiments, the VHH comprises the amino acid sequences of SEQ ID NO: 1. In further embodiments, a VHH conjugated to an antigen or pro-inflammatory agent may have the format of a DNA or RNA molecule encoding the specified conjugate.
In some embodiments, the VHH binding to MHCII further comprises a sortase recognition sequence at the N-terminus or C-terminus. In some embodiments the sortase recognition sequence comprises the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the sortase recognition sequence comprises the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments a pro-inflammatory agent or an antigen is conjugated to the VHH via the sortase recognition sequence. In some embodiments, the pro-inflammatory agent further comprises a hydrolysable or non-hydrolysable linker. In further embodiments, conjugates are produced by means of genetic fusion, other ligation enzymes (e.g., butelase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).
In some embodiments, the composition comprises a conjugate comprising a single domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, wherein the VHH binds to CD11c. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to a pro-inflammatory agent, wherein the first VHH and the second VHH both bind to CD11c. In some embodiments, the VHH comprises the amino acid sequences of SEQ ID NO: 2. In further embodiments, a VHH conjugated to an antigen or pro-inflammatory agent may have the format of a DNA or RNA molecule encoding the specified conjugate.
In some embodiments, the VHH binding to CD11c further comprises a sortase recognition sequence at the N-terminus or C-terminus. In some embodiments the sortase recognition sequence comprises the amino acid sequence LPETG (SEQ ID NO: 29). In some embodiments, the sortase recognition sequence comprises the amino acid sequence LPETGG (SEQ ID NO: 43). In some embodiments a pro-inflammatory agent or an antigen is conjugated to the VHH via the sortase recognition sequence. In some embodiments, the pro-inflammatory agent further comprises a hydrolysable or non-hydrolysable linker. In further embodiments, conjugates are produced by means of genetic fusion, other ligation enzymes (e.g., butelase, OaAEP1, subtiligase, etc.), or chemical methods (e.g., N-terminal modification using 2-pyridinecarbaldehyde (2-PCA), etc.).
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to a pro-inflammatory agent, wherein the first VHH and the second VHH bind to different surface proteins on the APC. In some embodiments, the first VHH binds to MHCII and the second VHH binds to CD11c. In some embodiments, the first VHH binds to DEC205 and the second VHH binds to MHCII.
In some embodiments, the pro-inflammatory agent is selected from the group consisting of: TLR9 agonist, LPS, HMGB1 proteins, IL2, IL12, and CD40L.
In some embodiments, the antigen comprises a polypeptide, a polysaccharide, a carbohydrate, a lipid, a nucleic acid, or combination thereof. In some embodiments, the antigen is from a microbial pathogen. In some embodiments, the microbial pathogen is a mycobacterium, bacterium, fungus, virus, parasite, or prion. In some embodiments, the antigen comprises a SARS-CoV-2 spike protein.
In some embodiments, the antigen is a tumor antigen.
In some embodiments, the composition is a vaccine composition.
Other aspects of the present disclosure provide methods comprising administering to a subject in need thereof the composition described herein. In some embodiments, the composition comprises (i) a conjugate comprising a single domain antibody (VHH) conjugated to an antigen and a pro-inflammatory agent, wherein the VHH binds to a surface protein on an antigen presenting cell (APC); or (ii) a first conjugate comprising a VHH conjugated to an antigen and a second conjugate comprising a second VHH conjugated to a pro-inflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen presenting cell (APC). In some embodiments, the method is for inducing immune response to an antigen. In some embodiments, the antigen is from a microbial pathogen and the method is for treating infection caused by a pathogen. In some embodiments, the method is therapeutic or prophylactic. In some embodiments, the antigen is a tumor antigen and the method is for treating cancer.
In some embodiments, the administration is intravenous. In some embodiments, the subject is human.
The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
The present disclosure, in some aspects, provides compositions comprising one or more conjugates (also referred to as “adducts” in the examples and figures) comprising a single domain antibody fragment (nanobodies/VHHs) conjugated to an antigen (e.g., an antigen to which immune tolerance is needed, such as a self-antigen or an exogenous enzyme used for therapy) and/or an anti-inflammatory agent, wherein the VHH binds to a surface protein on an antigen presenting cell (APC), methods of using such compositions for inducing immune tolerance to the antigen, and methods of using such compositions for treating autoimmune diseases. In some embodiments, the composition comprises a conjugate comprising a VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and an anti-inflammatory agent, wherein the VHH binds to a surface protein on an antigen presenting cell (APC). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen presenting cell (APC).
Other aspects of the present disclosure provide compositions comprising one or more conjugates comprising a VHH conjugated to an antigen (e.g., an antigen to which immune response is needed, such as an antigen from a pathogen or a tumor antigen) and/or a proinflammatory agent, wherein the VHH binds to a surface protein on an APC, methods of using such compositions to induce immune response to the antigen, and methods of using such compositions to treat infection (e.g., by a pathogen) and cancer. In some embodiments, the composition comprises a conjugate comprising a VHH conjugated to an antigen (e.g., an antigen to which immune response is needed, such as an antigen from a pathogen or a tumor antigen) and an proinflammatory agent, wherein the VHH binds to a surface protein on an antigen presenting cell (APC). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune response is needed, such as an antigen from a pathogen or a tumor antigen) and a second conjugate comprising a second VHH conjugated to an proinflammatory agent, wherein the first VHH and the second VHH bind to one or more surface proteins on an antigen presenting cell (APC).
The conjugates of the present disclosure comprise a single domain antibody (also referred to as nanobody or VHH). A “single domain antibody fragment,” a “nanobody,” or a “VHH,” as used herein, refers to an antibody fragment consisting of a single monomeric variable antibody domain. It is known that Camelids produce heavy chain-only antibodies (e.g., as described in Hamers-Casterman et al., 1992, incorporated herein by reference). The single-domain variable fragments of these heavy chain-only antibodies are termed VHHs or nanobodies. VHHs retain the immunoglobulin fold shared by antibodies, using three hypervariable loops, CDR1, CDR2 and CDR3, to bind to their targets. Many VHHs bind to their targets with affinities similar to conventional full-size antibodies, but possess other properties superior to them. Therefore, VHHs are attractive tools for use in biological research and therapeutics. VHHs are usually between 10 to 15 kDa in size, and can be recombinantly expressed in high yields, both in the cytosol and in the periplasm in E. coli. VHHs can bind to their targets in mammalian cytosol. A VHH fragment (e.g., NANOBODY®) is a recombinant, antigen-specific, single-domain, variable fragment derived from camelid heavy chain antibodies. Although they are small, VHH fragments retain the full antigen-binding capacity of the full antibody. VHHs are small in size, highly soluble and stable, and have greater set of accessible epitopes, compared to traditional antibodies. They are also easy to use as the extracellular target-binding moiety of the chimeric receptor described herein, because no reformatting is required.
In some embodiments, the VHH used in the conjugates described herein binds to a surface protein on an antigen presenting cell (APC). An “antigen presenting cell (APC)” refers to a cell that displays antigen complexed with major histocompatibility complexes (MHCs) on their surfaces, a process known as antigen presentation. T cells may recognize these complexes using their T cell receptors (TCRs). Almost all cell types can present antigens in some way. They are found in a variety of tissue types. As used herein, the term “antigen presenting cells” refers to professional antigen-presenting cells including, without limitation, macrophages, B cells, and dendritic cells. Antigen-presenting cells play important roles in effective adaptive immune response, as the functioning of both cytotoxic and helper T cells is dependent on APCs. Antigen presentation allows for specificity of adaptive immunity and can contribute to immune responses against both intracellular and extracellular pathogens. It is also involved in defense against tumors. Some cancer therapies involve the creation of artificial APCs to prime the adaptive immune system to target malignant cells. Additionally, APCs also play a role in immune tolerance by presenting self-antigens to T cells, e.g., as described in Best et al., Front Immunol. 2015; 6: 360, incorporated herein by reference.
In addition to the MHC family of proteins, other specialized signaling molecules on the surfaces of both APCs and T cells are also required for antigen presentation. In some embodiments, the conjugates described herein comprise a VHH that binds to a protein on the surface of an APC, thus engaging the APC. Non-limiting examples of surface proteins on APCs that can be targeted by the VHH in the conjugates described herein include, without limitation: Major histocompatibility complex II (MHCII), Integrin, alpha X (CD11c), Lymphocyte antigen 75 (DEC205, also referred to as CD205), Dendritic Cell-Specific ICAM-3-Grabbing Non-Integrin 1 (DC-SIGN), C-Type Lectin Domain Containing 9A (CLEC9a), Integrin, alpha E (CD103), C-X3-C Motif Chemokine Receptor 1 (CX3CR1), Cluster of Differentiation 1a (CD1a), and EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80, also referred to as EMR1).
In some embodiments, the VHH in the conjugates described herein binds to one surface protein on an APC (e.g., without limitation, MHCII, CD11c, DEC205, DC-SIGN, CLEC9a, CD103, CX3CR1, CD1a, or F4/80). In some embodiments, the VHH in the conjugates described herein is bi-specific or multispecific. In some embodiments, the VHH in the conjugates described herein binds one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) surface proteins in an APC. Any known VHHs that bind to surface proteins on APCs can be used in accordance with the present disclosure.
In some embodiments, the VHH binds to MHCII. VHHs that bind to MHCII have been described, e.g., in U.S. Pat. No. 9,751,945, incorporated herein by reference. The amino acid sequence of an example of a VHH that binds to MHCII is provided in Table 1.
In some embodiments, the VHH in the conjugates described herein comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VHH in the conjugates described herein comprises an amino acid sequence that is 80%, 85%, 905, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VHH in the conjugates described herein comprises the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the VHH binds to CD11c. VHHs that bind to CD11c have been described, e.g., in Bannas et al., Front Immunol. 2017; 8: 1603, incorporated herein by reference. The amino acid sequence of an example of a VHH that binds to CD11c is provided in Table 1.
In some embodiments, the VHH in the conjugates described herein comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VHH in the conjugates described herein comprises an amino acid sequence that is 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the VHH in the conjugates described herein comprises the amino acid sequence of SEQ ID NO: 2.
In some embodiments, any one of the VHHs in the conjugates described herein comprises additional sequences such as a sortase recognition sequence (e.g., as described in U.S. Pat. No. 9,751,945, incorporated herein by reference). Enzymes identified as “sortases” from Gram-positive bacteria cleave and translocate proteins to proteoglycan moieties in intact cell walls. Among the sortases that have been isolated from Staphylococcus aureus, are sortase A (SrtA) and sortase B (SrtB).
In some embodiments, a recognition sequence of a sortase further comprises one or more additional amino acids, e.g., at the N or C terminus. For example, one or more amino acids (e.g., up to 5 amino acids) having the identity of amino acids found immediately N-terminal to, or C-terminal to, a 5 amino acid recognition sequence in a naturally occurring sortase substrate may be incorporated. Such additional amino acids may provide context that improves the recognition of the recognition motif.
Sortases have been classified into 4 classes, designated A, B, C, and D, based on sequence alignment and phylogenetic analysis of 61 sortases from Gram positive bacterial genomes (Dramsi S, Trieu-Cuot P, Bierne H, Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol. 156(3):289-97, 2005. These classes correspond to the following subfamilies, into which sortases have also been classified by Comfort and Clubb (Comfort D, Clubb R T. A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect Immun., 72(5):2710-22, 2004): Class A (Subfamily 1), Class B (Subfamily 2), Class C (Subfamily 3), Class D (Subfamilies 4 and 5). The aforementioned references disclose numerous sortases and recognition motifs. See also Pallen, M. J.; Lam, A. C.; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3), 97-101. Those skilled in the art will readily be able to assign a sortase to the correct class based on its sequence and/or other characteristics such as those described in Drami, et al., supra. The term “sortase A” is used herein to refer to a class A sortase, usually named SrtA in any particular bacterial species, e.g., SrtA from S. aureus. Likewise, “sortase B” is used herein to refer to a class B sortase, usually named SrtB in any particular bacterial species, e.g., SrtB from S. aureus.
In some embodiments, the sortase used for producing the conjugates described herein is a sortase A (SrtA). SrtA recognizes the motif LPXTG (SEQ ID NO: 25), with common recognition motifs being, e.g., LPKTG (SEQ ID NO: 26), LPATG (SEQ ID NO: 27), LPNTG (SEQ ID NO: 28). In some embodiments LPETG (SEQ ID NO: 29) is used. However, motifs falling outside this consensus may also be recognized. For example, in some embodiments the motif comprises an ‘A’ rather than a ‘T’ at position 4, e.g., LPXAG (SEQ ID NO: 30), e.g., LPNAG (SEQ ID NO: 31). In some embodiments the motif comprises an ‘A’ rather than a ‘G’ at position 5, e.g., LPXTA (SEQ ID NO: 32), e.g., LPNTA (SEQ ID NO: 33). In some embodiments the motif comprises a ‘G’ rather than ‘P’ at position 2, e.g., LGXTG (SEQ ID NO: 34), e.g., LGATG (SEQ ID NO: 35). In some embodiments the motif comprises an ‘I’ rather than ‘L’ at position 1, e.g., IPXTG (SEQ ID NO: 36), e.g., IPNTG (SEQ ID NO: 37) or IPETG (SEQ ID NO: 38).
In some embodiments, the sortase used for producing the conjugates described herein is sortase B (SrtB), e.g., a sortase B of S. aureus, B. anthracis, or L. monocytogenes. Motifs recognized by sortases of the B class (SrtB) often fall within the consensus sequences NPXTX (SEQ ID NO: 39), e.g., NP[Q/K]-[T/s]-[N/G/s], such as NPQTN (SEQ ID NO: 40) or NPKTG (SEQ ID NO: 41). For example, sortase B of S. aureus or B. anthracis cleaves the NPQTN (SEQ ID NO: 40) or NPKTG (SEQ ID NO: 41) motif of IsdC in the respective bacteria (see, e.g., Marraffimi, L. and Schneewind, O., Journal of Bacteriology, 189(17), p. 6425-6436, 2007). Other recognition motifs found in putative substrates of class B sortases are NSKTA (SEQ ID NO: 44), NPQTG (SEQ ID NO: 45), NAKTN (SEQ ID NO: 46), and NPQSS (SEQ ID NO: 47). For example, SrtB from L. monocytogenes recognizes certain motifs lacking P at position 2 and/or lacking Q or K at position 3, such as NAKTN (SEQ ID NO: 46) and NPQSS (SEQ ID NO: 47) (Mariscotti J F, Garcia-Del Portillo F, Pucciarelli M G. The Listeria monocytogenes sortase-B recognizes varied amino acids at position two of the sorting motif. J Biol Chem. 2009 Jan. 7. [Epub ahead of print])
In some embodiments, the sortase used for producing the conjugates described herein is class C sortase. Class C sortases may utilize LPXTG (SEQ ID NO: 25) as a recognition motif.
In some embodiments, the sortase is a class D sortase. Sortases in this class are predicted to recognize motifs with a consensus sequence NA-[E/A/S/H]-TG (Comfort D, supra). Class D sortases have been found, e.g., in Streptomyces spp., Corynebacterium spp., Tropheryma whipplei, Thermobifida fusca, and Bifidobacterium longhum. LPXTA (SEQ ID NO: 32) or LAXTG (SEQ ID NO: 48) may serve as a recognition sequence for class D sortases, e.g., of subfamilies 4 and 5, respectively subfamily-4 and subfamily-5 enzymes process the motifs LPXTA (SEQ ID NO: 32) and LAXTG (SEQ ID NO: 48), respectively). For example, B. anthracis Sortase C, which is a class D sortase, has been shown to specifically cleave the LPNTA (SEQ ID NO: 33) motif in B. anthracis BasI and BasH (Marrafini, supra).
In some embodiments, a variant of a naturally occurring sortase may be used. Such variants may be produced through processes such as directed evolution, site-specific modification, etc. For example, variants of S. aureus sortase A with up to a 140-fold increase in LPETG (SEQ ID NO: 29)-coupling activity compared with the starting wild-type enzyme have been identified (Chen, I., et al., PNAS 108(28): 11399-11404, 2011). In some embodiments a sortase variant comprises any one or more of the following substitutions relative to a wild type S. aureus SrtA: P94S or P94R, D160N, D165A, K190E, and K196T mutations. An exemplary wild type S. aureus SrtA sequence (Gene ID: 1125243, NCBI RefSeq Acc. No. NP_375640) is shown below:
Sortase tagging can be used to install reactive chemistry moieties (e.g., click chemistry handles) onto a VHH, e.g., as described in U.S. Pat. No. 9,751,945, incorporated herein by reference. The click chemistry handle can be used for conjugating the VHH to other agents (e.g., antigens, anti-inflammatory agents, and/or proinflammatory agents). In some embodiments, the sortase recognition sequence is at the N-terminus of the VHH. In some embodiments, the sortase recognition sequence is at the C-terminus of the VHH.
In some embodiments, a reactive chemistry moiety is installed onto the VHH via a sortase mediated tagging (referred to as “sortagging”). Click chemistry handles are chemical moieties that provide a reactive group that can partake in a click chemistry reaction. Click chemistry reactions and suitable chemical groups for click chemistry reactions are well known to those of skill in the art, and include, but are not limited to terminal alkynes, azides, strained alkynes, dienes, dieneophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols, and alkenes. For example, in some embodiments, an azide and an alkyne are used in a click chemistry reaction. Additional click chemistry handles suitable for use in the methods of protein conjugation described herein are well known to those of skill in the art, and such click chemistry handles include, but are not limited to, the click chemistry reaction partners, groups, and handles described in [1] H. C. Kolb, M. G. Finn, K. B. 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All references cited above are incorporated herein by reference for disclosure of click chemistry handles suitable for installation on proteins according to inventive concepts and methods provided herein.
Other tags can also be added to the VHH via sortagging. Examples of suitable tags include, without limitation, amino acids, peptides, proteins, nucleic acids, polynucleotides, sugars, carbohydrates, polymers, lipids, fatty acids, and small molecules. Other suitable tags will be apparent to those of skill in the art and the invention is not limited in this aspect. In some embodiments, a tag comprises a sequence useful for purifying, expressing, solubilizing, and/or detecting a polypeptide. In some embodiments, a tag can serve multiple functions. A tag is often relatively small, e.g., ranging from a few amino acids up to about 100 amino acids long. In some embodiments a tag is more than 100 amino acids long, e.g., up to about 500 amino acids long, or more. In some embodiments, a tag comprises an His6, HA, TAP, Myc, Flag, or GST tag, to name few examples. In some embodiments a tag comprises a solubility-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, a Strep tag, or a monomeric mutant of the Ocr protein of bacteriophage T7). See, e.g., Esposito D and Chatterjee D K. Curr Opin Biotechnol.; 17(4):353-8 (2006). In some embodiments, a tag is cleavable, so that it can be removed, e.g., by a protease. In some embodiments, this is achieved by including a protease cleavage site in the tag, e.g., adjacent or linked to a functional portion of the tag. Exemplary proteases include, e.g., thrombin, TEV protease, Factor Xa, PreScission protease, etc. In some embodiments, a “self-cleaving” tag is used. See, e.g., PCT/US05/05763.
The conjugates described herein comprises a VHH conjugated to a second molecule. In some embodiments, the VHH comprises a sortase recognition motif and is conjugated to the second molecule via click chemistry. In some embodiments, the conjugate of the present disclosure comprises a VHH conjugated to one molecule. In some embodiments, the one molecule conjugated to the VHH is an antigen. In some embodiments, the one molecule conjugated to the VHH is an anti-inflammatory agent or a proinflammatory agent. In some embodiments, the conjugate of the present disclosure comprises a VHH conjugated to two molecules. In some embodiments, the conjugate of the present disclosure comprises a VHH conjugated to an antigen to which immune response is needed (e.g., an antigen from a pathogen or a tumor antigen) and an anti-inflammatory agent. In some embodiments, the conjugate of the present disclosure comprises a VHH conjugated to an antigen to which immune tolerance is needed (e.g., a self-antigen or an exogenous enzyme used for therapy) and a proinflammatory agent. Examples of methods for conjugating two molecules to a VHH are shown in
In some embodiments, an anti-inflammatory agent or a proinflammatory agent is conjugated to the sortase recognition motif of a VHH via a linker. In some embodiments, a linker is a non-hydrolysable linker (i.e. non-cleavable). Non-limiting examples of non-hydrolysable linkers include N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimidomethyl cyclohexane-1-carboxylate (MCC), maleimidocaproyl (MC), and derivatives thereof. In some embodiments, a linker is a hydrolysable linker (i.e. cleavable). Non-limiting examples of hydrolysable linkers include hydrazone, hydrazide, disulfide, 4-(4′-acetylphenoxy)butanoic acid (AcBut), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) and N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), valine-citrulline (VC), valine-alanine (VA), phenylalanine-lysine (FK), and derivatives thereof. A hydrolysable linker may be a self-immolating linker (i.e. self-cleaving), such as a pH-sensitive linker (e.g., hydrazone). A pH-sensitive linker may be used, for example, to release an anti-inflammatory agent or a proinflammatory agent conjugated to a VHH upon a shift in acidity of the physiological environment, such as when the VHH is delivered to a desired destination (e.g., an APC or intracellular compartment thereof). In some embodiments, the linker is a self-hydrolyzing hydrazone linker as shown in
In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to a self-antigen and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the self-antigens described herein may be used. In some embodiments, the self-antigen is myelin oligodendrocyte glycoprotein (MOG), or a fragment thereof (e.g., amino acids 35-55 of the MOG protein. In some embodiments, the self-antigen is citrullinated fibrinogen. In some embodiments, the self-antigen is insulin. In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to a protein used in a protein replacement therapy or a gene therapy (e.g., an enzyme such as Factor IX or Factor VIII or an adeno-associated virus (AAV) derived protein) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1).
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH and the second VHH both bind to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a self-antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH and the second VHH both bind to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the self-antigens described herein may be used. In some embodiments, the self-antigen is myelin oligodendrocyte glycoprotein (MOG), or a fragment thereof (e.g., amino acids 35-55 of the MOG protein. In some embodiments, the self-antigen is citrullinated fibrinogen. In some embodiments, the self-antigen is insulin. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a protein used in a protein replacement therapy or a gene therapy (e.g., an enzyme such as Factor IX or Factor VIII or an adeno-associated virus (AAV) derived protein) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH and the second VHH both bind to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1).
In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to a self-antigen and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the self-antigens described herein may be used. In some embodiments, the self-antigen is myelin oligodendrocyte glycoprotein (MOG), or a fragment thereof (e.g., amino acids 35-55 of the MOG protein. In some embodiments, the self-antigen is citrullinated fibrinogen. In some embodiments, the self-antigen is insulin. In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to a protein used in a protein replacement therapy or a gene therapy (e.g., an enzyme such as Factor IX or Factor VIII or an adeno-associated virus (AAV) derived protein) and an anti-inflammatory agent (e.g., dexamethasone), wherein the VHH binds to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2).
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH and the second VHH both bind to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a self-antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH and the second VHH both bind to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the self-antigens described herein may be used. In some embodiments, the self-antigen is myelin oligodendrocyte glycoprotein (MOG), or a fragment thereof (e.g., amino acids 35-55 of the MOG protein. In some embodiments, the self-antigen is citrullinated fibrinogen. In some embodiments, the self-antigen is insulin. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a protein used in a protein replacement therapy or a gene therapy (e.g., an enzyme such as Factor IX or Factor VIII or an adeno-associated virus (AAV) derived protein) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH and the second VHH both bind to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2).
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH and the second VHH bind to different surface proteins on the APC. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune tolerance is needed) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein first VHH binds to DEC205 and the second VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a self-antigen and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein the first VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the self-antigens described herein may be used. In some embodiments, the self-antigen is myelin oligodendrocyte glycoprotein (MOG), or a fragment thereof (e.g., amino acids 35-55 of the MOG protein. In some embodiments, the self-antigen is citrullinated fibrinogen. In some embodiments, the self-antigen is insulin. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a protein used in a protein replacement therapy or a gene therapy (e.g., an enzyme such as Factor IX or Factor VIII or an adeno-associated virus (AAV) derived protein) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent (e.g., dexamethasone), wherein first VHH binds to DEC205 and the second VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1).
In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to an antigen (e.g., an antigen to which immune response is needed) and a proinflammatory agent, wherein the VHH binds to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to an antigen from a pathogen (e.g., a SARS-CoV-2 protein such as the spike protein) and a proinflammatory agent (e.g., IL2), wherein the VHH binds to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the antigens from pathogens described herein may be used. In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to a tumor antigen and a proinflammatory agent (e.g., IL2), wherein the VHH binds to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the tumor antigens described herein may be used.
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune response is needed) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent, wherein the first VHH and the second VHH both bind to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen from a pathogen (e.g., a SARS-CoV-2 protein such as the spike protein) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., IL2), wherein the first VHH and the second VHH both bind to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the antigens from pathogens described herein may be used. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a tumor antigen and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., IL2), wherein the first VHH and the second VHH both bind to MHCII (e.g., the VHH having the amino acid sequence of SEQ ID NO: 1). Any one of the tumor antigens described herein may be used.
In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to an antigen (e.g., an antigen to which immune response is needed) and a proinflammatory agent, wherein the VHH binds to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to an antigen from a pathogen (e.g., a SARS-CoV-2 protein such as the spike protein) and a proinflammatory agent (e.g., IL2), wherein the VHH binds to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the antigens from pathogens described herein may be used. In some embodiments, the composition described herein comprises a conjugate comprising a VHH conjugated to a tumor antigen and a proinflammatory agent (e.g., IL2), wherein the VHH binds to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2).
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune response is needed) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent, wherein the first VHH and the second VHH both bind to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen from a pathogen (e.g., a SARS-CoV-2 protein such as the spike protein) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., IL2), wherein the first VHH and the second VHH both bind to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the antigens from pathogens described herein may be used. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a tumor antigen and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., 112), wherein the first VHH and the second VHH both bind to CD11c (e.g., the VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the tumor antigens described herein may be used.
In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune response is needed) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent, wherein the first VHH and the second VHH bind to different surface proteins on the APC. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune response is needed) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., IL2), wherein the first VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen (e.g., an antigen to which immune response is needed) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., IL2), wherein first VHH binds to DEC205 and the second VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1). In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to an antigen from a pathogen (e.g., a SARS-CoV-2 protein such as the spike protein) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., IL2), wherein the first VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1) and the second VHH binds to CD11c (e.g., VHH having the amino acid sequence of SEQ ID NO: 2). Any one of the antigens from pathogens described herein may be used. In some embodiments, the composition comprises a first conjugate comprising a first VHH conjugated to a tumor antigen and a second conjugate comprising a second VHH conjugated to a proinflammatory agent (e.g., IL2), wherein first VHH binds to DEC205 and the second VHH binds to MHCII (e.g., VHH having the amino acid sequence of SEQ ID NO: 1).
An “antigen,” as used herein, refers to a molecule that induces an immune response in a subject. An antigen of interest may be or may comprise, for example, a polypeptide, a polysaccharide, a carbohydrate, a lipid, a nucleic acid, or combination thereof. An antigen may be naturally occurring or synthetic.
In some embodiments, an antigen is an antigen to which immune tolerance is needed. In some embodiments, such an antigen is a self-antigen (also referred to as “autoantigen”) or an agent that has the capacity to initiate or enhance an autoimmune response, causing autoimmune diseases. It is thus desired to induce immune tolerance to such self-antigens. In some embodiments, the compositions described herein are used to induce immune tolerance (e.g., antigen specific immune tolerance) to self-antigens. Induction of immune tolerance (e.g., antigen specific immune tolerance) reduces antigen-specific immune responses to the antigen, which, in some embodiments, alleviates the severity of autoimmune diseases.
In some embodiments, the self-antigen used in accordance with the present disclosure is selected from the group consisting of: myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein, citrullinated fibrinogen, insulin, chromogranin A, GAD65, desmoglein 1 (DSG1) and desmoglein 3 (DSG3), acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and ribonucleoproteins.
In some embodiments, the self-antigen comprises myelin oligodendrocyte glycoprotein (MOG) or an antigenic fragment thereof. Myelin oligodendrocyte glycoprotein (MOG) is a membrane-embedded surface protein of the central nervous system (CNS) myelin sheath. Antibodies targeting MOG have been consistently found in the sera of patients suffering from autoimmune diseases such as acquired inflammatory demyelinating disorders of the CNS (e.g., as described in Nessier et al., EBioMedicine. 2019 October; 48: 18-19, incorporated herein by reference). Autoimmune diseases associated with MOG antibodies include, without limitation, acute disseminated encephalomyelitis (ADEM), optic neuritis (ON), transverse myelitis and brainstem encephalitis. In some embodiments, the self-antigen in the composition described herein is full length MOG. In some embodiments, the self-antigen in the composition described herein comprises a MOG fragment (e.g., amino acids 35-55 of MOG, MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 49)).
In some embodiments, the self-antigen comprises fibrinogen or an antigenic fragment thereof. Fibrinogen (coagulation factor 1) is a major player in thrombus formation; it is cleaved by thrombin to form fibrin, which is the most abundant component of a blood clot. Fibrinogen plays an important role in coagulation and cardiovascular diseases (CVDs). Additionally, fibrinogen is a proinflammatory factor in autoimmune and inflammatory diseases such as rheumatoid arthritis, vasculitides, inflammatory bowel disease, multiple sclerosis, chronic obstructive pulmonary diseases, kidney disorders, and posttransplantation fibrosis and in several types of cancer (e.g., as described in Arbustini et al., Circulation. 2013; 128:1276-1280, incorporated herein by reference). In some embodiments, the self-antigen is citrullinated fibrinogen. In some embodiments, the self-antigen in the composition described herein comprises a fibrinogen fragment (amino acids 79-91 of fibrinogen, citrullinated, QDFTNCitINKLKNS (SEQ ID NO: 50)). Anti-citrullinated protein antibodies (ACPA) are specifically and frequently detected in sera of patients with rheumatoid arthritis (e.g., as described in Takizawa et al., Ann Rheum Dis. 2006 August; 65(8): 1013-1020).
In some embodiments, the self-antigen comprises myelin proteolipid protein or an antigenic fragment thereof. Myelin proteolipid protein has been shown to be involved in autoimmune demyelinating disease, e.g., as described in Tuohy et al., Neurochem Res. 1994 August; 19(8):935-44, incorporated herein by reference.
In some embodiments, the self-antigen comprises insulin or an antigenic fragment thereof. In some embodiments, the self-antigen comprises insulin alpha chain GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 51)). In some embodiments, the self-antigen comprises insulin beta chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 52)). Insulin is involved in rare autoimmune diseases including insulin autoimmune syndrome and type B insulin resistance syndrome (e.g., as described in Censi et al., Ann Transl Med. 2018 September; 6(17): 335, incorporated herein by reference).
In some embodiments, the self-antigen comprises chromogranin A or an antigenic fragment thereof. Chromogranin A is associated with autoimmune gastritis (e.g., as described in Peracchi et al., European Journal of Endocrinology (2005) 152 443-448, incorporated herein by reference).
In some embodiments, the self-antigen comprises glutamic acid decarboxylase 65-kilodalton isoform (GAD65) or an antigenic fragment thereof, which is known to be associated with autoimmune diseases of the central nervous system, neurological autoimmune diseases. Type 1 diabetes, autoimmune thyroid disease, and pernicious anemia (e.g., as described in McKeon et al., Muscle Nerve. 2017 July; 56(1):15-27, incorporated herein by reference).
In some embodiments, the self-antigen comprises desmoglein 1 (DSG1) and/or desmoglein 3 (DSG3) or an antigenic fragment thereof. DSG1 and DSG3 are involved in skin autoimmune disease, e.g., as described in Amagai et al., Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86(5):524-37, incorporated herein by reference.
In some embodiments, the self-antigen comprises acetylcholine receptor (AChR) or an antigenic fragment thereof. Antibody-mediated autoimmune response to acetylcholine receptor causes myasthenia gravis, e.g., as described in Lindstrom et al., J Neurobiol. 2002 December; 53(4):656-65, incorporated herein by reference.
In some embodiments, the self-antigen comprises muscle-specific tyrosine kinase (MuSK) or an antigenic fragment thereof. MuSK has been shown to be involved in neuromuscular junction autoimmune diseases, e.g., as described in Vincent et al., Cuff Opin Neurol. 2005 October; 18(5):519-25, incorporated herein by reference.
In some embodiments, the self-antigen comprises a ribonucleoprotein or an antigenic fragment thereof. Ribonucleoproteins are involved in autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and Mixed connective tissue disease (MCTD), e.g., as described in Whittingham et al., Aust N Z J Med. 1983 December; 13(6):565-70; and Newkirk et al., Arthritis Research & Therapy volume 3, Article number: 253 (2001), incorporated herein by reference.
Other non-limiting examples of such autoimmune antigens and associated autoimmune diseases include: pancreatic beta-cell antigens, insulin and GAD to treat insulin-dependent diabetes mellitus (type I diabetes); collagen type 11, human cartilage gp39 (HCgp39) and gpl30-RAPS for use in treating rheumatoid arthritis; myelin basic protein (MBP), proteolipid protein (PLP) to treat multiple sclerosis; fibrillarin, and small nucleolar protein (snoRNP) to treat scleroderma; thyroid stimulating factor receptor (TSH-R) for use in treating Graves' disease; nuclear antigens, histones, glycoprotein gp70 and ribosomal proteins for use in treating systemic lupus erythematosus; pyruvate dehydrogenase dehydrolipoamide acetyltransferase (PCD-E2) for use in treating primary biliary cirrhosis; hair follicle antigens for use in treating alopecia areata; and human tropomyosin isoform 5 (hTM5) for use in treating ulcerative colitis. These examples are not meant to be limiting. One skilled in the art is able to identify the autoimmune antigen associated with the autoimmune disease of interest.
In some embodiments, the antigen comprises a protein used in a protein replacement therapy or a gene therapy, e.g., without limitation, Factor IX, Factor VIII, insulin, and AAV-derived proteins. These examples are not meant to be limiting. One skilled in the art is able to identify the proteins of interest that are used in protein replacement therapies or gene therapies. Inducing immune tolerance against these proteins reduces the destruction of the proteins by the immune system, leading to longer lasting therapeutic effect.
In some embodiments, the antigen used in accordance with the present disclosure is an antigen to which immune response is needed. For example, in some embodiments, such antigen is naturally produced by and/or comprises a polypeptide or peptide that is genetically encoded by a pathogen, an infected cell, or a neoplastic cell (e.g., a cancer cell). In some embodiments, an antigen is produced or genetically encoded by a virus, bacteria, fungus, or parasite which, in some embodiments, is a pathogenic agent. In some embodiments, a pathogen is intracellular during at least part of its life cycle. In some embodiments, a pathogen is extracellular. It will be appreciated that an antigen that originates from a particular source may, in some embodiments, be isolated from such source, or produced using any appropriate means (e.g., recombinantly, synthetically, etc.), e.g., for purposes of using the antigen, e.g., to identify, generate, test, or use an antibody thereto). An antigen may be modified, e.g., by conjugation to another molecule or entity (e.g., an adjuvant), chemical or physical denaturation, etc. In some embodiments, an antigen is an envelope protein, capsid protein, secreted protein, structural protein, cell wall protein or polysaccharide, capsule protein or polysaccharide, or enzyme. In some embodiments an antigen is a toxin, e.g., a bacterial toxin. In some embodiments, the antigen is a viral antigen. Exemplary viruses include, e.g., SARS-CoV-2, Retroviridae (e.g., lentiviruses such as human immunodeficiency viruses, such as HIV-I); Caliciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses, hepatitis C virus); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (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); Bunyaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (erg., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae; Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), EBV, KSV); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses). In some embodiments, the antigen comprises a Beta Coronavirus protein such as the spike protein (e.g., full-length or receptor binding domain (RBD)), envelop protein, membrane protein, or nucleocapsid protein. In some embodiments, the antigen comprises a SARS-CoV (e.g., SARS-CoV-1 or SARS-CoV-2) protein such as the spike protein (e.g., full-length or receptor binding domain (RBD)), envelop protein, membrane protein, or nucleocapsid protein. Examples of Beta Coronavirus proteins that may be used as an antigen in accordance with the present disclosure are provided in Table 2.
In some embodiments, the antigen is a bacterial antigen. Exemplary bacteria include, e.g., Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, Campylobacter sp., Enterococcus sp., Chlamydia sp., Haemophilus influenzae, Bacillus anthracia, Corynebacterium diphtheriae, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Actinomyces israelii and Francisella tularensis.
In some embodiments, the antigen is a fungal antigen. Exemplary fungi include, e.g., Aspergillus, such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Blastomyces, such as Blastomyces dermatitidis, Candida, such as Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Coccidioides, such as Coccidioides immitis, Cryptococcus, such as Cryptococcus neoformans, Epidermophyton, Fusarium, Histoplasma, such as Histoplasma capsulatum, Malassezia, such as Malassezia furfur, Microsporum, Mucor, Paracoccidioides, such as Paracoccidioides brasiliensis, Penicillium, such as Penicillium marneffei, Pichia, such as Pichia anomala, Pichia guilliermondii, Pneumocystis, such as Pneumocystis carinii, Pseudallescheria, such as Pseudallescheria boydii, Rhizopus, such as Rhizopus oryzae, Rhodotorula, such as Rhodotorula rubra, Scedosporium, such as Scedosporium apiospermum, Schizophyllum, such as Schizophyllum commune, Sporothrix, such as Sporothrix schenckii, Trichophyton, such as Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton violaceum, Trichosporon, such as Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, and Trichosporon mucoides.
In some embodiments, the antigen is from a parasite. Exemplary parasites include, e.g., parasites of the genus Plasmodium (e.g. Plasmodium falciparum, P. vivax, P. ovale and P. malariae), Trypanosoma, Toxoplasma (e.g., Toxoplasma gondii), Leishmania (e.g., Leishmania major), Schistosoma, or Cryptosporidium. In some embodiments the parasite is a protozoan. In some embodiments the parasite belongs to the phylum Apicomplexa. In some embodiments the parasite resides extracellularly during at least part of its life cycle. Examples include nematodes, trematodes (flukes), and cestodes. In some embodiments antigens from Ascaris or Trichuris are contemplated. In various embodiments, the antigen can originate from any component of the parasite. In some embodiments the antigen can be derived from parasites at any stage of their life cycle of the parasite, e.g., any stage that occurs within an infected organism such as a mammalian or avian organism. In some embodiments the antigen is derived from eggs of the parasite or substances secreted by the parasite.
In some embodiments, the antigen is a tumor antigen. In general, a tumor antigen can be any antigenic substance produced by tumor cells (e.g., tumorigenic cells or in some embodiments tumor stromal cells, e.g., tumor-associated cells such as cancer-associated fibroblasts). In some embodiments, a tumor antigen is a molecule (or portion thereof) that is differentially expressed by tumor cells as compared with non-tumor cells. Tumor antigens may include, e.g., proteins that are normally produced in very small quantities and are expressed in larger quantities by tumor cells, proteins that are normally produced only in certain stages of development, proteins whose structure (e.g., sequence or post-translational modification(s)) is modified due to mutation in tumor cells, or normal proteins that are (under normal conditions) sequestered from the immune system. Tumor antigens may be useful in, e.g., identifying or detecting tumor cells (e.g., for purposes of diagnosis and/or for purposes of monitoring subjects who have received treatment for a tumor, e.g., to test for recurrence) and/or for purposes of targeting various agents (e.g., therapeutic agents) to tumor cells. For example, in some embodiments, a chimeric antibody is provided, comprising an antibody of antibody fragment that binds a tumor antigen, and conjugated via click chemistry to a therapeutic agent, for example, a cytotoxic agent. In some embodiments, a tumor antigen is an expression product of a mutated gene, e.g., an oncogene or mutated tumor suppressor gene, an overexpressed or aberrantly expressed cellular protein, an antigen encoded by an oncogenic virus (e.g., HBV; HCV; herpesvirus family members such as EBV, KSV; papilloma virus, etc.), or an oncofetal antigen. Oncofetal antigens are normally produced in the early stages of embryonic development and largely or completely disappear by the time the immune system is fully developed. Examples are alphafetoprotein (AFP, found, e.g., in germ cell tumors and hepatocellular carcinoma) and carcinoembryonic antigen (CEA, found, e.g., in bowel cancers and occasionally lung or breast cancer). Tyrosinase is an example of a protein normally produced in very low quantities but whose production is greatly increased in certain tumor cells (e.g., melanoma cells). Other exemplary tumor antigens include, e.g., CA-125 (found, e.g., in ovarian cancer); MUC-1 (found, e.g., in breast cancer); epithelial tumor antigen (found, e.g., in breast cancer); melanoma-associated antigen (MAGE; found, e.g., in malignant melanoma); prostatic acid phosphatase (PAP, found in prostate cancer). In some embodiments, a tumor antigen is at least in part exposed at the cell surface of tumor cells. In some embodiments, a tumor antigen comprises an abnormally modified polypeptide or lipid, e.g., an aberrantly modified cell surface glycolipid or glycoprotein. It will be appreciated that a tumor antigen may be expressed by a subset of tumors of a particular type and/or by a subset of cells in a tumor.
In some embodiments, the tumor antigen is selected from the group consisting of: MAGE family members, NY-ESO-1, tyrosinase, Melan-A/MART-1, prostate cancer antigen, Her-2/neu, Survivin, Telomerase, WTi, CEA, gp100, Pmel17, mammaglobin-A, NY-BR-1, ERBB2, OA1, PAP, RAB38/NY-MEL-1, TRP-1/gp75, TRP-2, CD33, BAGE-1, D393-CD20n, cyclin-A1, GAGE-1, GAGE-2, GAGE-8, GnTVf, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-C1, MAGE-C2, mucin K, NA88-A, SAGE, sp17, SSX-2, SSX-4, surviving, TAG-1, TAG-3, TRAG-3, XAGE-1b, BCR-AB1, adipophiln, AIM-2, ALDH1A1, BCLX(L), BING-4, CALCA, CD45, CD274, CPSF, cyclin D1, DKK1, ENAH, EpCAM, EphA3, EZH2, FGF5, glypican-3, G250, HER-2, HLA-DOB, hepsin, IDO1, IGF2B3, IL12Ralpha2, intestinal carboyxyl esterase, alpha-foetoprotein, kallikrein 4, KIF20A, Lengsin, M-CSF, M-CSP, mdm-2, Meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secerine 1, SOX10, STEAP1, telomerase, TPBG, mesothelin, Axl, and VEGF.
In some embodiments, the antigen is a whole cell, a whole parasite, a whole virus, a whole bacterium, or a whole nanoparticle, exosome, or microparticle comprising one or more antigens. In one example, a VHH may be conjugated to a beta islet cell and delivered under non-inflammatory conditions in order to induce beta islet cell tolerance in the course of organ or tissue replacement therapy. In yet another example, a VHH may be conjugated to a parasite and delivered under inflammatory conditions in order to induce a strong immune response against multiple parasite antigens at once.
As shown herein, the conjugates comprising VHH conjugated to an antigen to which immune tolerance is needed (e.g., a self-antigen), when administered to a subject under non-inflammatory conditions is more effective in inducing antigen-specific immune tolerance to the self-antigen. In some embodiments, the non-inflammatory condition is provided by attaching an anti-inflammatory agent to the same conjugate comprising the VHH and the antigen. In some embodiments, the non-inflammatory condition is provided by co-administering a VHH conjugated to an anti-inflammatory agent in addition to the VHH conjugated to a self-antigen.
An “anti-inflammatory agent” refers to a substance that reduces inflammation in the body. Anti-inflammatory agents block certain substances in the body that cause inflammation. Any anti-inflammatory agents known in the art can be used in accordance with the present disclosure. In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In some embodiments, the steroidal anti-inflammatory agent is selected from the group consisting of: dexamethasone, prednisone, prednisolone, triamcinolone, methylprednisolone, and bethamethasone. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory agent. In some embodiments, the nonsteroidal anti-inflammatory agent is selected from the group consisting of: aspirin, celecoxib, diclofenac, ibuprofen, ketoprofen, naproxen, oxaprozin, piroxicam, cyclosporin A, and calcitriol. In some embodiments, the anti-inflammatory agent used in accordance with the present disclosure is dexamethasone.
In some embodiments, the anti-inflammatory agent is an anti-inflammatory cytokine. An “anti-inflammatory cytokine” refers to a cytokine that inhibits the synthesis of IL-1, tumor necrosis factor (TNF), and other major proinflammatory cytokines and reduces inflammatory response. In some embodiments, the anti-inflammatory cytokine is selected from the group consisting of IL-10, IL-35, IL-4, IL-11, IL-13, and TGFβ.
The present disclosure, in other aspects, provides that the conjugates comprising VHH conjugated to an antigen to which immune response is needed (e.g., an antigen from a pathogen or a tumor antigen), when administered to a subject under inflammatory conditions is more effective in inducing antigen-specific immune response to the antigen. In some embodiments, the inflammatory condition is provided by attaching a proinflammatory agent to the same conjugate comprising the VHH and the antigen. In some embodiments, the non-inflammatory condition is provided by co-administering a VHH conjugated to a proinflammatory agent in addition to the VHH conjugated to an antigen. In some embodiments, the proinflammatory agent is selected from the group consisting of: TLR9 agonist (e.g., CpG ODN), LPS, HMGB1 proteins, IL2, IL12, and CD40L. In some embodiments, the pro-inflammatory agent is IL2.
Some aspects of the present disclosure provide methods of comprising administering to a subject in need thereof: (i) a conjugate comprising a VHH conjugated to an antigen to which immune tolerance is needed (e.g., a self-antigen) and an anti-inflammatory agent, wherein the VHH binds to a surface protein on an APC (e.g., MHCII or CD11c) or a (ii) a first conjugate comprising a VHH conjugated to an antigen to which immune tolerance is needed (e.g., a self-antigen) and a second conjugate comprising a second VHH conjugated to an anti-inflammatory agent, wherein the first VHH and the second VHH bind to one or more (e.g., same or different) surface proteins on an APC. In some embodiments, when two VHHs are administered, they are administered in the same composition, or in different compositions (e.g., sequentially). In some embodiments, the method is for inducing an immune tolerance to an antigen. In some embodiments, the method is for treating an autoimmune disease.
An “autoimmune disease” to a disorder that causes abnormally over activity of the immune system, which attacks and damages its own tissues. Non-limiting examples of autoimmune diseases include: rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Myasthenia Gravis (MG), Graves' Disease, Idiopathic Thrombocytopenia Purpura (ITP), Guillain-Barre Syndrome, autoimmune myocarditis, Membrane Glomerulonephritis, Type I or Type II diabetes, juvenile onset diabetes, multiple sclerosis, Reynaud's syndrome, autoimmune thyroiditis, gastritis, Celiac Disease, Vitiligo, Hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathies, experimental autoimmune encephalomyelitis, immune neutropenia, and immune responses associated with delayed hypersensitivity mediated by cytokines, T-lymphocytes typically found in tuberculosis, sarcoidosis, and polymyositis, polyarteritis, cutaneous vasculitis, pemphigus (e.g., Pemphigus vulgaris, Pemphigus foliaceus or Paraneoplastic pemphigus), pemphigold, Goodpasture's syndrome, Kawasaki's disease, systemic sclerosis, anti-phospholipid syndrome, and Sjogren's syndrome. In some embodiments, the autoimmune disease is selected from the group consisting of: multiple sclerosis, type II diabetes, Pemphigus vulgaris, myasthenia gravis, lupus, celiac diseases, and inflammatory bowel disease (IBD). In some embodiments, the autoimmune disease is selected from the group consisting of: autoimmune encephalomyelitis, acute disseminated encephalomyelitis (ADEM), optic neuritis (ON), transverse myelitis and brainstem encephalitis, rheumatoid arthritis, vasculitides, inflammatory bowel disease, multiple sclerosis, chronic obstructive pulmonary diseases, kidney disorders, posttransplantation fibrosis and in several types of cancer, autoimmune demyelinating disease, insulin autoimmune syndrome, type B insulin resistance syndrome, autoimmune gastritis, autoimmune diseases of the central nervous system, neurological autoimmune diseases, Type 1 diabetes, autoimmune thyroid disease, pernicious anemia, skin autoimmune disease, myasthenia gravis, neuromuscular junction autoimmune diseases. Different self-antigens may be used in the conjugates for treating different autoimmune diseases. One skilled in the art is able to identify the appropriate self-antigen to use.
Other aspects of the present disclosure provide methods of comprising administering to a subject in need thereof: (i) a conjugate comprising a VHH conjugated to an antigen to which immune response is needed (e.g., an antigen from a pathogen or a tumor antigen) and a proinflammatory agent, wherein the VHH binds to a surface protein on an APC (e.g., MHCII or CD11c) or a (ii) a first conjugate comprising a VHH conjugated to an antigen to which immune response is needed (e.g., an antigen from a pathogen or a tumor antigen) and a second conjugate comprising a second VHH conjugated to a proinflammatory agent, wherein the first VHH and the second VHH bind to one or more (e.g., same or different) surface proteins on an APC. In some embodiments, when two VHHs are administered, they are administered in the same composition, or in different compositions (e.g., sequentially). In some embodiments, the method is for inducing an immune response to an antigen. In some embodiments, the method is for treating infection caused by a pathogen (e.g., a microbial pathogen such as the ones described herein). In some embodiments, the methods is for treating cancer.
The cancer may be a primary or metastatic cancer. Cancers include, but are not limited to, adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, biliary tract cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, glioblastoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphocytic and myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenstrom macroglobulinema, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neck cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myeloproliferative disorders, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary cancer, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous neck cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, choriocarcinoma, hematological neoplasm, adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal tumors and germ cell tumors, or Wilms tumor. In some embodiments, the cancer is lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, brain and central nervous system cancer, skin cancer, ovarian cancer, leukemia, endometrial cancer, bone, cartilage and soft tissue sarcoma, lymphoma, neuroblastoma, nephroblastoma, retinoblastoma, or gonadal germ cell tumor.
In its broadest sense, the terms “treatment” or “to treat” refer to both therapeutic and prophylactic treatments. If the subject in need of treatment has a disease (e.g., autoimmune disease, infection, or cancer), then “treating the condition” refers to ameliorating, reducing or eliminating one or more symptoms associated with the disease or the severity of disease or preventing any further progression of disease. If the subject in need of treatment is one who is at risk of having a disease (e.g., infection or cancer), then treating the subject refers to reducing the risk of the subject having an infection cancer or preventing the subject from developing an infection or cancer.
A subject shall mean a human or vertebrate animal or mammal including but not limited to a rodent, e.g., a rat or a mouse, dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, and primate, e.g., monkey. The methods of the present disclosure are useful for treating a subject in need thereof.
In some embodiments, the compositions described herein are pharmaceutical compositions. Pharmaceutically compositions that may be used in accordance with the present disclosure may be directly administered to the subject or may be administered to a subject in need thereof in a therapeutically effective amount. The term “therapeutically effective amount” refers to the amount necessary or sufficient to realize a desired biologic effect. For example, a therapeutically effective amount of a composition associated with the present disclosure may be that amount sufficient to ameliorate one or more symptoms of a targeted disease (e.g., autoimmune disease, infection, or cancer). Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular pharmaceutically compositions being administered the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular therapeutic compound associated with the present disclosure without necessitating undue experimentation.
Subject doses of the compositions described herein for delivery typically range from about 0.1 μg to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time there between. In some embodiments a single dose is administered during the critical consolidation or reconsolidation period. The doses for these purposes may range from about 10 μg to 5 mg per administration, and most typically from about 100 μg to 1 mg, with 2-4 administrations being spaced, for example, days or weeks apart, or more. In some embodiments, however, parenteral doses for these purposes may be used in a range of 5 to 10,000 times higher than the typical doses described above.
In some embodiments, a composition the present disclosure is administered at a dosage of between about 1 and 10 mg/kg of body weight of the mammal. In other embodiments composition of the present disclosure is administered at a dosage of between about 0.001 and 1 mg/kg of body weight of the mammal. In yet other embodiments, the composition of the present disclosure is administered at a dosage of between about 10-100 ng/kg, 100-500 ng/kg, 500 ng/kg-1 mg/kg, or 1-5 mg/kg of body weight of the mammal, or any individual dosage therein.
The compositions of the present disclosure are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic ingredients.
For use in therapy, an effective amount of the composition associated with the present disclosure can be administered to a subject by any mode that delivers the therapeutic agent or compound to the desired surface, e.g., mucosal, injection to cancer, systemic, etc. Administering the pharmaceutical composition of the present disclosure may be accomplished by any means known to the skilled artisan. Suitable routes of administration include but are not limited to oral, parenteral, intravenous, intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, rectal and intracerebroventricular. In some embodiments, the composition is administered intravenously (e.g., via injection or infusion).
The pharmaceutical compositions of the present disclosure, when desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
In addition to the formulations described previously, the compositions may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.
The compositions of the present disclosure and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
The pharmaceutical compositions of the present disclosure contain an effective amount of a therapeutic compound of the present disclosure optionally included in a pharmaceutically-acceptable carrier. The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
The pharmaceutical compositions of the present disclosure may be delivered with other therapeutics for treating a disease (e.g., an autoimmune disease, infection, or cancer).
Autoimmunity results from the recognition of self-antigens by components of the adaptive immune system. This explains the link of most autoimmune diseases with particular allelic variants of Class II MHC products that present the offending self-antigen. To treat autoimmune disease, induction of antigen-specific tolerance would be a highly desirable goal. Regardless of pathology, antigen presenting cells (APCs) are essential in the induction of disease, and conversely, APCs can be tolerogenic if they encounter antigen under non-inflammatory conditions. Described herein are nanobodies that recognize Class II MHC products, present on all APCs, which can be enzymatically conjugated to self-antigens such as a myelin oligodendrocyte glycoprotein (MOG) fragment in a preclinical model of autoimmune disease, experimental autoimmune encephalitis (EAE). Administration of these adducts under non-inflammatory conditions provides long-lasting protection against EAE. Similar adducts prevented hyperglycemia in a mouse model of accelerated type I diabetes and rheumatoid arthritis. Self-antigens were conjugated not only to nanobodies, but also a dexamethasone derivative, attached via a cleavable hydrazone linker. Co-administration of a Class II-MHC specific nanobody carrying the MOG peptide with that same nanobody, modified with the cleavable dexamethasone derivative, halted progression of disease in animals that were already symptomatic. While the precise target cell population responsible for therapeutic efficacy remains to be identified beyond the fact that these must be Class II MHC-positive cells, there is practical utility of these findings. The application of such antibody-drug conjugates in various inflammatory conditions should be considered as a viable treatment option.
Approximately 10% of the human population suffers from an auto-immune condition, accompanied by mild to life-threatening symptoms. In only select cases is there a plausible explanation for how disease is initiated. Immunotherapy of cancer using checkpoint blockade, while highly successful for a select set of malignancies, entails the risk of provoking autoimmunity by releasing the breaks on immune homeostasis. It is a clear example of an experimental trigger that uncovers the presence of harmful self-reactive cells, held in check until checkpoint blockade is applied.
Current treatments for autoimmune diseases include general immunosuppression, which blunts responses across the entire spectrum of antigens. This exposes patients to an increased risk of infection and possibly even malignancies. Because autoimmune diseases are often organ-specific, the immune component includes antigen-specific elements, either as triggers, as targets, or some combination of the two. This is perhaps best illustrated by various preclinical models of autoimmunity, where pathology can be elicited by administration of a defined antigen under the appropriate stimulatory conditions. For certain human autoimmune diseases, the antigens that induce pathology and recognized in the course of an autoimmune response are known. Examples include islet antigens in the case of type-1 diabetes, components of the myelin sheath in multiple sclerosis, and citrullinated antigens in the case of arthritis.
Nanoparticles composed of peptide loaded MHC products have been used to elicit a form of both Class I and Class II MHC-restricted tolerance. A further striking example is the ability of red blood cells, modified with a self-antigen, to induce a profound state of antigen non-responsiveness. This trait has been attributed to the exceptional turnover rate in comparison with other cell types, and the need to eliminate red blood cell remnants without causing an inflammatory response. The phenomenon of tolerogenic elimination of cell remnants is not limited to red blood cells, as transfusion of chemically modified, apoptotic peripheral blood lymphocytes can also dampen auto-immune responses.
Reported herein is the development and characterization of alpaca-derived single domain antibody fragments (nanobodies/VHHs) that recognize Class II MHC molecules. These nanobodies target all Class II MHC-positive cells, including antigen presenting cells (APCs). At one-tenth the size of conventional immunoglobulins, the small size of nanobodies ensures excellent tissue penetration and rapid clearance from the circulation. This makes VHHs ideal vehicles for targeted delivery of payloads of interest, such as antigenic peptides or small molecule drugs. Furthermore, an engineering strategy that used sortase A, a S. aureus-derived transpeptidase was established. It enabled site-specific modification of these VHHs at their C-terminus. Sortase-modified Class II MHC-specific nanobodies were used as imaging agents for positron emission tomography, the results of which were consistent with a short circulatory half-life, paired with excellent targeting properties. These methods likewise allowed the installation of a wide range of antigens involved in infectious and autoimmune disease. There is broad consensus that engagement of antigen presenting cells under non-inflammatory conditions can lead to tolerance, whereas administration under inflammatory conditions, for example in the presence of adjuvants, can elicit a strong protective response against foreign antigens. Valency, aggregation state and dose of the antigen are additional parameters that can make the pendulum swing from tolerogen to immunogen. The distribution of a diverse set of APCs over different anatomical sites and its dynamics pose a challenge for the identification of the relevant tolerogenic APC in vivo. The purpose of this study was not so much to pin down a particular APC (sub)set responsible for tolerance induction, but rather to demonstrate the efficacy of using VHHs to target the Class II MHC-positive cell population in different settings, including the targeted delivery of dexamethasone, an immunosuppressive small molecule. The clinical use of purified dendritic cells loaded with self-peptides is a matter of record, but there would be a practical advantage to avoiding cell-based therapies if a purely proteinaceous preparation could be administered to the same effect. Indeed, findings indicated that the combination of a Class II MHC VHH-peptide adduct, in combination with the same VHH conjugated to dexamethasone was remarkably effective at arresting progression of EAE in animals with overt signs of disease.
WK6 E. coli containing the plasmid encoding corresponding VHHs were grown to mid-log phase at 37° C. in Terrific Broth plus ampicillin and induced with 1 mM IPTG overnight at 30° C. Bacteria were harvested by centrifugation at 5,000×g for 15 minutes at 4° C. and then resuspended in 25 mL 1×TES buffer (200 mM Tris, pH 8, 0.65 mM EDTA, 0.5 M sucrose) per liter culture and incubated for 1 hour at 4° C. with agitation. Resuspended cells were then subjected to osmotic shock by 1:4 dilution in 0.25×TES buffer and incubation overnight at 4° C. The periplasmic fraction was isolated by centrifugation at 5,000×g for 30 minutes at 4° C. and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl, and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole, and 10% glycerol and then loaded onto a Superdex 75 10/300 column in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. The peak fractions were recovered and rebounded to Ni-NTA to be depleted of LPS (<2 IU/mg). Bound VHHs were washed with 40 column volumes of PBS+0.1% TritonX-114 and eluted in 2.5 column volumes endotoxin-free PBS (Teknova) with 500 mM imidazole. Imidazole was removed by PD10 column (GE Healthcare), eluting in LPS-free PBS. Recombinant VHH purity was assessed by SDS/PAGE and LC-MS.
The peptides were synthesized on 2-chlorotrityl resin (ChemImpex) following standard solid phase peptide synthesis (SPPS) protocol or ordered on GenScript. For GGG-Cy5, GGGC (SEQ ID NO: 61) (7.0 mg, 24 μmol) was dissolved in DMSO (Sigma Aldrich) (400 μL) and was added to Cyanine 5 maleimide (Lumiprobe) (5.0 mg, 7.8 μmol). The resulting mixture was gently agitated at room temperature until LC-MS analysis show no remaining starting material. The ligated product was then purified by RP-HPLC and lyophilized. LC-MS calculated for GGG-Cy5: C47H62N8O8S2[M+H]+ was 898.44, found 898.56. The resulting powder was stored at 4° C.
For GGG-Dexamethasone (DEX), in the first reaction, dexamethasone (Sigma Aldrich) (25 mg, 64 μmol) and N-β-maleimidopropionic acid hydrazide (ThermoFisher) (40 mg, 135 μmol) was dissolved in 3.0 mL of dry MeOH (Sigma Aldrich) and one drop of TFA was added to the solution. The resulting mixture was agitated overnight at room temperature. The MeOH was then evaporated, the precipitate dissolved in DMSO (1.0 mL), purified by RP-HPLC and lyophilized. LC-MS calculated for DEX-maleimide: C29H37FN3O7 [M+H]+ was 558.26, found 558.32. The resulting powder was stored at −20° C. In a second reaction, DEX-maleimide (20 mg, 36 μmol) and GGGC (SEQ ID NO: 61) (21 mg, 72 μmol) were dissolved in 5% 0.1 M NaHCO3 in DMSO (1.0 mL). The resulting mixture was agitated at room temperature until completion of the reaction. Once no starting material was left, the reaction was directly purified by RP-HPLC and lyophilized. LC-MS calculated for GGG-DEX: C38H53FN7O12S [M+H]+ was 850.35, found 850.21. The resulting peptide was stored at −20° C. and re-dissolved in PBS before at the right concentration before sortase ligation.
C-Terminal Sortagging (Using LPETGG (SEQ ID NO: 43) of VHH or GFP with GGG-Carrying Moieties
Sortagging reactions were carried out in 1 mL mixture containing Tris HCl (50 mM, pH 7.5), CaCl2) (10 mM), NaCl (150 mM), triglycine-containing probe (500 μM), GGG-containing probe (100 μM), and 5M-Sortase A (5 μM). After incubation at 4° C. with agitation for 1.5 hours, unreacted VHH and 5M-SrtA were removed by adsorption onto Ni-NTA agarose beads. The unbound fraction was concentrated and excess nucleophile with an Amicon 3,000 kDa MWCO filtration unit (Millipore). Reaction products were analyzed by LC-MS for purity and stored at −80° C.
All animals were housed in the animal facility of Boston Children's Hospital (BCH) and were maintained according to protocols approved by the BCH Committee on Animal Care. C57BL/6J (CD45.2+), B6.SJL-Ptprc (CD45.1+), NOD/SCID, BALB/c, B6/2D2, NOD/BDC2.5, Balbc/DO11.10, CD11c-DTR, μMT−/−, Batf3−/−, LAG3−/−, and FoxP3-DTR mice were either purchased from the Jackson Laboratory or bred in house. MHCII-GFP and PD1−/− mice were bred in house. OTI Rag2−/− and HLA-DR4-IE-transgenic C57BL/6 IAb null mice were purchased from Taconic.
Cells were harvested from spleen, lymph nodes, or other organs and were dispersed into RPMI1640 through a 40-micron cell strainer using the back of a 1 mL syringe plunger. Cell mixture were subjected to hypotonic lysis (NH4Cl) to remove red blood cells, washed twice in FACS buffer (2 mM EDTA and 1% FBS in PBS) and resuspended in FACS buffer containing the corresponding fluorescent dye-conjugated antibodies. All staining was carried out at 1:100 dilution and with Fc block for 30 minutes at 4° C. in dark. Samples were washed twice with FACS buffer before further analysis. All flow data were acquired on a FACS Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Antibodies used in this study are listed in Table 3.
Female C57BL/6 mice (10-12 weeks of age) or other mouse lines with C57BL/6J genetic background were immunized with Hooke kits: an emulsion of MOG35-55 in CFA and PTX in PBS according to the manufacturer's instructions (Hooke laboratories). Mice were scored daily, starting on day 7 post-immunization by an investigator blinded to the experimental treatment of individual mice. Mice were assigned to different experimental treatments randomly and cohoused together to eliminate inter-cage variability. All treatments were carried out on at least 3 mice and in at least two independent experiments, as indicated in the figure legends. All animals were included in the analyses. Clinical score is defined as follows: 1, limp tail; 2, partial hind leg paralysis; 3, complete hind leg paralysis; 4, complete hind and partial front leg paralysis; and 5, moribund. Easy access to wet food and water was provided for the experimental mice throughout the disease progression. Unless indicated otherwise, for prophylactic treatment, 20 μg sortagged VHH-antigens were intravenously administered 7 days prior to induction of EAE. For therapeutic treatment, 20 μg VHHMHCII-OVA323-339, VHHMHCII-MOG35-55, or 20 μg VHHMHCII-MOG35-55 mixed 20 μg VHHMHCII-DEX were administered on the day of EAE when the mice exhibited symptoms defined as clinical score of 1, 2, and 3 as indicated. At day 30 post-EAE induction or when mice reached clinical score of 4, mice were sacrificed by asphyxiation and then perfused with 5 mM EDTA in PBS. Spinal cords were isolated and fixed in 10% (wt/vol) formalin solution (Sigma), embedded in paraffin, sectioned at 20 μm, and stained with H&E or Luxol Fast Blue (Harvard Medical School Rodent Histology Core Facility). Stained sections were imaged at 4× and 10× magnification. Isolation of the immune cells that infiltrate the spinal cord was carried out by homogenizing the spinal cord, followed by 38% Percoll (Sigma) gradient separation (100% Percoll is 1.123 g/mL). Isolated cells were plated in 48-well plates and treated with 50 ng/mL PMA (Sigma) and 500 ng/mL ionomycin (Sigma) for 2 hours at 37° C. in complete RPMI media, followed by the addition of 10 μg/mL Monensin (Sigma) and incubated for 2 more hours. Cells were then surface stained, fixed, and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific, 00-5523-00) according to the manufacturer's protocol. Intracellular and Foxp3 staining were performed according to the manufacturer's protocols and cell samples were then used for flow cytometry.
For cytokine storm analysis, blood samples were taken 5 hours post therapeutic treatment with 20 μg VHHMHCII-MOG35-55, VHHMHCII-OVA323-339, or 20 μg VHHMHCII-MOG35-55+20 μg VHHMHCII-DEX on the first day these EAE mice reached clinical score of 3. Blood was collected in EDTA containing tubes and plasma was isolated via repeated centrifugation (500 g, 5 min, 4° C.). Plasma was stored at −80° C. until further analysis of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). TNF-α (ThermoFisher, 88-7324-22) and IL-6 (ThermoFisher, 88-7064-22) ELISAs were conducted according to manufacturer's protocol.
CD8 T-cells were depleted by administering 400 μg of anti-CD8a depleting antibody (clone 2.43, BioXCell) intraperitoneally twice weekly beginning 2 weeks prior to prophylactic treatment with VHH-antigen and throughout the EAE observation window. Macrophage subsets were ablated by injecting 300 μg anti-CSF1R (clone AFS98, BioXCell) every other day from 2 weeks prior to prophylactic treatment up to the end of the experimental set up. To deplete DCs, 100 ng DTX (Sigma) was administered intraperitoneally into CD11c-DTR mice 2 days prior to VHH-antigen administration. For depleting Tregs, FoxP3-DTR mice were injected with 3 doses of 1 μg DTX (Sigma) intraperitoneally at day −9, −8, and −1 prior to prophylactic treatment with VHH-antigen and weekly afterwards until the end of observation window. Cellular depletions were confirmed by flow cytometry of PBMCs or splenocytes.
Splenic and iLNs-derived CD4 T cells from 2D2 mice were enriched by negative selection using magnetic beads (Miltenyi Biotec, 130-104-453) and labeled with Violet CellTrace (ThermoFisher Scientific, C34571) as per the manufacturer's protocol. 500,000 of these 2D2 CD4+ T cells were transferred into CD45.1+ mice. Transfusion of 20 μg VHHMHCII-OVA323-339, 20 μg VHHMHCII-MOG35-55, equimolar of MOG35-55 peptides, or 100 μg MOG35-55 peptides mixed with 25 μg anti-CD40 (SouthernBiotech) and 50 μg PolyI:C (Sigma) as adjuvant was carried out the day after adoptive transfer. At day 3, 5, and 10, mice were sacrificed and spleens, iLNs, and blood were collected and analyzed by flow cytometry. Some 2D2 T cell adoptively transferred mice were also challenged on day 3 or 10 with 100 μg MOG35-55 in CFA subcutaneously. Mice were sacrificed 7 or 5 days later as indicated in the respective experimental set up. Spleens, iLNs, and blood were harvested and analyzed by flow cytometry.
Cells were sorted and lysed in RLT lysis buffer (Qiagen) supplemented with β-mercaptoethanol. RNA was the isolated using a RNeasy Micro kit (Qiagen) according to the manufacturer's protocol. 20 ng of RNA were used as input to a modified SMART-seq2 protocol. The resulting library was confirmed using a High Sensitivity DNA Chip run on a Bioanalyzer 2100 system (Agilent), followed by library preparation using the Nextera XT kit (Illumina) and custom index primers according to the manufacturer's protocol. Final libraries were quantified using a Qubit dsDNA HS Assay kit (Invitrogen) and a High Sensitivity DNA chip run on a Bioanalyzer 2100 system (Agilent). All libraries were sequenced using Nextseq High Output Cartridge kits and a Nextseq 500 sequencer (Illumina). Sequenced libraries were demultiplexed using the bcl2fastq program and the resulting Fastq data were trimmed and cropped with Trimmomatic. Alignment to the mouse mm10 reference genome and gene expression counts were carried out using Kallisto. Principal Component Analyses (PCA) were carried out in R. To test for differential gene expression from our RNA-seq data and differential chromatin accessibility in individual loci, the DEseq2 method was used. Volcano plots and heatmaps were generated in Python 3.6 using NumPy 1.12.1, and Matplotlib 2.2.2. For functional analyses, Gorilla (Gene Ontology Enrichment Analysis and Visualization Tool) was used to find enriched Gene Ontology (GO) terms in the up-regulated and down-regulated subsets of the top 500 most differentially expressed genes.
Spleen and inguinal lymph nodes were harvested from 7-9-week-old BDC2.5 mice. Cells were resuspended in complete RPMI (RPMI supplemented with 2 mM glutaMAX, 10 mM HEPES, non-essential amino acids, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, 10% heat-inactivated FBS) supplemented with 0.5 μM p31 peptide (BDC2.5 mimotope, GenScript) and plated in tissue culture dishes at 1 million cells/mL. After four days, cells were harvested, washed twice and resuspended in PBS. 5 million cells were adoptively transferred into 9-12-week-old female NOD.SCID mice via retro-orbital injection. Saline, 20 μg VHHMHCII-p31, or VHHMHCII-MOG35-55 were infused into the mice a day or 5 days later as indicated. Blood glucose measurements were carried out every other day for 2 weeks and weekly for up to 1-2 months. Mice were considered diabetic when their blood glucose level exceeded 260 mg/dL for two subsequent weeks as measured by using the Active meter (Accu-Chek) (range 20-600 mg/dL) with corresponding Aviva Plus test strips (Accu-Check).
Mice were sacrificed via asphyxiation at the 2-month endpoint or when blood glucose levels exceeded 600 mg/dL for two subsequent weeks. The pancreas was fixed for further immunohistochemistry analysis, i.e. H&E staining (Harvard Medical School Rodent Histology Core Facility). In a separate cohort of mice, spleens, inguinal/pancreatic lymph nodes and pancreas were harvested at day 14 post adoptive transfer for flow cytometry analysis.
Spleen and lymph nodes were collected from DO11.10 mice. CD4+ T cells from these mice were enriched by negative selection using magnetic beads (Miltenyi Biotec, 130-104-453). APCs were obtained by irradiating DO11.10 splenocytes at 2000 rad. Differentiation of these naïve CD4 T cells into Th1 phenotypes was induced by culturing them as follows: 200,000 CD4+ T cells and 2 million APCs were co-cultured in complete RPMI containing 0.3 μM OVA323-339 (GenScript), 5 ng/mL IL12 (PeproTech), and 10 μg/mL anti-IL4 mAb (R&D Systems) for 3 days. Cells were then harvested, washed, and counted. A total of 2 million Th1 DO11.10 T cells were injected intravenously into BALB/c recipients. One day following T cell transfer, recipients were immunized subcutaneously with 100 μg OVA in CFA (Sigma-Aldrich). At day 11, heat aggregated OVA (HOA) was injected into the left paw of the mice and paw thickness was measured daily up to day 18. Mice were then sacrificed, and their paws were removed and fixed in 10% (wt/vol) formalin solution (Sigma), embedded in paraffin, sectioned at 20 μm, and stained with Toluidine Blue (Harvard Medical School Rodent Histology Core Facility). Stained sections were imaged at 4× and 10× magnification. Popliteal lymph nodes were also collected and cells were restimulated in vitro with 1 mg/mL OVA in complete RPMI for 3 days for IFN-γ production. IFNγ was measured using the Mouse IFN-γ ELISA Set (BD Biosciences, 555138) per manufacturer's protocol. Sera was also collected at D18 end point for ELISA assays to measure anti-OVA and anti-OVA323-339 antibody responses. 96-well plates were coated with 10 μg/mL of OVA or GFP-OVA323-339 (generated by sortagging GFP-LPETGG(SEQ ID NO: 43) with GGG-OVA323-339) proteins in PBS overnight at 4° C. and incubated in blocking buffer (0.05% Tween20+2% BSA in PBS) before addition of serum samples. Incubation with tested serum was for 3 hours at room temperature. Plates were washed four times with PBS, incubated with goat anti-mouse IgG-HRP (SouthernBiotech) at 1:10,000 in blocking buffer for 1 hour, and developed with 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate reagent (Sigma). The reaction was stopped with 1 M HCl and absorbance was read at 450 nm.
Spleen and lymph nodes were collected from OTI Rag2−/− mice. CD8+ T cells from OTI Rag2−/− were enriched by negative selection using magnetic beads (Miltenyi Biotec, 130-095-236) and labeled with Violet CellTrace as the manufacturer's protocol. 500,000 CD8+ T cells were transferred intravenously into CD45.1+ mice. Transfusions of 20 μg VHHMHCII-OTI or VHHMHCII-ORF8 were carried out the day after adoptive transfer. Mice were challenged on day 10 with 25 μg OTI peptide in CFA (Sigma) and then sacrificed 5 day later for analyses. Spleens, iLNs, and blood were harvested and splenocytes were analyzed by flow cytometry.
Two million splenocytes were plated in 96-well round-bottomed plates and treated with Cell Stimulation Mixtures (eBioscience) and Brefeldin A (eBioscience) for 3 days at 37° C. in complete RPMI [RPMI 1640, 10% (vol/vol) heat-inactivated FBS, 50 μM β-mercaptoethanol, 100 U/mL Pen/Strep, 1× Gibco MEM Non-Essential Amino Acids Solution (Life Technologies), 1 mM Sodium pyruvate, 1 mM HEPES] supplemented with 1 mg/mL OVA peptides. Supernatant was collected and utilized for ELISA to measure Interferon gamma (IFNγ) production. IFNγ was measured using the Mouse IFN-γ ELISA Set (BD Biosciences, 555138) per manufacturer's protocol.
OB1 is a 17-mer B cell epitope derived from OVA. C57BL6/J recipient mice were intravenously injected with 20 μg VHHMHCII-OB1, equimolar amount of OVA proteins, or PBS at day 0. Subsequent boosts were carried out on day 7 and day 14. Serum samples were collected pre-immunization and 7 days after the last boost. For OVA-specific and OB1 peptide-specific ELISA, 96-well plates were coated with 10 μg/mL of OVA or GFP-OB1 proteins in PBS overnight at 4° C. and incubated in blocking buffer (0.05% Tween20+2% BSA in PBS) before addition before addition of serum samples. Incubation with tested serum was for 3 hours at room temperature. Plates were washed four times with PBS, incubated with goat anti-mouse IgG-HRP (SouthernBiotech) at 1:10,000 in blocking buffer for 1 hour, and developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate reagent (Sigma). The reaction was stopped with 1 M HCl and absorbance was read at 450 nm.
DR4-IE mice were immunized with 400 μg of human PLP175-192 (hPLP175-192) emulsified in CFA subcutaneously. The mice also received 300 ng of Pertussis toxin intravenously on days 0 and 3. At day 7, mice were given second boost subcutaneously with 400 μg of hPLP175-192 emulsified in Incomplete Freund's Adjuvant (IFA). Mice were weighed and scored daily starting on day 7 after immunization. The clinical score system was carried out similarly as the EAE model in C56BL/6J mice. On the first day a mouse reached a clinical score of 3, either 2 μg anti-human MHCII VHH (VHHhMHCII) carrying an irrelevant peptide control or 20 μg VHHhMHCII-hPLP175-192 mixed with 20 μg VHHhMHCII-DEX was administered intravenously. Flow cytometry of the spinal cords was described as above.
All data represented at least two independent experiments. All statistical analyses were performed using Prism 6. Statistical methods used are indicated in the corresponding legend of each figure. Statistically significant differences are indicated by asterisks as follows: *p<0.05; **p<0.01; ***p<0.001.
A Single Dose of VHHMHCII-MOG35-55 Provides Durable Protection Against Induction of Experimental Autoimmune Encephalomyelitis (EAE).
Described herein were the generation and characterization of an alpaca-derived single domain antibody (i.e. VHHMHCII) that recognizes a wide range of mouse Class II MHC molecules, including I-Ab and I-Ad. This VHH was engineered to carry a sortase recognition motif—LPETGG (SEQ ID NO: 43)—to allow its site-specific ligation (
Immunization of C57BL6 mice with MOG35-55 under inflammatory conditions, i.e. in the presence of complete Freund's adjuvant (CFA) and pertussis toxin (PTX), within 10-14 days elicits experimental autoimmune encephalitis (EAE), a multiple sclerosis-like condition. Prior administration of MOG35-55, delivered to MHCII+ APCs under non-inflammatory conditions, was predicted to interfere with the induction of EAE. To determine a possible dose of VHH-peptide adduct that might interfere with onset and severity of symptoms, 3 doses of 20 μg VHHMHCII-MOG35-55 adduct were administered intravenously (i.v.) 7 days prior to induction of disease. This treatment completely suppressed induction of EAE, whereas mice that received the identical amount of VHHMHCII conjugated to an irrelevant peptide (VHHMHCII-OVA323-339), or MOG35-55 peptide linked to a VHH of irrelevant specificity (VHHGFP) progressed to EAE (
To explore the durability of protection induced by VHHMHCII-MOG35-55, a single dose of VHHMHCII-MOG35-55 was administered one or two months prior to induction of EAE with the MOG35-55/CFA/PTX cocktail. Delayed onset, if not complete suppression of EAE, was observed (
Splenic CD11c+ DCs are APCs Associated with Induction of Antigen-Specific Tolerance
To explore possible mechanisms of VHHMHCII-mediated induction of tolerance, VHHMHCII-Alexa 647 (
Intravenous, but not subcutaneous or intraperitoneal injection of VHHMHCII-MOG35-55 protected against induction of EAE (
To determine whether delivery by VHHMHCII of more than just the minimal epitope can likewise induce tolerance, VHHMHCII-MOG17-78 was generated and used to treat mice 7 days prior to challenge. VHHMHCII-MOG17-78 likewise protected against induction of EAE (
Administration of VHHMHCII-MOG35-55 Elicits a Burst of Proliferation, Followed by Attrition, of MOG35-55-Specific CD4 T Cells.
To investigate the impact of VHHMHCII-MOG35-55 adducts on T cells of defined antigen specificity, 2D2 TCR transgenic mice were used as a source of monoclonal CD4+ T cells that recognize the I-Ab-MOG35-55 complex. Congenically marked, Violet CellTrace-labeled 2D2 CD45.2+ CD4+ T cells were transferred into CD45.1 recipients, followed by injection (i.v.) of VHHMHCII-peptide adducts a day later. The number of 2D2 cells in spleen, inguinal lymph nodes (iLNs), and blood was tracked for 10 days. Mice that received VHHMHCII-MOG35-55, 2D2 CD4+ T cells underwent an initial burst of expansion, followed by contraction 5 days after injection, as determined by the absolute number of 2D2 cells recovered from spleen, iLNs, and blood as well as whole body imaging using non-invasive positron emission tomography (PET) imaging for CD4+ cells (
MOG-Specific 2D2 CD4 T Cells Upregulate Co-Inhibitory Receptors Upon Administration of VHHMHCII-MOG35-55.
To corroborate these results, the transcriptome of 2D2 T cells in VHHMHCII-MOG35-55 recipients was examined. 2D2 CD4 T cells at different divisional stages were sorted (
Administration of VHHMHCII-MOG35-55 Induces MOG35-55-Specific Regulatory CD4 T Cells.
To uncover a role for regulatory T cells in VHHMHCII-MOG35-55-mediated tolerance, Tregs was eliminated in Foxp3-DTR mice by administration of DTX (
Next, the ability of the VHH-antigen adducts to interfere in other autoimmune conditions was tested. For type-1 diabetes (T1D), the aggressive BDC2.5 T-cell adoptive transfer model that mimics autoreactive T-cell-mediated destruction of β-cells was used. Transgenic CD4 T cells that carry the BDC2.5 T-cell receptor recognize pancreatic β cells and can be activated ex vivo with the mimotope p31. In NOD/SCID mice, such activated BDC2.5 T cells cause hyperglycemia within 8 days after transfer. p31 was conjugated to VHHMHCII (
Arthritis can be induced in BALB/c recipients by intravenous transfer of ex vivo activated Th1 DO11.10 T cells that recognize OVA323-339, followed one day later by a footpad injection of OVA/CFA emulsion and a challenge 10 days later by heat-aggregated ovalbumin (HAO) (
Combined, these results confirm the ability of VHHMHCII-antigen adducts to reduce the harm inflicted by activated, autoreactive CD4 T cells. The underlying mechanism(s) must be conserved across mouse MHC haplotypes.
To determine whether CD8 T cell responses are affected by administration of VHHMHCII-antigen adducts, the OVA-derived CD8 T cell epitope SIINFEKL (the OTI peptide restricted by H-2Kb) was attached to VHHMHCII (
Co-Delivery of VHHMHCII-MOG35-55 and VHHMHCII-Dexamethasone Increases Therapeutic Efficacy.
The impact of VHHMHCII-MOG35-55 administration to mice already symptomatic for EAE was then explored. Injection of VHHMHCII-MOG35-55 into mice that had developed a clinical score of 1 (limp tail), halted progression of EAE in 9 out of 16 mice (
The polyclonal nature of the evoked T cell response and the rather superficial clinical scoring system imply heterogeneity in the diseased cohort, which may explain why not all animals that received VHHMHCII-MOG35-55 responded similarly. It was then tested whether it might be possible to co-deliver an immunosuppressive drug to avert a cytokine storm. The immunosuppressive corticosteroid dexamethasone, attached via a self-hydrolyzing hydrazone linker to VHHMHCII, was delivered to Class II MHC+ cells (VHHMHCII-DEX;
A VHH that recognizes a wide range of human Class II MHC molecules (VHHhMHCII) was developed. This VHH was prepared in a sortase-ready format and modified with several self-antigens of human origin (
Human MOG97-108 peptide (TCFFRDHSYQEE (SEQ ID NO: 53)), hPLP175-192 peptide (YIYFNTWTTCQSIAFPSK (SEQ ID NO: 42)) and DEX to VHHhMHCII were attached (
A frequent target of autoantibodies in RA patients are post-translationally modified antigens such as Fibrinogen a that carry citrulline, a modified arginine residue. Hence, VHHhcu was modified with citrullinated Fibα79-91 (QDFTNCitINKLKNS (SEQ ID NO: 50),
To explore the mechanism of VHHMHCII-mediated tolerance induction, VHHMHCII-Alexa647 was constructed to follow the biodistribution of the VHHMHCII adducts. 20 μg VHHMHCII-Alexa647 was administered intravenously to Class II MHC-GFP mice. At 1.5 hours after injection, the majority of VHHMHCII-Alexa647 was captured by a splenic MHCII-GFP+ cell population in vivo (
In the EAE model intravenous, but not subcutaneous or intraperitoneal, injection of VHHMHCII-MOG35-55 conferred protection against induction of EAE (
The induction of antigen-specific tolerance is an aspirational goal in the treatment of auto-immune diseases. This is a particularly high bar to clear if one considers the presence of pathology and pre-existing autoimmunity at diagnosis. Auto-immune destruction of target cells is already well on its way before symptoms arise. Therapy must therefore deal not only with existing autoimmunity but also with the possibility of epitope spreading beyond the initiating insult. Any type of prophylactic treatment will be of limited value unless susceptible populations can be unambiguously identified, and then only if the risk of eliciting unwanted side effects is acceptably small.
In addition to curbing inflammation, wholesale immunosuppression has been the backstop in the treatment of autoimmunity, which comes with an increased risk of infectious disease. While antibiotic treatment can mitigate this drawback at least in part, the search for a more targeted approach to blunt undesirable immune reactions remains a priority. Most auto-immune diseases are T cell-mediated; T cell activation involves professional antigen presenting cells. If antigen presenting cells (APCs) acquire antigen in an inflammatory environment, upregulation of costimulatory molecules as well as the production of the proper mix of cytokines contribute to T cell activation. Tolerogenic dendritic cells are devoid of such costimulatory signals and consequently antigen presentation under non-inflammatory conditions promotes a state of non-responsiveness or tolerance. This concept has driven the exploration of tolerogenic dendritic cells. Dendritic cells can be sub-divided into subsets with distinct functional capacities, for example the ability to engage in antigen cross-presentation is a property mostly ascribed to the DC1 subset. The identification of surface receptors involved in antigen acquisition has identified DEC205, DC-SIGN and Clec9a as particularly relevant for entry of antigen into cross-presentation pathways. While pursued primarily as strong inducers of desirable immunity, such as anti-tumor responses, their ability to induce regulatory T cells as a means of reducing unwanted responses is considered no less important.
This rather narrow focus on dendritic cells has overshadowed earlier work in which antigens were targeted to Class H MHC products, expressed on all antigen-presenting cells, through the creation of anti-Class II MHC antibodies conjugated to self-antigens, It is, after all, the Class H MHC peptide complex that is the call to arms for the CD4 T cell compartment. For this reason, autoantigens were delivered under non-inflammatory conditions to Class II MHC-positive cells, a strategy that does not differentiate among the various APC subsets, but is efficacious nonetheless. Ideally, interventions ought to be antigen-specific and as simple as possible, both from a manufacturing and application perspective.
This data establishes that a MOG35-55-modified VHH that recognizes Class H MHC products can protect mice against the induction of EAE. A single injection of 10 micrograms of the adduct afforded protection that lasted for at least two months following administration of the nanobody-peptide adduct. Administration of the same VHHMHCII-MOG35-55 adduct in animals that already show symptoms of EAE (score 1, 2, or 3) halted progression, and even partially reversed the severity of the symptoms. When treating animals with EAE symptoms, only a subset responded, whereas the remainder showed rapid exacerbation, followed by death attributable to a cytokine storm. In symptomatic animals an inflammatory environment already exists, and delivery of the VHHMHCII-MOG35-55 adduct to APCs only added fuel to the fir. To overcome this acute response, a VHHMHCII-dexamethasone adduct was co-delivered, which dramatically improved survival, with no deaths.
Administration of nanobody-peptide adducts in the presence of anti-CD40 and poly dIdC as adjuvants strongly potentiated antibody responses against them. Administration in a setting where there is a chronic inflammatory response would be possible only if appropriate countermeasures were available, as in the case of the VHHMHCII-dexamethasone adduct.
The pharmacokinetic properties of nanobodies make them attractive for the construction of antibody-drug conjugates (ADCs). Nanobodies have a much shorter circulatory half-life than full sized antibodies, thus minimizing systemic exposure to compounds that are toxic. Their targeting properties are excellent, ensuring that once on site, self-immolating linkers will release the payload predominantly at the intended site. Full-sized immunoglobulin-based ADCs continue to circulate for periods up to weeks and release payloads directly into the bloodstream upon hydrolysis of the linkers via which the drugs are attached. The VHHMHCII-dexamethasone adduct thus has the desired properties of excellent targeting, as verified by non-invasive imaging, short circulatory half-life and ease of modification. The cellular targets recognized by VHHMHCII include all Class II MHC-positive cells. Even if the APCs responsible for induction of tolerance and for provoking a cytokine storm are distinct, the Class II MHC-based targeting approach would obviously cover both. Nanobody-drug adducts have yet to find the broad range of applications of their full-sized counterparts, but these data show it is an opportunity not to be discounted.
As to the mechanism that underlies the remarkable ability of anti-Class II nanobodies to induce tolerance against an attached payload, many possibilities can be excluded, based on the response seen in knock out mice or upon depletion of certain sets of cells. It is still unknown whether a single type of APC can be tolerogenic if targeted under non-inflammatory conditions, while provoking a strong response if antigen is encountered in an inflamed environment.
A single-domain antibody fragment (nanobody or VHH) that binds MHC class II antigens (VHHMHCII) was isolated and characterized with nanomolar affinity. To adapt this vaccine platform for SARS-CoV-2, a recombinant protein consisting of a fusion between VHHMHCII and the SARS-CoV-2 receptor-binding domain was generated (VHHMHCII-SpikeRBD) (
To confirm the immunogenicity of VHHMHCII-SpikeRBD, C57BL/6J mice were intraperitoneally primed with 20 ug of adjuvanted (poly dIdC and anti-CD40 monoclonal antibody) SpikeRBD, adjuvanted VHHMHCII-SpikeRBD, or adjuvant alone and were subsequently boosted with the homologous vaccine at post-prime as indicated (
A functional correlate of serological response was next evaluated by assaying the neutralization capacity of the resulting sera against vesicular stomatitis virus (VSV) pseudo-typed with the SARS-CoV-2 Spike glycoprotein. The sera obtained from mice immunized with the VHHMHCII-SpikeRBD fusion outperformed those from mice immunized with SpikeRBD only (
A robust CD8+ T cell response is important for the clearance of virus infected cells. Therefore, mice were immunized with a single dose of either VHHMHCII-SpikeRBD or SpikeRBD, each in the presence of adjuvant (
To distinguish between CD4+ and CD8+ T cells as the source of IFNγ, a flow cytometry assay was conducted, followed by intracellular cytokine staining. Most of the inflammatory cytokines were observed to arise from a CD8+ T cell response, based on the incubation of splenocytes with a mixture of peptides 42, 47, 48, and 49 (
Moreover, a strong CD8+ T cell response is observed with merely a single immunization and arises within 7 days post-immunization. This strongly suggests that VHHMHCII-SpikeRBD is capable of providing protective immunity against the SARS-CoV-2 infection, as the immunized cohort demonstrates a T cell response relatively early while waiting for the slower humoral response to emerge. Together, these data indicate the superiority of directly targeting APCs via Class II MHC.
With the scale of the SARS-CoV-2 (COVID-19) pandemic, immunization with three or more doses is likely to be impractical, yet a strong CD8+ T cell response may be possible by immunization with a single dose. Therefore, an experiment was conducted in which animals received two successive doses of VHHMHCII-SpikeRBD (
The experiments described in
It was then explored whether the VHHMHCII-SpikeRBD vaccine preparation could survive room temperature storage and lyophilization, yielding a final ‘dry’ product at room temperature, without loss of potency. All methods of storage that were tested produced equivalent levels of total IgG, in addition to other Ig isotypes previously observed (
Another key consideration is whether a VHHMHCII-SpikeRBD vaccine would perform well for all age classes, in particular for aged individuals. The VHHMHCII-SpikeRBD vaccine was therefore tested in aged mice (72 weeks old, equivalent to human age 56-69 years old), wherein it demonstrated a robust total antibody response against the SpikeRBD (
All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.
Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
Where websites are provided, URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses. The actual web addresses do not contain the parentheses.
In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.
This Application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 63/033,710 entitled “NANOBODY (VHH) CONJUGATES AND USES THERE OF,” filed on Jun. 2, 2020, and of U.S. Provisional Application Ser. No. 63/154,455 entitled “NANOBODY (VHH) CONJUGATES AND USES THERE OF,” filed on Feb. 26, 2021, the entire contents of each of which are incorporated herein by reference.
This invention was made with government support under P01DK011794 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/035428 | 6/2/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63154455 | Feb 2021 | US | |
| 63033710 | Jun 2020 | US |
